Characterization of phytoplasma associated with big bud and virescence disease of okra in India

Okra plants exhibiting symptoms of big bud and virescence were observed at Salem district of Tamil Nadu, India. To confirm the causal agent, total genomic DNA was extracted from ten symptomatic and three asymptomatic leaf samples of okra and subjected to PCR subjected to PCR assay using the 16S rRNA and SecY gene specific primers. The resulted PCR amplicons of 16S rRNA (~ 1.8 kb) in PCR, Nested-PCR (1.2 kb) and SecY gene (1.7 kb) were successfully amplified from all the ten diseased plant samples. No amplification was observed from the samples collected from symptomsless plants. Pairwise comparison of the 16S rRNA and SecY gene sequences of okra phytoplasma isolates showed maximum nucleotide identity of 99.4 to 100% with ‘Candidatus Phytoplasma australasiaticum’ strain, (formerly known as ‘Candidatus Phytoplasma australasia’). Further, virtual RFLP analysis of 16S rRNA gene sequence of F2nR2 region of okra phytoplasma isolates showed a similarity coefficient 1.0 with the 16SrII-D subgroup (accession number Y10097). This study represents the first report of a ‘Candidatus Phytoplasma australasiaticum’ 16SrII subgroup D related phytoplasma is associated with okra big bud and virescence disease in India.