【作者】 田雪亮；

【导师】 杨家荣；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2010， 博士

【摘要】 松材线虫与伴生细菌关系密切,伴生细菌能够增加线虫繁殖速率,并增强线虫的致病性。拟松材线虫与其伴生细菌之间可能也存在类似关系。为了揭示(拟)松材线虫伴生细菌多样性和生态作用,本文采用16S rDNA基因克隆文库和宏基因组技术结合KEGG代谢途径重建,对2种线虫的伴生细菌多样性和生态功能进行研究。这对全面认识(拟)松材线虫伴生细菌的多样性和生态作用具有十分重要的意义。主要研究结果如下:1.优化Nycodenz密度梯度离心并结合SDS裂解法富集松材线虫伴生细菌,并构建伴生细菌宏基因组Fosmid文库,并对文库克隆末端进行测序初步分析松材线虫伴生细菌种群结构,结果表明文库质量较高,文库包含的细菌以Gammaproteobacteria为优势种群。2.采用16S rDNA基因克隆文库和宏基因组454测序对中国种群松材线虫伴生细菌多样性进行研究,结果表明伴生细菌群落包含4类群细菌:Alphaproteobacteria、Betaproteobacteria、Gammaproteobacteria和Bacteriodetes,其中Gammaproteobact- eria为优势种群。3.采用可培养法和16S rDNA基因克隆文库对松材线虫M型种群、R型种群和入侵种群、土著种群的伴生细菌多样性进行研究,结果表明M型种群松材线虫伴生细菌群落中Gammaproteobacteria比例低于R型种群,且缺少被认为具有致病性的Pseudomonas sp.,这可能是造成M型致病性较低的一个原因;土著种群和入侵种群松材线虫伴生细菌种类部分相同,但又存在一定差异,这表明松材线虫在入侵过程中将原产地细菌也带入到新地,造成协同入侵,同时也能够从新地获得有利于自身生存的细菌来提高适生性。4.采用宏基因组Solex测序和KEGG代谢途径重建分析松材线虫伴生细菌的代谢特征和生态功能,结果表明伴生细菌代谢途径以碳循环和氨基酸代谢为主,外源有毒物质降解也是重要的代谢途径,如苯甲酸类及其衍生物等。伴生细菌宏基因组中参与松萜类物质降解途径的基因远远多于松材线虫基因组,形成完整的降解途径。这表明伴生细菌能够助于松材线虫降解松萜类物质,提高线虫自身适生性。伴生细菌还能够合成一些松树体内含量较少的氨基酸,可能为松材线虫提供营养。5.采用培养法和16S rDNA基因克隆文库分析了中国种群和日本种群拟松材线虫伴生细菌多样性及对拟松材线虫繁殖的影响,结果表明伴生细菌能够促进拟松材线虫繁殖。2个种群拟松材线虫伴生细菌属于4个类群:Alphaproteobacteria、Betaproteobacteria、Gammaproteobacteria和Bacteriodetes,但各类群细菌比例存在差异。不同地理种群拟松材线虫伴生细菌的多样性的差异可能因地理位置不同而造成的。6.以拟松材线虫基因组及其伴生细菌宏基因组重建代谢途径,比较分析拟松材线虫和伴生细菌代谢途径的差异,结果表明拟松材线虫和伴生细菌都能够降解松萜类物质,但线虫基因组中参与松萜降解途径的基因数量少于伴生细菌宏基因组,线虫对松萜物质降解不彻底,可能需要利用伴生细菌协同作用降解。伴生细菌也能够降解苯甲酸和儿茶酚等松树体内常见的物质,这可能是细菌对松树体内环境的一种适应。本研究针对伴生细菌多样性和生态功能进行系统研究,揭示了松材线虫入侵过程中、不同致病类型松材线虫间伴生细菌种群结构变化,阐明了伴生细菌的代谢途径特征,从代谢水平上揭示伴生细菌可降解松树体内的松萜类、苯甲酸类保护性物质,并为线虫提供氨基酸营养。松材线虫可能利用伴生细菌的这一特性来减轻寄主的抗性反应,从而提高自身的适生性。拟松材线虫伴生细菌种群结构与松材线虫类似,且对拟松材线虫的生态作用与松材线虫类似。更多还原

【Abstract】 It was reported that Bursaphelenchus xylophilus was closely related to its symbiotic bacteria, and suggested that the bacteria played ecological effects on the nematode, including increasing pathogenicity and promoting propagation of the nematodes. The same relationship may exist between bacteria and B. mucronatus. Metagenomic technology applied to microbiology overcomes the defect of cultural method, and can reveal the ecological role of microorganisms at gene and metabolism level. In this study, the diversity and ecological role of bacteria associated with the two species nematode were revealed by metagenomic method and reconstruction of metabolic pathway. This is contributed to comprehensive understanding the ecosystem of baceteria associated with B. xylophilus and B. mucronatus. The major results were followed.1. Enrichment of bacteria using density gradient centrifugation with Nycodenz and lysis with sodium dodecyl benzene sulfonate (SDS), construction of Fosmid library of bacteria associated with B. xylophilus and analysis of the composition of bacteria using end sequencing the clone were revealed that the clone of fosmid library was stable and the bacterial communities were dominated by bacteria affiliated with Gammaproteobacteria.2. Analysis of bacteria associated with B. xylophilus from China by 16S rDNA gene clone library and 454 sequencing showed that the bacterial groups contained 4 classes: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Bacteroidetes, in which Gammaproteobacteria was predominant group.3. Using cultural method and 16S rDNA gene clone library, comparasion of the diversity of bacteria associated with various populations B. xylophilus, M-forms and R-forms, invasive population and native population, revealed that the proportions of Gammaproteobacteria in bacterial community associated with M-forms B. xylophilus were lower than that in R-forms nematode. Moreover, absence of Pseudomonas sp.that is regarded as pathogen was one probable factor for R type nematode with low pathogenicity. The universality and specificity of bacteria associated B. xylophilus showed that some bacteria were remained during the transmission and spread of the nematode, and some new members were recruited from new ecological location. The variation of bacterial composition in the process of B. xylophilus invasion could possibly contribute to fitness of the nematode to new environment and population establishment and expansion of the nematode.4. The ecological role of bacteria to B. xylophilus was revealed by metagenomic sequencing and reconstruction of metabolic pathway. The result showed that the major metabolic pathways of bacteria were carbon cycle, amino acid metabolism and xenobiotics biodegradation that included benzoic acid and ramifications. The number of genes that took part in pathway of pinene degraduation in genome of B. xylophilus was less than that in metagenome of bacteria. This reflected that B. xylophilus probably depended on degrading pinene of bacteria, thus could be improved the adaptability of themselves. Additionally, the bacteria could synthetize some amino acids that were low content in pine trees and could meet the needs of nematode.5. Cultural method and 16S rDNA gene clone library were used to identify and characterize the composition of bacterial community associated with B. mucronatus of Chinese and Japanese population and the effect of bacteria on reproductive parameter of the nematode was also investigated. The results showed that bacteria could promote the propagation and development of B. mucronatus. The bacteria associated with two population nematode were both devided into 4 groups: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Bacteroidetes, but the proportion of each bacterial group was different. This difference maybe came from the different geographic location.6. Metabolic pathway of B. mucronatus and its associated bacteria were reconstructed using KEGG database based on genome of the nematode and metagenome of the bacteria, and then were compared. The results revealed that B. mucronatus and its associated bacteria could degrade pinene. However, the numbers of gene that took part in metabolic pathway of pinene degradation from genome of nematode were lower than that from metagenome of bacteria. This reflected that B. mucronatus could not completely degrade pinene and required cooperation with its associated bacteria. The associated bacteria could degrade familiar substance in pine trees, such as benzoic acid and catechol et al. This maybe was the result of fitness for bacteria to environment in pine trees.This study focused on the diversity and ecological role of bacteria associated with B. xylophilus and B. mucronatus, revealed the changes of bacterial composition in the process of invasion and various forms of B. xylophilus, clearified the characteristic of metabolic pathway of associated bacteria, and uncovered that the bacteria could degrade pinene in pine trees and could provide some amino acid for nematode. The composition of bacterial community associated with B. mucronatus was similar to that of B. xylophilus and the ecological role of bacteia to B. mucronatus was also like that of B. xylophilus.更多还原