Molecular Characterization of .phytoplasma Associated With Bermuda Grass (Cynodon Dactylonl.) And Periwinkle (Catharanthus Roseus L.) From Western Uttar Pradesh

Abstract

Phytoplasmas are cell wall less Mollicutes that colonize plant phloem sieve tube elements and insect gut wall. They are known to cause devastating losses in crops and natural ecosystems worldwide. They are transmitted from one plant to another by phloem-feeding insects primarily leaf hoppers, plant hoppers and psyllids. Bermuda grass (Cynodon dactylon L.) and Periwinkle (Catharanthus roseus L.) are the experimental hosts for Phytoplasma infection. I 0 Bermuda grass and 20 Periwinkle plant samples were collected during the survey of incidence of phytoplasma infection from roadside ofSVPUA & T, ground in front of College of biotechnology, Food processing unit, SVPUA & T, HRC, Pallavpuram Phase I & II and Kankerkhera, Meerut. The typical symptoms of Phytoplasma observed during the survey in Bermuda Grass and Periwinkle were: whitening of leaves, phyllody (production of leaf like structures instead of flowers), virescences (development of green flowers), yellowing of leaves and little leaves. The total DNA was extracted by taking midrib of leaves, stems and flowers of infected plants using CT AB method and association of phytoplasma was confirmed by Polymerase Chain Reaction (PCR) particularly by nested PCR in which two primers sets were used sequentially, PI/P7 followed by RIIR2 and bands of 1800bp and 1200bp were obtained in infected samples The result was further validated by another set of primersSecAI/ SecA3 and ITufulrTufu where 830bp and 850 bp bands were obtained. PCR result revealed that out of 10 Bermuda grass plants only 6 gave positive result and out of20 infected Periwinkle plants only 6 gave positive result. Only PI/P7 PCR product of grass was eluted and cloned in pTZ57RIT vector which was followed by confirmation using plasmid DNA isolation and colony PCR approaches. All the other PCR products of Bermuda Grass and Periwinkle were directly sequenced.The sequence obtained from Pl/P7 & SecA from Bermuda grass were submitted to Genbank, NCBI with accession no. KF234570 & KF234571 respectively. Similarly, sequences obtained from Pl/P7, SecA, and Tuf from Periwinkle were also submitted to Genebank, NCB! with accession no. KF437510, KF234572 and KF23457, respectively. The sequenced data was further validated by BLAST analysis and phylogenetic tree was constructed using MEGA5.2.1 software .. In iPhyC/assifier sequenced results ofPI/P7 ofboth the samples revealed that strain of phytoplasma infecting Bermuda grass is 'Candida/us Phytoplasma cynodontis' which belongs to 16Sr XIV-A and strain of phytoplasma infecting Periwinkle is 'Candida/us Phytoplasma asteris' which belongs6;Siorl- {3