Optimizing Polymerase Chain Reaction (PCR) conditions for detection of Weligama coconut leaf wilt disease–phytoplasma in 𝘗𝘳𝘰𝘶𝘵𝘪𝘴𝘵𝘢 𝘮𝘰𝘦𝘴𝘵𝘢

Abstract

Weligama coconut leaf wilt disease (WCLWD) was first reported from Weligama in Matara district of Sri Lanka in 2006. It is a phytoplasma disease and therefore, is expected to be transmitted by phloem feeding insect vectors. Proutisa moesta has been identified as a putative vector of WCLWD. Polymerase Chain Reaction (PCR) conditions to detect phytoplasma in 𝘗. 𝘮𝘰𝘦𝘴𝘵𝘢 has not been optimized yet. Therefore, experiments were conducted to compare two methods of DNA extraction and to determine the minimum number of 𝘗. 𝘮𝘰𝘦𝘴𝘵𝘢 needed for detection of WCLWD phytoplasma in the insect body. 𝘗. 𝘮𝘰𝘦𝘴𝘵𝘢 were collected from field cages at Weligama (viruleferous insects) and Bandirippuwa estate, Lunuwila (aviruleferous insects). Two methods were used to extract DNA and out of which CTAB extraction method yielded higher concentration and high quality DNA than that from the modified CTAB extraction method (which has an overnight incubation). DNA extracted from 15 viruleferous 𝘗. 𝘮𝘰𝘦𝘴𝘵𝘢 per sample yielded a multiplication with lower cycle threshold values than 15 aviruleferous 𝘗. 𝘮𝘰𝘦𝘴𝘵𝘢 per sample in real time PCR using primers and probes specially designed for WCLWD. No amplification was observed when the samples were subjected to normal PCR using the primers PC399 and P1694 which are specific for phytoplasma detection.