解淀粉芽孢杆菌杀线虫活性高效筛选模型的建立及应用

Establishment and application of efficient nematicidal screening model in Bacillus amyloliquefaciens

【目的】食线虫细菌对线虫的侵染机制报道甚少,限制了生防细菌的在农业实践中的广泛应用。本研究中,我们构建了一种对细菌杀线虫活性进行快速高效筛选的方法来快速寻找与线虫侵染功能相关的基因。【方法】将培养在固体平板上的野生型解淀粉芽孢杆菌FZB42及其突变株接种于5mL液体快杀培养基中,37℃培养24h后,在24孔板中分别加入200μL菌液和50-60条L4龄秀丽隐杆线虫(Caenorhabditis elegans),记录变化明显的时间点及线虫存活数,将相对于原始菌株杀线虫能力明显下降的突变株选出,并进行多次复筛。同时以传统的固体平板杀线虫法作为对照。【结果】用液体快杀法在21h时筛选出了与原始菌株相比杀线虫活性显著下降的2株突变株F1和F2,与传统的固体平板杀线虫法结果一致,且极大地缩短了筛选时间。【结论】该研究为下一步鉴定食线虫细菌中的毒力基因、进而阐明病原细菌侵染宿主的分子机制提供了良好的生物材料,并缩短了研究周期。

[ Objective] The mechanisms of nematophagous bacteria against nematodes remain unclear, limiting the use of biocontrol bacteria in the agriculture. Therefore, we constructed a rapid and efficient screening model to quickly identify new candidate genes involved in nematode infection. [ Methods] The wild-type Bacillus amyloliquefaciens FZB42 as well as more than 400 random mutants were inoculated into 5 mL liquid bioassay medium. After growth at 37℃ for 24 h, 200 uL bacterial culture and 50 - 60 Level 4 age nematodes were added to 24-well plates, and then the survival rates of nematodes were determined at different time points. Through several rescreening, we selected the mutant strains whose nematicidal activities significantly decreased compared with the wild-type strain. Meanwhile, the conventional bioassay of solid plate was used as control. [ Results] Two mutants （F1 and F2） with obvious decreased nematicidal activities were selected from the random mutation library by liquid bioassay, consistent with the result of conventional solid plate bioassay. By comparing to 168 h-screening in each round of the solid plate bioassay, the method of liquid bioassay required only 24 h. The result indicated that the liquid bioassay greatly reduced the experimental time. [ Conclusion] Our current study has successfully constructed a rapid and efficient method to bioassay the nematicidal activity, which could also lay the foundation for further cloning the candidate genes involved in the microbial infection against nematodes.