First Record of Watermelon Crinkle Leaf-Associated Virus 2 Infecting Watermelon in Open Field in Italy

Watermelon (Citrullus lanatus L.) is an important horticultural crop inItaly, with a production of around 600,000 metric tons per year (third pro-ducer in Europe). Viruses are considered a limiting factor in watermelonproduction, especially those transmitted by efficient vectors such as white-flies and aphids. In July 2023, foliar symptoms on 100% of watermelonplants (cv. Samba), including curling and wrinkling, yellow mottling, andchlorosis, were observed in a commercial field of about 4,000 m2 at Eboli(Salerno Province, South Italy; coordinates: 40.2983°N, 14.5799°E). Somefruits were also symptomatic, showing circular lesions and deformations.Overall, symptoms resembled those described for watermelon crinkle leaf-associated viruses (WCLaV-1, Coguvirus citrulli, and WCLaV-2, Coguvirushenanense; genus Coguvirus, family Phenuiviridae) (Hernandez et al. 2021;Hendricks et al. 2022; Mulholland et al. 2023). Total RNAs were isolatedfrom pooled leaf tissues of 10 symptomatic plants using the Viral Gene-spinTM Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, SouthKorea) and used for construction of RNA-seq libraries, which were se-quenced on an Illumina Novaseq 6,000 platform with paired-end readslength of 101 bp. The HTS yielded 31,125,612 raw reads, while aftertrimming 28,868,089 reads were recovered. Viral genome assembly wasperformed with the two algorithms implemented in the SPAdes program(version 3.15.3; Bankevich et al. 2012). Two sets of filtered contigs wereobtained: 3,184 contigs with the algorithms implemented in Metaspades, and3,369 contigs with algorithms implemented in RNAviral. BLASTn/BLASTxanalyses of the contigs were carried out against online databases (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Two contigs, obtained from the assem-bly of 7,520 reads for RNA1 and 4,585 reads for RNA2, respectively,showed the greatest nucleotide identities with the WCLaV-2 genome. Thesecontigs represented full-length genomic sequences of the correspondingvirus. No other viral contigs belonging to other viruses were generatedby the HTS library. The HTS results were confirmed by RT-PCR on eachof the 10 watermelon samples using the WCLaV-2-specific primersWCLaV-2vRP (59-GTCTCACATTCCTGCACTAACT)/WCLaV-2cRP (59-ATCGGTCCTGGGTTATTTGTATC) targeting 968 bp of the RdRP andWCLaV-2vMP (59-GACTTCAGAACCTCAACATCCA)/WCLaV-2cMP (59-CAAGGGAGAGTGCTGACAAA) targeting 562 bp of the MP (Hernandezet al. 2021). Amplicons of the expected size were sequenced, and sequenceswere 100% identical to the corresponding regions obtained by HTS se-quencing, confirming presence of WCLaV-2 in the watermelon plants. Afterverifying the sequences at the 59 and 39 ends, the sequences of the twogenomic ssRNA segments consisted of 6,679 nucleotides (nt) for RNA1 and2,729 nt for RNA2. These sequences were deposited in GenBank with theaccession numbers PQ869160 (RNA1) and PQ869161 (RNA2). Accordingto the BLASTn analysis, the RNA1 was highly similar (99.94% sequenceidentity; 100% query coverage) to the Ju-01-WCLaV2 isolate (LC636073)from Brazil, while the RNA2 was highly similar (96.23% sequence identity;99% query coverage) to the KF-15 isolate (NC_079049) from China.WCLaV-2 was first reported in watermelon in China (Xin et al. 2017) andthen in the United States (Hernandez et al. 2021), Brazil (Maeda et al. 2022),and Australia (Mulholland et al. 2023). Although it was recently detected insome watermelon commercial seeds in Italy (Minutolo et al. 2024), thisreport confirms for the first time the spread of the virus in open field wa-termelon crops in southern Italy. Studies to assess the incidence and impactof disease on watermelon production, the identification of potential vectors,and the role of the seeds in virus transmission should be conducted to aid thedevelopment of the best management practices with the aim to contain thedamages caused by the virus