Use of secA Gene for Characterization of Phytoplasmas Associated with Sugarcane Grassy Shoot Disease in India

Surveys of commercial sugarcane varieties were conducted to the phytoplasma disease incidence in eight major sugarcane growing states of India (Uttarakhand, Uttar Pradesh, Maharashtra, Bihar, Assam, Chhattisgarh, Haryana and Tamil Nadu) during 2014–2015. Leaves from 24 symptomatic sugarcane plants of eight varieties showing grassy shoot and chlorosis symptoms, and of 8 non-symptomatic plants were collected and analyzed for phytoplasma presence using 16S rRNA and secA gene-specific primers. Amplification of 1.8- and 1.2-kb products using nested primers (P1/P7 and R16F2n/R16R2) of 16S rRNA gene and 880- and 480-bp products using secA gene-specific primer pairs (SecAfor1/SecArev3 and SecAfor2/SecArev3) was obtained for all the 24 symptomatic sugarcane samples. Pairwise sequence comparison, phylogenetic and in silico RFLP analysis of partial 16S rRNA and secA gene sequences of eight strains of sugarcane grassy shoot phytoplasma representative of the eight states confirmed the association of ‘Candidatus phytoplasma oryzae’-related strains (16SrXI-B) with symptomatic sugarcane varieties. The study confirmed that secA gene-specific primers could be employed for molecular characterization of phytoplasmas associated with sugarcane grassy shoot phytoplasmas belonging to 16SrXI group.