Variations And Molecular Mechanisms In Fecundity And Pathogenicity Of Pine Wood Nematode(Bursaphe Ienchus Xylophilus)

As invasive species, pine wood nematode (Bursaphelenchus xylophilus) have caused serious damage to pine forests in many countries around the world, especially in China. However it has not caused extensive damage in the native place, North America. When the nematode invades into a new environment, it will make an adaptive evolution inevitably. In this paper, the study found that after pine wood nematode invading into China, the fecundity and pathogenicity was significantly enhanced; we found sod-3of the local populations in China had a higher expression level than the local populations in United States; and the DNA sequence of the local populations in China is longer than the local populations in United States (894bp longer within intron). Later, we verified the sod-3gene in many places in China, the United States, and Japan and found there are also the phenomenon of growth in sod-3gene. So we guess on the process of invasion about pine wood nematode, some genes mutated, regulation of transcription changed, and effected phenotypes to adapt in evolution.The results are as follows:1. We studied the genetic basis of alien invasive species-Bursaphelenchus xylophilus, and established the inbred lines of two local populations of China (Zhejiang province) and the United States (Pennsylvania), and compared the differences of fertility before and after the breeding of the inbred lines. The results showed that eight strains were established in the inbreeding of the local populations of United States and four strains were established in the inbreeding of the local populations of China. There was no significant difference of inbreeding on the fecundity of pine wood nematode. The population fecundity of the local populations of China is better than that the local populations of United States before inbreeding (wild type), meanwhile the population fecundity of the local populations of China is better than that the local populations of United States after inbreeding. The breeding of the inbred lines paves the rode for further research about genetic mechanism on the invasion of Bursaphelenchus xylophilus. 2. When doing some pathogenicity experiments about local populations in China and the United States, we found the local population in China is stronger in pathogenicity than the local population in the United States, and under the action of some drugs the pathogenicity also showed difference. We inoculated the inbred lines of local populations of United States and the local populations of China to black pines (pinus thunbergii), with three drugs respectively:hydrogen peroxide (H2O2)100mM, paraquat (PAR)100mM, ascorbic acid (ASA)200mM for wild pine virulence experiment. The experimental results show that the inbreed line of local populations in China are stronger than the inbred line of local populations in United States in pathogenicity.200mM ASA has some negative effect on pathogenicity to the pines for both of the inbred lines; though H2O2also has some negative effect on pathogenicity to the pines for both of the inbred lines, in comparison with the inbreed line of local populations in China, the inbred line of local populations in united states were almost lost the virulence to black pines; While under the effect of paraquat, the pine trees were dead at the same time, this suggests PAR has no interaction relationships with Bursaphelenchus xylophilus. This experiment shows that under the effect of H2O2, the two species show significant differences in pathogenicity to the black pines that the inbreed line of local populations in China and the inbred line of local populations in united states, and this difference is most likely to be caused by superoxide dismutase (SOD-3).3. We analyzed the transcriptional levels in TGF-β signaling pathway, insulin signaling pathway and ROS signaling pathway, found that it showed a significant difference in ROS signaling pathway between the local population in China and the United States. We did cDNA real-time quantitative PCR about reproduce-related genes and reactive oxygen species(ROS)-related genes, which include TGF-β signaling pathway (.daf-2,daf-12,daf-16), insulin signaling pathway (daf-1, daf-7, daf-8) and ROS signaling pathway(sod-1, sod-3, gpx-1, cat), on the inbreed line of local populations in China and the inbred line of local populations in united states. We found that there were no significant differences in the inbreed line of local populations in China and the inbred line of local populations in united states about TGF-β signaling pathway and insulin signaling pathway, but when we did cDNA real-time quantitative PCR about ROS signaling pathway genes,found that sod-3of the inbreed line of local populations in China had a higher expression level than the inbred line of local populations in United States, but the hydrogen peroxide enzyme gene(cat) was down-regulated instead, eventually which can cause the accumulation of H2O2, conducive to the harm to the host and successful invasion.4. We did genomic DNA electrophoresis analysis about sod-3gene and cat gene in many areas of China, Japan and the United States, finding that sod-3gene sequences of Chinese and Japanese local population are longer than the local population of the United States. So we sequenced the DNA and cDNA of sod-3, and found that the only individual bases’differences in levels of cDNA, and no differences in amino acid levels, but at the DNA level, sod-3gene’s DNA of the inbreed line of local populations in China were longer than the inbred line of local populations in United States up to894bp, while the894bp are all in intron, this DNA seqmengt is formed by reverse repeat sequences, and its function is not very clear, which needs further research.