Multilocus gene characterization of a 'Candidatus Phytoplasma trifolii'-related strain (16SrVI-D) associated with witches' broom and stunting disease of Gelbionis coronaria (L.).

During November-December of 2019, severe witches' broom along with little leaf and stunting symptoms was observed in Glebionis coronaria at Indian Institute of Sugarcane Research, Lucknow, Uttar Pradesh with an average disease incidence of 20%. An amplicon of ~ 1.3 kb of 16S rRNA gene was amplified in symptomatic C. coronarium plants using universal phytoplasma-specific nested primer pairs (P1/P7 followed by 3Fwd/3Rev). Additionally, in another host, Catharanthus roseus, growing in near beds and the leafhopper species Hishimonus phycitis, amplification of similar amplified products were obtained using the similar nested PCR primer pairs. Further, phytoplasma presence was confirmed by amplifying non-ribosomal genes including secA, rp and secY in the symptomatic G. coronaria, Catharanthus roseus and Hishimonus phycitis using gene-specific primers of secA, secY, and rp genes. The comparison of 16S rRNA, secA, rp, and secY gene sequences through BLASTn, phylogeny and in silico RFLP analysis of C. coronarium, C. roseus, and the leafhopper species Hishimonus phycitis identified the presence of 'Candidatus Phytoplasma trifolii'-related strain (16SrIV-D subgroup) in all the plants and leafhopper samples. These findings demonstrated the utility of multilocus genes for accurate identification of phytoplasma strains in the plant and insect samples. This is the first report of 16SrVI-D subgroup of phytoplasmas associated with witches' broom, little leaf and stunting disease in Glebionis coronaria.