Multilocus sequence typing of diverse phytoplasmas using hybridization probe-based sequence capture provides high resolution strain differentiation

Abstract

Phytoplasmas are insect-vectored, difficult-to-culture bacterial pathogens that infect a wide variety of crop and non-crop plants, and are associated with diseases that can lead to significant yield losses in agricultural production worldwide. Phytoplasmas are currently grouped in the provisional genus ‘Candidatus Phytoplasma’, which includes 49 ‘Candidatus’ species. Further differentiation of phytoplasmas into ribosomal groups is based on the restriction fragment length polymorphism (RFLP) pattern of the 16S rRNA-encoding operon, with more than 36 ribosomal groups (16Sr) and over 100 subgroups reported. Since disease symptoms on plants are not associated with phytoplasma identity, accurate diagnostics is of critical importance to manage disease associated with these microorganisms. Phytoplasmas are typically detected from plant and insect tissue using PCR-based methods targeting universal taxonomic markers. Although these methods are relatively sensitive, specific and are widely used, they have limitations, since they provide limited resolution of phytoplasma strains, thus necessitating further assessment of biological properties and delaying implementation of mitigation measures. Moreover, the design of PCR primers that can target multiple loci from phytoplasmas that differ at the sequence level can be a significant challenge. To overcome these limitations, a PCR-independent, multilocus sequence typing (MLST) assay to characterize an array of phytoplasmas was developed. Hybridization probe s targeting cpn60, tuf, secA, secY, and nusA genes, as well as 16S and rp operons, were designed and used to enrich DNA extracts from phytoplasma-infected samples for DNA fragments corresponding to these markers prior to Illumina sequencing. This method was tested using different phytoplasmas including ‘Ca. P. asteris’ (16SrI-B), ‘Ca. P. pruni’ (16SrIII-A),‘Ca. P. prunorum’ (16SrX-B), ‘Ca. P. pyri’ (16SrX-C), ‘Ca. P. mali’ (16SrX-A), and ‘Ca. P. solani’ (16SrXII-A). Thousands of reads were obtained for each gene with multiple overlapping fragments, which were assembled to generate full-length (typically >2 kb), high-quality sequences. Phytoplasma groups and subgroups were accurately determined based on 16S ribosomal RNA and cpn60 gene sequences. Hybridization-based MLST facilitates the enrichment of target genes of phytoplasmas and allows the simultaneous determination of sequences corresponding to seven different markers. In this proof-of-concept study, hybridization-based MLST was demonstrated to be an efficient way to generate data regarding ‘Ca. Phytoplasma’ species/strain differentiation.

Can leafhoppers help us trace the impact of climate change on agriculture?

“…To investigate the phytoplasma associated with strawberry green petal disease (SbGP) in Eastern Canada, twelve plants showing typical symptoms of the disease between the two growing seasons (Supplementary Table S3) (Brochu et al, 2021) and one asymptomatic plant were collected from three strawberry fields located at Capitale-Nationale, Québec, Canada. Genomic DNA was extracted from the leaf midrib using a CTAB-based method (Murray & Thompson, 1980) and used as a template for phytoplasma characterization by multilocus sequence typing as previously described (Pusz-Bochenska et al, 2022). The sequences of the 16S, cpn60UT, tuf, secY, secA, nusA, and rp phytoplasma taxonomic markers generated using this method from the twelve symptomatic samples were used to determine phylogenetic relationships with MEGA v. X (Kumar et al, 2018) and the neighbour-joining method with 1000 bootstraps.…”

A decade of hidden phytoplasmas unveiled through citizen science

“…Phytoplasmas (‘ Candidatus Phytoplasma’), are phloem-limited and insect transmitted pathogens associated with a vast number of plant diseases affecting many commercially important crops (Kumari et al 2019; Pusz-Bochenska et al 2022). In the last decade, countries like Brazil, India, and China have reported hundreds of new hosts affected by phytoplasmas, evidence of the risk that these pathogens represent in tropical and warm regions (Canale et al 2020; Rao 2021; Wang et al 2022).…”