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 in Italy, with a production of around 600,000 tons per year (third producer in the Europe). Viruses are considered a limiting factor in watermelon production, especially those transmitted by efficient vectors such as whiteflies and aphids. In July 2023, foliar symptoms on 100% of watermelon plants (cv. Samba), including curling and wrinkling, yellow mottling and chlorosis, were observed in a commercial field of about 4000 m2 at Eboli (Salerno province, South Italy; coordinates: 40°29'83''N 14°57'99''E) (Figure S1). Some fruits 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, Coguvirus henanense; genus Coguvirus, family Phenuiviridae) (Hernandez et al., 2021; Hendricks et al., 2022; Mulholland et al., 2023). Total RNAs were isolated from pooled leaf tissues of 10 symptomatic plants using the Viral Gene-spinTM Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Inc., South Korea) and used for construction of RNA-seq libraries, which were sequenced on an Illumina Novaseq 6000 platform with paired-end reads length of 101 bp. The HTS yielded 31,125,612 raw reads, while after trimming 28,868,089 reads were recovered. Viral genome assembly was performed with the two algorithms implemented in the SPAdes program (v. 3.15.3; Bankevich et al., 2012). Two sets of filtered contigs were obtained: 3,184 contigs with the algorithms implemented in Metaspades, and 3,369 contigs with algorithms implemented in RNAviral. BLASTn/BLASTx analysis of the contigs were carried out against online databases (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Two contigs, obtained from the assembly of 7520 reads for RNA1 and 4585 reads for RNA2 respectively, showed the greatest nucleotide identities with the WCLaV-2 genome. These contigs represented full-length genomic sequences of the corresponding virus. No other viral contigs belonging to other viruses were generated by HTS library. The HTS results were confirmed by RT-PCR on each of the 10 watermelon samples using the WCLaV-2-specific primers WCLaV-2vRP (5'-GTCTCACATTCCTGCACTAACT)/WCLaV-2cRP (5'-ATCGGTCCTGGGTTATTTGTATC) targeting 968 bp of the RdRP and WCLaV-2vMP (5'-GACTTCAGAACCTCAACATCCA)/WCLaV-2cMP (5'-CAAGGGAGAGTGCTGACAAA) targeting 562 bp of the MP (Hernandez et al., 2021). Amplicons of the expected size were sequenced and sequences were 100% identical to the corresponding regions obtained by HTS sequencing, confirming presence of WCLaV-2 in the watermelon plants. After verifying the sequences at the 5' and 3' ends, the sequences of the two genomic ssRNA segments consisted of 6679 nucleotides (nt) for RNA1 and 2729 nt for RNA2. These sequences were deposited in GenBank with the accession numbers PQ869160 (RNA1) and PQ869161 (RNA2). According to the BLASTn analysis, the RNA1 was highly similar (99.94% sequence identity; 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) and then in USA (Hernandez et al. 2021), Brazil (Maeda et al., 2022) and Australia (Mulholland et al., 2023). Although it was recently detected in some watermelon commercial seeds in Italy (Minutolo et al., 2024), this report confirms for the first time the spread of the virus in open field watermelon crops in southern Italy. Studies to assess the incidence and impact of disease on watermelon production, the identification of potential vectors and the role of the seeds in virus transmission should be conducted to aid the development of the best management practices with the aim to contain the damages caused by the virus.