松材线虫内生细菌与宿主种群增殖和致病力变异的相互关系及作用机制研究

The pine wood nematode Bursaphelenchus xylophilus is one of the most dangerous quarantine pests of China, which can cause destructive pine wilt disease. The pathogenesis of pine wilt disease has been unclear. Recently, the roles played by the bacteria carried by B. xylophilus in pathogenesis have attracted much attention. However, the previous researches mostly focused on exophytic bacteria of B. xylophilus, it is still unknown that which kind of exophytic bacteria has the key function in the pathogenesis and virulence variance. Within the animal, the microbial communities which are dominated by bacteria are closely associated with their host. While study about the interrelationship between B. xylophilus and its endophytic bacteria has seldom been reported. Therefore, this research studied the endophytic bacteria of B. xylophilus from distinct regions and pines, aiming to figure out the existence universality, population diversity and dynamic pattern of endophytic bacteria inside B. xylophilus. By studying the variation of endophytic bacteria of B. xylophilus living in the host pine as well as on Monochamus alternatus at different ages, it was revealed that the roles played by endophytic bacteria of B. xylophilus in different lifecycle stage of the nematode and the change of living environment. At the same time, the interrelationship between the change of endophytic bacteria categories and the virulence variance for B. xylophilus, which differed from its virulence, was analyzed. Revealing the function and mechanism of dominate endophytic bacteria undergoing in B. xylophilus reproduction and virulence. Studies about the interaction between B. xylophilus and its endophytic bacteria is helpful to uncover the roles and status of endophytic bacteria showing in B. xylophilus growth and virulence variance, it also provide a guide for studies about the pathogenesis of B. xylophilus, which has a great significance for efficient control of pine wilt disease.

松材线虫病是我国重大检疫性森林病害，其致病机理至今不明。近年伴生细菌在松材线虫致病过程中的作用引起人们关注，然而研究大多为松材线虫的体表细菌。目前体表细菌在松材线虫致病过程中的作用还存在诸多问题难以回答。以细菌为主体的内生微生物群与动物关系密切。因此，深入开展松材线虫内生细菌研究，对于全面揭示伴生细菌与松材线虫生长繁殖和致病力的关系具有重要意义。本研究将对来自不同地区和松树上的松材线虫内生细菌展开研究，以探明松材线虫内生细菌存在的普遍性、种群多样性及其变化规律；对在寄主松树或天牛体上不同发育阶段的松材线虫内生细菌变化状况展开研究，以探讨松材线虫内生细菌在线虫生活周期转型及生存环境转换过程中的变化及其作用；针对不同毒力松材线虫探讨内生细菌种类变化与其致病力变异的相互关系；深入揭示特异性内生细菌对松材线虫种群繁殖和致病力影响的作用机制；以阐明内生细菌在松材线虫生长发育和毒力变异中的作用和地位。

松材线虫是引起我国重大检疫性森林病害——松树萎蔫病的病原，其致病机理至今不明。近年松材线虫伴生细菌在松材线虫致病过程中的作用引起人们关注。然而以往关于松材线虫体表细菌的研究尚难以回答细菌是否对松材线虫生长发育及其致病力具有影响。以细菌为主体的内生微生物菌群与动物关系密切，而关于松材线虫体内细菌与宿主关系的研究此前国内外未见报道。2011年本研究团队通过透射电镜在松材线虫体内首次观察到细菌的存在。本研究在此基础上，通过对来自浙江、安徽、江苏、云南、广东和湖北六省以及黑松、马尾松、黄山松、思茅松和湿地松病死木中的松材线虫体内细菌进行分离鉴定，明确了松材线虫体内细菌存在的普遍性、主要种类及其变化规律；病木中繁殖型和扩散型松材线虫体内可培养细菌含寡养单胞菌属，而松墨天牛携带的松材线虫未分离到该属菌；繁殖型松材线虫体内细菌利用碳源能力低于扩散型线虫的体内细菌；繁殖型线虫中寡养单胞菌属和Achromobacter等的相对含量较高，而Citrobacter和Vibrio等在扩散型线虫中相对含量较高；阐明在线虫生活周期及生存环境转换过程中体内细菌的种群变化规律；对不同毒力松材线虫伴生细菌群落多样性的高通量测序分析发现，强毒松材线虫的丰富度指数显著高于弱毒虫株；松材线虫体内优势细菌嗜麦芽窄食单胞菌对松材线虫在单异活体培养下的产卵量、卵孵化率和繁殖量以及接种寄主松树后繁殖量和病程发展存在不同影响，对松材线虫毒力有一定增强作用；对不同处理松材线虫的转录组测序表明，嗜麦芽窄食单胞菌对松材线虫致病相关基因表达和生命代谢活动具有重要作用，揭示了优势体内细菌对松材线虫生长发育及致病力影响的内在机制。对松材线虫与细菌、真菌的同源基因序列比对结果暗示细菌和真菌在松材线虫遗传进化过程中可能起着重要的作用。研究结果阐明松材线虫体内细菌与宿主种群繁殖与毒力变异关系，对深入分析松材线虫致病机理具有重要的科学意义。