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Environmental
Microbiology
Isolation,
identification,
and
biocontrol
of
antagonistic
bacterium
against
Botrytis
cinerea
after
tomato
harvest
Jun-Feng
Shi
a,∗,
Chang-Qing
Sun
baInstituteofAgriculturalProductStorageandFreshKeeping,ShanxiAcademyofAgriculturalScience,Taiyuan,China bInstituteofCropSciences,ShanxiAcademyofAgriculturalScience,Taiyuan,China
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received16May2016 Accepted1March2017 Availableonline3June2017 AssociateEditor:FernandoAndreote
Keywords: Antagonisticbacterium Antifungalactivity Identification Tomato
a
b
s
t
r
a
c
t
Tomatoisoneofthemostimportantvegetablesintheworld.Decayafterharvestisamajor issueinthedevelopmentoftomatoindustry.Currently,themosteffectivemethodfor con-trollingdecayafterharvestisstorageoftomatoatlowtemperaturecombinedwithusage ofchemicalbactericide;however,long-termusageofchemicalbactericidenotonlycauses pathogenresistancebutalsoisharmfulforhumanhealth andenvironment. Biocontrol methodforthemanagementofdiseaseaftertomatoharvesthasgreatpracticalsignificance. Inthisstudy,antagonisticbacteriumB-6-1strainwasisolatedfromthesurfaceoftomato andidentifiedasEnterobactercowaniibasedonmorphologicalcharacteristicsand physiolog-icalandbiochemicalfeaturescombinedwithsequenceanalysisof16SrDNAandropBgene andconstructionofdendrogram.Effectsofdifferentconcentrationsofantagonistic bac-teriumE.cowaniisuspensiononantifungalactivityaftertomatoharvestwereanalyzedby myceliumgrowthratemethod.Resultsrevealedthatantifungalactivitywasalsoenhanced withincreasingconcentrationsofantagonisticbacterium;inhibitoryratesof1×105 colony-formingunits(cfu)/mLantagonisticbacterialsolutiononFusariumverticillioides,Alternaria tenuissima,andBotrytiscinereawere46.31%,67.48%,and75.67%,respectively.Byusinginvivo inoculationmethod,itwasfurtherconfirmedthatantagonisticbacteriumcouldeffectively inhibittheoccurrenceofB.cineraeaftertomatoharvest,biocontroleffectof1×109cfu/mL zymoticfluidreachedupto95.24%,andantagonisticbacteriumE.cowaniihasbiocontrol potentialagainstB.cinereaafterharvestoffruitsandvegetables.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Introduction
Accordingto thedatabase ofFood and Agriculture Organi-zation of the United Nations, tomato yield, an important
∗ Correspondingauthor. E-mail:sjfty@126.com(J.Shi).
economic product, inChinaranks first inthe world. How-ever, tomatodecayafterharvesthasbeenamajorissuein the developmentoftomato industry.Currently, thecontrol of tomato diseases after harvest is mainly through pre-vention and cureusing chemicals. Nevertheless,long-term
http://dx.doi.org/10.1016/j.bjm.2017.03.002
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
use ofchemicides leads tomany drawbacks suchas envi-ronmental pollution, effects on human health, and drug resistance.Withincreasingawarenessonfoodsafety, restric-tionontheuseofchemicidesisalsoincreasing.1 Biological
control methods have been developed in laboratories and commerciallyapplied.2Astudyhasdemonstratedthat
antag-onisticmicrobestocontroldiseasesisanewalternativewith great potential. In the biocontroltechnique for preventing pathogenicdecayaftertheharvestoffruitsandvegetables, themainactionis throughantagonistic interaction among microbes,by changingthe microbiological environmenton fruitsurfacetopromotereproductionofantagonistic bacte-riaandinhibitingthegrowthofpathogenicmicroorganisms, consequentlyreducingdecay.Duetoitsexcellentbiocontrol effect,noharmtohumansoranimals,noenvironmental pol-lutionorresidue,andmaintenanceofgoodqualityagricultural products,this methodisconsidered tobeoneofthe most importantmethodsforreplacingchemicides.3–5 Tilldate, a
numberofantagonisticmicroorganismshavebeenselected from>10typesoffruitssuchasorange,apple,peach, pear, kiwifruit,andfreshjujube,andbiocontrolorganismsisolated are mainly small filamentous fungi,6 bacteria,7,8 yeast,9,10
etc. At present, some companies outside China have suc-cessfully selected antibacterial agents, and some of them havebeendevelopedasproductsforcommercialusesuchas American“Aspire” (Candidaoleophila strain-182), “Biosave-100”, “Biosave-110,” and “Yield Plus” (Cryptococcus albidus), etc.
Researchers have foundthat the mainpathogens caus-ingthe decayafterharvestwere botrytiscausedbyBotrytis cinerea,alternaria caused byAlternaria tenuissima, and fruit decaycausedbyFusariummoniliforme.BecauseB.cinereacan toleratelowtemperature,producelargesporeyield,andhas shortperiodofonsetoffruitdiseases,botrytisisthe most severe disease aftertomato harvest, and hence prevention and cure of botrytis is a key approach for the protection anddevelopmentoftomatoproductsworldwide.11Recently,
there are some reports12–14 regarding biocontrol againstB.
cinereaoftomato.IthasbeenreportedthatCryptococcus lau-rentiieffectivelyreducedB.cinereainpost-harvesttomatoand post-harvestdecaycaused byPythium12;Wang et al. found
thatinoculationof1×108cfu/mLcellsuspensionofseayeast Rhodosporidiumpaludigenum reduced disease rate oftomato by42.1%, when comparedwith control group5 days after inoculationat25◦C.Duanet al.,13 showedthatinoculation
ofsuspensionof1×106cfu/mLPaenibacilluspeoriaeBC-39can
effectively controlB.cinerea inpost-harvesttomato;5days afterinoculationat25◦C,therateofprotectiveeffectagainst decaywas 88.4%and the rateoftherapeuticeffect against decaywas79.9%.Moreover,rateofweightlosswas signifi-cantlylowerthanthecontrolaftertomatoesweresoakedin thepreparedsuspension.14Althoughsomeeffectivebacterial
biocontrolagentshavebeenreported,thenumberofidentified biocontrolbacteriaafterharvestisevidentlyinsufficient com-paredwithotherfieldsofbiocontrolmeasures.Inthisstudy, botrytiswasusedasatargetagentafterharvestoftomatoes, andtheselectedB-6-1strainwithantibacterialactivitywas successfullyisolated.B-6-1strainwasidentifiedaccordingto themorphologicalcharacteristics,physiologicalandbiological features,andbysequenceanalysisof16SrDNAandropBgene
anddendrogramconstructionmethods.Thebiocontroleffect ofantagonistic bacterial strain afterthe harvestof tomato wasexploredtoprovideevidenceforthepreventionof botry-tisintomatoandtodevelopresearchonbiocontrolagentsof tomato.
Materials
and
methods
Materials
Theseedsoftomato(LycopersiconesculentumMill.,hezuo909) were from Changzheng tomato seed testing ground at Shanghai.TheseedswerepickedupfromXiaodiandistrict, TaiyuancityinShanxiProvinceandwereusedforisolation, inoculation, and preservative experiments. Cherry tomato seeds (L. esculentum var. cerasiforme, Hongshengnv) were fromKeFeng SeedIndustryCo.,Ltd(Changchuncity ofJilin Province)andwerepickedupfromXiaodiandistrict,Taiyuan city in Shanxi Province for the selection of antagonistic bacteria.
Antagonistic bacteria were isolated from the surface of tomatoesandpurified.TomatoeswereboughtfromXiaodian district,TaiyuancityinShanxiProvince.Pathogensincluding B.cinerea,A.tenuissima,andF.moniliformewereisolatedfrom diseasedtomatoes,identifiedbyHenle–Kochlawand molec-ularmethods,andpreservedinthelaboratory.
Experimentalmethods
Isolationandselectionofantagonisticbacteriafromthe surfaceoftomatoes
Tomatoeswerekeptmoistatroomtemperaturefor20days. AccordingtothemethodofJanisiewiczandRoitman,15 five
randomly selected tomatoes were placed into a 1000-mL beakerand0.2Mphosphatebufferwasadded.Itwasensured thatthesurfaceofthebufferwashigherthanthetomatoes. Washingsolutionwasdiscardedafterbeingshakenat100rpm for10min,andlaterphosphatebufferwasaddedfollowedby ultrasoniccleaningtwicefor30s.Thesolutionobtainedafter washing wasdilutedto10-,100-,and 1000-fold,and100L fromeachsolutionwassmearedonpotatodextroseagar(PDA) culturemediumandculturedat26◦Cfor2–3days;allsingle colonieswereculturedonPDAplatesusingstreaking inocula-tionmethod.
Singlecolonieson PDAculturemediumwerepickedfor shaking culture at 200rpm and 28◦C for 24h, and made into1×l010cfu/mLsuspension;thesurfacesofcherry
toma-toes were sterilized for2minand washed clean using tap water,andthenairdriedundersterileconditions.Awound (3mm×3mm)wasmadeonthesurfaceofatomatousinga sterilizedinoculationneedle,50-Lantagonisticbacterial sus-pensionwas inoculatedinto the wound2hlater,and then 15L1×106spore/mLpathogenicsporesuspensionwas
inoc-ulated.Sterilewaterwasusedasacontrolandtomatoeswere airdried,placedinplasticboxes,andkeptmoistforculture at26◦C.Rateofincidencewasmeasuredafter6days(tomato haddiseasewhenobviousbacterialgrowthanddecaywere observed), and themeasurements were repeatedthrice for every30tomatoes.
Identificationofantagonisticbacteria
DNAextraction
Sodiumdodecylsulfate-proteinaseKlysismethodwasused forDNA extraction,cetyltrimethylammonium bromidewas usedforprecipitationofcellfragmentandpolysaccharide,and isopropanolprecipitationwasusedforextraction.
16SDNAgeneamplification
Bacterial 16SrDNA wasamplifiedaccording tothe method of Coenye et al.,16 primers were as follows: 27 forward:
5-AGAGTTTGATCC TGG CTCAG-3 and 1541reverse:5 -AAG GAG GTG ATC CAC CC-3. Polymerase chain reaction (PCR)amplificationconditionwasasfollows:pre-denaturation was performed at 94◦C for 5min; denaturation at 94◦C for 40s; annealing at 55◦C for 50s; extension at 72◦C for 1min 15s; the total number of cycles was 35;termination wasdoneat72◦Cfor10min.PCR:0.8LTaq(5U/L);10L 10× PCR buffer (Mg2+ Plus); 8L deoxynucleotide
triphos-phate (dNTP) mixture (2.5mM each); 2.5ng template DNA; 2L primer F1 (10moL/L); 2L primer R1 (10moL/L); ddH2Owassupplementedupto100L.DNAfragmentswere
recoveredandpurified,andsequencemeasurementwas per-formed by Shanghai Invitrogen Biotechnology Co. Ltd. in Beijing.
ropBgeneamplification
ropB genewas amplifiedaccordingtothe methodofBrady et al.,17 forward primer of CM7-f: 5
-AACCAgTTCCgCgTT-ggCCTg-3 and reverse primer of CM7-r: 5 -CCTgAACAAC-ACgCTCggA-3. PCR amplification reaction condition: pre-denaturationwasperformedat95◦Cfor5min;denaturation at95◦Cfor35s;annealingat55◦Cfor1min15s;extensionat 72◦Cfor1min15s;thetotalnumberofcycleswas30; termina-tionwasdoneat72◦Cfor7min.ropBgenesequencereaction volumewas100L:0.8LTaq(5U/L);10L10×PCRbuffer (Mg2+Plus);8LdNTPmixture(2.5mMeach);2.5ngDNA
tem-plate;2LF1primer(10mol/L);2LR1primer(10mol/L); ddH2O was supplemented up to 100L. DNA fragments
were recovered and purified, and then sequence measure-mentwasperformedbyShanghaiInvitrogenBiotechnology Co.Ltd.
Homologyanalysisandconstructionofdendrogram
AftersequencingDNAgenes,homologicalBLASTwas com-paredintheNationalCenterforBiotechnologyInformation database.MEGA4.0neighbor-joiningmethodwasusedforthe constructionofdendrogram,and1000timesofsimilaritywere calculated.Dendrogramnodeshowedthatthevalueof boot-strapwas>50%.18
Analysisofphysiologicalandbiochemicalparameters
Physiologicalandbiochemicalmeasurementswereperformed fortwobacterialstrainsaccordingtothe9theditionofBergey’s ManualofDeterminativeBacteriology.19
BiocontroleffectofantagonisticbacteriaEnterobacter cowaniiB-6-1
Antibacterialactivityofdifferentconcentrationsof antagonisticbacterialsolution
AfterE.cowaniiwaspurifiedusingbacteria-platemethod,a singlecolonywasinoculatedinto100mLlysogenybroth(LB) culturemediumforshakingcultureat200rpmand30◦Cfor 24h;concentrationwasmeasuredusingdilutionplatemethod and then1×105,1×106, 1×107,1×108,and 1×109cfu/mL
bacterialsuspensionswerepreparedusingsterilewater.PDA culturemediumwasaddedandsolidifiedonacultureplateof diameterwithin9cm,and30Lofdifferentconcentrationsof antagonisticbacterialsuspensionwasaddedseparatelyand evenlysmearedontheplate.Afterairdrying,adiameterof 5mmpathogenic fungiwas inoculatedatthe centerofthe plate,andtheantagonisticbacteriawerereplacedwith ster-ile water asa control. Cultured ata constant temperature of28◦C,the diameteroffungal colonywasmeasuredafter 5–7days.Inhibitionratewascalculatedandeverytreatment was repeated thrice, and experiments were also repeated thrice.Inhibitionrate(%)=(dC−dT)/(dC−5)×100,wheredC is the diameter (mm) of fungal colony in the control and dT is the diameter (mm) of fungal colony with different treatments.
ConfrontationcultureofE.cowaniiB-6-1strainand pathogenicfungiaftertomatoharvest
AfterantagonisticbacteriumB-6-1strainwasinoculatedin astraightlineatadistanceof2.25cmfromthecenterona diameterof9cmPDAplateusinganinoculatingloop accord-ingtothemethodofWalkeretal.,20fungalpathogenssuch
as F. verticillioides, A. tenuissima, and B. cinerea were inoc-ulated at the center of the plate at a diameter of 5mm, respectively, andcultured inthe darkat26◦C for5–7 days. Measurement was performed until one side of pathogens grewtowardtheedgeofthecultureplate.Inhibitoryeffectof antagonisticbacteriumonfungalpathogenswascalculated. Experimentswererepeatedthriceandthreerepeatswere per-formed each time. Inhibitionrate (%)=(diameter ofcolony farfromantagonisticbacterium−diameterofcolonycloseto antagonisticbacterium)/diameterofcolonyfarfrom antago-nisticbacterium×100.
BiocontroleffectofB-6-1strainonB.cinerea
Healthytomatoeswerepickedandtheirsurfaceswere ster-ilized using 2%NaClO. A wound(3mm×3mm)was made at the equatorial site, with two woundsper fruit. Antago-nistic bacteriumE.cowanii B-6-1strain wasinoculated into the LB culturemediumforshaking cultureat200rpmand 30◦C for 24h, and different treatment solutions were pre-paredasfollows:A:zymoticfluidof1×107cfu/mL;B:zymotic
fluidof1×108cfu/mL;C:zymoticfluidof1×109cfu/mL;D:
supernatantcollectedafterfilteringbacteriumusing0.22-m filtermembrane;E:bacterialsuspensionof1×107cfu/mLafter
centrifugingthezymoticfluidandwashingtwicewith ster-ile water; F: culturemedium of1×107cfu/mL sterilized at 121◦Cfor20min;Control:sterilewater.Avolumeof40Lof
Table1–Incidencerateandthediameteroflesionafter
inoculationofthewoundsoftomatoeswithantagonistic
bacteriaandyeastagainstB.cinerea.
Strains Incidencerate(%) Diameteroflesions(mm) CK 100.00±0.00a 4.27±0.15a B-1 0.00±0.00f 0.00±0.00f B-6-1 0.00±0.00f 0.00±0.00f B-12 4.67±1.76f 0.90±0.20ef B-13 41.33±7.08cd 2.50±0.29cd B-15 71.04±4.33b 3.43±0.23abc B-20 28.67±3.47de 1.78±0.12de Y-7 75.34±2.91b 3.67±0.24ab Y-10 63.50±2.88bc 2.89±0.43bc Y-18 11.98±3.61ef 0.93±0.18ef Y-19 48.57±3.17cd 2.87±0.07bc Y-12 0.00±0.00f 0.00±0.00f SD=standarddeviation.
Datainthetableareexpressedasmean±SD.Differentlowercase lettersinthesamerowindicatesignificantdifferenceatP<0.05 levelbyDuncan’snewmultiplerangetest(P<0.05).
theabovesolutionwasinoculatedintothewoundof toma-toes,airdried,and2hlater15L1×105spore/mLofB.cinerea
sporesuspensionwasinoculated,and storedmoistat26◦C for6days.Theincidenceofdiseaseswasrecordedand cal-culated,which was repeated thrice forevery 20 tomatoes. Grading criteria forfruit diseases21 were asfollows: Grade
0,nodisease;Grade1,lesionareaaccountsfor<5%offruit area;Grade3,lesion areaaccountsfor6–10% offruitarea; Grade5,lesionareaaccountsfor11–25%offruitarea;Grade7, lesionareaaccountsfor26–50%offruitarea;Grade9,lesion areaaccountsfor>50% offruitarea.Diseaseindex=
(the number of rotten fruits at all levels×the representative value of the grade)/(the total number of fruits×the high-est representative value)×100; control efficiency=(control disease index-treatment of disease index)/control disease index×100.Statisticalanalysis
Statisticalanalysisofexperimentaldatawasanalyzedusing SAS9.0software.AnalysisofvariancewasusedforDuncan multiplevarianceanalysis.AvalueofP<0.05wasconsidered significant.
Results
Selectionofantagonisticbacteria
Aftercomparisonandidentificationofthetomatosurface,the rateofincidenceoflesioninthecontrolgroupwas100%. Com-paredwiththe control, 11 strains(6 bacterial and 5yeast) isolatedhadsignificantantagonisticeffect(P<0.05;Table1). Threestrainshadthebestantagonisticeffectandtheywere B-1, B-6-1,and Y-12.AftertreatmentwithB-6-1strain, the incidenceratewas0%andcancompletelyinhibitB.cinerea (Fig.1).
Identificationofantagonisticbacterium
Colonialmorphology
Morphological features of B-6-1 strain are as follows: The body of B-6-1 strain was short, rod-shaped, with a size of0.6–1.0m×1.2–3.0m. B-6-1strainon thecultureplate appeared round with irregular edges, light yellowish, and semi-transparent,withabulgedsurfaceandwassmoothand appearedsemi-liquid.
Physiologicalandbiochemicalfeatures
B-6-1 strain was negative for Gram staining with anaero-bicgrowthanditcannothydrolyzestarchbutcanhydrolyze nitritereductase.Methylred reactionwasnegative,contact enzymegrowthtestwaspositive,andoxidasereactionwas negative.ResultsareshowninTable2.
Homologyanalysisanddendrogramconstructionof16S rDNAinB-6-1strain
Thewholelengthof16SrDNAamplifiedusing27Fand1541R primerswas1441bp.Homologyanalysiswasperformedafter sequence BLAST,andDNAMENsoftwarewasusedfor mul-tiple sequence alignment.Homologyofsequences ofB-6-1 strainandtypestrainE.cowaniiT (AJ508303),Salmonella sub-terraneaFRC1S,SalmonellaentericaATCC,SalmonellabongoriBr, andE.cloacaeLMGwas99.6%,98.4%,98.1%,97.9%,and97.6%, respectively.
Bacillus licheniformis was used as an outgroup. MEGA4.0 softwarewasusedtoconstructB-6-1and related16SrDNA dendrogrambyneighbor-joiningmethod(Fig.2).B-6-1andE. cowanii(registrationnumberAJ508303)clusteredatthesame branch,andclusteringbootstrapsupportratewas97%;itwas initiallyconsideredthatthisantagonisticstrainwas Enterobac-ter.
HomologyanalysisanddendrogramconstructionofropBof B-6-1strain
ToaccuratelyidentifyB-6-1strain,ropBgenewasfurther ver-ified. Thelength ofthe fragment sequence ofCM7 primer amplificationwas752bp.Homologyanalysiswasperformed after sequence BLAST, and DNAMEN software was used for multiple sequence alignment of ropB gene. Homology with E. cowanii was 98.9%, followed by homology with E. cloaca (CP002886) and E. nimipressuralis at 92.3%;homology withE. cancerogenus(AJ56694) was92.2%,homology withE. asburiae(AJ543727)was91.9%,homologywithE. nimipressur-alis(AJ566948)was91.6%,andhomologywithEscherichiacoli (JN707682)wasthelowestat91.4%.
DendrogramofB-6-1strainandropBgenewasconstructed usingneighbor-joiningmethod(Fig.3).B-6-1strainandtype strainCIP107300(registrynumberAJ566944)ofE.cowanii clus-tered atthesame branch,andclusteringbootstrapsupport ratewas100%.Ithasbeenreportedthathomologyof16SrDNA sequenceofbacterialcoloniesreachedupto>97%,andhence itcanbeconsideredasisogeny.22Therefore,combinedwith
bacterialmorphology,physiologicalandbiochemicalfeatures, 16SrDNAandropBgenesequenceanalysis,B-6-1straincanbe identifiedasE.cowanii.
CK
B-6-1
Fig.1–InhibitoryeffectofantagonisticbacteriaB-6-1strainonB.cinerea.
Table2–PhysiologicalandbiochemicalcharacteristicsofB-6-1.
Characteristics Results Characteristics Results
Catalase + Oxidase −
Methylredreaction − Nitratereduction +
-Galactosidase + Argininedoublehydrolysis −
Lysinedecarboxylase − Ornithinedecarboxylase −
Citricaciduse + H2Sproduction −
Urease − Tryptophandeaminase +
Indoleproduction − VPreaction +
Gelatinase − Inositol +
Melezitose − Glycerin +
Mannitol + Raffinose +
Erythritol − Sorbitol +
d-Arabinose − Starch −
␣-Methyl-d-mannoseglycosides − Glycogen −
␣-Methyl-d-glucoside − Xylitol − N-Acetyl-glucosamine + Gentiobiosyl + Amygdalin + d-Turanose − Arbutin + d-Lyxose + Aesculin + d-Tagatose − Salicin + d-Fucose + Cellobiose + l-Fucose − Maltose + d-Arabitol − Lactose − l-Arabinitol − Melibiose + Gluconate + Sucrose + 2-Keto-gluconate w Trehalose + 5-Keto-gluconate − Inulin − -Methyl-d-xyloside − l-Arabinose + d-Galactose + d-Ribose + d-Glucose + d-Xylose + d-Fructose + l-Xylose − d-Mannose +
Adonalcohol − l-Sorbose +
l-Rhamnose + Dulcitol +
97 65 71 87 99 0.02
E. cowanii CIP 107300T(AJ508303)
B-6-1
E. cloacae LMG 2683T(Z96079)
Salmonella enterica ATCC 700720T(NR074910) Salmonella subterranea FRC1T(AY373829)
Salmonella bongori BR 1859T(AF029227) Escherichia/Shigella dysenteriaeT(X96966)
Escherichia/Shigella flexneriT(X96963) Bacillus licheniformis CICC 10101T(AY859477)
Fig.2–Dendrogrambasedon16SrDNAsequencesofstrainB-6-1constructedusingneighbor-joiningmethod.
Note:Thenumberatthenodemeansthepercentageofoccurrencein1000boot-strappedtrees;thescalebarmeans0.5% sequencedifference;“T”intheupperright-handcornerindicatesthetypeofstrain.
49 E. nickellidurana LMG 23000T(AM076893)
E. asburiae ATCC35953T(AJ543727)
E. cloacae EcWSU1(CP002886)
E. cancerogenus ATCC 33241T(AJ566943)
S. enterica subsp. enterica Dublin strain D25(JQ728847)
Escherichia coli E141(JN707682)
E. nimipressuralis ATCC 9912T (AJ566948)
E. cowanii CIP 107300T (AJ566944) B-6-1 27 41 96 39 100 0.01
Fig.3–DendrogrambasedonropBgenesequencesofstrainB-6-1constructedbyneighbor-joiningmethod.
Note:Thenumberatthenodemeansthepercentageofoccurrencein1000boot-strappedtrees;thescalebarmeans0.5% sequencedifference;“T”intheupperright-handcornerindicatesthetypestrain.
InhibitoryactivityofpathogensaftertomatoharvestbyE. cowaniiB-6-1
EffectofE.cowaniiB-6-1onthegrowthofthreefungal pathogensaftertomatoharvest
AllofdifferentconcentrationsofB-6-1bacterialsuspension inhibitedthegrowth ofF. verticillioides,A.tenuissima,andB. cinerea. Inhibitory effects among different pathogens were different: inhibitory effect on B. cinerea was the strongest, followed by a stronger effect on A. tenuissima, and the weakest effect on F. verticillioides. At a concentration of 1×105cfu/mL,the inhibition rate ofE. cowanii against the
myceliumofB.cinereawas67.48%.Theantagonisticbacterium at1×109cfu/mLcouldcompletelyinhibititsgrowth(Table3). ConfrontationcultureofB-6-1withfungalpathogensafter tomatoharvest
ResultsofconfrontationcultureofB-6-1andfungalpathogens F.verticillioides,A.tenuissima,andB.cinereaareshowninTable4. Growthofpathogenswassignificantlyinhibitedwhenthree
Table3–Inhibitoryeffectofdifferentconcentrationsof
antagonisticbacteriaonthreefungalpathogens
(P<0.05).
Concentration (cfu/mL)
Inhibitionrate(%)
F.verticillioides A.tenuissima B.cinerea
1×105 46.31±1.14b 67.48±1.39b 75.67±1.49b
1×106 48.12±2.13b 68.29±1.24b 76.53±1.63b
1×107 48.72±3.03b 69.38±1.99b 78.00±0.39b
1×108 53.33±1.03b 74.53±2.25ab 80.12±1.90b
1×109 69.75±2.08a 78.32±1.91a 100.00±0.00a
SD=standarddeviation.
Datainthetableareexpressedasmean±SD.Differentlowercase lettersinthesamerowindicatesignificantdifferenceatP<0.05 levelbyDuncan’snewmultiplerangetest(P<0.05).
Table4–Resultsofconfrontationcultureofantagonistic
bacteriaE.cowaniiB-6-1withfungalpathogens.
Typesofpathogens Inhibitionrate(%)
A.tenuissima 34.59±0.52
B.cinerea 35.79±1.13
F.verticillioides 26.57±0.95 SD=standarddeviation.
Datainthetableareexpressedasmean±SD.Differentlowercase lettersinthesamerowindicatesignificantdifferenceatP<0.05 levelbyDuncan’snewmultiplerangetest(P<0.05).
typesoffungal pathogenswereclosetothesideof antago-nisticbacterium,andobviousinhibitionzonewasobserved at the junction between antagonistic bacterial and fungal pathogens.InhibitoryeffectsofB-6-1onB.cinereaandA. tenuis-simawerestrongerbuttheeffectonF.verticillioideswasweaker.
BiocontroleffectsofdifferentconcentrationsofB-6-1 antagonisticbacterialliquidsonB.cinerae
Afterinoculationoftreatmentsolutionofdifferent concen-trations ofE. cowanii, incidence ofdisease was calculated. Withincreasingconcentrationsofculturemedium,the dis-easeindexoftomatobotrytisdiseasewasreduced.Disease indexoftomatobotrytisdisease inthe controlwas93.38%, whilethat of1×109cfu/mL zymotic fluid was only 4.44%;
biocontrolratereachedupto95.24%andtheincidencerate significantlydecreased.Ontheotherhand,biocontroleffects ofbacterialsuspensionandzymoticfluidweresimilar;no sig-nificancewasobservedbetweenthetwotreatments.Filtrate controleffectreached77.40%,andheatkillfluidontreatment hadnoobviouseffectontomatograymold(Table5).
Discussion
Currently,manyantagonisticbacterialstrainsforinhibiting botrytis disease inpost-harvest fruitshave been used.23–27
Acremonium breve has inhibitoryeffect on apple and grape fruit;Bacillussubtilishasinhibitoryeffectonbotrytisdiseaseof strawberryandcherry;Candidaguilliermondii,C.oleophila,and C.sakecan inhibitB.cinereaofpear,nectarine,andtomato fruits; Cryptococcuslaurentii, C. flavus, and C. albidus inhibit botrytisdiseaseofpost-harvestapple,strawberry,tomato,and
pearfruits;MetschnikowiafructicolaandM.pulcherrimainhibit botrytisdiseaseofappleandgrapefruits;Pseudomonascepacia, P.corrugate,andP.syringaehaveinhibitoryeffectsonpearfruits andapplefruits;Rhodotorulaglutinisinhibitsbotrytisdiseaseof strawberry,pear,andapplefruits;Trichosporonpullulans sup-pressesB.cinereaofcherryfruits.Thenumberofantagonistic bacteriaonpost-harvesttomatowaslessthanothertypesof fruits.Inthisstudy,thediscoveryofE.cowanii,whichhasnot beenreportedearlier,providesmorechoiceformicrobiological controlofpathogensinpost-harvesttomato.
Intheecosystemoffruitdiseasesandmicrobiology,when fruitdiseasesaresevere,pathogensoccupyafavorable posi-tion, whereas antagonistic bacteria is at inhibitory status. Therefore,fruitsdidnotdevelopdiseaseorhavemilddisease wheneffectiveantagonisticbacteriacontrolthedevelopment offruitdiseases,andselectionofantagonisticbacteriafrom these fruits is easier toproduce better inhibitory effectof antagonisticbacteria.28Inthisexperiment,fruitsusedfor
iso-lation ofantagonistic bacteriawere randomlyselected and keptmoistatroomtemperatureforaperiodoftime.Fruits without diseases were selectedforthe isolationof antago-nisticbacteriatomakeantagonisticbacteriabetteradaptto theecologicalenvironmentofpathogens.Manyresearchers haveselectedantagonisticbacteriabycombininginvitroand in vivomethods. In vitro selection is moreconvenient and rapidforexaminingtheinhibitoryeffectofstrains.However, instrainsselectedforinhibitoryeffect,invitroconditionmight notproducethesameinhibitoryeffectasinvivoexperimental condition.29Inthisstudy,invivoselectionmethodwasdirectly
used toobtain good bacterialstrain toavoidinconsistency betweeninvitroandinvivoexperiments.
Enterobacteria widely exist in nature, and it has been reportedthatsomestrainsinEnterobacterhaveverygood bio-controleffects.E.cloacaehasverystrongantagonisticeffects onPythiummyriotyluminvitro.30Yangetal.,31isolatedE.
cloa-caefromriceseedlingroot,sprayingitszymoticfluidonthe leavesofriceatbootingstagecansignificantlyimprove antag-onisticeffectonbacterialblight,andbiocontroleffectreached up to38%.In1995, Hintonand Bacon32 foundthat E.
cloa-caefromcornnotonlyeffectivelycolonizedcornseedlingbut alsodistributed onthemiddlecolumnand thecortexcells ofmaizeroot.Thebacteriumhaveamarkedlyantagonistic actiononF.moniliformeandseveralothertypesofcornfungal pathogens.Yang33foundE.cowaniiduringtheisolationof19
Table5–InhibitoryeffectsofvarioustreatmentsolutionofantagonisticbacteriaE.cowaniionB.cinereaofpost-harvest
tomatoes.
Treatment Diseaseindex Biocontroleffect(%)
Fermentationbroth(1×107cfu/mL) 71.14±3.15b 23.82±1.81d
Fermentationbroth(1×108cfu/mL) 48.89±1.76c 47.64±1.02c
Fermentationbroth(1×109cfu/mL) 4.44±0.68e 95.24±2.07a
Bacterialsuspension(1×107cfu/mL) 49.42±3.59c 47.08±0.40d
Filtrationfluid 21.11±1.79d 77.40±1.03b
Heatkillfluid 94.23±2.86a –
Control 93.38±1.93a –
SD=standarddeviation.
Datainthetableareexpressedasmean±SD.DifferentlowercaselettersinthesamerowindicatesignificantdifferenceatP<0.05levelby Duncan’snewmultiplerangetest(P<0.05).
endophyticbacterialstrainsfromdifferenttissuesandorgans ofcole,andstudieshaveshownthatthisstrainhascertain inhibitoryeffectsonfungalpathogensofcole.Fu34isolated
endophyticbacterialstrainE. cowaniifm50fromMiscanthus floridulusandCymbopogoncaesius,whichhavehigher biolog-icalactivitiesofnitrogenaseasanenzymeandperoxidase, andpromotesgrowthofrice.Bradyetal.,35isolatedE.cowanii
strainfromeucalyptuswithbacterialblightandbudblight, Heinferred thatE. cowaniimightexist ineucalyptusas an endophyticbacteriaandhasantagonisticeffect.Ontheother hand,itisreportedthatglycoproteininEnterobactercloacaecan inhibitnormalgrowthofpulmonarycells.36
Inthisstudy,filtrationliquidofE.cowaniiB-6-1caninhibit theoccurrence ofB.cinerae,which issimilar tothe results of a previous study on E. cloacae. Mukhopadhyay et al.,29
foundthatinNBorPDBculturemedium,filtrationliquidof E.cloacaehasinhibitoryeffectsonRhizoctoniasolani,Pythium myriotyum,Gaeumannomycesgraminis,andHeterobasidion anno-sum,demonstratingthatantifungalmetabolitesexistinthe liquid.E.cowaniiB-6-1heatkillingbacterialsolutiondidnot haveinhibitoryeffects,demonstratingthatantibacterial sub-stancesdonottoleratehigh temperature.Thesimilarity of antibacterialsubstanceE.cowaniiB-6-1andantibacterial sub-stanceproducedbyE.cloacaewasthatE.cloacaefiltrationliquid didnot have antibacterial activity after heatingfor10min at60◦C or boilingfor5min.On theother hand, inhibitory effectsoftheantagonisticbacteriumcorrelatedwiththe con-centrationofculturemedium:thehighertheconcentration, thebettertheinhibitoryeffect,whichisconsistentwiththe resultsofpreviousresearch.7
Insummary, B-6-1strain wasisolated from the surface oftomato,andidentifiedasE.cowaniiaccordingtobacterial morphology,culturefeatures,physiologicalandbiochemical characteristics,and16SrDNAandropBgenesequence analy-sis.E.cowaniiB-6-1notonlyhasstronginvitroinhibitoryeffect onF.verticillioides,Atenuissima,andB.cinereaaftertomato har-vestbut alsoeffectivelyinhibits theoccurrenceofbotrytis. Theviabilityatlowtemperatureandbiocontrolefficiencyin combinationwithotherpreservationmeasuresandbiocontrol mechanismsofE.cowaniiaredirectionsforfutureresearch.
Conflicts
of
interest
The authors declare that there is no conflict of interest regardingthepublicationofthispaper.
Acknowledgment
TheauthorsaregratefultoPostDocFundofShanxiAcademyof AgriculturalScience(No.BSH-2015JJ-003)andShanxiProvince ScienceandTechnologyResearchProject(No.20140311025-2).
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