Diverstiy And Ecological Role Of Bacteria Associated With Bursaphelenchus Xylophilus And B. Mucronatus

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.