真菌分離以及培養(yǎng)

〔越南進境人參果炭疽病病原菌的分別鑒定2023年第3期植物檢疫胡佳王衛(wèi)芳¨〕1.病原菌分別[12h熒光與12h黑暗循環(huán)交替)，病菌純化后保存?zhèn)溆谩?、病原菌培育特征和形態(tài)觀測取病果病部組織進展徒手切片，顯微觀看分生孢子盤和分生孢子的形態(tài)特征；觀看病菌的培育特性(PDA25oC，12h熒l2h黑暗循環(huán)交替)7d的菌落中挑取分生孢子團，配制成孢子懸浮液，承受載片懸滴法，觀測附著孢的形態(tài)及大小。3、病原菌18SrDNAPDA25oC12h熒~／12h黑暗循環(huán)交替條件下培育5d，挑取菌落邊緣穎菌絲，承受常規(guī)CTAB法提取病原菌基因組DNAPCR18SrDNA通用引物GeoA2和Geol1【9J。反響體系為25總體積，ddH2O16．375，10倍PCR緩沖液(含MgC12)25IxL，dNTP(25mmol／L)2L，GeM1(10I~mol／L)1LGeoA2(10lxmoL／L)1LTaq(5u／lxL)0125LDNA2L94℃預變性2min94oC45s55oC45s72oC2min3072~C延長10min4℃保存。PCR產(chǎn)物送上海英駿生物GenBankBLAST比較。4、致病性測定(Citrusreticulata)、蘋果(Maluspumila)果實，經(jīng)70％酒精外表消毒后晾干備用。從在PDA、25oC、12h熒光／12h黑暗循環(huán)交替條件下培育7d的菌落上挑取分生孢子團，用無菌水配成分生孢子懸浮液(100倍鏡下50—60個孢子)菌水作比照。用保鮮膜分別包裹接種果和比照果，封口，放置經(jīng)70％酒精消毒過的塑25oC、12h熒光／12h黑暗條件下保濕培育，定期觀看病癥進展過程。2的方法重分別病原菌，做Kohn法則確認是否同種致病菌?！蔡O果炭疽菌低毒性菌株的篩選及控病效果的爭論〕炭疽菌培育參照方中達等的方法在含有Ad后用25OmL內(nèi)有玻璃珠、滅菌的三角燒瓶中(11了個抱子/mL)，置20min，使抱子團充分分散，涂平板12個待用。涂平板仍參照方中達的方法pH3~10PDAPH4，pH4~5，〔東北農(nóng)業(yè)大學農(nóng)學碩士學位論文南瓜疫病菌(尸徹toPhthor。aP:iciLeon.)毒素致病機理及抗源篩選爭論〕生物測定法包括幼苗浸漬法、離體葉片浸漬法、葉片針刺、涂抹和噴霧測定、植物愈傷組織測定法、離體根冠細胞測定法、線粒體測定法、葉綠體測定法、細胞膜測定法等。ATP酶法、PAL酶法、核糖核酸和脫氧核糖核酸酶法、SOD酶法、POD酶法、MOD酶法。代謝水平的生物測定有種苗偶聯(lián)能量的測定、苯丙烷類代謝途徑的測定、C02暗固定測定法等。與抗性有關的重要的酶類有細胞壁水解酶、苯丙氨酸解氨酶(PAL)、過氧化物酶(POD)、多酚氧化酶(PPO)等，植保素為一些酚類和黃酮類化合物。2.2.1液體培育基制備Plich’S培育液:I000mlKH2PO45g，MgSO4;·7H2O2.0g，天門冬酰胺1.0g，VB10.1g5g25g;PH6.5。胡蘿卜汁培育液:200g302層紗布過濾后用蒸餾水定容至100Oml)6.5。2:2粗毒素的提取產(chǎn)毒培育CA247dsmm菌塊，接入100mlphch’s250m!525℃條件下振蕩培育巧d，培育液經(jīng)定性濾紙過濾，濾液經(jīng)細菌濾膜真空抽濾得無菌培育液。粗毒素的提取在無菌培育液中參加(NH4)SO4;置飽和度達85%(0oC)11000r/min離心15min，沉淀溶于去離子水中，42420℃下保存?zhèn)溆?。P.CpSl’cj產(chǎn)毒力量的影響根長生長抑制法用在兩種培育基培育得到的無菌濾液分別接種，接種方法是將感病品種(DX一l)的種子用清水漂洗汰除病、劣和雜粒，加水浸泡8小時后，倒掉水并用水沖洗幾遍再放到2526℃恒溫條件下培育。待主根長為Znun時，胚向下擺在事先已鋪有用無菌濾液浸濕的紗布的白瓷盤內(nèi)，每白瓷盤放50粒種子，再在種子上面掩蓋一層用濾液浸濕的濾紙，于溫度為26℃的483比照。生長抑制率(%)X100幼苗浸漬法完全開放，其次片真葉1/2開放的幼苗，經(jīng)無菌水處理后，移入分別盛有兩種無菌濾液的三p。caPsl’ci無菌水、空白培育液做比照，2426℃刻牛下，光照處理下，每5次重復，兩天后觀看其發(fā)病病癥。四〔辣椒疫霉菌產(chǎn)毒培育方式和提取方法的爭論李海燕趙偉全第16卷第1期黑龍江八一農(nóng)墾大學學報13 年3月〕Plich’s培育液在振蕩培育的方式下，辣椒疫霉菌〔Phytophthoracapsici〕產(chǎn)毒量最高。Phytophthoracapsici毒素的最正確方法為硫酸銨沉淀法試培育基Plich’s培育液：KH2PO40.5g，MgSO4·7H2O2.0g1.0g5.0g，VB10.01g25g1000mL。胡蘿卜汁培育液：稱取胡蘿卜200g，去皮切成小塊放入組織搗碎機內(nèi)搗碎裂分鐘，三層紗1000mL。辣椒葉煎汁培育液：取穎辣椒葉200g，加水煮30min，過濾，濾液加蒸餾水定容至1000mL。產(chǎn)毒培育條件承受胡蘿卜〔CA〕5d8mm的150mL250mL810瓶，525525〔HZQ－X100型〔120r/min〕15d。取出培育液經(jīng)三層定性濾紙抽濾，再經(jīng)微孔濾膜〔0.45μm〕真空抽濾，得無pH6.54℃冰箱中待用。毒素提取方法的選擇取上述s無菌培育濾液〔每份0，按以下五種方法進展提取。100mL〔MW8000～1000010mmol/LNaHCO3～1mmol/LEDTA30min，用雙蒸水充分洗滌4℃20%聚乙二醇〔PEGs,MW20230〕溶液中濃縮〔4℃冰箱中。100mL無菌培育濾液的燒杯放在一裝有冰水混合液的大燒杯內(nèi)，置于磁力攪拌器上緩慢攪拌，邊攪拌邊緩緩參加硫酸銨固體〔每100mL培育濾液加硫酸銨55.9g〕85%10min,使溶液充分混合，4℃過夜。丙酮沉淀法：將丙酮提前預冷至-20℃，將樣品濾液置冰浴中用磁力攪拌器緩慢攪拌，邊攪拌邊緩緩參加預冷丙酮〔每0L樣品加丙酮0，加完后連續(xù)攪拌～5，使溶液充分混合，4℃過夜。乙醇沉淀法：方法同丙酮法，只須將丙酮換為無水乙醇即可。聚乙二醇沉淀法：在冰浴條件下向100mL無菌毒素培育濾液中參加預冷至4℃的50%聚乙二醇溶液〔每0L濾液加G0，邊加邊用磁力攪拌器緩慢攪拌，持續(xù)攪40min使溶液充分混合，4himacCR21高速低溫離心機離心15min〔4℃，10000×g〕,棄去上清液，將沉淀用少量無菌雙蒸水復溶，再離心除去變性蛋白。424h6h透析液一次。3mL1.5mLEppendorf管中，-20℃保存。毒素濃度的測定BradfordG-250染色法。毒素生物活性的檢測〔1997〕6葉期茄門甜椒幼苗的第3、第4片葉，待測樣品經(jīng)透析去鹽后，用無菌的一次性注射器在葉反面沿葉脈注25μL樣品，分別以蒸餾水、未接菌的培育液為比照，每處理注射10片葉，24h后測定葉片壞死的嚴峻程度，以葉片的病情指數(shù)作為毒素的相對活性。病情指數(shù)＝〔各葉片壞死嚴峻率之和／調(diào)查總?cè)~片數(shù)〕×100%五、膠孢炭疽菌菟絲子?；投舅氐某醪綘幷搫⒒?20卷第3期20239月四川農(nóng)業(yè)大學學報膠孢炭疽菌菟絲子?；涂僧a(chǎn)生有致病力的外毒素,毒素引起菟絲子產(chǎn)生類似于接種分生孢子所引起的病癥。菟絲子蔗糖培育液、改進的Czapek-Dox培育液和搖瓶液體發(fā)酵培育基均可產(chǎn)毒。毒素經(jīng)10030min后仍保持較高的致病率,5mol/L4h后致pH,難溶于多種有機溶劑,透析試驗結(jié)果說明毒素為大分子物質(zhì),推想毒素的主要成分是蛋白聚糖類物質(zhì)培育條件菌種接種于PDA培育基平板中心,在28℃恒溫培育箱中培育7d后,在菌落邊10mm30mL250mL三角瓶,28℃旋轉(zhuǎn)搖床振蕩培育10d3個重復,dH2O和(或)滅菌的各種培育液以及未經(jīng)處理的粗毒素液等比照。培育基A.改進的Czapek-Dox培育液[4];B.馬鈴薯蔗糖培育液(PSB):取40g去皮馬鈴薯,按PDA培育基的配制方法,濾液參加蔗糖4·0g,定容到200mL,pH自然;C.菟絲子蔗糖培育液(CSB):取40 g 鮮菟絲子種子,其它同PSB;D.搖瓶液體發(fā)酵培養(yǎng)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%,黃豆粉5·0%,菟絲子粉1·0%,CaCO30·4%,pH值6·5~7·5或自然,以自來水配制。培育溫度設置旋轉(zhuǎn)搖床溫度為22℃、24℃、26℃、28℃和30℃,承受改進的Czapek-Dox培育液(下同)。pH值用1mol/LHCl1mol/LNaOHpH4·0~10·0的改進的Czapek-Dox培育液。粗毒素液的配制菌株接種于搖瓶液體發(fā)酵培育基,120h,培育物用定性濾紙過濾,1次。取濾4000r/min15min1次。上清液即為粗毒素液。另取發(fā)酵濾10030min,即為滅活分生孢子和菌絲體的粗毒素液。粗毒素液的致病性試驗承受保濕平皿噴霧接種法六、Occurrence,isolationandbiologicalactivityofphytotoxicmetabolitesproducedinvitrobySphaeropsissapinea,pathogenicfungusofPinusradiataAnnalisaCabras,MariaA.MannoniEuropeanJournalofPlantPathology(2023)115:187–193Springer2023FungalculturesPureculturesofS.sapineawereisolatedfromwoodofnaturallyinfectedadultplantsofradiatafromSardinia(Italy).Singlesporeisolatesweregrownonpotato–dextrose–agarat25Cfor7daysanddepositedinthefungalcollectionoftheDipartimentodiProtezionedellePiante,Extractionandpuri?cationoffungalmetabolitesTheculturesofS.sapinea(10l)wereobtainedasdescribedbyEvidenteetal.(1996).Combinedcultureswere?ltered,acidi?edtopH4.00with2NHCl,thenextractedwithethylacetate(4x2.5l).Theorganicphaseswerecombined,driedoverNa2SO4andevaporatedunderreducedpressureat40C.Theorganicextractwasappliedtoasilicagelcolumn(160g,80cmhigh,5cmid),elutedwithagradientofchloroform-iso-propanol(100:1?1:2v/v).Fractions(15mleach)weremonitoredbyTLCanalysisandtheresultinghomogeneousfractionscombined.Fractionsweretestedforbioactivityagainsttomatocuttingsasdescribedbelow.ActivefractionsT2andT4werepuri?edbypreparativesilicagelTLC,withchlo-roform-iso-propanol(100:1and25:1v/v,respec-tively)asthesolventsystem.ThebandsatRf0.70and0.39,respectively,visualisedbyUVlightat254nm,wereremovedfromtheplates,extractedwithethylacetateandevaporated.Themaincomponentof fraction T4 was further puri?ed 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