High-Resolution Identification of Phytoplasmas with Multiplexed Multilocus Sequence Typing Using Hybridization...

We recently described a method for PCR-independent sequence determination of a set of seven taxonomic markers for phytoplasmas and determined its utility for high-resolution strain differentiation of phytoplasma groups, including 16SrI, 16SrIII, 16SrX, and 16SrXII. Here we describe a protocol for high-throughput strain characterization with multiplexed hybridization reactions and a data analysis method that assembles five protein-coding gene sequences and concatenates them into a single phylogenetic marker of approximately 7.7 kb. In combination with the RFLP typing of the determined 16S and cpn60 sequences, this method provides detailed sequence data on phytoplasmas of known or unknown taxonomic affiliations and can differentiate closely related but distinct phytoplasmas within the same group, for example, readily differentiating the ribosomal subgroup 16SrI phytoplasmas 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma triticii'.