microbial interaction in soil

Mi,crob1ial lnteractions

in Soi 1

Jan Dirk van Elsas, Ling Tam, Roger D. Fin/ay; Ken Killham1 and Jack T. Trevors

CONTENTS

7. i Introduction ........ .... ... ..... 1¡ ... ....... . . ......... ... . . > ... ...... ~ .......................... ............. . ........ . .. . .. 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

7.3.2 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 Sertíling- Tile Paradigm of Signaling betwoon

7.4.3 U e af and. Betrn.yal \vith QS m Soil ...................................... 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'áctions io Soil ............................................. 197 7.5.1 Mctabolic Ir1teracti:ons in Soil ................................................. 197 7.5.2 Antagonisti.c InterJetiort in Soil ............................................. 200 7.5." Predatory Jnterttction in Soi1-8de1Jovibrio and

Myxobt_.lCteric1 ................... ? ...... . ... ... ...... .. ,. ............. ..... " .. . ..... ........ .., ...... ,. .. 202· ~i.5.4 Bacterial Interaction witi1 Fungí ........................................... 203 1 .. 5.5 Fu11gaJ Intentctior1s witb Bac teria, ..... ,. ....................... ............ 2fl5

7.6 Coneludirig Remarks ···· · · ·~ · ~· ········8····· ·~ ········· · · · ··············.o!, .............................. 206 Referen.ce ..................... ................ ...... ..... ... .................................................................... .., ....... , ............. 201·

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Microbíal loteractions in SoH 161

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 o·ne aaotber, ~o thc o.rganisms rnay be fu.nctio11ally eparate. Environ1nenta1 coi1dition (e.g. nutrient limítation, low temperatures or the pre 'ence of toxic oompounds) that do not allow rrücrobi~ll growth and cell division favor a ne utral intemctio:.o.

Com111emalism i. 11 unidU:ectionaI iriteractioo bet\veen rr1icroorg:aoisa1 • in whích 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 i11 which the interacting orgacis.ms receive tnutual benefits fro·m ttm partnership ( uch a in gymhios-is, sytl8rgism or sy1itl'ophy [ero ' -feedi.ng]). Mtttua1 S)tmbiosi., can be a · o~~uloo uec·tosymbio is," in whicl1 the symbiont occur on the extemal wface of the host. IDramples include internctions of, for in~"'tance, nitrogen-fixJng rhizobia with pl~Ln·cs.. \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-aíion. 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.ator 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 prey 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 reµeat . 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 fungí. and organisms like Rhisopltydiw11 and Phyrridiu1rt th.ar can paras itize alg-ae.

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Microbial tnt'eractio:ns in Soil 185

o.x.ygeu, com.p:ared to cells in lower, ofte11 oxygen-depJeted layers. 'Ihu.s1 ever1 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 certaín anribioci.cs. Moreover, the disposal <>f yroducts of cellttlar metabolism oommonly varies ,baween dilferent Jayem .of 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>Slno-1 a:rity, oxygen linútation and bigb cell density (&ee SecOOD: 7~4.2} ru:e aro.ong t11or·e ti1at are differentially expressed .. Hor.nogeireous microbla:l amernbl.age will tbu . sho\v ciear tI:atification, and &ucb st:ratífteation 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 bíofilms ( 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) clienúcal compounds., as will be di cussed ío Section 7.4.2. The regulation of a range of bact:e.rial function:s is related to ce11-to~celJ commuoieation, and ofteo sucb regulati.oo. oco.Cür& 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 growt11 ill biofilms and the ab-ility of cell populations to respon:d to changes ro nutrie11t co11cerJtmtions f 5). Parallel forms of sigo.aling tak~ pla.ce in. fu.agi and are disc·WiSed 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 co11dítions 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 tw·o-eompooent sensor/response {regulatory) systern • allm\' an. a.dequate ;rei poru;e to the

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Microbial lnteractions in Soil 189

\Vítltin the n1icrobial as embl11ge t.o tbe QS n10lecules can be charru..'1erized as n commurrity re pon e. The detectíon 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 abíli.ty to produce QS signaling rnolecute 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.erved 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

.of varying co.mplexity and regularory factors. In b:rief, ecreted peptide a.utoind.ucer increase in. co11c.entn1tio11 as a function of cell popul~ú: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~1oryJ .. ation prucesi acf,ivates this respon e regulator, allowing .it to bit:td to DNA and alter the tr1lns.<:ri.ption of QS-co·ntrolled 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 \VÍthio. microbini populati.ons. Pheno'types that ttre a ociate.c1 ~rith A.HL p1'0d1tctinn an.d sen'Siog include biolu1ninescence , pro(l~1ction of virulence f~tetor • conjugative transfer of Ti pla mid · in Agrobactl~riutn ttltnefaciens, antibiotic produc"tio·n, starvation resJ>ou :e (Rliizobiurtr. legu.t1.7i.tto-sarun1, Vibr.io · pp.) • . motility and biofilm formatior1 (7 ,8] . Only orne of tl1ese p1•oces 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 ·of bacteria with their bioti.c or a·biotic 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:l ly. 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 ºl' 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


Microbíal lnteractions in Soif 193

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 AHú. are .actuaJ1y muJti.purpose molecules that can perfo:m1 differe.nt ecological roles i'o accordan.ce \\l:Í.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

Fungí 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, eücilors 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 eº sig.o.aling mecha:uism , involving pheromonei which facilítate the intern.cti.on of ca:mpatjble gametes and de,relopmen.tal ho·rm.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 aritoregrúu:tors [ló]. T11ese signals convey in.fonnation on envir:onm,ental 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 í:.tnd myceuiL J\ s yet" we still have mther limited knowledge of th~ m-0lecular mecl1anisms of reg·ulati.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 fungL

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Microbial lnteractiorrs in Soil 197

e.o enabie the colonization of 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 un Wáilted 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~ í 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 .of micrQorgaa_i . ms involved in selected:

• Metabolic in.leractions in ·oíl • Ant.ugonistic ínteractio-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 s·tn.tcture aud compooition of a microbial community formir1g an a sembtage. So-me tnicrobial processes ate can·ied 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 assemblage. MJcroorgani ms in soíl 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 patblva·y. resulti,,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.etails) 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 speci·e.s are pres.en.t in tl1e urne physical locatio:n and are



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 emerging. l t 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 compounds is formed by the bacteriocins. Bacteríocins are proteinaceou compounds produced by bacteria, which are toxic for úghtly-related bacteria. Thus, bacteriocins mediate, in a negative way, the interactions between closely-knit organisms. The best studied example is offered by Escherichia coli, in whjcb at least 25 different bacteriocin (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 lysís and tran ported to susceptible celJs, where they may degrade the DNA, inhibit protein synthesis or destroy membrane integrity. They do not lyse their own cells, as these produce an immunity protein that binds to and inactivates the toxin. 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 conditíons that allow crowding. The microhabítat 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 na


7.5.5 f UNGAL INTERACTIONS WITH BACTERIA

Conversely, son1e fungí have evolved metabolic capabilitie to take advantage of the bacteria with which they sbare the habitat. For instance, soil fungí uch as Pleurotus ostreatus, Lepista 1iuda, Agaricus bru111resce11s and Coprinus quadrífidus 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 mechanism) in their vicinity and initiate specialized direc tional hypbae that can grow towards a bacteria! colony. The fungi 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 colon 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 fungí and bacteria! colonies gives the fungi a considerable advantage in obtaining nutrients e pecialJy under tbe nutrient-limiti ng

FIGURE 7.6 Mycetium of Hebe/on1a cn1s1u/i11ifonne after colonization of a potas ium feldspar surf ace fo r seven months. Thc ample wa prepared by fixation and critica! point drying followed by gold coating and analysi by canning electron micro copy. Hyphae (H) and bacteria (B) are visible. Sea le bar= 1 O µn1. The hyphaJ surface contact i mediated by a fi lm 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 fungí. In: Fungí;,, Biogeoche111ical Cycle.r. Bd. by G. M. Gadd, Cambridge University Pres . Cambridge UK, pp. 28- 30, 2006. Wilh permi sion.)

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. Spñn,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 ñu1gj on • oil bacrei·iat 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, pµ,. 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 SymbúJbacteriunl. therrnopltilunl~ an un.cultivable bacterium tbat depends on mlcrablnl cornmensailsm. Nucleic Acíds Research, 32, 4937-4944. 2004.

25. 'Wiener~ P., Anti1Jiotic prodl1ction ín a s¡latially s1mct11red er1vi1omn.ool, EcoffJg)' l....etters, 3, 12:2-l.30, 2.000.

26 . . Dea.oon, J., /tungal Bl.oletgy. 4lh ed., Blackwell. Oxfard. 2006.. 27. Cronin, D .• Moenne-l occoz, Y., Fenton. A., Dunne. C., Dowling. D ..... and

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, 251-261~ 1999.

29. Düwid, W., Biology aod global diB'tributiori of n:1yxohactéria in scil •, FEMS Mlcrobi<>logy Revi~vs. 24, 403-427. 2000.

30. Johao"son, J. F., Ptrul, L. R., and F.i1n.ta.y • . R. D., MicrohlaJ. intetaction in rhe mycorrblro. phere and theír significance fur ~nal>ie agriculwre, FEMS Micrqbi.altrg)~ &oltJgy. 4S, 1- 13, 2004.

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:..311. 2002.

32. Jargeat .. P., Co. seno, C., Ola'h, B ... Jauneao, A.~ Bonfilute. P., Ba:tut, l1. , 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. 6876-6884, 2004.

33. Chri teru;~n. B. B .• H~aageuseo, J. A .. J .• Heyd-0ro .. A,. and Mofu1, S . . Meraholic com1ne.osruisrr.is o.nd 001.upetitiou iu a two~4vµecies mlcro:b:ial: c:on.s.o:rtium. Applil!d and Envl.rt11u11entál Mi.c.~tology. 68.. 2495-~ 2002.


210 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 fungu Gandida albicaru i mediated by farnesol, Applied anti E11viro11111en1al Microbiology, 67, 2982-2992, 2001.

35. McLean~ R. J. C., WhiteJey. M.. Stickler. D. J., and Fuqua. W. C .. Evidence of autoinduoer acti ity in oatoraHy occurring bíofilms, FE~1S Microbiology Letter . 154, 259- 263. 1997.

36. Miller, M . . B .. and Bas ler, B. L .. Quorum seo ing in bacteria. An11ual Revieu1 of Microbiolo8Ji, 55, 165- 199 2001.

37. 01 o.n. S. and Hansson, B. s .. Action poten·tial-like acrivily fou11d in fungal 1t1ycelia is enBiti ve r.o stimulation, .Na1111l 1rissensch11fte11, 82, 30-31, l 995 ..

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CONTENTS

Plant-Associa.ted

Bacteria Lifestyle and Molecular lnteractions

Jan Sarensen and Angela Sessitsch

8.1 [ntrod,uct:i:o·r1 ..... ~ ........ "~ ....... ,., .... .., ............... ~ ....... ............ , ... " ....... t;• .... ~.-; ""' ··~ ... . -~ ....... ~ .. ~ ,, . .. .. . . . !!< ....... t!I ,., .. 211 8.2 ""fhe 'R1lizosphe·re as a Habit~a.t .......... ., .. ........ ................ ... '"" ........................... ~ .......... ~···~ ·~ 213

8.2.1 Rh.iZOS"phere Ba-cteria ··· · · · ······ · · ~" ~ "' ..................... ", ....... 4 .. ~ ........ .. ... . . . , • • 214 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. ... ,. .. ..... .......................................... 11> ........ ... _ . ........ ..,, 22.'J, .8.3. 1 llbj7,..op'lane B-acteria .............. ~ ....... ........................... 1'i . ..... "' ......................... . . .. 221 8.3,2 Root Surface: Colo.nization and Growth ................................. 222

8.4 'f "he End~plan.t .Habitat .......... ~ .. ..... ~ ...... .. ..... ,.. . ·~ ....... ... """ º. -~ .... .......... ~ • •• ,. ........... \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 Fíx.ation ......................................... 230

8.5 ConcJ·uding Remwks ......... ~ ..... .. ............................... ª . ............. , ..... . . ~ •• J ........ ~ . . ...... ,,.-¡,. 23 l

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 hí1ve -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 ínteraction \Vith lhe plant may be bene.ficial, neutral, or bannful. Differenc oíl 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 iníluenced 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, soi l 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 \vbích 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, g¡e.atly detennines their co11wuníty structure. Differe11t soíls are known to contain higbJy differing n1jcrofloras, and fro.01 these diverse 111icrobial pool , planr exudare select specific ubpopuJation whicb colonize the rhizos¡,here, rhizoplane or endoplant babitat . This may Jead-dcpendiog on tbe bulk ·oí l 1nicroflora-to varying microbial population colonizing 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 capacitíe to respond adaptively to tbe upply of water, oxygen and nutrients, as well as to .the. biotic interactions of co1r1perition, antagoni m and

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Plant-Associated Bacteria-t ifestyle and Mo•ecular 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 Últeniction · 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 arttagorúsm (p.lant pathogens). Tbis treatíse 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 specüic · .e · • ] di' di · n ' d · m1orm.at1on. me .u ng :ome recent · cove11es. ~oot-assoc1a.te tnJ.CJ.'OOl'p:ll-

·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()eitíon)~ 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 reeeíved '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.ons 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),

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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 rhízo 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-fixíng genera Al.<JSf)iril/un1. and Rhiz:obiu1n. 'fhe diversity of Pse.udo1nonas 'PP· in rhizo phere amples (herc defined as the cell com1nunity extracced from oíl adhering to the root) is discus ed briefly in the fo llowing 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 cultívation-based m.ethods, thnt P eudv1nt>11as spp. are generally 1nore abundanr ín rhizosph.ere tl1a.n in bulk o.i ls [l]. The stimulatory effect of the rhízosphere crf several. crop plants ou oil p. eudon1onads has thus been dem.oa 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 affiüacion of the clone to Pseudo1nonas pp. was dc1nonstrared by colony h.ybridiz.ation u ing lhe Pseuclon1orias-. pecific oligonucleotide 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 . Prominent PCR products were separated by DGGE ( ee Chapter 14 for an explanarion of the tecbaíque) 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

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Plant-Assocíated 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 soíl. Pseudc1nonas 'PP· were among tb_e abl1ud,ant genera. as judged from the database homologies obtained. Spe.cifically, apart from. several Bacíllus-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 root·coloniziog b.ae:teria in the yoong rlti7.;.0spl1ere. Soluble exmlate C'Omponent include a "'W'Íety of common monomeric compou.nds (sugars., amino acids and fatty acids)t wber-eas carboxylic aci.d (citrate« malate, succin,are a11c1 oxalate) bave be:en rep.art~ to be partícular1y 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 sµeci: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)ª 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 detnonstrated 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 speciñc organic components CM still be import.ant for tbe act11aI e statllB of Pseuti.ommlCJ~Y ceils intltl.b:iti:ng this h.abitat

Tbe significance of nim>ge.n (N) and phosphoros (P) limítation in soil .Md 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 straín. · limitatio'fl be.carne apparent a C-rich poly·mers f:rom the barley re.sidue.s were degraded.

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216 Modern Soil Mícrobiology

The rhizosphere (barley) demoostrated signiñcant N li11úlation, \vhereas P limitation wa not observed. Tb_is work was the first idenli fication of a major outrient limitalioo (N) of pote otial sígnificance 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'bi lities 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 ammoniurn and common amino acids (e.g. glutaJnáte). l r is stil l unknowu whether pecific N cotnponents ia exuda.tes regulate tJ1e growth of l)set1do-1110 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 jluorescen 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.enitriñcation. but al o a number .of 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. Nevenheless. there i. still a poor understanding of the effect of oxygeo level in oíl 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- ensitive P. fluorescen.s reponer strajn took place in \\1etted (85'1o of the oíl' water holding capaciry [\VHC]) but not in uowetted (60% WHC) barley rhiiosphere oil and in compacted bulk oil. Thjs study demonst:rated thal water and 1exturaJ 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.).

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Plant·Associated Bacteria- Lifesl)tfe and Molecular lnteractlons

8.2~3 MICROBIA~L ANTACiONtSM AND BtoLOGfCAL CoNTROl.

(BIOCONTROt) OJ PtANT PATtiOGfNS

217

Chaprer 19 p:rovides a ftúl 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 fungí 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 surf ace, The biooontrol agent ma:y here tttilize an essential growth sub~t:rnte more efñcieutl.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'tlogí , Indeed. the cataboüe pro.files of p•eudomonads 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 clwícal 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.bícb 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}foverdín, p. eudobactin and fe.rriba:c.tin. Thi& resulrs in rhe effective biological control of plant p'l:tth~en ucl1 as Fusar0lu.m oxysporurn an.d P)ithiWt1. sple1itlens. Soro.e .P ~11donwnas , trains are ex.tremely efficient in tb:eir .í:ron . equeatering, as they produce lárge amouots O:f efrective siderophores lYr are able to Utke up a range of düfe:ren:t Fe-siderophore complexes. Similarly, Serraría 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.ydrolytic cell~walt d.egrading ex:oe112yn1es u.cl1 as chitinase, whicl1 can antagoniz.e plant-patho¡enic fun.gi in tl1e rl1iz-0sphere. Rhiwsphere Enterobacter (Pmttaea) and Pseu<lomo11a.f spp. further produce a number of antifungal metabolites like 2.4-díacetyl pbloroglucinol (Phi), pb.enazine (Phz), pytlluteorin (Plt) or pyrrolnit:rin (Prn), ea,cb of which can. itlbibit a r:mge o'f pátbogeni.c fungí . In. particnlar, several


218 Modern Soil Microbiology

Pseudo11w11as bioconlrol traio bave been rudied inten í 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 híghly 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) inftuence Lhe production of Phi in P. flu<>rescens CHAO. Furtber, both the genoty·pe a_nd age of the host pla11t can n1odulate phi gene expression, w·herea root infection by P.)1thi1111i stit11u.latcs pJ1l expression [10].

Bacteria] productio11 of bioStrrfactatll rrtolec11le , notably cyclic lipopeptides (CLP.Y) , .in tbe rtúzo 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 f ungi in rhe rhizo phere [ 11 ). The rhizosphere-oolonizing P. jf11orescen train DR54, whicb 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 metal 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 PvdS·type 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 raúonary 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 fungal metnboljtes. A well-studied exampJe is the producLion of phcnazine (Pzn) in everaJ Pse11domonas bíocontrol strains. e.g. P. aureofaciens strain 30-84

[121. 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~talk~') betweer1 the RpoS sigma factor a.nd QS control bas been docu1Denterl in P. pr,tida W·CS358 in the rhizo pbere [14],

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Plant-Associated Bacteria- Lífeslyle 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 µresent 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. gáS 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 stirn·u.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-íology 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 províde 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.a·ve heen receotly r1 ·viewed (20J. See Figure 8.1 and Figure 8.2 for a depi.ction of th~e rnethod . ·r·be combiu.ed techniques have allo,ved tb.e tracking of single bacterial cells and tbeir pro1íferation oo tlte rbizoplane. resuJting :in profound ínsigbt 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.


Plant-Associated Bacteria- Lifestyle and Molecular lnteractions 225

including heavy-metal-accumulating plants. The i olates ha ve included a wide range of both Gram-positive and Gram-negative bacteria, and currently compri e more than J 29 pecies [31 ]. Pseudo1nonas, Bacill11s, Enterobacter and Agrobac1eriur11 ha ve been fou nd Lo be the mo t abundant genera isolated as endophytes from agricultural crops [31].

Cultivation-jndependent 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] ídenti 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 rhizospbere, phyllosphere and endosphere núcroenvironments of roots and shoots differed greatly in tbe presenceas determined by tbe analysis of isolates-of antagonists against plantpathogenic fungí. Most of such antagonists are commonly derived from the rhizosphere and the endo. phere of roots. Molecular as well as cultivationbased 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). Cultivation-independent 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 anúbacterial activity [32].

Besides the endophytes that colonize the intercelluJar spaces and va cu lar 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: Azorhizobíu1n, 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

Copy•ighled material

Plant-Associated Bacteria- Liíestyle 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 µm. (b) High-magnification view of panel a howing bacteria colonizing the intercellular space within rice ti ue. Bar = 1 O µm. (e) Bacteria colonizing the iotercellular pace of the third leaf of rice. Bar= 1 µm . (d) Section f rom rice coleoptile, howing bacteria colonizing the intercelluJar space. Bar = 1 µm . (e) Cross section

from lhe lip of a fourth rice hoot. howing Linte in va ion. Bar = t O µm . (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 = 1 O µm . (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.)

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 Phyw¡iathology. 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, Ap¡¡lie-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 jlwré.'iCens report,er ba.cteria in tile barley J'hi2oopbere. .~ppiied ami Etnrironmental ft.11.crobiology, 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 rhízosp:bere co1npetence of fluorescen.1 Pseud.omonas spp, Co11adian Jea.mal. of Micro~ biolog)', 41, 126-L35, 1995.

lú. Norz. R., Maurh.o'fer, M .. Scllneider-Keel. U., ~Duíl"y, B, K., Hans, D .• l.Uld Défngo, 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 thé :rlúwsphere, 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 Publishers., ew York. pp. 147- 172, 2004.

12. Pierson. L. S., Wood, D. W.1 ru1d Piet1>011, B. A., 1Homo erin.e lactoJ1e-medi01ed 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 Défago, G., Degmdation of pruhogeu quorum-sensing molccules by soil bacteria: A prevcntive and curative b:iological oontrol m.echantilm, 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 Défago, 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


Microorganisms Cycling Soil Nutrients and Their Diversity

Ji 1n 1. Prosser

CONTENTS

~-1 I11t;r<:>tit1~tí<>fl · ······~ · ··········· · · ·,·· · ·· · ·· · ···· ·······,···· · · ············· ··~···~·~····~ · ··,··~·-· ~ 9.2 Phylogenyf F\1nction nn<i Dive:rsity ... .................................................. 239

9.2.l The Sigoificance of Prokaryotic Spec:ies for ro. .• - • 2· ~9 .l:"'WICÜOD •••••••• .., ......... ,. ............... ... ...... ..... ...... ........... ,. ......... ., ............ .. .. -........... ............. J .

9. ~ 2 U ncul ti\.rated Organisms ............................... ..... ................. ~ .... 240: 9.23 A.ssesaing Lwks bet\veen Pbylogen.y and FUncdoo ......... ~ ... •· 24-0

9 " -:i. 'l F · . l D' · · . ,,,,./\ ~.J.~ t.mct1ona 1versity .... ~ .... .. .... 4 ... . .. .................. ............... , . ....... . "' . .. .. t-<\l • ., ili,,. ,U'fV.

9,3 Tl1e N:itrogen Cycle .......... "' ........ ~·····--.11·"·~ ·~·- ... -. .• ~ .............. , ....... if • .,. ........... tt · ···~······· 245· 9.3. l Nl'tr·1fi"'0 1·1· An ?.i ~ . ~ ,v ....... ...... ......... ... ~ ... .. ... ~ ....... .................. ~ .................................. .!11 .. -e.••••;o¡• ••• e w.if'.J

9.3 .. 1.1 .. r~1e Pl:-oces ..... "' .. .. ...................... ..... ~ ...... ..... .: .......... "' ••••.. ~ .·-·'!' · ··· 245

9 .. 3 .. 1 .. 2 1irbe 0~.Jnj ·[})S ... ......... "' ... ......... . ... . . ........ . ............. ..... ro. .. . . ... . , ........ .... .. 2.46 9 .. 3o;1 .. 3 Molecllla:r Ecology ......................... .. , .... ~ ,¡ .... .. , ......... w • • - ..... a. .. 11. .... .. ...... r¡¡ ... ~,, . 248 9.3.1 .. 4 Funotional .Diversity ............ ... ............................. ..... 248· 9.3.1.5 Evidence for Functiooal Diversity ftom

&vironmemal Seque.ne.es ......... ., ............................. 249 9.3.1.6 Links between Nitritication R.ares arul

Functional Di·versity ........................... .,. ....... ... .. .!c"l!ll•····· .. ~ · .. ···~ 25·1. 9.3.2 D·enitri1ficttti.on ....... ...... ~ ... ., ................. ...... .......... ..... -~ ..... .. ... ... ~,., ...... ..... ~ ........... .... , .... 11. lt~ · 251

9 .3 .. 2 ... 2 "'rhe Qr-gani_sm.s ................ . ·'*·· ........ i. .. ti •lli•• · · ........ ... ..... . . " •• • , .... ~ -·-· ···"' 25.3.

9·.4 Tu.e Carbo·n Cycle .... , ............ ........ .. .......... ~ ...... ~ ... ., .... ~ . .,,~ ... , ..... , ..... ~ ..... . """'H .. tl•·,···~ ........ 25.4 9.4. l Degractatio-n of Cbi,tin ............... - ........................ ............. ,. .. ... 25:6 9 .4.2 Degradati.011 of Orgaoo¡mllutants ................................... .,. ...... 2S7 9 .4.3 Tran.sformation of C Compounds in the Rbizospbft-re ........... 258 9.4.4 Methane Production and Oxidatlon .... ..................................... 258


M icroorganistns Cycling Soil Nutrients and Their Díversity 241

í;:-proteobacteria and ín a separa.te group, ll1e Nilrospf.1ia. ~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 bigb-level taxonomic grottps. T·bis 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 and nltrir.e oxidntion (horizontal ge11e transfer) may have led t.o their appearance in severa! distinct p.hylogeaeLic group . Regurdless of the

h ' ']. b .... ...: .r.: • "-·" • ~.:a l.. mee an1sms, so1 proce!\se. suc · as ru:urncatloo can ut; carnc;u oot vy mic:roorga11i 1ns with a differe.nt geoetic backgi~o1¡n.d and, therefore. with different physiological cb.aracteTistic . Sigr1ificant l'hyíriologicaJ and fut1ctional diversity llterefore ex.ist and pote11tially increas~ fbe range of er~vi.ro.nmetltai condition. under wh:jch .nitrificatinn 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 abí lity to detem1ine which org".tnism ~ are c.:'trcying out pfilticular soil procesi es, and for the impact of environmental chm1ge on rnicrobia1 c.omn1urúties ru1d ecosy"tetn proces es tba:t will be 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.avíar. 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.ylogeay and function. In míxed oommuníties. with many organi~ms able to perform differ~ot fu11ctians M(i m1my able to i:>erf orm 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 techniques~ moot of th.e infurmat:ion On tbe e.cosy tero functiO'O of ditfere.nl microbiaJ group is stilJ deri,red from. phy~iolog:icul 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

Copynghtcd material

Microorg.1nísms 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.úc nitrogen that are fo,und in tbe soil. F11nction.al microbial groups that. 'tt"lm&fo:.rm tltese inoqµ1uJc 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 rrRlf1CATJO·N

9..3.1 .1 The Process

Nitrifica.tion i the oxidation of reduced f orms of nitro gen to oitntt.e. Th~e n.10 ,t commoo. reduced form of nitrogen. i ammo·nia, and nitrifica:tion typic.ally

Denttrificatton

NJl:rogen · Nítrogenase fixatlon nu

AnlrnaJ,s .... <-·· - PlatitS

Dead animal, plant and rnEcrobia1 blomass

Decomposítlonl / AmmonificattoV A .· .

NHg .:..~ª .~uoo ~ NO _ . NUnm ox'ldlllion ~' NOaAmmoola monooxyganase · 2 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 Mí 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

Microorganisms Cycling oíl Nutrients and TI1eir Oiversity 249

is a broad- ·pectrum oxjda e wbich oxidizes methane, cnrbon monoxide, and a range of organic compouod . Many ammonia oxidizers are ureolyúc, enabliog growth \Vilb urea as ole oítrogen ource. Under anaerobic conditjon , ammonia oxidizer can denitrify; nicrous o íde 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. So·me relationship exi L bet\veeo autnlOnJa 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 strain . • in. particular Nitro· son1.orias europaea. ft · relevance to natural amrnooia oxid izer~ i unclear nnd. alti1ough originaJJy i 'Olated. frorn oi l, N. euro¡Jaea L"I poorly represenled in soi l ammonia 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 oí l 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 Sequen e.es

A number of 'tudic · ha ve u ~d 1nolecular technique ' to characterizc communitie in different environ1nents or follo\\tiug enviro1unental per1urbations (Table 9.1 ). The e pro ide inforrnation on tbe influence of en vi ron 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 inlJueace 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 Í" due ro ionjzatioo of amn1onia, \vhicb diffu e freely into tbe cell , to amrnonium. \vbjcb requires energy-dependent, ac1j e traosport mechanism . Activity in acíd oils may result from urf ac 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,

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 N20 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 t"tlrlte Mtricoxlde Nftrws axíde reductase roouetasa reduct:ase re:duct-ase

N03 NO a NO okz ¡ · V f JJ - ! • · ~o ·~

narG nirS norB nosl. nap.A nlrK nor'Z.

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 anal ysi .• 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.es 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 funetíonal genes. However, man;1 organ1sms

coatttin so.me. 'but 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 functionaJ 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:termioatioD of relative a.bund.ances wi.tbout absolute quanti'fication of eacb fu_nctiortal gi'<>up. e .. g. by u ing real-tinie PCR. Quantiñca.tion i aidedt


Microorganisms Cycling Soil Nutrients and Theír 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 hydrol~za bonds between N-acetyi-o~glllcO -2-amin.e residu ~ to forn1 oligon1ers a11d dimer which can be taken up by the ceU. providing sources of carbon 3·nd nitrogen. Ttte broad. distribution of chi tinolytic actívity prevents l6S ·rRNA geDe-based analy ·:is of cb.itinulytic organism . However. cbitína e enzymes fall witbin a. number of cJ.aues, a_11d functional gene prímers can be rlesign.ed fat tb.e analy i of organiS'ms with different fomls of the cbitinase genei .

Such pri1n.er t1ave been devclo¡>ed for c.h.i.tioases belo·nging to Class 18, group A. wlticl1 are commortl;" reco'.vered frt)m ·oil. Alrrplific:lti.01l. of ~bltinase genes from so.iJ • wúng 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 by .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~ chítitU\ 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 Ü EGRADATfO.N Of Ü 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 isoto·pe 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 t3C-1abeled compounds. lt provídes direct evidence of a.q-similntio·n of lnbeted.

compound · and. t.herefore lias tremendous value in detetmining whicl1


Microorgan ísms 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 átnm-Onia oxlclizer pecies 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:ta.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, 1431-1441 ~ 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, A¡1plie.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 Microbiology, 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>iron11imt.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'jdizing 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.


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