The Study On Endogenous Bacterial Diversity Inside Asian Citrus Psyllid, Diaphorina Citri Kuwayama

Asian citrus phyllid (ACP), Diaphorina citri Kuwayama, vectors Huanglongbing (HLB) causing substantial economic loss to the citrus industries. Study on the diversity of endogenous bacteria inside ACP and the effective factors will lay a foundation for further research on the interaction between the endogenous bacteria and HLB pathogen,Candidatus Liberibacter, inside ACP bodies. This may be helpful to develop methods to control HLB. In the present paper, ACP endogenous bacterial community and the effect of temperature as well as instars were studied using in vitro culture method and molecular biological technique. The similarities between the psyllid’s endogenous bacteria community and the bacteria communities in deferent parts of host plants were also analyzed. The main results were as follows:Three reported methods and a kit were compared on their efficiency in extracting DNA from the psyllids for 16S rDNA PCR. The results showed that the total DNA extracted with the reported methods presented low integrity. The DNA extracted with the FastDNA kits showed high purity and integrity, and could be well amplified with 1μL in concentration of 12-15 ng/μL. Moreover, its PCR amplification products qualified for cloning and sequencing.To study ACP endogenous bacterial diversity, the total DNA was extracted from ACP adults by FastDNA kits. Eubacterial 16S rDNA were amplified and cloned. A total of 100 cloned 16s rDNA fragments were sequenced and subjected to alignment and Phylogenetic tree analysis. The results reveal that the 100 sequences could be divided into eight groups with 98-100% identity between members in each group. The first group contained 34 clones with 99-100% homology to Syncytium endosymbiont of D. citri (Accession no.AB038368); The second group included only 1 clone with 100% homology to Mycetocyte endosymbiont of D. citri (Accession no.AB038367) and 80-86% homology to the first group. The third group also included one clone with 100% homology to Wolbachia sp. (endosymbiont of D. citri) (Accession no. EF433793). The fourth group contained 16 clones with 100% homoloty to Wolbachieae endosymbiont of Nilaparvata lugens (Accession no.GU124506) and 98-99% homology to the third group. The fifth group contained 15 clones with 99-100% homology to Wolbachia sp. (endosymbiont of D. citri) (Accession no. EU914940). The sixth group was composed of one clone with 91% homology to Wolbachia endosymbiont of D. citri (Accession no.EU914940). The seventh group contained one clone with 99-100% homology to Uncultured Pantoea sp. In soil samples (Accession no.AY942953). The eighth group included 31 clones with 99-100% homology to Secondary synbiont of D. citri (Accession no.EU570830).Orthogonal experiment [L9 (34)] revealed that TSB culture medium (pH 7.0) with the addition of silkworm haemolymph could be used to isolate more cultural endogenous bacteria from ACP. Based on the differences of colony morphology, the culturable endogenous bacteria of ACP were collected from optimized TSB medium and purified. The bacteria subsequently identified based on their 16S rRNA genes and physiological and bio-chemical properties. They were Staphylococcus xylosus, a possible novel species in Staphylococcus sp., Bacillus subtiis, Enterobacter sp., Curtobacterium oceanosedimentum, Micrabacterium testacyeum, Stenotrophomonas maltophilia, Curtobacterium citreum.The determination coefficients of standard curves for Syncytium endosymbiont and Wolbachia sp.(endosymbiont) in D. citri were 0.998 and 0.996, respectively. This showed that template concentration had a good linear relationship with Ct value, and the regression equation was Ct=-3.2530 x log (conc)+ 34.960 for Syncytium endosymbiont and Ct= -3.2077 x log (cone)+36.206 for Wolbachia sp.(endosymbiont). Average content of Syncytium endosymbiont and Wolbachia sp.(endosymbiont) in a psyllid showed significant difference between nymph and adult. Treatment with high temperature (35℃) increased the number of Syncytium endosymbiont in the psyllid. The average number of Wolbachia endosymbiont reduced significantly in the adults after treated 1 d, but significantly increased after 1 d、5 d treated with high temperature (35℃).To analyse the relation between the bacteria community inside ACP and those in ACP host plants, the bacteria were isolated from ACP and root zone soil, roots, branches, leaves of its host plants using the optimized TSB culture medium. The isolated bacteria were identified based on 16S rRNA genes. Analysis indicated that similarity coefficient between ACP endophytic bacteria communities from different host plants (0.3848) were significantly higher than those of bacteria communities in soil in root zone, roots and branches of the host plants, indicating the high stability of endogenous bacteria community in ACP. Citrus leaf endophytic bacteria community was also stable with similarity coefficient of 0.353 between the leaf bacteria communities from different host plants. Similarity coefficient was low between bacterial communities in root zone of different host plants. The similarity coefficient was low between bacteria communities from different parts of host plants.