First Report of ‘Candidatus Phytoplasma trifolii’-Related Strain Associated with Vaccinium Reddish Witches’-Broom Disease of European Blueberry in Lithuania
European blueberries (Vaccinium myrtillus L.) can be found across the Northern Hemisphere, particularly in cool, temperate forests. These shrubs produce dark blue berries that are rich in vitamins, antioxidants, and anthocyanins making them valuable for both human consumption and food supplements. Blueberries play a crucial ecological role as a food source for various animals (Pires et al., 2020). In Oct 2023, symptomatic plants were observed in the Aukštagiris Geomorphological Reserve (54°44′N.; 25°21′E.), Lithuania. These plants exhibited symptoms characteristic of phytoplasma infection, including reduced leaf size with a reddish color, witches’-broom structures, and stunted growth. Samples were collected from 5 asymptomatic and 20 symptomatic plants. The disease was designated as Vaccinium reddish witches'-broom (VacRWB). Total DNA was extracted from all samples and used as a template for nested PCR using the universal primer pairs P1/P7 and R16F2n/R16R2 to amplify a 1.2 kb fragment of the phytoplasma 16S rRNA gene (Lee et al., 1998)., Primers Tuf340/Tuf890 and Tuf400/Tuf835 were used to amplify a 0.5 kb fragment of the phytoplasma elongation factor Tu (tuf) gene (Makarova et al., 2012). PCR amplicons were obtained from 18 symptomatic plants. Enzymatic RFLP analysis of the 1.2 kb 16S rDNA revealed that the phytoplasma is a member of 16SrVI-A and a ‘Candidatus Phytoplasma trifolii’-related strain. Three R16F2n/R16R2 and three Tuf400/Tuf835 amplicons were cloned, sequenced and were respectively identical. BLAST analysis of obtained sequences against the NCBI nt database showed 99.44% to 100% identities for 16S rRNA gene and 96.42% to 99.49% identities for tuf gene sequences compared to previously identified ‘Ca. P. trifolii’strains (first hits: PP237769 (matched 1250/1250 bp) and JQ824231 (389/391 bp), respectively). iPhyClassifier (Zhao et al., 2009) was used to generate in silico RFLP profiles of the 16S rDNA sequences, revealing an identical pattern (similarity coefficient = 1.00) to the reference pattern of subgruop 16SrVI-A (acc.: AY390261). Furthermore, the enzymatic and virtual RFLP profiles (using 14 endonucleases) matched each other. Phylogenetic analysis of the obtained VacRWB gene sequences confirmed the classification. Selected sequences were deposited in GenBank (NCBI) under Acc. nos PQ585819 (16S rRNA gene) and PQ594182 (tuf gene). Previously, a phytoplasma strain of group 16SrVI was reported in association with V. myrtillus plants in Austria (Borroto Fernandez et al., 2007). In Lithuania and other parts of Europe, infections by phytoplasmas of the 16SrIII-F subgroup are commonly associated with wild blueberries (Valiunas et al. 2004), often exhibiting slightly different symptoms (e.g., without reddish coloration and more severe stunting). To our knowledge, this is the first report of a ‘Ca. P. trifolii’-related strain infecting European blueberry in Northern Europe, including Lithuania. Recently, a ‘Ca. P. trifolii’-related strain (LingbSY) has been identified in lingonberries in Lithuania (Dėlkus et al., 2024). The susceptibility of both wild blueberries and lingonberries to related phytoplasma strains suggests the potential involvement of a shared insect vector, which warrants further investigations. These diseases pose a significant threat to forest ecosystems and could potentially spread to cultivated blueberries and lingonberries emphasizing the need for a comprehensive control strategy.