First Report of a 16SrII D Group Phytoplasma Associated With Witches' Broom Disease of Soybean (Glycine max) in Maharashtra, India
Soybean (Glycine max L. Merr. Fabaceae) a native plant of South Asia, is an important source of vegetable oil and proteinaceous feed for cattle, poultry, and humans worldwide. Soybean is also considered a good source of omega 3 fatty acids, vitamin B12, amino acids, minerals, isoflavones, and probiotics in the form of fermented soy products. In India, soybean is a commercially important crop as 11.94 million metric tons of seeds and 1.69 million metric tons of edible oil were produced in 2013 according to FAOSTAT (Food and Agriculture Organization, Statistical Division) United Nations. Maharashtra is the second largest producer of soybean seeds in India and Vidarbha is a hotspot for soybean cultivation. Leaves of symptomatic soybean plants showing typical witches' broom symptoms, which included stunting, shortened internodes, and intense proliferation of leaf and flower buds, were collected from fields of Akola district (Vidarbha region) Maharashtra, during the monsoon season (August and September) of 2015. A total of 197 symptomatic soybean samples of cultivar JS 335 were collected from 26 fields of Akola district. To estimate disease incidence, ~70 to 100 plants from 10 to 15 randomly selected rows in each surveyed field were visually recorded for disease symptoms. Disease incidence was ranged from 2 to 10% in these fields. To confirm the association of phytoplasma, 25 mg of leaf tissue from 10 representative symptomatic and one asymptomatic plant were used for total DNA extraction using MO BIO PowerPlant Pro DNA Isolation Kit (Carlsbad, CA) The phytoplasma 16S rRNA gene was detected in all 10 symptomatic samples using PCR assay with phytoplasma universal primers P1 and P7 (Smart et al. 1996) No amplification was observed in the DNA isolated from the asymptomatic plant. All 10 PCR fragments were sequenced directly using primers 343R, 704F, 907R, 1028F, and 1492R (Baker et al. 2003) The obtained 16S rRNA gene sequences were identical to each other which showed 100% homology with a strain PpYc (Candidatus Phytoplasma australasia, Y10097, 16SrII D) when compared using the EzTaxon 16S rRNA database. The representative sequence was deposited in GenBank with accession no. LT558766. The virtual RFLP (Zhao et al. 2009) pattern derived from the R16F2n/R16R2 part of 16S rRNA sequence (LT558766) was found identical to the reference pattern of the 16Sr group II, subgroup D (Y10097) with a pattern similarity coefficient of 1. Earlier, 16SrI group phytoplasma reported from New Delhi, India, was identified to be associated with soybean witches' broom disease (Kumar et al. 2014) However, peanut witches' broom group phytoplasma (Ca. P. aurantifolia, 16SrII C) was recorded on soybean only from Malawi and Mozambique (Lava Kumar et al. 2011) To our knowledge, this is the first report of 16SrII D phytoplasma strain associated with witches' broom disease of soybean from India. Soybean is the number one oilseed crop in terms of both area and production and plays a significant role in improving the socioeconomic conditions of small and marginal farmers of central India, making this disease study more significant.