The first report on the occurrence of Flavescence doree phytoplasma affecting grapevine in vineyards of Montenegro and an overview of epidemic genotypes in natural plant reservoirs

Abstract Flavescence dorée (FD) is a severe and epidemic grapevine yellows disease, affecting most of the major European viticultural regions and classified as a quarantine pest. It is transmitted primarily by the ampelophagous Neartic deltocephalinae leafhopper, Scaphoideus titanus. The causative agent of the disease is FD phytoplasma (FDp), Vectotypes II and III, member of the 16S rRNA taxonomic subgroups V-C and V-D. Recent evidence indicates that FDp is native to Europe. Since 2018, vineyards in Montenegro have been subject to a preventive surveillance for the presence of FDp. In July of 2021, 56 samples of symptomatic grapevines were collected from vineyards in the major wine-growing regions. Using conventional nested PCR on the 16SrV group-specific FD9 marker (secY gene) and PCR-RFLP analysis of the 16S gene, the 16SrV-C phytoplasma subgroup was identified in a single grapevine from the Godinje location of the Crmnica wine subregion. The isolate was identified as Vectotype III, Map-FD3/VmpA-III genotype M51/III-c11 after characterizing the epidemiologicaly informative map and vmpA genes. In October of the same year, additional grapevines and potential reservoir or natural host plants of FDp and related phytoplasmas were sampled in the Godinje location. No new FDp-infected grapevines were found, indicating that this was likely an incidental transmission by natural, indigenous vectors and not an outbreak. However, one clematis and six alders were found carrying 16SrV group phytoplasmas and were subjected to map/vmpA typing, along with isolates of previously collected clematis plants in the Nudo wine region, and alder trees in non-viticultural areas and the potential wine region of Montenegrin North. The clematis plant in Godinje shared the same FDp Vectotype III as the infected vine, suggesting that clematis was the likely source of infection. On the other hand, alders in Godinje harbored VmpA-II cluster genotypes of either Map-FD2 M148 or AldY (alder yellows) M52, or multiple map gene sequences. Finally, all three FDp clusters of epidemic genotypes were found in clematis and alder reservoir plants in various regions of Montenegro. According to Map/VmpA Vectotype classification, clematis was identified as a potential source of FD3/III-c, and alders of FD1/III-f and FD2/II-d cluster genotypes. These findings call for stringent management and monitoring of S. titanus in Montenegrin vineyards to prevent FDp epidemic outbreaks