液化澱粉芽孢桿菌在作物病害防治的開發與應用

Development and Application of Bacillus amyloliquefaciens on Plant Diseases Biocontrol

郭建志(Chien-Chih Kuo) ； 陳俊位(Chien-Wei Chen) ； 廖君達(Chung-Ta Liao) ； 陳葦玲(Wei-Lin Chen) ； 蔡宜峯(Yi-Fong Tsai)

《臺中區農業改良場特刊》 121號 (2014/03) Pp. 69-86

摘要

液化澱粉芽孢桿菌（Bacillus amyloliquefaciens）為芽孢桿菌（Bacillus spp.）的一種，屬革蘭氏陽性菌，具有周生鞭毛、好氣性、活動力強及無莢膜的細菌。大多存在於土壤、水、空氣及植物體內外。近年來，對於液化澱粉芽孢桿菌防治作物病害的研究日益增多。有關防治作物病害的機制包括可以產生多種抗生物質如iturin A、surfactin與fengycin等，可抑制病原菌的生長；菌絲可纏聚在植物根圈並誘導植物產生防禦反應，進而抵抗病原微生物的入侵，可分泌多種分解酵素如蛋白質分解酵素（protease）、纖維分解酵素（cellulase）、澱粉分解酵素（amylase）與脂肪分解酵素（lipase），某些菌株具有溶磷活性（phosphate-solubilizing），有利於作物吸收養分，促進植株生長；複合揮發性物質（volaticle organic compounds，VOC）的產生；具有植物根圈促生細菌（plant growth promoting rhizobacteria，PGPR）功能，並誘導植物產生抗病反應。目前研究學者多以gyrB基因進行液化澱粉芽孢桿菌序列分析與比對，可有效地鑑定與區分枯草桿菌群中相似度高的芽孢桿菌菌種。本場自轄內作物之田間土壤中，分離並篩選數十株具有防治潛力之芽孢桿菌，利用培養基與多種植物病原真菌與細菌拮抗測試、耐熱活孢子數測定、生物活性等測試，篩選出1株對數種病原真菌Fusarium spp.與病原細菌具有優異之拮抗效果。此菌株經由細菌自動鑑定系統（Biolog system）、16S rRNA與gyrB gene序列分析後，鑑定為液化澱粉芽孢桿菌（Bacillus amyloliquefaciens Tcba05）。利用PCR技術可自Tcba05菌株之核酸中，增幅出伊枯草菌素A（iturin-A）、表面活性素（surfactin）之專一性DNA片段，顯示Tcba-5菌株具有產生抗生物質之能力。經由少量搖瓶量產測試後，委由中興大學進行750 l醱酵試量產試驗。其菌量經4天醱酵後可達10^10 cfu/ml，內生孢子亦達10^9 cfu/ml。經過5個月儲放後，其菌量仍可維持在10^9 cfu/ml。後續針對豇豆萎凋病與甘藍黑腐病進行防治評估，依據溫室與先期田間試驗結果，本場所篩選之液化澱粉芽孢桿菌B. amyloliquefaciens Tcba05菌株液態製劑及TCB9407，101-007與TC2-8菌株，均可有效降低萎凋病菌之罹病度達40%、甘藍黑腐病之罹病度達30-40%，顯示此菌株具有病害防治之潛力，後續將進行其他蔬菜作物病害之防治評估，並完成商品化登記之要件，以利加速推動生物農藥製劑商品化之時程。

關鍵字

液化澱粉芽孢桿菌 ； 生物農藥 ； 抗生物質

並列摘要

Bacillus amyloliquefaciens is one kind of Bacillus spp. It is belong to the B. subtilis group of aerobic, Gram-positive, peritrichous flagella, endospore-forming, no capsule and rod-shaped microorganisms. Research on the plant pathogen control by B. amyloliquefaciens is progressively increased. Bacillus amyloliquefaciens could produced various enzymes, included cellulose, protease, lipase and amylase. Some strains have phosphate-solubilizing activity. The lipopeptides produced from B. amyloliquefaciens can also influence the ecological fitness in terms of root colonization and also have a key role in the beneficial interaction with plants by stimulating host defence mechanisms. In this study, we isolated a dozens of microorganisms which have antimicrobial effect from soil. We select the Bacillus sp. Tcba05 strain which could inhibit the mycelium growth of Fusarium oxysporum by the antagonistic test. Used the Biolog system, 16S rRNA and gyrB gene identified Tcba05 strain as Bacillus amyloliquefaciens. The iturin-A and surfactin specific DNA fragment could be amplified by PCR technique. Results showed that B. amyloliquefaciens Tcba05 could produce the antibiotics. We commissioned National Chung-Hsien University produced 50 l and 750 l Tcba05 fermentation broths, the total colony count could reach to 10^10 cfu/ml after 4 days ferment, endospores number could reach 10^9 cfu/ml, the total colony count still can maintain to 10^9 cfu/ml after 5 months. We evaluated the bio-control effectivity of Tcba05 on fusarium blight of asparagus bean in greenhouse and infected field. The disease severity could reduce 50% and 40%, respectively. The 4 strains of B. amyloliquefaciens TCB9407, 101-007, TC2-8 and Tcba05 fermentation broth used in seed treatment can effectively reduce black rot infection rate. Application of different broths on cabbage in the field, cabbage black rot disease severity could reduced 30 to 40%. The results showed that the Tcba05 fermentation broth could effective reduce the fusarium wilt and black rot disease severity, respectively. It expects to commercialize with Bacillus amyloliquefaciens agent as soon as possible.

並列關鍵字

Bacillus amyloliquefaciens ； Biopesticide ； Antibiotics