Evidence for the association of mastrevirus, cucumovirus and phytoplasma with chickpea stunt disease and their putative insect vectors in India

Abstract

Symptoms of severe stunting, little leaf, yellowing and axillary shoot proliferation were recorded on chickpea plants in three northern states of India (New Delhi, Uttar Pradesh and Rajasthan) during January–March, 2017–2018 with disease incidences of 6–56%. Nested PCR assays with phytoplasma-specific universal primer pairs (P1/P7 and 3Far/3Rev) and 16S rRNA gene sequence analysis confirmed the association of ‘Candidatus Phytoplasma trifolii’ in the symptomatic chickpea variety Pusa Bheema at New Delhi. The in silico RFLP and phylogeny analyses of 16S rRNA sequence of the chickpea stunt (CpS) affected samples confirmed the association of 16SrVI-D subgroup phytoplasma. Similar phytoplasma strain (16SrVI-D subgroup) was detected in the leafhopper species, Hishimonus phycitis collected from symptomatic chickpea fields at New Delhi. Association of icosahedral virus particles of ~25–30 nm diameters were seen under electron microscope in CpS affected samples as well as in the aphids, Myzus persicae (Sulzer) collected from the symptomatic CpS samples at New Delhi. Both the plant and aphid samples were tested positive with the polyclonal antibodies (pAbs) of cucumber mosaic virus (CMV) in DAS-ELISA and ISEM tests. PCR assays using coat protein (CP) gene specific primers for CMV and the resultant sequence comparison also confirmed the association of CMV with the CpS affected symptomatic chickpea plants and M. persicae. The CpS affected samples collected from all the three states were further tested positive with chickpea chlorotic dwarf virus (chickpea chlorotic dwarf virus, CpCDV) CP gene specific primers in PCR assays. The 2.7 kb DNA sequence resulted from the rolling circle amplification (RCA) from the CpS affected chickpea samples of New Delhi revealed a highest sequence similarity of 99% with the CpCDV isolate from Pakistan. These results suggested a wider occurrence of CpCDV in chickpea crops in all the three states and a mixed infection of phytoplasma [‘Ca. P. trifolii’ (16SrVI-D subgroup)] and two plant viruses (CMV and CpCDV) associated with CpS disease at New Delhi.