Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31742
標題: 鏈黴菌PMS-702防治作物病害的功效與其抑菌主要代謝物治黴色基素之鑑定
Control of Crop Diseases with Streptomyces padanus PMS-702 and Identification of Fungichromin as Its Major Antifungal Metabolite Related to Suppress Plant Pathogens
作者: 石信德
Shih, Hsin-Der
關鍵字: biocontrol
生物防治
Streptomyces padanus
antifungal substance
fungichromin
bioassay
Rhizoctonia solani
Phytophthora infestans
polyene macrolide
鏈黴菌
抑菌代謝物
治黴色基素
生物檢定分析
立枯絲核菌
番茄晚疫病
多烯類大環內酯
出版社: 植物病理學系
摘要: 從台灣各地農田及栽培介質中分離獲得200株放線菌,196株屬於鏈黴菌屬(Streptomyces),其餘4株放線菌分屬於Actinomadura sp.、Microbispora sp.、Herbidospora sp.及Streptosporangium sp.。取鏈黴菌PMS-101、PMS-502及PMS-702菌株分別與Acremonium diospyri、Colletotrichum gleosporioides、Fusarium oxysporum f. sp. conglutinans、F. oxysporum f. sp. niveum、F. oxysporum f. sp. lactuctum、F. oxysporum f. sp. raphani、F. proliferatum、Pestalotiopsis eriobotryfolia、Pythium myriotylum、P. aphanideratum、Rhizoctonia solani等11種植物病原真菌進行對峙培養測定,結果PMS-702菌株具有最佳的拮抗能力。進一步取PMS-702再與其他20種植物病原真菌與9種植物病原細菌進行對峙培養測定,結果顯示PMS-702對於各種植物病原真菌均具有不同程度的拮抗能力,但是對於白絹病菌與植物病原細菌則均不具有拮抗能力。利用傳統鑑定方法發現PMS-702菌體的細胞壁含L-二氨基庚二酸(L-diaminopimelic acid; L-DAP),全細胞中不含特殊糖類,屬於Chemotype IC型,是Streptomyces 屬內的一個種。它在ISP2 (International Streptomyces Project Medium 2), ISP3及ISP4培養基上生長及產孢情形良好,在ISP2及ISP4培養基上可以產生黃色色素,但不產生黑色素;其營養菌絲呈灰黃色至橘黃色;氣生菌絲為灰黃褐色或淺灰色。在掃描式電子顯微鏡觀察PMS-702菌株,發現孢子鏈生呈螺旋狀排列,孢子數目超過20個,表面平滑。PMS-702菌株可利用的醣類有:D-glucose, D-fructose, D-xylose, D-mannitol, cellulose;可分解starch, casein, 及hypoxanthine。將PMS-702菌株之形態、生理、生化特徵及16S rRNA全長度基因序列分別與S. galbus CCRC12166及S. padanus CCRC12168等菌株的特性比對後,確定PMS-702菌株為Streptomyces padanus Baldacci. et al.。 利用葡萄糖-糖蜜培養基(GMM)、葡萄糖-黃豆粉-甘油醇煎汁(GSG)、幾丁質煎汁(MCB)、黃豆粉-魚粉-幾丁質煎汁(SFC)、黃豆粉-葡萄糖煎汁(SMG)及胰酪蛋白-酵母抽出物-葡萄糖煎汁(TYG)等六種培養基分別培養 Streptomyces padanus PMS-702,發現在振盪培養第7天後的菌絲乾重以在GSG、GMM及SMG生產的PMS-702菌絲乾重較為良好,其中又以在SMG生育最佳。利用香蕉黃葉病菌作為評估PMS-702在不同營養源中生長的抑菌功效指標菌種,結果發現PMS-702在SMG中培養7天的10倍稀釋濾液,可完全抑制孢子發芽。挑選SMG配方進行4天批式醱酵生產試驗,然後利用植株、果實或切離葉生物檢定醱酵培養液,結果發現S. padanus PMS-702的培養液對於萵苣褐斑病(由Acremonium lactucum引起)、芒果炭疽病菌(由Colletotrichum gloeosporioides引起)、白菜炭疽病(由 C. higginsianum引起)、柑桔綠黴病 Penicillium digitatum引起)、甘藍露菌病(由 Peronospora brassicae引起)及桃果實疫病(由Phytophthora citrophthora引起)均具有防治的效果。綜合上述研究成果,證明 S. padanus PMS-702是一株具有研發成為植物保護製劑潛力的菌種。 利用對峙培養檢測Streptomyces padanus PMS-702菌株對立枯絲核菌(Rhizoctonia solani)的拮抗作用,結果顯示其對於6種不同作物的7個立枯絲核菌菌株,均具有拮抗能力。以PMS-702製劑粉衣處理甘藍種子,可以有效減少立枯絲核菌第四融合群引起甘藍幼苗立枯病的發生。利用數種溶劑分別萃取PMS-702的醱酵濾液及菌絲層,結果以乙酸乙酯萃取之活性物質最多,且該兩部分萃取物均具有抑制R. solani, AG-4的生物活性。隨後萃取二者混合之全醱酵液獲得初萃取物,經矽膠管柱層析法(Silica Gel Chromatography),利用不同比例溶劑分別得到4個沖提區分(PM1-PM4)。進一步由這些沖提區分純化出1、2、3及4號等四種化合物。其中具有抑菌活性的1號化合物,係為非結晶性淡黃色粉末,主要紫外光吸收光譜分佈於 356-357, 337-338, 340,及320-322 nm。綜合1號化合物之紅外線光譜、核磁共振光譜及質譜儀圖譜等相關分析資料,顯示其歸屬於多烯類大環內酯(polyene macrolide),即為治黴色基素(fungichromin; C35H58O12)。其他不具有生物活性的2號化合物,經鑑定為sterol glycoside;此外由PM2沖提區分,尚可純化出屬於類黃酮類的daidzein;至於4號化合物為色素物質。治黴色基素對於立枯絲核菌具有抑制作用,其最小抑菌濃度為72 μg/ml,本研究係首次發現從S. padanus PMS-702可以產生抑制立枯絲核菌的治黴色基素。 利用對峙培養方式評估Streptomyces padanus PMS-702對茄科晚疫病菌之拮抗功效,結果顯示S. padanus PMS-702對於來自番茄或是來自馬鈴薯的10個供試的菌株均具有抑制作用。利用PMS-702製劑分別於農委會種苗場及農試所進行三次田間番茄晚疫病防治試驗,結果顯示PMS-702製劑可以有效防治番茄晚疫病。利用PMS-702培養液純化的四種萃取物質,sterol glycoside, daidzein, fungichromin及4號化合物各200 ppm,分別探討它們對於晚疫病菌的孢囊產生及游走孢子釋放之影響,結果發現僅治黴色基素具有完全的抑菌功效。不同濃度的PMS-702培養濾液及治黴色基素,均有不同程度抑制番茄晚疫病菌孢囊產生、孢囊發芽、游走孢子釋放及靜止子發芽的效果,其中抑制效果隨著兩者稀釋倍數提高而下降。利用光學及電子顯微鏡觀察S. padanus PMS-702、PMS-702培養濾液及治黴色基素對晚疫病菌的影響,發現PMS-702菌株可使晚疫病菌的孢囊表面呈現泡狀皺縮;此外PMS-702培養濾液與治黴色基素可使孢囊內原生質凝聚或破裂,致游走孢子無法產生或釋放。若游走孢子釋放後接觸到PMS-702濾液或治黴色基素亦會呈現細胞質凝聚及瓦解的現象,此外病原菌菌絲表面結構也會遭受其破壞。本研究結果證明polyene macrolide之治黴色基素是PMS-702製劑抑制番茄晚疫病菌的主要活性物質,其對游走孢子釋放的最小抑菌濃度(minimum inhibitory concentration ; MIC, 大於90% 抑制能力)為5 ppm。
More than 200 strains of Actinomycetes were isolated from farmland soils and agriculture wastes in Taiwan. Among those, 98% of the strains were identified as Streptomyces spp. The other 2 % strains belonged to the genus Actinomadura, Herbidospora, Microbispora, and Streptosporangium. Thirty isolates of plant fungal pathogens and nine strains of plant bacterial pathogens were used to bioassay the antagonistic ability of Streptomyces sp. PMS-702 on potato dextrose agar plates. Acremonium diospyri, A. lactucum, Alternaria brassicicola, Athelia rolfsii (Sclerotium rolfsii), Botrytis cinerea, B. elliptica, Colletotrichum dematium, C. higginsianum, C. gloeosporioides, Fusarium oxysporum f. sp. cubense, F. oxysporum f. sp. conglutinans, F. oxysporum f. sp. lactucum, F. oxysporum f. sp. lilii, F. oxysporum f. sp. niveum, F. oxysporum f. sp. raphani, F. moniliforme, F. proliferatum, F. solani, Mycosphaerella pinodes, Phellinus noxius, Penicillium digitatum, Pestalotiopsis eriobotryfolia, Phytophthora capsici, Ph. citrophthora, Ph. infestans, Ph. palmivora, Ph. parasitica, Pythium aphanidermatum, P. myriotylum, and Rhizoctonia solani AG-4. were inhibited by PMS-702 with varying degrees. However, the PMS-702 was not able to inhibit Scleroium rolfsii and nine plant bacterial pathogens. The results of spore morphology, cell wall chemotype, cultural and physiological characterization and the molecular characteristics suggested that the strain PMS-702 is identified as Streptomyces padanus Baldacci, et al. Six media, Glucose-Molasses medium (GMM), Glucose-Soybean meal-Glycerol Broth (GSG), Modified Chitin Broth (MCB), Soybean meal-Fish meal-Chitin Broth (SFC), Soybean meal-Glucose Broth (SMG) and Tryptone-Yeast extract-Glucose Broth (TYG), were evaluated for culturing Streptomyces padanus PMS-702 in shaking flasks. Higher biomasses of S. padanus PMS-702 mycelia were harvested from GSG, GMM, and SMG. Among them, the SMG broth was much more suitable for growth of S. padanus PMS-702. The culture filtrate of PMS-702 grown in SMG for 7 days was able to completely inhibit spore germination of indicator fungus, Fusarium oxysporum f. sp. cubense. In the scale-up production, S. padanus PMS-702 was grown in a 5L fermentor containing 3 L of SMG for 4 days. Then whole plants, fruits or detached leaves were used to bioassay its suppressive ability. The culture broth of S. padanus PMS-702 was effective in controlling lettuce brown spot caused by Acremonium lactucum, mango anthracnose caused by Colletotrichum gloeosporioides, Chinese cabbage anthracnose caused by C. higginsianum, peach fruit rot caused by Phytophthora citrophthora, orange green mold caused by Penicillium digitatum, cabbage downy mildew caused by Peronospora brassicae. These results suggested that S. padanus PMS-702 is a potential agent for developing a bioprotectant for controlling plant fungal diseases. Streptomyces padanus strain PMS-702 was an antagonist of Rhizoctonia solani AG-4, the causal agent of damping-off of cabbage. Treatment of cabbage seeds with the culture filtrate of S. padanus strain PMS-702 was effective in reducing incidence of damping-off of cabbage. The major active ingredient from the culture filtrate of S. padanus strain PMS-702 was purified by silica gel column chromatography and identified as the polyene macrolide, fungichromin, by one dimentional (1H NMR, 13C NMR) and two dimentional (HMQC, HMBC) nuclear magnetic resonance and Mass (FAB-MS) spectral data. Bioassay studies showed that fungichromin had a strong antifungal activity against R. solani AG-4, and its minimum inhibitory concentration (over 90% inhibition) was found to be 72 g/ml. This is the first report of fungichromin from S. padanus as an active ingredient for the control of Rhizoctonia damping-off of cabbage. Streptomyces padanus strain PMS-702 was a potential biocontrol agent of tomato late blight, caused by Phytophthora infestans. Laboratory and field tests indicated that Streptomyces PMS-702 formulation was effective in reducing tomato late blight. Four compounds, sterol glycoside, daidzein, fungichromin and unknown compound 4 were obtained from culture filtrate of S. padanus PMS-702. Among them , fungichromin was completely effective in reducing sporangial sporulation and zoospores released from zoosporangia. The advanced experiments proved that broth culture filtrate and fungichromin were significantly effective in inhibiting sporangial sporulation, sporangial germination, zoospores released from zoosporangia and cytospore germination of Ph. infestans, respectively. These effects were negatively related with the dilution rate of culture filtrate and fungichromin. The phenomena under observations of light microscope and scanning electron microscope indicated that fungichromin was able to incite substantial plasma agglutination, cell malformation and cell collapse of the treated sporangia and zoospores of Ph. infestans. It did also cause zoospore membrane rupture and plasma leakage. The minimum inhibitory concentration of fungichromin against zoospore release (over 90% inhibition) was at 5 ppm. These results suggested that fungichromin played an important role in the mode of action of PMS-702 formulation for controlling tomato late blight.
URI: http://hdl.handle.net/11455/31742
Appears in Collections:植物病理學系

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