Potential role of Euscelis incisus Kirschbaum and Dicranotropis hamata Boheman in the transmission of ‘Candidatus Phytoplasma solani’ to grapevine

INTRODUCTION In Euro-Mediterranean regions, Bois noir phytoplasma (BNp) strains ('Candidatus Phytoplasma solani', subgroup 16SrXII-A) (Quaglino et al., 2013) are transmitted to grapevine by Hyalesthes obsoletus Signoret, a polyphagous vector living preferentially on nettle (Urtica dioica L.), bindweed (Convolvulus arvensis L.), mugworth (Artemisia vulgaris L.), and chaste tree (Vitex agnus-castus L.) inside and/or around vineyards (Langer and Maixner, 2004; Sharon et al., 2015). Grapevine represents a dead-end host for the BNp, which is only incidentally transmitted by H. obsoletus from other host plants (Weintraub and Beanland, 2006). In vine-growing areas where H. obsoletus is absent, the presence of BNp implies the existence of alternative vectors. Recently, Reptalus panzeri Low and Reptalus quinquecostatus Dufour have been reported as natural vectors of BNp in Serbian and France vineyards, respectively (Cvrkovi\u107 et al., 2014; Chuche et al., 2016). Several studies reported that other Cixiidae and Cicadellidae have been captured within or near BN-diseased vineyards and found to contain BNp (Batlle et al., 2000; Palermo et al., 2004). Based on such information, it appears reasonable that additional insect species could be capable of spreading the disease (Mori et al., 2015). In the present study, transmission trials have been carried out to investigate the possible capability of transmitting BNp to grapevine of Cixiidae and Cicadellidae species prevalent in a vineyard in Franciacorta (Lombardy region, North Italy). MATERIALS AND METHODS Specimens of the 7 prevalent BNp-harboring insect species, captured at 3 sampling days (June 11 and 25; July 7, 2015) in a BN-affected vineyard in Franciacorta, have been forced to feed on phytoplasma-free grapevine (Chardonnay) plants under controlled conditions. Dead insects were maintained at -30\ub0C. \u2018Ca. P. solani\u2019 was detected by nested PCR-based amplification of stamp gene (Fabre et al., 2011) using as templates the total nucleic acids extracted from both the insect specimens (grouped in pools) and the petioles of grapevine plants collected at October 2015 and July 2016 (plants were kept in insect-free greenhouse for one year).\u2018Ca. P. solani\u2019 detected in insects and plants were characterized through nucleotide sequence analyses of stamp amplicons. RESULTS AND DISCUSSION Stamp gene amplification detected \u2018Ca. P. solani\u2019 in H. obsoletus , Euscelis incisus, and Dicranotropis hamata specimens and their host grapevine plants, sampled in July 2016, one year after the transmission trials. Even if some E. incisus, and D. hamata insects were found uninfected, their host grapevine plants were positive to stamp gene amplification. This can be due to the analysis of insects in pools, where the phytoplasma titer could be diluted and too low for nested PCR-based detection. Only plants hosting H. obsoletus, were found infected by 'Ca. P. solani' also in October 2015, at the end of the season in which transmission trials were carried out. No amplification was obtained from control plants, kept in controlled conditions without insects (Table 1). Moreover, identity analysis of stamp gene nucleotide sequences evidenced that the same 'Ca. P. solani' strain, identical to that prevalent in the examined vineyard in Gussago (Sanna et al., 2016), was found in the insect pools and in the grapevine plant employed in the same transmission trial (data not shown). Such results indicated that E. incisus and D. hamata are able to transmit 'Ca. P. solani' to grapevine. In previous studies E. incisus were found positive to 'Ca. P. solani' (\u160kori\u107 2013) and able to transmit Stolbur from infected clover to healthy clover (Valenta et al., 1961); D. hamata was found infected by 'Ca. P. solani' only in Franciacorta vineyards (Sanna et al., 2016). To the best of our knowledge, this is the first study reporting E. incisus and D. hamata as vectors able to transmit 'Ca. P. solani' to adult grapevine plants. Further studies are needed to investigate the transmission efficiency in open field and to study accurately the ecology of such insects