Description of microbiota associated with 'Candidatus Phytoplasma solani' insect vectors to grapevine: is microbial resource management a possible strategy for their biological control?

In the present study, we described the bacterial diversity and community structure of the 'Candidatus Phytoplasma solani' infected and non-infected insect vectors (Cicadella viridis, Dictyophara europaea, Euscelis incisus, Euscelidius variegatus, Philaenus spumarius, and Psammotettix alienus), newly discovered in northern Italy alongside the main vector Hyalesthes obsoletus and a known host but non-vector, Cicadella viridis. A total of 300 individuals were captured with sweep entomological net and pooter in mid-June 2018 and transferred to the laboratory in 100% alcohol for molecular analyses. ‘Ca. P. solani’ was detected by nested PCR-based amplification of the stamp gene using as templates the total nucleic acids extracted from the insect specimens. Depending on the result of this PCR, specimens were separated into the “infected” and “non-infected” groups. A total of 14 individuals per species (7 specimens per group) were sent to Personal Genomics (Verona, Italy) for Illumina sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene. The high-throughput sequencing data were processed with Qiime pipeline, and the OTU-picking was carried out by comparison with the GreenGenes database. Chloroplast and mitochondria were filtered as well as rare OTUs (i.e., OTUs with reads < 10). For each species and group, alpha-diversity and beta-diversity, as well as abundance based on phyla and genera were calculated using the Phyloseq package in R and compared between different species and groups. Comparison by T-test of alpha-diversity indices (Observed and Shannon) showed a significant difference between the infected and non-infected groups for E. incisus and E. variegatus species, indicating a greater amount of diversity in the infected group. Likewise, the bacterial community composition (beta-diversity) was completely distinct between the infected and non-infected groups of E. incisus and E. variegatus, while no distinction was present for H. obsoletus and C. viridis specimens. Interestingly, bacteria belonging to the phylum Bacteroidetes and the genera Sulcia and Wolbachia were significantly reduced in the infected samples. As Wolbachia is known for its intimate relationship with its insect hosts and can be relevant for biocontrol of pests, the reduction of the abundance of bacteria of this genus and its possible relevance for the fitness of the insect will be considered in future works.