Suspected phytoplasma symptoms of little leaf, yellowing, chlorosis, phyllody, witches’ broom, and stunting were observed on ten different ornamental plant species at New Delhi, Andhra Pradesh, Haryana, Bengaluru, and Pune, India, during March to July 2016. To investigate the possibility of phytoplasma etiology, PCR assays were performed using universal primer pairs (P1/P7 followed by 3Far/3Rev) specific to the phytoplasma 16Sr RNA gene. First round PCR amplification with primer pair P1/P7 did not yield expected 1.8 kb product of 16S rRNA region from any of the 17 symptomatic samples. However, 1.3 Kb amplicons were observed in nested PCR assays with 3Far/3Rev primer pair in symptomatic leaf samples of Hibiscus rosa-sinensis L. (Pune isolate), Saponaria officinalis L. (Pune isolate), and Allamanda cathartica L. (Delhi isolate). No amplifications were observed in any of the other tested symptomatic and non-symptomatic plant samples either in first round or second round of nested PCR assays with phytoplasma specific primer pairs. Pairwise sequence comparison of 16S rDNA sequences of the five positive phytoplasma strains of A. catharica, H. rosa-sinensis, and S. officinalis in the present study revealed 99–100% sequence identities with strains of ‘clover proliferation’ (16SrVI) group. Phylogenetic and virtual RFLP analysis of 16S rDNA sequences of the five identified phytoplasma strains belonging to three ornamental species further confirmed their clustering and grouping with member strains of ‘clover proliferation’ subgroup D. This is the first record of the phytoplasma association of ‘clover proliferation’ subgroup D with H. rosa-sinensis, S. officinalis, and A. cathartica in the world.
Nine vegetable plants species exhibiting phytoplasma suspected symptoms of white/purple leaf, little leaf, flat stem, witches' broom, phyllody and leaf yellowing were observed in experimental fields at Indian Agricultural Research Institute, New Delhi from December 2015 to July 2016. Total DNA extracted from the three healthy and three symptomatic leaves of all the nine vegetables were subjected to PCR assays using phytoplasma specific primers P1/P7 followed by R16F2n/R16R2 and 3Far/3Rev to amplify the 16S rDNA fragments. No amplifications of DNA were observed in first round PCR assays with primer pair P1/P7 from any of the symptomatic samples. However, phytoplasma DNA specific fragments of ~ 1.3 kb were amplified from L. (two isolates), vr L. (one isolate) and L. (one isolate) by using 3Far/3Rev primer pair and 1.2 kb fragment was amplified from L. (one isolate) by using R16F2n/R16R2 primer pair. No DNA amplification was seen in other symptomatic vegetable samples of tomato, carrot, cucurbit, bitter gourd and species utilizing either P1/P7 primer pair followed by 3Far/3Rev or R16F2n/R16R2 primer pairs. Out of three leafhopper species collected from the symptomatic vegetable fields, only was found positive for association of phytoplasma. No DNA amplifications were observed in healthy plant samples and insects collected from non-symptomatic fields. Comparative sequence comparison analyses of 16S rDNA of positive found vegetable phytoplasma strains revealed 100% sequence identities among each other and with phytoplasma strains of 'clover proliferation' (16SrVI) group. Phytoplasma sequences, virtual RFLPs and phylogenetic analyses of 16S rDNA sequence comparison confirmed the identification of 16SrVI subgroup D strain of phytoplasmas in four vegetables and one leafhopper (HP) species. Further virtual RFLP analysis of 16S rDNA sequence of the vegetables phytoplasma strains confirmed their taxonomic classification with strains of 'clover proliferation' subgroup D. Since, feeding on symptomatic vegetable species in the study was also tested positive for the 16SrVI phytoplasma subgroup-D as of vegetables; it may act as potent natural reservoir of 16SrVI-D subgroup of phytoplasmas infecting vegetable and other important agricultural crops.
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