First Report of 'Candidatzts Phytoplasma asteris' (subgroup 16SrI-X) Associated with Bottle Gourd Virescence and Phyllody Disease in India

The bottle gourd (Lagenaria siceraria [Mol] Standl.), member of Cucurbitaceae family, is a common and very popular vegetable grown in most parts of tropical and subtropical India and consumed in almost every household throughout the year. In India, 1.81 million tons of bottle gourd production was recorded from a cultivation area of 103.23 thousand hectares in 2015. Bottle gourd plants with typical virescence and phyllody symptoms, less vigor and stunted growth, were observed in fields in Pune district of Maharashtra state, during surveys in 2012, 2013, and 2014 (March to May). Affected plants showed the peduncle bearing floral parts abnormally elongated with no fruit setting. The observed symptoms were reminiscent of a phytoplasma infection. The average visual disease incidence was 0.51% in the surveyed areas, covering approximately 12,000 plants. To detect the presence of phytoplasma, 25 mg of leaf tissue from 26 representatives symptomatic and four asymptomatic plants were used for total DNA extraction using CTAB method. A phytoplasma 16S rRNA gene (1.25 kb) was detected in all selected symptomatic plant samples when tested using PCR assay with primers P1/P7 followed by nested PCR using R16F2n/R16R2 primers. Additionally, 480 bp of phytoplasma partial secA gene (Hodgetts et al. 2008) and 440 bp of the partial tuf gene (Makarova et al. 2012) was amplified, purified, and sequenced to confirm the phytoplasma presence. The 16S rRNA, secA, and tuf genes did not amplify by PCR in asymptomatic bottle gourd plant. The obtained 16S rRNA gene sequences (LT594117 and LT594118) from symptomatic plants showed 99.6% identity with a reference strain OAY (‘Ca. Phytoplasma asteris,’ M30790) when compared using the EzTaxon 16S rRNA database (Kim et al. 2012). The virtual RFLP pattern derived using iPhyClassifier from the R16F2n/R16R2 fragment of 16S rRNA sequences was found most similar to the reference pattern of strain BTSpLT138_IX, Papaya Bunchy top phytoplasma (16SrI-X, JF781308), with a pattern similarity coefficient of 0.98 (Zhao et al. 2009). The virtual RFLP patterns with AluI and MseI were confirmed by actual RFLP analysis. Furthermore, the partial sequences of secA (LT617882 and LT617883) genes showed 88% sequence identity with Chrysanthemum yellows (AJS09571) and New Jersey aster yellows (AJS09572), confirming the association of ‘Ca. P. asteris’ (16SrI) with symptomatic bottle gourd plants using NCBI-BLAST. The tuf gene sequences (LT617884 and LT617885) showed a sequence identity of 100% with Eggplant dwarf phytoplasma (BAC76339) reported from Japan. Previously, the 16SrIII-Y phytoplasma strain of ‘Ca. P. pruni’ was reported to be associated with L. siceraria yellows and little leaf in Brazil (Montano et al. 2015). This study is the first record of subgroup 16SrI-X of ‘Ca. P. asteris’ phytoplasma strain associated with virescence and phyllody disease symptoms in bottle gourd in India. This disease may become a serious threat to bottle gourd cultivation as it causes 100% yield loss due to floral abnormality and sterility. Also, bottle gourd is a regular and widely cultivated crop for a large community of farmers in India, making it more vital to study epidemiology of the new disease. © 2017, American Phytopathological Society. All rights reserved.