Identification and Nematicidal Activity Assay on Root-knot Nematode of a Bacillus Strain | 对根结线虫高毒力芽胞杆菌的鉴定及其活性测定

Chinese. 为筛选高效安全的杀虫资源，从海底淤泥中分离到一株对根结线虫(Meloidogyne spp.)高毒力的芽胞杆菌YBf-10，PCR扩增16S rRAN基因，经测序、序列比对分析和系统进化树构建，发现其与坚强芽胞杆菌(Bacillus firmus)Z1-7菌株16S rDNA同源性为99%，初步确定所分离的菌株为坚强芽胞杆菌。将该菌株培养至芽胞成熟，离心取上清，10倍稀释后进行生物测定，发现其对北方根结线虫二龄幼虫有很高的毒力。处理24 h校正死亡率达到50%以上，72 h达到100%。对根结线虫虫卵进行毒力测定表明，作用48 h后能显著抑制虫卵孵化达到80%以上。在培养过程中分不同时段取样，并用所取样品上清进行生物测定，发现从稳定期开始表现出了对北方根结线虫的毒力，在整个稳定期毒力持续增强，直到衰亡期后期，毒力达到最高，表明坚强芽胞杆菌所产生的杀线虫活性物质主要是在稳定期合成的。将发酵上清80℃处理30 min毒力无明显变化，通过饱和硫酸铵沉淀上清中蛋白，该蛋白对线虫无明显毒力，但是去蛋白后的上清对线虫仍然具有与未经处理上清相似的杀线虫活性，表明坚强芽胞杆菌产生的杀线虫活性物质是一种非蛋白类的小分子化合物。研究结果提示，本研究所分离的坚强芽胞杆菌在稳定期能够大量合成对线虫具有毒杀活性的小分子化合物，对根结线虫表现出极高毒力，为利用该菌株开发植物寄生线虫生防制剂提供了杀虫资源。

English. For screening of high efficiency and safe nematicidal resource, a Bacillus strain YBf-10 which exhibits extreme toxicity to root-knot nematode was isolated from marine sediment. The 16S rRAN gene of this strain was amplified by PCR, then sequenced and analysed with Blast in NCBI. The result indicated that it showed 99% identity with the 16S rDNA of Bacillus firmus strain Z1-7. It means the isolated Bacillus strain is B. firmus. The YBf-10 strain was cultured in LB medium until spores were mature, the supernatant was harvested by centrifuged. The supernatant was diluted by 10-folds, and bioassay to 2nd stage juveniles (J2) of Meloidogyne hapla was conducted. The results showed that YBf-10 exhibited extreme toxicity, and the adjusted mortality reached to 50% at 24 h, and even up to 100% at 72 h. The supernatant could obviously suppress hatching of eggs of M. hapla to 80% at 48 h. For ascertaining the synthetic phase of the nematicidal substance, samples were collected at interval of culturing times, the supernatant of the samples was prepared by centrifugalization, and assayed toxicity to J2 of M. hapla. The result indicated that, from onset of the stationary phase, it exhibited obvious toxicity. The toxicity enhanced persistently during all the stationary phase, this meant that the nematicidal substance was synthesized at stationary phase of YBf-10 strain. The nematicidal activity of supernatant treated at 80℃ for 30 min showed no obvious change. The protein in supernatant was prepared by ammonium sulfate precipitation with 30% relative saturation, and exhibited no toxicity to J2 of M. hapla. Whereas, the supernatant removed protein exhibited the same nematicidal activity with the raw supernatant. The result indicated that the nematicidal substance produced by YBf-10 was micromolecular compound, not protein. On the basis of above research, we conclude that the Bacillus strain which exhibits toxic to nematode is characterized as Bacillus firmus, and it synthesizes micromolecular nematicdidal compound at stationary stage. The compound exhibits extreme virulence to root knot nematode, and supplies nematicidal resource for exploring biological control nematicide with the YBf-10 strain.