Molecular characterization, vector identification and sources of phytoplasmas associated with brinjal little leaf disease in India

Brinjal little leaf (BLL) is a widespread disease of phytoplasma etiology in India that induces severe economic losses. Surveys were conducted in eight brinjal-growing states of India during July 2014 to September 2015 and eighteen BLL samples showing little leaf, phyllody and witches' broom symptoms were collected for phytoplasma identification. Presence of phytoplasmas was confirmed in all the eighteen BLL samples using polymerase chain reaction with phytoplasma-specific primer pairs (P1/P6, R16F2n/R16R2). Pair wise sequence comparison and phylogenetic relationship of 16S rRNA gene sequences of BLL phytoplasma strains confirmed that sixteen out of eighteen BLL strains belonged to clover proliferation phytoplasma (16SrVI) group and two BLL strains (GKP-A and GKP-B) from Gorakhpur, Uttar Pradesh, were classified under 16SrII group. Further virtual RFLP analysis of 16S rDNA sequences allowed finer classification of BLL strains into 16SrII-D and 16SrVI-D subgroups. BLL phytoplasma strains belonging to 16SrVI-D subgroup were found as the most widespread phytoplasma strains associated with BLL disease in India. 16SrVI-D subgroup phytoplasma association with two symptomatic weed species viz. Cannabis sativa subsp. sativa at Noida, Uttar Pradesh and Portulaca oleracea at IARI fields, New Delhi was also confirmed by nested PCR assays with similar set of phytoplasma-specific primers, pairwise 16S rDNA sequence comparison, phylogeny and virtual RFLP analysis. Out of five identified leafhopper species from BLL-infected fields at Noida, Uttar Pradesh and Delhi, only Hishimonas phycitis was identified as carrier and natural vector of 16SrVI-D subgroup of phytoplasmas by nested PCR assays, sequence comparison, phylogeny, virtual RFLP analysis and transmission assays.