First Report of Tomato Leaf Curl New Delhi Virus Infecting Several Cucurbit Plants in China

In autumn 2022, a novel and devastating viral disease affecting cucurbits emerged in Ningbo (Zhejiang province), Haimen (Jiangsu province), and Shanghai, China, causing an approximate 650-hectare infestation and resulting in nearly US$15 million in economic losses. The incidence rates of infection reached up to 72.5% on muskmelon (Cucumis melo L. ssp melo), oriental melon (Cucumis melo L. var. agrestis), pumpkin (Cucurbita moschata), luffa (Luffa acutangula), and squash (Cucurbita pepo), and were highly associated with the presence of whitefly (Bemisia tabaci). Infected plants exhibited symptoms such as dwarf stunting, reduced leaf size, leaf chlorotic patches, malformation, fruit deformation, leaf downward rolling, and yellowing (Figure 1). To identify the pathogen, forty cucurbit leaf samples were collected from Haimen (18), Ningbo (19), and Shanghai (3) and tested for cucurbits chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and Begomovirus using RT-PCR or PCR. All samples tested negative for CCYV and CYSDV using species-specific primers; however, 29 out of 40 samples tested positive (see Supplementary Table 1) for Begomovirus using the degenerate primer pairs PA/PB (Deng et al. 1994). PCR products from seven samples, representing different regions and hosts, underwent Sanger sequencing. The nucleotide sequences of these products showed 98.2-99% identity to tomato leaf curl New Delhi virus (ToLCNDV) by BLASTn. Subsequently, the 29 positive cucurbit samples were confirmed using ToLCNDV-specific primer pairs NDVAF/NDVAR and NDVBF/NDVBR (Jyothsna et al. 2013) for DNA-A and DNA-B, respectively. The DNA-A and DNA-B genome sequences of ToLCNDV isolates from Haimen (Haimen4), Ningbo (Ningbo6), and Shanghai (Shanghai1) were obtained using the primer pairs NDVAF/NDVAR, A1961F/A2645R (covering complete DNA-A sequences), NDVBF/NDVBR, and B1613F/B2579R (covering complete DNA-B sequences，see Supplementary Table 2). No amplicon was produced with primer pairs UNA101/UNA102 and beta01/beta02 (Supplementary Table 2) for detecting Alphasatellite and Betasatellite DNAs, respectively. The complete DNA-A genome sequences (2739 bp) of Haimen 4 (accession no. OP585369), Ningbo 6 (accession no. OP585370), and Shanghai 1 (accession no. OP683993) isolates exhibited 99.5-99.6% nucleotide identity to each other, and their highest nucleotide sequence identity (99.3-99.4%) was shared with the DNA-A of ToLCNDV-Zhejiang isolate (accession no. OP356207) from tomato in Zhejiang Province, China. The complete nucleotide sequences (2693 nt) of DNA-B for Haimen 4 (accession no. OP683995), Ningbo 6 (accession no. OP683996), and Shanghai 1 (accession no. OP683994) isolates showed 99.0-99.1% identity to each other, and their highest nucleotide sequence identity (~99.1%) was shared with the DNA-B of ToLCNDV-Zhejiang isolate (accession no. OP356208).All ToLCNDV isolates from mainland China, including the Zhejiang isolate and the three isolates in this study, shared 98.3-98.7% nucleotide sequence identity and 98.2-98.4% with the DNA-A genome of the severe isolate (accession no. HM159454) from tomato in New Delhi, India, and the DNA-B genome of the India:Delhi:Cucumis:2012 isolate from cucumber in New Delhi, India, respectively. However, the genome sequence identities between mainland and Taiwan isolates (accession nos. GU180095 and GU180096) were below 93%, suggesting that mainland China isolates of ToLCNDV are more closely related to the India isolate than to the Taiwan isolate.To fulfill Koch's postulates, infectious clones of the Haimen 4 isolate were constructed and agroinfiltrated into muskmelon, oriental melon, pumpkin, luffa, and squash plants. In brief, two plasmids, containing 1.56-mer DNA-A and 1.4-mer DNA-B genome sequences, were constructed using enzyme digestion and ligation, transformed into Agrobacterium tumefaciens strain GV3101, respectively, and then co-agroinfiltrated into cucurbit plants. Initial symptoms appeared in the new leaves at 7 days post-inoculation (DPI), followed by severe leaf curling, dwarfing, stunting, reduced leaf size, and chlorotic leaf patches at 18 DPI. The presence of DNA-A and DNA-B of ToLCNDV in inoculated plants was confirmed by PCR using primer pairs A1961F/A2645R and B1613F/B2579R, respectively. Collectively, the pathogen of this emerging disease has been identified as ToLCNDV. ToLCNDV was first reported on tomato in India and is now the most predominant and economically significant disease affecting cucurbit and solanaceous crops in Southeast and East Asia, the Middle East, and the Mediterranean Basin (Moriones et al. 2017). In China, ToLCNDV was initially reported on oriental melon in Taiwan (Chang et al. 2010) and subsequently on tomato (Lycopersicon esculentum) in Zhejiang province (Li et al. 2022). To the best of our knowledge, this is the first report of ToLCNDV infecting muskmelon, pumpkin, luffa, and squash in China. Further investigations on the epidemiology of this viral disease in China are needed.