枯草芽孢杆菌C-D6对辣椒炭疽菌附着胞形成的抑制作用研究

Bacillus subtilis C-D6 as a potential biocontrol agent against appressorium formation of Colletotrichum capsici

摘要:

【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 kD，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。

Abstract:

[Objective] To explore new pathways for biocontrol of anthracnose by investigating the inhibitory effect of Bacillus subtilis C-D6 against appressorium formation of Colletotrichum capsici. [Methods] The antifungal activity of B. subtilis C-D6 against C. capsici was evaluated by dual-culture method. Optimal medium for production of antifungal active substances was determined in shake flask culture. The antifungal proteins were purified by ammonium sulfate precipitation, followed by Sephadex G-75 column chromatography and anion-exchange chromatography. The molecular mass of the purified proteins were determined by SDS-PAGE. [Results] B. subtilis C-D6 showed high antagonistic activity and inhibitory effect of appressorium formation against C. capsici. The optimum medium for the production of antifungal substances of C-D6 strain was YPD and 14 h after inoculation, the culture filtrate had a significant inhibitory effect on appressorium formation of C. capsici. An antifungal protein, with a molecular mass of 32 kD, was purified from culture filtrates of C-D6 strain and the purified protein exhibited obvious inhibitory activity against appressorium formation of C. capsici. [Conclusion] Our results suggest that B. subtilis C-D6 could be a potential bioagent for control of anthracnose caused by C. capsici.