An epidemiological study aimed to identify potential insect vectors was carried out on a Vermentino and Chardonnay vineyard in north Sardinia (Italy) which was affected by \u201cBois noir\u201d phytoplasma disease. The Auchenorrhyncha populations were monitored from April to November in 2003 and 2004, using sweep net. The following insects were captured: Delphacidae (Laodelphax striatellus), a Cercopidae, Cicadellidae Deltocephalinae (Agallia ribauti, Eupelix cuspidata, Euscelis lineolatus, Euscelidius variegatus, Exitianus taeniaticeps, Goniagnathus guttulinervis, Neoaliturus fenestratus, N. guttulatus, Phlepsius intricatus, Psammotettix alienus and Tamnotettix zelleri) and Typhlocybinae (Empoasca vitis and Zyginidia scutellaris). In 2003 the molecular analyses (PCR and RFLP), carried out on insects samples, were aimed to assess the presence of the 16SrXII phytoplasma group, while in 2004 the detection was also extended to other groups.
In total, 121 DNA samples belonging to 9 species of leafhoppers were amplified and digested with the MseI restriction endonuclease. In 2003 the DNA was extracted and amplified in direct PCR with the universal primers R16F2/R2, then a nested PCR with the specific primers R16(I)F1/R1 (Lee et al., 1995) was carried out. The amplified products were digested with the MseI restriction endonuclease, electrophoresed in agarose gel at 2% and visualised in a GEL DOC. In 2004 the extracted DNA was amplified in direct PCR with P1/P7, followed by two nested-PCR. The primers used in the first round were F1/B6 and in the second R16F2/R2 (Duduk et al., 2004). The amplified products were digested with TruI, RsaI and SspI, and then after electrophoresis in polyacrylamide gel at 5%, were visualised in an UV transilluminator.
Results showed that the following species were infected: G. guttulinervis, in September was infected by a 16SrXII-A phytoplasma, Stolbur group (Garau et al., 2004); E. lineolatus in the preimaginal state in May and in the adult form in June and L. striatellus in October were infected by a 16SrI-C phytoplasmas, Aster yellows group. P. alienus was infected in different samples by phytoplasmas 16SrI-C and 16SrI-B. A Cercopidae was positive to 16SrX-A phytoplasmas, Apple proliferation group, in July. P. alienus is here reported as a new natural host for two aster yellows phytoplasma subgroups, 16SrI-C and 16SrI-B.
Preliminary detection on samples from Chardonnay plants showed presence of 16SrI-B phytoplasmas, which indicate that the grapevine is probably involved in the lifecycle of this prokaryote in this specific environment. No positive results were obtained from spontaneous flora for the pathogens under investigation
