Microbial Interaction in Soil

Mi,crob1
ial lnteractions
in Soi 1
Jan Dirk van Elsas, Ling Tam, Roger D. Fin/ay;
Ken Killham1 and Jack T. Trevors
CONTENTS
Introduction ........ ............
~
178
7.2 Ecological Categmies of M.icrobial Interactio:os in Soil ................... 't80
7.3 M'icrobia! As eJt1blages, Island ' and MicrobiaJ Interactioru. ............ t 82
7.3.1 Tne Ishmd Coneept of Soil aod lmpiications for
7. i
1 . . . . . . . . . . .
7.3.2
. . . . . . . . . . . . . . . > ... . . . . . .
. . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . .
........ .
.. .
..
Microbial A emblages and Gene Expression ....................... 184
7.4 Molecular Mec'hanisn1s Underlying Microbial lnteractioUB............... 185

7.4.J Molecular Sen.sing ar1d Sig:naling ...............,.......... ,.................. I 85
7.4.2 Quorum Sertling- Tile Paradigm of Signaling betwoon
7.4.3
U e af and. Betrn.yal \vith QS
mSoil...................................... 191
7.4.4 Altemative Punctions for AHL .............................................. 193

7.4.5 Environmen.tal Se,osing Me,cl1ani ms iu Fllllgi ....................... 193
7.5 T)rpes of Microbial lntet'ctions io Soil ............................................. 197
7.5.1 Mctabolic Ir1teracti:ons in Soil ................................................. 197
Antagonisti.c InterJetiort in Soil ............................................. 200

7.5." Predatory Jnterttction in Soi1-8de1Jovibrio and
7.5.2
Myxobt_.lCteric1 ...................
202
~i.5.4 Bacterial Interaction witi1 Fung ........................................... 203
1.5.5 Fu11gaJ Intentctior1s witb Bac teria, ..... ,. ................................... 2fl5
7.6 Coneludirig Remarks ~ ~ 8 ~ .o!, .............. . .............. 206
Referen.ce ..................... ..................... ............................................................................. , .......,. ........ . 201
? . . . . . . . . . . ... . . . . . . .. ,. . . . . . . . . .. . . . . . . . . " . . . .. . . . . . . . . . . . .., . . . . . . ,. ..
117
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Microbal loteractions in SoH
16 1
Neulrali.fm can be cb.a:rac tetized as the lack of interactio:n(s) between

microbial population . For ex.tllllple, microbial populatio:ns may be spa.cially
distru1t (occur 011 diffcr:ent 'islands," see below) ar population.'i may ocrur in
very low nu111ber that do .not contact o:r sense other populatio11( ). The
rnicrobial proces e rr1ay oot overlap or affect one aaotber, ~o thc o.rganisms
rnay be fu.nctio11ally eparate. Environ1nenta1 coi1dition (e.g. nutrient limtation, low temperatures or the pre 'ence of toxic oompounds) that do not allow
rrcrobi~ll growth and cell di vision favor a ne utral intemctio:.o.
Com111emalism i. 11 unidU:ectionaI iriteractioo bet\veen rr1icroorg:aoisa1
in whch one microb.ia.1 population benefits from another oue while the latter is
not affected (33). For exarnple, one rnicrobial popul.a.tion may produce growtb
factors- such as vitan11ns and/or amino acids- that o.re u ed by the other
microbial p<rpuJation. to tlle sole beilefit of the latrer.
Mi1tualism is an internction i 11 which the interacting orgacis.ms receive
tnutual benefits from ttm partnership ( uch a in gymhios-is, sytl8r gism or
sy1itl'ophy [ero ' -feedi.n g]). Mtttua1 S)tmbiosi., can be a o~~uloo uectosymbio is," in whicl1 the symbiont occur on the extem al wface of the host.
IDramples include internctions of, for in~"'tance, nitrogen-fixJng rhizobia with
pl ~Lncs.. \vbich donate bound nitroge11 n.nd re.ceive bound carboo in rerurn;
See Ch.apter 8 for a.further ex ten ion of this i sue. Anothe:r irnportant category
of muruali tic int.emction are the myoorrhizal . ymbios.es between. planes ru1d
fu.ngi. described in Chapter 5. Syntrophy is a form of metabolic collaboi-aion.

An essen.tiat inetabolite m.ay be produced by ori.e microorg-.miSll'l whlch etmnot
be prod.uc.ed b)' another ooo. Synergi m is a facultative interaction. in wh.icb
both microbial population. nre c:il1 capable of growfu and n irvival iJ1 th.e
absence of the othe:r. For example, on.e population may produce a metabolic
compound tha,t tbe other popu]ntion cao further metabolize to a compou:nd t'.Wlt
both popula.tions can utilize.
Parasitisni!Pred<filon are processes in whicb one organi .m. gain an
advantage at tbe expense of anotber one (pa.rasitism), or con,, wnes the t1ther
one {predatioo), obviou ly to the detriment of tbe latter. Por in t.anoe~ the
pred.a tor organi in .mJiy enguJf. attack or dige.~t the prey organisn1, sucb a
ou.tlined in Chapter 6 for soi1 protozoa. Thi , can resuJt in a cyclic internction.
in which the prey is overtaken by me pre-dator~ resulting in a d.ecline ~o:f the
pre y densiry. whlch in nirn induce: a ub. eque.nt deeline of the predator
popi1laticltt As the prey popuJ.atioo. increases again as a resnlt of the retluced
predatory pressure, the predator population will incr~ttse and rhe cycJe reeat .
Examples of parasitism/pre<iation in oil include bacteria that ai'e dige.sted
by soil protozo:n ( ee Cbapter 6) or by other bacteria (see Sectioo 7..5 ..3). fungi
that are degraded by other fu ng. and organisms like Rhisopltydiw11 and
Phyrridiu1rt th.ar can paras itize alg-ae.
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Microbial tnt'eractio:ns in Soil
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o.x.ygeu, com.p:ared to cells in lower, ofte11 oxygen-depJeted layers. 'Ihu.s ever1

1
cells of l'b.e same species can oocur in different physiologicaJ tates over veey
small ,clistan:ees witbin n. microbial asse.n1blage in. soil. This cancept is mghly
relevant for our consielerotion of inter:icti\1e p:r~ and. prod'llction of
econdary metabolites, including certan anribioci.cs. Moreover, the disposal <>f
yroducts of cellttlar metabolism oommonly varies ,baween dilferent Jayem .o f
a biofilm. In. suinm-ary, a matu.re microbial biofilm moay con~ of varlous
layers, which are linked by channels. Conditions will vaey 'between ccll la}re:r
at di:fferent depths and \.vil.l tberefore drive c1ifferentia1 ge.ne expres,s.ion. Por
instnnce, genes for the i~1lation of ftagelJ.ac"{frlven motility. c.eLtuJru e>Slno1a:rity, oxygen lintation and bigb cell density (&ee SecOOD: 7~4.2} ru:e aro.ong
t11ore ti1at are differentially expressed .. Hor.nogeireous microbla:l amernbl.age
will tbu . sho\v ciear tI:atification, and &ucb st:ratfteation may even be n1ore
pror1ounced.:in J1eterogenooW! on~ . The latter type of biatilm has been poorly
tudied to date, but structure-function rela.tionSh:ips ha.ve be~n describ.ed in
biofil.rru in. edime11ts coroposed of s.ulfate 'reducer an.d methanog:ens.~ as well
a in uitrifying 0011sartia containing mttlti.species bacteria.
Cell-to--cell in:teraeti(}Th,q within bofilms ( uel1 as signaling) play i.mpommt
roles in the differentitltion processes of tire individual cells, and su,eb interactjon ' wilI coortlintlte group behavior. Cell-to-cell cootintmieation in bacteria
occurs through secre:t:ed. (diffusibte) cliencal compounds., as will be di cussed
o Section 7.4.2. The regulation of a range of bact:e.rial function:s is related
commuoieation, and ofteo sucb regulati.oo. oco.Cr& u1 ca.ses wben

higher ceil cotl'centrati.ons 'provid.e an eoologica'I advant.age i11 t.h.e natural
envirorunent. Diffusible signa! molecules bave a direct role in ac.tivating cell
growt11ill biofilms and the ab-ility of cell populations to respon:d to changes ro
nutrie11t co11cerJtmtions f5). Parallel forms of sigo.aling tak~ pla.ce in.fu.agi and
to ce11-to~celJ
are discWiSed in Section 7.4~5.
7.4 MOLECULAR MECHANISMS UNDERlVING MICR081Al

INTERACTIONS
7.4,. 1
MOLECULAR SENSINC ANO S1GNALING
Key features of tl1e genotnes of i11icroorganism iu 001nplex .b:abitats like soi\

are the p:resence of a large nwnbe:r of di verse oo.nscrry mecbanmms tha:t emible
the e organisms to m.orutar tite co11dtions prevailing m their SUn'OWdi_ngs
(e.g. tbe conce.ntration of a range of aompoonds s.uch. as PO-h divers~ sm.Trces
of C an.d N. temp.e.rnture an.ci pH). Microorganisms in ~'tlil bave mus evolved
pecific genetjc .y tems to sense their biotic and abi.atic envirooments and
respond to pc:rceived s.ignals. The e systems ~ the so-c.allet.l two-eompooent
sensor/response {regulatory) systern allm\' an. a.dequate ;rei poru;e to the
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Microbial lnteractions in Soil
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\Vtltin the n1icrobial as embl11ge t.o tbe QS n10lecules can be charru..'1erized as

n commurrity re pon e. The detecton of lhe accu1nulated .i.gnaling
iuole-eules by a specific c.eJJula:r receptor, the AHL receptor protein. elicits
a sign.aling ca ca.ele an<i activa.tes gene expre sion. The exib'tence of complex
.regulatory nerv orks .in th.e celJ tbat are associated with QS mggests tlult ~e
sy"tem.s, in creating positive feedback loop , may be utjlized fot the l'llpjd
amplification of exogcr1ous ignals (7). Quorum ensing tl1us relie c)n two
proteins, an AHL ynthase ancl an AHL receptor protein [7].
The abli.ty to produce QS signaling rn olecute bru; bee11 demonstrated foc
meinbers of the a.-, f3- and y-subcla.sses of the P.roieobacterla [7], an.c:l the
igJl.al respon e mechanism J1a. been obs.e rved in over 30 species. A.') snnwn
in Figtire 7.1, Grnm-positive bacteria a.re also responsive to cell papulation
densitie . They oc-rete ptoces ed peJJtide ignaling molecule usually b)! a
dedicated K f P-binding cassette exporter protein (ABC) [8]. Tbis sigruillng
U:b-stru.cture i a pbo phorylation/dep.hospl1orylation tw<rcomponent c~cade
.o f varying co.mplexity and regularory factors. In b:rief, ecreted peptide a.utoind.ucer increase in. co11c.entn1tio11 as a function of cell popul~:ion density.
A ".en.sor prorein. sensor kinase, detect.~ t:he peptide signru and inten1cts
\vitt1 these. i11irJa.ting a series of poosphorylation events that cttlmjnate in th:e
pho phoryfa.tio11 of a cog11n:te response regulator 1)roteir1. Th.e phosp~1ory J ..
ation prucesi acf,ivates this respon e regulator, allowing .it to bit:td to DNA
and alter the tr1lns.<:ri.ption of QS-controlled gene( .).
What phenotypes are C\Jn:trolled by QS? Bacteria use QS comm1.micatio'.o
to regul~1te a very diverse arrny of p.hysiological. activitie \Vthio. microbini
populati.o ns. Pheno'types that ttre a ociate.c1 ~ri th A.H L p1'0d1tctinn an.d sen'Siog
include biolu1ninescence , p ro(l~1c ti on of virulence f~tetor conj ugative
transfer of Ti pla mid in Agrobactl~riutn ttltnefaciens, antibiotic produc"tion,
starvation resJ>ou :e (Rliizobiurtr. legu.t1.7i.tto-sarun1, Vibr.io pp.) .motility and
biofilm formatior1 (7 ,8] . Only orne of tl1ese p1oces es are r.elevant fur ooil, ruttl
Ta.ble 7.2 lists the phe11otypes rnost t1elevant fo,r this llabitat. lmportantly maoy
AHL-regulated genes are invol ed in rl1e ecological .inrera.ctions o f bacteria
with their bioti.c or abiotic environment. such as in the case of tbe productioo
of aotibiotics and cbitirutses. bio'fi!1n forn1ation, rruionary phase response~
motility an{1 lig11t producrjon. Spcci tica:lly. QS often directs the inte1'tl.ctions
between bacteria and eukaryotic 11osts (e.g. patl1ogooe: i ). Per orp nism, rr1ore
than one :Phenotype may be controlled by QS. For insranc.e. Stnorhlmbit1ni
t1zeliloti l1a l' t<) 5% of itS genes belongirrg tO differ.e:n:t regulcms u:nd:er
QS control.
'What i the eco1og:icaJ signific:ance of QS in oiJ? By regulatin:g gioup
proces..es uch a . :ymbiosi , virulence. oompetence for transformu1ion,,
coujugation, antibiotic pro,d:uction, 1notilil)1 sporu.l.arion and 'bicr.til:m fo:r:mation
[35); QS ~eems to provicle a meeh;1ni m i.n ooil by \Vhich bacteria orch.estrnte
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Microbal lnteractions in Soif
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to bacterial AHLs. Fo.r example, tb:e expres ion of over 100 plant genes
was found to be u;preguJated at leasl fourfold (16). Hence, plant rooo 4<listen ..
to bacrerial communicatioo, a.nd in tun) S\Vitch o-n u r.ange of pecific
physiological response including tre aJ1d defe11se (iod.uce,d y te111ic
re ist.anc.e --:lSR) response . See al. o Chapter 20.
7.4~4 A tTERN,ATJVE
FuNcrtoNs FOR AHILs
Very recer1tly, it Wtls reported that the canoriical QS autoi.nducet of t:he AHL
type al o pass.es additional f'U.nctions. Specifically. some AHLs were found to
act Iike antibiotics, inhibiting the g;rowtb of . ensitive organimns. In addition. a
ideropbore-like action (~equestering of iron} was found a ano:ther potential
m.ecl1arti m of action. He11ce. ir i possible thal AH. are .actuaJ1y muJti.purpose molecules that can perfo:m1 differe.nt ecological roles i'o accordan.ce
\\l:.th tbe need of the producing org.anJ ".OJ. in jrs local environment. Tties-e roles
are likely linked to lhe activicy of tite org11nis:m in it:s nabitat.
7.4.5
ENVJRONMENTAL SiNStNG M ECHANISMS IN f UNGI
Fung occur botb a unicellu.Jar and 01ulticelltU.ar filamentous forros and also
need to peroeive environmeotal cue rutd regulate their responses appropriately. Clearly. the problems faced by a multicellular filamenrous tn:ycellutn
are not exactly the same as tbo e fared by unicellular organis:ms such a
bacteria and yeasts, but 011le ir1r,ere ting parallel exlst. These lirie dis-cussed,
below w1d. referred to Jn Table 7.3.
Fungal mycelia are reg,ulatecl b;,r ophisticated cbemosensory mechani. m
whlch play importanr. roles in the interaction" wit11 o.ther organ.Lmu:J. Por
instance, \\'it.h pla11t ho ts, ecilors and inbibito:rs are important; \ ith
co1npetiton, antibiotics inftuence tl1e interaction.s; arld in tungal I?athoge11
mycotoxins are involved. Addit:ionJillly. there are "in-hoo e sig.o.aling mecha:uism , involving pheromonei which faciltate the intern.cti.on of ca:mpatjble
gametes and de,relopmen.tal horm.ones wb.ich regulate tbe formation or
,m.aint1::n111ice of di:fferentiated .m.ul'ticeIJ.ular struct:ures. B.amples of th.ese are
given belo\.v. A f11rtber broad category of in-bouse ignals c.onsist of tm array

of endogenous cues that bave collecti\~ely be.en t~rm.ed aritoregru:tors [l].
T11ese signals convey in.fonnation on envir:onm,e ntal oonditions or the status of
c.ell within a fu:ngal colon.y o:r mycelium ~u1d are transmitted as extracellular
met.abolites. Autoregulatory sigoals ens11re coordinated functicm and are
involved in ger111i.11ation, morphogene.~i , asexual and sexual development
and.in dimorphism of pec'ies exi ting both ,ru sio.gle cells :.tnd myceuiL J\ s yet"
we still have mther limited knowledge of th~ m-0lecular mecl1anisms of
regulati.on and o:f the aerual ignal mola.'11le. involved. Ma-ny siudies ha.ve
beeo cou.ducte.d \Vith tnodel specie , whicb. are not alwnys typ.icaJ oil fu ngL
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Microbial lnteractiorrs in Soil
e.o enabie the colonization of
197
fresli~
nutri.en.t rich substta.tes. During tbis
process and o ther.' , progrnmmed or environntenta.lly induoed c-ell deam may
oocltr. Cel.l d.entb can occLrr through autopllftgy1 apAptO$is or .nur:os.ls. The
fir$1: two prooease~ are programmed and genetically re.gulated. wher-eas the
third i envirnomei1tal1y indu:eed. ProgramJtled cdl duatli allows for the
ren)OVal of unWilted cell ", n1akir1g w.ay for cellular remod.eli:ng and
differen.ti.ation [20].
7.5 TYPES OF MICROBIAI. INTERACTIONS IN SOIL

Given the fact that life in sml is ooncen.ttate.cl within micro1patial ~artcts.: Ot
b.ot spots, ch.e ecological and evolutionary need for mi.craorgllllisms to lnteract
with oti1er organism~ Iikely ro be greaL As d1scus~ed. d.epending on th.e
microhabit.at type, a multitude of interactions between. or.ganiStllS that 'hare
tbe ame microbabitat is :po sib.le L1~2l ,22]. Here we di euss relevant aspeen!
of tbe ecology .o f micrQorgaa_i . ms involved in selected:

Metabolic in.leractions in ol
Ant.ugonistic nteractio-ns in uOil
Pred.atory interactions in oil
Internction witb. fru1gi
I11teractio1:}S witb ba.cteria
7.5.1
M ETABOLl:C INTERA.CTlONS IN SOll
Mic-robial rr1et:aboJjc activity is the key determinam of tbe stn.tcture aud

compooition of a microbial community formir1g an a sembtage. So-me
tnicrobial processes ate canied out by juS"t 011e patficul-ar gro:up of miaoo:rgatris'fns~ resulting in a largely hcrmogen.oous assembla..,get w:b~rea other
processes cannot be performed by single microbial g:rou11Js. The la.tter
processes are thus dependent oo a functio.o.drlv-en microbial ab..~mblage
\vbich eonslsts of n1ore tllan on:e species, ~ming a b.etero~eneous assem-
blage. MJcroorgani ms in sol can. int.eract metaboli.cally in: ditfereo:t ways.

Two o:r more miaoorganisn:ts can oantribute different e1.ements of a comu1on
metaboJ.ic patblvay. resu lti,,g in the net synthesi.s or degf'adation o.f speci.tic
compoonds, to the beneftt of all patrlcipants in tb:i metabollt (syntrophic)
network. Nitrlflcatwn , the couversion of ummonia to nitrate via nitrlte iJ:1
differe11r steps (see Cl1aptet 9 for furtber d.e tails) is on:e example of sucb
cooperatioo. This process need the cnmbined a.ctlon of OOJn'lOnia-oxiditing
(e.g. Nitroson1on.r;;s spp.) and nitrir.e-oxidiziog (e .g. Nitrobm:ter spp.) bacteria.
ldeally, bo.th bacterial specie.s are pres.en.t in tl1e urne physical locatio:n and are
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201
f ungi may be associated with defense of territory, since the release of

enzymes to degrade polymers represents an investment of resources.
Competition with other organisms for breakdown products can first be
prevented by tight feedback regulation of depolymerizing enzymes and the
u e of wall-bound enzyme for the final stage of polymer breakdown, che
most easily used monomers are this less avaiJable to competing microorganisms. Another secondary defense of territory mechanis m is the production of
antibiotics or other suppressive metabolites [26].
As further outJined in Chapter 8 and Chapter 18, clear evidence for the
ecological role of antibiosis in soil co1nes from studies on the biological
control of plant pathogens. For instance, Pse11do111onas fluorescens Fl 13 is
able, by the production of 2,4-diacetyl phloroglucinol (DAPG), to control the
patato soft rot pathogen Envinia carotovora ssp. atroseptica as a result of
its capacity to produce DAPG. Evidence for the role of DAPG production
was obtained by controls wilh mutants in which DAPG produccion was
inactivated [27]. Sorne higher organism may even u e the antibiotic power
of bacteria to their own advantage. One revealing recent finding was the fact
that specific ants use antibiotic producers (streptomycetes) to ward off key
fungal pathogens from their communities, and evidence far similar strategjes
in other higher organisms (e.g. specific beetles) is emergi ng. lt seems that
bacteria! antibiotic production ha emerged during evolution to erve specific
biological aims, relevant not only far antibiotic producers but also for other
organisms with which these form as ociations.
The second clas of inhibitory compo unds is formed by the bacteriocins.
Bacteroci ns are proteinaceou compounds produced by bacteria, which are
toxic for ghtly-related bacteria. Thus, bacteriocins mediate, in a negative
way, the interacti ons between closely-knit organisms. The best studied
example is offered by Escherichia coli, in whjcb at least 25 different
bacteri ocin (cal led "coljcins") have been described (28]. Colicin are
produced when competition for nutrients is fierce, i.e., under condjtions of
low nutrient availability per cell (al o called "crowding"). Tbe colicins are
released by cell lyss and tran ported to susceptible celJs, where they may
degrade the DNA, inhibit protei n synthesis or destroy membrane integrity.
They do not lyse their own cells, as these produce an immunity protein that
binds to and inactivates the toxi n. This sophisticated mechanism to interact
with closely-related organisms is thought to play an importa.nt role in
interactions within the microhabi tats in soil in conditons that allow
crowding. The microhabtat structure of soil may promote the coexistence
of different types of bacteriocin producers [22), which parall.els the effect of
soil structure on antibiotic producers. Hence, the spatial strucrure of the soil
environment has majar implications for the level of interactions between
m1croorgan1 m .
Co y ighted m
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205
7.5.5 f UNGAL INTERACTIONS WITH BACTERIA

Conversely, son1e fung have evolved metabolic capabilitie to take advantage
of the bacteria with which they sbare the habitat. For instance, soil fung uch
as Pleurotus ostreatus, Lepista 1iuda, Agaricus bru111resce11s and Coprinus
quadrfidus can anack oil bacteria (e.g. Pseudo1no11as and Agrobacteriu11i)
and use the cells a a ource of nutrient . The fungi sen e the bacteria! colonies
(by sorne unknown chemoattractive mec hanism) in their vicinity and initiate
specialized direc tional hypbae that can grow towards a bacteria! colony. The
fu ngi ecrete pecific compound in advance of the directional hyphae that
lyse the bacteria! cells. After penetrating tbe bacteriaJ colony, the clirec tional
hyphae produce a coralloid mass of as imilative hyphae. After colonization,
there is no furtber proliferation of hyphae in the vicinity of the bacterial
co lon y. Nutrients in ex ces of the immediate fungal requirement are
translocated through the directional hyphae for utilization elsewhere. The
bacterial colonies disappear within a period of time as short as 24 h. This
interaction between fung and bacteria! colonies gives the fungi a considerable
advantage in obtaining nutrients e pecialJy under tbe nutrient-limiti ng
FIG URE 7.6 Mycetium of Hebe/on1a cn1s1u/i11ifonne after colonization of a potas ium
feldspar surface fo r seven months. Thc ample wa prepared by fixation and critica!
point drying followed by gold coati ng and analysi by canning electron micro copy.
Hyphae (H) and bacteria (B) are visible. Sea le bar= 1On1. The hyphaJ surface contact
i mediated by a film of extracelJular mucilage (arrow) nnd bacteria are seco in Lhe
mucilage. (From Finlay. R. D. and Ro ling. A. lntegrated nutrient cycles in forest
ccosysten1: the role of cctomycorrhizal fun g. In: Fung;,, Biogeoche111ical Cycle.r. Bd.
by G. M. Gadd, Cambridge University Pres . Cambridge UK, pp. 28- 30, 2006. Wilh
permi sion.)
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Microbial loteractions in Soil

19. Cben, H., Pu.jita, M.~ Feng1 Q., Clnrdy, J., and Fink, G. R.., Tyroso1 is a quorum
sen.- ing :molc~le In. Cantflda albka!1s, PNAS, 101 , St/48- S0.52, 2004.
20. Lu_, B. C. K., Progrotrul'Wd ceJl (leath in fungi, In The Myc~ GM\lfth
Differemiaiion and St!:\11aJity, Kti;es, U. and F'a'Cher. R.. Bds., 'VoJ. I. Spn,ger,
Ber1ln. 2006.
21 . De :Boer, W., Folm1u1, L. B., SumulerbeU. L. c.. au.d 13-oddy, L . kiving in u
fung:al 1.vorld: impnct of u1gj on oil bacreiiat oiche d~'elopn1ent. PBMS
MicrobilJlngy Review , 29. 795~81 1 , 2005.
22. DouglM, A. E., truLegies in a.nt.agonistic and ooape.r.ttive interuetio~ In
M1crobial EvoJuri,011, Miller, R. V, aod Day. M. J., Bds.. ASM P~ess,,
Wa 'hingtoo,. OC, p,. 275-289, 2004.
23. Wolfaardr., G. M., Lawr:ence., J. R., Ro.~ R. D. Cal<fwell. S. J., an.d
Caldwell, D. E., ~lfulrieellular orgruiization in. a degradative biofibn oom:mu-n.ity, A.pplied tJ1ui Enviromiren.tai .~icrobiology, 60,. 434 446, 1994.
24. Ueda, K., Yamasbita,. A.. lshika'\V~ J. Sbimada, M.. Wmsujj. T. Morimum.
K.. lkeda, H., Hauo:ri, M ., and B.e:ppu.. r., Gen:.ome equeooe of SymbJbacteriunl. therrnopltilunl~ an un.cultivable bacterium tbat depends on mlcrablnl
cornmensailsm. Nucleic Acds Research, 32, 4937-4944. 2004.
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EcoffJg)' l....etters, 3, 12:2-l.30, 2.000.
26. .Dea.oon, J., /tungal Bl.oletgy. 4lh ed., Blackwell. Oxfard. 2006..
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0 0an1. F., Eoological interaction of a biocontrol Pseuiioniona.s fttJl)r.eacencS
str.ai.11 produciug ~4 din:ciecyl pb1orogl'\lcir1ol \\itb tbe s'O.ft rot potatn p.atl1og,;e:n
En.vinl.a carotovoro sub.sp. o:troseptica, FEMS Mi.crobio/Qgy- E.cowgyt 23,
95- 106, 1997.
28. Pagte, L. ru:id Hogeweg, P., Colicin djverfilr:y: a result af eoo..evollltionary
dyn:amlcs. JounmJ o;f'.l1tel1N!ncal Bioi<Jgy, 196, 251 -26 1~ 1999.
29. Dwid, W., Biology aod global diB'tributiori of n:1yxohactria in scil , FEMS
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30. Johao"son, J. F., Ptrul, L. R., and F.i1n.ta.y .R. D., MicrohlaJ. intetaction in rhe
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3 J. Bianco:tto~ V. an<l Bonfante, P.. Arouseula:r mycrurhix.al fungi: a speciali :e.d
nicbe for rhizospher:ic and endooolhtlar bacteria, Amorde Van ,leeuWf!n.Jioik.
81, 3{)5:..31 1. 2002.

32. Jargeat.. P., Co. seno, C., Ola' h, B ... Jauneao, A.~ Bonfilute. P., Ba:tut, l and
Becard, O., Isola:tion. froo. living cap.acitioo, a11d. genome mr1ctur:e of HCtr:ntii
daJ11,v G:lumerl.bacter gi.ga~ornrom.'' tbe endooeilulro- ha.ctmiu:m of the.
n1yeorrhizcl fungus Glgasporti. margarita. Joumcd of Bm:ttrriology, 186.
1
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6876-6884, 2004.
33. Chri teru;~n. B. B. H~aageuseo, J. A.. J. Heyd-0ro.. A,. and Mofu1, S. .Meraholic
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Applil!d and Envl.rt11u11entl Mi.c.~tology. 68.. 2495-~ 2002.
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Modern Soil M icrobiology
34. Horoby, J. M., Jensen, E. C. Lisec, A. D., Tasto, J. J., Jahnke, B., Shocmaker,
R., Du auJt, P., rutd Nickerson. K. W., Qaorum sen ing in the dirnorphic
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Microbiology, 67, 2982-2992, 2001.
35. Mc Lean~ R. J. C., WhiteJey. M.. Stickler. D. J., and Fuqua. W. C.. Evidence of
autoinduoer acti ity in oatoraHy occurring bofilms, FE~1S Microbiology
Letter . 154, 259- 263. 1997.
36. Miller, M . .B. and Bas ler, B. L.. Quorum seo ing in bacteria. An11ual Revieu of
Microbiolo8Ji, 55, 165- 199 2001.
37. 01 o.n. S. and Hansson, B. s.. Action potential-like acrivily fou11d in fungal
1t1ycelia is enBiti ve r.o stimulation, .Na1111l 1rissensch11fte11, 82, 30-3 1, l995.
1
Copyrig'1tcd matcnal
Plant-Associa.t ed
Bacteria Lifestyle and
Molecular lnteractions
Jan Sarensen and Angela Sessitsch
CONTENTS
8.1 [ntrod,uct:i:or1. . . ~ ........ "~ .......,., ..... , ...............~ ..................,... "....... ~.-; ~ -~ ~ ~
211
8.2 ""f he 'R1lizosphere as a Habit~a.t .......... ., . ........................... '" ........................... ~..........~~~ 213
8.2.1 Rh.iZOS"phere Ba-cteria ~" ~ "' .....................",....... ~
214
t; ....
""'
... .
.......
..
4 ..
, , . . . .. . . . !!< ....... t!I ,., ..
. . . . . . .. . . ... . . . ,
8.2.2 Nutrient Availa:bility and Metabolimi/Orowtb ...................... 215

8.2.3 Microbiat Ant<.igon.i m and Biological Control
(Bioconrro1) of Plan,t Parhogeus. ............................................. 217
8.2.4 Developnu:mt of Plant Resista.ne-e to Patlt\>gens ..................... 219
8.2.5 P'fant Growth. Pro:moting Effects .............................................. 220
8.3 The Rhi.zoplane as a Habit.at. ... ,. .................................................
22.'J,
.8.3. 1 llbj7,..op'lane B-acteria.............. ~ ..................................
221
8.3,2 Root Surface: Colo.nization and Growth ................................. 222
8.4 'f"he End~plan.t .Habitat .......... ~ ....... ~ ........ ... .. ,. . ~ . . .. ... """ . -~.............. ~ ,. ...........\O~ ........... ~ . .'22.4
8.4. 1 Ettdopbytic Bacteria (lncluding Rbizobia) .............................. 2,24
8.4.2 Root and Veg-etative .Plant Tissue Colouization .................... 226
S.4.3 Nodule Formatiot1 and N Fx.ation ......................................... 230
8.5 ConcJuding Remwks ......... ~ . .................................... . ........ ..... , ..... . . ~ ~ ,,.-,. 23 l
11> . . . . . . .. ..._ . . . . . . . . . ..,,
1'i . . . . . . "' . . . . . . . . . . . . . . . . . . . . . . . .. . . . .
J ........
. . ......
8.1 INTROOUCTION
Plants in oil offer a highly specific environnient to naturally, oocurring oil
microbial c:-0mmui;rlties. Henee., over evoluti-OJ:Hll'Y time, soil micro:o:rgani&w
h.uve developed a rar\ge of sttategies thttt h1ve -enabled. them to in~aet 'witb
,plant.~. 1'he cotnpositior1 of plant~ass-Ociated microbiai oom:mmtlti;e-s is bi,gbly
211
212
Modern Soil Microbiology
in1portant for the performance of tbe plant, as the nteraction \Vith lhe plant
may be bene.ficial, neutral, or bannful. Differenc ol bacteria interact at
differcnt di tances and witb varying degrce: of iatin1acy with plant . They
may
1. Ljve in the soil infiuenced by the rools (Le. the rhizo. phere)
2. Colonjze the root surface (i.e. tbe rhizoplane)
3. Colonize the inrercellular spaces or vascular ti sue in. ide plant'\
(endoplant habitat)
The rh.izo phere has been defined as tbe con1part1nent of soil \Vhtch is
inluenced by plant roots and by the oompounds the e relea. e. Root depo its
a11d exudates represent important

source of sub trate avajlable to botb
"
rhizo, phere and rhizoplane microorganjsm , and thu exert a great influ.ence
on the structures of the n1icrobiaJ communities that are pre. ent. Furthennore, it
has been hown that the con1position of these ub trate can di ffer ~ ubstantially between plru1t species as \Yell as bet\veen culti ars. and al o depeod on
the developmental stage of the plant. In additjon. che compo ition of root
exuda/es may be affected by soil parameter , including pH, soil te ture,
nutrienl avai labiliry or limitation, and by expo ure 10 plant pathogens. [n
addjtion to the rather t:raightforward effect of root e udace . there are other
effec1s oo microbes in the rbizo. phere \vbch re.!i ult frorn spalial and temporal
cha:nge. in the roots and. tbeir physiology, ca11 ed by factor such as dju rnal
variatio.n , root aging, .lateral root e.me.rgence le~1diog to temporal wounds, and
plain root wounding.
De.spitc lhe strong effecrs of plru1t roor. aod their exudares on the rootassociated. n1icrobia1 popul.ations, the soil microflora il'. elf represent the
pri1nary ource of plan1-a &<>ciared microorganisn1s ru1d, therefore, ge.atly
detennines their co11wunty structure. Differe11t sols are known to contain
higbJy differing n1jcrofloras, and fro.01 these diverse 111icrobial pool , planr
exudare select specific ubpopuJation whicb colonize the rhi zos,here,
rhi zopl ane or endoplant babitat . This may Jead-dcpendiog on tbe bulk
ol 1nicroflora-to varying microbial population colon izi ng a particular
plant pecies or cultivar. Furtbermore, due to electjon by plant exudate
compo ilion, specific bacteria tnay be tiinuJaled to become active and gro\v,
leading to a lower di ver ity of microorganism as \\1elJ as to a bigher microbial
biomass in the rhizo phere soil cornpared to the bull< oil.
Not urpri ingly, tbe root-as ociated microbial con1munity sub equently
al o develop a functional diver ity acccrrding to different type of rnetaboli rn
and capacite to respond adaptively to tbe upply of water, oxygen and
nutrients, as well as to .the. biotic interactions of co1r1perition, antagoni m and
Copyrigritcd material
Plant-Associated Bacteria-t ifestyle and Moecular lnteractions
213
synergisrn between the microbes. With tbe aew metbooologies that bnve been
developet.i over rec..~nt years (se(} Chapter J l throu.gh Cbapter 16), tt has
becotne ciear tlmt ~1.>eeific compounds in root exudates (e.g. limiirng nutrie11m,
antir.nicrobial metaholites, hormo.nes an.d. communication signals) often play
1najor r'Oles in. the microbial interactio~ in the root zonet incfuding those
between the plant root and tbe microarganisn1s. Chapter 7 alread;y diseussed
tlle pecitics. of st1n:le of the lteniction between microorg-ani.snm in ooilA
It is the airo of thi chapter to _present an overvie\v of the mierobial
diver it)' that i a. . oeiated \Vith the ptant. io particulat at the ront hrterface.
includlng n trentise of selected key f11nctional traits df relevaot root microo.rganism . In pa.rticuJar and. where pos ible, we .. hall attentpt to i<i.entify lih~e
fu.nctionaJ tmits, includin,g their trtolecular dete:rn1inant . Bmptta i will be
placed on rhizosphere. hacteria. whereas f11ngi wilJ be dealt witb anly where
tlley appear as targets of baeter.ial arttagorsm (p.lant pathogens). Tbis treatse
of selected b,acterinl groups kno~rn for their beneficial d'fe.Cf,s wilJ provide a.
useful oombination to the reruier witb respe_,Qt to both gene:ml and specic
.e
me].udi'ng :ome recent di

n~oot-assoc1a.te
'
d tnJ.CJ.'OOl'p:ll
m1orm.at1on.
cove11es.
imui and th'.e interactions they are involved in will be treated e-parate.ly in the
mree following sections according to their physical oompartments: tire ront
exterior (rtlizoophere) che root urfaee (rhizaplane). and the plant :interior
(ertdoplant).
8.2 THE RHIZOSPHERE AS A. HAOITAT

The mjcrobiaJ. commumty in tbe rhizo .vhere develops prlmarily tilrotlgh the
relea, e of orgrutic ub. trote from th.e root (a prcJCess called rltizodtp()eiton)~
which mny amount to lOo/&-40% of the plant's photooynthate. In light of t.he
import.11n.ce of 1.hl.zosphere n1ic:roorganJsm to tbe various . teps in n:utrient
cycling processes (e.g. N cycling by nlinera1ization. :oitri'fication and de.nitrification), it is \lnderstandable why the bacteria! commumtiea in the
rh.izosphere have reeeved ' O much attention in agronomy (\vith respeot to
ct1ltivation nnd oil managern:ent) over ..everal d.eca(les. More receutly. the use
(}f p1ant-beneficial bacteria .as oil or plat1t .eed inocula~ e.g. tori protection
against pJant pathogens and fo;r hmmona.l planr growth stlmulmioo. (see
Ch;ipter 2Q), I1a ' greatly "timula.ted ne\\' re.searcl1 in microbic"ll rhiz~~here
interactioo .. In the follo\ving. we hall present ex.amples al rocm;i;t; DN.A-based
diver ity studie in tb.e rhizosphere and studies u ing reporter bacteria. to
revea! n.utrient (C, N. P. and oxygen) limi.tati.o ns to bacteria~ In addition,
e\1idence from stud:ies on inoculant plant.-beneticial bacteria. \Vhich airo to
identify the xnolecular de~nninants of anmgoni 'm agaitmt fungj (bioc~ntro1),
Copynghtcd material
214
Modern Soi l Microbiology
tbe development of re istance ro plant pathogens, and planl gro\vth promotion,

will be discussed.
8.2.1
RHtZOSPHERE BACTERIA
The young rhzo pherc. conlains an extensivc microftora encon1pa sing

saprophytic and plant-benetlcial bacteria, which are allracred by the release
of exudate corr1ponents. n1ucigel and loughed-off root cortcx cells. 11le
rnJcrobes are often .inclttded :in the gelatioous 1nucigel layer that cover the
root. 1.'he sloughed.-off cells a.re lost fro111 tbe root cap, after \v.hich U1ey can
becon1e heavily colonized and sbO\Y autolysis. Ainong the fast-growing and
early-coloniz.ing bacteria attracted, by tbe exudate are rnembers of genera such
as Bac;llu and .Pse11domo11as, but also of the N-fixng genera Al.<JSf)iril/un1.
and Rhiz:obiu1n. 'fhe di versity of Pse.udo1nonas 'PP in rhizo phere amples
(herc defined as the cell com1nunity extracced from ol adhering to the root) is
discus ed briefly in the following paragraph, \Vhile t.hat. of A:;ospirillu1n ru1d
Rlrizobiu111 spp. i di cu sed in subseque11t ection .
Much information ha. been obtained on the di ver ity of Pseudo111onas pp.
from soil and the rbizo phere using conventional culLivation technique . but
receot ludie hnve al o c learly illu trated tbe opportunitie offered by the
new DNA- and RNA-based technique to de cribe the abundance and
diversity of indigenous Pseudo1nonas population in natural oil and rbizosphere1. Molecular detection of Psettdo1no11a spp. among environmencal 16S
ribo or11al RNA gene clones has recently confirmed the numerou ob ervari.ons, made by cultvation-based m.ethods, thnt P eudv1nt>11as spp. are
generally 1nore abundanr n rhizosph.ere tl1a.n in bulk o.ils [l]. The stimulatory
effect of the rhzosphere crf several. crop plants ou oil p. eudon1onads has thus
been dem.o a trated, a tbe number of clones phylogenerically related to
Pseudo1110.nas spp. was higber in rbiz.ospher:es of both Loliu1n Jeren1ie
(ryegra s) and 1nfolii1f11 repe11 (\vbite c1over) as con1pared to co1Tesponding
bulk oil [2]. Clone equencing was nor performed in the latter . rudy. but
affiacion of the clone to Pseudo1nonas pp. was dc1nonstrared by colony
h.ybridiz.ation u ing lhe Pseuclon1orias-. pecific oligon uc leotide probe PSMc;.
On the ba i of partial sequencing of 16S rRNA g-ene clones. plant roots have
further been , hown to have a seJective effect tO\vnrds thc y-Proteobacteria"'
leading to a predon1inance of Psei1dom1Jrias spp. in the rhizo. phere.
Finally, Duineveld et al. f3] made an interesting attempt to co111pare t:he
relative abundances of 16S ribosomal RJ\iA gene~ and con'e .ponding I6S
rRNA fragment in Chrysantliem.itm rbizo pbere soil . Prom inent PCR
products were separated by DGGE ( ee Chapter 14 for an explanarion of
the tecbaque) and excised for equencing. i11 order to identify both active and
total. populatio.n.s a revealed by abundant .1 6S rRNA gene and J6S rRNA
Copyrigritcd matcnal
Plant-Assocated Bacteria- Lifestyle and Mole<:ular lnteractions
215
attz.plicons, re.spectively. 11:1e analysi demons:trated th:e oocurrenee of fewer

DGOB band (lower diversity) in the pro.files from th.e rhiZcoopbeire rus
compared to tho e from che bulk sol. Pseudc1nonas 'PP were among tb_e
abl1ud,ant genera. as judged from the data base homologies obtained.
Spe.cifically, apart from. several Bacllus-relnted. bands., at least 3 011t of 12
Of the banda were related t<l PseudoF1t.01UIS pp.
8.2.2
N UTIUENT A VAll>\BlllfY ANO M ETABOLJSM/GROWfH
Root ext1dates have long been considered to repre.sent the major crurcc" o:f
carbon (C), upportiog t'he growfh of rootcoloniziog b.ae:teria in the yoong
rlti7.;.0spl1ere. Soluble exmlate C'Omponent include a "'W'ety of common
monomeric compou.nds (sugars., amino acids and fatty acids)t w ber-eas
carboxylic aci.d (citrate malate, succin,are a11c1 oxalate) bave be:en rep.art~
to be partcular1y abundan.t and im.portant for bacreriaJ growth in tlle rhizosphere. This was sh.own. by tbe fa.et tltat Pseu.domon.a Jtlutauts una.ble to
utilize sugars are good root colo.n.izer wriile rnutants uuable t() tttili~e
orgnnic/carboxytic acids are not [4].

Tbe exudate e co1nposition anc1 the distribution of di:tferent e oompo:unds
in tbe rhizosp?iere :is undou.btedly very important 'for oompetition between and
selection Of microbes, as documented in studies usin.g seci:tic reperrter
bacte.ria to seose C availability io. the rhi:zosphere ( ee Cbapre:r 17 and
CbapteI 11 for a description of bio en ora and reporter gei1es, rwpectively).
Inoculant lux-repurter (biolwninescence) gene-equipped Pseudomnna-s p:p.,

wb.ich .nad beeo. b:riefly tarved (tor carbon) cletlrly responded to exudare C.
Wheat root. ex.udate elicited a respon ~e com.pamble to tbat of a .rednci.ng
sugar mooo1n.er (gf.uco e). ratber than that of eommon a.mino a.cid (glt1tamate)
or carbox.ylic acid ( u.ccinate) oomponents in roor exuda:t~ [5]. TI1e cells we;re
also sbo\Vll to be capa:ble of discriminating between the composition uf root
exudltte8 from plants grO\Vll with or withottt nerbicid.e treatmetlt. Other studies
u.sing Pseudo1;w1ias reporter bacteria have detnonstr ated C limita:tion in bulk
soil, but not ir1 the rhizosphere. WhiJe C may not always be limiting in the
rhizosphere, tl1e composition ttnd availability of specic organic components

CM still be import.ant for tbe act11aI e statllB of Pseuti.ommlCJ~Y ceils intltl.b:iti:ng
this h.a bitat
Tbe significance of nim>ge.n (N) and phosphoros (P) limtation in soil .M d

tbe rhiz.osphere bas aJ o bcen ex.a.mined t1.siog . imilar blolumt.aesee:-nt
Ps.eudovwrias report-er ll1il11s [6,7]. ln bulk oil. neither N 110.r P limil~tio11
co\1ld be ob~ ervecf, in contril$t to tbe ob ervati-oru; above of C limitaticrn in tbis
habitat:. However. ameodn1ent of soil with barley stra.w changed the life
conditio.n for rhe iutroduced, Pset.ulonwn_,r stran. limitatio'fl be.carne
apparent a C-rich polymers f:rom the barley re.sidue.s were degraded.
Copynghted material
216
Modern Soil Mcrobiology
The rhizosphere (barley) demoostrated signicant N li11lation, \vhereas P

limitation wa not observed. Tb_is work was the first idenli fication of a major
outrient limitalioo (N) of pote otial sgnificance for the growth and activity of
p eudomonad in tbe rbiz-0sphere. An important recent development i
represented by the so-called doub.le reporters, which can addres change in
autrient availabiJities u. ing a single reporter strain . Thu. , tlle concomilant
application of several repo,rters addre-S&in,g C, N a11d P c:tVfLila'bilities in the
ar11e :habitar will be use:ful for fuwre di&ections of the nutrient conditions tbat
are .key to tbe growt:b and survivaI of .Psel1don1011as pp. in the rhizosphere.
T.he N reporter strai:n used in the above tudy reacted to a 'li111itation of
both ammoni urn and common amino acids (e.g. glutaJnte). l r is stil l unknowu
whether pecific N cotnponents ia exuda.tes regulate tJ1e growth of l)set1do1110 11a s spp. in the, rbizo phere. Specific reportee bacteria Lhat rcspond to
individual ainjno acids how grear proinise for the identi fica tion of sucb
growth -l inl iting compound. . Inductioo of a respoo e in a ly ine-responsi ve
Pseudomonas putida reporter strain was demonstrated in thc rh izo phere of
n1aize, but oot in bulk oil. In addition a tryptophan-re pon ive reponer ~trajn
howed ignificant induction in older root egn1ent with lateral root
fonnation, but not at the root tip. Finally, a Pseudo1nonas jluor escen reponer
tr'din \Vas recently u edro show t:hat tbe regulac:ion of the uplake of putre cine.
a cornn1on polyamine in root exudate (tolttato), wa important for growrb and
thus competitive abiljry in the rhizo pbere.
Oxygen availability in. oil j offundrunental in1pon ance to the expres ion
of evera.1 traics in Pseudo1nonas sp,p. notably d.enitrication. but al o a
number .o f redox-regu lnted traits like che production of fluore cer1t siderophores a.nd t1ydroge11 cyaoide (HCN). High co.nsun1plion and lin1ited
supply of oxygen may be expected in the r'hizosphere1 i.n organic nggregates
(hot spots), or in highly compacted soil dte.l:i. Neven heless. there i. still a poor
understanding of the effect of oxygeo level in ol on the diversity of
microbiaJ communi ties. incloding tbe selective value of anaerobic trajt:s in
Pseudo111on.as spp. In the first attempt to determine the avai lability of oxygen
by reporter bacteria, induction of a low-oxygen- e nsitive P. fluorescen.s
reponer straj n took place in \\1etted (85'1o of the ol' water holding capaciry
[\VHC]) but not in uowetted (60% WHC) barley rhiiosphere oil and in
compacted bulk oil. Thjs study dem onst:rated thal water and 1ext uraJ
condrtion common in ~ oi l easily promote low-oxygen, and thu denitrif ying,
conditions in both rhizo phere a:a.d bulk soin . More work ba ed on reponer
strains i needed to e.lucida.te the role of redox-regulated phenotype in oiJ.
Usjng a nitrate reductase-deficient roura11t of a denitrifying P. fluorescens
. train, it was recently demonstrated thar nitrare reduction can confer a
selective advantage in the n1aize rhizospb.ere (Ler11anceau, P, pers. com1n.).
Copyrigritcd material
PlantAssociated Bacteria- Lifesl)tfe and Molec ular lnteractlons
217
8.2~3 MICROBIA~L ANTACiONtSM AND BtoLOGfCAL CoNTROl.

(BIOCONTROt) OJ PtANT PATtiOGfNS
Chaprer 19 p:rovides a ftl ttccotmt of bioco-otroJ agents., their productio11l use

and registnttion., Many application of ageots for the biolog:it"'al ooatrol of r:oot..
in'fecring fung are d:ependeot oo the efficier1cy of tbe illteractions betwem, the
biooontrol str:ain and the roots. Hei1ce. we here examine the mode of action
and effect of uch agents a ,infiuenced. by the unique co11ditioru_ in
the rb.i.zo pi1ere.
One cla sical, indirect mechanism of bacteria! antagoni m to"vard plant~
pathogooic fungi is the competition for es entlal nuttients during colonization
of tbe plant surface, The biooontrol agent ma:y here tttilize an essential growth
sub~t:rnte more efcieutl.y tliau ttre p:a.th<>gen. wltich is tbu.s 0111:ccrmpeted. Clear
competition betwee11 a P. pu.tida strai11 and. .Pj11,fm11n spp. was evidenc.ed for
pea seed exudate coinpoun:cl_s that trigger spora:ngium germination. This
resuJted in the prote.ction. of the peu agruru t these f'tlog , Indeed. the cataboe
pro.files of peudomonads and Pytliiwr1 ultinu1rn. are largely similar. snggesting
lliat oompetition for es~ entia1 nutrients is likely to be an impumlttt m,ech.ani m
in biocontr.ol [8].
At1otber clwcal mechtmis.m in antagonlsn1 of pl.ant pathogen_s is the
comp.lexation of ferric iron by ideraphores (org:an:ic moleeules of different
itructu:re8 that ch~late iro-n~ produced by .many roicrobes). Fe limit:ation i mo t
prominent. in neutrru or alkaline soil in w.bcb its oolubility (Pe3 +) i very
lo\v, The tnicroorga.ni "tns tb.ar JJroduce the most efticient sideropl10Jes rem.uve
tbi ~. essential micronutrieot fr-om competing organisms. and are tltus antagon~
i.~.c to these competitor [9]. Many fiuoresc:ent PsffudortwntM trailli excrete
siderophores uch as pyochelin. p}foverdn, p.eudobactin and fe.rriba:c.tin. Thi&
resulrs in rhe effective biological control of plant p'l:tth~en ucl1 as Fusarlu.m
oxysporurn an.d P)ithiWt1. sple1itlens. Soro.e .P ~11donwnas , trains are ex.tremely
efficient in tb:eir .:ron .equeatering, as they produce lrge amouots O:f efrective
siderophores lYr are able to Utke up a range of dfe:ren:t Fe-siderophore
complexes. Similarly, Serrara spp. produce enterobactin und aerobac'tin
sid_erophores under iron limitation, leading to aatag:oru tic action against
i1nporrant plant-pathogenic fungi llk.e Rhi:i'.o to1ii.a solanl an,d. Scleratinia
sclerotiorum,
Etzterobacter (renamed Pant.tJ.et.::). Pseudonzcnaa, Serrat1.a rutd Bur/ih:0l~
tleria spp. are common ::>roducers of b.y drolytic cell~walt d.egrading
ex:oe112yn1es u.cl1 as chitinase, whicl1 can antagoniz.e plant-pathoenic fun.gi
in tl1e rl1iz-0sphere. Rhiwsphere Enterobacter (Pmttaea) and Pseu<lomo11a.f
0
spp. further produce a number of antifungal metabolites like 2.4-dacetyl

pbloroglucinol (Phi), pb.enazine (Phz), pytlluteorin (Plt) or pyrrolnit:rin (Prn),
ea,cb of which can. itlbibit a r:mge o'f ptbogeni.c fung . In. particnlar, several
218
Modern Soil Microbiology
Pseudo11w11as bioconlrol traio bave been rudied inten ely to revea! che
molecular detennioants of tbeir antifungal metabolile production. In the 'veJI
tudied P. jluorescens traiti CHAO, the production of PhJ and HCN repre ents
a hghly efficient biologieal control mechani ro, which erve here as a key
exaniplc illu trating che complex regulation of biocontrol acrivity in tl1e
rhizosphere. Abiotic :factors such as che type and leve] of the carbon source
and the Jevel oJ oxygen, Fe, and other metals (Zn, .Mo. Cu) inft uence Lhe
production of Phi in P. flu<>rescens CHAO. Furtber, both the genotype a_nd age
of the host pla11t can n1odulate phi gene expression, wherea root infection by
P.)1thi1111i stit11u.latcs pJ1l expression [10].
Bacteria] productio11of bioStrrfactatll rrtolec11le , notably cyclic lipopeptides
(CLP.Y) , .in tbe rtzo p}1ere, has recendy gained consjderable attention. CLP are
wetting agents Lhat facil itate tbe colooiz.ation by P. jfuorescen. of floret surfa.ces
in broccoH and ugar beet eedJJng roots, but che production of CLP hru aJso been
a ~ igned an itnportant role in tbe biocont:rol of planr-pnlhogenic fungi in rhe
rhizo phere [ 11). The rhizosphere-oolonizing P. jf11orescen trai n DR54, wh icb
produce the CLP visco iaamide, couJd reduce the disease (da1nping-oft) in sugar
bt.""ets induced by Rltiwcronia solani and increase plant emergence. CLP, may acc
as so-called io11ophores. creatiog ion channel in the fungal cell wall. A role as
m etal chclatol'8 imilar to lha t of iderophores has aJ o been ob erved . Thc results
point to a role of CLP in microbial antagonism in the rnizo phere. bu1 it has not
been e tabtisbed \Vhetbertbe CLP act alone or in yncrgi m with other bacteria]
compounds. uch as antibiotics or extncellular enz.yrne . Such ynergism i
ugge red by evidence obt:ained wi th Pseildornonas train DSS73, \Vb.ich
p.roduces lhe CLP ampbisin.
In Pseudo1nonas biocontrol stralns, Che ex_pression of antagonistic traits is
often under global transcriptionaJ control .uivol.ving a nutI\ber of differen:t
ig11la factors. One exatnple i . formed by the PvdStype sigi11a factors
invol,,ed in the control of siderophore production. AI1orher one is provided
by the RpoS-type igma factors, wbicb are in volved in the stre . and s raonary
pha e controlled production of antifungaJ rnetaboLite uch as Phi , Pl t or P111.
Intriguingly, sorne bacteria also exh1bit quorum sen ing QS) cootrol (see
Chapter 7) of the synthe is of siderophores, exoenz.yrne and anti fu ngal
metnboljtes. A well-studied exampJe is the producLion of phcnazine (Pzn) in
everaJ Pse11domonas bocontrol strains. e.g. P. aureofaciens strain 30-84
[1 21. Direct experimental evidence for QS-regulated biocontrol in thc tornato
rhizosphere comes from Lhe ob ervation tb.at the Pin producer P. clzlororaphis
PCL 1391 lose its ability ro protect tbe planr against a Fusariu1n pathogen in
the presence of otber bacteria. as a result of the degrndation of the QS
ignalLing rnolecule by these bacteria (13].
Mutual interaction ("ero s~tal k~') betweer1 the RpoS sigma factor a.nd QS
control bas been docu1Denterl in P. pr,tida WCS358 in the rhizo pbere [14],
Copyrig'1tcd matcnal
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Plant-Associated Bacteria- Lfeslyle and Molecular lnteractions
221
production in the rhizosphere, it i . furth.er important to id:entify tbe phmtderi ...ed mole.eult:i_r sigruils rha.t may indu.ce bacteria! hormone produetlon.
Exudates from maize (Zea mays) roots may stimulat:e the product:ion of IAA. in
P. ff.lwrescetts ir1. vitf'O, but little is known as yet ahout the actual c:ompounds in
th.e exudates tba.t induce bacteria! honuone production in fue r'.hizospl1ere.
8.3 THE RHIZ.OPLANE AS .A HABITAT

In. the rhizopla,ne, the microbial com:munity dlat is locally resent has direc:t
canta.et to tl1e root epiltielial cell and 1nay thu ex:pel'iellre the bi~ t impact
of the activity of ttle pla.Dt cells., in.cluding the tran port of water. gS and
n1.1trients ar1d the exudation of organJc compounds. Moch as fur the rmm~
sphere, the rrospect of eed. or root inocuiation with plant-heneficial bacteria:
ha stirnu.lated a large 11un1beT of studi.es whicb are ail:ned at un.derstm1ding tbe
early events of b~icterial colonization a.nd proliferation on the root snrface.
Actually ~ many of the "rhiz.osphereu studies, inclttding, fo-r instanc~ the
rxtlcro copie images of early root~colonizing inoculants, are i11<leed :tttdie,s
of events that take place in the rhizoplane. In tire following. we 8.hall. present
ex.amples of suco studies tnat used detaiJed imagiJ1g by ad,1anced, .microscapy
of rblzopfane oolottlz.ation. 'Further, orne recent assessments of the molec111ar
detern1iruwts. o:f bacterial coloni1..ation and growth 011 the root stirfa.cet
ineluding biofiln1 formati<>n, \vill be present:ed.
8.J .1
RHlZOPLANE BACTERIA
The ignific~u1t ad.\ltmcement of tluore cence micro copy. in particular

confocal laser scanning microscopy (CLSM) to rud.y both sir1gle cells nnd.
whole microbial populations on .root urface has been une crf the most
inlportant asset ' of p1ant- oil microb-ology for the last deca!Je. Over time~
m..'lny s:tudies nave tise-0 combination of fluoresc.ent tain (probes), e.g.
taxono:mic probes .for a sele.cted rrain or group of indigeno:us o:r inoculate.d
microorgariiSin together '>Vith. p.hysiological probes for their specific cellular
activity. Chapter 11 and Chapt:er 12 provde extensive discumons of fuese
techniques, and, a:pproacl1es such n . fluorescence staioing by cell s:ur:fu~
tar;geting antibodie ' or RNA-targeting oligonacleotide p:robes in cambillation
wi.th CLSM b.ave heen receotly r viewed (20J. See Figure 8.1 and Figure 8.2
for a depi.ction of th~e rnethod . rbe combiu.ed techniques have allo,ved tb.e
tracking of single bacterial cells and tbeir pro1feration oo tlte rbizoplane.
resuJting :in profound nsigbt into the spatial and temporal patterns of early
root colonization. For instance, observa:tio,ns can be made fronJ tJ1e .ear!y
binding of (i11ocula11c) ceU to a eed onward to cheir tirm establishment on the
root surface.
1
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Plant-Associated Bacteria- Lifestyle and Molecular lnteractions
225
including heavy-metal-accumulating plants. The i olates have included a wide

range of both Gram-positive and Gram-negative bacteria, and currentl y
compri e more than J29 pecies [31 ]. Pseudo1nonas, Bacill11s, Enterobacter
and Agrobac1eriur11 have been fou nd Lo be the mo t abundant genera isolated as
endophytes from agricultural crops [31].
Cultivation-j ndependent analyses have shown that endophytic bacteria)
communitie are characterized by limited complexities as compared to those
found in the rhizosphere, with differing populations inhabiting potato stems
and roots (29,32]. Berg et al. (27] denti fied different bacteria! communities in
the interior of potato root and shoots in young as well as in ftowering plants;
bowever, the populations in these microenvironments were similar in
senescent plants. Furthermore, the rhi zospbere, phyllosphere and endosphere
ncroenvironments of roots and shoots differed greatly in tbe presenceas determined by tbe analysis of isolates-of antagonists against plantpathogenic fung. Most of such antagonists are commonly derived from the
rhizosphere and the endo. phere of roots. Molecular as well as culti vationbased analyses have already indicated that the plant genotype pJays an
important role in detennining tbe community structure (32] as well as tbe
activi ty of bacteria! endophytes [30). Culti vation-i ndependent analysis
confirmed Lhat, at least ac the species level, endophytes represent a subset of
tbe rhizosphere microfiora [32). Nevenheless, different subsets of strains are
esta.bJished in the e different niches. Generally, members of aJJ major bacterial
domain have been found to be able to colonize tbe plant interior. However, if
plants undergo stress, thjs may influence the endophytic microbial community
structures. For example, potato plants which were highly stressed by light
deficiency hosted populations of reduced complexity in comparison to nonstressed plants, which \vas probably due to tbe production of various stress~
induced enzymes and metabolites witb anbacterial activity [32].
Besides the endophytes that colonize the intercelluJar spaces and va cular
tissues, others can live intracellularly in the plant, in speciaJly developed and
specialized root organs. These mainJy include the organisms classically called
"rhizobia," which occupy nodules of leguminous plants, and Franlda spp.,
whicb are associated witb woody plants. Tbe bacteria forming nodules (on
roots and stems) have che capacity to establish a symbiosis with legumes.
During this symbiosis, the bacteria inhabit the nodules, where they reduce
atrnospheric nitrogen, making i.l available to the plant. The rhizobia fall into
four deep phylogenetic branches within the a-Proteobacteria: Azorhizobu1n,
Brad)'rhizobit1n1, Mesorhizobiunz and the RJ1izobium-Sinorhizobiu111-Allorhizobium group. Gray and Smith [33] empbasized the need for a conceptual
distinction between bacteria that enhance plant growth through very close
organelJe-based relationship . while re iding intrace11ularly within specialized
structures (nodules), and tho e lhat colonize ex.tracellular ti sues and ex.i t in
Copyighled material
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Plant-Associated Bacteria- Liestyle and Molecular lnteractions
229
(a)
(c)
(d) __
(f)
(a) Bocteria invaded the shoot ti s ue, an unexpanded third lcaf (indicated by thc
two top arrow ). orne bacteria outside the tissue (lowc t arrow). Bar = 10 m. (b)
High-magnification view of panel a howing bacteria colonizing the intercellular
space within rice ti ue. Bar = 1Om. (e) Bacteria colonizing the iotercellular
pace of the third leaf of rice. Bar= 1 m . (d) Section from rice coleoptile,
howing bacteria colonizing the intercelluJar space. Bar = 1 m . (e) Cross section
from lhe lip of a fourth rice hoot. howing Linte in va ion. Bar = t O m . (f) Lower
cross section from the ame founh leaf tip of panel e. Bacteria entered tbe young
fourth leaf aod colonizcd the intcrcelJular spacc (arrow). Bar = 1O m . (From
Elbeltagy , A., Ni hioka, K., Sato, T. , Suzulci, H.. Ye, B.. Hamada .T., Isawa, T..
Mil ui. H.. and Minami awa, K., Endophytic colooi1..ation and in planta nitrogen
fixation by a Herbaspirillt1111 p. i olated from wi ld rice pecie , App/ied and
Environ1nental Microbiology. 67. 5285- 5293, 200 1.)
book.
book.
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Plant-Associated Bacteria- l lfestyle and Molecular lnteractions
233
4. Lugtenber& .B. B. J.. DekkefS, L. C., aod Bloomberg. G. V., Motecular

<JeterroinaotS of rhizosp.here colonization by Pseudomonas, Arumal Re~1iew of
Phywiathology. 39, tt.61-490, 2001 .
5. Yeomao , C. Portoo:us, F., Paterson. E., Mehiirg, J\ . A., and Kiflbam, K.,
As~>mnent of lux-marked Pseudan1JJ11f1S fluoresceru far reporting on o.rgamc
carbon con1pounds., FEMS Microbiol.og)' Letters, 176, 79-83, 1999.
6. Jen,sc,1i, L. E. ru1d Nybroe, O. Nitrogcn availahility to Pse1tdon1tJnasftUfJrescens
DF57 is lir:nited. during det.':Qmpo. ition of bade:y straw in bulk oil aru:1 in the
barley rhizospbere, Aplie-d a1id Envin)lllltemal Microbiolog}'. 65. 4320-4328,
1999.
7. Krngelwid. L., Ho bond.. C., and Nybroe, o .. Distrlb.ution of met:abolic aetivit)'
and phosphate starvation response of lux-tagged Pseudornonas jlwr.'iCens
report,er ba.c teria in tile barley J'hi2oopbere. .~ppiied ami Etnrironmental ft.11.cro-
biology, 63, 492(}..4928, l 997.

8. Ellis, .R. J., Timms-Wilson, T. M . and Bailey. M. J., Ide-n:ti.fication of canserved
traits in tluo:rescent p eudom.ona.ds with antifungal ttctivity., En.virorrmemal
Micrahi.alogy, 2, 274-284, 2000.
9 . Raaijmakers. J. M., van der Slui . 1.. Ko ter, M . Bakker. P. A. H. M., Welsbeek,
P. J.., and Scbi:ppers. ,B., Utiliz.1tion crf l:reterolo;gous sideropooros and rhzosp:bere
co1npetence of fluorescen.1 Pseud.omonas spp, Co11adian Jea.mal. of Micro~
biolog)', 41, 126-L35, 1995.
l. Norz. R., Maurh.o'fer, M.. Scllneider-Keel. U., ~Dul"y, B, K., Hans, D. l.Uld
Dfngo, O.. Binlic fncto.rs a:ffecting ex:pressi01t of tbe 2.4~diaoetytphloroglu,clno1
biosyu.thesis gene phlA in Pseulio111onas fluore~cens biooo11troJ train CHAO i.n
th :rlwsphere, Pltytopathol.ogy, 91, 873-88 l, 2001.
11. Nyb.roe, O . anrl S~rensen. J . Production of cyclic lipopeptides by 1Ju~nt
pseudomonads, Jn Pseudornonas. '.Rru;nos, J.-L.. F...d. Bi,(>sytrthesi.v of Macror1totecuies a11tl Moleculct.r Metab,olisrn. VoL 3, Kluwer Aca.demic/Plenum
ew York. pp. 147- 172, 2004.
12. Pierson. L. S., Wood, D. W.1 ru1d Piet1>011, B. A., 1Homo erin.e lactoJ1e-medi01ed
Publishers.,
gt:.,>iw
regulatioo io pla.n1-aSJ1ocia.ted bacteri.a. A.nruuU R:eview vf Plzytopa1Jw-ll>gy,
36, 207:...225, 1998.

13. Molirm, L., Constaotinescu, F., Reimrnann. C., Dutry, B. and Dfago, G.,
Degmdation of pruhogeu quorum-sensing molccules by soil bacteria: A
prevcntive and curative b:iological oontrol m.ec hantilm, F'EM:S Mk't'oblolcgy
Ecology. 45 , 71- 81, 2003.
14. Bernmi, I. and Venruri. V. Regulation of the tV-acyl ho.muserine laccone,de:poodent quorum-sensing yitem m rb:izo. phere .PseudoJnJJnQS purida
WCS.358 and cross-talk with. tbe st.at:i.onro:y-p.tH1- e RpoS sigina factor and. the
globa l .regul:itor GncA, l.\{Jplied and En~Jiro1irne11tal A1icr(Jbwwgy, 70 ,
5493-5502, 2004.
15. NQtt. R., Mau:rht>fe:r, M., Dttbach. H., Haas, D., an.d Dfago, G.~ Fusmie acid
proo.uci11g su'ains of Fusarilnrt oxysp<>rum aJ:ter 2,4-diacecylplllnrog,!Jtcin.ol
biosyn~is gcu.e expr~~crn in Pseudanwnas fl1curesce-ns CHAO ilt vil:ro aad
book.
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Microorganisms Cycling
Soil Nutrients and
Their Diversity
Ji1n 1. Prosser
CONTENTS
~- 1 I11t;r<:>tit1~t<>fl ~ , , ~~~~ ,~- ~
9.2 Phylogenyf F\1nction nn<i Di ve:rsity ... .................................................. 239
9.2.l
The Sigoificance of Prokaryotic Spec:ies for

. -
.ro.
l:"'WICOD
. , .........,. ............... ... ...... ..... ...... ........... ,..........., ................-........................ 2J~9.
9. ~2
U nculti\.rated Organisms .....................................................~.... 240:

9.23 A.ssesaing Lwks bet\veen Pbylogen.y and FUncdoo .........~... 24-0
. l D'1versity
. . ..~ ...... ....
" -:i. 'l F t.mct1ona
., ili,,. ,,,,./\
,U 'fV.
9~.J.~
4 ... .
. . ......... . . . . . . . . . . . . . . . . . . . . . . . . ,
.. .. . . . . " ' .
.. . .
t-<\l
9,3 Tl1e N:itrogen Cycle .......... "' ........ ~--.11"~~- ...-. . ~ ..... ........ ,.......
tt~ 245
,vAn ..................... . ... ..... ... ....... ........... ....... .................................. -e.;o e?.i
. 1fi"' 0 11
w.if'.~
J
9.3. l Nl' tr
if .,. ...........
.!11 . .
9.3 . 1.1 ..r~1e Pl:-oces ..... "' ............................... ~ ............:.......... "'..~ .-'!' 245
9 . 3. 1..2 1irbe 0~.Jnj [})S ... ......... "' ... ......... . ... . . ........ . ............. .....
2.46
ro. .. . . . . . , ........ .... ..
9 . 3o;1. 3 Molecllla:r Ecology ........................... ,. . ~

~,, . 248
9.3.1..4 Funotional .Diversity ................................................. 248
9.3.1.5 Evidence for Functiooal Diversity ftom
, .... .. , ......... w - ..... a. .. 11. .... .. ...... r ...
&vironmemal Seque.ne.es ......... .,............................. 249

9.3.1.6 Links between Nitritication R.ares arul
Functional Diversity . ......................... .,. ....... . . ...!c"l!ll. ~ . ~ 251.
9.3.2 Denitri ficttti.on ............ ~ ... .,. ....................................-~....... ..... ~,.,...........~ .......... ....,....1 . lt~ 251
1
9 .3 . 2 . . 2 "'r he Qr-gani_sm.s ............. . .'* ........
i . .. ti
lli ........ ... ...... . " , .... ~ -- "'
25.3.
9.4 Tu.e Carbon Cycle....,...................... . ...... ..~ ...... ~ ... ., ....~ ..,,~ ... ,.....,..... ~......"""'H ..tl,~........ 25.4
9.4. l Degractatio-n of Cbi,tin ...............- .....................................,. ..... 25:6
9.4.2 Degradati.011 of Orgaoomllutants ....................................,....... 2S7
9.4.3 Tran.sformation of C Compounds in the Rbizospbft-re ........... 258
9.4.4 Methane Production and Oxidatlon ......................................... 258
book.
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M icroorganistns Cycling So il Nutrients and Their Dversity
;:-proteobacteria and n a separa.te group, ll1e Nilrospf.1ia.
241
~fJ:w. ,
ammonla and
nitrite ox:idirer are uot closely relared~ in terms of evolnti-00. and each
fun~tiooal grot1p i found in ever-dl bi gb-level taxonomic grottps. Tb is
di ver ity may bave :rri en thro11gb evolution and divergence of aJl aunnonia
a11d njtrire ox.idiur from comm.on. ar1cestor~. The .processes may even t1ave
evolved several. tinies. Alte:mative1y. ttansfer of genes required fo:r ammnnia

a nd nltrir.e oxidntion (horizontal ge11e transfer) may have led t.o
their appearance in severa! distinct p.hylogeaeLic group . Regurdless of the
....u...:rncatloo
.r.:
l..
'
meeh an1sms,
so1']. proce!\se. sucb as ru:
can "-"
ut; carnc;u oot vy
mic:roorga11i 1ns with a differe.nt geoetic backgi~o1n.d and, therefore. with
different physiological cb.aracteTistic . Sigr1ificant l'hyriologicaJ and fut1ctional diversity llterefore ex.ist and pote11tially increas~ fbe range of
er~vi.ro.nmetltai condition. under wh:jch .n itrificatinn can lake pla.oe. For
mnny pbylogenetically determined group the functional di\~rsity li mucf1
greater. For example, approximately 50% of phylogenetic grou.p ' wimin the
bacteri.i;i. ru:i:d archaea contain org~roisn~ wbJcb can carry out den:lm.Hcmion.
Tbj nas implication for ot1r ab lity to detem1ine which org".tnism ~ are
c.:'trcying out pfilticular soil procesi es, and for the impact of environmental
chm1ge on rnicrobia1 c.omn1urties ru1d ecosy"tetn proces es tba:t will be
~.:a
discu sed belo\v.
9.2.3.2 Who D-0es What1
The di tribu.tion of ecos}'-stem ftmction runong different phyJogenetic grtlltps

has impticatio11s for the ways in wh.ich mic:robial communities, and theif
ecosystem fu.nction , are mea.su.red. ar1d cl1aracteri.z.ecl. and for their eoological
beb.avar. Jf a functicm is resnicted to a ~ingle 16S rRNA-based phylogetletic
group, its diversity can be determi.ned by :.m..al)1sis of l6S rRNA genes.
However, tbis situatjon is me, and I6S rRt"IA gene equen:ee data. are
genero.Jly of limited \lalue in pred.icting function. Tbis t~1ise a f:nnitamenta1
que tioo whea o.ttempti.ng to rel(;!te pb.y logeay and function. In mxed
oommunties. with many organi~ms able to perform differ~ot fu11ctians M(i

m1my able to i:>erform the ame fuJlCti()n but. under dift'erent conditions, wbo i
doing wh_ar.'1
A number of approaches have been used to ad~ ttm qaestioo; mauy of
the molecular techniques empJoyed are described in otiler c.bapter-s. Des.pite

the enormous advanc~ it1 molecular techniq ues~ moot of th.e infurmat:ion
On tbe e.cosy tero functiO'O of ditfere.nl microbiaJ group is stilJ deri,red from.
cb.ancterizution of pure c1tl111res of enviro:nmental isolates.

The coa:cert1, of cot1rse, is tba.t these i ola.tes ma,y not be truly representative
of <rrgani ms playing major roles in oil processes., and rr1any gtoup- have
no cultivated reptesentative. Nevertheless. rm.alysi of a labomto1y culmre
phy~iolog:icul
Copynghtcd material
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Microorg.1nsms Cycling Soil , utrients and Tileir Diversity
245
if \Ve are to oomprehend the 8ignificance of che enormou oon1plexity and

cijversity of bacteriul and arehaeru co1nmunities.
9.3 THE NITROCEN CYCLE

Tbe major proresses in the oil nitrogen cycle are ill\-Ist:rnte-0. in Figure 9.1. Tbe
oomplexicy of the cycJe result , in pan. from tf.1e. many t'orms of iooJ:'gm.c
nitrogen that are fo,und in tbe soil. F11nction.al microbial groups that. 'tt"lm&fo:.rm
tltese inoq1uJc forou can. ;in a few cases, be .characterized u ing 168 rRNA,
ge:nes, but function.al genes are generally more informati:ve. Key ex.ample are
provided iil Figu:re 9.1. However, tl1e ~.test co.mpl.exit-y li-e$ wit:bin the
organic nitrogen pool. whicb i derived from dead animal, p:lant, nnd mier-0biaJ
biomass, Vrsry little is known of the functiooal divetsity of orgnni,sms
deoomposing thi material. Tbe discussion below tberefore fucuses oo ra~o
nitroger1-cyc1ing processes tflJlt have beeo tudied io deptb: n.itri.fica:rioo
aod denitrification.
9.3.1
N rrRlf1CATJON
9..3.1 .1 The Process
Nitrifica.tion i the oxidation of reduced forms of nitrogen to oitntt.e. Th~e n.10 ,t

commoo. reduced form of nitrogen. i ammonia, and nitrifica:tion typic.ally
Denttrificatton
NJl:rogen Ntrogenase
fixatlon
nu
AnlrnaJ,s .<.. -
PlatitS
Dead animal, plant and

rnEcrobia1 blomass
Decompostlonl /
AmmonificattoV
NHg .:..~ .~uoo~ NO _ . NUnm ox'ldlllion ~' NOaA
Ammoola monooxyganase
Nltri.m o,'(ldor~
s.moA
r.ote
Nitn'flcauon
FIGURE 9.1 The terrestrial nitrogen cycle. inehidin.g enzytl'WS ootaly~ng pmicu1ur
tntl:IEformations and M ocia.ted futi.ctioo-.aJ genes th:n b:ave 'Wen used for a:naJysis of
divcrsit:y within funetiona.l g.roups.
Copynghtcd material
book.
book.
book.
Microorganisms Cycling ol Nutrients and TI1eir Oiversity
249
is a broad- pectrum oxjda e wbich oxidi zes methane, cnrbon monoxide, and
a range of organic compouod . Many ammonia oxidizers are ureolyc,
enabliog growth \Vilb urea as ole otrogen ource. Under anaerobic
conditjon , ammonia oxidizer can denitrify; nicrous o de i a by-product
of ammonia oxidation (Figure 9.1). Tbls ,1er 'atiliry greatJy increa e tbe
potential envi.ror1rnental impact of nitrifters on .ruJ eco.,y"ten1. and their lirik
to other biogeochernical cycllng proce ses. Some relationship exi L bet\veeo
autnlOnJ a oxidiz.er phylogeoetic group tlie er1viror1111eul in \.vhich tbey are
found"' aod their Jl1y iolog1cal chArttci:eristic . PredjctabJy, repre.Geotatives of
mruine grclttp are halotoleran'l bu.t tbere are also links bctwcen pl1ylogeny
and urea e activity and tolerance ro bigb amm.onia con entrorions (6).
U'nfortunatcly, the lnck of availal'.Yility of cultivnted ammonia oxidriers ha
rest ricLed phy iological tudiei to relatively few strai n. in. particular Nitro
son1.orias europaea. ft relevance to natural amrnooia oxi d izer~ i unclear nnd.
alti1ough origi naJJy i 'Olated. frorn oi l, N. euroJaea L"I poorly represenled in
soi l ammon ia o jdjzer clone librarie- . ar1d no ide.n1ical 16S rRl A cque11 e
ha ever been reported. Nitro ospirtr ~equence. are frequentl)' much more
abundant in oil , but little is known of tbe pbysiology of tbi orgaoism.
Sirojlarly, there i. e idence thal lhe oitrite o idjzer Ni1ro:,Jira may be of
impon.anee in ol envi.ronrnents, but Jaboratory rudie have focu ed on
Nirrobacter. Thi empha izes the importa11ce of rnolecular tudie. in deterrnining organj 1n , Lhal aJe key to .c>i l eco 'Y tern fu:nction, but al o highlight.
lhe need for better i olarjon tecb_nique;, . The lauer would enable a berrer
physiolog.ical characteri1ation a..nd. tb.e development of cultivarion-indepeadent rnethod. for determi11jog iti situ pl1ysiology.
9.3.1.5 Evide11ce for Fur1ctio.nal Diversity from Enviro.r1mental

Sequene.es
A number of 'tudic ha ve u ~d 1nolecular technique ' to characterizc commu-
nitie in different environ1nents or follo\\tiug enviro1unental per1urbations

(Table 9.1 ). The e pro ide inforrnation on tbe influence of en viron mental
factors on community trucrure and indirect clues to tbe ecopby io1ogical
charncteri t.ic. of different pbylogeneric group-. One ex.a.rnple i the inlJ ueace
of oil pH on am.mooia o idizers f7] . Ammonia oxjdizer :ire inhibited in
liquid culture at pH aJu below 6.5 and in acid oils. This " due ro ionjzatioo
of amn1onia, \vhicb diffu e freely into tbe cell, to amrnonium. \vbjcb requires
energy-dependent, ac1j e traosport mechanism . Activity in acd oils may
result from urfac gro ..vth, biofilm formatioo. or the u e of urea, ralher .lhan
amm.onia. Hov.,cver. cultj\1aLior1-ba ed approache could aot deLermine
\Vbether acidopl1ilic trains were . elected in acid oil undergojng autotrophic
r1,itrification. Ho.,: ever, dena.turiog gradianr. gel electTophore i DGGE
Copyrightcd materia,
book.
book.
book.
Microorganisms Cycling Soil
utrients and Their Diversrty
253
Deiii.trification return ' nitrogeo. to tbe atmosphere ai1d therefure t.'-Ontributes tt>
tlte loss of nitrogen-ba ed fertili.zers in agricultural syste-01$. l tl wa.~ew-ate:r
treattn.ent systems it iB .required .for the re.i11oval of o.x:idiz.ed nitrogen.
following oxjdotion of ammouia to nitrare by nitrifying IY.tcteria. The
proooss i al ~o importat\t bec-au e of the role of N2 0 in tb.e greenhoUS:e
elTect an.d lhe destruction of strJtospheric ozone.
Redu~tion of nitrate to nitrogen ga in\ olves fou:r redttction. prooes.ses;
Nitra:le
roouetasa
reductase
NO
NO
N03
Mtricoxlde
reduct:ase
t"tlrlte
narG
nap.A
nirS
nlrK
okz V
JJ
- !
norB
nor'Z.
Nftrws axde
re:duct-ase
~o
nosl.
Two recluctase ha\1e. ooen cha.racte:ri. ed for each of the Cmt tb:ree step of
denitriJicat:ion., and there is evidence for the exj tenc-e of otbe:~. 1bis furthe:r
co:mplicates analysi. as it cnet111 . that e ven for a singJe proeess more than one
. et offunctional ge"ne prime'rs may be reqttired. A further co.m.pJjc:at;io;a is that
gen.e s as ociated wirh orn.e of t11e steps.. e.g. :nitrou Dxide redu.ctio:n, ca1'l 'be
encoded on a plasmid.
9.3.2.2 The Organisms

The ability to {lenitrify is distributed Ylidely \vtthln bath bacteria and urcbaen.
Approxi .nmtely 50% o.f the cultivaced ph.yl.i posse8s denitrifiers. 'l"his cnuld.be
due to a. eorrunon an.eest.or, existing before th.e plit betwee.n ru-cllaea and
buctetia, w:ttl ub eqru.mt lo-. c>f denitrifi.ctLtion gene"' fr<rm o.me groups1 or
tltrougl1 ho:r-izon.tal gene trnris-fe-r evei1ts~ As a oon~quen.oe. it L nnt po .ible 'to
de&ig11 16S rRNA ger1e primers targeting ali der1inifier ; hence~ mol-0cular
tudies ha\"e focu ed o:n the funetonal genes. However, man;1 organ1sms
coatttin so.me. 'b ut n.ot. ali of the e11zymes in tbe deuitri fication patlrway. ruid.
detection of a particular gen.e does not neces nriJy mean that t'h:e host Ol"pl'.ti8.m
can completely reduce nitt"'Jle oo nitrogen gas.
9.3.2.3 Mole.c.-ular Ecology

A range of primer h.ave beeo used to study deo.itrifier ecology, lmserl on tbe
sequence of tbe fu nctionaJ gene encoding en.zyrnes for eacb af the
denitrifict1tion rea,ctions [l O]. One problem. f:toweve:r, is tb.e n.eed to use
more than 011e primer set to detect ali der1i'trifier: . Thl, prevents the
de:termioatioDof relative a.bund.ances wi.tbout absolute quanti'fication of eacb
fu_nctiortal gi'<>up. e ..g. by u ing real-tinie PCR. Quantica.tion i aidedt
book.
book.
book.
Microorganisms Cycling Soil Nutrients and Ther Oiversity
257
Globally, it is the seoond ro.ost abundant organic oompouttd, after celluJo e,

and its degradatioa is carr:ied. out primarily by bacteria and fun.gi. A ll
chlitinolytic org:anisms po..~ess chtlina"'ve, ~hich h ydrol~za bonds between

N-acety i-o~glllcO -2-amin.e residu ~ to forn1 oligon1ers a11d dimer which can
be taken up by the ceU. providing sources of carbon 3nd nitrogen. Ttte broad.
distribution of chitinolytic actvity prevents l6S rRNA geDe-based analy :is of
cb.itinulytic organism . However. cbitna e enzymes fall witbin a. number
of cJ.aues, a_11d functional gene prmers can be rlesign.ed fat tb.e analy i of
organiS'ms with different fomls of the cbiti nase genei .
Such pri1n.er t1ave been devclo>ed for c.h.i.tioases belonging to Class 18,
group A. wlticl1 are commortl;" reco'.vered frt)m oil. Alrrplific:lti.01l. of ~bltinase
genes from so.iJ wng tbese. prim.ers, and pbylogen:etic nncl; eti. of sequenees
ha.ve been used w determine the effet.~ of the apptieation of ewage sludge on
the degrad:ation of cb:itin buried in. the soi! (i11 litter l>ags) and on tke oolonizin.g
chitinolytic oom111unities .[12]. Sl'udge increase.d the mt-es of degradation and
increased the nurnbers of actinobacteri.a,. many of whicb are chitinolytic. lo
a sub equ.e-11t litter bag experiment, chitin:olytic communities were dominated
b y .actinoba.cteria aod 'howed. an increase in activity .afrer -lu.dge treatmen~
but a d.ecrease in diversity of chitinolyt.ic organi ms. In addition._ ome of
th~ chtitU\ e gene equenees formed a. novel clu ter witf1 no previoa ly
characteriz.ed chltina. e genes. The re ult therefore indicare a :redtlciio:n
in pbylogenetjc di'ver it)1 in oil fullowing .ludge tret1tn1en~ whieh ma)l
red.u.ce the f1Jnctiomil dlversity of the sy&tem. Tb:ey also indlc.aL-e fu.net:ional
redundancy i11 th.e community, v.1th respect to chltinolytic activity~ b11t not
nec.e sarily with respect to other phy~ iological chnracteristic of the
chitinolytic community.
9.4.2
EGRADATfO.N Of RCANOPOLLUTANTS
~n1e
fu.nctional diver ity of degrader~ of son1e orgnnopollutant . i hlgh, but

other~ are onry ti.egrade.d. by resrricted groups of organiams. Biocllemical
degradation path,vays of ome organopollutant, are well studied
( 'ee Chapter 20). However. molecttlar t:ecllltiques are, fot tbe ft:rat time,
enabling the detenninatian of ~'hic.h organi sm are involverl in degrarlatiom.

by both culturable and 11on-cu1turabte mia'oorgani ms. Indirect evidence
oomes from. chrulges in microbial community tructures during degmdation,
bllt more direct evidence ea11 be obtained using sffi/JJs isutope pro/1i1ig (S.IP).
Stable isotope prubing is explamed in detail in Chu>ter 15 and :i based an
molecuJar analy is of 13C-labeled. marke.r foU.owing amen.dment of soil with
3
t C-1abeled compounds. lt provdes direct evidence of a.q-similntion of lnbeted.
compound and. t.h erefore lias tremendous value in detetmining whicl1
book.
book.
book.
Microorgan sms Cycling Soil Nutrient:s and Their Drversity
26'1
9. Webster, G. Enlhley, T. M., Preitng, 'f . B. Smith, Z., and Prosser, J. L~ I..inb
betwoon tnm-Onia oxlclizer peci es composition, .functioool ~r ity and
ni;trifiearion kinetlcs in grassland ooils., E11vironmentCLl Mlcrobicrlogy1 7.
676-684, 2005.
10. Philippotc, L aud H.aliin, S., Fin.ding the missing link between diversity an:d
activity u.dng denitrifyiug bacteria as a rnodel fun.cti.01ml cO:ntlllt1nicy. Onre.111
Opi11wn in Microbiology. 8. 234-239, 2005.
l l. Rich. J. J. and Myro!d, D. D.. Com1nurtity compo.sit:t a.n ami a:eti:vities of
dooitrifying bacteria frun1 adjacem ag,ricultaral ci~ dparian so:il, an.d creek
seditnent in Oregon. USA. Soil 8i.ol4gy and .Biod!.e-ml,stry, 36, 143 1- 1441 ~ 2004.
12. M&calfe., A. C., Kr ek. M., Gooday, G. \V., Pro '~r. J. l., and WeIJfngt:on.
E. ~1. H., Molecular analy i of a bacterial cbitinolytic oo.mmunlty tu a11 ttplao.d
pasture, A1plie.d rmd Euviro11.1n1tn1al Mi.cr'Obiol<Jgy, 6:8, 5042-5050, 2002.
13. Rangel-Castro, J. L, Pr'O er. J. l., Kilib.am, K... Nl-co-1. G. W., M~eharg. A., Ostle,
N. A11dersou, l. C .. Sctirogeottr, C. M., aud lnesmt, P., Sta.ble isoto:pe p:roblng
aoolysis of the i11ftuence of Ii miog on root exudate ufili,za.tion hy scil
microo_r:ganisnis, .Errvirorimental Mic robiology, 7, 828-838., 2005.
14. 'l'reusch, A. H., Lt..'ini.nger, S., SchLepex, C., Kietzin, A., Klook, H.-P ., and
Scbttster. S. C., Novel geues fo;r nitrlte reductase and a:m.<r.relal'ed proteins
indicate tt role of uocu.luvate.d. m.es:opbilic cren.arci:l:aeota in nltroge-n cycliug,,
E111>ir on11imt.al M1crobiology , 7, 198:5-1995, 2005.
15. K.Onneke, M., Beru:hru:d, A. E., De La r cn:re, J. R.., WaJ:loo;r, C. B., Smhl. D. A.
nnd Waterbury, J. B. lsola:tioo of an aototrcrpbic nro:rn.oni.a~o'X'j dizing marine
archaeon, Na.ture, 437. 543-546, 2005.
16. Niool. G. N. and Schleper. C., 'fb.e role of atcbaea in amrn.onia oxidatio~
Trends in Microbiology, 14i 207- 212, 2006.

Microbial Interaction in Soil

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Mi,crob1

Microbial A emblages and Gene Expression ....................... 184

7.4 Molecular Mec'hanisn1s Underlying Microbial lnteractioUB............... 185

U e af and. Betrn.yal \vith QS

7.4.4 Altemative Punctions for AHL .............................................. 193

Antagonisti.c InterJetiort in Soil ............................................. 200

Microbal loteractions in SoH

Neulrali.fm can be cb.a:rac tetized as the lack of interactio:n(s) between

fu.ngi. described in Chapter 5. Syntrophy is a form of metabolic collaboi-aion.

Microbial tnt'eractio:ns in Soil

o.x.ygeu, com.p:ared to cells in lower, ofte11 oxygen-depJeted layers. 'Ihu.s ever1

commuoieation, and ofteo sucb regulati.oo. oco.Cr& u1 ca.ses wben

are discWiSed in Section 7.4~5.

7.4 MOLECULAR MECHANISMS UNDERlVING MICR081Al

MOLECULAR SENSINC ANO S1GNALING

Key features of tl1e genotnes of i11icroorganism iu 001nplex .b:abitats like soi\

Microbial lnteractions in Soil

\Vtltin the n1icrobial as embl11ge t.o tbe QS n10lecules can be charru..'1erized as

Microbal lnteractions in Soif

FuNcrtoNs FOR AHILs

ENVJRONMENTAL SiNStNG M ECHANISMS IN f UNGI

given belo\.v. A f11rtber broad category of in-bouse ignals c.onsist of tm array

Microbial lnteractiorrs in Soil

e.o enabie the colonization of

nutri.en.t rich substta.tes. During tbis

process and o ther.' , progrnmmed or environntenta.lly induoed c-ell deam may

7.5 TYPES OF MICROBIAI. INTERACTIONS IN SOIL

of tbe ecology .o f micrQorgaa_i . ms involved in selected:

I11teractio1:}S witb ba.cteria

M ETABOLl:C INTERA.CTlONS IN SOll

Mic-robial rr1et:aboJjc activity is the key determinam of tbe stn.tcture aud

blage. MJcroorgani ms in sol can. int.eract metaboli.cally in: ditfereo:t ways.

Microbial lnteractions in Soil

f ungi may be associated with defense of territory, since the release of

Microbial lnteractions in Soil

7.5.5 f UNGAL INTERACTIONS WITH BACTERIA

Microbial loteractions in Soil

Mlcrobi<>logy Revi~vs. 24, 403-427. 2000.

81, 3{)5:..31 1. 2002.

Modern Soil M icrobiology

, , . . . .. . . . !!< ....... t!I ,., ..

8.2.2 Nutrient Availa:bility and Metabolimi/Orowtb ...................... 215

Modern Soil Microbiology

a11d exudates represent important

Plant-Associated Bacteria-t ifestyle and Moecular lnteractions

me].udi'ng :ome recent di

8.2 THE RHIZOSPHERE AS A. HAOITAT

Modern Soi l Microbiology

tbe development of re istance ro plant pathogens, and planl gro\vth promotion,

The young rhzo pherc. conlains an extensivc microftora encon1pa sing

Plant-Assocated Bacteria- Lifestyle and Mole<:ular lnteractions

attz.plicons, re.spectively. 11:1e analysi demons:trated th:e oocurrenee of fewer

N UTIUENT A VAll>\BlllfY ANO M ETABOLJSM/GROWfH

orgnnic/carboxytic acids are not [4].

Inoculant lux-repurter (biolwninescence) gene-equipped Pseudomnna-s p:p.,

rhizosphere, tl1e composition ttnd availability of specic organic components

Tbe significance of nim>ge.n (N) and phosphoros (P) limtation in soil .M d

Modern Soil Mcrobiology

The rhizosphere (barley) demoostrated signicant N li11lation, \vhereas P

PlantAssociated Bacteria- Lifesl)tfe and Molec ular lnteractlons

8.2~3 MICROBIA~L ANTACiONtSM AND BtoLOGfCAL CoNTROl.

Chaprer 19 p:rovides a ftl ttccotmt of bioco-otroJ agents., their productio11l use

spp. further produce a number of antifungal metabolites like 2.4-dacetyl