Multilocus gene typing, mixed infection of phytoplasma strains associated with rose genotypes and confirmation of their natural reservoir sources

Disease incidence of 12.5 to 87.5% of phytoplasma-suspected symptoms of phyllody, flower malformation, little leaf and flat stem were recorded in rose genotypes of four states (Delhi, Manipur, Uttar Pradesh and Maharashtra) of India during surveys made from 2018 to 2020. Phytoplasmas association was confirmed in nested PCR assays in all the 16 symptomatic Rosa × hybrida and Rosa damascena genotypes using primer pairs of 16S rRNA, secA and rp genes. Sequence comparison, phylogenetic and in silico RFLP analysis of 16S rDNA sequences allowed the identification of the rose-infecting phytoplasma strains into two different ribosomal groups and subgroups, namely 16SrI-B and 16SrII-D. Utilization of the rp gene–specific primers for 16Sr I (rpF1/rpR1 and rp(I)F1A/rp(I)R1A) and 16SrII groups (rp(II)F1/rp(I)R1A and rp(II)F2/rp(I)R1A) could confirm the mixed infection of 16SrI-B and 16SrII-D subgroups of phytoplasma in symptomatic rose genotypes Pusa Muskan, Pusa Manhar, Haseena, Deepak and MS Randhawa at New Delhi. Leafhoppers collected from rose fields at New Delhi (Empoasca motti and Hishimonus phycitis) and Uttar Pradesh (H. phycitis) were tested positive with 16SrII-D subgroup of phytoplasma and suggested these as putative vectors. Besides, five species of weeds from New Delhi and four species from Uttar Pradesh growing in the symptomatic rose fields were also tested positive for phytoplasma using 16S rRNA gene–specific primer suggestive as alternate hosts. The report of association of 16SrI-B phytoplasma subgroup–related strain with Coccinea sp. and Galinsoga sp. is first record in the world and Poa annua is the first report of phytoplasmas host in India.