The Diversity Of Bacteria Associated With Bursaphlenchus Xylophilus

Pine wilt disease, a dewastating disease occurred in pine trees, is one of the most important forest disease in China. Pine wood nematode(PWN) Bursaphelenchus xylophilus is the pathogen of pine wilt disease, but the mechanism of the PWN pathogenicity have no consensus. At present, symbiotic or parasitic bacteria were reported to be associated with many plant pathogenic nematodes, but the relationship between PWN and its associated bacteria was not understood. In this study, the metagenomic fosmid library for the bacteria associated with B. xylophilus were constructed, and the diversity of bacteria associated with Bursaphlenchus xylophilus from various geographic populations were compared. The main results and conclusions are as follows:1.Direct and indirect Lysis methods were used to extract the total DNA. The concentration and purity of total DNA extracted by the two methods were excellent. DNA concentration extracted by direct lysis method was higher than by indirect lysis method, for it included PWN genomic DNA. Direct lysis method may cause some DNA degradation or shearing. Two kinds of methods can amplified 16S rRNA gene fragments of bacteria well.2.The metagenomic fosmid library for the bacteria associated with B. xylophilus consisted of 19200 clones with an average fragment insert size of 40 kb. The size of insert fragment of the clones was range from 30 kb to 45 kb, thus the total size of the fosmid library was estimated at 7680000 kb. Fosmid stability assays indicated that the insert DNA was stable during propagation in the fosmid system. The end sequencing for randomly selected 96 clones was compared to NCBI with Blast program. The results showed that the percentage of B. xylophilus sequence was 5.2%, but that of the other bacteria sequences was 64.6% and that of no significant similarity sequence was 14.6%. The analysis of the bacterial diversity revealed that Stenotrophomonas was dominant group; Sphingomonas, Cupriavidus and Pseudomonas were secondly dominant communities. The fosmid library supplied the fundation for analysis of ecological role of bacteria and the interaction between the nematode and bacteria.3.The 16S rRNA gene library for the bacteria associated with B. xylophilus showed that 24 OTUs were obtained, which affiliated to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Bacteroidetes. The dominant bacteria were belonged to Gammaproteobacteria, especially Stenotrophomonas maltophilia in this class dominated the library. The diversity of bacteria associated with the PWN is high and the ecosystem of the PWN and associated bacteria was opening.4.The 16S rRNA gene library for the bacteria associated with B. xylophilus from various geographic populations showed that the bacteria associated with 4 B.xylophilus populations all belonged to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Bacteroidetes. Some bacteria were commonly represented in all populations, such as Sphingomonas sp.,Pseudomonas sp. and Flavobacterium sp., whereas some bacteria were special to each population, it is reasonable to assume that the diversity of bacteria associated with various populations may be influenced by ecological location and itself physiological property.