Interactions in the Root Environment: An Integrated Approach: Proceedings of the Millenium Conference on Rhizosphere Interactions, IACR-Rothamsted, United Kingdom 10–12 April, 2001

223 Pages·2002·7.04 MB·English

by  J. K. Lithgow

INTERACTIONS IN THE ROOT ENVIRONMENT :
AN INTEGRATED APPROACH
Interactions in the Root Environment:
An IntegratedApproach
Proceedings ofthe Millenium Conference on RhizosphereInteractions,
IACR-Rothamsted, United Kingdom, 10-/2April,2001
Editedby
DAVID S. POWLSON
GEOFF L. BATEMAN
KEITH G. DAVIES
JOHN L. GAUNT
and
PENNY R. HIRSCH
ReprintedrfomPlant andSoil,Volume232,Nos.1-2(2001).
•1).." .~
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Contents
INTERACTIONS IN THE ROOT ENVIRONMENT :
AN INTEGRATED APPROACH
ProceedingsoftheMillenium ConferenceonRhizosphere Interactions,
IACR-Rothamsted,UnitedKingdom. 10-/2April, 2001
Foreword
Section I.Signallingand recognition intherootenvironment
I.AnalysisofN-acyl homoserine-lactonqeuorum-sensingmoleculesmadebydifferentstrains and
biovarsofRhizobium leguminosarum containingdifferentsymbioticplasmids
J.KLithgow, Y.E.Danino,J.Jones& J.A.Downie 3
2.Signallingbetweenarbuscularmycorrhizalfungi andplants:identificationof ageneexpressed
duringearlyinteractionsbydifferentiaRlNA displayanalysis
H.Roussel, D.vanTuinen,P.Franken,S.Gianinazzi& Y.Gianinazzi-Pearson 13
3.Methods for studying thenematophagousfungus Verticillium chlamydosporium in theroot
environment
P.R.Hirsch,S.D.Atkins,T.H.Mauchline,e.O.Morton,KG.Davies& B.R.Kerry 21
4.Doesallelopathyofferrealpromiseforpracticalweedmanagemenatndforexplainingrhizosphere
interactionsinvolvinghigherplants?
M.A.Birkett,K Chamberlain,A.M.Hooper& J.A.Pickett 31
Section2.Rootstructureandfunction
5.Root proliferatio,nnitrateinflow andtheircarboncostsduringnitrogencaptureby competing
plants inpatchysoil
D.Robinson 41
6.Thenutritionalcontrolofrootdevelopment
B.Forde& H.Lorenzo 51
7.Effectofphosphorusavailabilityon basalrootshallownessincommonbean
H.Liao,G.Rubio,X. Yan,A. Cao,KM. Brown& J.P.Lynch 69
8.A comparisonofstructur,edevelopmenatndfunctioninclusterrootsofLupinus albus L. under
phosphateand iron stress
J.Hagstrom,W.M.James& KR. Skene 81
Section 3.Biologically-mediatedprocessesinthe rootenvironment
9.Rhizospherecarbonflowmeasuremenatndimplications:fromisotopestoreportergenes
K Killham& e.Yeomans 91
10.Soil andrhizosphereashabitatsfor Pseudomonas inoculants:newknowledgeondistribution,
activity andphysiologicalstatederivedfrommicro-scaleandsingle-cellstudies
1.Serensen,L.E.Jensen& O.Nybroe 97
11.Sulphurandphosphorustransportsystemsinplants
EW. Smith 109
12.Incorporatingrhizosphereprocessesinto field-scale(agro)ecosystemmodels
J.Arah 119
13.Plant-mediatepdrocessesstoacquirenutrients:nitrogenuptake byrice plants
GJ.D.Kirk 129
Section 4.Biodiversityinthe rootenvironment
14.Plants and fertilisers asdriverscohfangeinmicrobialcommunitystructureand function insoils
A.G.O'Donnell,M. Seasman,A.Macrae,I.Waite& J.T.Davies 135
15.Soil fungi: diversity anddetection
P.Bridge& B. Spooner 147
16.On therelationshipsbetweennematodes,mycorrhizalfungi and plant:sfunctionalcomposition
ofspecies andplanpterformance
L.Brussaard,T.w.Kuyper& RG.M.deGoede 155
17.Bacterialdiversity of therhizosphereof maize(Zea mays) grown intropicalsoil studied by
temperaturgeradientgelelectrophoresis
N.C.M.Gomes, H.Heuer, J.Schonfeld,R.Costa, L.Mendonca-Hagler& K. Smalla 167
18.Monitoringtemporal and spatial variationrihnizospherebacterialpopulationdiversity:A com
munityapproachfor theimprovedselectionofrhizospherecompetenbtacteria
VJ Goddard,MJ.Bailey, P.Darrah, A.K.Lilley & I.P.Thompson 181
Section5.Exploitation ofrhizosphere interactions
19.EnhancingthebiocontrolefficacyofPseudomonasfluorescens Fl13 by alteringtheregulation
andproductionof2,4-diacetylphloroglucinol
I.R Delany,D.E Walsh,I.Ross, A.M.Fenton,D.M.Corkery& E O'Gara 195
20.Plant andrhizosphereprocessesinvolved inphytoremediatioonfmetal-contaminatesdoils
S.P.McGrath,FJ.Zhao& E.Lombi 207
21.An integratedapproachfor theevaluationof biologicalcontrol of thecomplexPolymyxa
betaelBeetNecrotic YellowVeinVirus, bymeans of seeindoculants
R Resca, M.Basaglia, S.Poggiolini,P.Vian, S.Bardin,D.E Walsh,CM .EnriquezBarreiros, 215
E O'Gara,M.P.Nuti, S.Casella& D.Peruch
PlantandSoil 232: 1-2,2001.
©2002KluwerAcademicPublishers. PrintedintheNetherlands.
Foreword
Mechanisms controlling the multipleinteractions emerging knowledge and maximise opportunities for
between plant roots, other organisms and the soilcross-fertilisationbetween disciplinse.The contribu
environmentarecurrently arousing great scientificin tions to the conference were arranged into fiveareas
terest. Increased understanding of thiensteeractions of activeresearc,hand the papers inthis special issue
is highly relevant to thedevelopmentof strategies aregrouped according.ly
forbiological control ofsoil-borne pests and diseases
and the more precise regulation of nutrient supply to
plants.Rapidexpansion of knowledge is occurring in
Signallingandrecognitionintherootenvironment
the area of chemical signalling and sensing in living
organisms andthere isnocwonsiderablescopeforthe
Organisms large and small need tcoommunicatein
newideastobeextendedtorthhiezosphere.Newtech
order to intera,ctbut a multiphase medium such as
niques, based, for example, on advancems oinlecular
soil provides obvious problesm. Nevertheless, inter
biology and imaging, offeropportunitiesfor greatly
actions do occurandroots,micro-fauna and filament
expanding our knowledge of signalling, recognition
ous microorganisms, althougchonstrainedbythe soil
andinteraction intherootzone.
particles, grow or move through soil spa.cVeoslatile
The papers in this Special Issue were contrib
signal moleculescandiffuse through these airspaces,
uted at the RothamsteMdillennium Conferenceon
and the soil water acts as a conduit for both microor
'Interactionsin the RootEnvironment'held in April
ganisms and solublecompounds.Plants can commu
2000.Scientific breakthroughs and potential applica
nicate with each other and influence the behaviour of
tionsofincreasedknowledgeionfteractionisntheroot
both pathogens and beneficial microorganisms, using
zonearedispersedthroughmanyscientdifiiscciplines.
chemicalsignals. Similarly, soil bacteria and fungi
The specific aim of this Conference was to integrate
communicatewith each othe,rand can also influence
new findingsfrom research covering a wide range of
plants.The sessioncovered manyof these aspects in
approaches,environmentsanddiscipli.nes
cluding allelopathy and plant interactions with ,fungi
Scientificexperimentstoestablish theprinciples of
nematodesandbacteria.
crop nutrition began at Rothamsted in 1843and their
findings have had a dominant influence on agricul
tural practice during the 20th cen.tuCruyrrent work
at IACR-Rothamsted and IACR-Long Ashton spans Root structure and function
soilmanagement andcrop nutrition, plant physiology,
metabolism and development, crop aenndvironment A plant root system is a highly orgasneidstructure
protection,agronomy and cropmanagementand bio which, according to speci,eshas evolved to serve
mathematics related to agriculture. At the dawn ofits primary functions of anchorage, storage and the
the 21st century it is timely to review scientific pro acquisition of water and nutrie.nTtshe relationship
gress relevant to the rhizosphere in view of the ad between root structure and function needs to becon
vancesinmethodologythataremaking itsstudymoresidered notonlyatthecellular le,vbeultalso interms
tractabl.eIt seems likely thaitncreasedunderstand of the architecture of the whole root system. Root
ing of rhizosphere processes will be key to a betterdevelopment is plast,icbeing strongly influenced by
fundamental understanding roofot-soil-microbein environmentaflactorssuchastheavailability ofnutri
teractions in both natural amndanagedecosystem.s ents, water and oxyge.nThese environmental factors
Exploitation of this understanding may well provide can modify the pattern odfeploymentof roots or
new ways of managing agricultural crops in the new even trigger the formation of specialised structures
century in ways that are boathgriculturallyand en (e.g.,aerenchymaorproteoid roost).Recent advances
vironmentally sustinaable.The conference,and the are throwing light on the mechanisms by which en
papers presented here, seek tofaciliitnatteegrationof vironmental signals are translated into developmental
responses byroot.s
2
Biologicallymediatedprocessesinthe root theroleofmicro-organsimpopulationdviersityinsoil
environment ecosystems,thesignificanceofchangesinpopulation
structure, andtheapplicationof different tehcniques
This session examinedthe interaction ebtweenthe tofurtherourunderstandingotfhiscomplexsubject.
root and soil processes ni the rhizosphere.It con
sidered theconsequencesandopportunitiesotexploit
our understanding ofthese interactions inhtree key Exploitationofrhizosphereinteractions
areas: plant-mediatedprocesses to acquire ntirogen,
sulphurandphosphorus;hteinfluenceofcarboninputs This session considered how our understanding of
in therhizosphere onmicrobialdiversity,dynamics interactionsin the rhizospherecan be exploited in
andfunction;andcomputersimulationof soil-plant themaintenance ofsustainableplant productionsys
microbeinteraction.s tems.Topicsincludedanoverview ofthecurrentstate
of biological control,particularly the use of applied
biocontrol agents, modellingthe spreadof micro
Biodiversityintherootenvironment organisms in soil and on roots and its relevanceto
disease management,bioremediation of plolutedsoil
Fertileagriculturalsoils containat least 108 bacteria and restoration of solifertility,and the commercial
g- l soil, comprising numerousdifferent taxnoomic exploitationofmicrobialnioculants.
groups,as well as many fungi andothereukaryotic
micro-organisms.Thecontributionof themajorityof
thesespeciesto plantgrowhtandsoilprocessesisun The guesteditorsfor this SpecialIssue werePro
known. Thefunctionaland geneticdiversity of soil fessor David Powlson, Drs Geoff Bateman, Keith
microbialpopulations are notnecessarily the same, Davies, John Gaunt and Penny Hirsch of IACR
andtheirrelativeimportance in maintaining usstain Rothamsted nad Dr PeterBarlow of IACR-Long
ablesoil systems isunclear.This session examined Ashton.
PlantandSoil 232: 3-12,2001. 3
©2002KluwerAcademicPublishers. Printed intheNetherlands.
Analysis ofN-acyl homoserine-lactonequorum-sensingmoleculesmade
by differentstrains and biovars ofRhizobiumleguminosarumcontaining
differentsymbioticplasmids
J.K.Lithgow],V. E.Danino, J. Jones& J.A. Downie!
John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK t Present address: DepartmentofMolecularBiology
andBiotechnology,UniversityofSheffield, SheffieldSI02TNICorrespondingauthor"
Keywords:Rhizobiumleguminosarum,plasmids,conjugation,quorum-sensinga,cyl-homoserinelactone
Abstract
StrainsofRhizobiumleguminosarumuseacelldensity-dependengetneregulatorysystemtoassesstheirpopulation
density. This is achieved by thaeccumulationofN-acyl-homoserinelactones (AHLs) in theenvironmentduring
growthof the bacteria and these AHLs stimulate the induction of various bacterial genes that are up-regulated in
thelate-exponential andstationary phases ofgrowth. Agenetwicealll-lycharacterisesdtrainofR.leguminosarum
biovarviciae was found to have four genes, whose products synthesise different.AWHeLshave analysed AHL
production by four genetically distinct isolateRs.olfeguminosarum, three of b.vviciae and one of bv.phaseoli.
DistinctdifferenceswereseeninthepatternofAHLsproducedbytvhieciabev.strainscompared withbpvh.aseoli
andtheincreased levelsanddiversity of AHLs found in.vbicviae strains canbeattributed torthhielgene, which
is located on the symbiotic (Sym) plasmid and is up-regulated when the bacteria are grown in the rh.izosphere
Additional complexity tothe profileofAHLs isfound toabsseociatedwith highly transmissible plasmipdRLlJI
ofR. leguminosarumbv.viciae, butthisisnotobserved withsome other strains, including thosecarrying different
transmissible plasmid.sIn addition to AHLs produced by the products of genes on the symbiotic plasmid, there
is clear evidence for the presence of other AHL production. Eloxcpiression levels and patterns of AHLs can
change markedly indifferent growth me.dTihaese results indicate that there isa netwoqrkuoorfum-sensingloci
in different strains oRf. leguminosarum and these loci mayplayarole in adapting torhizospheregrowth and
plasmidtransfer.
Introduction carrying an N-linked acyl group on the terminal
(non-reducing)sugar (Downie 1998.)Nod-factors are
the principaldeterminantsof host range of a given
BacteriaofthegenuRshizobiumcanformasymbiotic
relationship with various leguminous plant resulting Rhizobium species and specificity idseterminedby
nodgene products that modify the Nod factor by the
in the development of nitrogen-fixing root nod.ules
addition ofvarioussubstituents suchasacetate, sulph
The symbiosis involves cell-to-celclommunication
ate or sugars such as fucose or arabinose (Downie,
between host plant and infecting bacterium. Plant
1998). The genes for Nod-factor production and ni
roots secrete flavonoids or iso-flavonoids, which in
trogen fixation are carried on large symbiotic (Sym)
duce the expression of rhizobianlod (nodulation)
plasmids insome strains (Schlaman e.t,a1l998).
genes (Schlaman etaI.,199.8T)his results in the pro
As well as communicatingwith the host plan,t
duction of bacteriallipo-oligosaccharidenodulation
(Nod) factor,s which are usuallyoligomersof four Rhizobium cellscommunicatewith each other using
or fivefJ l-4-linkedN-acetylglucosamineresidues small molecules called N-acyhlomoserinelactones
(AHLs). AHLs are widely used by Gram-negative
bacteriafor 'Quorumsensing',a mechanism of cell
* FAXNo :[phone].
E-mail:[email protected] desity-dependenrtegulation ofgene expressi.oAnHL
4
structuresacnvaryinthelengthoftheacylchain and by 30H,C14:1-HSL (GrayetaI.,1996) was shown to
natureofthesubstitution(H,OH,or0)atthe3C po beindirectbecausethisAHLdid notdirectlyactivate
sition(SwiftetaI.,1996;WilliamsetaI.,1999).Most expressionof therhiABC genesby RhiR (Rodelaset
AHLs are thoughttobediffusibleacrosshtebacterial aI.,1999).
membraneand the AHL concentration threeforere Lithgow et aI., (2000)charactersied thecinR and
flectsthe celldensity of a givenbacterial ppoulation. cin!genesrequiredfortheregulationandsynthesisof
Athighcelldensities,AHLs activateatranscriptional 30H,C14:1-HSL.ThecinRandcin! genesarethought
regulatoryprotein a( LuxR-type regulator), which to belocated on theR. leguminosarum chromosome.
bindstospecificpromotersandregulatesgeneexpres In ahierarchicalsignalling network,thecinR! locus
sion(FuquaetaI.,1996).In mostcases,thisregulator and 30H,C14:1-HSL regulateexpression of rhi!, and
also induces expression of the AHL-synthase gene at least oneother AHL productionlocus on pRL1JI
(encoding a LuxI-type AHL synthase); the resulting thoughttobeinvolvedinconjugalSyrn-plasmidtrans
increaseni AHL production resultsin positivefeed fer. In addition, thecinR! locus regulates naother
backregulation(Fuquaet aI., 1996).AHL-regulated non-pRL1JI AHL productionlocus elsewhere in teh
phenotypesinGram-negatviebacteriaarediverse and R.leguminosarum genome(LithgowetaI.,2000).
includebioluminescenceni Vibriofischeri (Eberhard Production of multiple AHLs has also been de
etaI., 1981;Kuo et aI., 1994),virulence fcatorpro scribed inRhizobium etli. Some of theseAHL s are
ductioninPseudomonasspp.(Gambelloet aI., 1939; synthesised and regulatedby theRail and RaiR pro
WinsonetaI.,1995),andplasmidtransferinAgrobac teins,respectiveyl (RosemeyeretaI.,1998).Asurvey
teriumtumefaciens(FuquaandWinans,1994;Piperet ofplant-associatebdacteriagaveevidenceofrmultiple
al.,1993;ZhangetaI.,1993). AHLproductionbyR.leguminosarumbv.trifoliiand
ThefirstAHL to bedescribedni Rhizobium spp. Sinorhizobium meliloti (ChaetaI., 1998).The Sym
wasN-3-hydroxytetradecenoyl-L-homoserinaectlone plasmid,pNGR234a ofRhizobium sp.NGR234,also
(30H,C14:1-HSL), producedby R. leguminosarum carriesatra!gene,whichisstrongyl homologoustoA.
(Gray et aI., 1996; Schripsema et aI., 1996). tumefaciens tra! that regulatesTi-plasmidtransferby
30H,C14:1-HSL was initially known as 'small bac AHL-mediatedquorumsensing(FreibergetaI.,1997).
teriocni',becausetiwasshown to inhibit the growth A common theme emergingfrom identification
of some R. leguminosarum strainscarrying certain of AHL -productionand regulatorygene loci is that
Sym-plasmids,suchaspRLlJI (Hirsch, 1979).Gray many(butnot all) of these genesarelocated onindi
et al. (1996) showed that 30H,C14:1-HSL does not genous plasmids.Variousstudies havedemonstrated
kill sensitivecells of R. leguminosarum, but inhibist thepresence ofmultipleplasmidsin rhizobia1strains
growth,possiblybyinducinganearlyonsetofstation (HynesandFinan1998).In this work, wehavemade
ary phase. Strainscarrying pRL1JI actuallyrepress use of strains ofR. leguminosarum that have dif
30H,C14:1-HSLproduction(BrewinetaI.,1980,Wijf ferent genetic backgroundsincluding differenttypes
felmanetaI., 1983)and theerforedo not inhibittheir and numbersof large indigenousp1asmids.Wehave
own growth.30H,C14:1-HSL was shown to regulate usedmutanstdeleted forplsamids(orlargeregions of
the rhizosphere-expressedgnees(rhiABC) of pRL1JI plasmids)andpreviously-constructedtrsainsthathave
(Grayet aI., 1996),which caninfluencethenodula acquiredrtansferableplasmids.By analysing the dif
tion efficiencyofR. leguminosarumbv.viciae (Cubo ferentAHLsmadeby thesestrainswehavebeenable
etal., 1992).Expression of therhiABCgenes is reg tocorrelatetheproductioonfvariousAHLs withsome
ulated by the regulatoryproteinRhiR (Cubo et aI,. plasmid-borneloci.
1992;GrayetaI,.1996).RodelasetaI.,(1999)showed
thatR. leguminosarum bv. viciae producesmu1itple
types of AHL molecules in addition ot 30H,C14:1 Materialsandmethods
HSL. An AHL synthasegene, rhi!, was found up
streamofrhiAonpRLlJI.RhiIdirectsthesynthesisof Bacterialstrainsare describedni Table 1.Rhizobium
multiple AHLs including N-hexanoyl-L-homoserine strains were grown in tryptone yeast extract(TY)
lactone(C6-HSL) andN-octanoyl-L-homoserinealc medium(Beringer, 1974) or in MOPS minimalme
tone(Cg-HSL)anditsi theseAHLsthatactivateRhiR dium (Thorneand Williams, 1999), supplemented
toinduce therhiABC(andrhil) genes(RodelasetaI., withmannitol(1% w/v),sodiumglutamate(5mM) and
1999).The observedactivation ofrhiABCexpression vitamins (biotin,thiamine and DL-pantotheniccaid).
5
Table1.Rhizobiumstrains used
Strain RelevantCharacters Reference
248 R.leguminosarumbv.viciaecarrying Hirschetal.(1980)
pRLlJI (pSym)
300 R.leguminosarumbv.viciaecarrying Hirschetal.(1980)
pRLlOJI (pSym)
6015 Derivative of300deleted fortnhoed-rhi JohnstonetaI.(1978)
gene region onpRLIOJI
6015/pRLIJI Derivative of6015carryingpRLIJI Johnstonetal.(1978)
60I5/pRL3JI Derivative of6015carryingpRL3JI, Brewinetal.(1982)
whichoriginatesfromR.leguminosarum Hirsch etal.(1980)
strain 306
8002 R.leguminosarum bv.phaseolicarrying Johnstonetal.(1982)
pRP2JI(pSym) Lambetal.(1982)
8401 Derivative of8002lackingpRP2JI Lambetal.(1982)
8401/pRLIJI Derivative of840IcarryingpRLIJI Downie etal.(1985)
TOM R.leguminosarumbv.viciae carrying Winarno andLie(1979)
pRL5JI (pSym) Brewinetal.(1980)
RBLl387 Derivative of248lackingpRLlJI PriemandWijffelman
(1984)
Bacterialgrowth wasmonitoredbymeasuringoptical from thechromatographytank, dried in air and then
densityat 600 nm(OD600) using an MSE Spectroplus re-chromatographeidn the samedirection.The plate
spectrophotometer. was removed,dried in air and thenoverlaidwith a
Assays of30H,CI4:I-HSL productionwere done thin filmofLuria-Bertanisoftagar (0.7%, w/vs)eeded
usingthesmallbacteriocinassaydescribedbyWijffel withC.violaceum CV026. Afterovernightincubation
man et a.l,(1983).OtherN-acylhomoserinelactones at30°C, AHLs were locatedby detectionofpurple
wereassayedfollowingbacterialgrowthfor 48 h in spotsagainsta whitebackground.
TY medium, at which point thOeD600 hadreached
0.8- 0.9.In MOPS minimalmedium,thebacteria
were grown for 72 h or 120 h, when thOeD600 Results
hadreached0.2 or 0.8respectivel.yThe cells were
removedbycentrifugationa,ndtheAHLs wereextrac Choice ofstrainsforanalysisofN-acylhomoserine
ted fromculturesupernatantswith dichloromethane lactones
as describedby Winson et al.,(1995). AHLs were
analysedby thin-layerchromatography(TLC) using Four strainsofR. leguminosarum werechosenon the
Chromobacterium violaceum CV026 as the AHL in basis that they weriesolatedfromdifferentareas and
dicatororganism(McCleanet al., 1997)C.V026 can are geneticallydistinctbased on DNA andenzyme
beusedasabiosensorforexogenousAHLs becauseit polymorphisms(Young, 1985,J.P.w.Young,personal
producesthepurplepigmentviolaceinin responseto communication).Each carriesa differentsymbiotic
AHLswithshort acylchains.Culturesupernatantasnd plasmid(Table 1).R. leguminosarum bv.viciae strain
syntheticAHL standards(as0.1mgmI-1solutionsin 248 istheoriginalhostofthewell-characterisesdym
acetonitrilew)ere spotted (2- IOILl) ontoaluminium bioticplasmidpRLlH, that is highlytransmissible.
backedRPI8 reverse-phaseTLC plates (Merck)and Strain 300 has adifferentSyrn-plasmid,pRLlOH that
dried in astreamofair.Sampleswereseparatedwith is not transmissible. R. leguminosarum bv. viciae
60% (v/v) methanolin water as themobilephase. strain TOM wasisolatedin Turkey and isunusualin
Once thesolventfront hadmigratedtowithin2em of that itcannodulatecv.Afghanistanpeas; strain TOM
the topofthechromatogramt,heplatewasremoved carriesthetransmissiblesymbioticplasmidpRL5JI.R.
leguminosarum bv.phaseoli strain 8002 wasisolated