萵苣黑斑病菌之鑑定及其微生物防治試驗

關鍵字: 萵苣黑斑病;lettuce black leaf spot;形態鑑定;ITS序列分析;寄主範圍測試;A. lactucae;B. amyloliquefaciens;黃豆粉-糖蜜培養基;花生油;乳化劑;Iturin A ... Skipnavigation 中文 English 中興大學機構典藏NCHUInstitutionalRepository 中興大學機構典藏DSpace系統致力於保存各式數位資料（如：文字、圖片、PDF）並使其易於取用。

NCHUInstitutionRepository 農業暨自然資源學院 植物病理學系 請用此HandleURI來引用此文件： http://hdl.handle.net/11455/31990 標題: 萵苣黑斑病菌之鑑定及其微生物防治試驗IdentificationfortheCausalAgentofLettuceBlackLeafSpotandItsMicrobialControlExperiments 作者: 高郁琇Kao,Yu-Hsiu 關鍵字: 萵苣黑斑病;lettuceblackleafspot;形態鑑定;ITS序列分析;寄主範圍測試;A.lactucae;B.amyloliquefaciens;黃豆粉-糖蜜培養基;花生油;乳化劑;IturinA;Surfactin;morphologicalcharacteristics;ITSsequencinganalysis;hostrangetest;A.lactucae;B.amyloliquefaciens;Soybeanmeal-Molassesmedium;peanutoil;emulsifiedagent;IturinA;Surfactin 出版社: 植物病理學系所 引用: 行政院農業委員會。

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自栽培區取得之結球萵苣苗盤其罹病率高達98%且缺株率達16%。

由罹病株分離獲得之菌株皆歸屬於鏈格孢菌屬(GenusAlternaria)真菌，隨後依柯霍氏法則測定病原性，證實分離獲得之26株Alternariasp.菌株皆可引起萵苣黑斑病，且病徵與田間原罹病株相仿。

將Alternariasp.AL1-2及AL3-11兩菌株分別接種於不同栽培種之萵苣、番茄、馬鈴薯、甜椒、甘藍、白菜、茼蒿、紅莧菜、菠菜、蕹菜、北蔥、胡蘿蔔、胡瓜以及西瓜等作物測試其寄主範圍，結果發現該病原菌僅危害萵苣，於接種7天後即會導致萵苣葉片黃化乾枯進而造成植株死亡；此外，其對於番茄及甘藍則引起疑似過敏性反應。

分析Alternariasp.AL1-2及AL3-11菌株之核醣體去氧核醣核酸之轉錄內區間(internaltranscribedspacer,ITS)序列，發現兩菌株之序列與A.solani較為相近，將其與財團法人食品工業研究所生物資源保存及研究中心提供之A.solani(BCRCNo.32123)序列比較，兩者之相似度為99%。

該病原菌於蔬菜瓊脂(V8juiceagar)平板上之菌落呈黑褐色，具輪紋，產孢梗大多具單一產孢點，少數則具2-4個產孢點，每一產孢點僅產生單一分生孢子，分生孢子大小平均為82-90×13μm，呈長橢圓形或長卵形，具6-10個橫隔膜及0-3個縱隔膜，大部分分生孢子具單一個喙，少部分則具兩個喙，喙(beak)屬於基部較寬而尖端纖細(narrow-taper)之形式且長達163-176μm。

病原菌菌絲生長、分生孢子發芽及感染萵苣之最適溫度皆為24℃。

綜合上述病原菌之形態、生長特徵、ITS序列分析及其對萵苣之致病性，進而與國內外相關文獻比較，該萵苣黑斑病之病原菌係屬於一新發現種，因此命名為AlternarialactucaeKao&Huangsp.nov.。

測試15株拮抗菌株對萵苣黑斑病菌之拮抗效果，結果發現本研究分離獲得之BacillusamyloliquefaciensBA01菌株對萵苣黑斑病菌之拮抗效果最佳，其於馬鈴薯牛肉煎汁瓊脂(PNA)平板上對A.lactucaeAL1-2及AL3-11菌株之菌絲生長抑制率分別達57%及56%；將BA01菌株培養於牛肉煎汁培養液(NB)中四天，其醱酵液會誘使病原菌之發芽管膨大，進而抑制病原菌分生孢子發芽率達80%以上。

由結球萵苣切離葉生物分析法評估B.amyloliquefaciensBA01菌株之防病能力，結果發現BA01菌株可抑制病斑擴展率達89%。

本研究利用結球萵苣切離葉評選BA01菌株合適之培養基配方，發現以黃豆粉-糖蜜培養基(SYM)培養BA01菌株時，其100倍稀釋醱酵液可抑制病原菌AL1-2及AL3-11菌株危害萵苣之百分率分別為36%與41%；進一步測試SYM培養基添加植物油對BA01菌株防病能力之影響，結果發現將BA01菌株培養於添加2.5%(w/v)花生油之SYM培養基，其醱酵液之防治成效最佳，200倍稀釋醱酵液對病害之抑制率可達46%以上；此外，於SYM培養基中添加0.125%(w/v)乳化劑亦可顯著提升BA01菌株之防病能力，其200倍稀釋醱酵液對病害之抑制率可達56%以上。

在溫室，將上述兩種BA01菌株之250倍稀釋醱酵液施用於結球萵苣植株，結果兩者皆可顯著降低病害之發生，其中尤以於接種病原菌前一天施用者之防治效果最佳。

利用高壓液相層析儀(HPLC)分析BA01菌株之代謝產物，發現BA01菌株具有產生IturinA及Surfactin等抑菌物質之能力。

本研究所研製之兩種BacillusamyloliquefaciensBA01菌株醱酵液證實具有進一步研發成為微生物源植物保護製劑產品之潛力。

Duringspringseasonin2011,adiseasepreviouslyunreportedinTaiwanwasobservedoncommercialplantingsoflettuce(LactucasativaL.)atErluninYunlinCounty.Alotofblack-brownspotsshowedonleavesofdiseaedheadlettuceplants.Whenspotsaresonumerous,theycoalescetoformareasextendingwholeleafandresultinginpoorstandingofheadlettucewithleafyellow,dryinganddeath.DiseaseincidenceandtherateofpoorstandingofheadlettuceseedlingsoccurredincultivationtraysatErlunwere98%and16%,respectively.AspeciesofAlternariawasconsistentlyisolatedfromdiseasedportionsoftheinfectedleaveson2%(w/v)wateragar.Sporesuspensions(105conidia/ml)ofthepathogenwereusedtoinoculatefourteencropstoconfirmitshostrange.Theresultsshowedthatleafspotsweresimilartotheoriginalsymptomsdevelopedonlyoninoculatedplantsoftenlettucecultivars.However,inoculatedtomatoandcabbageplantsdidshowhypersensitive-likereaction.Otherinoculatedplantsremainedsymptomless.BasedonribosomalDNAinternaltranscribedspacer(ITS)sequencinganalysis,theITSsequenceofthepathogenwassimilartoA.solani.TheITSsequenceidentitybetweenthepathogenandA.solani(BCRCNo.32123)obtainedfromFoodIndustryResearchandDevelopmentInstitutewas99%.OnV8juiceagar,coloniesofthepathogenaredark-brownwithconcentricrings.Mostofconidiophoreswithsingleconidiogenoussiteandafewwith2-4conidiogenoussites.Conidiabornesinglyfromeachconidiogenoussiteontheconidiophores,long-ellipsoidorlong-ovoid,6-10transverseseptaand0-3longitudinalsepta,1-2beaks,82-90×13μm.Beaksarenarrow-taperedandbecomeupto163-176μmlong.Theoptimumtemperatureformycelialgrowth,conidialgerminationandinfectiononlettuceofAlternariasp.isolatesAL1-2andAL3-11was24℃.TheresultsindicatedthatthefungusmaybeanewspeciesofAlternariaonLactucasativaintheworldandwenameditAlternarialactucaeKao&Huangsp.nov..FifteenantagonisticbacteriawereevaluatedfortheireffectsonmycelialgrowthandconidialgerminationofA.lactucaeisolatesAL1-2andAL3-11.Amongtestedbacteria,BacillusamyloliquefaciensisolateBA01wasaneffectivebiocontrolagentwhichwasabletoinhibit55%ofmycelialgrowthand80%ofconidialgerminationanddidalsocausegermtubesswellingofA.lactucae.ApplicationofdetachedheadlettuceleafforevaluatingtheefficacyofBA01oncontrollinglettuceblackleafspotshowedthatBA01wasabletoreduce89%ofdevelopmentofblackspotondetachedheadlettuceleaf.ThefermentedbrothofBA01culturedinSoybeanmeal-Molasses(SYM)mediumwasanalyzedbybioassaymethodofdetachedheadlettuceleaffordiseasecontrol.100-folddilutionoffermentedbrothofBA01culturedinSYMmediacouldinhibit36%and41%ofdevelopmentofblackspotcausedbyA.lactucaeisolatesAL1-2andAL3-11,respectively.AddingdifferentplantoilsintoSYMmediatoevaluatetheireffectsonthecontrolefficacyofBA01,theresultsindicatedthatfermentedbrothofBA01culturedinSYMmediaamendedwith2.5%(w/v)peanutoildidshowtobethemosteffectiveincontrollinglettuceblackleafspot.Inhibitionrateof200-folddilutionofthefermentedbrothwasmorethan46%.Inaddition,wealsofoundthatSYMmediaamendedwith0.125%(w/v)emulsifiedagentcouldenhancetheabilityofBA01forcontrollinglettuceblackleafspot.Inhibitionrateof200-folddilutionofthefermentedbrothreachedmorethan56%.Ingreenhousetest,sprayingtwofermentedbrothsofBA01at250-folddilutiontoheadlettuceplantsonedaybeforeinoculatingwiththepathogen(105conidia/ml)didshowtobethemosteffectiveincontrollingthedisease.AnalysisofantifungalcompoundsproducedfromculturalfiltrateofBA01byhighpressureliquidchromatography(HPLC)showedthatBA01couldnotonlyproducesurfactinbutalsoproduceiturinA.ThisstudydemonstratesthatthetwofermentedbrothsofB.amyloliquefaciensBA01havethepotentialtobedevelopedintomicrobialplantprotectantsforcontrollinglettuceblackleafspot. URI: http://hdl.handle.net/11455/31990 其他識別: U0005-0207201315064400 顯示於：植物病理學系 顯示文件完整紀錄   TAIR相關文章 國立交通大學 - /國立中興大學 - 萵苣萎凋病菌的生理小種鑑定與防治試驗/黃久菱;Huang,Ju-Ling國立中興大學 - (植物病理學會刊第13卷第4期,p357)萵苣萎凋病之防治試驗/黃久菱;黃振文國立中興大學 - 杏鮑菇褐斑病菌之鑑定、生物特性、偵測與防治/陳錦桐;Chen,Jin-Tong國立中興大學 - 西瓜蔓割病菌的生物學及其防治試驗/黃振文;Huang,Zhen-Wen農業試驗所 - 萵苣萎凋病菌的生理小種鑑定與抗病品種篩選/洪爭坊;張碧芳;黃久菱;萬宥伶;黃振文國立中興大學 - 台灣萵苣萎凋病菌生理小種分子鑑定技術之開發/賴柏蓉;Lai,Po-Jung國立中興大學 - 萵苣褐斑病菌的形態、病原性及存活/林秀儒;Lin,Hsiu-Ju GoogleScholarTM 檢查 在IR系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。