植物根际益生细菌代表性菌株贝莱斯芽孢杆菌FZB42对松材线虫的抑杀性

Inhibition of pine wood nematode Bursaphelenchus xylophilus by rhizobacterium Bacillus velezensis FZB42

摘要:

[目的] 由松材线虫导致的松树萎蔫病是松树的毁灭性病害，也是我国最主要的林业病害之一。本研究测评了在农业上广泛使用的、我国微生物肥料行业主要菌种资源之一——贝莱斯芽孢杆菌，对松材线虫的潜在抑杀性能。[方法] 选用贝莱斯芽孢杆菌的代表性菌株FZB42为材料，测定对不同条件下的菌液上清、不同菌株的菌液上清、细菌素plantazolicin的提取物以及菌体直接接触等方式，对松材线虫死亡率/存活率的影响，并构建FZB42生物膜合成缺损菌株，测定比较其对松材线虫存活率的影响。[结果] 相比于Landy培养基，使用LB培养基得到的菌液上清，对松材线虫具有明显抑杀性，培养48 h的菌液上清比培养24 h的菌液上清抑杀性更强，同时，菌液上清的抑杀效能随浓度升高及处理时间增长而有所增加。其中，使用培养48 h后收集的LB菌液上清处理48 h，松材线虫死亡率可高达约50%。之前报道对秀丽隐杆线虫有杀线虫活性的细菌素plantazolicin，经不同突变株上清以及plantazolicin提取物测试，被证实对松材线虫无抑制作用。直接接触实验表明，尽管FZB42的生物膜形成作用会刺激松材线虫提高抗胁迫能力，但整体而言，菌体接触对松材线虫亦有明显抑杀效果。[结论] 本研究通过严格细致的测试证明，贝莱斯芽孢杆菌FZB42对松材线虫具有抑杀性，该抑杀作用的分子基础和作用机理有待深入挖掘，但其与plantazolicin无关。作为安全性好、研究开发程度高的一类生防菌株，贝莱斯芽孢杆菌在防治松材线虫病方面的潜在价值值得关注。

Abstract:

[Objective] Pine wilt disease caused by the pine wood nematode Bursaphelenchus xylophilus is a destructive disease of pines and one of the most dangerous forestry diseases in China. Bacillus velezensis strains are widely used in agriculture as microbial agents for promoting plant growth and the major bacterial resources in China's microbial fertilizer industry. In this study we evaluated the inhibition of Bu. xylophilus by Ba. velezensis. [Methods] Ba. velezensis FZB42 was used as a representative strain in the study. The inhibition was determined using supernatants of FZB42 cultures under different conditions, the supernatants of different mutants, the extract of plantazolicin, and the direct FZB42 contact on the mortality/survival rate of Bu. xylophilus. An FZB42 mutant deficient in biofilm formation was constructed and its effect on the survival rate of Bu. xylophilus was also determined.[Results] Compared with Landy medium, the supernatant of FZB42 cultured in LB medium had a significant inhibitory effect on Bu. xylophilus. The supernatant collected at the 48^th hour after inoculation was more inhibitory than that collected at the 24^th hour. The inhibitory efficacy improved with the increase of supernatant concentration and treatment time. Using LB culture supernatant collected at the 48^th hour to treat nematodes for 48 h could result in a mortality rate of the nematodes as high as about 50%. The tests with the supernatants of different mutants and the culture extracts showed that plantazolicin, a bacteriocin reported to have a nematocidal activity against Caenorhabditis elegans, had no inhibitory effect on Bu. xylophilus. Direct contact experiments also showed that FZB42 significantly decreased the survival rate of Bu. xylophilus, although the bacterial biofilm formation could enhance Bu. xylophilus resistance to stress. [Conclusion] We demonstrated with laborious but strict assays that Ba. velezensis FZB42 had an inhibitory effect on the pine wood nematode Bu. xylophilus. The molecular basis and mechanism of the inhibitory effect need to be further explored, but it has proved to be not related with plantazolicin. As a group of biocontrol strains with good safety, profound basis research and high degree of development, the potential application value of Ba. velezensis in the control of the pine wood nematode deserves our attention.