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【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】 There are few reports on the mechanism of nematode infection by the nematophagous bacteria, which limits the widespread use of biocontrol bacteria in agricultural practice. In this study, we constructed a rapid and efficient screening method for nematicidal activity of bacteria to rapidly find genes involved in nematode infectivity. 【Method】 The wild-type Bacillus amyloliquefaciens FZB42 and its mutant strain cultured on solid plate were inoculated into 5mL liquid fast kill medium and cultured at 37 ℃ for 24 hours. Then, 200μL bacterial liquid and 50- Sixty L4 Caenorhabditis elegans were selected and the time point of change and nematode survival were recorded. Mutants with significantly lower nematicidal ability than the original strain were selected and repeatedly screened. At the same time to the traditional solid plate nematode method as a control. 【Result】 Two strains F1 and F2 with significantly lower nematicidal activity than the original strain were screened by liquid quick kill method at 21h, which was consistent with the traditional solid plate nematicidal method and greatly shortened the screening time. 【Conclusion】 This study provides a good biomaterial and shortens the research cycle for the next step in identifying virulence genes in nematophagous bacteria and further elucidating the molecular mechanism of pathogenic bacteria infecting hosts.