Towards improving phytoplasma taxonomy using phylogenetics and phylogenomics: A focus on Australian 16Sr II phytoplasmas

Phytoplasmas are unculturable, pleomorphic Mollicutes that infect plant and insect hosts. As such, the taxonomy of this group has relied on molecular phylogenetics rather than the polyphasic approaches often used for culturable bacterial species. Phytoplasmas are classified into groups and subgroups based on sequence similarity and RFLP analyses of the 16Sr gene. Routinely, a novel phytoplasma group is identified and classified into the provisional genus ‘Candidatus Phytoplasma’ when the 16Sr gene shares less than 97.5% sequence similarity with any previously described ‘Candidatus Phytoplasma’. RFLP analysis of the 16Sr gene is then used to further assign a subgroup to a phytoplasma strain. There is some doubt however, around the robustness of this classification scheme. The number of subgroups can be inflated when using an RFLP-based approach where subgroups are differentiated based on a single nucleotide polymorphism without the support of meaningful biological or ecological data. Additionally, the 16Sr gene presents two challenges to phytoplasma taxonomy. The 16Sr gene shows limited phylogenetic resolution between closely related strains and is duplicated in the phytoplasma genome, where these two copies can show sequence heterogeneity. Due to these limitations, phytoplasma taxonomy has moved to a multilocus-based approach where additional genes, more variable than the 16Sr, are being evaluated. In this study, we investigate the usefulness of combining different genomic resources to explore the taxonomic relationships of members within the ‘Candidatus Phytoplasma’ genus. The results of this study will contribute to the understanding of phytoplasma evolution and genetic diversity within Australia, and to identify robust markers for improved monitoring, disease management, and diagnostics of these plant pathogens.