Phytoplasmas associated with Flavescence dorée (FDp) grapevine disease are quarantine pathogens controlled through mandatory measures including the prompt eradication and destruction of diseased plants, and the insecticide treatments against the insect vector, the ampelophagous leafhopper Scaphoideus titanus. In the present study, a multidisciplinary approach has been applied to investigate the FDp ecological cycle in a test vineyard agro-ecosystem in Canton Ticino, south Switzerland. Despite the scarce population density of S. titanus, a regular trend of new infections (3.4% of the total vines) through the years was observed. The leafhopper Orientus ishidae was found as the most abundant among the captured insect species known as phytoplasma vectors (245 out of 315 specimens). The population of O. ishidae was evidenced prevalently (167 specimens) in the south-western side of the vineyard and within the neighbouring forest constituted mainly by hazel (Corylus avellana) and willow (Salix spp.). These plant specieswere found infected by FDp related strains (30% of analysed trees) for the first time in this study. Interestingly, O. ishidae was found to harbour FDp related strains in high percentage (26% of the analysed pools). In addition, 16SrV phytoplasma group was detected for the first time in the insect Hyalesthes obsoletus and a FDp related strain in Thamnotettix dilutior, present in low populations within the test vineyard. Molecular characterisation and phylogenetic analyses of methionine aminopeptidase (map) gene sequences of FDp and related strains, here identified, revealed the great prevalence of the map-type FD2 in grapevines (97%) and in O. ishidae pools (72%). Such a map-type was found also in hazel and in T. dilutior, but not in S. titanus. Moreover, map-types FD1 and FD3 were identified for the first time in Switzerland in several host plants and phytoplasma vectors, including grapevine (FD1), S. titanus (FD1) and O. ishidae (FD1 and FD3). Based on the data obtained in this study, it is reasonable to hypothesise that the ecological cycle of FDp could be related not exclusively to the grapevine-specific feeding diet of S. titanus, but it could include other insect vector(s) and/or plant host(s). Further studies will be needed to prove the role of O. ishidae as vector able to transmit FDp from wild plants (e.g. hazel) to grapevine.