Identification of epidemic “flavescence dorée” molecular variants in Emilia-Romagna and Veneto regions

The study of the genetic variability of phytoplasmas is a fundamental tool to clarify their epidemiology and to implement an effective monitoring and management of their associated diseases. “Flavescence dorée” (FD), a threatening disease of grapevine associated to phytoplasmas, is distributed within the most important European wine-producing areas, and has severe effects on both vineyard productivity and landscape management. FD is a quarantine disease in Europe, mainly transmitted by the ampelophagous leafhopper Scaphoideus titanus and, despite the efforts to contain the pathogen dissemination, the disease is still epidemic in several viticultural areas of Northern Italy. Based on sequence and restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene two FD ribosomal groups were described as present in Italy during the last century: 16SrV-C and 16SrV-D. However, the sequencing of non-ribosomal loci, such as secY, map and rpsC, allowed the identification over the years of several variants within the FD phytoplasma populations. A multilocus analysis approach was carried out on symptomatic FD-infected samples collected in Emilia-Romagna and Veneto regions, in different areas where the disease is spreading in the last 2-3 years. The geographic distribution of the two strains was confirmed to be different, with areas (Treviso province) with the prevalence of FD-C and others (Verona and Modena provinces) with only FD-D strains presence. Interestingly, FD-D phytoplasmas were identified for the first time also in a vineyard located in Modigliana (Forlì-Cesena province) and surrounded by forests. The molecular analyses allowed the identification of genetic variants among FD-D populations in both regions, that is quite a novel finding for these phytoplasmas that showed since their first identification in 1996 very little variability. In particular, the sequencing of secY amplicons highlighted the presence of the same SNP variation in samples of cultivars Teroldego and Trebbiano collected in Verona province and in grapevines cv Sangiovese located in Modigliana (Forlì-Cesena province). On the other hand, among FD-C phytoplasma strains, the highest variability was shown on rpsC gene, that highlighted 5 restriction profiles after RFLP analyses on samples cv Glera collected in Treviso and Belluno provinces. These findings suggest that the epidemiology of the disease is still to be monitored and is now involving different vectors/plant hosts species that are very likely responsible for the emergence of these FD variants. Therefore, a continuous and capillary monitoring of the FD strains associated with disease on the areas where it is present is necessary for application of the most appropriate and stringent controlling measures aimed to avoid the epidemic spreading of these emerging pathogenic variant strains.