侧孢短芽孢杆菌与枯草芽孢杆菌共培养抗菌活性增强的生态学机理

Bacillus sp. is widely used in the field of biological control of plant diseases and insect pests for the antibiotics producing ability. But there is limited published information concerning on the co-metabolism of Bacillus bacterium. Brevibacillus laterosporus BL-21 and Bacillus subtilis HNDF2 were isolated from Heilongjiang Province farmland soil and had antagonistic activity against Fusarium spp., The antifungal activity of co-cultures with the two is obviously better than mono-cultures, but the ecology mechanism is not clear. This project aims to study the potential of induction mode in antifungal discovery from Brevibacillus laterosporus co-cultured with Bacillus subtilis HNDF2 by biological activity assay, clear the threshold value of HNDF2 population density and metabolite concentration that can stimulate BL-21 producing antibiotics, analysis dynamics of microbial in co-culture system by flow cytometry, detect relationship between carbon and nitrogen metabolism and co-culture active metabolites according to the biological characteristics of two species of Bacillus, find signal molecules using proteomics and metabolomics, separate, identificate and bioinformatics analysis new antibacterial substances. Finally, all of the results should be integration analysis to reveal the co-culture ecology mechanism of enhanced antifungal activity.

芽孢杆菌属细菌能产生多种抗生物质，在植物病虫害生物防治领域应用较为广泛，但是对其共培养代谢产物的研究报道较少。侧孢短芽孢杆菌BL-21和枯草芽孢杆菌HNDF2是从黑龙江省农田土壤中分离到的生防菌株，二者共培养的抗菌活性比单菌培养有明显增强，但是其共培养抗菌活性增强的生态学机理不清楚。本项目拟通过生物活性检测法研究枯草芽孢杆菌HNDF2诱导方式不同对侧孢短芽孢杆菌BL-21产抗的影响，明确HNDF2刺激BL-21产抗的群体密度和代谢产物浓度阈值；采用流式细胞仪分析共培养体系中微生物的消长动态；根据两种芽孢杆菌的生物学特性，检测微生物碳氮代谢与共培养活性代谢产物产生的关系；利用蛋白质组学和代谢组学寻找种群间或种群内信号分子、分离鉴定新的抗菌活性产物，并进行生物信息学分析；将各项结果整合分析，以揭示拮抗菌共培养抗菌活性增强的生态学机理。

本项目利用直接共培养和间接共培养方法，完成了枯草芽孢杆菌HNDF2不同诱导方式对侧孢短芽孢杆菌BL-21产抗的影响，结果表明：BL-21产抗菌物质的活性受HNDF2 群体密度和代谢产物的影响。当HNDF2的营养体密度为1×107 CFU/mL，芽孢密度为1×105 CFU/mL时对BL-21产抗的促进作用最强。HNDF2细胞壁的肽聚糖成分没有刺激BL-21产抗的作用。HNDF2刺激BL-21产抗的代谢产物不耐高温，其碳代谢产物有显著的抑制或阻遏BL-21产抗的作用，其中甘露糖代谢产物对BL-21产抗的抑制作用最明显；而其氮代谢产物则对BL-21产抗的效果有显著增强作用，其中酵母粉代谢产物刺激BL-21产抗的效果最显著。对HNDF2外泌的刺激BL-21的物质进行了分离及质谱分析，结果表明HNDF2外泌的分子量11651.2 D的蛋白质对BL-21产抗有显著刺激效果，生物信息学分析表明该蛋白与解淀粉芽孢杆菌Bacillus amyloliquefaciens subsp. plantarum str. FZB42的假定蛋白相匹配。两种微生物接种时间不同，导致共培养体系中两种微生物的动态消长规律及主体代谢产物发生显著变化。当BL-21先接或与HNDF2同时接入时，BL-21是优势菌，共培养主产物是较大分子的抗菌蛋白和抗菌肽；当BL-21比HNDF2较晚接入时，HNDF2是优势菌，共培养主产物是较小分子的脂肽类物质。对HNDF2与BL-21同时接种的共培养代谢产物进行了分离和类型分析，发现和纯培养相比，共培养液的抗菌蛋白和抗菌肽的产量和种类有明显增多，有新物质产生。已经从共培养代谢产物中分离出两种相对分子质量分别为1 304.604和1 292.730的新型小肽类抗菌物质。从HNDF2先接种的共培养液中通过酸沉醇提的方法得到了较纯培养多的脂肽类抗菌物质。对共培养代谢产物的抗菌机理进行了研究，结果表明共培养代谢产物通过抑制植物病原真菌孢子的萌发、菌丝体的生长、破坏细胞壁和细胞膜结构等多种方式起到抗真菌的作用。上述研究结果为深入研究微生物共培养以及开发新型生物农药提供了科学依据。