Role of Vitex agnus-castus as host plant of Hyalesthes obsoletus and 'Candidatus Phytoplasma solani' in North Italy

Nettle (Urtica dioica) and bindweed (Convolvulus arvensis) are the main host plants of Bois noir phytoplasma (BNp, 'Candidatus Phytoplasma solani, 16SrXII-A) and its major insect vector, Hyalesthes obsoletus, in central and western Europe. In Israel, chaste tree (Vitex agnus-castus) is the main host plant of H. obsoletus, but does not harbor 'Ca. P. solani'. Thus, it is employed in "push and pull" strategy to reduce the vector population in vineyard agro-ecosystems. However, in eastern Europe, chaste tree was found as a key source plant for 'Ca. P. solani' transmission to grapevine. The present study aimed to investigate (i) the interaction between chaste tree and H. obsoletus through survival, attractiveness and oviposition trials carried out comparing the behavior of H. obsoletus in chaste tree versus nettle and grapevine, and (ii) the capability of chaste tree to harbor ‘Ca. P. solani’ in North Italy through transmission trials. In survival trials, H. obsoletus adults, captured on nettle in open field at July 04, 2016, and June 27, 2017, were maintained on chaste tree, nettle and grapevine (cv Chardonnay) plants under controlled conditions (8 trials per plant species; 30 insect specimens per trial). Data analysis was carried out using the Kaplan-Meier survival test. Attractiveness trials were carried out in (i) laboratory conditions [bioassays using two choice olfactometer between chaste tree vs grapevine; chaste tree vs nettle; nettle vs chaste tree. Each test was performed on 40 H. obsoletus adults (20 females and 20 males), captured on nettle in open field; data analysis by Chi square test]; (ii) semi-field conditions [choice test performed by releasing 20 H. obsoletus adults, captured on nettle in open field, in cages where chaste tree and grapevine, chaste tree and nettle, nettle and chaste tree plants were maintained under controlled conditions. Observation of insect adults position was done at 1, 4 and 8 hours from the release. Data analysis was carried out by Chi square test]; (iii) field conditions [capture of H. obsoletus adults by chromatic traps positioned on chaste tree, grapevine and nettle plants, maintained in vases placed at 5, 10 and 20 m from natural nettles harbouring large vector populations. Captures were observed 7 days after the nettle mowing. Data were analyzed by ANOVA and Tukey test. In oviposition trials, 100 H. obsoletus adults (50 females and 50 males), captured on nettle in open field at June 21, 2016, and July 14, 2017, were maintained under controlled conditions in chaste tree and nettle plants in vases (50 L) (4 and 8 trials per plant species in 2016 and 2017, respectively). In February 2017, young H. obsoletus were extracted from the soil by Berlese funnel and analyzed by stereomicroscope. Data were analyzed by Chi square test. In transmission trials, 250 H. obsoletus adults, captured on nettle at the borders of BN-affected vineyards, were forced on 16 phytoplasma-free chaste tree plants under controlled conditions in July 2017. Eight phytoplasma-free chaste tree plants without insects were used as controls. Dead H. obsoletus were maintained at -30°C. Total nucleic acids, extracted from the insects and from leaf samples collected from the chaste tree plants in October 2017, were used as templates in nested PCR reactions carried out for the species-specific amplification of stamp gene. Identified 'Ca. P. solani' strains were characterized by stamp gene nucleotide sequence analysis. Obtained data showed that (i) H. obsoletus adults survive on chaste tree and grapevine for one week; (ii) both females and males of H. obsoletus prefer chaste tree than grapevine; (iii) H. obsoletus is able to produce eggs and overwinter as young form on chaste tree, even if with lower efficacy compared to nettle; (iv) H. obsoletus is able to transmit 'Ca. P. solani' to chaste tree (8 plants out of 16 were found infected by the same phytoplasma strain identified in H. obsoletus specimens). The obtained results allowed to increase the knowledge about the role of Vitex agnus-castus as host plant of Hyalesthes obsoletus and 'Candidatus Phytoplasma solani' in North Italy. Further studies are needed to determine the actual role of chaste tree in the BN epidemiology.