First report of Flavescence Dorée Phytoplasma Infecting Grapevines ( <i>Vitis vinifera</i> L.) in Bulgaria

Grapevine yellows (GY) are serious diseases associated with genetically different phytoplasmas, distinguishable only by molecular diagnostics. Two pathologies, Bois noir (BN) and Flavescence dorée (FD), are the most critical GY for viticulture in Europe (EU). BN is associated with a phytoplasma of the 16Sr XII-A subgroup (Ca Phytoplasma solani), which is widespread in almost all Mediterranean wine-growing countries. FD is in the A2 list of quarantine pests in EU and is associated with phytoplasmas belonging to 16Sr V-C and V-D subgroups. In Bulgaria, only the presence of BN and its vector Hyalesthes obsoletus Signoret has already been reported, and FD on grapevine has not yet been detected, but its vector Scaphoideus titanus Ball was found in the country (Avramov et al., 2011). More recently, Clematis vitalba and Ailanthus altissima infected with 16Sr V-C subgroup phytoplasmas were found in Bulgaria near vineyards (Genov et al., 2018). At the end of September 2025, in the North Central Bulgaria region (43°34'25"N 25°19'12"E), a 254 hectare vineyard was surveyed and symptoms resembling GY were observed in approximately 20% of the inspected vines The affected plants manifested leaf discoloration (yellowing/reddening), downward rolling and fragility of the petiole; drying of inflorescences, non-ripening and drying of clusters; lack of lignification; and soft and drooping shoots. Seven samples from symptomatic vines of six varieties (Chardonnay, Gamza, Aligote, Pinot noir, Cabernet Sauvignon and Merlot). were collected. Leaf veins were ground to powder with liquid nitrogen and total DNA was extracted using CTAB protocol. The DNA extracts were first amplified by nested PCRs using the universal primer pair P1/P7 followed by 16r758f/M23Sr for broad phytoplasma detection, then with R16 (I) and (V) F1/R1 for group assignment (Angelini et al., 2001). RFLP (Restriction Fragment Length Polymorphism) analysis was performed with the restriction enzyme TaqI on 16r758f/M23Sr amplicons to identify the phytoplasma subgroup. PCR and RFLP analyses of all the seven samples were positive for presence of phytoplasma from 16Sr V-D subgroup. FD identity of phytoplasmas was then confirmed by specific real-time PCR (Angelini et al., 2007). A further nested PCR amplifications targeting group 16Sr-V phytoplasmas was conducted of the non-ribosomal secY and secY-map genetic loci (Arnaud et al., 2007). Finally, the 16r758f/M23Sr, secY and secY-map amplicons were sequenced by commercial company (BMR Genomics srl., Padova, Italy) in order to assign the exact genotype. Sequence data were deposited in the GenBank database and compared by BLAST search with the available published sequences. The same phytoplasma genotype was recovered from all 7 sampled vines across all markers, allowing us to classify it as M54 map genotype (Mehle et al., 2010; Malembic-Maher et al., 2020). (acc. No PZ338007-PZ338013, 16r758f/M23Sr sequences; acc. No PZ338014-PZ338020, SecY sequences; acc. No PZ338021-PZ338027 secY-map sequences). To the best of our knowledge, this is the first report of Flavescence dorée phytoplasma infecting grapevine in Bulgaria. The finding was communicated to the Bulgarian Food Safety Agency (BFSA) and published in the EPPO database (EPPO, 2026).