Real-time PCR technique-based detection of coconut root (wilt) phytoplasma

Phytoplasmas are unculturable, phloem-limited plant pathogens and are associated with coconut root (wilt) disease. Recently, real-time polymerase chain reaction (PCR) methods were employed for the detection of plant pathogens. In the present study, a specific and efficient real-time PCR-based detection system for coconut root (wilt) phytoplasma was developed using double-stranded DNA intercalating dye, SYBR Green. Primers were designed to specifically amplify a 218 bp fragment from the 16S ribosomal DNA region. A melting curve analysis was programmed at the end of the cycling reaction. A unique melting peak at 80.71 degrees C was observed for infected coconut samples. The healthy and no-template control had a lower or no melting peak. The PCR products were also sequenced and results were analysed using Blastn. The sequences showed similarity with sugarcane white leaf disease phytoplasma, with the nucleotide identities being more than 98%. This technique is highly sensitive for the detection of phytoplasma in coconut.