南极沉积物来源抗菌细菌的筛选及抑菌物质的鉴定

Screening of antimicrobial bacteria from Antarctic sediments and identification of antagonistic substance

摘要:

【背景】科学研究表明，由于南极环境条件特殊，微生物资源丰富，有望筛选出功效显著的抗菌微生物。【目的】以黄瓜枯萎病致病菌木贼镰刀菌(Fusarium equiseti)为指示菌，从南极沉积物中分离筛选具有拮抗作用的细菌菌株并对其抑菌物质进行初步鉴定。【方法】应用平板对峙法和琼脂扩散法分别对样品和发酵液进行初筛和复筛，筛选出对F. equiseti抑菌效果最强的菌株，基于形态学、生理生化、分子生物学分析，对该菌株进行鉴定。之后，对目标菌株发酵上清中的抑菌物质进行抑菌谱研究，并对其抑菌成分进行温度和pH的稳定性检测，通过硫酸铵沉淀的方法初步鉴定发酵液中的抑菌物质。【结果】从南极沉积物样品中共分离纯化出62株细菌，有5株具有较好的抑菌效果，其中抑菌效果最强的菌株鉴定为枯草芽孢杆菌斯氏亚种(Bacillus subtilis subsp. spizizenii)，命名为JYM35。抑菌谱检测结果显示，菌株JYM35对丝瓜枯萎病致病菌层生镰刀菌(Fusarium proliferatum)和辣椒枯萎病致病菌F. equiseti具有较强的抑菌效果，对长豆褐腐病致病菌笄霉属(Choanephora)有较明显的拮抗作用，对水产致病菌副溶血弧菌(Vibrio parahaemolyticus)和溶藻弧菌(V. alginolyticus)也有一定的拮抗作用。菌株JYM35发酵上清中所含的抑菌物质热稳定性强且耐碱不耐酸，硫酸铵沉淀后可初步确定其抑菌物质隶属蛋白类。【结论】菌株JYM35是一株产蛋白类活性物质的广谱型抑菌菌株，对枯萎病致病菌的拮抗作用最强。因此具有较好的开发利用价值。

Abstract:

[Background] Scientific research shows that due to the special environmental conditions in Antarctica and the abundance of microbial resources, it is expected to screen out highly effective antimicrobial microorganisms. [Objective] To isolate and screen out antimicrobial strain from Antarctic sediments and to make preliminary identification of antibacterial substance, by using the pathogen of cucumber fusarium wilt named F. equiseti as indicator fungus. [Methods] The plate-based confrontation method was used to screen the strain from sediment samples and fermentation broth, with the strongest antimicrobial effect on Fusarium equiseti. Based on morphological, physiological, biochemical, molecular biology analysis, the strain was identified. Then the antimicrobial spectrum of antimicrobial substance in the fermentation broth was studied, and the stability of the antimicrobial component was detected under different temperatures and pH conditions, and the antimicrobial substance in fermentation broth was identified by the method of ammonium sulfate precipitation. [Results] A total of 62 bacteria were isolated from the Antarctic sediment samples, and 5 were with better antiseptic effects, of which the most effective strain was identified as Bacillus subspedestion (Bacillus subtilis subsp. spizizenii) and named JYM35. The result of antimicrobial spectrum showed that JYM35 is of strong antagonistic effect on the pathogen of towel gourd fusarium wilt named F. proliferatum and pathogen of pepper fusarium wilt named Fusarium equiseti, and has antagonistic effect on brown rot of long-bean named Choanephora, and also has certain antimicrobial effect on aquatic pathogen named Vibrio parahaemolyticus and V. alginolyticus as well. The antagonistic substance contained in the fermentation broth of JYM35 has the characteristic of high thermal stability, and it is resistant to alkali but not to acid. Through ammonium sulfate precipitation, it can be preliminarily determined that the antagonistic substance belongs to proteins. [Conclusion] Strain JYM35 is a broad-spectrum antimicrobial strain that produces protein-like active substance and has the strongest antagonistic effect against fusarium wilt.