Characterization of phytoplasmas associated with sesame (Sesamum indicum) phyllody disease in North India utilizing multilocus genes and RFLP analysis

Abstract

Sesame (Sesamum indicum L.) phyllody infected fields of three states, viz. Delhi, Uttar Pradesh and Bihar in India were surveyed during July-October 2013. There was 15-35% incidence of sesame phyllody (SP) and witches’ broom (WB) disease in different fields. The etiology of SP and WB disease was confirmed using nested polymerase chain reaction with phytoplasma specific primers (P1/P6 and R16F2n/R16R2) for amplification of a fragment of ~1.5 and ~1.25 kb of 16SrRNA gene. Amplification was also done for sec A, tuf and groEL genes either in nested PCR or direct PCR using previously published primers. Sequencing of amplified product of 16SrRNA, secA, tuf and groEL genes was done. The BLAST analysis and phylogenetic analysis of 16Sr RNA and secA gene sequences revealed that phytoplasma associated with SP and WB isolates in all the three states belonged to ‘Candidatus Phytoplasma asteris’ (16SrI) and ‘Candidatus Phytoplasma aurantifolia’ (16Sr II) groups. However, tuf and groEL gene specific primers could not distinguish SP and WB isolates into different group level status. In silico restriction enzyme digestion of 1.25 kb product of 16SrDNA gene sequence of SP and WB phytoplasma with BamHI, BfaI, EcoRI, HhaI, HpaII, RsaI, AluI, HaeIII, TaqI restriction enzymes and virtual iphyclassifier analysis further classified SP and WB isolates into 16Sr I-B and 16Sr II-C subgroups. Our study confirmed that SP and WB isolates from Uttar Pradesh belonged to 16Sr I-B and 16Sr II-C subgroups. However, isolates from Delhi and Bihar belonged to 16Sr I-B subgroup.