Abstract
Phytoplasmas are plant-pathogenic bacteria that impact agriculture worldwide. The commonly adopted classification system for phytoplasmas is based on the restriction fragment length polymorphism (RFLP) analysis of their 16S rRNA genes. With the increased availability of phytoplasma genome sequences, the classification system can now be refined. This work examined 11 strains in the 16SrI group within the genus ‘Candidatus Phytoplasma’ and investigated the possible species boundaries. We confirmed that the RFLP classification method is problematic due to intragenomic variation of the 16S rRNA genes and uneven weighing of different nucleotide positions. Importantly, our results based on the molecular phylogeny, differentiations in chromosomal segments and gene content, and divergence in homologous sequences, all supported that these strains may be classified into multiple operational taxonomic units (OTUs) equivalent to species. Strains assigned to the same OTU share >97% genome-wide average nucleotide identity (ANI) and >78% of their protein-coding genes. In comparison, strains assigned to different OTUs share < 94% ANI and < 75% of their genes. Reduction in homologous recombination between OTUs is one possible explanation for the discontinuity in genome similarities, and these findings supported the proposal that 95% ANI could serve as a cutoff for distinguishing species in bacteria. Additionally, critical examination of these results and the raw sequencing reads led to the identification of one genome that was presumably mis-assembled by combining two sequencing libraries built from phytoplasmas belonging to different OTUs. This finding provided a cautionary tale for working on uncultivated bacteria. Based on the new understanding of phytoplasma divergence and the current genome availability, we developed five molecular markers that could be used for multilocus sequence analysis (MLSA). By selecting markers that are short yet highly informative, and are distributed evenly across the chromosome, these markers provided a cost-effective system that is robust against recombination. Finally, examination of the effector gene distribution further confirmed the rapid gains and losses of these genes, as well as the involvement of potential mobile units (PMUs) in their molecular evolution. Future improvements on the taxon sampling of phytoplasma genomes will allow further expansions of similar analysis, and thus contribute to phytoplasma taxonomy and diagnostics.
Highlights
Phytoplasmas are a group of insect-transmitted plant-pathogenic bacteria that reduce yields of diverse crops worldwide (Lee et al, 2000; Hogenhout et al, 2008; Bertaccini and Lee, 2018)
AYWB was based on Sanger sequencing (Bai et al, 2006) and DY2014 was based on Illumina (Cho et al, 2019b), while NJAY was based on one single Roche/454 library (Sparks et al, 2018) and OY-V was based on one Illumina paired-end library and one Roche/454 GS FLX mate-pair library (Kakizawa et al, 2014)
The widely used classification system for phytoplasmas that is based on the sequencing and restriction fragment length polymorphism (RFLP) analysis of their 16S rRNA genes does not provide sufficient resolution or accuracy
Summary
Phytoplasmas are a group of insect-transmitted plant-pathogenic bacteria that reduce yields of diverse crops worldwide (Lee et al, 2000; Hogenhout et al, 2008; Bertaccini and Lee, 2018) These obligate parasites are related to the animalpathogenic mycoplasmas (Chen et al, 2012), and both groups of these wall-less bacteria were assigned to the class Mollicutes under the phylum Tenericutes (Brown, 2010). The commonly used system for phytoplasma classification was first established in the 1990s (Lee et al, 1993, 1998) This system utilizes a defined set of 17 restriction enzymes to perform restriction fragment length polymorphism (RFLP) analysis on a 1.25 kb PCR product derived from their 16S rRNA gene (Gundersen and Lee, 1996). With the development of an easy-to-use web-based tool iPhyClassifier (Zhao et al, 2009), this system has been well-adopted by the phytoplasma research community
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