Phylogenetic analysis of “rose witches'-broom” phytoplasma from cultivated Rosa damascena in India representing a new subgroup V-B1 in 16S rRNA gene group V

Damask Rose (Rosa damascena; Family Rosaceace) is one of the most expensive essential oil bearing crops of many countries including India. A previously undescribed “rose witches'-broom” infestation was detected and exhibited symptoms of little leaf, apical proliferation and chlorosis during winter season in the experimental farms of CSIR-CIMAP, Lucknow (India). Samples from the healthy and infected plants were collected and indexed by PCR using the generic primer pairs P1/P6 and R16F2n/R16R2. The nested PCR product was cloned, sequenced and phytoplasma detected. The 16S rRNA gene sequence revealed that present phytoplasma showed maximum similarity of 97–98% with Candidatus Phytoplasma balanitae (HG937644), Balanites triflora' witches'-broom phytoplasma (BltWB) (AB689678) and Periwinkle yellows phytoplasma (EU375835), as well as other members of 16SrV group. Phylogenetic analysis of the 16S rRNA gene sequences of phytoplasma from Damask rose clustered with 16SrV phytoplasma group. However, computer-simulated RFLP analysis revealed unique profile of the phytoplasma sequence from rose with BamHI, HpaI and MseI and distinguished it from Periwinkle phytoplasma, Ca. P. Balanitae, Balanites triflora' witches'-broom phytoplasma and all previously described ‘Candidatus Phytoplasma’ of 16SrV groups. Further, the pattern similarity coefficient value was 0.55, lower than 0.85 with the representative phytoplasmas classified previously in 16SrV groups. Taking into consideration the unique plant host, RFLP profile and the restricted geographical occurrence in addition to the 16S rRNA gene sequence, the present phytoplasma is proposed to be rose witches'-broom phytoplasma representing a novel taxon 16SrV-B1. This is the first record of phytoplasma infection on Damask Rose from India.