真菌分离以及培养

〔越南进境人参果炭疽病病原菌的分别鉴定2023年第3期植物检疫胡佳王卫芳¨〕1.病原菌分别[12h荧光与12h黑暗循环交替)，病菌纯化后保存备用。2、病原菌培育特征和形态观测取病果病部组织进展徒手切片，显微观看分生孢子盘和分生孢子的形态特征；观看病菌的培育特性(PDA25oC，12h荧l2h黑暗循环交替)7d的菌落中挑取分生孢子团，配制成孢子悬浮液，承受载片悬滴法，观测附着孢的形态及大小。3、病原菌18SrDNAPDA25oC12h荧~／12h黑暗循环交替条件下培育5d，挑取菌落边缘颖菌丝，承受常规CTAB法提取病原菌基因组DNAPCR18SrDNA通用引物GeoA2和Geol1【9J。反响体系为25总体积，ddH2O16．375，10倍PCR缓冲液(含MgC12)25IxL，dNTP(25mmol／L)2L，GeM1(10I~mol／L)1LGeoA2(10lxmoL／L)1LTaq(5u／lxL)0125LDNA2L94℃预变性2min94oC45s55oC45s72oC2min3072~C延长10min4℃保存。PCR产物送上海英骏生物GenBankBLAST比较。4、致病性测定(Citrusreticulata)、苹果(Maluspumila)果实，经70％酒精外表消毒后晾干备用。从在PDA、25oC、12h荧光／12h黑暗循环交替条件下培育7d的菌落上挑取分生孢子团，用无菌水配成分生孢子悬浮液(100倍镜下50—60个孢子)菌水作比照。用保鲜膜分别包裹接种果和比照果，封口，放置经70％酒精消毒过的塑25oC、12h荧光／12h黑暗条件下保湿培育，定期观看病症进展过程。2的方法重分别病原菌，做Kohn法则确认是否同种致病菌。〔苹果炭疽菌低毒性菌株的筛选及控病效果的争论〕炭疽菌培育参照方中达等的方法在含有Ad后用25OmL内有玻璃珠、灭菌的三角烧瓶中(11了个抱子/mL)，置20min，使抱子团充分分散，涂平板12个待用。涂平板仍参照方中达的方法pH3~10PDAPH4，pH4~5，〔东北农业大学农学硕士学位论文南瓜疫病菌(尸彻toPhthor。aP:iciLeon.)毒素致病机理及抗源筛选争论〕生物测定法包括幼苗浸渍法、离体叶片浸渍法、叶片针刺、涂抹和喷雾测定、植物愈伤组织测定法、离体根冠细胞测定法、线粒体测定法、叶绿体测定法、细胞膜测定法等。ATP酶法、PAL酶法、核糖核酸和脱氧核糖核酸酶法、SOD酶法、POD酶法、MOD酶法。代谢水平的生物测定有种苗偶联能量的测定、苯丙烷类代谢途径的测定、C02暗固定测定法等。与抗性有关的重要的酶类有细胞壁水解酶、苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)等，植保素为一些酚类和黄酮类化合物。2.2.1液体培育基制备Plich’S培育液:I000mlKH2PO45g，MgSO4;·7H2O2.0g，天门冬酰胺1.0g，VB10.1g5g25g;PH6.5。胡萝卜汁培育液:200g302层纱布过滤后用蒸馏水定容至100Oml)6.5。2:2粗毒素的提取产毒培育CA247dsmm菌块，接入100mlphch’s250m!525℃条件下振荡培育巧d，培育液经定性滤纸过滤，滤液经细菌滤膜真空抽滤得无菌培育液。粗毒素的提取在无菌培育液中参加(NH4)SO4;置饱和度达85%(0oC)11000r/min离心15min，沉淀溶于去离子水中，42420℃下保存备用。P.CpSl’cj产毒力量的影响根长生长抑制法用在两种培育基培育得到的无菌滤液分别接种，接种方法是将感病品种(DX一l)的种子用清水漂洗汰除病、劣和杂粒，加水浸泡8小时后，倒掉水并用水冲洗几遍再放到2526℃恒温条件下培育。待主根长为Znun时，胚向下摆在事先已铺有用无菌滤液浸湿的纱布的白瓷盘内，每白瓷盘放50粒种子，再在种子上面掩盖一层用滤液浸湿的滤纸，于温度为26℃的483比照。生长抑制率(%)X100幼苗浸渍法完全开放，其次片真叶1/2开放的幼苗，经无菌水处理后，移入分别盛有两种无菌滤液的三p。caPsl’ci无菌水、空白培育液做比照，2426℃刻牛下，光照处理下，每5次重复，两天后观看其发病病症。四〔辣椒疫霉菌产毒培育方式和提取方法的争论李海燕赵伟全第16卷第1期黑龙江八一农垦大学学报13 年3月〕Plich’s培育液在振荡培育的方式下，辣椒疫霉菌〔Phytophthoracapsici〕产毒量最高。Phytophthoracapsici毒素的最正确方法为硫酸铵沉淀法试培育基Plich’s培育液：KH2PO40.5g，MgSO4·7H2O2.0g1.0g5.0g，VB10.01g25g1000mL。胡萝卜汁培育液：称取胡萝卜200g，去皮切成小块放入组织捣碎机内捣碎裂分钟，三层纱1000mL。辣椒叶煎汁培育液：取颖辣椒叶200g，加水煮30min，过滤，滤液加蒸馏水定容至1000mL。产毒培育条件承受胡萝卜〔CA〕5d8mm的150mL250mL810瓶，525525〔HZQ－X100型〔120r/min〕15d。取出培育液经三层定性滤纸抽滤，再经微孔滤膜〔0.45μm〕真空抽滤，得无pH6.54℃冰箱中待用。毒素提取方法的选择取上述s无菌培育滤液〔每份0，按以下五种方法进展提取。100mL〔MW8000～1000010mmol/LNaHCO3～1mmol/LEDTA30min，用双蒸水充分洗涤4℃20%聚乙二醇〔PEGs,MW20230〕溶液中浓缩〔4℃冰箱中。100mL无菌培育滤液的烧杯放在一装有冰水混合液的大烧杯内，置于磁力搅拌器上缓慢搅拌，边搅拌边缓缓参加硫酸铵固体〔每100mL培育滤液加硫酸铵55.9g〕85%10min,使溶液充分混合，4℃过夜。丙酮沉淀法：将丙酮提前预冷至-20℃，将样品滤液置冰浴中用磁力搅拌器缓慢搅拌，边搅拌边缓缓参加预冷丙酮〔每0L样品加丙酮0，加完后连续搅拌～5，使溶液充分混合，4℃过夜。乙醇沉淀法：方法同丙酮法，只须将丙酮换为无水乙醇即可。聚乙二醇沉淀法：在冰浴条件下向100mL无菌毒素培育滤液中参加预冷至4℃的50%聚乙二醇溶液〔每0L滤液加G0，边加边用磁力搅拌器缓慢搅拌，持续搅40min使溶液充分混合，4himacCR21高速低温离心机离心15min〔4℃，10000×g〕,弃去上清液，将沉淀用少量无菌双蒸水复溶，再离心除去变性蛋白。424h6h透析液一次。3mL1.5mLEppendorf管中，-20℃保存。毒素浓度的测定BradfordG-250染色法。毒素生物活性的检测〔1997〕6叶期茄门甜椒幼苗的第3、第4片叶，待测样品经透析去盐后，用无菌的一次性注射器在叶反面沿叶脉注25μL样品，分别以蒸馏水、未接菌的培育液为比照，每处理注射10片叶，24h后测定叶片坏死的严峻程度，以叶片的病情指数作为毒素的相对活性。病情指数＝〔各叶片坏死严峻率之和／调查总叶片数〕×100%五、胶孢炭疽菌菟丝子专化型毒素的初步争论刘慧,20卷第3期20239月四川农业大学学报胶孢炭疽菌菟丝子专化型可产生有致病力的外毒素,毒素引起菟丝子产生类似于接种分生孢子所引起的病症。菟丝子蔗糖培育液、改进的Czapek-Dox培育液和摇瓶液体发酵培育基均可产毒。毒素经10030min后仍保持较高的致病率,5mol/L4h后致pH,难溶于多种有机溶剂,透析试验结果说明毒素为大分子物质,推想毒素的主要成分是蛋白聚糖类物质培育条件菌种接种于PDA培育基平板中心,在28℃恒温培育箱中培育7d后,在菌落边10mm30mL250mL三角瓶,28℃旋转摇床振荡培育10d3个重复,dH2O和(或)灭菌的各种培育液以及未经处理的粗毒素液等比照。培育基A.改进的Czapek-Dox培育液[4];B.马铃薯蔗糖培育液(PSB):取40g去皮马铃薯,按PDA培育基的配制方法,滤液参加蔗糖4·0g,定容到200mL,pH自然;C.菟丝子蔗糖培育液(CSB):取40 g 鲜菟丝子种子,其它同PSB;D.摇瓶液体发酵培养基:蔗糖2·0%,(NH4)2SO41·0%CaCl2·2H2O0·1%,K2HPO40·1%,MgSO4·7H2O0·03%,蛋白胨0·1%,玉米粉1·0%,可溶性淀1·0%,黄豆粉5·0%,菟丝子粉1·0%,CaCO30·4%,pH值6·5~7·5或自然,以自来水配制。培育温度设置旋转摇床温度为22℃、24℃、26℃、28℃和30℃,承受改进的Czapek-Dox培育液(下同)。pH值用1mol/LHCl1mol/LNaOHpH4·0~10·0的改进的Czapek-Dox培育液。粗毒素液的配制菌株接种于摇瓶液体发酵培育基,120h,培育物用定性滤纸过滤,1次。取滤4000r/min15min1次。上清液即为粗毒素液。另取发酵滤10030min,即为灭活分生孢子和菌丝体的粗毒素液。粗毒素液的致病性试验承受保湿平皿喷雾接种法六、Occurrence,isolationandbiologicalactivityofphytotoxicmetabolitesproducedinvitrobySphaeropsissapinea,pathogenicfungusofPinusradiataAnnalisaCabras,MariaA.MannoniEuropeanJournalofPlantPathology(2023)115:187–193Springer2023FungalculturesPureculturesofS.sapineawereisolatedfromwoodofnaturallyinfectedadultplantsofradiatafromSardinia(Italy).Singlesporeisolatesweregrownonpotato–dextrose–agarat25Cfor7daysanddepositedinthefungalcollectionoftheDipartimentodiProtezionedellePiante,ExtractionandpurificationoffungalmetabolitesTheculturesofS.sapinea(10l)wereobtainedasdescribedbyEvidenteetal.(1996).Combinedcultureswerefiltered,acidifiedtopH4.00with2NHCl,thenextractedwithethylacetate(4x2.5l).Theorganicphaseswerecombined,driedoverNa2SO4andevaporatedunderreducedpressureat40C.Theorganicextractwasappliedtoasilicagelcolumn(160g,80cmhigh,5cmid),elutedwithagradientofchloroform-iso-propanol(100:1fi1:2v/v).Fractions(15mleach)weremonitoredbyTLCanalysisandtheresultinghomogeneousfractionscombined.Fractionsweretestedforbioactivityagainsttomatocuttingsasdescribedbelow.ActivefractionsT2andT4werepurifiedbypreparativesilicagelTLC,withchlo-roform-iso-propanol(100:1and25:1v/v,respec-tively)asthesolventsystem.ThebandsatRf0.70and0.39,respectively,visualisedbyUVlightat254nm,wereremovedfromtheplates,extractedwithethylacetateandevaporated.Themaincomponentof fraction T4 was further purified by preparativereversephase TLC withwater-ethanol(1:1v/v)asthesolventsystem.ThebandsvisualisedbyUVlight(254nm)atRf0.53and0.59wereseparatelyremovedandextractedwithacetonitrile.ThePlantPathologyJournalTheKoreanSocietyofPlantPathology七、IsolationandPartialCharacterizationofPhytotoxicMycotoxinsProducedbySclerotiniasp.,aPotentialBioherbicidefortheControlofWhiteClover(Trifoliorumrepens)PlantPathol.J.20(1):52-57(2023)Yeon-KyuHong*,Bong-ChoonLeeFungalculture.TheSclerotiniasp.cultureusedinthisstudywasoriginallyisolatedfromnaturallyinfectedT.repensrootwhichwascollectedfromthefieldoftheNationalYeongnamAgriculturalExperimentStation(NYAES),Milyang,Korea.Theorganismwasmaintainedonhalf-strengthpotatodextroseagar(1/2PDA;Difco,Detroit,MI)slantsinsmallvialsundermineraloilat4oC(Zhangetal.,1996).Fortoxinproduction,thefunguswasgrownin250Erlenmeyerflaskcontaining200mlofCzapekDoxbrothmedium(24gmediumanddistilledwaterto1L;Difco,Detroit,MI).Cultureswereincubatedattemperaturesof28±1oConarotaryshakeroperatingat150rpmfor7days.Isolationandpurificationoftoxins.After7daysofgrowth,theculturefluidwasobtainedbyfiltratingthroughthreelayersofcheesecloth,andcentrifugedat8,000rpmfor20min.Culturefiltrateswereconcentratedto10%oftheiroriginalvolumebyusingaflashevaporatorat60oC(Steineretal.,1971;Stierleetal.,1992).TheinitialseparationofactivesubstancesfromtheculturefiltratefollowsthesolventfractioningprocedureshowninFig.1.Dualsolventsystemsusedn-hexane,chloroform,ethylacetate,n-butanol,andwateraslinearseparationsystem(Fig.2).Briefly,thesameamountofeachsolventwasequaledandmixedbyshakingfor3mininaseparationfunnelwith150mloftheculturefiltrateoraqueouslayer,andseparationwasmadebetweenthesolventandtheaqueouslayers.Eachextractwasthenevaporatedusingaflashevaporator,andtheresiduewasweighedandcollectedinvialsusingmethanol.Inordertodetectbiologicalactivity,eachcomponentwaspreparedandsubjectedtoaleafbioassaybyplacingin2%aqueousethanolsolutioncontaining0.05%Tween20asawettingagent.CompoundsIandIIwerepurifiedbymeansofliquid-liquidextractionandC18opencolumnchromatography(300×30mm,i.d)(Fig.4).Todeterminethepurity,thepurifiedtoxinIandtoxinIIwereanalyzedbyRP-HPLC(Hitachi,Japan).TheanalyticalRP-HPLCcolumnwasaTOSOHODS-120T(150×4.6mmi.d,Japan),andtheflowratewassetat0.7mL/minbyusingalineargradientsolventsysteminitiatedwith15%methanolto85%methanolfor50minwithmonitoringat254nmundertheseRP-HPLCconditions.ProceedingsoftheXInternationalSymposiumonBiologicalControlofWeeds4-14July1999,MontanaStateUniversity,Bozeman,MontanaUSANealR.Spencer[ed.]. pp.125-130 (2023)八、IsolationandPartialCharacterizationofPhytotoxinsProducedbyExserohilummonoceras,aPotentialBioherbicideforControlofEchinochloaSpeciesWENMINGZHANGandA.K.WATSONCulturing.TheE.monocerascultureusedinthisstudywasoriginallyisolatedfromnaturallyinfectedEchinochloaspeciesleavescollectedinthePhilippines.Theorganismwasmaintainedonhalf-strengthpotatodextroseagar(1/2PDA;Difco,Detroit,MI)slantsinsmallvialsundermineraloilat4°C(Zhangetal.,1996).Fortoxinproduction,thefunguswasgrowninl-LRouxbottlescontaining200mlofModifiedFriesmedium(100gsucrose,2gcaseinhydrolysate,1.5gNaNO3,1gK2HPO4,0.5gKCl,0.5gMgSO4,0.01gFeSO4,anddistilledwaterto1L)(Tuite,1969).Cultureswereincubatedatlaboratorytemperature(25±2° C)onarotaryshakeroperatingat150rpm.Isola