Inferring Tripartite Associations of Vector-Borne Plant Pathogens Using a Next-Generation Sequencing Approach

Phytoplasmas are a group of plant-pathogenic, cell-wall-less bacteria vectored primarily by leafhoppers (Hemiptera Cicadellidae), one of the most diverse families of insects. Despite the importance of documenting associations between phytoplasmas, their insect vectors, and plant hosts to prevent disease outbreaks, such knowledge is currently highly incomplete and largely neglects the diversity of the system in natural areas. Here, we used anchored hybrid enrichment (AHE) to recover the DNA of five plant genes (rbcL, matK, ITS1, ITS2, and trnH-psbA) in 58 phloem-feeding leafhoppers from around the world that had previously tested positive for phytoplasma infection. Using BLASTn and a strict filtering approach, we assigned taxonomic classifications to the plant sequences and tested for cophylogenetic signals between potential Deltocephalinae leafhopper vectors and their associated plants. We observed incongruence between plant and insect phylogenies. Many leafhopper species, including presumed grass specialists, fed on distantly related plant lineages; 66% of sampled leafhoppers fed on plants from at least two different orders. By disentangling phytoplasma-leafhopper-plant interactions, we identify locations at risk of phytoplasma disease outbreaks. Furthermore, the observed wide diet breadth raises questions about how phytoplasma infection may manipulate the feeding preference of their insect host and helps fill the gaps in understanding the ecology and diversification of the tripartite association.