Watermelon Crinkle Leaf-Associated Virus 1 and Watermelon Mosaic Virus Mixed Infecting Watermelon ( <i>Citrullus lanatus</i> ) in Indiana

Watermelon (Citrullus lanatus) production in the USA was valued at $787 million in 2023, with most of the production concentrated in Florida, California, and Georgia. In the Midwest, Indiana leads watermelon production with a value of $49 million (USDA-NASS, 2024). In August 2024, watermelon plants (cv. Guardsman) showing virus-like symptoms, including mosaic, yellowing, leaf deformation, and wrinkling, were observed in southern Indiana (Knox County). No insect vectors were observed in the fields during the site visits. Samples were collected from three plants in each of four separate watermelon fields. A preliminary serological screening using ImmunoStrip® tests (Agdia, Elkhart, IN) for the general potyvirus group and melon severe mosaic virus was performed. Only the ImmunoStrip® tests for the potyvirus group showed positive results. Total RNA was extracted from a symptomatic representative sample from each field using the Spectrum Plant Total RNA kit (Sigma-Aldrich, St. Louis, MO). A pool of total RNA from four individual samples was then submitted for high-throughput sequencing (HTS) using an Illumina NovaSeqX platform as 150 bp paired-end reads (Psomagen, Rockville, MD). The library was prepared using the TruSeq Stranded Total RNA with Plant Ribo-Zero kit. HTS generated 58 million reads. Sequence reads were trimmed with BBDuk software, de novo assembled using SPAdes v3.15, and submitted to the NCBI. Viral contigs were identified using Blastx against the full non-redundant database (as of November 2024). Watermelon crinkle leaf-associated virus 1 (WCLaV-1) was assembled into three contigs: 6.6 Kb viral polymerase (RNA1, 47,009 reads; coverage 1,122X; acc. PQ621806), 1.4 Kb movement protein (RNA2, 6,932 reads; coverage 1,193X; acc. PQ621807), and 1.3 Kb nucleocapsid (RNA2, 5,300 reads; coverage 1,006X; acc. PQ621808). The highest level of nucleotide identity detected ranged from 98.0-99.9% compared with WCLaV-1 isolate Pi-01 from Brazil (LC636068-9). Additionally, a nearly full-length watermelon mosaic virus (WMV) genome (~10 Kb) with >99% identity compared with WMV isolate C05-270 from France (EU660585) was assembled (17,871,776 reads; coverage 24,638X; acc. PQ855772). Putative mycovirus sequences were also detected through HTS; however, further analysis was not conducted due to the incompleteness of the sequences and the limited number of hits. The occurrence of each virus in the individual samples was further validated by RT-PCR using sequence-specific primers designed in this study. WCLaV-1 was detected in 2/4 watermelon samples, while WMV was detected in 2/4 samples, with one sample being infected by both viruses. Three amplicons were sequenced using Nanopore Technology (Plasmidsaurus, Eugene, OR), compared to homologous sequences in GenBank, and uploaded to the NCBI under accessions PV030975-PV030977. Interestingly, HTS did not detect WCLaV-2, commonly detected in mixed infections with WCLaV-1. Although WCLaV-1 and WCLaV-2 were previously associated with symptoms (Xin et al. 2017), the effect on growth and yield is still unknown (Hendricks and Roberts 2023). To the best of our knowledge, this is the first report of WCLaV-1 and WMV infecting watermelons in Indiana, highlighting the need for additional research on the impact of single and mixed infections of these viruses on cucurbit crops.