Broader knowledge of phytoplasma diversity and phylogeny will be crucial to uncovering the evolutionary mechanisms that drive host shifts and evolution in this system, and for providing a framework to understand the potential of previously undiscovered phytoplasmas as future threats to agriculture. Recent discoveries of new phytoplasmas in natural areas worldwide suggest that the diversity and ecology of this ancient lineage of highly specialized bacteria remain poorly known. However, as has already been well documented for other groups of organisms, natural habitats worldwide continue to harbor large numbers of undiscovered taxa. Most prior research on phytoplasmas has been conducted from the relatively narrow perspective of plant disease epidemiology in agro-ecosystems with a focus on the detection and characterization of phytoplasmas obtained from diseased plants. Our results suggest that screening phloem-feeding insects using qPCR and AHE sequencing may be the most efficient method for discovering new phytoplasmas.Īssociations between hemipteran insects and phytoplasmas have evolved over the past ~350 million years and vary from commensal to parasitic from the insect’s perspective. 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. This suggests that AHE is more efficient for obtaining phytoplasma sequences. 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. 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). 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. 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.

16sr dna sequence analysis