Molecular characterization and phylogeny of phytoplasma associated with bunchy top disease in its new host Okra (Abelmoschus esculentus) in India reveal a novel lineage within the 16SrI group

Abstract

Okra plants with bunchy top disease were found to be prevalent during the period of August–October 2009 in New Delhi, India. The common symptoms observed were shortening of internodes, aggregation of leaves at the apical region, reduced leaf lamina, stem reddening, fruit bending, phyllody and stunting of plants. The disease incidence ranged from 2–60% accompanied by significant reductions in production of both flowers and seeds. Nested polymerase chain reaction targeting phytoplasma specific 16S rDNA and rp genes revealed all symptomatic plants to be positive for phytoplasma. Homology searches depicted its closest identity to phytoplasmas of 16SrI ‘Candidatus Phytoplasma asteris’, like the Sugarcane yellows and Periwinkle phyllody phytoplasmas. Profiles for 16S rDNA obtained with 10 restriction endonucleases, differed in TaqI sites for two phytoplasma isolates (BHND5 & 10) from the standard pattern of 16SrI-B subgroup, the latter was seen in the case of isolate BHND1. Restriction fragment analysis of rp genes with AluI, Tsp509I matched with patterns of the rpI-B phytoplasmas. Phylogenetic reconstruction of rp genes revealed okra bunchy top phytoplasma (BHND1) as a divergent isolate, the subsequent sequence analysis of which showed the presence of a novel BslI site. These significant differences suggest that multiple phytoplasma strains are affecting okra, one of which is a diverging lineage within the 16SrI-B group while others represent a new 16SrI subgroup not reported so far. Additionally, this is the first report of a phytoplasma associated disease in okra plants worldwide.