Analysis Bacterial Diversity Of Diaphorina Citri And Phylogenetic Of Endosymbiont Wolbachia From Citrus Cultivation Area Of China

The citrus psyllids (Diaphorina citri Kuwayama) is the main insect vectors of the HLB (Citrus huanglongbing) pathogens, which mainly damage the Rutaceous plants. The internal of insects is a complex micro-ecosystem, in which a large and varied microbial community inhabits. This community plays important roles in evelopment, digestion and absorption of nutrition of their host, and colonization resistance against invasion of exotic microbes and so on. In this study, We did some research on the microbial diversity of citrus psyllids (Diaphorina citri) collected from some wild lands in my country which vectors Liberibacter sp, Using 16S rDNA-based molecular approaches of denaturing gradient gel-electrophoresis(DGGE) and restriction fragment length polymorphism(RFLP). We try to detect andanalyse phylogenetic relationship of the endosymbiont Wolbachia in Diaphorina citri, expecting to find out the bacteria which may play an important influence on the growth of citrus psyllids,and provide a new ideas and theoretical basis for the prevention and treatment of citrus psyllids.The primary results were as follows:The analysis of microbial diversity of citrus psyllids was conducted using 16S rDNA-based of restriction fragment length polymorphism (RFLP) method with bacterial universal primers.And ligation of the 16SrDNA fragement to the pMD19-T, then tranfor it to E.coli strain JM109 competent cell.The 16S rDNA clone librarys were constructed. The 16SrDNA fingerprints of the microorganisms were analyzed by isolated plasmid and digested with Haeâ…¢, MspI and RsaI enzymes respectively. A total of 185 postive clones were analyzed by RFLP, and 31 unique RFLP patterns were obtained with three restriction enzyme (Haeâ…¢, Mspâ… and Rsaâ… ).Sequenced the 31 clones, and aligned the sequences with GenBank database. It showed that they were belonged to six different families of bacteria, including Pseudomonadaceae, Enterobact -eriaceae, Xanthomonadaceae, Burkholderiaceae, Rickettsiaceae, Rhizobiaceae. The microorganisms can be identified to bacterial genera bacteria including Pseudomonas sp, Ralstonia sp, Pantoea sp, Serratia sp, Wolbachia sp, Stenotrophomonas sp, and Carsonellia ruddii, Candidatus Liberibacter asiaticus, Syncytium endosymbiont, Secondary endosymbiont. There are also seven different uncultured bacterias. The dominant bacteria in the citrus psyllids belonged to Syncytium endosymbiont and accounted for 31 % of the total 16S rRNA clones. The molecular method of PCR of denaturing gradient gel electrophoresis (PCR-DGGE) analysis based on the sequence of 16S rRNA V3 region gene was used to the microflora analysis of the psyllids citrus from different geography and different host plants. By 16S rRNA V3 region gene DGGE method, seventeen distinct bands were obtained from 16S rDNA amplificons. The bands were purified and sequenced.The sequences were aligned with GenBank database and the result showed that they were Pseudomonadaceae, Rickettsiaceae, Enterobcteriaceae,Xanthomonadaceae, Staphylo- cocceae and Bacillaceae family microorganisms. The microorganisms can be identified to genera bacteria including Bacillus sp, Staphylococcus sp, Wolbachia sp, Pantoea sp, Pseudomonas sp and Syncytium endosymbiont. Syncytium endosymbiont is the dominant bacteria group based on the strength of the bands from DGGE, existing in all citrus psyllids samples in this study. Wolbachia and Pantoea sp also exist in all the samples. The Cluster dendrogram analysis of DGGE bands showed that the difference of the bacterial community is relatively small in the citrus psyllids come from the same host plants.The endosymbiont Wolbachia infection of Diaphorina citri from five regions was detected with PCR amplification using primer pairs designed from the 16S rDNA, ftsZ and the wsp gene of Wolbachia,three genes were cloned and sequenced, aligned the sequences with GenBank database.Then phylogenetic analysised the three genes information of Wolbachia.The result confirmed the five regions citrus psyllids were infected by Wolbachia.The three genes phylogenetic trees show that the Wolbachia infecting citrus psyllids in our country belong to the supergroup B.The wsp gene phylogenetic further showed that the Wolbachia belong to the Con strain. The homology of 16SrRNA, ftsZ and wsp gene sequences in different geography populations is up to 99%, which indicated Wolbachia in different geography Diaphorina citri almost have no remarkable difference.The result showed that the rich microbiota diversity existed in the citrus psyllids and the species and amount of the bacteria are different in the citrus psyllids of different host plants and different geography.The result confirmed citrus psyllids were infected by Wolbachia, and which belong to the Con strain of supergroup B. The result expected to provide a theoretical foundation about the relationship between endosymbiont and insect, the interaction between the microorgnism and candidatus Liberibacter in citrus psyllids.And expected to provide a new ideas about control of citrus psyllids.