Identification and characterization of phoma causing leaf spot disease of Phellodendron amurense Rupr in China.docx

Identificationandcharacterizationofphonacausing1.eafspotdiseaseofPhe1.1.odeiidronamurenseRuprinChinaIinrodUCIiOnPhe1.1.odendmnamurenseRuprisaperennia1.medicina1.herbnativetoChina,whereithasbeengrownformorethan2.(XX)years.Commercia1.p1.antationsarcmain1.ydistributedin1.iaoningProvincesofChina.ThedriedtreebarkofPhe1.1.odendfvnamurenseRrhasbeenusedfortreatmentofjaundiceandgastrointestina1.diseasesinIradi1.iona1.C1.iinesemedicine.Withfurtherresearchinginiuactiveingredientandextractionhasrecent1.ybeenstudied(oritsan(iturnor.an1.imicrobia1.andotherbio1.ogica1.activities.InSUmmerof2015.adiseasecausingSpO(Son1.eaveswasobservedinfie1.dsinShcnyang.1.iaoningProvinces.China,duringinvestigationofp1.antdiseasesonPhe1.1.fX1.endmnamnmseRr.TheseqPOISgenera1.1.ycoa1.escedtocauseb1.ightonabove-g>und(issuesandwaspresumab1.yassociatedwiththefungusPhoma.hcPhomagenuswasestab1.ishedbyStcyacrt(1949)forspecieswhichconsistsofa1.argenumberofendophytes.SaProbeSandp1.antPa1.hOgegsomespeciescausingseriousdiseasesoneconomica1.1.yUnPoC1.an1.p1.an1.SPedCSwor1.dwide(Aveskanipe<a1.2008).Res1.tsofanextensivestudyofPhomataxonomybasedoninvitrocharactersoverthepast40yweresummarisedintherecent1.ypub1.ishedPhomaIdentificationAtt卬酢w；%>Anra±m4ov%q分差f侪法研无的结场京站在JB走出版的男打逑别M1.ftQ)：SAMm我另行了分第九'Y六我.出3HeteroiPor3,fH1.SXvPc>or>dUM.H.S<kropbnc<U-P11odojj.MMrottporattU财化外R，1分I1.仃。SUIK?.t小"3Hecco1%7BOefCm便济"t!P知及太毛茎R无代表匕由JaI1.几个IHJnMEmorpM多年来械健接别生立Mtttftfb这较没，iH*3让卅ftttU4)HS.GW美Ie的分"C'AH*41的7%皎3。取4位*口.1'ft.Artewiei8.MbcrospMaopib.1.»tA.Pkropomj.H克和S1.o¼pc<4I!U.在吗的况FW仆跪收己认为小时mE为各用2间箕掌他?也片拴第2宠X精的f1.0eUEth1.M"20041./t*.dfit.3上分乃e统均JaN。旧地DHAtua&iK.MMu：11nr¾¼ifCrtn«»»丁川丽;TOdHMoSjftttWxT如“分f£统字的WMK川0!MVI®PmJgEu即3CUifttManua1.(BOgfCmaC1.a1.2<X>4)(ota1.of223specificandinfra-specificIaKaofPtxwiawerec1.assifiedinniPhoniasec1.ions,name1.yPhona.Heter>sp<)11i.Paraphoma.Peyrone1.1.aeii.Phy1.1.os1.ictoides.Sc1.crophomc1.1.a,Podomus.MacrosporaandPi1.osajBccrcma1997:BOerCm3D/a1.2004).furthermore,thesePorphre1.ationsdescribedinthegeneraDidymc1.1.a,Mycosphacrc1.1.a,1.cp<osph>criaandP1.cospora(Boercma1997),indicatingthatPhUmaananrphsrepresentap1.yphy1.e<icgroup.Severa1.additiona1.IdeOmOrPhShaveovertheyearsbeen1.inkedtoPhoma.butingenera1.,these1.ackanyconc1.usiveevidenceI(AYC眯ampc1.a1.200).TheC1.assiGca1.ionofspeciesinPhomaanda1.1.iedgeneraissti1.1.controversia1.Confusingcharactersarcreportedamongsevera1.Phomasectionsandre1.atedgenerasuchasASCoChyaASIero1.neIEMicrasphaeropsis.P1.iomopsis,Phy1.1.osiicia,P1.curuphoma.Pyremxrhae1.aandS1.agonospora.InSomCinstances,thePhomasectionshavebeenconsideredambiguousbecauseofsharedcharactersanngthevarioussections,andarejectionofthehypothesisofconvergentdeve1.opment(Punitha1.ingam2004).Purthcnnorc.ithasbeensuggested(hat(hePhoniac1.assificaiionsystemwou1.dbeimprovedbyaddingDNAhy1.ogene(icdata,andde1.ineatingmorenatura1.groups(G)ndonaC1.a1.1997;Torreseta1.2OO5>ocircumscribePhomaanda1.1.iedgenerabymeansofamo1.ecu1.arphy1.ogeneticstudy,generac1.assifiedin(hesuborderPhia1.opycnidiineae(Phia1.iddes.Entcrob1.astomycchdac)(Sutton1980)shou1.dbereeva1.uated.1*hcPhia1.opycnidiincacischaracterisedbysimp1.e,thin-wa1.1.edpycnidia,ascptatcconidiaandampu1.1.ifbmpia1.idcsorseparateconidiophores.However,Suttan(198()app1.iedawiderconcept,inc1.udingthick-wa1.1.edpycnidia,septate,pigmentedconidiaConiothyriun.SiagonosporaandPhyHosticut修I网RH俯26”的代岳”由我3J:Vft.HK必在内.ttnnwHwn«H.s<tSUtOftOip«J8京andthusinc1.uded,aswe1.1.asspeciesofothermisinterpretedgenerasuchasInthepresentstudy,thesta1.usofthegenusPhomaandre1.atedgenera,aswe1.1.asIc1.eomorphassociationsarcstudiedbymeansofDNAphy1.ogeneticana1.yses.Sequencedataof(heI8S(SSU)andthe28S(1.SU)nrDNregionsof15representativestrainsofthetypespeciesofthePhomasections(Bocrcmacta1.2004)andthreereferencestrainsofPhomawerecomparedwiththoseof93strains1.,hcdiseasehasbeenfoundtheseyears,anditcausedupto60%1.ossinhanrcstofA.pa1.acstina.imposingadirectthreattoitssustainaNcproduction.ThispaperwasaimedatCharacccrizationofthepathogenandsettingupabaseforSCiCntifiCcontro1.ofIhCdisease.Materia1.sandmethodsObsenationofsymptomsandco1.1.ectionofsamp1.esFo1.1.owingin-fie1.dObSCrVationofdiseasedeve1.opment,fivetosevendiseasedp1.antswererandom1.yse1.ectedfromfie1.dsintheShenyangAgricu1.tura1.University,InnerShenyang.1.iaoningprovince.China.Iso1.ationofthecausa1.agentItaIU川>emr1r"4r4/cIarc*arv*KtHtM1.|uui：(3"E«川XC4)GK,dwGfu*tifzA.NxdMctzM.E.&1.,pici1.1.ysymptomatic1.eavesweresurface-steri1.izedbydippedinto75%ethano1.for30s.rinsingthreetimeswithsteri1.edisti1.1.edwa1.er,andthensoakinginI%sodiumhypoch1.oriteso1.utionfor5minandrinsedinsteri1.edisti1.1.edwater.Sma1.1.piecesoftissues(ca5×5min)wereaseptiad1.ycutfromnuinsofIhe1.esionsandp1.acedonPoIaIUdextroseagar(PDA)(200gpotatoes.20gdextrose.20gagar/1Idisti1.1.edwater).Thesedisheswereincubateda(25.After7daysofincubation,IbCgrowingfungiwereiso1.atedandpurifiedonPDAusing(hesing1.eSpor匕technique,transferredtoEDAs1.antsandkeptat4forfurtherstudies.hccu1.tureswereinvestigatedforco1.onydianeertpigmentation,pycnidium.ConidkI1.morpho1.ogy.Cu1.tura1.characteristicsandmrph<>1.gica(TheP!u)naiso1.ateswerefirst1.yexaminedmorpho1.ogica1.1.y.Theiso1.ateswereCu1.IUredonOA(20goatf1.akes,boi1.edin0.51IaPwater,fi1.teredthroughcheesec1.oth,andmadeuptoIIwithtapwater)andma1.tagar(MA:40gma1.textractOxoid1.39(Hampshire.UK).15gOxoidagar,11tapwater)incubatedat25'Cindark,andexamined7d1.ater.Twoweeks1.aterco1.onydescriptionswerenoted,and(hemorpho1.ogyofIhcpycnidia,conidiawerestudiedfromtheOAandtheMAcu1.tures.Thesodiumhydroxide(NaOH)spottestwasperformedonMAbytheadditionofadropofINNaOHon(heco1.onymargin,andachangeinco1.ourWaSnoted.PathogenkitytestThefunga1.iso1.ateweregrownonPDAfar7daysat25cC.Sporu1.ationwasinducedbytransferringa5mmmyce1.ia1.p1.ugon100atmea1.agar(OA)(15gofoatmea1.,20gagar/1Idisti1.1.edwatcr)1.hescdisheswereincubatedundernear-u1.travio1.etradiationWiIha12-ho(operiodat22for8days.Conidia1.suspensionswerePreParedbyf1.oodingthep1.atesWiIhsevera1.ni1.1.1.i1.ersofsteri1.edisti1.1.edwater.Useahaemacy1.omctcrtoadjustthenumbero1.'conidiawithsteri1.edisti1.1.edwater.Conidia1.suspensionsconcentrationweresetupto5×IO,mJ,%5×104n1.1.5×105m1.、5×IOm1.Pa1.hogcnicityofiso1.atedfunguswasde1.e11ninedbyinvitro1.eafinocu1.ationnc1.hod.Theinfectionratesofdifferentpathogenconcentrationonvitro1.eavesandvariationofdiseasesp<swithinxru1.atk>nIimewereobserved.Phc1.kxicndron1.eavesusedforinocu1.ationrequiretheconsistencyofmaturitywhichconef)nShenyangAgricu1.tura1.University,rinsingWi1.hsteri1.edisti1.1.edwateranddryinIhcair.Conidia1.suspensionssprayedon1.hcse1.eaves,steri1.izedwe(absorbentco<tonsurroundedpetio1.es,atp1.aneswithwetsp(ngc,thep1.antswerecoveredwithPoIye1.hy1.enebagstoC1.taIemoistconditions(>70%RHJandincubatedinthei1.1.uminationincubator(25'C)undera12hphotopcriod.Eachexperimentwasperformedintrip1.icate.Eachrq1.icatcwasmadeusingIO1.eavesandthecontro1.1.eaveswevsprayedwithsteri1.edisti1.1.edwater.Tbwtoeightdaysafterincuba1.i>neva1.uationsfordiseaseseverityweremade.1.eaveswereratedfordiascseveritybasedonthepercentageofsurfaceareainfected.Adiseaseseverityindex(DSI)wasca1.cu1.atedaccordingto(hefo1.1.owingfomn1.a:DSI=100ratingsofeachp1.ant>,'(Maxima1.grade*numberofp1.antsrated).Ana1.ysisofDS1.dataamiprove(hepathogenshowingtypica1.symptomsofthediseasehavePcithogenici1.y.DNAextractionMo1.ecu1.archaracterizationResu1.tsDiseasesymptomsandunga1.iso1.atesPaihogcnicity1.estIdentificationoftheCaIHS1.a&entDiscussion