Comparison of Traditional and Next-Generation Approaches for Uncovering Phytoplasma Diversity, with Discovery of New Groups, Subgroups and Potential Vectors

SIMPLE SUMMARY: Phytoplasmas are bacteria transmitted by insects that cause severe diseases in many plants, including crops, worldwide. Most phytoplasma research focuses on the epidemiology of phytoplasma-associated diseases in agriculture, and relatively few efforts have been made to survey phytoplasma diversity in natural areas. We compared traditional methods for detecting and identifying phytoplasmas with a new method based on next-generation DNA sequencing and found that the next-generation method performs as well, or better, for identifying phytoplasmas in DNA extracted from plant-feeding insects. Using this method, we report several new country/region records and insect associations for known phytoplasmas, three new designated phytoplasma subgroups and three possible new groups. ABSTRACT: Despite several decades’ effort to detect and identify phytoplasmas (Mollicutes) using PCR and Sanger sequencing focusing on diseased plants, knowledge of phytoplasma biodiversity and vector associations remains highly incomplete. To improve protocols for documenting phytoplasma diversity and ecology, we used DNA extracted from phloem-feeding insects and compared traditional Sanger sequencing with a next-generation sequencing method, Anchored Hybrid Enrichment (AHE) for detecting and characterizing phytoplasmas. Among 22 of 180 leafhopper samples that initially tested positive for phytoplasmas using qPCR, AHE yielded phytoplasma 16Sr sequences for 20 (19 complete and 1 partial sequence) while Sanger sequencing yielded sequences for 16 (11 complete and 5 partial). AHE yielded phytoplasma sequences for an additional 7 samples (3 complete and 4 partial) that did not meet the qPCR threshold for phytoplasma positivity or yielded non-phytoplasma sequences using Sanger sequencing. This suggests that AHE is more efficient for obtaining phytoplasma sequences. Twenty-three samples with sufficient data were classified into eight 16Sr subgroups (16SrI-B, I-F, I-AO, III-U, V-C, IX-J, XI-C, XXXVII-A), three new subgroups (designated as 16SrVI-L, XV-D, XI-G) and three possible new groups. Our results suggest that screening phloem-feeding insects using qPCR and AHE sequencing may be the most efficient method for discovering new phytoplasmas.