Preliminary Study On The Differences Of Associated Bacteria Of Bursaphelenchus Xylophilus With Different Pathogenicity And Pseudomonas Fluorescens Regulating Gene Expression Of Pine Wood Nematode

Pine wilt disease（PWD）as a destructive pine disease caused by Bursaphelenchus xylophilus（Steiner&Buhrer）nickle has become one of the most serious global tree diseases causing great damage to the coniferous forests,ecological environment and economy in the world.In recent years,more and more studies have proved that the associated bacteria and some key genes of B.xylophilus play important roles in its pathogenesis.Therefore,it is of great significance for PWD control to study the relationship between B.xylophilus and its associated bacteria,as well as the key genes of B.xylophilus.In this study the bacteria carried by B.xylophilus from the two different sites（F and Q samples）were investigated based on metagenomic analysis.Results showed that,the distribution of the bacteria from the two samples was significantly different.At the phylum level,Proteobacteria as the largest phylum in both the two samples,which accounted for about 85%in F sample and 65%in Q sample.At the family level,Enterobacteriaceae and Rhizobiaceae were dominant in F sample with the relative abundance of around 55%,while the two bacterial families only accounted for 15%in Q sample.Moreover,Burkholderiaceae was only detected in F sample.PICRUSt analysis based on KEGG database showed that the bacteria carried by B.xylophilus from F sample had stronger abilities in amino acid metabolism,carbohydrate metabolism,energy metabolism,drug metabolism-cytochrome p450,membrane transport,replication and repair,translation.In addition,Bug Base analysis predicted that the bacteria from F sample had stronger pathogenicity and capability of antioxidant stress.Furthermore,the feeding ability and pathogenicity of F and Q samples were tested,andresults indicated that the B.xylophilus of F sample had stronger feeding ability and pathogenicity,which was consistent with the results of PICRUSt analysis based on KEGG database and Bug Base analysis.Our research indicated that the pathogenicity of B.xylophilus could be closely related to its associated bacteria,and Enterobacteriaceae,Rhizobiaceae and Burkholderiaceae belonging to Proteobacteria phylum were important pathogenicity-related bacterial families,which is valuable for the study on the pathogenesis of B.xylophilus.In order to further understand the influence of associated bacteria on B.xylophilus,the transcriptome analysis of sterile B.xylophilus and B.xylophilus carring Pseudomonas fluorescens were carried out,and the feeding ability of two species of B.xylophilus was compared.Results of differential expression gene annotation showed that P.fluorescens could regulate the gene expression of B.xylophilus,and a total of218 down-regulated genes and 89 up-regulated genes were found.Some up-regulated genes were related to pathogenicity,growth,development and neurotransmission hinting that these genes might increase the pathogenicity of B.xylophilus.Based on KEGG enrichment analysis,more difeerentially expressed genes were enriched in the Glycolysis/Gluconeogenesis pathway and its related other energy metabolism pathway,which showed a difference in energy metabolism between sterile B.xylophilus and B.xylophilus carring P.fluorescens.It provides a new experimental basis for elucidation of the synergistic pathogenic mechanism of B.xylophilus and its associated bacteria.Besides,the pectin lyase gene BxPel2 of B.xylophilus was cloned and expressed.A 750 bp Bx Pel2 coding pectin lyase from B.xylophilus was successfully cloned.The recombinant pectin lyase of B.xylophilus was expressed in Escherichia coli and purified through Ni²⁺affinity chromatography.SDS-PAGE analysis showed that the relative molecular weight of pectin lyase was about 28 k Da,which was consistent with the expected molecular size.The content and enzyme activity of recombinant protein were determined,and results showed that the concentration and enzyme activity of were 0.464 mg/m L and 98.832 U/mg,respectively.This study provides a reference for further elucidating the functions of pectin lyase from B.xylophilus and PWD control based on this enzyme.