A Workflow for Phytoplasma Discovery and Characterization in Potential Insect Vectors: From Non-destructive DNA Extraction and Vouchering to Next-Generation Sequencing.

Insect vectors remain unknown for most currently described phytoplasmas, and even known vectors are often understudied because voucher specimens essential to corroborating the species identification are rarely preserved and made available for subsequent analysis. However, because phytoplasmas often concentrate in the bodies of potential insect vectors that feed on infected plant phloem, recent studies have shown that screening DNA extracted from such insects is an efficient means for detecting and characterizing phytoplasmas in natural and anthropogenic ecosystems. Given uncertainties in morphology-based identification of insects and ongoing changes in insect taxonomy, an improved workflow is needed to integrate taxonomy, molecular biology, and epidemiology in host-pathogen research. Here we describe a newly established workflow to: (1) collect, handle, and preserve voucher specimens of potential vectors of phytoplasma; (2) non-destructively extract DNA from potentially infected insect specimens; (3) efficiently screen insect specimens for the presence of phytoplasmas; and (4) conduct downstream NGS analysis for multilocus characterization of phytoplasma strains and associated insect gut contents.