Insects in the Bois noir pathosystem of neighbouring viticulture regions along Croatian-Hungarian state border

In the frame of the bilateral Croatian-Hungarian project, viticulture regions with widespread Bois Noir (BN) and potential vector species were identified. Several areas adjoining state borders potentially having similar BN pathosystems were targeted for molecular characterization of pathogens in grapevines, weeds and insects. The total of 91 insect samples collected in 2010 was tested for phytoplasma presence. Most samples (71) consisted of individual Hyalestes obsoletus adults, out of which 25 were caught in the vineyard of Vukanovec (Međimurje) with Urtica dioica as a dominant weed of known importance for BN epidemiology. The rest of the insects were collected from other vineyards in Međimurje, Zagreb and Baranja with Convolvulus arvensis as a dominant weed. Insect populations screened in Međimurje included also Reptalus cuspidatus, whilst Baranja vineyards harboured more diverse insect populations including R. quinquecostatus, Anaceratagallia ribauti, Lygus rugulipennis, Cixius sp., Philaneus spumarius, and Laodelphax striatella, besides the above mentioned species. Total DNA was isolated by using MagNA Pure LC Instrument and MagNA Pure LC DNA Tissue Isolation Kit II (Roche) mostly from individual insects. Templates obtained from this magnetic-bead technology procedure were used for conventional direct and nested PCR employing P1/P7 (Deng and Hiruki, 1991 ; Schneider et al., 1995) and R16F2n/R2 (Gundersen and Lee, 1996) generic phytoplasma primers, respectively. Identification of stolbur phytoplasma DNA was performed by RFLP after digestion with the enzyme Tru1I (Fermentas). BN-positive insect samples were subjected to Tuf-typing (Langer and Maixner, 2004). In parallel, the DNA was tested by real-time PCR using modified Pelletier et al. (2009). BN phytoplasma DNA was amplified from 20 individual insects in conventional PCR-RFLP and real-time PCR experiments. The individual insects harbouring BN DNA were H. obsoletus from Vukanovec (2), Jastrebarsko (9) and Baranja (8), as well as a pooled sample of Laodelphax striatella from Baranja. Tuf-types were obtained for 15 H. obsoletus. Only one H. obsoletus from Vukanovec, a pathosystem dominated by U. dioica, was found to harbour VK-Type I. The rest was infected by VK-Type II, including the other positive H. obsoletus from Vukanovec, indicating that VK-Type II is more common and geographically more widespread along the Croatian-Hungarian border. Finding both VK-Types in insects caught in close proximity (Vukanovec) indicate that this ecological niche may involve both Urtica and Convolvulus type BN pathosystems. This research was performed in the frame of the Hungarian – Croatian project (No. HR- 12/2008) “The role of wild plant species in the epidemiology of grapevine phytoplasmoses“, supported by the National Office for Research and Technology from the Fund for Research and Technology Innovation in Hungary, and the Croatian Ministry of Science, Education and Sports.