Microbial Ecology Of The Rhizosphere

2 Microbial ~cology of the Rhizosphere HARVEY BOLTON, Jr., and JAMES K. FREDRICKSON Balteile Pacifie .\'orl/1I"cSI La60ralories, Richland, Washinglon

LLOYD F. ElLIOTT Agricultllral Research Sen'icc, U.S, Department of Agricu/llIre, Con'ailis, Oregon

I.

INTRODUCTION

Microbial ecology of the rhizosphere refers to the study of the interactions of microorganisms 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 and influenced 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 the physical localion around lhe root (sphcre). The rhizosphere has been further subdivided by some researchers into the eClorhizosphcre, or an outer rhizosphere, and the endorhizosphere, ar an inner rhizosphere, where invasion and colonization of root cortical cells by soil microorganisms occurs (Balandreau and Knowles, 1978; Dommergues, 1978). Portions of lhe rhizosphere can also be called lhe mycorrhizo.\;7he:e ,;'hen lhere are mycorrhizal fungi associaled with /OOIS (Linderman, 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 rhizáplane (Clark, 1949), It is often functionally or experimentally difficult to distinguish lhe rhizoplane from the rhizosphere. ln this re\'iew, lhe term rhizosphere will encompass bOlh the rhizosphere (endorhizosphere and ectorhizosphere) and lhe rhizoplane. The rhizosphere is lhe physical location in soil where plants and microorganisms interacl. II has been estimated that one ",heat plant (Triricum aeslÍ\'III1l) can produce a total rool length of 71,000 m, which canstitutes a large surface area ",hen dispersed throughout the soil (Pa\·lychenko. 1937), The interest in rhizosphere microbiology derives from the abilily af the sáil microbiot,a to influence ·plant growth and vice versa. The study of rhizosphere processes requires a multidisciplinary approach and is extremely challenging because of the complexity of this system, 27

Bollon el aJ.

28

Schroth and Weinhold (1986) stated that "... those who enjo)' studying orderly systems amenable to quantitative analysis are likely to consider rhizosphere investigations as a masochist"s delight:" A general definition of ecology is lhe study of both ecosystem structure and function (Odum. 1971). Ecosystem structure involves (1) the composition of the biological 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 ex istence, sllch as temperature and light. Ecosystem function involves (1) energy flow through the ecosystem and biogeochemical cycling and (2) biological or ecological regulation, including both regulalion of organisms by environment and regulation of environment by organisms (Odum. 1962). The ecology of microorganisms in·the rhizosphere is also the study of stru911re and fllnction. An unâerstanding of the basic principies of rhizosphere microoial ecology. including 1he fllnction and diversit)' of the microorganisms that reside there, is necessary before soil microbial tcchnologies can be applied to the rhizosphere. The purpose of this chapter is to introdllee the reader to some general principies and processes that occur in the rhizosphere. The reader is also directed to several olher excellent reviews on rhizosphere microbial ecology (Balandreau and Knowlcs, 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 rod·shaped 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 rhizosphere and how microorganisms can influenee the growth of planls atÍd other mieroorganisms will be diseussed. Finalll', the researeh needed in rhizosphere eeologl' to aid the development of rhizosphere microbial teehnologies and examples of potential leehnologies wiU be diseussed.

II.

THE RHIZOSPHERE EFFECT

A.

Introduction

The rhizosphere effeel is a slimulatioll of microbial growlh surrounding the root because of the release of organic compound;, (Fig. 1; ElIiott et aI., 1984). An understanding of the types of organic (ompounds a\'ailable for microbial growth in lhe rhizosphere and ho\\' various phnical, chemical, and bioJogieal factors influenee lhe release of these compounds from lhe root is necessarl' both to understand the stimulation of microbial gro\\'th and acti\ity in lhe rhizosphere and 10 develop rhizosphere soil microbial technologies. A wide variely of organic compo\Jnds of planl origin have been found in the rhizo;,phere. A standardization of letms was adopted lo avoid eonfusion when discussing lhe sources and namcs of various clas;,cs of organic compounds a\'ailable for microbial grow th. The organic materiaIs from plant roots were classified by Rovira and associates (1979) as follow5:

1.

2.

lh)

Ewdlilcs: low molecular weight cornpound;, (i.e., sugars, amino acids) lhal leak from intacl ceUs Sccrclioll5: compounds lhat are aClively released from rool edis

Bolton el aI.

30

3.

4. 5.

Planr mucilage: there are four sources from various parts Df the rOOl including a. Secretions by the golgi bodies of the root cap cells b. Hydrolysates Df the primary cell wall Jocaled between the roo I cap and the epiderrnis c. Secrelions by epidermal cells and roOl hairs Wilh primary walls d. Compounds resulting from the microbial degradation and modification of dead epidermal cells Mucigel: gelalinous material on lhe root surface composed of plant mucilage, bacterial cells, melabolic products, and colloidal organic and minera! malerial Lysales: material released lhrough the lysis Df older epidermal cells

Locations on the plant root at which these organic substrales may be released are presented in Figure 2. ., There has long been interest ih root-derived organic C in the rhizosphere because of the enhancemenl of microbial growth on and near the roo!. ln fact, Hillner coined lhe ferm rhizosphere in response to obscrvations oE enhanced microbial growth surrounding lhe roaIs of legumes, which was assumed to be caused by the excretion of organic materiais (Cu ri and Truelove, 1986). It has been postulated thal the re!case of organic C from the planl root is in response to injury or microbial attack, or from naturally occurring Ieaky pIdnl mel]lbranes. However, it has aIso been suggested Ihal the planl has evolved C leakage to stimulat~ an active rhízosphere microflora. The microflora can, in tum, promole plant growlh by enhancing soi1 organic marrer rransformations, mobilizing inorganic nUlrienls, pró-

__ Rool SOII

~ 'i-:

N~;~~~i~f

Epldermal and Cortical CeJls lysed and lnvaded by Bacleria

5 d

20 mm

-

':\ Micro Ofganisms wilh Microbial and Plant Mucilages

J

=::

J

3c

4

5,,,,,,",, '"" .-, "''' } --1&2

-.-

J - H I - - - - - - - - - - - - - 3b Sloughed Rool Cap Cells 3a

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

Microbial Ecology of lhe Rhizosphere

31

ducing growth-promoting substances, acting as antagonists against pathogens, and by other mechanisms treated elsewhere in this book. The rele ase of organic C compounds íTOm the root into the rhizosphere can be,an appreciable 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 released into the soi!, presumably from autolysis ofthe root conexo Barley (Hordeum vu/garis L.) 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 the total "CO, fixed by the plant was lost from the roots of both barley and"wheat grown in nonsterilesand. Native plant gras~ species can also exude significant quantities of their fixed C. AgropyrOIl crisrarwll, A. smirhii, and Boute/oua gracilis roots released 8, I7, and 15'7c, respeclÍvely, of the total C fixed by the plant inlO the rhizosphere during a 90-day growth period (Biondini et aI., 1988).

B.

Nature of Organic Carbon in lhe Rhizosphere

A wide variet)' of soluble organic compounds that are produced by the plant may be released into the rhizosphere. The nature of plant-derived compounds found in the rhizosphere is dependent on plant species, growth conditions, rooting medium, and stage of plant de\'Clopment. Amino acids, sugars, organic acids, proteins, pol)'saccharides, growth-promoting and growth-inhibitingsubstances, ali have been reported as root exudates (H ale et aI.. 1978). Different classes of compounds ha\'C been identified as root exudates from a "ide variet)' of plant species (Table 1). The diversity of compounds that are present in the rhizosphere probably affects the compasition and activit)' af the microbial population that develops in the rhizosphere. Carboh)'drates derived from roots are one of the major sources of C and encrg)' for microbial growth and metabolism ir! the rhizosphere (Foster and Bowen, 1982). Glucose is often cited as a major root exudate from various plant species. Corn (Zea mays L.) grown 36 days 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 compounds were different understerile and nonsterile conditions, dcmonstrating that the microorganisms present COU Id utilize the organic exudates or alter root exudation palterns. Twehe different amino acids were detected in ihe root exudates ofaxenic blue grama seedlings (Boll/cfoua graci/is), but onl)' eight could bc identified (Bokhari et aI., 1979). The nature and abundance of organic campounds probably has a major influence on the t)'pes of microorganisms that colonize the rhizosphere. Most of the studies to date have addressed the gross flux of C from the plant root into the rhizosphere at specific stage. of plant growth and for limited periods. Few s)'stematic studies have been made of the spatial and temporal C flow from roots into the rhizosphere. An understanding of microbial stimulation and seJection processes in the rhizosphere throughou~ the growth cyc1e of the plant will require long-term studies of C rele ase b)' roots and of lhe temporal and long-term effects of this release on the associated microflora.

C.

Factors lhat Affect Organic Carbon Release in lhe Rhizosphere

lt is well established that different plant species release different organic com-

pounds into their rhizospheres. Ea'rly work by Rovirâ (1956) demonslrated lhat

32 Table 1

Bolton el aI. Organic Compounds Detccted

Class of organic compound

a~

Plant Rool

E~"dates

Exudàle components

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, glycine. 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, huIyric, 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, /lrnl,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, hydrocyanic ;,cid. gIYC<J~idcs, saponin (glucosidcs), Organic phosphnrm cnrnpnunlh, ncmatnLic cyst or cgg-halching faclors, ncmalo(k atlr;ICl;ml~, fungai rnycclial gro.....lh slilllulanls, mycclium gro\\"lil inhihitoTs, ZOO"POll' attractant!'., ~pon: and scicroliutn germinalinn s(imulants and inilibilor>, tJaçfl'ríal srirnulanls and inhihirors. parasiric wCl'd

Sugars

g{'rrnin"tiorl ~limu1ators

Ihcrc was a substanti:i1 eliffcrcnce iII the root exudalion I'allerns of oals (111'<'1/(1

5alil'lm/) and pe,ls (J'irtllll mli.l'wn) groWlJ in sand. Pcas excrcled 22 arnino COrnpounds, oats cx('[clcd 14 amino compollnds, and both cxcrctcd fructose and glu· cosc, Thc qllantit)' anel cOlllposilioIl of roo! exudales fmm Irce scedlings can also c1iffer. Smit~ (1969) found eliffcrenl cxlldation pallems amollg se\"enll spceies of pinc (I'il/us nank5ian(l, p. lall/berlill/la, P. radiala, J~ rigida) and hlack loeusl (Robi.nia pscudoacacia). 1\'umerous olher studies havc demonslrated \"arialion in exudation pallems from differcnt planl spccies (Kalznelson el aI., 1955; Ro\"ira, 1'159; Ro\'ira anel Harris, 1961; Vancura, 19(4). The stage of planl c!c\eIOpmenl can also affect lhe composition Df compoullds rcJcased b)' roots into the rhizosphcre. As lhe planl ages, both lhe quantil)' and composition of organic eornpounds Ihal are exudcd often ehangc (Bale et aI., 1978; Vancura et aI., 1977). The amount of amino N-eontaining cofnpounds was greater in the exudates of younger and mature blue grama planls than in lhe exudales of plants at an intermediale stage of de"elopment, and mature blue grama p/anIs exereted signifieantly more sugar Ihan young ones, demonstraling Ihat the amount

Microbial Ecology of lhe Rhizosph

33

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

Stcrilc Root eXlldatc Sugars Glucose

Nonsterile

(J.Lg'g dry root) 7370

3900' 1760'

Arahinnsc huclOso

2~~0

I~OO'

SlIcrosc

1590

1720 NSb

2040 3810 270

3100 NS 4710 NS 190NS 530 NS 470 NS 280NS 110 NS 140 NS 50NS

Chgallic acids Oxalollcctic acid Fumaric acid

Malic acid ('ilric acid Succinic íJcid Iknwic ;,cid Aconitic acio Tarté1rk (Kid Glularic ;,ôd Amitlo Jcid~ GIlIlamic acid r\ Sptlrt ir aciel

Alanine GI)'cino y-Aminohulyric acid

2~70

~80

320 200 100 70 30 71 63 59 39 30

Scrinc

26

Arginin<.'

31 12 lO 17

Ci!utaminc Valinc I,('ucine

126' 52 NS 44 NS 32 NS 18 NS 22 NS 15 NS 18 NS 12 NS lO NS

·Sif,llifiC;lTl1ly dlf(c:rcnl at (1:.:0.05. "1'\S, nol ~j~nirl(ânIJ) (llffl'rcnL SOI"C('. Krafh7~k t'l a\. (1984).

(lf sugar rxuckel "'n \'ar)' with lhe phenological si age of lhe pIam (Bokhari el aI., 1979). C~rboh)'drates \\'cre more dj\,crse and abundant fTOm lhe rool exudates of a 3-t"cek-old '\lgar rnapJe (Accr sacclwrlil71) scedling than a 55-year-old malure Iree, although a greater di\ersity anel amounl of amino N compounds and organic acids werc relcaseel from the uns\lbcrized lips of lhe mature lree rools (Smilh, 1970). The col11position anel aCli\'it)' of the rhizosphere microflora is likel)' lo be altered as a function of time because of changes lhat occ\lr in chc cxudation paetcrns of roots as plants i1gc. Thc same factors that intJucnce planl growth and development wiJl affccl rool exudalion. Tempcralure, irradiancc, sai! moislure content, soil and plant 1)1I1rient slalus, and rOOl injury ar slress ean al\er the <\mounl and composition of root exudalcs. Increases (Rovira, 1959; Vancura, 1967) and decreases (Martin and Kemp, 1980; Vancura, 1967) in lemperalure will increase root exudation. A decrease in light usually decreases exudalion, presumably because of a net decrease in

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 lO wet 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) in· creased under P stress, but decreased under N slress. Mechanical stresses, Dr lhe friction between rools and the porous medium through which lhe rools grow, increase rool exudalion. Barber and Gunn (1974) noled an increase in lhe amounl of amino N-conlaining compounds'and carbohydrates, from 5 lo 9% of lhe tolal dry malter contenl of barle)' rools, when pIanIs were grown in Iiquid cullure containing glass beads when compared with ,liquid culture alone. When agitated in a sand suspension, wheat and pea roots released, in 1 hr, approximatel)' the sarne amount of amino N compounds that was 'released during a 2-week growth period in quies· cent solution culture (Ayers and Tho,rnton, 1968). Foliar applications of fertilizers ând pesticides can also affçct the quantity and composition of TOOt exudates (Hale et aI., 1978). Foliar application of N increased the amino acid and decreased the sugar content of root exudates, whereas a de· crease in the amino acid content and an increase in sugars occurred with a foliar P treatment (BaJasubramanian and Rangaswami, 1969). Organic compounds applied to 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 transformation product were exuded by the roots of coleus (Co/eus b/umei Benth), indicating that organic compounds applied to Jeaves may also be transformed during translocation' and before exudation. Foliar, application of streptomycin did not affeet lhe lotaI quantity of rhizosphere micTOorganisms, but lhe fraction that was gram-negativc bacteria was reduced after the application (Davey and Papavizas, 1961). This demonstrated thal chemicals applied to leaves can potcntialll' alter the comrnunity structure of the rhizosphere, Hormones applied to the leaves can also influencc the rhizosphere microflora. Foliar spraying of Phaseo/us aurcus with up to 100 ppm indoleacetic acid increased the rhizosphere microbial population ovcr that of control plants (Singh, 1982). These results suggest lhat the rhizosphere microflora may be manipulated by the foliar application of chcmicals. Selective stimulation of li microbial isolate that can utilize a specifie compound mal' be achicvcd' bl' foliar application 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 be translocated and released into the rhizosphcre. its colonization of the rhizosphcrc may be enhanced. The presence of microorganisnis inthe rhizosphere wilf increase root exudation. Barber and Martin (1976) found that 5-10% of the photosynthetically fixed C was exudated frombarley roots under sterile conditions, but when microorganisms werc 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,

Microbial Ecology of lhe Rhizosphere

35

respecliveJy (Biondini el aI., 1988). Prikryl and Vancura (1980) fouiJd that the amounl of wheal rool exudale almosl doubJed when Pseudomonas putida .....as presenl in lhe rooling solution, campared wilh growlh under sterile condilions. The reason for Ihis slimulalion af rool exudation by rhizosphere microorganisms is not well underslood. One explanation is that the microorganisms are rapidly metabolizing the available C leaked from the root, thereby creating a concentration gradient leading to funher Jeakage. Microorganisms may also make rools more leaky, eilher by ph)'sically damaging lhe roOts or by producing pIanl hormones or secondary metaboliles Ihal affecl rooI physiology.

D.

Sites af Organic Carbon Release in the Rhizosphere

The 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 aamino groups Ihal reacl wilh lhe ninhydrin lo produce a purple color for free a-amino groups or a yellow cólor for substiluled a-amino groups) excreled from broad bean (Vicia [aha) seedling rools and found specific regions of enhanced exudation. AI first, lhe seedling roais were uniformly excreling ninhydrin-positive subslances, bul with addilional roo I growth, the region behind the rool tip was lhe primary site of exudalion. 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 of excrelion ofninhydrin·posilive substances. Release of ninhydrin-posilive substances occurred along lhe enlire lcnglh of laleral roots as lhey deveIoped. Pulse labeling of planls Wilh 14COZ and subsequenl radiographic examinations of lhe rools háve greatly aided in determining the localions aI which exudalion occurs (McDougarl'and Ro\'ira, 1970; Rovira, 1973). Determination ar lhe exudalionlocation has offered insighl inlo rhizosphere ecology and whelher or not microbial colonization of rool surfaces is enhanced aI Ihese "hol SpOIS" of C leakage. The major sile of C released from seminal wheal roais inlo lhe soil was lhe zone of rool elongation (Rovira, 1973). Much of lhe I"C-labeled malerial was insoluble polysaccharide 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 a major source of exudate. As lhe number of lilleral wheal rools increases, 50 does exudation. This suggesl's lhal exudalion is either from laleral roollips or lhe region at which the lateraIs emerge {rom the main root (McDougall and Rovira, 1970). Occasionally, fixed C can be rapidly Iranslocated and exuded by planl roots. McDougall and Rovira (1970) found discrete arcas of radioacli\'ily ai lhe apices of lhe laleral rools, 1-2 min after pulsing lhe atmosphere wilh l"CO Z' bUl zones of radioaCli\'ily along lhe primar)' roo I and some of the laleral roais were more diffuse afler 2 hrs.

m. A.

THE PHYSICOCHEMICAL ENVIRONMENT Df THE RHIZOSPHERE Introduclion

ln addition 10 C inpuI, there are severa I other planl-induced physical and chemical alteralians of lhe' rhizosphere that affecl the composilionand aClivities of rhizo-

Bolton et aI.

36

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

B.

Physica! Factors

Roots generally grow in soil along the path of least resistance, such as through pores 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 equivalent to their own volume. The soil displaced by root growth causes a ione of compactión around the root in which the soil bulk density increases, and particles tend to orient themselves with their most narrow dimensions parallel to the root (Foster and Bowen, 1982). Soil minerais near .lhe root surface can also be altered, compared with the bulk soil, as a consequence of increased weathering and disaggregation, Amorphous iron and aluminium oxides can also accumulate,- resulting in smaller pores in the soil néar the root (Sarkar el aI., 1979). The tortuosily, ar the palh thal nutrients and water must follow lo arrive at the root, increases in the rhizosphere because the average pore sizc or porosity and pore diameters decrcase in the rhizospherewhen compared with those of bulk sai I. The flow of water from the soil to the root creates.a water potential gradient from 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 the rhizosphere of mesophytic plan/s, whereas "'ater potentials as 'low as -4.00 MPa· may occur inthe rhizosphere ofxerophytes (Foster and Bowen, 1982). Therefor'e, water potential varies fromthe bulk soil to the root surfaee and is a major factOr controlling the composition anil activity of rhizosphere microorganisms. Diurnal fluctuations in plant transpiration will create short-term (i.e., hours) fluctuationsm water potential of the rhizosphere soil as a function of time. Successful rhizosphere microflora must be able to withsland not only low water potentials, but also wide fluctuations that occur over short periods.

C.

Chernica! Faclors

Plant cells secrete not onl)' organic compounds that influence microbial growth, but they also secrete inorganic subslances. Both organic and inorganic compounds can affect the chemical environment of the rhizosphere, thus indirectly affecting the rhizosphere microbiota. Roots can 'selectively absorb and transport ions, thereby altering the chemical composition of the soil solution in the rhizosphere. The pH, Eh' and concentration 'of nutrients and soluble C will be different irí the rhizosphere from those in the bulk soi!. Soluble C released from plant roots into the rhizosphere affecls not only microbial growlh and activity by supplying a C and energy sourc~, but can increase lhe solubilityof cations by complexaliõn. A gentle percolation of the 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 field·gro .....n barley,the soluble soil soJution coneentrations of Mn. Zn, and Cuin the rhizosphere changed. during the growing season, with thegreatest mobilization of these cations occurring earl)' in rhizosphere dev'elopmenl (Linehan et aI., 1985). Changes in rhizosphere pH, eompared with bulk sai!, results from the relcase of H' ar HC0 3 - ions by roots during ion uptake, by the évolution of CO 2 from root and microbial respiralion, and by the rele ase of organic and amino acids b)' roots (Marschner, 1986). As nulrients are absorbed by roots from the soi! soJution, a corresponding ion mUSI be released by the root into the rhizosphere to mainlain ionic balance. For the uptake of a cation, H+, and for the uptake of an anion " HC0 3 ' , are usualIy released (Marschner, 1986). The form of plant-availabJe ~ in the soil directly affeets rhizosphere pH, since the uptake of ammonium N results in a net excretion of H", whereas nitrate ~ uptake results in HC0 3 - excretion. Smiley (1974) showed differences of up to 2.2 pH units in wheat rhizosphere soi! for ammonium N-fertilized versus nitrate N·fertirized plants in the greenhouse and up to 1.2 unils difference in the field. Differences in rhizosphere pH were found among wheat varieties and plant genera. Rhizosphere pH can also influence the activity of plant pathogens. Infection of winter wheat and hyphal growth by lhe take·all fungus (Gaellmannomyces gramillis) was reduced in soils lhal were ferlilized with ammonium N compared with nitrate N (Smiley, 1978a,b). Gaeümallllomyces graminis was sensitive to the aeidic environment of the rhizosphere caused by ammonium N fertilization (Smile)' and Cook, 1973). These sludies showed lhal alleralions of lhe rhizosphere chemical environmenl direclly affccl microbial growlh and aclion in lhe rhizosphere. For some plants, it is not possible lo predict the effect that a mineral N form wilI have on rhizosphere pH. Rape (Brassica llapllS var. Emerald) grown aI highrooting densities in P·deficienl soi! wilh N supplied as N0 3 had a decrease in rhizosphere pH from 6.5 lO 4.1 (Grinsted el aI., 1982). The cation!anion balance of lhe plant tissue showed that more cations Ihan anions .....ere laken up during the cxperiment. It was postulaled thal efflux of H+ from lhe rool occurred to mainlain lhe ionic balance across the rool-soil interface and resulted in lhe lowering of rhizosphere pH (Hedlcy et aI., 1982). The saiI type in which lhe plants are grown can also influence lhe exlent to which rhizosphere pH differs from lhe bulk soi!. Hauler and Mengel (1988) found Ihat 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 the bulk soil. The buffering capacilY of the calcareous soil neulralized the pH effect in lhe rhizosphere. Thus. fertilizet, planl species, and soil type ali influence rhizosphere pH and the pH aI which lhe rhizosphere microflora musl sur\'i\'e, grow, and function. Rool and microbial respiration in lhe rhizosphere creales· a microenvironment lower in O 2 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 measure the rhizosphere's redox polential. It has been poslulated that anaerobic mi· crosites 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 -the rhizosphere of com (2. mays) , wilh denilrificalion aClivity decreasing rapidly a few

Bolton e! aI.

38

millimelers from lhe rool surface. These effecls were hypolhesized la be caused by the íncrease ín soluble organíc material present in the rhizosphere, which r~sulted in an increase in microbial respiration and a subsequent decrease ~n O 2 such that nitrate N was utilized as an eleqron aceeptor. A rhizosphere mieroorganism that is able to funetion with varying eleetron acceptors (i.e., a faeultative denilrifier) maS' be better adapted to surviving and eompeting under the possible fluctuating O 2 concenlralions Ihal can occur ín lhe rhizosphere. -

IV.

MICROBIAL PRESENCE AND GROWTH lN THE RHIZOSPHERE

A.

Inlroduction

The rhizosphere effeet refers to th~ enhanced microbial growth and populalion densities in the rhizosphere from the incre ase in soluble C and nutrients, when eompared wilh those of the bulk soil. Table 3 shows Ihat both higher populations and a greater díversíty of mícroorganisms, as determined by Iransmission eleetron microseopy, \Vere found doser to the plant root (Foster, 1986; Foster and Rovira, 1978). There can be considerable differences in the relatil'e abundance of Vê/rious taxonomie and nutritional groups of microorganisms bet\veen rhizosphere and nonrhizosphere soi! (Ta,ble 4). The ratio of the microbial population in the rhízosphere lo that of the bulk soil (the RIS ratio) has been used as a measurement of microbial enhancemenl in the rhizosphere. The enhanced groll'th of microorganisms in lhe rhizosphere depends on microenvironmental conditions and can extend over 2 mm or more {rom the root surfaee (Foster and Bowen, 1982). .

B.

Location of Microbial Growth

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

Table 3 Distinct Microbial Types Based on Ultrastructural Morphology and ToraI Numbcrs aI Differenr Disrances (ram Subrerranean C/over Roors (Trifolium subterral1eum L.) Determined by Transmission Electron Microscopy of Ultrathin Sections Distance (iLm) 0-1

0-5 5-10 10-15 15-20 Souree: Foster (1986).

Morphologically distinct microbial l)'pes 8 11 6 3 3

120 96 41 34,

13

Microbial Ecology af the Rhizosphere

39

Table 4 Wheat Rhizosphere and Nonrhizosphere Popularions of Some Major Taxonomic and Nulrilional Groups of Soil Microorganisms as Delermined by Plale Counls . Populalions Taxonomic groups Bacleria Acrinomyceles Fungi Prolozoa Microalgae NUlrirional groups Ammonifiers Gas-producing anaerobes Anaerobes Denilrifiers Aerobic ceBulose degraders Anaerobic ceBulose degraders Spore formers Azolobacler

Rhizosphere (Iog CFU/g) 9.08 7.66 6.08 3.38 3.70 8.70 5.59 7.08 8.10 5.85 3.95 5.97 <3.00

Conlrol soil (log CFU/g) 7.7(J>

6.85 b 5.0C!' 3.0C!' 4.43' 6.60b 4.48' 6.78' 5.oob 5.00' 3.48NSd 5.76NS <3.00

RiS ralio' 24.0 6.6 12.0 2.4 0.2 125.0 13.0 2.0 1260.0 7.0 3.0 1.6

'The R'S ralio is lhe ralio of populalions in lhe rhizosphere lO contrai soil beCore log Iransformation of lhe dala. 'Significanll)" differenl aI pSO.OOI. <Significanll): different aI pSO.OS. 'NS, nol significantl)' different. Sou,et: Rouall et a!. (1960).

modified and showed Ihal microbial colonizalion and dislribulion on lhe rhizoplane of several planl genera was nonrandom (Newman and Bowen, 1974). Sludics wilh Pinus radiara rools grown for 14 days showed Ihal colonization was limiled to a few microcolonies occurring on the rool surface and at epidermal (ell junctions. -Microbial colonization of the root surface correlated with the presencc of soi! organic matter, implying that organic matter served as the inoculum source for rhizosphere colonizalion (Bowen and Rovira, 1976). After several days, microbial disttibution was nonrandom, with maximum growlh occurring at junctions along root epidermal cells. Colonization of sloughed root cells was also significan\. Bowen (1979) inoculaled P. radiola roots uniformly with a thin layer of Pseudomonas ftuorescens and found lhatafter 2-days growth in slerile perlite, baclerial growth was enhanced along cell junctions. A 55-fold incre ase in bacterial numbers was found at the junclions of Eucalyplus calophylla rool cells, compared with neighboring areas (Bowen and Rovira, 1976). The proliferalion of bacleria at the junction of epidermal cells indicales thal this is an .area of maximum rool exudation (see previous seclion). Regions of accelerated root lysis between the root tip and base also resulted in enhanced regions of bacterial colonizaiion of 10-day-old seminal wheat roots (van Vuurde and Schippers, 1980). Thus, regions where exudates and rootderived organic material are available provide favorable microenvironments in which enhanced microbial growth and competition will occur. Competition for these microsiles and other microbial inleraclions are discussed in lhe following seclioo.

40

C.

Bolton et aI.

Microbial Colonizalion of Plant Roots and Growth Rates

Colonization of planl rools by microorganisms is nol well underslood. Several factors have been implicated as influencing colonization, including the ability of a microorganism lo adhere lo the rool. Polysaccharídes on the microbial cell surface . appear to be important for several microbial-plant associations such as crown gal by AgrobaclCriwll wmcfacicns (Douglas el aI., 1982, 1985; Matthysse el aI., 1981; Thomashow el aI., 1987) and lhe nodulation of legumes by Rhizobillm specLçs (Cangelosi el aI., 1987; Dazzo el aI., 1984; Leigh et aI., 1985; Smil el aI., 1987). A Pscl/domonas pl/lida slrain aggressively colonized kidney bean (Phascollls mlgaris) rools and was agglulinated by a glycoprolein from kidn-ey bean rools (Anderson el aI., 1988). Two transposon mutants of P. plIlida, which were not agglulinated by lhe glycoprolein, colonized bean rools lo a lesser degree than the wíld type. These mutants adhered to the root at levels.;20- 10 30-fold less than the wild t~'pe, sugge~t­ ing Ihat glycoproteín binding has a role ín their attachmenllo bean roais (Anderson et aI., 1988). Piri (fimbriae), surface proteinaceous strúctures ema-nating from the microbial cell, have been ímplícated in the attachmenl of Klcbsiella and EnIcrobaeler spp. (Haahtela and Korhonen, 1985; Haahtela et aI., 1985; Korhonen et aI., 1983, 1986) and for Bradyrhizobiunl japollielllll (Vesper and Bauer, 1986; Vesper et aI., 1987) to roots. Transposon mutants of B. japolliel/nl Ihat produced twice as many pilia!ed edIs allached at a 2.5-fold higher amount to soybean rool segmenls 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 longterm (i.e., 25 days) colonization ofradish roots under gnotobiotie conditions (James et aI., 1985). Binding was no! reJa!ed to éell hydiophobicity, bu! was enhaneed in the 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 may be 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-negalive rods, including Psel/doll/onas, Flal'Obaelcril/m, and A/caligenes spp. (Álcxa,nder: 1977). !nitia! rooI colonizers are often associated wilh soil organic malteT. Pseudornonads are frequent rhizosphere colonizers becausc the)' are associaled with organic malte r, are a nUlrilionally Cliverse group, and are a group wilh a rapid growth rale (Bowen, 1980). Vancura and Kunc(I977) selectil'ely inhibited bacteria and fungi with antibioties and measured soil respiration in and outside the rhizosphere; lhe}' found lhat baclerial acti\'ily in lhe rhizospherc was greater than fungaI. Rcspiralion of rhizosphere soi\ \Vas deçreased 6-18'7i and 20-45'1< in lhe presencc of cycloheximide (Actidione; fungaI inhibitor) and streptornycin (bacterial inhibitor), respectivcly (Vancura and Kunc, 1977). The abilit)' lo grow on both rools and residues was demonstrated wilh rootcolonizing Pselldomonas spp. which are delelerious to wheal rool growlh (Elliott and Lynch, 1984, 1985; Fredrickson and Ellioll, 1985a). These organisms produce a toxin lhat inhibils wheal root growlh (Bolton and ElIiott, 1989; Bolton ct aI., 1989; Fredrickson and ElIiolt, 1985b). The organisms were initially isoJaled from wheal rools, bUI were able lo colonize the rools of a wide variety of crop planls and crop

Microbial Ecology of lhe Rhizosphere

41

re~idues (Fredrickson el aI.. 1gS7). 'Howe\"er, rOOI growlh inhibitlon was somewhal plant-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 major partion of the lotaI bacterial papulation on lhe residue. The population of a deleterious Pselldol1l0llaS sp. inoculated into soi! increased 100- and lOOO-fold when soil was amended with 0.23 and 2.3'k ground wheat straw, compared with unamended soi! (Fredrickson et aI.. 1987). Stroo and co-wor~ers (1988) showed that a deleterious Pselldol1l0llaS sp. inoculated onto nonsterile wheat straw in the laboratory constituted over 80% of lhe total bacterial population. The introduced Pseudomonas sp. also sUn'h'ed lhtoughout lhe winter in hiEh numpers on b.arley residues in lhe field [i.e., lOó colon, fo'rming units (CFU)fg strawl (Stroo et aI., 1988). Populalions of both the introduced pseudomonad and total hacleria were higher 00 rcsidues under no-till management than in tilled plots. The Pseudomol1Gs sp. intro-' dueed onlo lhe barle)' residuealso colonized lhe roóts of the winter wheilt erop, demonstrating that bacteria that c
Table 5).

Table 5 Generation Times (hr) of Total \'iable \lacleria, Pscl/domo/las spp. anó Baei!!l/s spp. on Pinus radiala Roots and ín Unplanred Soil S~mple

Roo!' l'nplantect soi!

Talai viable bacleria

PSClIdomO/lIlS spp.

8aeillus spp.

7.2 [·U

5.2 77

>100

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

39

Bolton et aI.

42

Bo"",'t:n (1979) calculated generation times 01'1 roots for total microorganisms and 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 Ihis lime was much slower, and when ali the data were eompared, growth curves similar to 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 an initial rapid growth, referred lO as an 'inlensity factor, ieflecling the amount ar richness 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 grováh slowed, 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 is similar, there is a distincI difference, The rool surface 01'1 which the bacteria are growing is simultaneously changing ':~nd growing, which creates spccial probJems for calcuJaling microbial growlh rales, There are three approachcs for reporting microbial populations 01'1 roots. Microbial populalions are reportcd 01'1 the basis of per grams' dr)' weighl of root, per centimetcrs length of root, or as per surfaee area of rool. ln each method, the plant root is growing during the experimenl. If total rool lenglh, weighl. or surface area is used to compare microbial colonization, then the incre ase in these variables as a function of time must be taken. into consideration 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-

5 ..

4

..

õ O

E

3

~

::>

u.

()

2

C)

o Total Bacteria

O ..J

O

Pseudomonas spp.

O~-~--.-~--r----"""------,-

0,0

1.0

2.0

3,0

4,0

Time (days) """"'-ui total bacteria and Psrudomollos spp.

01'1 Pillus radialo roots 0.5-lanted in nonsterile soíl: a, íntensíty factor;

Microbial Ecolog)' of the Rhizosphere isms in laboratory media or plant solution culture are directly related to gro....1hin lhe rhizosphere. Thesé methods do not mimic the soil physical and cht:mical relations outlined earlier that can dramatically influence rhizosphere relationships a~d lhe resultant microbial responses. Researche.rs~ultimatelymust employ nonsterile soil for evaluating rhizosphere technologies, tX;cause the rhizosphere is opêrationally defined as a volume of soi!. In'addition, competition for space and nutrients .....jll be much greater in the presence of an indigenous soi! microbial population. HO\\Íever, experiments insimpler systems that are void of soi! are essential f6r underslanding the mechanisms of rhizosphere microbial dynamics.· A second misconceplion is that microbial growth and population dynamiCs in culture media are diréctly applicable lO the rhizosphere. Although the rhizosphere is an enriched· environmenl, compared with the bulk soi!, it is not comparable wlth a rich laboratory medium. Third is the notion that natural selection will fa\'or microbial'strains lhat benefil planl growlh. The traiis that allow an organism'to survive, compete, 'reproduce, and function in ihe rhizosrhcre are more important Ihan any beneficial effecls on plan! growth. The type of root also influences microbial growth. Seminal roots of wheal supporl a larger rhizosphere population of bacteria, actinomycetes, and fungi than nodal roots (Sívasilhamparam el aI., 1979): These differences were poseulaced to be causcd by analomical differences in the cortical root tissue. The seminal rools tended 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 rhizosphere for microbial growth. Also, the numbers of bacteria, actinomycetes, and fungi in the rhizosphere decreased, whereas populations on and in ehc root eissue increascd with plant age (Sivasithamparam et aI.. 1979). The microbíal ecology of the rhizosphere is also dependent on planl genotypes. Spring wheat lines Cadct and Rescue and two homologous chromosomal substitulion \ines, C-R5B and C-R5D, which ",ere identical with Cadel excert for lhe subSlitulion of chromosome 513 and 5D fTOm Rescue, respeclively. were choscn for study (Neal el aI., 1973). The substitutíon of chromosome 5B from Rescue into Cadel significaml)' altered the populalion size of lolal bacleria and several physioJogícal groups Df rhizospherc microorganisms (Tabie 6). These resuics demonstrare that 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 lhe Rhizosphere of Spririg \\'hea! Lines \\'heal Line Cadet Reseue C·R5B' C·RSO'

Bacleria 8.2b' 8.5a 8.5. 8.3b

CelluJol)'tic PeclinoJylie (Iog CFLJ'g dr)' soil) 3.7b 5.la 5.2. 3.5b

4.6d 6.8a 6.4b 5.8e

AmyJoJytic

Ammonifying

6.4c 7.6b 7.8a 6.6e

7.3e 8.1b 8.3. 7.2c

'DaI a in eaeh cofumn Ihal i. followed by a different letter is significant/y differenl (p:sO.05, n=3). bC·R5B and C·R5D 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 microbiologists and plant breeders and geneticists -éould provide novel methods to alter microbeplant interactions. These manipulations could provide a more thorough understanding of rhizosphere relations and co"uld produce desired results, such as increased crop production and enhanced resistance to soil-borne diseases: The carrying capacity (microbial colonization potential) of barley roots was determined by Bennelt and Lynch (1981) to be 10.7 log CFU/g dry roo!. When a Pseudomonas sp., a Mycoplalla sp., and a CurlobaCleriwlI sp. were inoculated separately at 6.0, 8.0, or 10.0 log CFU/g dry root on gnotobiotically grown barley plants, similar maximum population densities developed after abou14 days. These data suggest thatthe absolute.number of bacteria able to colonize the rhizosphere is at least partly independent of laxonomic or physiological groupings in lhe absence of compelilion. Most microbial colonization, s\jTvival, and growth studies conducted in the rhizosphere use dilulion-plating lecl\niques for lhe enumeralion of microbial populations. But hecause some microhes strongly adhere lo lhe root, lhere is almost never a complete removal of microorganisms from lhe rhizosphere, 'even wilh repeated shaking. Rovira and co-workers (1974) reported t~nfold greater baclerial numbers when eSlimated by direcl microscopy, compared wilh plale counls. The use of plate counls lo enumerate rhizosphere microorganisms will undereslimalc aclual numbers. O\her cultural methods may also be used lo selecl for or againsl specifie microbial phenolypes. Some of lhe rhizosphere microorganisms observed with eleclron microscopy are morphologically uni que and include 10bed and starshaped cel!s or eells wilh spiral arms or elongated segments (Foster, 1986). These ceI! morphologies are usually nol detected by standard cultural methods. This demonstrates that certain rhizoplane microorganisms may be obligatory rhizosphere colonists lhal cannot be cultured with traditionaltechniques or lhal cellular morphology diffcrs according to whether cclls arc growing in the rhizospherc or in an artificial medium. ln studying lhe rhizosphere, iI is importanllo realize that not ali of lhe microbial participanls can be idenlified or isolaled. Indeed, it has been demonslrated lhar microorganisms can exisl in a noneulturable slate in lhe environment, bul retain lheir viability (Colwell et aI., 1985). The evidence indicales that therc may be groups of rhizosphere microorganisms about which we know nOlhing. Oncc a rhizosphcre-competent microbe corne~ into contacl with lhe root environment, colonizalion of the roO! can occur. Organisms associatcd with soil organic material are a prime source of inoculum for rhizo~phere colonization. \Vater movement through the soil can also be a means whereby microorganisms are lransported to the vicinity of lhe root for rhizos'phere colonization to oceur. This mode may bc especially imporlant as a delivcry s)'stem for rhizosphere inoculants. Water percolating through a soil column increased the tntllSport of a Rhi:obilllll sp. (Breitcnbeck el aI., 1988; Madsen and Alexander, 1982) and a Pseudolllollas sp. (Madsen and Alexandcr, 1982) in laboratory experiments. An advancing wetting front also cnhanced the transport of Bradyrhi:obiulll japolliclIIll lhrough soil, indicating Ihal unsalurated 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.,

Microbial Ecology of lhe Rhizosphere

45

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 bacteria! transport and colonization of the rhizosphere in the field may lead to erroneous conclusions. Smith and co-workers (1985) demonstrated that transport of Escherichia coli by percolating water was grealer in intact soil cores than in sieved soil packed in columos. The relative behaviors of the added bacterium and a 0- tracer suggested Ihat bacterial transporl in the intacI soil cores occurred through soil macropores. avoiding adsorption to the soil matrix and transporl through smaller pores with a more tortuous path. Sie\'ing the soil destroyed the macroporosity present in the field. Macropores are Iess resistanl lo root penelration and would most likely allow bOI h an increased root densily in the macropore and the fio\\' of perco!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 hypothesized that J11O\ement of bacteria in the soil and lo other sections of lhe root is callsed by downward water flow (Chao et aI., 1986; Parke et aI., 1986) and by bacterial attachment to the root and movemenl as lhe roo I grows (Howie el aI., 1987). ln an experimental system developed by Howie and associales (1987). percolaling water was virtllal\y eliminated, with water movement occurring only toward the root. Bacterial movement on the roots occllrred as a function of time, with nonmotile 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 seed eolonization by 32 bacterial slrains representing Psel/domonas pI/lida, P. filloresam. and Serriltia spp. (Scher et aI., 1988). Allhough not conclusive, these stlldies suggesl that bacterial motility may not be a major factor influencing hacterial colonizarion of the roo!. Soil type can abo influence root-mediated microhial transport in soil (Trevors el aI., 1990). Downward movement of a genetically cngineered P. jlllorescells occurred only when whcal roots were present and when vertical 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 a loam. 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 the rhizosphere is useflll for rhizospherc ecoJogy and nutrient cycling research. A standard techniqlle for measuring the mass of microorganisms (micrograms of biomass C per gram soil) is \he chloroform fumigation \echnique (Jenkimon and powlson, 1976). This technique relies upon Iysing the majority of the sail microorganisms with chloroform vapor and measuring respiration during the mineralization of the released soluble compounds when the chloroform is removed. This technique has been used to quantify nutrienl content of the soil microbial biomass in the rhizosphere including C (Helal and Sauerheck, 1986; Merckx et aI., 1987; Merckx and 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 microbial biomass 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 because bacteria encased in the mucigel may survive the chlorofOrm fumigation (Martin and Foster, 1985). Errors also can result in systems with high soluble C contents, which Jenkinson and Powlson (1976) mention. . The rate of tritiated thymidine incorporatioo was used to quantify growth rates of 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 the microbial biomass was determined and used to calculate a growth rate. The specific growth 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 with cOO"entional growth rate measurements was eonducted. This technique has the advantage of providing a measurement of in vivo growth rates without relyiog on the culturability 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 to this technique. First, we do not know the efficiency of extracting DNA from the rhizosphere and purifying it to quantify the incorporated thymidine. Curreot research on the fate and effeets of genetically eogineered microorganisms has made great strides in improving the efficiency of DNA extraction from enviroomental samples (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 conversion factor based on four constants is required to ealculate growth rates. Christensen 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 thymidine base eontent of the DNA, the genomic size (i.e., the DNA content) per ceI!, and the specific activity of the thymidine incorporated into lhe DNA must be known. Bacterial synthesis of thymidine, a thymidine pool in the rhizosphere, or poor thymidine uptake kinetics will lead to isotope pool dilution and a low estimate of the growlh rale. Finally, the technique provides an overall growth rate for lhe enlire rhizosphere and offers no information on spatial dislribulions or individual groups of organisms. Dcspite its limitations, this technique could be a useful tool for determining in vivo growth and activity of rhizosphere microorganisms and could aid in identifying competitive strains for biotechnological applications.

E.

Enzymes in lhe Rhizosphere

A wide range of enzymes of both plant and microbial origin may be present in lhe rhizosphere, including oxidoreductases, hydrolases,lyases, and transferases (Lynch, 1990b). Enzymes from the rhizosphere microflora are the main cOneero here. Enzymes catalyze the breakdown of orgamc materiais (e.g., .cellulases, dehydrogenases), fertilizers (e.g., ureases), and organic nutrients to plant-available forms (e.g., phosphatases, sulfatases). The competitiveness of a rhizosphere colonisl may be enhanced 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 enzymatically enhances the cOJJversion ofnutrients from the organic to inorganic form or from the unavailable form to a fonn available for planl growlh. 11 has been directlv demonstraled 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 enzymes were detected by ultracytochemical tests in individual rhizosphere bacteria. Developments in immunocytochemical techniques should alio\\' demonstration and possibly localion of rhizosphere enzymes by boI h transmission and eJectron microscopy (Lynch, 1990b). II is assumed that enzyme activity is generally greater in lhe rhizosphere than in bulk soil because of the larger microbial populalion and lhe presence of roots. Neal (1973) compared phosphatase activily in unplanted soi! with soil planted with grasses and forbs representative of dominant, codominanl, increaser, or invader species. Only invader plants had a significant increase in phosphalase activity when compared wilh .the control soi!. Whelher the increase in phosphalase aClivity was a result of planl or soil microbial activiiy, or both. was not determined. The rhizosphere of rape (Brassica Ilapus) planted in a sandy loam soi! had a phosphalase aClivily 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 with ryegrass with unplanled soi!. ln general, the sulfatase and urease acti\'ity in the planted soil did not decrease during 5 monlhs, whereas aClivily in the unplanted soi! did decrease. Proleinase activity was highly variable. II was hypOlhesized thalthe planted soil continued to release enzymes from plant and microbial origino whereas lhe 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. The phosphalase aClivity in lhe inner and outer rhizosphere of maize, barley, and wheat was studied by Burns (1985). The outer rhizosphere (soi! removed from roo Is with genlle agilalion) always had phosphalase aclivilies lower than the inner rhizosphere (soi! removed from roots by vigorous agitation), demonstrating enhanced enzyme aClivity eIoser lo lhe root. Also, the rhizosphere of soybean grown in a sandy loam soil had higher dehydrogenase, urease, and phosphatase aClivities than an unplanted soil after 40 days (Reddy el aI., 1987) ln a hydroponic system, Gould et aI. (1979) delennined lhe relative contribution of the plant BOll/e/olla gracilis, an inoculated Pselldomoflas sp., and coinoculalion wilh lhe Pseudomollas sp. and an ameba (Acallt/lamoeba sp.) to tolal phosphatase activity. The presence of the bacteria or the bacteria and amebae increased the acid phosphatase aClivily in Solulion and aIso increased root phosphalase activity. These results suggest that rhizosphere microorganisms not only conlribule enzyme aClivity in the rhizosphere, but aIso slimulale enzyme production by intacl rools. ManipuIalions of rhizosphere microflora lo enhance the reJease of a beneficial enzyme would be one approach to enhance plant growlh. Also, the inoculalion of the rhizosphere with a microbe producing Jarge quantities of an enzyme 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

V.

BoIton et aI.

MICROBIAL EFFECTS ON PLANTS

Rhizosphere microorganisms are often of interest because the)' can have beneficial (e.g., N 2 fixation, mycorrhizae. biocontrol of plant pathogens, production of growth-promoting substances) or detrimental (e .g., disease, d~leterious rhizobacteria, immobilization of plant nutrients) effects on plant growth. It is necessar)' to understand the mechanisms by which rhizosphere microorganisms influence plant growth, to develop lechnologies that enhance their beneficial activilies and reduce detrimentaJ aclivities to crop plants. The converse is true for weed species (Le.; take advantage af thedetrimental activities of rhizosphere microorganisms to limit growth 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 othcr chaplers in this book for more delailed information on N 2 fixation (sec Chaps. 6 and 9), vesicular-arbuscuJar m)'corrhizae (see Chapo 13), ectomycorrhizae (se e Chapo 14), bioconlroJ of plant pathogens "'Íth fungi (see Chapo I I) and rhizobacteria (sec Chapo 10), production of growth-promoting sllbstances (see Chapo 12), disease (scc Chapo 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 predation), and neutral (neutralísm) interactions with the various members of the rhizosphere microbial community. For a more complete definition of these various interactions, the reader is referred to Chapter 1. The interactlons among the various microorganisms in lhe rhizosphcre not only can affcct the specific organisms of concern, but also other microorganisms and the plan!. Thc discipline of biological controJ of plant palhogens is founded on lhe principIes of microbial compelition, amensalism. parasitism, or predation, ar a combination thereof. These interactions are extremei)' important in the ecological stud)' of the rhizosphere, yet they are among the least well-understood areas in rhiiosphere ecology (Bowen, 1980). Bacteria occupy less than 10':'~ of the root surface. with most of this colonization occurring at regiom of C e"xUdation and a favorablc microen\'ironment, wbich are likely zones of enhanced activity alld interactions. The reader is referred to more in-dcpth discussions of microbial interactions in the rhizosphere for more detailed information (Bazin et aI., 1990; Curl and Harper, 1990; Cu ri and Truelovc, 1986). For this charter. IWO model systerns are discussed as exarnples of lhe potentiaJ fate and dfects of an inoculated or inlroduced microorganism on the nalive microflora or on coinoclllated slrains. One model s)'stem thal is useful for studying microbial interactions in the rhizosphcre and cffects on plant growth is the Rhizobilll1l-legume s)'stem. Rhizosphere microflora lhat do not affeet legume 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 sllbsequent 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 the·field. lt was hypdthesized that this \-.;a,s due to the produetion of a nonexcnitable protein. Field and greenhouse data indicated that inereased nodulation of·beans (Phaseolus vII/garis) by R. phaseoli occurred with coinoculation of Pselldomonas plltída (Grimes and Mount, 1984). However, bean yield and shoot weight were not significantly affeeted by coinoeulation, demonstrating that inereasing nodule number or infection by Rhizobillm spp.'may not 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 by an increased in nodule number, occurred with coinoculalion Df R. /lIgllminosarwn and a deleterious rhizobacterial PsclIdomonas sp. However, nodule and shoot dr)' weíghts were the sarne whether or not the PSClldofllonas sp. was eoinoeulated. Li and Alexander (1988) took a different approach to enhanee eolonization and nodulation by rhizobia. Antibiotic-producing bacteria, which were resistant to \he . antibiotic, were coinoculated with a Rhízobiwll sp. onto legume roots. Colonization and nodulation of the alfalfa and soybean rhizospheres were enhanced. Li and Alexander (1988) hypothesized that suppression of Rhizobilll/l spp. antagonisis by the antibiotie produced by on~ of the strains was respon'sible for the enhanced nodulation. Coinoculation Df legumes with both Rhi::ohiufIl spp.· and antibioticprodueing microorganisms is an area worthy of further study bccause of its potential for allering· microbial compelition in the rhizosphere. Fulirmann and Wollum (l9S9) dttected a decrease iI) lhe number of taproot nodules and in seedlillg emergente of soybean (G. 'max)ànd altcred nodulation competition among B. japoníCllfll strains when coinoculatcd wilh PselJdomolJas spp. lron availabilíty was implicated as a factor involvcd in the plant-B. japoníCllm-rhizosphere microflora interactions. Intact soil core microcosms have also been used as a model system for studying microbial interactions in the rhizosphere. The soil core microcosm design developed by Van Voris (1988) for e\aluating the fate and effects of chemieals was adapted 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 microcosms are a viablc option for obtaining preliminary information on the fate and effeels of introduced strains, inc!uding GE:'-1s. because tests and microorganisms can bc colltained in the laboratory (Cairns and Prall, 1986; Fredrickson elal., 1990; Omenn, 1986; Strauss ct aI.. 1986, Trcvors. 1988). The intact soil core microcosm represenls an intacl sample of lhe environm.:-nt and is uscful for delermining the (ate and c((eets o(an intrO<Íucecj strain on the structure and activilies o(soi( microbial communities (Fredrickson et aI., 1990). Reeenlly. soil core mierocosms wcre used to determine lhe fate and cffeels of transposon Tn5 l11UlanlS of Azospírillum lípoferllm wilh com (Z. mays)
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, and winler wheat ""as planted. More than 80% of lhe tolal pseudomonad population on the wheat rhizoplane was the introduced slrain ai the lhree-Ieaf stage of ""heat growch (Bolton eC a!., 1991b). The proportion of fluorescenl pseudomonads on lhe rhizoplane decreased from 24 to less lhan 1%' because of RCl inoculation. The inlroduced slrain was able to out-compete a significanl portion of the native soi! pseudomonads on lhe rhizoplane and also allered the bacterial composilion of the rhizoplane by decreasing lhe percenlage of fluorescenl pseudomonads. The populalion of RCl on lhe wheal rhizoplane was calculated to be approximately 40% of the total aerobic helerotrophs, indicating that RCl compeled favorably with other heterotrophic bacleria. Inoculalion wilh RCl also decreased species diversity on che rhizoplane, as measured by species evenness and equilability indices. This was because the introduced strain was prfponderant on the rhizoplane and the distribution of individuaIs among the various species was skewed. This sludy demonstrates that laboratar)' cullured organisms introduced to the rhizosphere can compete favorabl)' with the native soil microflora for colonization of lhe rhizosphere. The effecl of the introduced slrain Psclldo/J7onos ReI on various microbial populations on the rhizoplane decreased as the planl aged (Bolton el a!., 1991 b). Onl)' 42% of the total pseudomonads and 30/<- of the tOlal bacteria on the wheat rhizoplane were the introduced strain at the boot slage of wheat growth (Bolton et a!., 1991 b). Thís demonstrates the imporlancc of quantifying microbial interactions in. the rhizosphcre at various stages of plant growlh. As slated earlier, the rhizosphere is a dynamic habilal with organic C release and type varying as a function of plant age.

VII. A.

RHIZOSPHERE MICROBIAL TECHNOLOGIES Inlroduclion

Manipulalion of lhe rhizosphere to alter eilher lhe composilion or the aClivilies of soil microorganisms offers lhe opporlunil)' to develop several lechnologics. Most applicaticJns will require lhe establishmenl of inlrodllced inocula; lherdore, choosing a compelitive organism ""iII be a kc)' to sllccessful inlroduclion of microorganisms ínto the rhizosphere. The ínoculatíon of Jegllminous plants ""ith rhizobía and the biological controlof root palhogens are lwo lechnologies lhal are \Vell cstablished and already of commercial importancc in lhe United SI ales (sec Chaps. 6, 8, and 11). ln addition to lhe lopics discussed else""here in this book, there are several potential teehnologies based upon rhizosphere microbial processes. These incJude the use of delelerious rhizosphcre bactcria for the conlrol of ""eeds and lhe use of the rhizosphere and associatcd microflora for lhe' biodegradalio'n of organic conlaminants.

B.

\'\'eed ControI

Delelerious bacteria may be common componenls of the soil and rhizosphere microflora, and by manipulating lheir aclivily (e.g., increased toxin produclion) or

Microbial Ecology of lhe Rhizosphere

51

increasing their populations (Le., establishment of inocula). they may become effective weed conlrol ageors. The use of rhizosphere microorganisms as biological conlrol agents to prevent the infection of plants with soil-borne pathogens is an a~ea thal has received considerable attenlion and is a technolJgy that is curreritly being. applied to some extenl (Cook, 1985). The use of microorganisms, either pathogens or nonparasitic plant palhogens (exopathogens), has potential applications for the biological control of weeds (Cherringlon and ElIiott, 1987). Here. the focus is not on bioJogical co'ntrol of plant pathogens, but ralher, on the control of weedy plants by using microorganisms. Research emphasis has to shift from inhibiling or controlEng the growth and action of planl pathogens ar deleterious rhizobacleria to promoling their rhizosphere colonization, growth, survival, and the expression of their deleterious 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-caíonizing Pseudomonas spp. from the rhizosphere af downy brome (Bromus tectorum), which severely reduced lhe root growth of this weed, bUl nol of that winter wheat. Also, many soil-borne planl pathogens are very specific and aflen promole disease af only a single species or even cultivar. Research on lhe biocontrol of weeds mUSl firsl identify candidate microorganisms wilh lhe necessary attributes for rhizosphere compelence. These attributes should 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 organísm to be used effeclively at various 'slages of wced growth and developmenl. The microbial strains musl also be evalualcd for effecliveness and for lheir effecls on nontargel plants and major beneficial microbial species. A variely of potenlialIy phylopalhogenic bacleria and fungican be readily isolaled from the rools of several different weeds (Kremer et aI.. 1990). It is not always evident that these organisms are normal components of the rhizosphere microflora because their effects are often dampenedby competition with nonpathogenic bacteria and because lhey commonly are present in low numbers. As with olher rhizosphere microbial technologies, the ability of a specific microarganism to' effectively compele is likely to be a key faclor in promating its efficacy. Bacteria isolaled from lhe rhizosphere of seven economicalIy important weed species were predominantly gram·negalive (>99'it of alI isolates), consisting of fluorescent and olher pseudomonads, Erll'inia herbicola, Flal'obacterillm spp., and Acaligcnes spp. (Kremer et aI., 1990). These species have also been identified as the dominant microbial types present in the rhizosphere of crop plants (Bowen, 1980; Rouatt and Katznelson, 1961). Rclatively high praportions (i.e., 35-65%) of the rhizobacterial isolates from weed species could inhibit seedling growth of the pIant from which lhey were initially isolated in growth pouch and pot assays (Kremer et aI., 1990). This demonstrates lhat numerous naturally occurring microbes cxist in the rhizosphere of weeds lhal can have potenlially detrimental effects on their growth. Krcmcr and co-workers (1990) used both a microbial assay (Gasson, 1980) and seedling bioassays to determine if microbial antimclaboliles inhibitory to weed growth were being produced. Conflicting results were sometimes oblained because the inhibilion of the indicator organism (E. coli) and stimulalion of weed seedling growlh resulted from the sarne organismo The ulilization ofmicrobial assays is time-

52

Bollon el aI.

and labor-efficient, yet SpOI screening of mícrobes isolaled duringseedling bioassays musl slill be conducled lO ensure unambiguous results and lo improve lhe chance Ihal useful slrains are nol missed. Approximalely 60 and 75% of lhe bacleria isolaled from lhe roots of several weeds produced antibiolics effeclive againsl a baclerium and a fungus, respeclively (Kremer el aI., 1990). The addilion of Fe",J reduced lhe inhibilory effecl of these microbial assays, implying Ihal anlimicrobíal aClivity may have been caused by a siderophore or thal an antibiolíé produced was regulaled by Fe concentration. From lhese results, ii was suggesled Ihal lhe rhizosphere microbial populalion could be manipulated in favor of deleterious rhizobacleria (Kremer el aI., 1990). These investigalors suggesled Ihal successful candidales musl be aggressive rool colonizers, produce specifie phytotoxins against lhe hosl and not nonlargel planls, be able lo compele wilh other rhizo:;phere colonists, and be able lo synlhesize or utilize other baclerial siderophores. Anolher sludy investigaled lhe biological conlrol of downy brome by manipulaling lhe rhizosphere (Kennedy el aI., 19YI). Downy brome is
Microbial Ecology of lhe Rhizosphere

C.

53

Enhanced Drganic Contaminant Degradation in the Rhizosphere

Because of lhe enhanced aClivily and growth of microorganisms in the rhizosphere, there is a considerable potential for enhancing the biodegradalion of organic contaminants present ín soil near the plant roO!. Manipulatíon of lhe plant community or of the associated rhizosphere microflora, or both. has potential as a relatively passive and inexpensh'e technology for remediating soil contaminated with organics. Ahhough manipulation of the rhizosphere specifically for bioremediation .has not been developed as a technology. the results from several studies encourage funher ínvestigation: Hsu and Bartha (J 979) demonstratcd enhanced minera!ization of two organophosphate insecticides (Diazinon and parathion) in the rhizosphere of bush bean (Phaseolus mlgaris). Increases of approximately S and 10% ín the mineralization of [1'C1Diazinan and ["C)parathion. rcspectively. were found in soil with a bush bean rhizosphere. The viable counts (lf heterotrophic microorganisms in the planted and control soils werc 'similar, although therc was no distinction between bulk and rhizosphere soil. These resll1ts sllggest that the plani enhanceél mineralization of the pesticides eilher through a general enhancemcnt in the activity ofthc soi! microbial community or bccausc of a sc1cction for a spccific microbial community that was C3fli1blc of degrading rhcsc pcsticidcs (Hsu ,md Banha. 1979). The rate of ring c1eavage of parathion was also cnhanccd in the rhizosphere of rice (Oryza sfIIÍ\'a cv. Supriya) compared with unplantcd soil (Reddy and Sethunathan. 1983). Flooding of lInfllamed sail had little cffect on mineralization, with less than 5(ié ofthe 1"Clparathion being evolved as "CD: during 15 days.ln soil planted to rice, lInflocidcd sail e,"oh'ed 97< of the ["Clparathion as "CD!. whereas flooding the soil resulted in a "CO! evolution of 22%. This latter increase in parathion mineralization in the rice rhizosphere W:IS hypothesized to be caused by the enhanced growlh of this ri ce variely in flO(llkd soi!. Both root and shoot biomasses were threcfold higher afler 15 days af grawth, when compared with those under nonfloocled conditions. ln tum. this increase in the biomass af the roo Is and shools ma)' havc cnhanced lhe rhizosphcrc microtlora. The extel1t of dcgradation of severa! polycyclic aromatic hydrocarbons (PAHs) lI'ilS açceJerated in soil rlantcd wilh lkcp-roc)(cd prairic grasses over that af an lInplanted soil (Aprill and Sims. 1990). The PAHs in this study exhibited no downward mobility in soil cores after II-l days. The greater redllction in extractability of the PAHs from lhe planted soil was hypothesized lO be caused by their enhanced degnldation in the rhizosphere or by Iheir increased incorporalion into hllmic material. The PAHs were not l'C-labelcd. IhllS measllrements of their mineralization was not possiblc. More reccnlly. the biodegradation of trichloroethylene (TCE) was shown to be significanlly higher in rhizosphere soi!. cÇlmpared with nonrhizosphere sai! (Walton and Anderson. 1990). Soil was eollcctcd from a former TCE disposal site. The rhizosflhere soil was colleeled from the rooting. zone of the four dominant plant sflecics prcsenr ar rhe disposal site. and nonrhizosphere soi! was collected from non"egetated arcas within and outside the dispasal site. The TCE was lost more quickJy from lhe headspace of rhizosphcre soi! slurries than from nonrhizosphere soils. When ["C)TCE was added to rhizosphere and nonrhizosphere soils, a threefold

54

80110n el aI.

increase in l'C0 2 occurred after 30 days in the rhizosphere soil compared with the control. Therhizosphere soils had a four- to sevenfold higher microbial biomass than the nonrhizosphere soils. 1t was hypothesized that the increased biomass in the rhizosphere soi! enhanced TCE mineralization. AIl mechanisms of TCE degradation yel known are forluitous or comelabolic reaclions. No pI anIs were grown in lhe soils during the TCE mineralizalion experiments, precluding plant uplake of lhe organic agen!. However, iI is unclear whelher the TCE mineralization noled in lhe soil sample would also occur in lhe field with activeIy growing planls. An obvious neXl slep would be to determine TCE fate in lhe rhizosphere during active pIam groil·th both in conlrolled laboratory condilions and in lhe field. An addilional advantage of using planl-microorganism combinations for lhe remedialion of contaminated soils is lhat lranspiration from lhe plant will enhance lhe movemenl of soluble contamin~.nls lO lhe planl rool where lhey can be degraded by rhizosphere microflora. Àlso, lhe planl root may be useful as a delivery system lo lranspor! contaminant-degrading microorganisms to lhe compound of interest WilhoUl disturbing or mixing lhe soi!. Seed coaling or inoculalion of secdling rOaIs may allow an added conlaminanl-degrading slrain lo grow along wilh lhe rool and conlacl an increasing volume of sai!. One limitalion lo lhis approach is lhat less 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 by lhe plan!. For volalile organic solvenls such as TCE, plant lranslocalion may actually resuIt in release of TCE lo lhe atmosphere, much in the sarne way lhal airstripping is used lo lransfer volatile organic compounds from an aqueous (i.e., groundwaler) lo a gaseous (i.e., almosphere) phase. AIso, lhis lechnology would probably be bel ter using plants wilh high-rooling densities, such as grasses. VIII.

A.

SUMMARY

Research Needs in Rhizosphere Microbial Ecology

The rhizosphere is a dynamic microbial niche. The success of rhizosphcre soil microbial lechnologies will é1epend on isolaling and understanding the mechanisms by which microorganisms influence plant growlh as lI'ell as a basic underslanding of the traits lhat eonslitute a compelitive rhizosphere colonizer. A microbial isolale lhal carries out a useful process (\f function in lhe rhizosphere will be useless unless the organisms can successfully compele in the field and express the desired Irai!. Inlegraled muItidisciplinary research is needed to undersland lhe complex interaclions of biotic, chemical, and physical processes that inleract to define lhe environment at lhe root surfaee. There are great varialions in lhe environmenlal conditions along lhe root surface and radially from lhe rool surface into lhe bulk soi!. Microbial-plant root interactions must be sludied at smallcr scales lo undcrsland the numerous concunenl processes in the rhizosphere. There is currently a Jack of informalion on the dislribution of microorganisms on root surfaces, lhe ir relalive melabolic activities, and now Ihey inleract la affeet plant growth and lhe growlh and funclion of other microorganisms. Model s)'stems must be carefully chosen and experimenls designed lo ansll"er specific fundamental questions on why certain microorganisms 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 Olher compelilors, and resislance lO inhibilion by olher pOlenlial rool colonizers. The abililies lo ulilize a unique organic rool exudale or lo selectively increase lhe release of organic C from rools are other traits that may be imporlant. Genetic manipulalion of the plant or rhizosphere microorganisms is a po" erful 1001, wilh considerable pOlential for exploring lhe microbial factors that influence Iheir 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, "nonphenazine-producing mulanls and lhe phenazine-producing wild Iype were used 10 show lhal phenazine was produced in lhe rhizosphere and was effeclive in reducing the disease. Such an approach may be useful for addressing lhe role of anlibiolic production in microbial compelilion in lhe rhizosphere. Transposon mUlagenesis has been used lo obtain mulanls of a planl growlh-promoting P. jluore5cens Ihal were characterized as Agg-, lhe inabilily lo be aggluli,nated by a rool surfaceassociatcd glycoprolein. The Agg- mulants exhibited significant!y lower leveIs of rool binding (Anderson el a!., 1988) and colonizalion (Tari and Anderson, 1988) Ihan did lhe parenl slrain. Once trails have been idcnlified Ihal influence the pOlcntial of an organism lo colonize lhe rhizosphere, lhe organisms mighl be genetically manipulaled to enhance these abilities. Tradilional rcsearch in rhizosphere microbial ecology has focused on increasing Ihc producti"ity of crop planls. This research has ob"ious me ri Is and should continue. New approaches to weed conlrol, which use rhizosphere microorganisms and enhanced organic contaminanl degradation in the rhizosphere, have the potential to become useful technologies for soh'ing several environmental problems.

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