Multiplex PCR for Discriminating Host Plant Associations of Hyalesthes obsoletus (Hemiptera: Cixiidae), a Key Vector and Driver of 'Ca. Phytoplasma solani' Epidemiology

Given the ecological and epidemiological specialization of Hyalesthes obsoletus, the principle vector of 'Candidatus Phytoplasma solani', the primary objective of this study was to develop molecular tools for discriminating three host plant associations of the vector populations: (i) Convolvulus arvensis-Urtica dioica (Ca-Ud), (ii) Vitex agnus-castus (Vac), and (iii) Crepis foetida (Cf). The genetic diversity of the nearly full-length mitochondrial COI gene (1467 bp) was analyzed and compared among previously reported and newly collected individuals of the three host plant associations on a wide geographic range. Multiplex PCR was designed and evaluated for discriminating H. obsoletus host plant associations based on the size of amplified fragments: 1084 bp for the Cf association, 645 bp for the Ca-Ud association, and 355 bp for the Vac association. Examples of the epidemiological value of combining data on the genetic characteristics of the vector and the pathogen are provided. The method is intended to facilitate an accurate identification of the vector's phylogenetic lineage, natural host plant preference, and epidemiological transmission routes of 'Ca. P. solani'. When applied to H. obsoletus specimens collected from cultivated plants within an agroecosystem and combined with 'Ca. P. solani' genotyping, the method should provide valuable information on disease epidemiology, source(s) of emergence, and transmission routes.