Multilocus analyses on grapevine ‘bois noir’ phytoplasmas from Italy and Serbia.

Introduction ‘Flavescence dorée’ (FD) is a quarantine phytoplasma in EU and inspite the reduction of its impact in affected European viticultural areas, it is still of relevant importance, considering the ability of phytoplasmas associated with this disease to differentiate new strains in short periods of time. Therefore knowledge about FD strains differentiation is of major relevance towards the correct disease management. Strains were differentiated on 16S ribosomal gene and on other molecular markers (Martini et al., 2002; Botti and Bertaccini, 2007; Arnauld et al., 2007). In this work molecular characterization of a number of FD strains from diverse grapevine growing areas was performed on secY (traslocase) and tuf (elongation factor Tu) genes. Materials and Methods During 2011 grapevine samples were collected in Emilia-Romagna region (North Italy) in areas where FD epidemic was increasing. As reference strains in periwinkle elm yellows, strain EY1 (‘Candidatus Phytoplasma ulmi’, 16SrV-A) and FD strain FD-AS (16SrV-C) were used. Reference strains in grapevine were FD Veneto 8/08 and Emilia Mo2/08 (16SrV-D) and Tuscany 6, REV2, REV7, and Serbia 86/09 (16SrV-C). After total nucleic acid extraction PCR/RFLP analyses on 16S ribosomal gene plus spacer region using primers B5/P7 (Padovan et al., 1995; Schneider et al., 1995) in seminested and M1/V1731 (Martini et al., 1999) in nested reactions on P1/P7 amplicons were carried out. To distinguish between 16S ribosomal subgroups TaqI (Fast, Fermentas, Lithuania) at 65°C for 10 minutes was employed on 300 ng of amplicon. The FD-D strains were further examined by RFLP analyses on SecY and tuf genes (Angelini et al., 2001; Contaldo et al., 2011) using TaqI and Tsp509I and AlfI respectively. Results and Discussion A total of 26 FD-D infected samples were selected after preliminary screening for further molecular characterization on the SecY and tuf genes. The RFLP analyses on SecY gene was carried out on 23 samples since 3 resulted not amplifiable on this gene. Two different profiles with Tsp509I and TaqI restriction enzymes were detected (Fig. 1) of which one is undistinguishable from reference strain Veneto 8/08 (profile I, Bertaccini et al., 2009) and from FD-88, the FD-D strain representative of the epidemic widespread in France and Northern Italy since 1990. Among the 23 samples examined 9 showed a profile that was clearly differentiable (profile II, Bertaccini et al., 2009). These results confirm the successful spreading of the FD-D strain identified in 2009 in the Lambrusco variety (Bertaccini et al., 2009) and confirm its distribution still restricted to Modena and Reggio Emilia provinces and to the same Lambrusco variety. The RFLP analyses on tuf gene was carried out on 21 FD-D infected samples. It is interesting to underline that the samples non amplified on this gene were not corresponding to those non amplified on SecY gene except in one case. RFLP analyses with AlfI on tuf amplicons from grapevine and reference strains substantially confirmed the differentiation of FD-C and FD-D phytoplasmas in 16S rDNA gene (Martini et al., 1999), however in some cases alternative grouping was observed. In particular strain FD-AS, 16SrV-C (in collection since 1970) showed profile identical to 16SrV-D strains; strain Tuscany 6, 16SrV-C was identical to reference strains EY1, 16SrV-A and finally Serbian strain 86/09, 16SrV-C was undistinguishable from strains REV2 and REV7, 16SrV-C but also from strain Re5 and Ra3 affiliated to subgroup 16SrV-D. These latter information indicates possibility of genetic rearrangement in the tuf gene of field collected FD strains as one of the mechanism involved in ‘flavescence dorée’ strain differentiation.