First Report of 16SrII-D phytoplasma associated with eggplant big bud (Solanum melongena L.) in India.

Eggplant is an important solanaceous crop grown extensively in the tropics and subtropics. India ranks second in global eggplant production after China. Infection of eggplant by phytoplasma reduces overall yield and market value of the crop. During a field survey conducted in the 2014 growing season in major eggplant-growing regions of Karnataka (Hunsur), characteristic big bud symptoms were observed with an average incidence of 12% in around 27 ha. Symptoms included young apical shoots with green, reduced leaves and hypertrophic calyxes. Three big bud samples were collected for further identification of associated causative agents by PCR. Healthy eggplant leaves and phyllody associated with Vinca rosea samples were used as negative and positive controls, respectively. Total genomic DNA was isolated from all samples using a GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO), and nested PCR was performed to amplify the 16SrRNA gene using universal phytoplasma primer pairs P1/P7 and R16F2n/R16R2 (Gundersen and Lee, 1996). The expected 1.25-kb amplicons from nested PCR were detected only with DNA from big bud symptomatic samples and Periwinkle samples. The nested PCR products were purified and sequenced from both directions using an ABIx370 Genetic Analyzer (Applied Biosystems, Waltham, MA). The resulting sequences were aligned using CodonCode aligner (Centerville, MA) and analyzed by BLASTn. Analyses revealed that all three big bud samples shared 100% sequence similarity with eggplant big bud phytoplasma (GenBank Accession No. JX441321.1). The in-silico RFLP pattern of the F2n/R2 primed region of 16SrDNA gene analyzed by iPhyClassifier (Zhao et al. 2009) revealed 100% sequence similarity with the reference strain RFLP pattern of the 16Sr group II, subgroup D (Accession No. Y10097.1). Based on 16SrDNA sequences and in silico RFLP analysis, the phytoplasma associated with big bud of eggplant was identified as a member of 16SrII-D group (‘Candidatus phytoplasma australasia’). The representative sequence was deposited as GenBank Accession No. KT259050.1. Association of 16SrII-D phytoplasma with big bud was previously reported from Oman (HQ423156) and Egypt (FR257482) (Al-Subhi et al. 2011; Omar and Foissac, 2012; Singh et al. 2012). 16SrII-D group phytoplasma is known to cause big bud on various vegetable crop plants worldwide and its occurrence on eggplant is of high importance as it reduces the overall productivity of the crop. To our knowledge, this is the first report of 16SrII-D group phytoplasma associated with big bud of eggplant in India