Morphological Observation And Effect Of Associated Bacteria On Gene Expression Of Bursaphelenchus Xylophilus

Pine wilt disease(PWD)which is caused by pine wood nematode(PWN),Bursaphelenchus xylophilus(Steiner & Buhrer) Nickle, is a devastating disease of pine trees. In recent years, more and more researches indicate that bacteria carried by PWN play an important role in pathogenesis of PWD, and PWD is a complex disease caused by both PWN and its associated bacteria. Therefore, future studies on the detection of pathogens of PWD and synergistic effect of these two pathogens are urgent in order to elucidate the mechanism of PWD and lay a theoretical foundation for prevention of this disease.In this paper, four dyes were studied for their staining effects on PWN. The results showed that rose bengal and water-soluble aniline blue had much better staining effects on PWN than methylene blue and crystal violet, which could stain the whole body of PWN. Methylene blue and crystal violet could only stain the head and tail of PWN.Besides, effects of three plant lectins named as concanavalin A, wheat germ agglutinin and peanut agglutinin on PWN were also primarily studied. Results indicated that PWN moved fast upon treatment by plant lectins, some PWN appeared in the shape of “S” or“8”, and gathered. With prolong of treatment time, the mortality of PWN increased. When PWN was treated for 10 min with 4.0 mg/m L concanavalin A, wheat germ agglutinin or peanut agglutinin, the mortality of PWN was 100%, 80% and 65%,respectively. This study will offer some clues for detection and prevention of PWN.In order to investigate the roles of bacteria associated with PWN, effect of Pseudomonas fluorescens Gc M5-1A on genomic expression of PWN was studied at the transcriptomic level. Results showed that differential expression of 178 genes was detected in PWN associated with Pseudomonas fluorescens Gc M5-1A compared with sterilized PWN, among which 68 genes could be blasted to homologous genes in Genbank. In the 68 differentially expressed genes, 6 genes were statistically significant,with 5 up-regulated genes and 1 down-regulated gene. The 5 up-regulated genes were lys-2 encoding lysozyme, sodh-1 encoding alcohol dehydrogenase 1, LOC100631470 encoding pyruvate dehydrogenase E1 component subunit beta, Phy H encoding phytanoyl-Co A dioxygenase and cyp-42A1 encoding Cytochrome P450 family protein.The down-regulated gene sft-4 encoded SFT-4 protein. The regulation of differential genes was verified by quantitative RT-PCR. The results indicated that the expression level of LOC100631470 and sodh-1 in PWN carrying Pseudomonas fluorescens Gc M5-1A was3.02 times and 2.65 times of that in sterilized PWN, respectively, while the expression level of sft-4 in PWN carrying Pseudomonas fluorescens Gc M5-1A was only 0.36 times of that in sterilized PWN. This result is constant with that of transcriptomic analysis. This study will offer a theoretical reference to elucidate the synergistic effect of PWN and its carrying bacteria in PWD.