苏云金芽胞杆菌基于改变番茄根际微生物群落结构和功能防治根结线虫病的机理研究

Root-knot nematode disease is a serious soil borne disease, which can cause more than 90% reduction of Chinese crops seriously. Now it has been reported many biocontrol strains against root-knot nematode. However about the biocontrol mechanism against root-knot nematode with rhizosphere microorganisms, there is no report in the world and it needs to be solved urgently. Preliminary works showed that a Bacillus thuringiensis NBIN-863 strain had above 58% field control activity against root-knot nematode. Genome sequencing found there was no nematocidal genes in this strain. PCR-DGGE analysis showed significant structure changes of rhizosphere microbial community. In this study, three treatments will be set up on the biocontrol group, the diease group and the healthy group of greenhouse tomato.With Illumina Miseq sequencing technology, 16S/ITS rDNA sequencing will be carried out to statistical analyze the structure changes of rhizosphere microbial community. By Biolog technology and Metaproteomics technology, carbon metabolism capability will be detected and some functional proteins inhibition of root-knot nematode and pathways will also be identified to analyze the function changes of rhizosphere microbial community. Finally, based on the above data, the mechanism of biocontrol of root-knot nematode disease based on Bacillus thuringiensis changed the structures and functions of rhizosphere microorganisms of tomato will be revealed. It will provide new micro-ecological control basis to control root-knot nematode disease and it has important theoretical and practical significance to improve the yield and quality of tomato.

根结线虫病是危害严重的土传性病害，严重时造成我国作物90%以上减产。目前已报道多种根结线虫生防菌，但生防菌基于根际微生物群落防治根结线虫病的机理，国内外尚无报道，迫切需要解决。前期工作：筛选到一株苏云金芽胞杆菌NBIN-863对番茄根结线虫病的田间防效高达58%，基因组测序发现没有毒杀线虫的相关基因，PCR-DGGE电泳图谱发现显著改变根际微生物群落结构。本研究拟对大棚番茄设生防组、发病组和健康组，利用Illumina Miseq测序技术进行16S/ITS rDNA测序，统计分析根际微生物群落结构的改变；利用Biolog技术和宏蛋白质组学技术检测碳代谢能力，鉴定分析抑制根结线虫的功能蛋白和通路，统计分析根际微生物群落功能的改变，揭示苏云金芽胞杆菌基于改变番茄根际微生物群落结构和功能防治根结线虫病的机理，为防治根结线虫病提供全新的微生态防治理论，对提高番茄产量和品质有重要的理论和实践意义。

根结线虫病是危害严重的土传性病害，严重时造成我国作物90%以上减产。目前已报道多种根结线虫生防菌，但生防菌基于根际微生物群落防治根结线虫病的机理，国内外尚无报道，迫切需要解决。前期工作：筛选到一株苏云金芽胞杆菌NBIN-863对番茄根结线虫病的田间防效高达58%，基因组测序发现没有毒杀线虫的相关基因，PCR-DGGE电泳图谱发现显著改变根际微生物群落结构。本研究对大棚番茄设生防组、发病组和健康组，利用Illumina Miseq测序技术进行16S/ITS rDNA测序，统计分析根际微生物群落结构的改变，结果显示Bt NBIN-863是根际土壤中的一种优势菌属，可以在番茄的整个生育期均很好的存活，从而持续不断的起到杀死根结线虫的作用。此外，Bt NBIN-863还影响了其他微生物的群落结构，Streptomyces, Alcaligenaceae, Legionella, Hirsutella, Acremonium和Candida的相对丰度都增加了。跟发病后期的发病组（FB）相比，防治组显著的降低了Fusarium的数量，这种病原真菌是导致番茄枯萎病的病原。根据防治组和发病组相对丰度的比值，有4种细菌和4种真菌报道过具有杀线虫活性的微生物种群数量均显著增加。Biolog ECO生态板实验显示，防治组的微生物代谢活性比发病组高，168h达到最高值，揭示出Bt NBIN-863能够改变含有根结线虫的病土中根际微生物的代谢活性，并使它们逐步增加到最大活性。根际土壤微生物的蛋白质组图谱显示，根据防治组/发病组的比值，对差异表达前44的蛋白质进行了分析。有31.8%的蛋白来自芽胞杆菌，11.4%的蛋白来自Aspergillus，4.5%的蛋白来自假单胞菌。一共鉴定出了11种杀虫晶体蛋白显著表达，这些杀虫晶体蛋白和其他二级代谢产物有助于控制线虫的虫口数量。此外还发现了一些跟病害发生相关的蛋白及与根结线虫病害伴随发生的复合病害的致病因子，并发现了与根结线虫病害发生相关的代谢通路。我们的工作揭示了一种关于Bt NBIN-863在根际土壤中防治根结线虫病害的“领导”机制。Bt NBIN-863不仅自己通过一些杀虫活性物质来杀死线虫，而且还吸引了一些其他杀线虫微生物聚集到一起共同杀线虫，此外还提出了一种Bt NBIN-863在土壤中如何防治根结线虫病害的新机制。