1、IekntifkatiOnandcharacterizationofPhoinafungico1.acausing1.eafspotdiseaseofPhe1.1.adendnHiarnurenseRuprinChinaA1.ntractPhf1.1.otictuImfXamtrcnseRraperennia1.herbinCnjxsicacfami1.y,chixIKCdforjaundiceandgastrointestina1.discaws.In2015.u1.eafspodiseaseWaSQbSCrVCdonp1.antsofP.etnuremeinaproductionfie1.
2、dofShenyang1.iaoningProvic.ChinaTbcdisschadaspeck1.edappea11i!xreiniIia1.1).Evemuu1.1.y.infec1.irusua1.1.yCaUMrdye1.1.owigofIiieka%,eundprvnu1.urudet1.iatin.Ihecau5a1.agentofinf&1.iunnh(swaxidenh11edasP/iojtmJint(ic(1.abasedmiHinrphoIogicM刖dCUkUra1.chaw1.cristies,pathogenicityCcMsarx1.phy1.ogenetica
3、11A1.ysix.Towrknow1.edge,thisisthefirstreportofakafspodiseaseonP.iwwvrsecausedbyP.fwigko1.ainChina.!nt11iuc1.inPhd1.micndrrffx(jjnu/rnxcRuprixaperennia1.medicina1.herbIU1.ivCtoChina,whereithasbeengrownfoemorethan2.0(Xyears.Commercia1.p1.unutknarcmuin1.diribcdin1.iaoningPiovinccofChina.TbCdriedrccbar
4、kofP.arnurensehasbeenusedforcrcatmeniofjaundiceaidgxUinteUina1.dsNiiw、inIradi1.iiiiu1.Chinesemedicine.Wi1.iifurthernreavhingini1.activeingredientaniextractionhasrecent1.ybeenQUdiCdfbriMa111.i(umnr.antimicrobia1.antfrveda!IheMedicina1.IktbGardeninShenyasigAgricu1.tura1.Univcrvi1.y(SYUXShenyanj.1.iann
5、ing,China.Sincethen,itsincidcrxx:inthesubsequentyear*Signifkunt1.yincrcnwdIn2015,thediseaseimposedUdirect(hrcuttoWMainabIcpduion.Asthediseasea1.csccdtocauseb1.ighton1.eaves(issuesandhcfungu.PHtna.WaSiso1.atedfromIbeSC1.egions.thsuspectedha(WaSthecawa1.PaIbOgen“thediseaM!.PhonwisVrryimportantpytopath
6、ngcn(Avcxkiimpcta1.201Q)andthetypespeciesRherhnrnmWsctubtp(1857)wasOi1.aMishedbySaocurdo1.ISSO),MuchresearcheshnsbeenCaIr1.edouttaconsistsofa1.argenumbercdophytcs.SaPfDbCSaidp1.antPaIhogCits.butIheCkUNiiiCakanofspecie%inPhtMm1.atx1.a1.1.iedgeneraissti1.1.cMi1.roveria1.Asubdivision1.theawxua1.gcnsPho
7、mathatkcurrencywide1.yapp1.ieddividesthegcwsintoninew1.innx,inc1.udingthesectionsPjMinm、Hcenswra.Macnnra.Paraphww.PGrwtdktea、PhyU(KiiicU)IdestPiJWia.PIetKnhrmtsandSe1.enMne1.1.tiBocicnu1997).Confusingcharactersarereportedamongsevera1.PhomtiMXIiUIarx1.IthtcdgtrteraurhasAscwhyUhPhtNnopxix、Phy1.kziim.P
8、kigphtNna.PyrtfntHrhatrRandSta)(NUMpt)n.KeuiI1.NJanextensiveMudyHe1.wtxnPkttmttandPhy1.kziictan1.ybasedMp1.ain,invitroChaKICWrV卜UftberIIMire.i1.IabeenuggeaedIha1.thePhnmaC1.aviHca1.innyxtcmWOU1.dbeimpenvedbyadding11Nphy1.ogeneticdata,a11iUc1.inca1.in:morenatura1.grkca1.ikn1.1.ca1.in.Ihep1.hgewasdivi
9、dedIIMDPhy1.kztida.Butt1.mg1.COCnbmXionofmoho1.ogica1.andmn1.ec1.irmethodsinchistudy,furtherMudiesshowedIha1.Ihcpathogenbe1.ongstoPhna.Materia1.sandmethodsObservationOfsymptom、andco1.1.ectiunofnmp1.eFO1.IoWinWinGc1.dob%cnuknnfdisc:Nedevckpity.InnerSbcnyang.1.iaoningprovince.China.MftS1.Ch4C1.rruMKQ5
10、IhO1.atiOntheCaUMdfed1.ttU“I咏rmctft20MBocrema,6.K.deGntecJ.Nwtoot,M.6.,&Kw,M.C.20049banqA*“*bEMraftMGCJtUrw.M1.InOOE:CABIPUdUNR,(Vpica1.1.yHymp1.oinatic1.eaveweresurtces1.eriIizedbydappedinto75%CU1.anD1.1.WCrCaseptica1.1.ycutfrommarginsofthe1.esionsadp1.acedonpoutodextroseagar(PDA)(20Ogpotatoes.20gd
11、extrose.20agnr/1.IJiMiI1.cdwa1.erTbcwdhhcwereincubatedMHena1.u1111.ightantiobserveddai1.y.After5day、ofincuhatin11.thegrowingfungiWCTCivb1.cdantipurifiedonPDAusingthesing1.esporetechnique,transferredtoPDAs1.antsandkeptuc4Xforfurtherstudies.Thecu1.tureswereinvestigatedforco1.onydiameter.PyCnidiUinandC
12、emidia1.InDrPiM1.k塔y.(Cu1.tura1.Charactcr1.Micsandmorpho1.ogica1.ThePhomaiso1.atewasfirM1.yexaminedmorpho1.ogica1.1.y.Theiso1.atewascu1.turedonPDAMrtnca1.garOAj(15gofO1.iUnCa1.20gagan,1.Idisi1.1.edWdICfJundnw1.tagar(MEA;40gma1.1.extractOxid.15gOX(Iidagar.111.apwa1.er)incubated31.25Cucoxitinuousi1.Iu
13、inuia1.ion.atdexaminedco1.onydiiimctcOntheOAax!(bePnAafter7d.Twoweek、1.ater,thetnrphn1.cgyofthePyCnid山andconidiaweirMitdicdfromtheOAandIhCMEAcu1.tures.Imagesoftheco1.onicsoftheiso1.atewerecapturedUstngacamcra.Theexperimentrepea1.edtwicewithRmrrep1.icates.DNCX1.ruc1.HMi.PC?RandNeqUenCinXDNAexIrac1.iu
14、n.(heMudiedu1.1.uxe%wetvgro11PD11edtumatroomIempcra1.urvuarMaryshakerfor7dayx.ECnCmiCDNAswereexIrxr1.aifrommyce1.iabygnTIAMGENI、AsecureP1.antKitDcconiingtothemanufacturersinanitions.1.argCSubuni(28S(1.SU)andInccnui1.transcribedspacer(ITS)wcvaa1.yzed.Pnmcrsused(oamp1.ifytheu,onudcarregionsWCrC1.R0R.,
15、1.R7andiTS41,S5.rcspccivc1.y.ThePCRsWCrCperformedinaS100oEThemu1.Cyc1.er(Biu-Kud1.aboraioriessJncBerke1.ey,CA.USA)inaIOudvo1.umeof251.Amp1.ificaticmsofPCRroc1.ionmixturecontainedTu-mi12.51.ddh951.t1.1.ofc。Chprimerand1.1.DNA(CmPIaIeCyc1.ingconditionsforu1.1.WCreaninitiu1.dcnu(uranonMcof5minM9,C.fo1.I
16、ovmxIby35cyc1.esof30sa(95C.theXmnCaIi忸stqsvrcperforndas45%a1.4Xtbr1.SU.50、a1.48furITS.211nnat72V.fo!1.mvedby72V.TiiePCRPnHhIeUWemHiw1.yzctihydectmphorcsisOn1%n革IrOSCge1.containing().1ndvhua1.i/cdunderUV1.ight.SequencingWgperformedbySangcnBiotechno1.ogy1.td(Shanghai.China).PinIogenctkana1.ysesIheMXue
17、ncigresu1.t%WeItrsubjectedIosimihi1.yau1.yixagainMUeGenBankNRbtabiiscsUMngB1.AST(httpMMWnChimIEnih.窣qv.iB1jST/),Tbchhcs1.cri11j:matcheswithannMa(cdgenusandxicswereSdCUCdaspotentia1.mabcsforeachsequenceandthephy1.ogciKiicana1.ysesweredonewithaconbida1.ignmentconsistingofIxxhtheITSad1.SUregions.A1.ign
18、mentviewedusingC1.USTA1.Xversion1.81andtheNeighbour-Joining(NJ)diskuceana1.ysiswaxCHXIUCWdusingMEGAversion4.0.PntbocnicitytestThefunga1.isdaccwasgrownonPDAfor7daysan25.SporuiationwasiduxdbyIransfcrringa5-mn)mycdia1.NUgontooatmea1.agar(OA)8day%.Ccmtdia1.、口*n3unxWtMtJrearudby11)dingIhep1.a1.esWiIh5Ei1
19、1.iIitCniofMeri1.cdiM11.c1.wa1.eranddjuMthenumbernfconidhbyhiicmacytomc1.er.Conidia1.suspensionsncencrutionWCrCsetuptoIx1.伊m1.1.Pf1.thogcnkiiyofiso1.atedfingusWagdcermifiedbyinviuo1.eafnokionnhod.P.Wmet1.eavesIriCdforinocu1.ationrequirethecsixU!yofnatun(ywhichCo1.kv1.edIiUmIhCMedxra1.HobCiatiknofSh
20、enyangAgrku1.1.ura1.U11i*vrsi1.y.1.iaoningProvince,China,rinsingwithsteri1.eJiMiIkdwitteranddryintheair.Conidia1.suspensionssprayedon(he1.eftpanofIxxhsurfaceswiththeconidiasuspensionunti1.run-offandPCffafaCdinui1.ioMc.Inocu1.ated1.eaverPhCCdondihcwithwsponge.Thecontro1.ICMnvrep80%R)andincubatedinhci
21、1.1.uminationincubator(25C)undera12hPheHoPCriOdResu1.tsDiseasesymptomsandhna1.ko1.atThediseaseinii1.yappearedonp1.ainsin1.ateJuneandrainfa1.1.occurredThetypica1.sym(onsbeganwithsma1.1.,ova1.-circu1.ar,yc1.1.ow-budedwithafaded-green1.u1.u.Seini-BunedPycnKiWwcrvobservedinhcIi1.rgeareaofIcmuiisandthece
22、tferueapfxrurvdPahInI1.ion.Fina1.1.y.Iheinfcc1.cd1.eavesdefo1.iatedear1.ier.(u1.turu1.charactcrinticMandnrpH)1.oiw1.Pycmdiaa1.waysw1.itary.PfIH1.UCede.pcnb3(eIdubobhte.g1.ibmu5,mwuring(50)65130(150)(65952XX220)mwithasing1.e,conspicuous,non*papi1.Iaicostio1.e.Pysidia1.hw/pa1.ebrown.PyUdOParCrWhyrna1.
23、ou、.CompoSCdOfiSodianKIrkce1.1.s.-51.ayer*.8.514.5(16)mthick,outer12IayCns1.tgbd),ptgnxed.ConidiaVariiib1.CinQIajKandsize.、u怩kboHIdn,a1.CrUbIUM：1.hn3kd.siiidoIasc1.a1.e.measuntig(5)5.57.58.5)34.5(5)mawith02minuteMtUteSVinitia1.1.yhya1.ine.Cociidh1.exudatesandCh1.amydosporeswereabsent.Co1.onicsonOA55
24、68mmdiamafter71.marginregu1.arena1.mycv1.umwhi1.e,oocsc10x1.1.y.Inuneedmyce1.iumgtven%o1.ivaceoustoD1.naceousnearI1.urCOkMIyctn:.Abuiii1.aiHb1.ackpycnkiiaarcscatteredOVCthemedium;reverseconc1.ourxs.CNoniuonMHA6575mmdiumafter7fp1.u4(gca1.andcu1.tura1.c1.arueten1.ic%wevimihrkIhespeciesRJungictt1.tiba
25、edonI1.iedecrip1.ingivenbyBDerenia(Bcfv11uCta1.2004)总界基，MEA.PDA,OAMo1.ecu1.arnu1.yxixTti11caunOfthecausa1.funu.ITSregionandI5Uregionwereamp1.ifiedbyPCRandsequenceddirect1.y.Rqxvcnutiv?sequencesofITSand1.SUhavebeendepositedinGcnBank(AccessionNo.GU2379OOandEU75427.cspecii*c1.y.Scquccofthest11iiwasa1.
26、ignedwithothersfromb1.astsu1.(s(Tab1.eI).Ticombinedihasetconsistedof160()CharaeteiNnr1.udingIhCa1.ignmentgap(S:andKU:1051).T1.iePhy1.oetWUCtreeofthecombinedgeneMxe11ceshas41。WCdthatstrainfromA.appnicninChinafannedaUniquCarx1.j1.suppo11cdCIadcQhMgroupedwiththeP.fungkotreferencestfainshtewacompeci1.tc
27、with!fwicoia.THbICIGBkm.SiminnaHdomiMphhaM1.oca1.ityITS1.SUCBS6J92PTAWVJ加侬也GU2379EUX4127CBSIIIW5PitNk1.fid“nthruKRO6I71BU7M*MCBS120486Pttyik坞NdadInniitMaN55836OKF2O63I4CBS1233，wfrdriafiaMFJ558356KFW6309CS2W7OPk、曲MIkTaTdWmuiuPJS3S3SSKF2O63IOCS37.TOPhNkMkra早n11MWFJ5835OKFK1.ft291CBS35?MPva*w力AIQaGU237
28、X37.ES124515PkiVKiiM(TM1tfGU237768CBS991.95PAvwwtwaniimuFM2744CBS654.”PAtawj,MwmrruFJ427O43CRS522/6卅叫由MmUr11fwnrtt4/KF251166GQ3875H2CS172.70尸(7W血,11wrnrw11fvwrfrrkVCisGU237H76CBS615.75rwrwnw1I-ICBS502.1.1.A*uwr，加em2ICttS377.92PaEArrfwuwIICBS557,X4auuuZhdanM*CBS-h100ITS51.RM991.CBSQ3.92/Aa3gc而ICBS506
29、91rt(WgniCateiiICBSW1.95PAomuomrtheMf100CBS654.77PAvwwtMini,inru100ICBS522.66A/wj血Hnnrnrw11r,wrarCBS1.?2.70PauMnachtyuntkwifoa100I1.CBS568.91A,6m11fimeti1001.CBS170.70A*4irwimv-CBS111635MMwiErubra100dayv.herea%IbecoiUru1.pkn1.5remainedaymp0ixcuMonPhcIMrwInwamwvnxcRrh;t%becomewide1.yCU1.IiV“tedinrec
30、entyear、avaChinesetraditiona1.herbinx11hcustChina.ToOUrknow1.edge,thisstudyisthefirstreportof1.eafsp(xonP.ItnxutettifcausedbyRfuco1.ainChina.Wcdcscnbct1.inJC1.ai1.IhCtypica1.SymP(OnMoninfectedp1.anbandIIXWphD1.Ugidc1.aracIerizatiunofI1.teiw1.a(e.andWecombinedInHPhDh1.gidc1.ttracte11Mic.sequencesofrD
31、NAITSand1.SU.andxthogcnici1.y(cssto(fcmosuatc(1.uIhccau%a1aen1.ofIiie1.eafpo!eixP.Tbeproperidcn1.ica1.iunaru1.CIU汽c(CriHianmightbehe1.pfu1.fnrgmwcrtoaccurate1.ydim即candxccsu11.ymanagethedisease.Accordingcopreviousrcfcfccs.RfungicdaumxIcobeca1.1.edPhoinaCbrysaiubcmie1.a(Aveane1.a1.20(19).()Wreaxx1.(o
32、)ba%edwever.refecIdaPhyIIoMiciaspecie%(VanderAa&VaneV2(MJ2).,hetfv.aCWnameIXProPmcdherefrthepresentspecie%.Kixs&Nakawnc(IWS)a1.readyfound(hatsevera1.fustgrowingAne1.imycesspecieswerePhykXECtiCaHydistinctfromthetypespecies,whichisc1.nracC(iMx1.byarathers1.owrou,hra1.e,adsuggestedthu(A.quen-inusbe1.on
33、gedoPMNna.ThkEndingWaSMIPPoNCdbyrcbu1.(sDbuHMdinIaicrMudics(Su1.1.ivan&White2000.Szcncivdnyifta1.2(105).Inthistudy.COnidiNandaeria1.hyphaewere1.arge1.yPfndUCCdinMEAandweremo1.gica)1.ysimi1.artothereportedprevious1.yforP.fungico1.a(QbankFungiDauihasc),MukigcncPhy1.ogciKiieana1.ystsinthetden(iHcationo
34、fPhumiisquiteIinportanitoavoidmtM(knti11caons.1.SUsequence*cangiveagoodindicationonIheIaini1.yIevdofaniso1.ate(MUdenbe以ta1.2(K9).andITSregionshowsthehighot1.eve1.CfingNPeCiiiCvariation(vr%kampda1.29).ThestrainwascIuMcredwithotherknown()11ininthephy1.ogeneticana1.ysesaxishowedhigh1.yo1.yhy1.cticWIIhm
35、P.fingia)ktmneighbour-1.iningtreebasedon1.SUandITS.Incnc1.u%in,Iheresu1.tsofidcn(iIica1.ionandCha1.uCWnZaUGns1.wedU1.atP.tungica%Ihene1.io1.ogii1.agentoftheIcafxpo1.diseaseonP.mun,nsrinChinz.IntheSUrVCyofponfie1.dsinSYAU.in2013.theincidenceofdhc*scdp1.antsofPh.fuia)kwasUik1.er30%.kis1.ur1.yinceascdin2014.withGCnigCof82%.Withtheinctvasingofcu1.(ivu1.icmyearax!fin1.ectionsurcesaccuniu1.Med.Ku11ictMnhasgreatpo1.en1.ia1.1.yIobecomeanimpor1ampathogenofP.11nuvnr.Convucn1.1.y.furtherregrvhRrequiredtnRiIif1.infomaiionre1.atingtopotentia1.contro1.measuresIIndCUhUGdrnc(iccsforIhcreductionofthedisease.
