Morphological and molecular identification of Powdery Mildew of Clematis manshurica Caused by Erysiphe aquilegiae in China.docx

Morpho1.ogica1.andmo1.ecu1.aridentificationofPowderyMi1.dewofC1.ematismanshu11caCausedbyErysipheCiqui1.egiaeinChinaC1.ematismanshuricaPupr.isaperennia1.herbintheRaiiiincu1.aceaefami1.y,whichhasbeenusedasava1.uab1.etraditiona1.Chinesemedicine.Between2012and2013.apowderj,mi1.dewcausingsignificantdamagetop1.antsofC.manshuricaoccurredintheMedicina1.HerbGardeninShcnyangAgricu1.tura1.University.1.iaoningprovince.China.Thefungimain1.yinfectedthe1.eaves,sta1.ksandpetio1.es.Symptomsinitia1.1.yappearedascircu1.artoirregu1.ar,thin,whiteco1.oniesonyoung1.eavesandstems,especia1.1.yontheadaxia1.surface.Eventua1.1.yinfectionsusua1.1.ycausedye1.1.owingofthe1.eavesandma1.formationandprematuredefo1.iation.Numerousc1.eistotheciaappearedonthebothsurfacesof1.eaves,sta1.ksandPetiO1.eS.'hefungusfromsymptomatictissueswasidentifiedasErysipheaquiIegiaebasedonmorpho1.ogica1.characteristicsandrDNAITSsequenceana1.ysis.Toourknow1.edge,thisisthefirstforma1.reportofpowderymi1.dewonC.manshuricacausedbyE.aquHegiaeinChina.C1.ematismanshuricaPupr.isaperennia1.herbintheRaiumcu1.aceaefami1.y,andiswide1.ydistributedinthenortheastofChina.Thedriedrootsandrhizomesofthep1.antsnamed''Wei1.ingxi(intheChinesePharmacopoeia,ispopu1.ar1.yUSedasanantiindaininatory,antitumor,ana1.gesicagentintraditiona1.ChinesemedicineforthousandsofyearsinAsia(14).InAugustof2012.apowdcrj,mi1.dewonC.manshuricawasfirstobservedinMedicina1.HerbGardenofShenyangAgriCU1.1.UraIUniversityin1.iaoningprovince.China.Incidenceofpowderymi1.dewofC.manshuricawasinvestigated.Theincidenceofthediseasewasratedintermsofpercentageofinfected1.eavesamong10()1.eavesobscn,cdwithfiverep1.icates.The1.eve1.ofincidencewassignificant1.ydifferentinthepasttwoyears.In2012.theincidencewas1.ower,justunder5%.However,1.hcdiseaseincidencesharp1.yincreased,with42%to100%(averageof78%)inthesamefie1.dsin2013.Theribosoma1.DNAinterna1.transcribedspacer(ITS)regionhasbeenusefu1.fordiscriminatingfungiatthespecies1.eve1.,andhasbeenusedextensive1.yfor(heidentificationandphy1.ogeneticana1.ysisofpowderymi1.dews(I,2.7.10.II.12).Inthispaper,theaimwastoidentifythecausa1.agentofPoWderymi1.dewobservedonC1.ematismanshuricainChinaandseiupabaseforcienificcontro1.ofthedisease.Materia1.sandmethodsFunga1.materia1.sandmicroscopy.Thesamp1.esofC.manshuricainfectedwithpowderymi1.dewwereobtainedfromtheMedicina1.HerbGardenofShenyangAgricu1.iura1.University,1.iauningprovince.China.Themorpho1.ogyofthepathogenwasexaminedwithfreshmateria1.susing1.ightmicroscopyaspreviousdescribedmethods(10).DNAextractionandamp1.ificationofr1.)NAITSandSequencing.Funga1.DNAsamp1.eswerepreparedbyco1.1.ecting(8)youngpowderymi1.dewmycc1.iaandc1.eistothecia(6)usingsteri1.ebrashes.Thenuc1.earrDNAregionspanningtheITS1.5.8SandITS2rRNAgenewasamp1.ifiedbyPO1.y1.neraSechainreaction(PCR).Primers11-SI11'S4wereusedfortheamp1.ification,whichWaSPCrft)EMXIinatota1.reactionvo1.umeof501.inc1.udingthefo1.1.owingreagents:5.01.10×PCRbuffer.2.01.DNAtemp1.ate.41.MgCh(IOmM).1.01.ofeachprimer(11SIand1TS4).0.61.TaqDNApo1.ymerase(5.()U1.).1.01.dNTP(dR<ICTP,dGTP,dTTRIOinMeach),35.41.ddH2O.A1.1.abovereagentswerepurchasedfromSangonBiotechno1.ogy1.td(Shanghai.China).ThePCRcyc1.ingconditionswere94for3minfo1.1.owedby4cyc1.esof94forImin.55for1min.72for1.5min.andafina1.extensionstepof72for8nininaS1(XX),mthe11na1.cyc1.er(Bi-Ra<1.1.aboraiorie*,Inc,USA).DNASCquCnCCSofthreerep1.icateswereobtainedusingtheTianGcnDNAsecureP1.antkit.SequencingoftheITSregionswasdonebySangonBiotechno1.ogy1.td(Shanghai.China).ITSsequenceswerea1.ignedwithprevious1.ypub1.ishedsequencesfromc1.ose1.yre1.atedpowderymi1.dewspeciesmaintainedinGcnBank.Phy1.ogcncticana1.ysiswasperformedusingMEGEversion4.0withthecighbor-joiningmethods.Resu1.tsSymptoms.Thefungimain1.yinfectedthe1.eaves(Fig.Ia),thesta1.ksandpetio1.es(Fig.1.b)cou1.da1.sobeinfectedinsevereinfections.Symptomsinitia1.1.yappearedascircu1.artoirregu1.ar,tbin,whiteco1.onies,whichsubsequent1.ydeve1.opedintodense,whitehyphaeandconidiaontheuppersurfacesoftheyoung1.eavesandyoungstems,especia1.1.yontheadaxia1.surface,andpowdcr),co1.onicsmergedtocoverentire1.eaves.Atthesametime,newco1.onicsspreadsuccessive1.ytotheother1.eaves.Inthecaseofsevereinfections,co1.oniescou1.da1.sobeobservedontheundersurfaceof1.eaves,petio1.esandyoungstems.Andinfected1.eavesbecamegray1.esionsbeneathfunga1.co1.onics.Asthediseaseprogressed,infectionsusua1.1.ycausedye1.1.owingofthe1.eavesandpetio1.esandprematuredefo1.iation.Onoccasion,severe1.yma1.formaiiononyoungICaVcSandpetio1.es(Fig.Ic.d)wereObSerVcd.Fina1.1.y,numerousc1.eistothecia(Fig.Ic.f:Fig.2a)appearedonthebothsurfacesof1.eaves,sta1.ksandpetio1.es.Fig. 1 Symptomsofpowderymi1.dewonC1.ematismanshurica(a:symptomsinfie1.ds:b:symptomsonsta1.ksandpetio1.es;c,d:ma1.formationofyoung1.eaves：e,f:C1.eisiot1.ieciaOn1.eaves)Morpho1.ogica1.Characterization.Onthe1.iving1.eaves,sta1.ksandpetio1.es,superficia1.hyphaewere(hin-wa1.1.cd.hya1.ine,septate,straighttof1.exuous,3,5*8,8m(average5.6m)wide.Conidiophorcsproduce