Real-time PCR technique for detection of arecanut yellow leaf disease phytoplasma

Abstract

The Yellow Leaf Disease, with phytoplasmal etiology, is a serious disease affecting arecanut palms and causing substantial yield loss. Phytoplasmas are cell wall less, unculterable, phloem limited plant pathogens generally detected using microscopy, serology and molecular techniques. Here we report a SYBR green based real time PCR approach for detection of arecanut yellow leaf disease phytoplasma. We designed efficient primers for SYBR green based real time PCR to overcome the problems in conventional PCR. Primers QPF2/QPR2, designed from highly conserved phytoplasma 16S rRNA gene was used to amplify DNA preparation from spindle leaf tissues of symptomatic palms using real time PCR. A unique melting peak at 82.3 ± 0.5 °C was observed for symptomatic arecanut samples. The PCR products were further purified, sequenced and analysed using BLASTn. The sequences showed 99 % nucleotide identity with Indian arecanut yellow leaf disease phytoplasma sequences in the database. Two representative sequences were also deposited in the Genbank database. The present study thus devised a platform for rapid and sensitive detection of phytoplasma associated with the arecanut yellow leaf disease.