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研究生:洪爭坊
研究生(外文):Cheng-Fang Hong
論文名稱:小星辰花苗炭疽病菌之生物特性及防治
論文名稱(外文):Biological characteristics and control of the causal agent of caspia seedling anthracnose
指導教授:黃振文黃振文引用關係
指導教授(外文):Jenn-Wen Huang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:62
中文關鍵詞:小星辰花炭疽病生物防治鏈黴菌治黴色基素
外文關鍵詞:caspiaColletotrichumanthracnoseStreptomycesbiological controlfungichrominLimonium
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西元2002年,台灣中部地區多處花卉育苗場與栽培區發現小星辰花(Limonium bellidifolium Dumort.)葉片佈滿棕褐色圓形至不規則形之壞疽斑,嚴重時病斑互相癒合,導致小星辰花整個葉片壞疽或整株死亡。取回罹病葉片進行組織分離,共獲得分生孢子為鐮刀形的Colletotrichum sp. Cas-01、Cas-03及分生孢子為長橢圓形的Colletotrichum sp. Cas-04、Cas-05、Cas-06、Cas-07、Sta-04及Sta-b3等八個菌株。將各菌株的分生孢子(105 conidia/ml)分別噴霧接種於假植15天的小星辰花幼苗上,七至十四天後,植株出現與田間相仿的病徵;進一步再取樣分離,證實各菌株均具有病原性。選取孢子形態為鐮刀狀之Colletotrichum sp. Cas-01與Cas-03菌株,以及致病毒性較穩定之Colletotrichum sp. Cas-06與Sta-04菌株,培養於馬鈴薯葡萄糖瓊脂培養基(potato dextrose agar, PDA)上;其中Colletotrichum sp. Cas-01與Cas-03菌株氣生菌絲少,菌落呈暗褐色,培養基背面有深褐色色素累積,會產生大量剛毛(seta),分生孢子堆淺黃色至鮭紅色,分生孢子無色透明,鐮刀形或新月形,大小22.0-30.0 × 3.0-4.5 µm;孢子發芽後形成之附著器(appressoria)深褐色至黑色,呈棒狀或圓形。Colletotrichum sp. Cas-06與Sta-04菌株的菌落在PDA上呈灰白至橄欖綠色,無菌核(sclerotia)或剛毛(seta),會產生鮭紅色的分生孢子堆。分生孢子無色透明,長橢圓形,兩端鈍圓或一端為截頭狀(truncated),大小11.3-17.5 × 3.8-6.3 µm。孢子發芽後形成之附著器(appressoria)深褐色至黑色,呈橢圓形或不規則形。此外,Colletotrichum sp. Cas-06與Sta-04菌株在PDA上會產生黑色球形的子囊果,子囊透明,棍棒狀,大小42.5-68.8 × 8.8-12.5 µm;子囊孢子透明,呈棍棒狀至紡錘形,略微彎曲,大小11.3-18.8 × 3.8-6.3 µm。利用Sheu氏等研發之C. capsici 的專一性CcInt引子配合ITS4引子,針對Colletotrichum sp. Cas-01及Cas-03菌株進行PCR,並無法增幅出專一性條帶。然而利用Mills氏等人研發之C. gloeosporioides專一性CgInt引子配合ITS4引子進行PCR後,則可由Colletotrichum sp. Cas-04、Cas-05、Cas-06、Cas-07、Sta-04與Sta-b3菌株增幅出大小約450 bp的專一性條帶,而由Colletotrichum sp. Cas-01及Cas-03菌株則仍無法增幅出任何條帶。進一步以ITS1與ITS2共同引子對,針對Colletotrichum sp. Cas-01與Cas-03菌株進行PCR,並將增幅之ITS1與5.8S rDNA部分序列解序後,與美國生物技術資訊中心(NCBI)資料庫進行比對,結果其與Colletotrichum dematium的序列相似度達99-100 %。綜合上述病原菌形態特徵與分子生物學上的佐證,確定引起小星辰花苗炭疽病之病原菌為Colletotrichum dematium (Pers.: Fr.) Grove與C. gloeosporioides (Penz.) Penz. & Sacc.,後者的有性世代為Glomerella cingulata (Stonem.) Spauld. & Schrenk。將C. dematium Cas-01及Cas-03菌株與C. gloeosporioides Cas-06及Sta-04菌株的分生孢子噴霧接種於十四種植物上,C. dematium Cas-01及Cas-03菌株僅在高溫高濕(28℃,RH 100 % 持續七天以上)下,造成小星辰花苗、蜀葵、茼蒿及太陽菊的壞疽病徵。而C. gloeosporioides Cas-06及Sta-04菌株則可危害小星辰花、藍雪花、檬果、茼蒿及太陽菊等植株。在不同溫度下,C. dematium Cas-01及Cas-03與C. gloeosporioides Cas-06及Sta-04菌株的菌絲生長最適溫度為29 ℃。C. dematium Cas-01與Cas-03菌株分生孢子發芽的最適溫度分別為20及24 ℃,而附著器形成的最適溫度亦分別為20與24 ℃。C. gloeosporioides Cas-06與Sta-04菌株孢子發芽最適溫度介於20-32 ℃,附著器形成的最適溫度為24 ℃。在持續高溫潮濕(28 ℃, RH 100 %)以及高接種源濃度(105 conidia/ml)的環境下,將使本病害的病勢發展趨於嚴重。將八種拮抗微生物與小星辰花炭疽病菌C. gloeosporioides Cas-06及Sta-04菌株進行對峙培養,其中Streptomyces padanus PMS-702菌株對C. gloeosporioides Cas-06和Sta-04菌絲生長的抑制寬度可達15 mm。進一步將S. padanus PMS-702培養在花生粉-葡萄糖培養液中,七天後取其培養濾液,處理C. gloeosporioides Cas-06和Sta-04的分生孢子,發現分生孢子出現腫大與細胞壁破裂等現象。以不同濃度S. padanus PMS-702培養濾液處理C. gloeosporioides Cas-06及Sta-04菌株的分生孢子,結果PMS-702培養濾液濃度在20 ml/L以上時,對兩菌株孢子發芽的抑制率可達100 %。而利用高效能液相色層分析儀進行S. padanus PMS-702培養濾液與治黴色基素的分析結果,兩者皆在第22-23分鐘處出現吸收高峰,顯示S. padanus PMS-702培養濾液中含有治黴色基素。以不同濃度治黴色基素處理C. dematium Cas-03菌株與C. gloeosporioides Cas-06 及Sta-04菌株的分生孢子,結果治黴色基素濃度在2.0 ppm以上時,抑制三個菌株孢子發芽的百分率可達98.2-99.5 %。進一步利用S. padanus PMS-702的培養濾液100 ml/L噴佈處理接種過C. gloeosporioides Cas-06與Sta-04菌株分生孢子(105 conidia/ml)的小星辰花苗,結果發現施用PMS-702培養濾液可有效減少小星辰花苗炭疽病的發生率達53.1-67.2 %。不同時期施用S. padanus PMS-702培養濾液防治小星辰花苗炭疽病的結果,顯示接種C. gloeosporioides Cas-06和Sta-04菌株分生孢子後隨即施用S. padanus PMS-702培養濾液的效果最佳,可降低罹病度達20.3-26.6%。
In 2002, plants showing symptoms of necrotic, reddish brown spots on caspia (Limonium bellidifolium) leaves were observed in central Taiwan. Eight Colletotrichum species, Cas-01, Cas-03, Cas-04, Cas-05, Cas-06, Cas-07, Sta-04, Sta-b3, were isolated from leaf lesions of diseased seedlings and adult plants. Conidia of the isolates were used to inoculate caspia seedlings kept at 100% RH for one day then grown in the growth chamber. After 7 to 14 days, numerous acervuli were produced on inoculated leaves from which the same pathogen could be consistently isolated. On potato dextrose agar (PDA), spore masses of six Colletotrichum sp. isolates Cas-04, Cas-05, Cas-06, Cas-07, Sta-04, and Sta-b3 were pinkish or salmon red with dense to sparse grayish aerial mycelium. Conidiomata did not produce setae. Conidia were hyaline, straight, cylindrical, and apices obtuse, 11.3-17.5×3.8-6.3 (14.8×4.9) µm. Appressoria were brown to dark brown in color and ovate to irregular in shape. Perithecia of Colletotrichum sp. isolates Cas-06 and Sta-04 formed on PDA produced asci with 8 single-celled, slightly curved ascospores. Spore masses of the other two Colletotrichum sp. isolates, Cas-01 and Cas-03, were pale buff to salmon red with sparse grayish aerial mycelium. Conidiomata produced abundant setae. Conidia were hyaline, falcate, fusiform, apices acute, 22.0-30.0×3.0-4.5 (25.6×3.5) µm. Appressoria were abundant, medium brown, clavate to circular. Further polymerase chain reaction was conducted with specific CgInt, CcInt, and universal ITS4 primer pairs for the identification of the eight Colletotrichum spp. isolates. A fragment about 450 bp was amplified from six Colletotrichum sp. isolates, Cas-04, Cas-05, Cas-06, Cas-07, Sta-04, and Sta-b3, by using C. gloeosporioides specific CgInt primer and ITS4 primer, but not from Colletotrichum sp. isolates Cas-01 and Cas-03. No specific fragments were amplified from Colletotrichum sp. isolates Cas-01 and Cas-03 by using CcInt and ITS4 primer pairs, either. ITS1 and partial 5.8S rDNA sequences of Colletotrichum sp. isolates Cas-01 and Cas-03 were amplified by using universal ITS1 and ITS2 primer pairs. The DNA sequences showed 99-100% identities to Colletotrichum dematium ITS1 and partial 5.8S rDNA sequences on NCBI GenBank database. Based on morphology, pathogenicity and molecular biological evidences, it was concluded that the disease on caspia seedlings was caused by C. dematium (Pers.: Fr.) Grove and C. gloeosporioides (Penz.) Penz. & Sacc. with the teleomorph Glomerella cingulata (Stonem.) Spauld. & Schrenk. For host range tests, fourteen plant species including African marigold, caspia, cape leadwort, Chinese cabbage, Chrysanthemum grinatum, egg-plant, garland chrysanthemum, hollyhock, lettuce, mango, pepper, sweet pepper, tomato, and transvaal daisy were inoculated with conidial suspension of C. dematium isolates Cas-01 & Cas-03 and C. gloeosporioides isolates Cas-06 and Sta-04 each respectively. The results showed that C. dematium isolates Cas-01 and Cas-03 infected caspia, Chrysanthemum grinatum, garland chrysanthemum, and hollyhock only under high temperature (28 ℃) and prolonged high humidity (RH 100 % over 7 days). On the other hand, C. gloeosporioides Cas-06 and Sta-04 could infect caspia, cape leadwort, Chrysanthemum grinatum, mango and garland chrysanthemum under high temperature (28 ℃) and short retention time of high humidity (RH 100 % over 24 hours). Optimum temperature for mycelial growth of C. dematium isolates Cas-01 & Cas-03 and C. gloeosporioides isolates Cas-06 & Sta-04 was at 29 ℃. Optimum temperatures for C. dematium isolates Cas-01 and Cas-03 conidial germination and appressorial formation were at 20 and 24 ℃, respectively. Optimum temperature for C. gloeosporioides isolates Cas-06 and Sta-04 conidial germination was between 20 to 32℃, and for appressoria formation was at 24 ℃. Results of pathogenicity test showed that the disease progressed rapidly under high temperature (28 ℃), high inoculum density (105 conidia/ml), and prolonged high humidity (RH 100 % over 24 hr) when caspia seedlings were inoculated with C. gloeosporioides isolates Cas-06 and Sta-04. However C. dematium isolates Cas-01 and Cas-03 infected them only under serious conditions (28 ℃, RH 100 % over 7days) and caused mild symptoms. For biological control of caspia anthracnose, eight antagonist isolates, Streptomyces almguist SS-05, S. chibaensis SS-06, S. xantholiticus SS-09, S. sioyaensis PMS-502, S. padanus PMS-702, Bacillus pumilus PMB102, B. thermoglucosidasius PMB207, and B. subtilis BS-001, were tested in vitro for their antagonisity. S. padanus PMS-702 was more effective in inhibiting mycelial growth of C. gloeosporioides isolates Cas-06 and Sta-04. PMS-702 was cultured in peanut meal-glucose broth for 7 days and its culture filtrate was used for control of caspia anthracnose. Conidia of C. gloeosporioides isolates Cas-06 and Sta-04 swelled and cell wall distorted after treatment with PMS-702 culture filtrate for 12 hours. When the concentration of PMS-702 culture filtrate was over 20 ml/L, conidial germination of C. gloeosporioides isolates Cas-06 and Sta-04 was completely inhibited. HPLC analysis of S. padanus PMS-702 culture filtrate was conducted for determination of the active components of S. padanus PMS-702 culture filtrate. Results of HPLC analyses showed consistent peak patterns with fungichromin at the 22-23 minute. Conidial germination of C. dematium isolate Cas-03 and C. gloeosporioides isolates Cas-06 and Sta-04 was almost completely inhibited when the concentration of fungichromin was over 2.0 ppm. Fungichromin might be as one of the major active antifungal components in PMS-702 culture filtrate for inhibiting the pathogen. In growth chamber tests, anthracnose disease severity of caspia seedlings was reduced 53.1-67.2 % after application with 100 ml/L of PMS-702 culture filtrate for 7 days. However, the disease severity was reduced about 20.3-26.6% when spraying S. padanus PMS-702 culture filtrate at 20 ml/L immediately after inoculation of C. gloeosporioides isolates Cas-06 and Sta-04.
前言-------------------------------------------------------------------- 1
材料與方法-------------------------------------------------------------- 3
供試菌株來源---------------------------------------------------------- 3
供試菌株之培養-------------------------------------------------------- 3
供試植株-------------------------------------------------------------- 3
接種源之製備與接種方法------------------------------------------------ 3
病害調查法------------------------------------------------------------ 4
小星辰花苗炭疽病菌之形態鑑定------------------------------------------ 4
利用分子生物技術鑑定小星辰花苗炭疽病菌-------------------------------- 4
一、利用專一性引子對進行聚合酶連鎖反應鑑定小星辰花苗炭疽病菌-------- 4
二、Colletotrichum sp. Cas-01與Cas-03菌株的ITS1及5.8 S 部分rDNA之PCR
增幅產物的濃縮-------------------------------------------------- 5
三、Colletotrichum sp. Cas-01與Cas-03菌株的ITS1及5.8 S 部分rDNA之回收
及序列比對------------------------------------------------------ 5
小星辰花苗炭疽病菌之寄主範圍測定-------------------------------------- 6
小星辰花苗炭疽病菌之生理特性------------------------------------------ 6
一、溫度對小星辰花苗炭疽病菌菌絲生長的影響-------------------------- 6
二、溫度對小星辰花苗炭疽病菌分生孢子發芽與附著器形成之影響----------- 6
小星辰花苗炭疽病之發生條件-------------------------------------------- 7
一、接種源濃度對病害發生之影響-------------------------------------- 7
二、保濕時間與病害發生之關係---------------------------------------- 7
三、溫度對病害發生之影響-------------------------------------------- 7
小星辰花苗炭疽病之防治------------------------------------------------ 8
一、不同拮抗微生物對小星辰花苗炭疽病菌之影響------------------------ 8
二、鏈黴菌S. padanus PMS-702之液態培養------------------------------ 8
三、鏈黴菌S. padanus PMS-702培養濾液對小星辰花苗炭疽病菌孢子發芽之影響
---------------------------------------------------------------- 9
四、利用高效能液相色層分析儀分析S. padanus PMS-702培養濾液及治黴色基素--------------------------------------------------------------- 9
五、治黴色基素對小星辰花苗炭疽病菌孢子發芽之影響------------------- 9
六、鏈黴菌S. padanus PMS-702培養濾液防治小星辰花苗炭疽病之效果評估- 9
七、不同施用時間對鏈黴菌S. padanus PMS-702培養濾液防治小星辰花苗炭疽病之效果評估-------------------------------------------------- 10
八、不同施用次數對鏈黴菌S. padanus PMS-702培養濾液防治小星辰花苗炭疽病之效果評估-------------------------------------------------- 10
結果------------------------------------------------------------------- 11
小星辰花苗炭疽病之病徵----------------------------------------------- 11
小星辰花苗炭疽病菌之鑑定--------------------------------------------- 11
一、病原菌之形態---------------------------------------------------- 11
二、利用分子生物技術佐證小星辰花苗炭疽病菌的鑑定-------------------- 11
三、小星辰花苗炭疽病菌之寄主範圍------------------------------------ 12
小星辰花苗炭疽病菌之生理特性----------------------------------------- 12
一、溫度對小星辰花苗炭疽病菌菌絲生長的影響-------------------------- 12
二、溫度對小星辰花苗炭疽病菌分生孢子發芽與附著器形成之影響---------- 13
病害發生條件--------------------------------------------------------- 13
一、接種源濃度對病害發生之影響-------------------------------------- 13
二、保濕時間與病害發生之關係---------------------------------------- 13
三、溫度對病害發生之影響-------------------------------------------- 14
小星辰花苗炭疽病之防治----------------------------------------------- 14
一、不同拮抗微生物對小星辰花苗炭疽病菌之影響----------------------- 14
二、鏈黴菌S. padanus PMS-702培養濾液對小星辰花苗炭疽病菌孢子發芽之影響-------------------------------------------------------------- 14
三、利用高效能液相色層分析儀分析S. padanus PMS-702培養濾液及治黴色基素
-------------------------------------------------------------- 15
四、治黴色基素對小星辰花苗炭疽病菌孢子發芽之影響------------------- 15
五、鏈黴菌S. padanus PMS-702培養濾液防治小星辰花苗炭疽病之效果-------------------------------------------------------------- 15
六、不同施用時間對鏈黴菌S. padanus PMS-702培養濾液防治小星辰花苗炭疽病
之效果--------------------------------------------------------- 16
七、不同施用次數對鏈黴菌S. padanus PMS-702培養濾液防治小星辰花苗炭疽病之效果--------------------------------------------------------- 16
討論------------------------------------------------------------------- 17
引用文獻--------------------------------------------------------------- 21
中文摘要--------------------------------------------------------------- 24
英文摘要--------------------------------------------------------------- 27
圖表------------------------------------------------------------------- 30
附錄------------------------------------------------------------------- 58
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