Characterization of phytoplasma associated with wilt disease in coconut and approaches for its sensitive diagnostics

Abstract

Coconut wilt associated with phytoplasma presence is a serious disease that threatens the coconut plantations in South India. Symptoms progress rapidly and cause complete destruction of coconut palm which results in severe economic loss to farmers. Survey in the areas of Thanjavur and Coimbatore districts revealed disease incidence upto 2.5 % and the affected palms exhibited unique symptoms, which differ from the root wilt disease symptoms reported so far. Nested PCR with universal primers and multilocus characterization of tuf and certain rp genes confirmed the presence of phytoplasmas. The 16S rRNA ribosomal gene sequence-based identification assigned the coconut wilt phytoplasma to the 'Candidatus Phytoplasma asteris' species. To achieve timely management of the disease and also to check its spread, Loop Mediated Isothermal Amplification (LAMP) and real-time LAMP diagnostics by targeting the 16S rRNA gene, were established for rapid and specific detection of phytoplasma presence. PCR with LAMP outer primers was carried out and sequence analysis confirmed the amplification of the 16S rRNA gene of phytoplasma. LAMP assay positive samples showed the color shift from violet to blue and was further confirmed by the ladder-like bands produced during the amplification. Diseased samples also generated a unique annealing peak at 87 ± 0.5 °C in the real-time LAMP assay. The LAMP protocol devised will be useful for quick and sensitive detection of this phytoplasma and it has potential application to detect phytoplasma presence in suspected coconut palms and to allow screening of nursery seedlings to ensure disease free planting.

Keywords: Identification; LAMP and real-time LAMP assay; Nested PCR; Palm phytoplasma.