A metagenomic investigation of phytoplasma diversity in Australian vegetable growing regions

Abstract

In this study, metagenomic sequence data was used to investigate the phytoplasma taxonomic diversity in vegetable-growing regions across Australia. Metagenomic sequencing was performed on 195 phytoplasma-positive samples, originating either from historic collections (n=46) or during collection efforts between January 2015 and June 2022 (n=149). The sampled hosts were classified as crop (n=155), weed (n=24), ornamental (n=7), native plant (n=6), and insect (n=3) species. Most samples came from Queensland (n=78), followed by Western Australia (n=46), the Northern Territory (n=32), New South Wales (n=17), and Victoria (n=10). Of the 195 draft phytoplasma genomes, 178 met our genome criteria for comparison using an average nucleotide identity approach. Ten distinct phytoplasma species were identified and could be classified within the 16SrII, 16SrXII (PCR only), 16SrXXV, and 16SrXXXVIII phytoplasma groups, which have all previously been recorded in Australia. The most commonly detected phytoplasma taxa in this study were species and subspecies classified within the 16SrII group (n=153), followed by strains within the 16SrXXXVIII group ('Ca. Phytoplasma stylosanthis'; n=6). Several geographic- and host-range expansions were reported, as well as mixed phytoplasma infections of 16SrII taxa and 'Ca. Phytoplasma stylosanthis'. Additionally, six previously unrecorded 16SrII taxa were identified, including five putative subspecies of 'Ca. Phytoplasma australasiaticum' and a new putative 16SrII species. PCR and sequencing of the 16S rRNA gene was a suitable triage tool for preliminary phytoplasma detection. Metagenomic sequencing, however, allowed for higher-resolution identification of the phytoplasmas, including mixed infections, than was afforded by only direct Sanger sequencing of the 16S rRNA gene. Since the metagenomic approach theoretically obtains sequences of all organisms in a sample, this approach was useful to confirm the host family, genus, and/or species. In addition to improving our understanding of the phytoplasma species that affect crop production in Australia, the study also significantly expands the genomic sequence data available in public sequence repositories to contribute to phytoplasma molecular epidemiology studies, revision of taxonomy, and improved diagnostics.

Keywords: ANI; Candidatus Phytoplasma australasiaticum; Candidatus Phytoplasma stylosanthis; draft genomes; metagenomic assembled genomes (MAGs); unclassified phytoplasmas.

Fig. 1.. Whole-genome comparisons for phytoplasma genome sequence data obtained for 178 samples. (a) ANI heatmap, generated by pyani version 0.2.10 using the ANIm algorithm, for all strains sequenced in this study alongside representative and publicly available genomes. Some clusters are highlighted using brackets. (b) ANI percentages and (c) alignment fractions (AF) in each pairwise comparison of samples that did not cluster with representative genomes in Fig. 1a. The genomes of representative strains and publicly available are shaded in grey. See colour gradient representing the percent identities in the heatmaps of (a) and (b) or the AF per genome in (c).

Fig. 1.. Whole-genome comparisons for phytoplasma genome sequence data obtained for 178 samples. (a) ANI heatmap, generated by pyani version 0.2.10 using the ANIm algorithm, for all strains sequenced in this study alongside representative and publicly available genomes. Some clusters are highlighted using brackets. (b) ANI percentages and (c) alignment fractions (AF) in each pairwise comparison of samples that did not cluster with representative genomes in Fig. 1a. The genomes of representative strains and publicly available are shaded in grey. See colour gradient representing the percent identities in the heatmaps of (a) and (b) or the AF per genome in (c).

Fig. 2.. Map of Australia showing the number of phytoplasma-positive samples collected per state or territory, with pie charts illustrating the proportions of ANI identified phytoplasma taxa identified per state or territory (see key below for descriptions of colour-coding). The scale on the right indicates the number of samples collected for each state or territory, with the number in brackets indicating the total number of ANI-identified samples per location within the map area. Abbreviations: ACT, Australian Capital Territory; NSW, New South Wales; NT, Northern Territory; QLD, Queensland; TAS, Tasmania; VIC, Victoria; WA, Western Australia.

Fig. 3.. Bar graphs indicating the relative abundances of (a) symptom types recorded for each plant host family analysed in this study (n=176 samples); (b) the ANI-identified phytoplasma taxa per plant or insect host family analysed in this study (n=178 samples); and (c) the symptom types recorded for each ANI-identified phytoplasma taxon analysed in this study (n=176 samples). Numbers in the bar graphs indicate the total number of samples. Colour legends are shown above each graph.