First report of Tomato leaf curl Karnataka virus associated with leaf curl disease of Zinnia elegans in India

Abstract

Tomato leaf curl Karnataka virus (ToLCKV) is a species of begomovirus which has a circular, ssDNA monopartite genome and is transmitted by the whitefly (Bemisia tabaci). Zinnia elegans is a popular ornamental bedding plant which is grown worldwide for its beautiful flowers. A few begomovirus species have been reported to infect Z. elegans, including Ageratum yellow vein China virus in Vietnam (Li et al., 2013), and Ageratum enation virus and Zinnia leaf curl virus in India (Kumar et al., 2010; Pandey & Tiwari, 2012). In March 2019 symptoms of excessive leaf curling with mild mosaic were observed on Z. elegans plants growing in a nursery in Bhopal, central India (Fig. 1). To investigate the aetiology of the disease total DNA was extracted from leaf samples from two diseased and one asymptomatic Z. elegans plant using a plant genomic DNA purification kit (Promega, USA), following the manufacturer's instructions. A begomovirus was detected from both diseased samples (but not the asymptomatic sample) by PCR using begomovirus-specific primers: CPIT-I/CPIT-T (AM180920/AM180921) (Singh, 2005), PALIv 1978/ PARIc 496 and PALIv 722/ PALIc 1960 (Rojas et al., 1993). The expected amplicons (~800 bp, 1.2 Kb and 1.2 Kb, respectively) were purified using a PCR Clean-up System (Promega, USA) and Sanger sequenced in both directions by Bioinnovations Connecting Technologies to Life (Mumbai, India). The consensus sequence from both samples was identical and the complete viral DNA-A genome of 2761 nucleotides was deposited in GenBank (Accession No. MK965196). Sequence analysis was done by BLASTn and showed that the DNA-A genome had the highest identity (96-97%) with ToLCKV (KX219744, HM007094, LN878123, U38239); Sunflower leaf curl virus (JX678965) and 94–95% with Tomato leaf curl virus (JX524172, KX831454). Phylogenetic analysis using neighbour-joining in MEGA v.7.0 showed a close relationship with isolates of ToLCKV and relationships with other begomoviruses (Fig. 2). The disease was successfully transmitted by B. tabaci from naturally infected Z. elegans to healthy seedlings of Nicotiana tabacum, Solanum lycopersicum and Z. elegans (each 3/3), symptoms being observed 30 days post inoculation. The test plants all tested positive by PCR using the begomovirus-specific primer pairs above. Positive amplicons were sequenced and the nucleotide sequences were identical to each other and the original isolate (MK965196). The present study demonstrates that Z. elegans is a host of ToLCKV. ToLCKV is a serious threat to the production of several economically important crops in India and has been reported at an incidence of >80% on soybean, sunflower and tomato in India (Malathi et al., 2017). Recently, ToLCKV and satellite molecules have been found associated with enation leaf curl disease of another ornamental plant, Pseuderanthemum reticulatum, in India (Ashwathappa et al., 2021). The whitefly transmission study provides additional evidence that ToLCKV causes the observed disease in Z. elegans and it is possible that ornamental plants may act as a reservoir of the virus. To the best of our knowledge, this is the first report of the association of ToLCKV with leaf curl disease of Z. elegans. The authors are thankful to the Vice Chancellor of Barkatullah University, Bhopal, India, for use of the facilities and the UGC, New Delhi, India for financial support under the UGC-BSR Research Start-Up grant.