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研究生:何炳璋
研究生(外文):Biing-Chang Ho
論文名稱:鏈黴菌StreptomycessioyaensisPMS502量產及其於病害防治之應用
論文名稱(外文):The mass production fo Streptomyces sioyaensis PMS502 and its application in disease control
指導教授:曾德賜
指導教授(外文):Dean Der-Syh Tzeng
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:2001
畢業學年度:89
語文別:中文
論文頁數:73
中文關鍵詞:鏈黴菌抗生素液態發酵甘藍幼苗猝倒病甘藍幼苗立枯病生物製劑
外文關鍵詞:Strepomyces sioyaensisantibioticliquid fermentationPythium aphanidermatumRhizoctonia solani AG-4biofungicide
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鏈黴菌 Streptomyces sioyaensis PMS502 量產及其於病害防治之應用
何炳璋
本研究之供試菌株 Streptomyces sioyaensis PMS502 係由本系病害管理研究室所提供,研究目的在探討營養因子對供試菌生長、抗生物質產生之影響,及供試菌對病原真菌可能之拮抗機制,希望能用於未來量產技術之改進與生物製劑應用之參考。利用 Czapek''s broth medium 為基礎培養基進行連續震盪培養,供試氮、碳素源中分別以硫酸銨及蔗糖最有利供試菌的生長,在 Czapek''s broth medium 培養中則不易偵測到抗生物質產生。利用 1% 燕麥煎汁培養基為基礎培養基可改善供試菌生長,特別是抗生物質生合成,伴隨抗生物質產生,培養基 pH 會隨之下降。九種供試穀物培養基中,燕麥煎汁培養基最有利於供試菌產生抗生物質,以 1 — 2% (w/v) 為最適之濃度,添加幾丁質與酵母粉對抗生物質產生亦有明顯促進效果。11 種供試碳素源中,添加幾丁質對產生抗生物質的促進效果最明顯,其餘碳素源除了纖維素外,對抗生物質產生或多或少具有抑制作用;添加 8 種供試氮素源對抗生物質生合成亦有抑制作用。進一步利用對供試菌生長及產生抗生物質皆具優勢之燕麥粉液體培養基,以 5 L 發酵槽進行初步量產。在發酵量產中,添加幾丁質與纖維素可促進供試菌生長與產生抗生物質。藉由發展中之液態發酵操作流程,生物活體的最高產量可達 4 × 108 cfu/ml。供試菌培養於燕麥及玉米煎汁培養基之培養濾液以 Sep-Pak C18 cartridge 管柱吸附後,可以 70% 甲醇溶液可將抗生物質流洗出來,利用 HPLC 進行分離純化,皆於延滯時間 13 — 14 分鐘可檢測出一個主要的吸收峰,並可於回收留洗液中測得拮抗活性,顯示兩培養基中所產生的抗生物質為同一物質。進一步探討抗生物質對病原菌的抑制機制,以 Sep-Pak C18 萃取之抗生物質處理 Alternaria brassicicola, Pythium aphanidermatum 及 Rhizoctonia solani AG-4,於光學顯微鏡下鏡檢,發現抗生物質具有殺死菌絲效果,與導致菌絲尖端有細胞內容物溶出的現象,並且可抑制分生孢子的發芽。P. aphanidermatum 及 R. solani AG-4 菌絲液經抗生物質處理後,明顯有電解質滲漏的現象,顯示細胞膜遭受嚴重的破壞。溫室試驗中,液態培養之供試菌以澆灌的方式施用,可明顯抑制由 P. aphanidermatum 引起之甘藍幼苗猝倒病及 R. solani AG-4 引起的幼苗立枯病的發病率,將培養菌液或濾液混拌於土中,亦可促進甘藍植株的生長。由上述實驗結果而推的證據顯示,S. soyaensis PMS502 確具有發展為生物製劑的潛力。
The mass production of Streptomyces sioyaensis PMS502 and its application in disease control.
Biing-Chang Ho
The main objectives of this investigation were to explore the nutritional requirement for the growth and antibiotic production and thus to provide useful information for the mass production of Streptomyces sioyaensis isolate PMS502 (kindly provided by professor J. W. Huang) for biofungicide application. The possible mode of action of the antagonistic effect of the tested isolate against pathogenic fungi was also investigated. In a shaking culture system (130 rpm, 30 ℃ in total darkness) in which Czapeks broth was applied as the basal medium, ammonium sulfate and sucrose were found among 8 nitrogen sources and 8 carbon sources, respectively, the best to support the growth of test bacterium. The antibiotic production however was hardly detectable from these Czapeks based cultures. The use of 1% oat decoction as a basal medium greatly improved the bacterial growth especially the antibiotic production. In accompany with the production activity, quite extencsive decrease of pH of the culture medium was generally observed. Oat decoction was among 9 grain-decoction broths tested the best to support the antibiotic production; the optimum concentration was around 1 to 2% (w/v). And the productivity was significantly enhanced by the supplementation of chitin and yeast powder. Chitin was among 11 carbon sources tested most effective in enhancing antibiotic production of the test bacterium. The rest of tested carbon sources, except cellulose, appeared to be inhibitory more or less to the antibiotic production in the oat decoction medium. The addition of 8-tested nitrogen sources was found to be inhibitory also. The superior efficacy of oat decoction broth in supporting the growth and antibiotic production of test bacterium was further demonstrated by scale up production trial in which a series of 5 L liquid fermentor system were used. And in the fermentor system, the growth and antibiotic production were both found enhanced by the addition of chitin and cellulose. By the established liquid fermentation operation protocol under development, the maximum yield of biomass has reached 4 × 108 cfu/ml. In order to illustrate the mode of action of the antifungal activity, the biochemical and biological characteristic of the antibiotic produced was further explored. The culture filtrate obtained from oat and corn decoction broth cultures were clarified by a Waters Sep-Pak C18 cartridge. The antibiotic retained on the column was eluted by 70% methanol. The followed high performance liquid chromatography (HPLC) by C18 reverse phase column demonstrated a major peak with expected antifungal activity at 13 — 14 min retention time. The identity of the major peak detected from both oat and corn decoction broth cultures indicated the presence of same antibiotic. The partial purified antibiotic preparation obtained after Sep-Pak C18 cartridge clarification was tested against Alternaria brassicicola, Pythium aphanidermatum and Rhizoctonia solani AG-4. The followed microscopy examination revealed the killig effect on tested fungal cells, the extrusion of cellular content at hyphal tip, and the inibition of conidia germination. Prominent electrolyte leakage was detected from mycelial culture of P. aphanidermatum and R. solani AG-4, shortly after antibiotic application, indicating the critical importance of membrane damage in the observed consequence. In greenhouse test, the drenching of broth culture of tested bacterium was shown effective in reducing the damping off infection of cabbage seedling by P. aphanidermatum and R. solani AG-4. By single application, the survival stand of tested cabbage seedling surveyed 2 weeks after seeding was greatly increased, and a prominent growth promotion effect on cabbage was observed. The evidences provided here indicated clearly the great potential of S. soyaensis PMS502 as a biofungicide.
目錄
壹、前言
貳、前人研究
參、材料方法
一、菌株來源、培養及接種源製備
二、藥品試劑來源及培養基種類
三、拮抗族譜之測試
四、接種原濃度對生長量與抗生活性之影響
五、營養因子對生長量及抗生活性之影響
(一)抗生活性之測試
(二)生長量之測定
(三)氮素源對生長量與抗生活性之影響
(四)碳素源對生長量與抗生活性之影響
(五)有機添加物對生長量與抗生活性之影響
(六)不同穀物培養基對抗生活性之影響
(七)燕麥粉煎汁培養基中之生長與抗生活性之時序關係
(八)不同燕麥濃度對抗生活性之影響
(九)添加不同濃度酵母粉對抗生活性之影響
六、初步量產測試
(一)燕麥、玉米及粉頭培養基添加酵母粉對生長量與抗生活性之影響
(二)攪拌速率對生長量與抗生活性之影響
(三)幾丁質及纖維素之添加對生長量與抗生活性之影響
(四)麥芽精、酵母精及酵母粉之添加對生長量與抗生活性之影響
七、抗生物質之初步萃取及生物分析
(一)抗生物質之萃取與純化
(二)高效液相層析 (HPLC)
(三)熱處理對抗生物質安定性之影響
(四)酸鹼值對抗生物質安定性之影響
八、抗生物質對植物病原菌之作用機制
(一)抗生物質對 Rhizoctonia solani AG-4與 Pythium aphanidermatum 菌絲體活性之影響
(二)抗生物質對 Alternaria brassicicola 孢子發芽之影響
九、PMS502 之生物防治應用性評估
(一)施用 PMS502 對甘藍幼苗猝倒病之防治效果
(二)施用 PMS502 對甘藍立枯病之防治效果
(三)施用 PMS502 對甘藍生長之影響
肆、結果
一、拮抗族譜之測試
二、接種原濃度對生長與抗生活性之影響
三、營養因子對生長及抗生活性之影響
四、初步量產測試
五、抗生物質之初步萃取及生物分析
六、抗生物質對植物病原菌之作用機制
七、PMS502 之生物防治應用性評估
伍、討論
陸、中文摘要
柒、英文摘要
捌、參考文獻
玖、圖表說明
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