First Report of Moroccan Watermelon Mosaic Virus (MWMV) infecting Cucurbita pepo in Slovenia

From June to August 2025, cucurbit plants showing mosaic, blistering, crinkling, yellowing, and leaf deformation were collected from 27 locations in Slovenia (Supplementary Figure 1, 2) during an official survey for viruses listed in Annex II of Commission Implementing Regulation (EU) 2019/2072. Six composite samples (pools), each comprising six individual samples collected from symptomatic cucurbit plants, were selected for high-throughput sequencing (HTS), including 34 Cucurbita pepo, one Cucumis sativus, and one unidentified cucurbit. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Germany) and sequenced using an Oxford Nanopore Technologies MinION Mk1B (PCR-cDNA barcoding kit SQK-PCB114.24, R10.4.1 flow cell; Supplementary material). Data were analyzed using the pipeline of Pecman et al. (2022), with basecalling performed using Dorado v0.7.2. Raw reads were deposited in the European Nucleotide Archive (accession PRJEB110990). Moroccan watermelon mosaic virus (MWMV) was detected in four pools, together with cucurbit aphid-borne yellows virus (CABYV), cucumber mosaic virus (CMV), watermelon mosaic virus (WMV), and zucchini yellow mosaic virus (ZYMV) (Supplementary Table 1). No cucurbit-infecting EU quarantine viruses, including lettuce infectious yellows virus, melon yellowing-associated virus, squash vein yellowing virus or begomoviruses, were detected. MWMV was confirmed by RT-PCR in seven individual C. pepo samples from four HTS-positive pools: D785/25, D816/25, D826/25, D840/25, D985/25, D1025/25, and D1026/25. All MWMV-positive samples were confirmed by RT-PCR to be co-infected with at least one additional virus: CABYV, CMV, WMV, and ZYMV (Supplementary Tables 1, 2). A near-complete MWMV genome assembled from pool P6_25 (NCBI PZ518257) shared 97.2% nucleotide identity with isolate OQ847413 from Morocco. For biological confirmation, sap from MWMV-positive samples, except D840/25 (showing faint bands in RT-PCR), was mechanically inoculated onto two plants of Chenopodium quinoa at the three- to five-true-leaf stage and three plants of C. pepo ‘Bianca di Trieste’ at the one- to two-true-leaf stage. Leaf tissue was homogenized in phosphate buffer and rubbed onto carborundum-dusted leaves. Four weeks after inoculation, newly developed leaves were pooled by host and tested by RT-PCR; MWMV was detected in C. pepo pools inoculated with D785/25, D826/25, D1025/25, and D1026/25, occurring alone in one C. pepo pool, in mixed infections with ZYMV in two pools, and in a mixed infection with WMV in one pool (Supplementary table 1), whereas no MWMV was detected in any C. quinoa pools. This is the first report of MWMV in Slovenia, extending its known distribution in Europe including previous reports from neighboring countries (Roggero et al. 1998; Jagunić et al. 2025). It also demonstrates that MWMV occurs in Slovenian cucurbit production in mixed infections with other major cucurbit viruses.