黄柏叶斑病病原菌鉴定.docx

黄柏叶斑病病原菌鉴定Identificationofpathogenicfungus(补充病菌科名)causing1.eafspotonphe1.1.odendron黄柏叶斑病病原菌鉴定AbstractPhe1.1.odendronisoneofthetraditiona1.ChineseIntroductionMateria1.sandmethodsIso1.ationofthecausa1.agentFo1.1.owingin-fie1.dobservationofdiseasedeve1.opment,fivetosevendiseasedp1.antswererandom1.yse1.ectedfromfie1.dsintheShenyangAgricu1.tura1.University.InnerShenyang,China.Typica1.1.ysymptomaticsteins,1.eavesandpetio1.esweresurface-steri1.izedbysoakingin1%sodiumhypoch1.oriteso1.utionfor5minandthenrinsingthreetimeswithsteri1.edisti1.1.edwater.Sma1.1.piecesof(issues(ca5×5mm)wereaseptica1.1.ycutfrommarginsofthe1.esionsandp1.acedonpotatodextroseagar(PDA)(200gpotatoes,20gdextrose,20gagar/11disti1.1.edwater).Thesedisheswereincubatedat25.After5daysofincubation,theco1.oniesweretransferredtofreshPDAdishesandincubatedindarkforundernear-u1.travio1.etradiationwithaI2-hphotoperio<iat25topurifytheiso1.ates.Thecu1.tureswereinvestigatedforco1.onydiameter,pigmentation,pycnidia,conidia!morpho1.ogyanddiffusib1.eantibioticmetabo1.ite'E'(扩散性抗生素代谢物)(Boeremaeta1.2004),whichwastestedbyp1.acingadropofconcentratedNaOHonedgeoftheco1.oniesonOAtoobservetheco1.orvariation.形态学鉴定：菌落直径、颜色、分生胞子器，分生池子形态。(培养过程中是暗处理还是光线接近紫外照射，为什么，有什么不同)PathogenicitytestThefunga1.iso1.ateweregrownonPDAfor7daysat25.Sporu1.ationwasinducedbytransferringa5-mmmyce1.ia1.p1.ugontooatmea1.agar(OA)(15gofoatmea1.,20gagar/11disti1.1.edwater).Thesedisheswereincubatedundernear-u1.travio1.etradiationwitha12-hphotoperiodat22for8days.Conidia1.suspensionswerepreparedbyf1.oodingthep1.ateswithsevera1.mi1.1.i1.itersofsteri1.edisti1.1.edwater.UseaIiaemacytometertoadjust(henumberofconidiawithsteri1.edisti1.1.edwater.Conidia1.suspensionsconcentrationweresetupto5x10X11"、5×IO4m,%5×105m,5×106m1.Pathogenicityofiso1.atedfunguswasdeterminedbyinvitro1.eafinocu1.ationmetho<i.Theinfectionratesofdifferentpathogenconcentrationonvitro1.eavesandvariationofdiseasespotswithinocu1.ationtimewereobserved.Phe1.1.odendron1.eavesusedforinocu1.ationrequire(heconsistencyofmaturitywhichcomefromShenyangAgricu1.tura1.University,rinsingwithsteri1.edisti1.1.edwateranddryintheair.Conidia1.suspensionssprayedonhcsc1.eaves,steri1.izedwetabsorbentcottonsurroundedpetio1.es,atp1.antswithwetsponge,thep1.antswerecoveredwithpo1.yethy1.enebagstocreatemoistconditions(>70%RH)andincubatedinthei1.1.uminationincubator(25C)undera12hphotoperiod.Eachexperimentwasperformedintrip1.icate.EaChrep1.icatewasmadeusing101.eavesandthecontro1.1.eavesweresprayedwithsteri1.edisti1.1.edwater.是否需要黑暗培养3d,培养第4天光照与黑暗时间各I2h,待叶片显著发病后调查病斑数及病情指数Towtoeightdaysafterincubationeva1.uationsfordiseaseseverityweremade,1.eaveswereratedfordiseaseseveritybasedonthepercentageofsurfaceareainfected.Adiseaseseverityindex(DS1.)wasca1.cu1.atedaccordingtothefo1.1.owingformu1.a:DSI=100×(ratingsofeachp1.ant)(Maxina1.grade×numberofp1.antsrated).Ana1.ysisofDSIdataandprovethepathogenshowingtypica1.symptomsofthediseasehavepathogenicity.Tab1.e1Sourceandabi1.ityofthefunga1.iso1.atesinc1.udedinthisstudytocause1.eafspotonhe1.1.odendronIso1.ateGenusSource(issueDiseaseSCVCriIyindCX2days4<hys6days8(bysDNAsequencingGenomicDNAsofiso1.ateswereextractedfrommyce1.iawhatwereobtainedbygrowingcu1.turesonPDAfor7dinPetridishes,andDNAwasextractedbyusingDneasyP1.antMiniKit（试剂盒）byQIGEN.PCR扩增序列：ITS,5.8S,1.SU,Resu1.tsDiseasesymptomsandfunga1.iso1.ates此处应有对病症的描述，出间病害情况，病斑照片全叶及局部斑点照片、菌落照片（培养基正反面）、抱子照片、泡子器照片Tab1.e1Iso1.ateSourcetissueCharacceni根据形态学初步确定genusPa1.hogcnici1.y1.estTab1.e1AbiIityofthefunga1.iso1.atesinc1.udedinthisstudytocause1.eafspotonhc1.1.odendronIso1.ateGenusDiseaseseverityindex2days4days6daysSdays此处应有对数据进行分析整理淘汰，有处理照片与致病照片对照。DNAsequencingPCR扩增图片，扩增后序列对比，系统发育树