First Report of Watermelon Crinkle Leaf-Associated Virus 1 (WCLaV-1) and (WCLaV-2) Infecting <i>Citrullus lanatus</i> in Arizona, U.S.A.

Watermelon (Cucurbitaceae: Citrullus lanatus (Thunberg) Matsumura & Nakai) is globally cultivated for its edible fruits. In Arizona, watermelons are planted (~2,000 ha/year) with a production value of ~$45 million/ year. In June and October 2023, virus-like symptoms consisting of yellow mosaic and mild leaf crinkling were observed on watermelons and zucchini squash plants grown in an experimental field (0.5 ha) located at the Yuma County Cooperative Extension farm, University of Arizona, Yuma (32.715435-114.700694). The incidence was estimated at ~10%. Whiteflies and aphids were found in the field. Total RNA was extracted from leaf tissues of randomly selected watermelons (n=18) and zucchinis (n=18) according with Tzanetakis et al. (2007). The 36 samples were equimolar pooled and used for cDNA library construction with the TruSeq Stranded Total RNA using the Ribo-Zero Plus rRNA depletion kit (Illumina). The cDNA library was sequenced on the Illumina NovaSeq 100M platform generating ~71M paired-end reads (151nucleotides (nt) /each). The de-novo transcriptome assembly was made using Trinity software (Grabherr et al., 2011) and the contigs were submitted to BlastX against the RefSeq virus genomes (NCBI). Reads of two coguviruses (Phenuiviridae, Bunyavirales): watermelon crinkle leaf-associated virus 1 (WCLaV-1) reads =284, and watermelon crinkle leaf-associated virus 2 (WCLaV-2), reads=808, were identified. The RNA1 and RNA2 segments of WCLaV-1 (1 to 17X) and WCLaV-2. (1 to 40X) were generated from the mapped reads, and each shared ≥ 96.4% nt identity with the corresponding virus reference genomes. Other cucurbit viruses were detected (Supplementary Table S1). The results were confirmed from each of the pooled samples (n=36) by RT-PCR using WCLaV-1 and WCLaV-2 specie specific primers (Hernandez et al. 2021), which amplify partial sequences of the RNA 1- encoded RNA-dependent RNA polymerase (RdRp) and RNA 2- encoded movement protein (MP) and nucleoprotein (NP) genes. Expected amplicons for RdRp, MP and NP of ~881bp, 538bp, 786bp (WCLaV-1) and ~968bp, 562bp, 449bp (WCLaV-2) were obtained from six watermelon samples (Positives: WCLaV-1, n= 3 and WCLaV-2, n= 3). Amplicons were Sanger sequenced (GenBank accessions, WCLaV-1-RdRP (PQ094220 to PQ094222), MP (PQ094223 to PQ094225) and NP (PQ094226 to PQ094228); WCLaV-2-RdRP (PQ094229 to PQ094231) MP (PQ094232 to PQ094234), NP (PQ094235 to PQ094237). Pairwise comparisons revealed ≥ 99.89 % identity with the RdRp, MP, NP genes of WCLaV-1 GenBank accession MW559074-MW559082, and 100% identity with RdRp, MP, NP genes of WCLaV-2 GenBank accession MW559083-MW559091. Additionally, a survey in June 2023 was conducted on different farms in Yuma County. A total of 22 watermelon plants showing yellow mosaic symptoms were collected and RT-PCR tested for WCLaV-1 and WCLaV-2. Three samples tested positive for WCLaV-1and seven for WCLaV-2. WCLaV-1 and WCLaV-2 were initially reported in China (Xin et al. 2017) and then in the United States in 2021 (Hernandez et al. 2021). To our knowledge, this is the first report of WCLaV-1 and WCLaV-2 infecting watermelon in Arizona, expanding its geographical distribution in the USA. The biology, epidemiology, and symptomatology of these coguviruses are still poorly understood. WCLaV-1 and WCLaV-2 have been reported co-infecting watermelon together with other virus species making it difficult to clarify its transmission, symptoms-related and potential risk for cucurbits.