Dual labeled probe based real time PCR method for detection of 16SrXI-B sub-group phytoplasma associated with coconut root wilt disease in India

Abstract

The Root Wilt Disease (RWD) is a non-lethal debilitating disease of coconut palms in India. Phytoplasma belonging to 16SrXI-B sub-group are associated with the disease. Rapid and specific detection protocol will greatly aid the current RWD management of replanting with disease-free seedlings. Here we report the development of a real-time PCR-based rapid and specific detection method for RWD phytoplasma. The phytoplasma 16S rRNA gene was used for designing primers and probe. The probe was dual-labeled with fluorescent molecule FAM at the 5′ end and quencher TAMRA at the 3’end. The real time assay was optimised for primer/probe, template DNA annealing temperature and cycling. The optimised assay could detect the phytoplasma in 85 % of the samples tested. Amplicon sequence confirmation ensured the fidelity of the assay. The assay developed here is suited for 16SrXI-B sub-group phytoplasmas.