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【目的】研究枯草芽孢杆菌(Bacillus subtilis)C-D6菌株对辣椒炭疽菌(Colletotrichum capsici)附着胞形成的抑制作用,探索炭疽病生物防治的新途径。【方法】通过对峙培养测定C-D6菌株的抗菌活性,应用摇瓶培养结合生物测定筛选产生抗菌活性成分的最适培养基,采用硫酸铵分级沉淀、Sephadex G-75凝胶柱层析和阴离子交换层析对抗菌蛋白进行分离纯化,应用聚丙烯酰胺凝胶电泳测定蛋白分子量。【结果】C-D6菌株在PDA平板上对辣椒炭疽菌显示明显的抑制作用,其YPD培养液能完全抑制该菌的附着胞形成。摇瓶培养的结果显示C-D6菌株产生抗菌活性物质的最适培养基为YPD培养基。C-D6菌株在该培养基中培养14 h后,所形成的活性物质可完全抑制辣椒炭疽菌的附着胞形成。从该菌的YPD培养液中分离获得一个分子量为32 k D,能明显抑制辣椒炭疽菌附着胞形成的抗菌蛋白。【结论】C-D6菌株的生防特征显示该菌株对防治辣椒炭疽菌引起的炭疽病具有潜在的应用价值。
【Objective】 The objective of this study was to investigate the inhibitory effect of Bacillus subtilis C-D6 on the attachment of Colletotrichum capsici and to explore a new approach to the biological control of anthracnose. 【Method】 The antibacterial activity of C-D6 strain was determined by confrontation culture. The optimal medium for producing antibacterial active ingredients was screened by shake flask culture and bioassay. The fractions were precipitated by ammonium sulfate fractionation, Sephadex G-75 gel column chromatography and anion Exchange chromatography on the antimicrobial protein separation and purification, the use of polyacrylamide gel electrophoresis protein molecular weight determination. 【Result】 The results showed that C-D6 strain showed obvious inhibition on pepper anthracnose on PDA plate, and its YPD culture medium could completely inhibit the attachment formation of the strain. The results of shake flask culture showed that the optimum culture medium of C-D6 strain for producing antibacterial active substance was YPD medium. After incubation of the C-D6 strain in this medium for 14 h, the resulting active substance completely inhibited the attachment of C. capsici. An antimicrobial protein with a molecular weight of 32 kD was isolated from the YPD culture of this strain, which can significantly inhibit the formation of anthracnose colonies. 【Conclusion】 The biocontrol characteristics of C-D6 strain showed that this strain has potential application in the prevention and control of Anthracnose induced by pepper anthrax.