First report of watermelon crinkle leaf‐associated virus 1 and 2 in different cucurbit hosts in Spain

In 2023, populations of the melon aphid (Aphis gossypii) collected from field-grown cucurbit crops exhibiting yellowing and mosaic virus-like symptoms in Murcia, Spain, were analysed by High Throughput Sequencing (HTS, RNA-Seq). Total RNA was extracted from eight samples, each comprising a pool of 40 aphids, using Tri-Reagent, and subsequently sequenced. The analysis revealed the presence of watermelon crinkle leaf-associated virus 1 (WCLaV-1), amongst other viruses, in one of the aphid samples collected from watermelon (de Moya-Ruiz et al., manuscript in preparation). WCLaV-1 was confirmed by RT-PCR using primers designed to detect the RNA-dependent RNA polymerase (RdRp) gene (Hernandez et al., 2021), and Sanger sequenced (STAB VIDA, Portugal). WCLaV-1 and watermelon crinkle leaf-associated virus 2 (WCLaV-2) are coguviruses first described in China in 2015 on watermelon (Xin et al., 2017) and subsequently reported in Australia, Brazil and the United States on watermelon and other cucurbits such as courgette and squash (Hernandez et al., 2021; Maeda et al., 2022; Hendricks & Roberts, 2023; Iriarte et al., 2023; Mulholland et al., 2023). In Europe, WCLaV-1 and WCLaV-2 have been described recently in Slovenia, and WCLaV-2 in Italy, affecting watermelon crops (Parrella, 2025; Vučurović et al., 2025). Both viruses are presumed to be transmitted by aphids; WCLaV-1 is also mechanically transmitted but with low efficiency (Mulholland et al., 2023; Xin et al., 2017). Their impact on the yield of cucurbit crops remains unclear. To assess the occurrence and distribution of WCLaV-1 and -2 in cucurbit production areas in Murcia, RT-PCR using primers targeting the RdRp region of each virus was used to test 10 diseased cucurbit samples (two samples from crops of courgette, cucumber, melon, pumpkin and watermelon), as described by Hernandez et al. (2021). Two partial RdRp fragments of each virus were amplified from watermelon samples collected during annual monitoring of cucurbit viral diseases in the Murcia region (Fig. 1). The corresponding 881 nt and 968 nt of the RdRp fragments from WCLaV-1 and WCLaV-2, respectively, were cloned and used to synthesize specific digoxigenin-labeled RNA probes. Dot-blot hybridization analysis of 50 samples from frozen samples of cucurbit crops collected between 2022 and 2024 revealed that WCLaV-1 and WCLaV-2 were present in 45% and 9% of watermelon samples with virus-like symptoms, respectively. Both viruses were also detected in melon and cucumber samples at a low frquency (WClaV-1: 4% and 9%, respectively; WCLaV-2: 7% and 1%, respectively). Two Spanish WCLaV-1 and WCLaV-2 isolates were selected randomly from watermelon samples from each season, together with a further isolate from melon and two from cucumber. Their partial RdRp genes were amplified and Sanger sequenced, and corresponding sequences were deposited in GenBank under Accession Nos. PV544223-PV544230 for WCLaV-1 and PV544217-PV544222 for WCLaV-2. Phylogenetic analysis of the partial nucleotide sequence of the RdRp was conducted using the maximum likelihood method in MEGA X. The results showed that Spanish isolates of WCLaV-1 clustered with an Asian isolate (KY781184), and were distinct from American, Australian and Iraqi isolates (Fig. 2A). Likewise, Spanish isolates of WCLaV-2 clustered with Asian (KY781187) and American (MZ325854) isolates, and were separate from Brazilian, Italian and other American isolates (Fig. 2B). Spanish isolates of WCLaV-1 and -2 grouped together regardless of the host. This is the first report of WCLaV-1 and WCLaV-2 affecting cucurbit crops in Spain, expanding the known geographic distribution and host range to cucumber and melon. This study highlights the need to conduct systematic and extensive monitoring in Spain to ascertain the distribution and prevalence of WCLaV-1 and WCLaV-2 in cucurbit crops. Additionally, further research on the ecological aspects and biological traits of WCLaV-1 and -2 are necessary to understand their epidemiology, transmission and impact on crop yield. We thank A. Kupczok, D. Karapliafis, and E. Matsumura (Wageningen University and Research, Netherlands) for their valuable assistance in aphid virome characterisation and helpful discussions and comments. This work was part of the AGROALNEXT programme, supported by MCIN with funding from NextGenerationEU (PRTR-C17.I1), in addition to the research project, PID2022-141108OB-I00, funded by MCIN/AEI/10.13039/501100011033/FEDER (EU).