Multilocus gene characterization of phytoplasmas in 16SrII-D subgroupassociated with Coreopsis grandiflora little leaf disease in India

In September 2018 symptoms of little leaf were observed in Coreopsis grandiflora located in flower nurseries at IARI campus, New Delhi. Little leaf and phyllody symptoms were also observed on nearby growing ornamental hedge plants belonging to Duranta genus and in a weed (Parthenium hysterophorus). Amplicons of ∼1.25 kb were consistently amplified with nested PCR assays using DNA extracted from the symptomatic Coreopsis, Duranta and Parthenimum samples, while no amplification was observed with any of the asymptomatic samples of the same species. Leafhoppers (Empoasca sp.) collected from the nurseries of Coreopsis were also found phytoplasma positive using the same nested PCR protocol. BLAST and pair wise sequence comparison of the R16F2n/R2 region of 16S rRNA gene of C. grandiflora little leaf (CGLL), Duranta sp. little leaf (DLL), P. hysterophorus phyllody (PHP) and Empoasca sp (Em) revealed 99.8% sequence identity with the ‘Candidatus Phytoplasma australasia’ sequence. Phylogeny and virtual RFLP analysis with iPhyclassifier confirmed the affiliation to ‘Ca. P. australasia’, subgroup 16SrII-D of all these phytoplasma strains. Amplicons of the expected size were also consistently amplified from these strains with primers specific to 16SrII group of SAP11, imp and tuf genes. All the amplified products were sequenced and confirmed the phytoplasma affiliation to 16SrII-D subgroup. This is the first report of 16SrII-D group phytoplasma presence in C. grandiflora showing little leaf disease.