人参灰霉病病原学及其致病机制研究.docx

硕士考佐卷攵人参灰霉病病原学及其致病机制研究Disserta1.ionforMaster,sDegreeEtio1.ogyandPathogenicMechanismsofGinsengGrayMo1.dCausedbyBotrytiscinerea独创性声明本人声明本论文是我个人在导师指导下完成的研究工作及取得的成果。尽我所知，除了文中特别标注和致谢提到之处外，本论文不包含其他人的研究成果和已经发表的文字表述，也不包含本人或他人为获得沈阳农业大学或其它教育机构的学位、证书等而使用过的材料.本论文时与我一同工作的同志为本研究所做的贡献均已做说明并表达r诚挚的谢意。研究生签名:时间：年月日导师签名:时间：年月日关于论文知识产权和使用授权的说明本人完全了解沈阳农业大学有关保留、使用本校毕业生学位论文的规定，即本论文知识产权单位为沈阳农业大学：沈阳农业大学有权保留本论文的纸质文本和电磁介质文本，有权允许他人杳阅和借阅本论文各种文本，有权采用影印、缩印或扫描等方式豆制、保存、汇编本论文，有权以不同方式在媒体上发表、传播本学位论文的内容。（保密的学位论文在解密后遵守此物议）研究生签名:时间：年月日导新签名:时间:年月日基金项目本论文得到了以下项目的资助：1 .农业部行业科技专项(201303111)：人参产业化技术研究与示范。2 .沈阳农业大学研究生创新项目(2015)目录摘要IABSTRACT3第一章人参灰寄病及灰葡萄胞致病机制研究进展61人参灰霉病的发生危害61.1 人参分布61.2 人参灰客病的发生危害72灰葡萄地研究进展72.1 灰毒捕的发生危害72.2 灰粉萄胞生物学特性72.3 灰前荀他的致病机制82.3.1 灰葡萄池的侵入过程82.3.2 灰菊尊抱诱导植物细胞程序性死亡92.3.3 灰葡萄抱毒素的致病作用92.3.4 灰韵萄抱草酸的致病作用92.3.5 灰葡萄胞活性氧的致病作用102.3.6 灰前萄泡细胞壁降解施的致病作用102.3.7 灰葡蓟抱果胶酣的致病作用I1.2.3.8 灰韵荀抱纤维素酶的致病作用12239灰酊萄他的信号转导123问题与展望13第二章人参灰寄病菌生物学及其致病性比较研究14I材料与方法141.1.材料141.2 人参灰律菌比较生物学研究141.3 灰霉菌致病性比较161.4 人参灰期病病原学鉴定161.4.1 人参灰卷菌形态学鉴定161.4.2 菌丝的培养与模板DNA的制备16.4.3PCR扩增及测序分析162结果与分析172.1 症状172.2 病原前鉴定172.2.1 形态学鉴定172.2.2 分子鉴定182.3 人参灰霉病菌生物学特性比较201.1.1 养基对病菌商丝生长的影响201.1.2 温度对病菌菌统生长的影响201.1.3 碳源对病菌菌丝生长的影响201.1.4 PH对病菌丝生长的影响22目杀1.1.5 觎源对病菌菌丝生长的影响231.1.6 光照对灰病菌的影响232.4不同灰葡萄胞菌株的致病力比较252.4.1 不同灰葡荀胞菌株对人参叶片的致病力比较252.4.2 灰葡萄也不同菌株对草莓果实的致病力272.4.3 灰葡萄抱不同菌株对番茄果实的致病力273结论与讨论29第三章人参灰霉菌ISSR标记体系的建立及遗传多样性分析311材料与方法311.1 材料311.1.1 供试菌株311.2 方法321.2.1 菌丝的培养与模板DNA的制备321.2.2 人参灰寄菌ISSR-PCR反应条件优化321.2.3 各引物退火湿度优化及引物筛选331.2.4 电泳谱带的记录及数据统计分析332结果与分析332.1 ISSR优化体系的建立与扩增效果332.2 ISSR引物的筛选与PCR扩增结果342.3 聚类分析结果与遗传相似性分析373结论与讨论39笫四章人参灰霉病施病组织细胞壁降解酶动态研究40I材料与方法401.1 供试材料401.2 活体外细胞壁降解醐的提取4113活体内细胞壁降解前的提取411.4 细胞壁降解的的纯化415细胞壁降解酣活性的测定方法411.5.1 多一半乳篇醛酸酶(PG)与聚甲基半乳憾酸酸曲PMG)活力测定411.5.2 按甲基纤维索的(Cx)与M葡荀精计两活力测定431.6人参灰霉菌细胞壁降解酹条件优化442结果与分析442.1 细胞壁降解拗对人参叶片的作用442.2 接种人参灰霉病的后叶片中细胞壁降解酣活性的变化452.3 活体外细胞壁降解酹产生.条件优化472.3.1 培养时间对细胞壁降解的产生的影响472.3.2 震荡条件对细胞壁降解的产生的影响472.3.3 温度对细胞壁降解解产生的影响482.3.4 PH对细胞壁降解的产生的膨响493结论与讨论50第五章细胞壁降解酶与人参灰霉病菌致病力相关性研究51I材料与方法511.1 材料511.2 灰霉菌致病性比较511.3 活体外细胞壁降解的的提取521.4 活体内细胞壁降解酶的提取521.5 细胞壁降解酣活性的测定方法522结果与分析532.1 灰霉菌致病性比较532.2 活体外灰葡萄抱细胞壁降解酶活性与致病性关系542.2.1 灰葡萄他PG活性542.2.2 PG与致病力的关系55223灰葡萄施PMG活性552.2.4 PMG活性与致病力的关系562.2.5 灰葡萄抱p的药糖甘隧活性56226B-前萄糖伴施活性与致病力的关系572.2.7 灰前萄mCx活性572.2.8 Cx活性与致病力的关系582.3 活体内灰葡荀胞细胞壁降解的活性与致病性关系582.3.1 PG与致病力的关系582.3.2 PMG与致病力的关系592.3.3 葡萄犍件施与致病力的关系592.3.4 Cx与致病力的关系602.4 灰葡萄胞生长量与致病力的关系602.4.1 灰葡萄胞菌丝生长602.4.2 灰葡萄抱菌丝干重613结论与讨论62参考文献63致谢69攻读硕士学位期间发表论文71ContentsAbstract(inChinese)1ABSTRACT3Chap1.er1.ReviewonGinsengGrayMo1.dandPathogenicMechanismofHotrytiscinerea61 OccurrenceanddenageOfGinsengGrayMo1.d61.1 IntroductionofginsengGrayMo1.d61.2 OccurrenceanddcmagcofGinsengGrayMo1.d72 ReviewonB.cinerea72.1 OccurrenceanddcmagcofB.cinerea72.2 Bio1.ogica1.characteristicsofH.cinerea72.3 PathogenicIneChaniSmofB.cinerea82.3.1 Penetrationofthehostsurface82.3.2 P1.antce1.1.deathinducedbyB.cinerea92.3.3 Toxins92.3.4 Oxa1.icAcid92.3.5 ReaciiveoxygenspeciesIO2.3.6 Cd1.wa1.1.degradingenzymeIO2.3.7 Pectinase112.3.8 Ce1.1.u1.ase122.3.9 Signa1.Transduction123 Prob1.emandprospect13Chapter 2 ComparisonoftheBio1.ogica1.CharacteristicsandPathogenicityofBotrytiscinereaiso1.atesfromGinseng141 Materia1.sandTnCthOdS141.1 Materia1.s141.2 Comparisonofthebio1.ogica1.Characieris(ics141.3 Comparationofpathogenicity161.4 Ginsengmou1.ddiseaseetio1.ogyidentification161.4.1 Ginsenggraymo1.dmorpho1.ogyidentification161.4.2 Thecu1.tivationofhyphaeandthepreparationoftemp1.ateDNA161.4.3 PCRanp1.it1.caionandsequencingana1.ysis162 Resu1.ts172.1 Symptom172.2 Identificationofpathogen172.2.1 Morpho1.ogica1.identification172.2.2 Mo1.ecu1.aridentification182.3 ComparisonoftheBio1.ogica1.Characteristics202.3.1 EffectofCu1.tureMediumonMycc1.ia1.GrowthbyB.cinerea202.3.2 EffectoiTemperaiureonMyce1.ia1.Growthbyfi.cinerea2()2.3.3 EffectofCarbonSourceonMycc1.ia1.GrowthbyB.cinerea202.3.4 EffectofpHonMyce1.ia1.GrowthbyB.cinerea222.3.5 EffectOfNitrogenSourceonMyce1.ia1.GrowthbyB.cinerea232.3.6 Effectof1.ightonMycc1.ia1.GrowthbyB.cinerea232.4 ComparationofPathogenicity252.4.1 ComparationOfpathogenicityofdifferentiso1.atesto1.eavesofginseng252.4.2 Comparationofpathogenicityofdifferentiso1.atestoCruitsofstrawberry272.4.3 Comparationofpathogenicityofdifferentiso1.atestofruitsoftomato273 Conc1.usionandDiscussion29Chapter 3 Estab1.ishmentofISSRReactionSystemofBotrytiscinereaandItsAna1.ysisofGeneticDiversity311 Materia1.sandmethods311.1 Materia1.s311.1.1 TestdIso1.ates311.2 Reagent321.2.1 Myce1.ia1.cu1.tureandpreparationoftemp1.ateDNA321.2.2 ISSRreactionsystemandreactionparameters321.2.3 Eachprimerannea1.ingtemperatureoptimizationandprimersscreening331.2.4 E1.ecirophoresisbandrecordsanddatastatistica1.ana1.ysis332 Resu1.ts332.1 ISSRoptimizethesystemestab1.ishmentandamp1.ificationeffect332.2 ISSRprimersscreeningandPCRan1.i11catedresu1.ts342.3 C1.usterandgeneticsimi1.arityana1.ysis373 Conc1.usionandDiscussion39Chapter 4 ConditionsandActivityAna1.ysisofCeBWa1.1.DegradingEnzymesProducedfromBotrytiscinereaofGrayMokiofGinseng401 Materia1.sandInCth(KIS401.1 Materia1.s401.2 ExtractionofCWDEsinVitro411.3 ExtractionofCWDESinvivo411.4 E1.ectrophoresisbandrecordsanddatastatistica1.ana1.ysis411.5 DeterminationmethodsofCWDEs411.5.1 DeterminationmethodsofPGandPMG411.5.2 DetenninationmethodsofCxand-g1.ucosidase431.6 ConditionsofCWDEsPrOdUCCdfromB.cinerea442 Resu1.ts442.1 Ce1.1.wa1.1.degradationenzymesonthero1.eOfginseng1.eaves442.2 ActivechangesofCWDEsof1.eavesonginsengafterinocu1.atingwithB.cinerea.452.3 ConditionsofCWDEsProducedfromB.cinerea472.3.1 Effectsofcu1.tureTimeonCWDEsFormation472.3.2 EffectsofOsci1.1.ationConditiononCWDEsFormation472.3.3 EffectsOfTeinperatureonCWDEsFonnation482.3.4 EHcctsofpHonCWDEsFormation493 Conc1.usionandDiscussion50Chapter 5 Ce1.1.Wa1.1.-degradingEnzymesofBotrytiscinereainRe1.ationtoViru1.enceinGinseng511 Materia1.sandMei1.ux1.s511.1 Materia1.s511.2 ComparisonofPathogenicityofB.cinerea511.3 ExtractionofCWDEsinVitro521.4 ExtractionofCWDEsinVivo521.5 DeterminationMethodsofCWDEs522 Resu1.ts532.1 ComparisonofPathogcnicityofB.cinerea532.2 Corre1.ationRe1.ationshipBetweenViru1.enceandCWDEsinViiro542.2.1 EnzymeActivityPGofB.cinerea542.2.2 Corre1.ationRe1.ationshipbetweenViru1.enceandPGinvitro552.2.3 EnzymeActivityPMGofB.cinerea552.2.4 Conc1.ationRe1.ationshipbetweenVccandPMGinvitro562.2.5 EnzymeActivity-g1.ucosidaseofB.cinerea562.2.6 Corre1.ationRe1.ationshipbetweenVccand-g1.ucosidascinViiro572.2.7 EnzymeActivityCxofB.cinerea572.2.8 Corre1.ationRe1.ationshipbetweenViru1.enceandCxinViiro582.3 Corre1.ationRe1.ationshipbetweenVccandCWDEsinVivo582.3.1 Corre1.ationRe1.ationshipbetweenViru1.enceandPGinVivo582.3.2 Corre1.ationRe1.ationshipbetweenViru1.enceandPMGinVivo592.3.3 Corre1.ationRe1.ationshipbchvccnVccand-g1.ucosidaseinVivo592.3.4 Corre1.ationRe1.ationshipbetweenViru1.enceandCxinVivo602.4 Corre1.ationRe1.ationshipbetweenVccandMassGrowthofB.cinerea602.4.1 Corre1.ationRe1.ationshipbetweenVccandMycc1.ia1.Growth602.4.2 Corre1.ationRe1.ationshipbetweenViru1.enceandDryWeight613 Conc1.usionandDiscussion62Reference63Ackonw1.edgements69Paperpub1.ishedduringmasterphase71摘要人参(P""ugi"segC.A.May)为五加科多年生草本药用植物，作为传统的名贵中草药，具有极高的药用和经济价值。近年来，国家对森林资源保护的重视不断加强，严禁砍伐森林种植人参，因此非林地农田栽参成为了目前我国人参产业的重点发展方向。由于人参栽培环境的变化.近年来农田裁参中灰霉病新流行成灾，严重阻碍了人参产业的健康发展，迄今国内外尚未见对人参灰猿病的系统研究报道0因此，本文依托农业部公益性行业科研专项(201303111)一人参病虫害灾变规律及安全防控技术集成研究，针对人参灰霉病菌的生物学比较、遗传多样性、致病机理进行了系统研究，主要研究结果如下：I.以16个东北地区灰寄病(B。WiSdnemaPe2菌株为研窕对象，比较了生物学特性及致病力的差异。结果表明，不同培养条件对不同菌株菌丝生长均有显著影响：供试菌株在PDA和PSA培养基上生长较好，PJARKPJA1.3,PY1.1.kPYC1.1.和PJ1.1.1.最适培养基为PS,FSY1.1、PJA1.1、PJA1.2、PB1.1.KIQA1.1.、PH1.1.I和1.SYF1.最适培养基为PDA“供试菌侏在525"C下均能生长，地适温度为20C,个别菌株FSY1.I、PJ1.1.2、PRH1.KPH1.1.1.和1.SYF2最适生长温度为25C,在pH311的能围内均能生长，最适生长PH为5,PYC1.I和1.SYF2菌丝最适生.长PH为6,PJARkPJA1.2、PJA1.3和PY1.1.1.最适生长PH为4。最适菌丝生长的碳源为麦芽做和葡萄做。最适菌丝生长的缸源为醉母膏、牛肉商和蛋白豚。所有供试灰霉菌株接种后均能引起人参、草寿和番茄发病，但来自相同寄主菌株间致病力存在差异，菌株致病力差异与菌株地域来源无明显相关性。2 .利用ISSR-PCR分子标记技术，刻人参灰意菌进行了遗传多样性分析。建立了标记体系：以UBC810为引物,在251.反应体系中，1.0UTaqDNA聚合旅,0.2mmo1.1.dNTPs,2.5mmo1.1.1MgCh,0.4mo11J引物，30ng模板DNA。对23个不同来源的灰葡萄胞菌株进行了ISSR遗传多样性分析，选用的13个引物共扩增出)个DNA片段，其中多态性位点为154个，占总扩增片段的81.1%.,经UPGMA聚类分析，供试的23株灰菊面地前的遗传相似系数在O53I.OO,在0.814水平上，可分为6个遗传谱系。不同来源的灰葡荀胞菌种内均表现出明显的遗传差异：遗传群组的划分与菌株的地理来源和寄主存在定的相关性：灰葡萄抱菌遗传宗谱与致病力间存在复杂的关系.两者之间不存在简单的对应关系.3 .对人参灰痂病罹病组织细胞壁降解酣动态及活体外细胞壁降解陶产生条件进行了系统研究，结果表明：经细胞壁降解的处理后的人参叶片形成典型的水溃状病斑。多聚半乳陋酸酸酶(PG)和果胶甲基半乳糖酹酸胸(PMG)活性在接种后第2天活性最高:而比葡苗糖甘酣和樱甲基纤维素陶(CX)在接种后第4天活性最高。其中PG活性最摘要高，其次为PMG,B-葡萄糖什曲和CX活性较低。未检测到多里半乳糖羟酸反式消除椀(PGTE)和果胶甲基反式消除醉(PMTE)的活性PG,PMG、CX和户葡萄储昔薛4种细胞壁降解酶，均有特定的最适培养条件，静置培养均利于各种细胞壁降解陶的产生：PG和PMG在培养12d时活性达到高峰，在培养9d时前笥葩昔版和CX活性最高：PG和PMG活性在15C时活性最高,20C为CX和伊葡萄糖年曲最适产的温度.细胞壁降解的的活性在PH为5时最高。4 .对人参灰霉病菌不同菌株的致病力与细胞壁降解辞活性相关性分析。结果表明，人参灰森病菌PG，PMG,Cx和p箭茴树许的4种细胞壁降解的是灰葡萄地的主要致病因子。在不同的培养条件下，强致病力菌侏生长速度较快.强致病力菌株在培养液中和罹病组织中产生的PG,PMG,Cx和0-葡面糖昔酶的活性,均高于弱致病力菌株.在果胶培养液中，强致病力菌株的PG和PMG活性分别为较弱致病力菌株的2.2和1.7倍：于人参叶片上，强致病力菌株所产生的PG和PMG活性最高分别可达2.1Umg和3.2Umg.弱致病力菌株PG和PMG活性最低可低至0.3Ung和1.4Umg,在纤维崇培养液中，强致病力菌株的Cx和快葡萄糖苻制的平均活性分别为弱致病力菌株的1.8和2.0倍；在人参叶片上，均较强致病力菌株所产生的Cx和小仙商糖甘的平均活性分别为较弱致病力菌株的5.5和2.1倍。对PG、PMG、CX和P-葡萄糖存髓活性与不同菌株在寄主上所致病斑平均扩展速率进行相关性分析，4种施的活性与病斑扩展速率均具有一定的相关性，并呈线性递增的关系，其中PG与致病力的相关性最强。证明了细胞壁降解的在灰葡萄饱菌致病中石重要的作用，其中PG起主导作用。关键词：人参灰霉病，比较生物学，遗传多样性，细胞壁降解陋，1.ry1.iscine1.TAbstractGinseng(PuxginsengC.A.May)isaperennia1.herbmedicina1.p1.antoftheAra1.iaCeaePanax.Asatraditiona1.medicine,ithasaveryhighmedicina1.andeconomicva1.ue.Thegovernmentpaidattentiontotheforestresourcesprotectionserious1.yandprohibiteddeforestationforp1.antginsengstrict1.yinrecentyears,themode1.ofnon-forestp1.antedhasbeenthedeve1.opmentofdirectionofginsengindustryinChina.Duetothechangesofthegrowthenvironmentofginseng.Ihegraymo1.ddiseaseincidenceandtheseverityoffarm1.andriseradica1.1.y,andrestrictedthehea1.thydeve1.opmentoftheginsengindustrysevere1.y.Supportedbythenationa1.Agro-scientificResearchinthePISSRIicInterestTheStudyofInfestationRegu1.arityandSecurityPrecautionaryTechno1.ogyoftheDiseaseandPcstonGinsengandbasedontheorigina1.foundationof1.aboratory,threeaspectshavebeenstudied:comparisonofthebio1.ogica1.characteristics,ana1.ysisofgeneticdiversity,pathogenicmechanism.Theresu1.tswereasfo1.1.ows:1. Differencesofbio1.ogica1.characteristicsandpathogenicityof16Botrytiscinereaiso1.atesfromnorthcastChinatoPanaxginsengwerecompared.Theresu1.tsshowedthattheeffectsofdifferentconditionsonthemyce1.iumgrowthofB.cinereastrainsweresignificant.Onthesamecondition,differencesweresignificantinmyce1.iumgrowthamongtestedstrainsexcept1.ight.Theoptimummediaforgrowthofa1.1.si11insinc1.udePDAandPSA.PSAwastheopti1111)mediumforthemyce1.iumgrowthofiso1.atesPJARI,PJA1.3,PYC1.IandPJ1.1.I.PDAwastheoptimummediumforthemyce1.iumgrowthofiso1.atesFSY1.I,PJA1.I.PJA1.2,PY1.1.I.PB1.1.I.PQA1.I,PH1.1.Iand1.SYFI,whi1.ePSAWaSoptimumforotherstrains.Tcinpcraiurcsformyce1.iumgrowthofeachtestediso1.atesrangesfrom5,Cto25'C.Theoptimumtemperatureofmyce1.iumg>wthoftheiso1.ateFSY1.,PJ1.1.2.PRH1.I.PH1.1.Iand1.SYF2whs25*C,whi1.ethatforothersWaS20,C.TherangesofpHformyce1.iumgrowthofthetestediso1.ateswerethesame,from3to11,Asformyce1.iumgrowthofPYC1.IandI.SYF2,thefavorab1.ep1.Iva1.uewas6.T1.>eoptimump1.Iofmyce1.iumgrowthoftheiso1.ates(PJARI,PJA1.2,PJAI,3andPY1.IJ)WaS4,whi1.ethatfortheotheriso1.ateswas5.Theoptimumcarbonsourcesofmyce1.iumgrowthwereg1.ucoseandma1.tose.T1.iefavorab1.enitrogensourcesofmyce1.ia1.growthoftestediso1.ateswereyeastextract,beefextractandpeptone.TherewasnosignificanteITectof1.ightonhetnyce1.iungrowthofH.cinereastrains.Theresu1.tsa1.soshowed(hata1.1.testediso1.atescou1.dcausegreymou1.donPanaxginseng,Strawbeny.tomato,respectivi1.y.However,therewasdifferenceinpathogenicityamongthe(IifTerentiso1.atesfromthesamehost,andthepathogenicitydifferencewasnotobvious1.yre1.atedtothe1.oca1.itiesofiso1.ates.Mostofstrongviru1.entstrainsweresc1.erotiumtype.2. UsingISSR-PCRmo1.ecu1.armarkers,geneticdiversityana1.ysisofginsenggreymo1.dandtagsystemisestab1.ished.TheoptimumconditionsforISSR-PCRwere1.0UaqDNAPOIynIeraSe*0.2mno1.1.,dNTPs.2.5mmo1.1.-1Mg*,0.4no1.-1.1primers.30ngtemp1.atesDNAin251.reactionsystemandUBC810aspremier.Thediversityof23B.cinereaiso1.ateswith13primerswasana1.yzed,andtheresu1.tsshowedthat190fragmentswereamp1.ified.154AbstractPoIyn1.orPhiC1.ociwereaccountedfor81.1%inthetota1.amp1.ifiedfragments.Thegeneticdiversityamong23B.CEereaiso1.ateswasana1.yzedaccordingtotheamp1.ifiedresu1.tsandamo1.ecu1.ardendrogramwasconstructed.Thesimi1.aritycoefficientof23B.cinereaiso1.ateswasat0.53-1.(X)andtheyweredividedinto6groupsatthe1.eve1.of0.814.inthispaper.theauthorhasestab1.ishedthebefittingISSR-PCRreactionsystemforB.cinereaandfoundtherewasmanifestinterspecificgeneticdiversityofB.cinereaiso1.ates.'I1.iistechniquecanbeusedtoana1.yzetheinterspecificgeneticdiversityofB.cinereainthefuturestudy.Geneticgroupdivisionandstraingeographica1.sourcehadcertainre1.evance.There1.ationshipbetweengenetic1.ineagesandpathogenicityofB.cinereawascomp1.icated.Itwasfoundthatthereisnocertaincorrespondingre1.ationshipbetweengenetic1.ineagesandpathogenicity.3. Conditionsforinvitroce1.1.wa1.1.degradationenzymes(CWDEs)andinocu1.ationofB.cinereaCWDEsactivitychangehascarriedonthesystemresearch,theresu1.tsshoweingthat:Aftertheginsengofgreymou1.dofCWDEstreatment,thetypica1.watersymptomwasformed.PGandPMGshowedmaximumactivitiesintheseconddayafterinocu1.ation.Cxand-g1.ucosidascshowedmaximumactivitiesinthefourthdayafterinocu1.ation.ButPGTEandPMTEwerenotdetected.BasedontheCWDEsproducedinvitroconditions,theresu1.tsrevea1.edthat4kindsofCWDEshadaspecificoptima1.cu1.tureconditions.Siaiionarycu1.1.urcmademorefortheproductionofCWDEscomparedtotheshakecu1.ture.TheactivitiesCxand-g1.ueOSidaSCreachedapeakwhentheiso1.atewascu1.tured9d,whi1.ePGandPMGreachedapeakwhentheiso1.atewascu1.tured12d.ItwasfoundthatthebestCUItUrVtemperatureforPGandPMGwasI5'CandthebestforCxand-gkvosidascwas20'C.InregardingtopHva1.ue.4kindsofCWDEsshowedmaximumactivitiesatcu1.turepH5.4. Basedonsixdifferentviru1.entstrainsandCWDEscorre1.ationana1.ysis,theresu1.tsindicatethatthehigh1.yviru1.entiso1.ateshadmoremyce1.ia1.growththanthemoderate1.yVtiso1.ates,withdifferentmedia.PathogenicfactorsofB.cinereawereinvestigatedinregardtofunga1.growthandtheproductionofthece1.1.wa1.1.-degradingenzymes.PG.PMG.-g1.ucosidascandCx.PGPMG,Cxand-g1.u