We investigated the endosymbiotic microbiota of the citrus psyllid Diaphorina citri that vectors Liberobacter sp., the causative agent of citrus greening disease. Eubacterial 16S rDNA fragments were amplified by PCR and cloned from the whole DNA of D. citri. RFLP analysis showed that the PCR products contained five major sequence types. Nucleotide sequences of the five types were determined and subjected to molecular phylogenetic analysis. The first sequence was a member of the γ-Proteobacteria, closely related to mycetocyte symbionts of other psyllids. The second sequence belonged to the β-Proteobacteria, related to Oxalobacter and Herbaspirillum. The third sequence, belonging to the γ-Proteobacteria, showed a significant similarity to the sequences of Arsenophonus spp., known as endosymbionts of a triatomine bug and a parasitoid wasp. The fourth sequence was almost identical to the sequences of Liberobacter spp. in the α-Proteobacteria. The fifth sequence showed a high similarity to the sequences of Wolbachia spp. in the α-Proteobacteria. In situ hybridization experiments using specific oligonucleotide probes confirmed that the first and second sequences originated from the mycetocyte and syncytium symbionts, respectively. The presence/absence of the five endosymbiotic bacteria in adult individuals of D. citri from an Indonesian natural population was examined by diagnostic PCR analysis. The mycetocyte symbiont and the syncytium symbiont exhibited 100% infection, whereas the Arsenophonus, Liberobacter and Wolbachia showed partial infections at a rate of 83.3%, 45.2% and 76.2%, respectively.
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