First report of Tomato leaf curl New Delhi virus in tomato in Nepal

Plant viral diseases are major challenges for vegetable production in Nepal and a recent outbreak of white fly-transmitted viral diseases has caused significant economic losses. Tomato leaf curl New Delhi virus (ToLCNDV) is a highly infectious and emerging begomovirus which causes devastating diseases of vegetable crops in the Indian subcontinent. Severely diseased tomato plants (c. 100) were observed in polytunnels in the Panchkhal area, Kavrepalanchok district in Bagmati province and in Besisahar municipality in the Lamjung district of Gandaki Province, Nepal in 2021 and 2022. The affected plants had symptoms including leaf curling (Figures 1, 2), yellowing, chlorotic mottling, and brown necrotic flecks in the interveinal areas (Figure 3). The incidence was around 100% in Kavrepalanchok and around 60% in Lamjung. Ten diseased leaves, one per plant from five plants each from Kavrepalanchok and Lamjung, were collected and tested postitive with an Immunostrip (Agdia, USA) specific to ToLCNDV. To confirm the diagnosis, DNA was extracted from the leaves using AMPure XP beads (Beckman Coulter, UK) (Kumaishi et al., 2022). PCR was conducted using ToLCNDV-specific primers ToLCNDV-up (5′-GAACTATGGTGAAGCGACCAGCAGA-3′) and ToLCNDV-do (5′-ACACAGGTCCTTAGGTACCTGG-3′) (Alfaro-Fernández et al., 2016). A single amplicon of c. 900 bp was produced from all the Immunostripe-positive samples of tomato, whereas no amplicon was amplified from healthy samples. Two PCR products each from Kavrepalanchok and Lamjung were purified using AMPure XP beads and submitted to the Beijing Genomic Center, Hong Kong and Nepal Academy of Science and Technology for bidirectional sequencing. Both consensus sequences were deposited in GenBank (Accession Nos. OQ646514 and OQ646515). BLASTn analyses of the sequences indicated that the isolate from Lamjunj district had the highest identity (96%) with a ToLCNDV isolate from bitter gourd in India (OM048966.1; Naik et al., 2022) and the Kabrepalanchok isolate had 98% identity with a ToLCNDV isolate reported on tomato in New Delhi, India (MW429271; Sarkar et al., 2021). Pairwise alignment showed 96% sequence identity between the two Nepalese isolates. ToLCNV was first detected in India in tomato (Padidam et al., 1995) and then in Bangladesh (Maruthi et al., 2005). To our knowledge, this is the first report of ToLCNDV in Nepal. Based on these results more intensive surveys targeting ToLCNDV must be undertaken on different crops and weeds to determine the host range and alternative hosts of this virus in Nepal. The authors would like to thank Tirtha Ghimire and Bindu Laxmi Shrestha for in supporting plant sample collections and Immunostrip tests, Feed the Future Integrated Pest Management Innovation Lab for supporting with Immunostripes and Diane Saunders at John Innes Centre for providing AMPure XP beads.