Evidence of mixed infection of phytoplasma and begomovirus associated with Withania somnifera and Capsicum annum plants from Uttar Pradesh, India

Abstract Symptoms of yellow discoloration, curling of leaves, and little leaves with excessive branching were observed on Withania somnifera and Capsicum annum plants, with an average incidence of 5 and 8%, respectively, in Hardoi District of Uttar Pradesh, India during 2016–17 and 2017–18. The little leaf and excessive branching suggested the possibility of phytoplasma association. However, the presence of whiteflies in the vicinity and leaf curling suggested begomovirus infection. Three leaf samples from Withania somnifera and Capsicum annum symptomatic plants and one from non-symptomatic leaf were used for DNA isolation and were subjected to PCR using P1/P6 primers and nested PCR R16F2n/R16r2 primers, respectively, for the detection of phytoplasma. The begomovirus coat protein-specific primer AV1F/AV1R was used to detect begomovirus infection. Nested PCR amplified the ∼1.2 kb amplicon in all six symptomatic leaves and no amplification was observed in non-symptomatic leaves. The CP region primer yielded ∼800 bp amplicons in all three symptomatic samples of each plant. Amplified products from both primers were eluted, purified, and sequenced. The phytoplasma sequence obtained from Withania somnifera (MH789552) shared the highest sequence identity (99.92%) with other isolates of a clover proliferation group (16SrVI-D) group of phytoplasmas. However, Capsicum annum plants shared the highest identity (99%) with the Ca. P. asteris-related (16SrI-B) group of phytoplasma. In silico RFLP analysis of the 1.2 kbp product of the 16S rRNA sequence of the W. somnifera and C. annum phytoplasma strains submitted to the pDRAW32 tool (https://www.acaclone.com/) and phylogenetic analysis through the MEGA 6.0 tool confirmed that it as a member of the 16SrVI-D subgroup and 16SrI-B subgroup, respectively. The CP gene sequence of the W. somnifera isolate (MW176071) showed maximum identity (99%) with several isolates of the Tomato leaf curl virus reported from various places in India. However, the C. annum isolate (MW420480) shared maximum identity with the Ageratum enation virus. The investigation confirmed the mixed infection of ToLCV and 16SrVI-D group phytoplasma in the W. somnifera plant and 16SrI-B alongwith AEV in C. annum plants.