Molecular Characterization of a ‘Candidatus Phytoplasma trifolii’ Strain Associated with Little Leaf and Decline of Ashwagandha from Eastern Uttar Pradesh, India

During a survey conducted in ashwagandha (Withania somnifera) fields grown at Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya, Uttar Pradesh, India, phytoplasma suspected symptoms of little leaf and decline were observed on ashwagandha plants was above 12%. The identity of the phytoplasma strain detected in all symptomatic ashwagandha samples was determined through PCR assays using phytoplasma 16S rRNA and secY genes specific primers. Both the amplified gene products were cloned, sequenced and the nucleotide sequence comparisons were made with sequences available in NCBI database. The 16S rRNA and secY genes sequences of this phytoplasma strain had 97.45 to 99.93% identity with several ‘Candidatus Phytoplasma trifolii’ strains reported from India. The phytoplasma identification was supported by the identity percentage to the reference strain (99.49%) and by the close clustering of the two phytoplasma strains studied with members of 16SrVI group in both the phylogenetic analysis of 16S rRNA and secY genes sequences. The virtual RFLP pattern generated for the phytoplasma from ashwagandha was identical (similarity coefficient 1.00) to the reference pattern of 16SrVI subgroup D.