2

Microbial ~ o l o y 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 coloniza-
tion 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 (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 rhizosphere
will encompass bOlh the rhizosphere (endorhizosphere and ectorhizosphere) and
lhe 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) 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 sil microbiot,a to influence plant
growth 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 orderly
systems 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 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 exis-
tence, 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 inthe
rhizosphere is also the study of stru911re and fllnction. An unerstanding of the
basic principies of rhizosphere microoial ecology. including1he fllnction and diver-
sit)' 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 princi-
pies 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 U I e 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 rhizosphere
and how microorganisms can influenee the growth of planls atd other mieroorgan-
isms 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 influ-
enee 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. Ewdlilcs: low molecular weight cornpound;, (i.e., sugars, amino acids) lhal
leak from intacl ceUs
2. 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 including
a. Secretions by the golgi bodies of the root cap cells
b. Hydrolysates Df the primary cell wall Jocaled between the rooI 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
4. Mucigel: gelalinous material on lhe root surface composed of plant mucilage,
bacterial cells, melabolic products, and colloidal organic and minera! malerial
5. 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 micro-
bial growth surrounding lhe roaIs of legumes, which was assumed to be caused by
the excretion of organic materiais (Curi 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 an active
rhzosphere microflora. The microflora can, in tum, promole plant growlh by
enhancing soi1 organic marrer rransformations, mobilizing inorganic nUlrienls, pr-
3a

Epldermal and Cortical
CeJls lysed and 5
lnvaded by Bacleria
Sloughed Rool Cap Cells
SOII
'i-:
d
- J 3c
':\ Micro Ofganisms
wilh Microbial and 4
Plant Mucilages
J
5,,,,,,",, '"" .-, "''' }
=:: --1&2
-.-
J-HI------------- 3b
__ Rool
20 mm
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 release of organic C compounds TOm 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 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, respeclvely, 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)'saccha-
rides, growth-promoting and growth-inhibitingsubstances, ali have been reported as
root exudates (Hale 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 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 com-
pounds 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 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 Rovir (1956) demonslrated lhat
32 Bolton el aI.
Table 1 Organic Compounds Detccted Plant Rool
Class of organic compound Exudle components
Sugars Glucose, fructose, galactose,
rhamnose, rihose, ra(finosc,
oligosaccharide
Amino compounds Asparagine, a-alanine, glulamine, aspartic acid,
Ieucine.'isolcucine, scrine, aminobulyric acid, gly-
cine. 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;
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,
polygalacturonase
Miscdlancous All,im. scopolclin, flu",escenl hydro-
cyanic ;,cid. saponin (glucosidcs), Or-
ganic phosphnrm cnrnpnunlh, ncmatnLic cyst or
cgg-halching faclors, ncmalo(k fun-
gai rnycclial gro.....lh slilllulanls, mycclium gro\\"lil
inhihitoTs, ZOO"POll' attractant!'., and
scicroliutn germinalinn s(imulants and inilibilor>,
tJafl'ral srirnulanls and inhihirors. parasiric wCl'd
g{'rrnin"tiorl
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 COrn-
pounds, 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. (1969) found eliffcrenl cxlldation pallems amollg se\"enll spceies of
pinc (I'il/us nank5ian(l, p. lall/berlill/la, P. radiala, 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
Table 2 Soluhlc Root Exudates of Maize Gro",n for 36 Days in SteriJe
and l"utricnt Solution Culture
33
Root eXlldatc
Sugars
Glucose
Arahinnsc
huclOso
SlIcrosc
Chgallic acids
Oxalollcctic acid
Fumaric acid
Malic acid
('ilric acid
Succinic Jcid
Iknwic ;,cid
Aconitic acio
Tart1rk (Kid
Glularic ;,d
Amitlo
GIlIlamic acid
r\ Sptlrt ir aciel
Alanine
GI)'cino
y-Aminohulyric acid
Scrinc
Arginin<.'
Ci!utaminc
Valinc
I,('ucine
Sif,llifiC;lTl1ly dlf(c:rcnl at (1:.:0.05.
"1'\S, nol (llffl'rcnL
SOI"C('. t'l a\. (1984).
Stcrilc
Nonsterile
(J.Lg'g dry root)
7370
3900'

1760'


1590 1720 NSb
2040 3100 NS
3810 4710 NS
270 190NS
530 NS
320 470 NS
200 280NS
100 110 NS
70 140 NS
30 50NS
71 126'
63 52 NS
59 44 NS
39 32 NS
30 18 NS
26 22 NS
31 15 NS
12 18 NS
lO 12 NS
17 lO NS
(lf sugar rxuckel "'n \'ar)' with lhe phenological si age of lhe pIam (Bokhari el aI.,
1979). \\'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 de-
crease 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 pI anIs 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 nd pesticides can also affct 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 dem-
onstrated 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 con-
trol 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 exuda-
tion. 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 metaboliz-
ing 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 a-
amino groups Ihal reacl wilh lhe ninhydrin lo produce a purple color for free a-amino
groups or a yellow clor 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 rooI 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 ofninhydrinposilive 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 hve greatly aided in determining the localions aI which exudalion
occurs (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. 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 poly-
saccharide and, presumably, sloughed-off rool cap cells. As s o ~ n in Figure 2,
sloughed rooI 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. Mc-
Dougall 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 radio-
aCli\'ily along lhe primar)' rooI and some of the laleral roais were more diffuse afler
2 hrs.
m. THE PHYSICOCHEMICAL ENVIRONMENT Df THE
RHIZOSPHERE
A. Introduclion
ln addition 10 C inpuI, there are severaI other planl-induced physical and chemical
alteralians of lhe' rhizosphere that affecl the composilionand aClivities of rhizo-
36 Bolton et aI.
sphere microorganisms. The rhizosphere 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 rhizsphere 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 micro-
bial 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 compactin
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 nar 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 sai I 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 ir 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
but can increase lhe solubilityof cations by complexalin. A gentle percolation of
the rhizosphere of maize and wbeal recovered unidentified soluble organic materi-
l\1icrobial Ecology of lhe Rhizosphere
37
aIs lhaI complexed Co, Zn. and Mn (Merckx el aI., 1986). ln fieldgro..... 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 volution of CO
2
from root
and 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, 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 Nfertirized 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 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 form
wilI have on rhizosphere pH. Rape (Brassica llapllS var. Emerald) grown aI high-
rooting densities in Pdeficienl 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 sai I 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 rhizo-
sphere 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 mea-
sure 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
38
Bolton e! aI.
millimelers from lhe rool surface. These effecls were hypolhesized la be caused by
the ncrease n soluble organc material present in the rhizosphere, which
in an increase in microbial respiration and a subsequent decrease 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 n lhe rhizosphere. -
IV. MICROBIAL PRESENCE AND GROWTH lN THE
RHIZOSPHERE
A. Inlroduction
The rhizosphere effeet refers to enhanced microbial growth and populalion
densities in the rhizosphere from the increase in soluble C and nutrients, when
eompared wilh those of the bulk soil. Table 3 shows Ihat both higher populations
and a greater dversty of mcroorganisms, 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 V/rious
taxonomie and nutritional groups of microorganisms bet\veen rhizosphere and non-
rhizosphere soi! (Ta,ble 4). The ratio of the microbial population in the rhzosphere
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 dislribule. Toe slalislieaJ leeonique deveJoped by
Greig-Smith (1961) to deteet patterns af vegetation in terrestrial eeosyslerns
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 .
Rhizosphere
Conlrol soil
Populalions (Iog CFU/g)
(log CFU/g)
RiS ralio'
Taxonomic groups
Bacleria 9.08 7.7(J>
24.0
Acrinomyceles 7.66 6.85
b
6.6
Fungi 6.08 5.0C!'
12.0
Prolozoa 3.38 3.0C!'
2.4
Microalgae 3.70 4.43' 0.2
NUlrirional groups
Ammonifiers 8.70 6.60
b
125.0
Gas-producing anaerobes 5.59 4.48' 13.0
Anaerobes 7.08 6.78' 2.0
Denilrifiers 8.10 5.oo
b
1260.0
Aerobic ceBulose degraders 5.85 5.00' 7.0
Anaerobic ceBulose degraders 3.95 3.48NSd 3.0
Spore formers 5.97 5.76NS 1.6
Azolobacler <3.00 <3.00
'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. -Micro-
bial 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) inocu-
laled 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 increase 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 epider-
mal 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 root-
derived 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 Bolton et aI.
C. 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. Polysacchardes 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 specLs
(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 wld type. These
mutants adhered to the root at levels.;20- 10 30-fold less than the wild
ing Ihat glycoproten binding has a role n their attachmenllo bean roais (Anderson
et aI., 1988). Piri (fimbriae), surface proteinaceous strctures ema-nating from the
microbial cell, have been mplcated in the attachmenl of Klcbsiella and En-
Icrobaeler spp. (Haahtela and Korhonen, 1985; Haahtela et aI., 1985; Korhonen et
aI., 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 twice
as many pilia!ed edIs allached at a 2.5-fold higher amount to soybean rool seg-
menls and colonized 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 (James
et aI., 1985). Binding was no! reJa!ed to ell 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. (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 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. Rcspi-
ralion of rhizosphere soi\ \Vas dereased 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 root-
colonizing 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
(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 deleteri-
ous 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 (1988) showed that a deleteri-
ous Pselldol1l0llaS sp. inoculated onto nonsterile wheat straw in the laboratory
constituted 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 residues
in 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 00
rcsidues 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 c<i\<mize the pTeceding crop T.esiducs c:n a1so
colonize lhe subsequenl crop (Stro el aI., 1988). Interesliigly, lhe no-lill seedcd
plants had higher numbers of the pseudomonads lhan lhe convenlionally seede/:l
planls. Accordingl)', iI ma)' be. possible to use crop residu'es as an inexpensive means
for delivering a.microbial inoculum to lhe rhizosphere.
The ability of Pselldomollas species to colonize and grow in lhe rhizosphere
varies greatly. Four relaled strains of Pseudomonas spp. inoeulated anta
Ellca/yplllS seiberi roots inereased, 2-, 62,- 141-, and 380-fold over a 4-day period
(Bo\\'en. 1980). Gro\\'lh of one of Ihese Pscudomonas spp. in lhe rhizosphere of
E. globulus was appro.ximately one-sixlh of E. seiberi, demonstrating lhat grO"'lh
rates of microorganisms in lhe rhizosphere depend on planl specics. This stresses
lhe need for individual microbial strains for their rhizosphere coloniza-
tion Vihen working I; develop rhizosphere microbial technologies for different
p\ants.
The gcneration, or doublng. lime Df microorganisms in lhe rhizosphere has
becn extensil'el)' studied to prol'ide insighl inlo lhe potential for manipulating
mieroorganisms in the rooI zone. Barber and lynch (1977) grew bar/ey inoculated
"jth rhizosphcrc organi5ms in solulion culturc. Gcneration times were 24 hr during
lhe first 4 da)'s of planl gro\\'th and increased to 103 hr during the 7- tO 16-day
perodo Pselldomonas spp. had a shorter generalion time when Bacilllls spp. in the
rhizosphere of Pinus radiora'seedlings (Table 5) (Bowcn and Rovira, 1976), which
suggesls lhat Pseudomonas spp. have a sclective ad\'anlage o\,er Bacillus spp. in
rhizoplane colonizalion. The gencration times for boIh iI) soil wcre rnuch lower (see
Table 5).
Table 5 Generation Times (hr) of Total \'iable \lacleria, Pscl/domo/las spp. an Baei!!l/s
spp. on Pinus radiala Roots and n Unplanred Soil

Roo!'
l'nplantect soi!
Talai viable bacleria
7.2
[U
PSClIdomO/lIlS spp.
5.2
77
8aeillus spp.
39
>100
-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 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 grovh
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 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 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
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 Bacteria
O Pseudomonas spp.
4,0 3,0 2.0
Time (days)
1.0

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....1hin
lhe rhizosphere. Thes methods do not mimic the soil physical and cht:mical rela-
tions outlined earlier that can dramatically influence rhizosphere relationships
lhe resultant microbial responses. must employ nonsterile
soil for evaluating rhizosphere technologies, tX;cause the rhizosphere is opration-
ally 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\\-
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 dirctly
applicable lO the rhizosphere. Although the rhizosphere is an enriched environ-
menl, 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, 'repro-
duce, 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 (Svasilhamparam 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 rhizo-
sphere 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 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 lhe
subSlitulion of chromosome 513 and 5D fTOm Rescue, respeclively. were choscn for
study (Neal el aI., 1973). The substituton of chromosome 5B from Rescue into
Cadel significaml)' altered the populalion size of lolal bacleria and several physio-
Jogcal 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 Bacleria CelluJol)'tic PeclinoJylie AmyJoJytic
Ammonifying
(Iog CFLJ'g dr)' soil)
Cadet 8.2b' 3.7b 4.6d 6.4c 7.3e
Reseue 8.5a 5.la 6.8a 7.6b 8.1b
CR5B' 8.5. 5.2. 6.4b 7.8a 8.3.
CRSO' 8.3b 3.5b 5.8e 6.6e
7.2c
'DaI a 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,
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 -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 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 popu-
lations. 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 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 star-
shaped 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 rhizo-
sphere 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 environ-
ment, 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 into contacl with lhe root envi-
ronment, colonizalion of the roO! can occur. Organisms associatcd with soil organic
material are a prime source of inoculum for colonization. \Vater move-
ment 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 percolat-
ing 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 cn-
hanced 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 bacte-
ria! transport and colonization of the rhizosphere in the field may lead to erroneous
conclusions. Smith and co-workers (1985) demonstrated that transport of Escher-
ichia 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 hypothe-
sized 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 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 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. fillo-
resam. and Serriltia spp. (Scher et aI., 1988). Allhough not conclusive, these
stlldies suggesl that bacterial motility may not be a major factor influencing hacte-
rial colonizarion of the roo!. Soil type can abo influence root-mediated microhial
transport in soil (Trevors el aI., 1990). Downward movement of a genetically cngi-
neered 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 stan-
dard 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
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 (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 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 DNAfrom the
rhizosphere and purifying it to quantify the incorporated thymidine. Curreot re-
search 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 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 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. Bacte-
rial 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 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 lhe
rhizosphere, 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 be
enhanced by its ability to enzymatically deeomposeroot cells and soluble C exudate.
Microbial Ecology of the
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 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 enzymes
were detected by ultracytochemical tests in individual rhizosphere bacteria. Develop-
ments 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 rooIs 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 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 tolal
phosphatase activity. The presence of the bacteria or the bacteria and amebae
increased 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 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 BoIton et aI.
V. 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, rhizobac-
teria, 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 (see Chapo
14), bioconlroJ of plant pathogens "'th 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 pre-
dation), and neutral (neutralsm) 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 vari-
ous 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). Bac-
teria occupy less than 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; Curi and Truelovc, 1986). For this charter. IWO model systerns are discussed
as 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 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 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,s
due 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.'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)'
weghts 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 Rhzobiwll sp. onto legume roots. Coloniza-
tion 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 of the strains was respon'sible for the enhanced
nodulation. Coinoculation Df legumes with both Rhi::ohiufIl spp. and antibiotic-
produeing 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 emer-
gente of soybean (G. 'max)nd altcred nodulation competition among B. japonCllfll
strains when coinoculatcd wilh PselJdomolJas spp. lron availabilty was implicated
as a factor involvcd in the plant-B. japonCllm-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 devel-
oped 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
(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 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<ucecj strain on the structure and activilies o(soi( micro-
bial communities (Fredrickson et aI., 1990). Reeenlly. soil core mierocosms wcre
used to determine lhe fate and cffeels of transposon Tn5 l11UlanlS of Azosprillum
lpoferllm wilh com (Z. mays) <!nd wheat (Benljen ct aI., 1989) and a root eoloniz-
ing Ps('({do/llollas sp. wilh whejt (Fredrickson et aI., 1(89). Thcre was no effccc of
the introduced Azospiril/llI11 /ipoferllln on the populations of R. /egwnno.\arum ar
nitrifiers in lhe rhizosphere of eilher corn ar \\'heat (Bentjen el aI., 1989). There
\\'as no discernible erfect of lhe inlrodueed PsclIdomOllaJ sp. on total aerobic
hcterotrophs on the rhizoplanc \\'heal at the boot stagc of growth (Frcdrickson ct
aI., 1989).
Rceent studies have compared results oblained with intaet soil core microcosms
incubaled in a laboratory and a growth chamber with those from lhe field to
determine the fate of an inlroduced whcat root colonizing Pseudomo/ws sp. RCI
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 popula-
lion 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 distribu-
tion 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!., 1991b).
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). Ths 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. RHIZOSPHERE MICROBIAL TECHNOLOGIES
A. 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, choos-
ing a compelitive organism ""iII be a kc)' to sllccessful inlroduclion of microorgan-
isms nto the rhizosphere. The noculaton of Jegllminous plants ""ith rhizoba and
the biological controlof root palhogens are lwo lechnologies lhal are \Vell cstab-
lished 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 mi-
croflora, 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 ~
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 control-
Eng the growth and action of planl pathogens ar deleterious rhizobacleria to pro-
moling 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-caonizing 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 microorgan-
isms 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 organsm 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 nonpatho-
genic 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 gramnegalive (>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 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) 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 mcrobes isolaled duringseedling bio-
assays 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 anlimicrobal aClivity may have been caused by a
siderophore or thal an antibiol 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 manipulal-
ing lhe rhizosphere (Kennedy el aI., 19YI). Downy brome is <ln espeeially Irouble-
some weed to lhe produetion of wheat because it is graminaeeous and grows at the
sarne time as does lhe crop anel beeause there is no seleetive herbicide for iI.
Kennedy and associales (1991) tested 1000 rhizosphere isolalcs from downy brome
for lhe abilily lO inhibil rool gro"'lh of do"'ny brome, l?ul not Ihat of winter ",heal
and ",ere able lO identify 81 isolales wilh lhe desired trails. Of these 81 Mrains, only
15 inhibiled downy brome, but nol winter wheal when grown in nonstcrilc soil in <I
growlh chamber. This demonslrated the rcquiremcnl for sereening numerous iso-
lales to identify potenlial bioeontrol strains and that lhe devclopmenl of rapid
sereen leehniques is highly desirable. The tcsling of nontargel planls must also
include mulliple eultivars ar varielies. Kennedy and eoworkers (1991) tested four
eultivars of winter ",heal and found thal some of lhe baetc'ria! slrains inhihited
downy brome and scleet wheat cultivars. This is similar to rcsults obtained by
Ellioll and Lynch (IY84) with delelerious rhizobaeleria Df winler wheal. Whe<lt
eultivars "aried widely in Iheir suseeplibility to root inhibilion by pseudomonads
isolaled from winter wheat roots. Of the 16 ",heat eultivars tested, inhibition Df rooI
gro"'th in seedling bioassays ranged from :16 to 8R'1r of the conlrol wilh one isolate
and 61-134% with a seeond ,train. Thcre p p e ~ r d lo he a Irend for lhe more
reeenl1)' developed whe<ll cultivars \O be inhibited to ii grealcr e.\lent than the older
ones.
The biological conlrol of downy brome by a rhizosphere f'sclldomollas sp. in
lhe field has recenll)' been demonslrated. Threc isolales Ihal inhihited downy
brome in seedling bioassa)'s and growth ehamber studies "'erc applied lO lhrec field
localions in Washington State, which had natural infestations of down)' brome. The
organisms were spraycd onlo lhe plots ai 10' CFU!m
l
and the plots werc planted to
winter wheal. Two isolates significantly reduced the number of do",ny brome planls
per pIai, the groVl'lh Df downy brome shoots, and lhe quanlit)' Df downy brome
seed. The decrease in seed produclion demonslrated that annuaJ applications of lhe
bioconlrol agent might reduce the quantity of sced available for subsequent infesta-
lions. The organisms had no detrimental eHeel on the nontarget erop plant and, in
facl, ai two of lhe Ihree Joeations, lhe yields of winter wheal were higher where lhe
biocontrol organism ""as applied.
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 community
or of the associated rhizosphere microflora, or both. has potential as a relatively
passive and inexpensh'e technology for remediating soil contaminated with organ-
ics. Ahhough manipulation of the rhizosphere specifically for bioremediation .has
not been developed as a technology. the results from several studies encourage
funher nvestigation:
Hsu and Bartha (J 979) demonstratcd enhanced minera!ization of two or-
ganophosphate insecticides (Diazinon and parathion) in the rhizosphere of bush
bean (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 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 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 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 sfII\'a cv. Supriya) compared with unplantcd soil (Reddy and Sethuna-
than. 1983). Flooding of lInfllamed sail had little cffect on mineralization, with less
than 5(i 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 en-
hanced 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 rooIs and shools
ma)' 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 an
lInplanted 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 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 mate-
rial. 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!. clmpared 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 groilth
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 lO lhe planl rool where lhey can be de-
graded by rhizosphere microflora. lso, 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 secd-
ling 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 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 would
probably 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 soil
microbial lechnologies will 1epend 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 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 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, "non-
phenazine-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 surface-
associatcd 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 geneti-
cally 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 con-
tinue. 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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