贝莱斯芽孢杆菌S6拮抗物质分离纯化及抑菌机理

Purification and Inhibitive Mechanism of Antagonist Substances from Bacillus velezensis S6 Strain

[目的]旨在明确新型生防细菌贝莱斯芽孢杆菌S6的拮抗物质和抑菌作用。[方法]拮抗物质的研究方法包括硫酸铵沉淀法、DEAE-Sephrose^(TM) Fast Flow阴离子交换、Superdex TM75凝胶层析及SDS-PAGE电泳分析;抑菌作用的研究方法包括菌丝生长速率法和孢子萌发法。[结果]贝莱斯芽孢杆菌S6拮抗物质对多种病原菌具有很好的抑制作用。采用80%饱和度(NH_4)_2SO_4沉淀法提取贝莱斯芽孢杆菌S6的发酵液粗提物,粗提物通过DEAE-Sephrose TM Fast Flow阴离子交换和Superdex^(TM) 75凝胶层析进行纯化,经SDS-PAGE电泳检测到单一的条带,其分子质量为20.1 kDa。贝莱斯芽孢杆菌S6的拮抗蛋白质量浓度为5、10 g/L时,对番茄早疫病菌的抑制率分别为71.13%、90.33%;质量浓度为1 g/L时,对早疫病菌的孢子萌发抑制率为60.42%,质量浓度达到10 g/L时,孢子几乎不萌发。拮抗蛋白处理的菌丝发生了扭曲、畸形、肿大。[结论]本研究结果为利用贝莱斯芽孢杆菌S6开发新的生防制剂奠定了基础。

[Aims] This paper aims to clear antagonistic substances and inhibitory effect era new bio-control bacterium Bacillus velezensis.S6. [Methods] The research methods of antagonistic substance included ammonium sulfate precipitation method, DEAE-SephroseTM anion exchange, Superdex^TM 75 chromatography and SDS-PAGE electrophoresis. The research methods of inhibitory effect included mycelial growth raw and spore germination rate. [Results] The antagonistic substance of B. velezensis S6 had good inhibitory effect to some plant pathogenic fungi. Crude substance was extracted from cell-flee culture broth of B. velezensis S6 by 80% （w/v）（NH4）2SO4 saturation precipitation method, crude substance was purified by DEAE-SephroseTM anion exchange and SuperdexTM 75 chromatography, the single band was observed by SDS-PAGE electrophoresis, its relative molecular mass was 20.1 kDa. The inhibitory rate of mycelial growth of Altemaria solani on tomato were 71.13 and 90.33% respectively at the concentration of antagonistic protein of 8. velezensis S6 of 5 and 10g/L, and the inhibitory rate of spore germination was 60.42% at the concentration of 1 g/L, the spores hardly germinated at concentration of 10g/L. The mycelia treated by the antagonistic protein were distortion, deformation and swelling. [Conclusions] The results laid foundation for developing new biocontrol agents based on B. velezensis S6.