Identification of an Endophytic Antifungal Bacterial Strain Isolated from the Rubber Tree and Its

14 Pages·2015·4.67 MB·English

RESEARCHARTICLE
Identification of an Endophytic Antifungal
Bacterial Strain Isolated from the Rubber
Tree and Its Application in the Biological
Control of Banana Fusarium Wilt
DeguanTan1☯,LiliFu1☯,BingyinHan1☯,XuepiaoSun1,PengZheng2,JiamingZhang1*
1 MOAKeyLaboratoryofBiologyandGeneticResourcesforTropicalCrops,InstituteofTropicalBioscience
andBiotechnology,CATAS,Haikou,HainanProvince,571101,China,2 LijiangTeachersCollege,Lijiang,
YunnanProvince,674110,China
☯Theseauthorscontributedequallytothiswork.
* [email protected]
Abstract
OPENACCESS BananaFusariumwilt(alsoknownasPanamadisease)isoneofthemostdisastrousplant
diseases.Effectivecontrolmethodsarestillunderexploring.Theendophyticbacterialstrain
Citation:TanD,FuL,HanB,SunX,ZhengP,Zhang
J(2015)IdentificationofanEndophyticAntifungal ITBBB5-1wasisolatedfromtherubbertree,andidentifiedasSerratiamarcescensbymor-
BacterialStrainIsolatedfromtheRubberTreeandIts phological,biochemical,andphylogeneticanalyses.Thisstrainexhibitedahighpotential
ApplicationintheBiologicalControlofBanana
forbiologicalcontrolagainstthebananaFusariumdisease.Visualagarplateassayshowed
FusariumWilt.PLoSONE10(7):e0131974.
doi:10.1371/journal.pone.0131974 thatITBBB5-1restrictedthemycelialgrowthofthepathogenicfungusFusariumoxysporum
f.sp.cubenserace4(FOC4).Microscopicobservationrevealedthatthecellwallofthe
Editor:XianlongZhang,NationalKeyLaboratoryof
CropGeneticImprovement,CHINA FOC4myceliumclosetotheco-culturedbacteriumwaspartiallydecomposed,andthe
conidialformationwasprohibited.TheinhibitionratiooftheculturefluidofITBBB5-1
Received:April9,2015
againstthepathogenicfunguswas95.4%asestimatedbytipcultureassay.Chitinaseand
Accepted:June9,2015
glucanaseactivitywasdetectedintheculturefluid,andthehighestactivitywasobtainedat
Published:July2,2015 Day2andDay3ofincubationforchitinaseandglucanase,respectively.Thefiltratedcell-
Copyright:©2015Tanetal.Thisisanopenaccess freeculturefluiddegradedthecellwallofFOC4mycelium.Theseresultsindicatedthatchit-
articledistributedunderthetermsoftheCreative inaseandglucanasewereinvolvedintheantifungalmechanismofITBBB5-1.Thepotted
CommonsAttributionLicense,whichpermits
bananaplantsthatwereinoculatedwithITBBB5-1beforeinfectionwithFOC4showed
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare 78.7%reductioninthediseaseseverityindexinthegreenhouseexperiments.Inthefield
credited. trials,ITBBB5-1showedacontroleffectofapproximately70.0%againstthedisease.
DataAvailabilityStatement:Allrelevantdataare Therefore,theendophyticbacterialstrainITBBB5-1couldbeappliedinthebiologicalcon-
withinthepaper. trolofbananaFusariumwilt.
Funding:ThisworkwassupportedbytheNatural
ScienceFoundationofChinaunderGrantnumber
31471561,theNaturalScienceFoundationofHainan
ProvinceunderGrantnumber309051,andthe
NationalNonprofitInstituteResearchGrantof
Introduction
CATAS-ITBBunderGrantnumberITBB110307.
Bananaisamongthemostimportantfoodandfruitcropsinmanydevelopingcountries[1].
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. However,diseasesandpestsbecamesevereproblemswhencertaingenotypeswerecultivated
PLOSONE|DOI:10.1371/journal.pone.0131974 July2,2015 1/14
ApplicationofanEndophyticBacterialStraininBiocontrol
asmonocultures[2],andlimitedtheincreaseofbananaproductioninthelastfewdecades[2].
Fusariumwilt(alsoknownasPanamadisease)isoneofthemostnotoriousanddestructivedis-
easesinbanana[3].Ithasbeenreportedinallbanana-producingcountries,includingAsia,
CentralandSouthAmerica,Africa,andAustralia[4].
ThepathogenofbananaFusariumwiltwasidentifiedasasoil-bornehyphomycete,Fusar-
iumoxysporumformaespecialiscubense(FOC)[4–6],andwasclassifiedintofourphysiologi-
calracesbasedonvirulencetohostcultivarsinthefield[7,8],includingFOC1,FOC2,FOC3,
andFOC4.FOC1infectsthecultivarsGrosMichel(AAAgroup),Silk(AABgroup),andPisang
Awak(ABBgroup);FOC2infectsBluggoe(ABBgroup)anditscloserelatives;FOC3infects
Heliconiaspp.;andFOC4infectsCavendishcultivars(AAAgroup)andallthecultivarssuscep-
tibletoFOC1andFOC2[8].
Before1960,thecultivarGrosMichelwasdominantandsuppliedalmostalltheexport
trade.However,thiscultivarwassusceptibletoFOC1,andtheaccidentutilizationofinfected
rhizomesorsuckerstoestablishnewplantationscausedthewidespreadofthedisease,andthe
world’sbananaindustrywasalmostdestroyed[3].Asaresult,Cavendishcultivarsthatwere
resistanttoFOC1werecultivatedtoreplaceGrosMichelworldwide[9].Thesecultivarsremain
tobethewell-performedclonesinthewesterntropics.However,theyarenotresistantto
FOC4identifiedintheeasterntropics,andcausedseriouscroplossesintheCavendishbanana
plantationsinAsia,Australia,andAfrica[10].Forexample,thebananaplantationsinChina,
includingHainan,Guangdong,Guangxi,Yunnan,andFujianProvinceshaveencounteredan
estimatedaveragediseaseincidenceof20to40%[11],withthehighestrateof90%[12–14].
AlthoughFOC4existsonlyintheeasterntropics,thereisalsoconcernoverthespreadingof
FOC4tothewesterntropics,sincebananaproductionispredictedtobedestroyedcompletely
inthatregionifFOC4invaded[15].
Tocontrolthedisease,fungicides,biocontrolagents,andresistantcultivarshavebeeninves-
tigated[2,10,13,16–23].Molecularbreedinghasalsobeenusedinthedevelopingofresistant
varieties[24,25].Significantmycelialgrowthordiseasecontrolwasachievedinsomeofthese
studiesinthelaboratoryand/orinthegreenhouse,butthefielddemonstrationsweremostly
notsatisfactory.
Wehaveisolatedanendophyticbacterialstrainfromtherubbertree,andidentifieditasa
Serratiamarcescensstrainwithaspecialintracellularsecretionapparatusandantifungalactiv-
ity.ThisstrainhadahighpotentialinthebiologicalcontrolofbananaFusariumwilt.
MaterialsandMethods
IsolationofthebacterialstrainITBBB5-1
ThebacterialstrainSerratiamarcescensITBBB5-1wasisolatedfromsterilizedyoungbranches
oftherubbertree(HeveabrasiliensiscloneReyan7-33-97)growninthegreenhouseatthe
InstituteofTropicalBioscienceandBiotechnology,ChineseAcademyofTropicalAgricultural
Sciences(CATAS),Haikou,China.Youngbrancheswereremovedfromhealthyrubbertrees,
segmentedtoapproximately4cm,andwashedwithdistilledwater.Thesegmentswerethen
rinsedin75%ethanolfor30s,andsterilizedin0.2%HgCl for8min,followedbywashing
2
withsterilizeddistilledwaterfourtimes.Thesegmentswerecutintothinslices(1–2mm)and
incubatedonagar-solidifiedLuria-Bertani(LB)medium[26]inthedarkat28°Cfor4–10days.
TheITBBB5-1strainthatproducedaredpigmentwasisolatedandmaintainedonLBplates
orstoredat-80°C.ThisstrainwasalsodepositedattheChinaGeneralMicrobiologicalCulture
CollectionCenterlocatedinBeijing,ChinaunderaccessionnumberCGMCC7416.
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Morphologicalcharacterization
ITBBB5-1bacterialcellsweremountedonglassslides,withorwithoutstainingbyGram-stain
reagents,andexaminedusingalightmicroscope(OlympusBH2,Japan).Photographswere
takenunderanoilimmersionobjectivelens(100X).
Forscanningelectronmicroscopy,cellsinlateexponentialgrowthwerecollectedfromsus-
pensionculturesinLBbrothbycentrifugation(5000rpm,5min),washedtwicewith0.01M
phosphatebufferedsaline(PBS,0.228gNaH PO ,1.15gNa HPO in1LddH O)andfixed
2 4 2 4 2
in0.5%glutaraldehydeand1%formaldehyde.Thecellsweredehydratedthroughaseriesof
acetonesolutions,spreadedoverglasscoverslips,criticalpointdried,andthendressedwith
gold.ThesampleswerethenobservedunderaJSM-35Cscanningelectronmicroscope(JEOL
Ltd.,Japan).
Fortransmissionelectronmicroscopy,cellswerecollectedfromLBplatesorsuspension
cultures,fixedwith2%glutaraldehydeand1%formaldehydedissolvedin50mMTris/HCl
buffer(pH7.4)at4°C,andharvestedbycentrifugationat5000rpmfor5min.Thecellswere
washedin50mMNa-cacodylatebuffer(pH7.0)andresuspendedin1%osmiumtetroxide
(aqueoussolution)overnightat4°C,followedbydehydratingthroughanacetoneseries.After
embedmentinSpurr’sresin,ultrathinsectionswerecutwithadiamondknife.Theslideswere
mountedonformvar/carbon-coatedslots,sequentiallystainedwithuranylacetateandleadcit-
rate,andfinallyobservedunderaJEOL1010transmissionelectronmicroscope(JEOLLtd.,
Japan).
DNAextractionandamplificationof16SrDNA
TheITBBB5-1strainwasculturedinLBbrothovernightwithshakingat28°Candharvested
bycentrifugation.GenomicDNAwasextractedusingtheUniversalGenomicDNAExtraction
Kit(Sangon,Shanghai,China),accordingtothemanufacturer’sinstruction.16SrDNAwas
0 0
amplifiedusingtheforwardandreverseprimers5-AGAGTTTGATCCTGGCTCAG-3
0 0
and5-AAGGAGGTGATCCAGCCGCA-3,respectively[27].Thefragmentwassequenced
atShanghaiSangonBiologicalEngineeringTechnology&ServicesCo.,Ltd.Thesequencewas
analyzedusingMacVectorsoftware(OxfordMolecular,Oxford,UK),andwasregisteredinthe
GenBankdatabase(JN896750).BLASTsearchusingthe16SrDNAsequenceasaquerywas
performedagainsttheGenBankdatabase.
Phylogeneticanalysis
Forphylogeneticanalysis,16SrDNAsoftherelatedbacterialstrainswereobtainedfromGen-
BankdatabaseandalignedusingClustalX3.0[28].Theprimerregionswereremovedfromthe
sequences.ThealignmentresultswereexportedtoMEGA4[29].Phylogenetictreesweregen-
eratedusingtheNeighbor-Joining(NJ),MinimumEvolution(ME),andMaximumParsimony
(MP)methodswith1000bootstrapreplicates.OnlytheMinimumEvolutionTreeisprovided.
TheevolutionaryhistorywasinferredusingtheMEmethod[30].Theoptimaltreewiththe
sumofbranchlength=0.06731820isshowninthispaper.Allpositionscontaininggapsand
missingdatawereeliminatedfromthedataset(completedeletionoption).Therewereatotalof
1293positionsinthefinaldataset.Thetreeswererootedwith16SrDNAsequencesfrommem-
bersoftheEnterobacteriaceaefamily,includingKlebsiellaplanticolastrainDR3(X93216),and
twootherstrains.
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Theplantpathogenicfungalstrainandsporepreparation
ThepathogenicfungalstrainFusariumoxysporumf.sp.cubenserace4(FOC4)thatcaused
severediseasesinbananawasprovidedbyDr.JunshengHuang,InstituteofPlantandEnviron-
mentProtection,ChineseAcademyofTropicalAgriculturalSciences(CATAS).Thestrainwas
maintainedonpotatodextroseagar(PDA)mediumat28°C.TheFOC4conidialsuspension
waspreparedbyincubatingFOC4inPDAbrothonashakerat200rpm,28°Cfor5days,fol-
lowedbyfiltrationwithtwolayersofgauzetoremovethemycelia.Theconidialconcentration
wasadjustedto1×106conidiaperml,andwasstoredat4°Ctillutilization.
VisualagarplateassayoftheantifungaleffectofITBBB5-1against
FOC4
TheITBBB5-1strainsuspensionandthesuspensionsofthecontrolstrainsEscherichiacoli
DH5αandAgrobacteriumtumefaciensEHA105werepreparedbyincubatingthestrainsinLB
brothwithshakingat250rpm,25°Cfor2days,followedbydilutionofthebacteriato108cfu
permlwithdistilledwater,andstorageat4°C.
Thevisualagarplateassay[31]wasusedtotesttheinhibitioneffectofITBBB5-1onthe
growthofthepathogenicstrainofFOC4.TheFOC4conidialsuspensionpreparedasdescribed
abovewasinoculatedinalineatthecenterofLBplates.Thenanaliquotofthebacterialsus-
pensionofITBBB5-1wasinoculatedintherightandleftlines2cmawayfromthecentralline;
LBmediumand/orthesuspensionsofE.coliDH5αandA.tumefaciensEHA105wereusedat
thepositionofITBBB5-1ascontrols.Theplateswereincubatedat26°C.Thewidthofthe
myceliallinewasmeasuredeveryday,andthesignificanceoftheinhibitioneffectwastested
withone-wayANOVAmethodat1%confidencelevel.
FormicroscopyoftheFOC4myceliaatthefrontierofthefungallineinthevisualagarplate
assay,thefrontiermyceliaweremountedonglassslides,andstainedwithanEhrlichhematox-
ylinandeosinstainingreagent(Leagene,Beijing)for20min,washedwithdeionizedwater
and1%ammoniumsolutionfor30seceach,andcoveredwithglassslips.Thesampleswere
observedandphotographedunderalightmicroscope(Axioskop40,Zeiss,Germany).
ToconfirmthelyticactivityofculturemediumofITBBB5-1,theFOC4myceliawere
mountedonglassslides,andtreatedwithfiltrateoftheculturefluidofthestrainITBBB5-1for
30minutes,andobservedundermicroscope.
QuantitativeassayoftheantifungalfunctionagainstFOC4
TheITBBB5-1strainwasculturedinLBbrothonashakerat250rpmand25°Cfor2days,
andcentrifugedat4,200gfor20mintoremovethebacteria.Thesupernatantwasfiltrated
with0.22μmfilterunits(Millipore,Bedford,USA)toremoveremainingbacteria.Thefiltration
wasstoredat4°C.TheinhibitioneffectofthefiltrateagainstFOC4wasquantifiedbyatipcul-
turemethod[32].Fivemlpipettetipswereusedasculturevesselsbysealingthetipwithparaf-
fin.100μloftheabovefiltratewasaddedto700μlPDAbrothinthepipettetips,using100μl
LBbrothascontrol.Eachtipwasinoculatedwith10μlFOC4conidialsuspensionpreparedas
describedabove,andincubatedat28°Cfor6days.Theparaffinwasremovedfromthetip,and
themyceliaofFOC4werecollectedbygentlecentrifugationat96gfor5min.Themycelia
wereweighedusingabalanceof0.1mgaccuracy(MettlerToledo,AL204).Theantifungalratio
wascalculatedbytheequation:(Weightofcontrol—weightoftreatment)/weightofcon-
trol×100%.Theexperimentwasreplicatedthreetimes,andthesignificanceoftheinhibition
wastestedwithone-wayANOVAat1%confidencelevel.
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AssayofchitinaseandglucanaseactivityofITBBB5-1
AsinglecolonyoftheITBBB5-1strainwasincubatedin3mlLBbrothat25°Cfor24hwith
shakingat250rpm.Fiveμloftheactivatedbacterialfluidwasinoculatedintoeach10mlcul-
turetubecontaining3mlofLBbroth,and30tubeswereused.Thetubeswereincubatedat250
rpmand25°C.Sampleswerecollectedat12hintervals,3tubeseachtime.Thesampleswere
centrifugedat4,200gfor10min.Thesupernatantwascollectedandfilteredthrough0.22μm
filterunits(Millipore,Bedford,USA)toremoveremainingbacteria.Chitinaseactivityofthe
filtratewasmeasuredasdescribedpreviously[33].Oneunitofchitinaseactivitywasdefinedas
theamountofenzymethatcatalyzedthereleaseof1μmolofN-acetylglucosamineperhourat
50°C.β-1,3-glucanaseactivitywasdeterminedasdescribedpreviously[34]withafewmodifi-
cations.Thereactionmixturecontained50μlfiltrate,250μllaminarin(Sigma)(2mg(cid:2)ml-1lam-
inarinin50mMsodiumacetatebuffer,pH5.0),and200μl50mMsodiumacetate(pH5.0).
Thereactionwasincubatedat38°Cfor3h,andthen500μlcopperreagentwasaddedinthe
reaction.Thereactionwasboiledfor10minandquicklycooleddowntoroomtemperature.1
mlofarsenomolybdatesolutionwasthenaddedintothereactionmixtureforcolordevelop-
ment.Theabsorbancewasmeasuredat500nmwithaspectrophotometer(Bio-tech,USA).
Oneunitofβ-1,3-glucanaseactivitywasdefinedastheamountofenzymethatcatalyzedthe
releaseof1μmolofglucoseperhour.
GreenhousetestofthebiocontrolfunctionofITBBB5-1againstFOC4
One-month-oldnurserybananaplants(varietyWilliams,MusaAAACavendishsubgroup)
wereboughtfromtheTissueCultureFactoryoftheCATAS.Theplantsweregrowninplastic
potsof15cmindiameterand10cmindepth,andwereclaimedtobediseasefree.Thebiologi-
calcontrolfunctionoftheITBBB5-1strainagainstFOC4wascarriedoutinthegreenhouse
oftheInstituteofTropicalBioscienceandBiotechnology,CATAS.
BeforeinfectionwithFOC4,theplantsweretreatedwith100mlITBBB5-1suspensionpre-
paredasdescribedabove,andrepeatedonce3dayslater.TheplantstreatedwithonlyLBbroth
dilutedtothesameratioasthebacterialsuspensionwereusedasacontrol(CK1).Fifteendays
afterinoculationwithITBBB5-1,eachpotwaswateredwith100mlFusariumconidialsuspen-
sion(1×106spores/ml)preparedasdescribedabove.Theplantsthatwerewateredwithonly
thePDAmediumwereusedasanothercontrol(CK2).Thetreatmentsandcontrolsweredone
intriplicateconsistingof10plantsperreplicate.Twomonthslater,thediseasesymptoms
wererecordedbasedonthefivegradescalefrom0to4asdescribedpreviously[35]:0-corm
completelyclean,planthealthy;1-isolatedpointsofdiscolorationinvasculartissue;2-discolor-
ationupto1/2ofvasculartissue,slightchlorosisinleaves;3-discolorationgreaterthan1/2of
vasculartissue,moderateorseverechlorosisinleaves;4-totaldiscolorationofvasculartissue,
plantdead.ThediseaseseverityindexwascalculatedusingtheformuladescribedbyHuang
etal.[13].Diseaseseverityindex=[∑(Class×Numberofthatclass)/(4×Totalnumberof
assessedplants)]×100.Basedondiseaseseverityindex,thecontroleffectwascalculatedasfol-
lows:Controleffect(%)=[(Diseaseseverityindexofcontrol—Diseaseseverityindexoftreat-
ment)/Diseaseseverityindexofcontrol]×100.Thesignificancewasanalyzedwithone-way
ANOVAtestat5%confidencelevel.
Fieldtrials
Tofurtherconfirmthebiologicalcontrolfunction,fieldtrialswereperformedinChengxi,Hai-
kouCity.Onemonth-oldnurseryplants(varietyWilliams,MusaAAACavendishsubgroup)
weretreatedwith100mlITBBB5-1suspensionpreparedasdescribedabove,andrepeated
once3dayslater.PlantstreatedwithonlyLBmediumdilutedasthebacterialsuspensionwere
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usedascontrol.FifteendaysafterinoculationofITBBB5-1,eachpotwaswateredwith100ml
Fusariumsporesuspensionpreparedasdescribedabove.Fifteendayslater,theplantswere
transplantedinthefieldthatwasfreeoftheFusariumdiseaseat2m×2mintervals.Eachtreat-
mentandcontrolcontained3blocksof10plants,andtheblockswerearrangedatintervals.The
restrictioneffectwassurveyed6monthslater.Thediseaseseverityindexandcontroleffectwere
calculatedasdescribedabove.Thesignificancewasanalyzedwithone-wayANOVAat5%con-
fidencelevel.
Results
LightandElectronMicroscopycharacterizationoftheendophytic
bacterialstrainITBBB5-1
ThebacterialstrainITBBB5-1wasisolatedfromsterilizedstemsegmentsoftherubbertree.
ItsclonesonLBmediumwereround,red,andopaquewithawet,convex,andsmoothsurface
(Fig1A).Conventionalphysiologicalandbiochemicalexaminationsrevealedthatthecells
wereGram-negative,motile,oxidase-positive,catalase-positive,andmethylred-negative.Light
microscopyshowedthatthecellswereredandrod-shaped,withsecretedredvesicles(Fig1B).
Observationofthepelletedcellsindicatedthattheredvesiclesfusedwitheachotherand
formedmuchlarger0.5–2μmvesicles(Fig1C).Accordingtothesefeatures,theITBBB5-1
strainwasinitiallyidentifiedasSerratiamarcescens.Thepigmentwasassumedtobeprodigio-
sinproducedbysomeS.marcescensstrains[36,37].
Scanningelectronmicroscopyshowedthatthecellswerecoccobacillus,andhadnumerous
shortandthinflagellasurroundingthecells(Fig1D).Transmissionelectronmicroscopy
revealedthatthecellshadatriple-layercellwall,inwhichtheinnerandouterlayershadlow
electrondensityandthecentrallayerhadhighelectrondensity.Additionally,thesurfacefim-
briae,whichwasimportanttopathogenicstrainsforhostinfection[38],wasabsent(Fig1E
and1F).Thisfeaturewasdifferentfromsimilarobservationsofsomehumanpathogenicand
environmentalstrainsofS.marcescens[38–41].
SequenceanalysisandphylogeneticclassificationoftheITBBB5-1
strain
Theamplified16SrDNAsequenceofITBBB5-1was1534bp,withaGCcontentof54.51%.
BlastsearchesresultedinthehighestsimilaritywithS.marcescensstrainEF208031,withan
identityof99%.PhylogeneticanalysesindicatedthatthefourSerratiaspecieswereclearlysepa-
rated.ITBBB5-1wasclusteredwithintheS.marcescenscladewithbootstrapsupportsof93%,
95%,and60%whenNJ,ME,andMPmethodswereused,respectively(Fig2;theNJandMP
treesarenotshown).Moreover,theITBBB5-1strainwasclassifiedinsubgroup2ofS.marces-
cens,togetherwiththeenvironmentalstrainsPseudomonassp.DHU-38(HM047515),S.mar-
cescensstrainL1(EF208031),andS.marcescensstrainPakistan:Lahore(FM179314).Thus,
ITBBB5-1conformedtoS.marcescensstrains.
AntifungaleffectofITBBB5-1againstFOC4
TheantifungalactivityoftheITBBB5-1strainagainstthepathogenicfungusofbananaFusar-
iumwiltFOC4wastestedwithvisualagarplateassay(Fig3).Therestrictioneffectwasnotsig-
nificantwhentheFOC4fungallinewasfarawayfromthebacteriallineduringthefirsttwo
daysofincubation(Table1).TheinhibitioneffectbecamesignificantatDay3,whenthefungal
linegrewclosertothebacterialline.Thewidthofthefungallinewasonly3.07cmatDay4
whenco-culturedwithITBBB5-1(Fig3A;Table1).Thegrowthofthefungallinewasrestricted
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Fig1.LightandelectronmicroscopyofS.marcescensstrainITBBB5-1.A,Morphologyofthecloneson
LBplates;B,lightmicroscopyimageofthebacterialcells(blackarrows)andtheextracellularvesicles(white
arrows)shownbytheiroriginalredcolor,scalebar=0.5μm;C,Lightmicroscopyofthepelletedbacterial
cells,whitearrowsindicatethefusedvesiclesinnaturalcolor,scalebar=2μm;D,Scanningelectron
microscopy,scalebar=0.5μm;E,Transmissionelectronmicroscopy,whitearrowindicatestheintracellular
structure,scalebar=0.5μm;F,Thecellwallstructureundertransmissionelectronmicroscope,scale
bar=0.1μm.
doi:10.1371/journal.pone.0131974.g001
betweenthetwoITBBB5-1linesinthefollowingdays,andthemycelialfrontierthatwasclose
totheITBBB5-1linebegantocollapseatDay7(Fig3B).Incontrast,theFOC4fungallinesin
thecontrolplateswere4.10,4.00,and3.97cminwidthatDay4whenLBbroth,E.coliDH5α,
andA.tumerfaciensEHA105wereusedascontrols,respectively(Table1).Thegrowthofthe
fungallineswasnotsignificantlyaffectedbyE.coliandA.tumerfaciens(Table1,Fig3C),and
theFOC4myceliaclimbedoverthebacteriallinesofE.coliandA.tumerfaciensandgrewto
almostafullplateatDay8(Fig3D).Microscopicobservationindicatedthatthecellwallofthe
Fig2.PhylogeneticclassificationoftheITBBB5-1strainbasedon16SrDNAsequences.
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Fig3.RestrictioneffectofITBBB5-1onthemycelialgrowthofFOC4.A-B,MyceliaofFOC4co-cultured
withITBBB5-1photographedatDay4(A)andDay8(B);C-D,MyceliaofFOC4co-culturedwithA.
tumerfaciensEHA105photographedatDay4(C)andDay8(D);E-F,MicroscopicobservationofFOC4
myceliagrownwithITBBB5-1(E)orEHA105ascontrol(F).Bluearrowsindicatethedecomposedcellwalls
inthemycelia;greenarrowsindicatetheconidia;scalebarsrepresent10μm.
doi:10.1371/journal.pone.0131974.g003
Table1. ThewidthoftheFOC4myceliallinesco-culturedwithITBBB5-1andcontrolstrains(cm).
Days 2 3 4 5 6 7 8
CK-LB 1.63±0.15a 2.97±0.06a 4.10±0.26a 5.10±0.53a 5.87±0.51a 6.60±0.62a 7.20±0.70a
CK-DH5α 1.67±0.06a 2.86±0.12a 4.00±0.10a 5.03±0.15a 5.90±0.17a 6.53±0.31a 7.63±0.21a
CK-EH105 1.60±0.10a 2.83±0.06a 3.97±0.15a 4.87±0.12a 5.77±0.12a 6.73±0.15a 7.70±0.26a
ITBBB5-1 1.53±0.15a 2.47±0.06b 3.07±0.12b 3.23±0.15b 3.27±0.12b - -
Note:“-”notmeasuredwhentherewasnofurthergrowth.TheinhibitioneffectofITBBB5-1againstthegrowthofFOC4wassignificantfromDay3as
testedwithone-wayANOVAmethodat1%confidencelevel,whilethegrowthofFOC4wasnotaffectedbyE.coliDH5α(CK-DH5α)andA.
tumerfaciencesEHA105(CK-EH105).Differentlettersindicatesignificantdifference.
doi:10.1371/journal.pone.0131974.t001
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frontiermyceliaclosetotheITBBB5-1linewaspartiallydecomposed,leavinginflatedand
light-stainedspotsinthemycelia,andtheconidialformationwasinhibited(Fig3E),whilethe
myceliainthecontrolplateswereuniformlystainedandtheconidialformationwasnotaffected
(Fig3F).
TheinhibitionfunctionoftheculturefluidofITBBB5-1wasquantifiedwiththetip-culture
method(Fig4A).TherewasalmostnogrowthfortheFOC4myceliainthetipcontainingthe
cell-freesupernatantoftheculturemediumoftheITBBB5-1strain,withanaveragemycelial
freshweightofonly3.3±0.58mg.Theaveragefreshweightofthemyceliainthecontroltips
was72.0±9.5mg.Theinhibitionratiowas95.4%,andstatisticallysignificantbyANOVAtest
(Fig4B).
ChitinaseandglucanaseactivitiesofthefermentativefluidofITBBB5-1
ChitinaseactivityofITBBB5-1fermentativefluidincreasedsteadilyintheinitial48hofincu-
bation(Fig5A),andkeptahighandstablelevelofapproximately9unitspermlofthefluid
from48hto72h.Theactivitythendeclinedslightlyat84h.β-1,3-glucanaseactivityofthefer-
mentativefluidwaslowerthanthatofchitinaseatalltestedtimepoints.However,thetemporal
dependentpatternoftheactivitywassimilar(Fig5A),whichincreasedintheinitial60h,and
declinedat84h.Thehighestglucanaseactivitywasapproximately3unitspermlofthefluid.
TheseresultsshowedthatITBBB5-1secretedextracellularlyticenzymeschitinaseandβ-1,
3-glucanase,andcoulddecomposethepathogenicfungiwithchitinandβ-1,3-glucanascell
wallcomponents.
MicroscopyobservationshowedthattheFOC4myceliatreatedwithfiltratedfermentative
fluidofITBBB5-1waspartiallydegraded(Fig5B),whilethecontrolmyceliatreatedwithonly
LBbrothremainedintact(Fig5C).Thisresultindicatedthatthelyticenzymessecretedby
ITBBB5-1contributedtoitsantifungalmechanism.
InhibitoryeffectofITBBB5-1againstbananaFussariumdiseaseinthe
greenhouse
ThepottedbananaplantsinthegreenhouseweretreatedwiththeITBBB5-1strainand
infectedwithFOC4totestwhetherITBBB5-1couldprotecttheplantsagainsttheFusarium
wilt.TheplantsthatwereinoculatedwithITBBB5-1beforeinfectionwithFOC4hadalower
diseaseseverityindexthanthecontrolplantsCK1thatwereonlytreatedwithLBmedium
beforeinfectionwithFOC4.CK1hadadiseaseseverityindexof59.2,andgrewsignificantly
weakerwithsmalleramountsofleavesthantheplantsprotectedbyITBBB5-1(Table2).The
protectedplantshadsimilarnumberofleavescomparedtothecontrolplantsCK2thatwere
freeofFOC4(Table2).ThecontroleffectofITBBB5-1againstFusariumwiltinthegreen
houseexperimentswas78.7%.
ControleffectofITBBB5-1againstbananaFusariumwiltinthefield
FieldexperimentsindicatedthatITBBB5-1significantlyprotectedbananaplantsfromdevel-
opingFusariumdisease.TheplantsthatwereinoculatedwithITBBB5-1beforeinfectionwith
FOC4hadadiseaseseverityindexofonly18.3after6monthsofinfection,whilethecontrol
plantstreatedwithonlyLBmediumbeforeinfectionwithFOC4developedmoresevereFusar-
iumdisease,withadiseaseseverityindexof61.7.ThecontroleffectofITBBB5-1against
Fusariumwiltdiseaseinthefieldwasapproximately70.0%(Fig6).
PLOSONE|DOI:10.1371/journal.pone.0131974 July2,2015 9/14
ApplicationofanEndophyticBacterialStraininBiocontrol
Fig4.QuantitativeassayoftheinhibitioneffectoftheculturefluidofITBBB5-1onmycelialgrowthof
FOC4.A,tipcultureofFOC4inPDAbrothwithadditionoffiltratedmediumofITBBB5-1(PDA+),orwithonly
additionofLBmediumascontrol(PDA-);B,mycelialweightofFOC4growninPDAmediumwith(PDA+)or
without(PDA-)ITBBB5-1culturefluid.
doi:10.1371/journal.pone.0131974.g004
Discussion
Therubbertreeisrichinendophyticmicroorganisms.Theeconomicclonesoftherubbertree
reproducebyvegetativepropagationknownasbudding,andthebarkofthematuretreeisreg-
ularlytapped.Thisagriculturalprocessprovidesconsistentwoundsandpathwaysformicrobes
toinvadeandspread.Wehaveisolated18endophyticfungalstrainsfromtherubbertree[42],
ofwhichthreestrainsdemonstratedinhibitiontothegrowthofthepathogenicfungusColleto-
trichumgloeosporioidesPenz.Sace,whichcausestherubbertreeanthracnose,andFusarium
oxysporumCubense,whichcausesthebananaFusariumwilt.Anotherendophyticfungus,Tri-
tirachiumsp.ITBB2-1exhibitedsaltresistanceandoptimumgrowthatasaltconcentrationof
Fig5.ChitinaseandglucanaseactivityofITBBB5-1.A,Temporaldependentvariationofchitinaseandβ-
1,3glucanaseactivityintheLBbrothofITBBB5-1;B,MyceliaofFOC4treatedwiththecell-freeculture
mediumofITBBB5-1after2daysofincubation;bluearrowsindicatethedegradedmycelia;C,Myceliaof
FOC4treatedwithonlyLBbroth.Scalebarsrepresent10μm.
doi:10.1371/journal.pone.0131974.g005
PLOSONE|DOI:10.1371/journal.pone.0131974 July2,2015 10/14