First Report of Watermelon Crinkle Leaf-Associated Virus 1 Naturally Infecting Watermelon (Citrullus lanatus) in Georgia, United States

Abstract

Watermelon (Citrullus lanatus) is one of the major vegetable crops grown in Georgia during the spring and summer seasons, contributing $180 million of farmgate value to the state’s economy (Georgia Farm Gate Value Report 2019). During the summer of 2021, watermelon plants with foliar symptoms such as yellow mottling, chlorosis, and wrinkling with thickened, bunchy, and upward curling were observed on commercial fields of Georgia, USA. A disease incidence of 15-20% in ~56 ac in Tift county and 10-15% in ~60 ac in Wilcox county was observed. The symptoms observed were similar to those described for watermelon crinkle leaf-associated viruses (WCLaV-1 and WCLaV-2) from Florida (Hendrick et al., 2021) and Texas (Hernandez et al., 2021). Symptomatic leaves from Tift (n=40) and Wilcox (n=20) counties were collected, surface sterilized with 0.1% bleach and used for total nucleic acid extractions using MagMAX 96 Viral RNA isolation kit (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s instruction without DNase treatment. The potential introduction of WCLaV-1 and WCLaV-2 into Georgia was tested by reverse-transcription-polymerase chain reaction (RT-PCR) assay using specific primers targeting RNA-dependent-RNA polymerase (RdRp) and movement protein (MP) genes of both viruses (Hernandez et al., 2021). The expected amplicon sizes for RdRp (~900 nt) and MP (~500 nt) genes of WCLaV-1 located on RNA 1 and RNA 2 segements, respectively, were observed in 39 of 40 (97.5%) samples from Tift and seven of 20 (35%) samples from Wilcox. However, WCLaV-2 was not detected in any of the tested samples. All 60 samples also tested negative for the whitefly-transmitted viruses prevalent in the region, including cucurbit chlorotic yellows virus, cucurbit yellow stunting disorder virus, and cucurbit leaf crumple virus using virus-specific primers (Kavalappara et al., 2021). A subset of the samples analyzed by RT-PCR were also tested by SYBR green-based real-time RT-PCR assay targeting MP gene of WCLaV-1 using primers WCLaV-1FP (5’TCCACAAGCTTGATGGA- GGG3’) and WCLaV-1RP (5’TCCCGAGTGAGGAAGCTAGT3’). The virus was detected in samples from both counties and the results matched with those obtained by the conventional RT-PCR assays (Suppl. Table 1). The presence of WCLaV-1 was further confirmed by sequencing (Genewiz, South Plainfield, NJ, USA) coupled with BLASTn analysis of amplicons resulted from the conventional RT-PCR from three randomly selected samples . The partial RdRp sequences (OL469153 to OL469155) were 99.3% and 99.9% identical to the corresponding sequences of WCLaV-1 isolates from China (KY781184) and Texas (MW559074) respectively. The partial MP sequences (OL469150 to OL469152) were 100% identical to those from China (KY781185) and Texas (MW559077). WCLaV-1 and WCLaV-2 were first discovered in Asia (Xin et al., 2017). Both viruses were subsequently reported from North and South Americas (Hendrick et al., 2021; Hernandez et al., 2021; Maeda et al., 2021), indicating their geographical expansion. Biological information, including vector relations, is unknown for both viruses and other members of the genus Coguvirus (family Phenuiviridae), to which they are provisionally assigned (Zhang et al., 2021). Further studies are also required to understand the biology and impact of both viruses on watermelon production and other crops, if any.

Persistent, and Asymptomatic Viral Infections and Whitefly-Transmitted Viruses Impacting Cantaloupe and Watermelon in Georgia, USA

“…WCLaV-1 was detected in one of the six watermelon samples analyzed by HTS. This virus was recently identified in Georgia on watermelon [6] and the samples from which it was detected showed symptoms of mosaic, crinkling, and bunching (Figure 2A), as described previously [8,30,31]. Based on the molecular features and phylogenetic reconstructions, WCLaV-1 has been provisionally assigned to the genus Coguvirus (family Phenuiviridae) [30].…”

“…Four different plant RNA viruses were identified after de novo assembly and BLASTn searches against the NCBI database. One of them was the recently reported watermelon crinkle leaf-associated virus 1 (WCLaV-1) [6] and the other three were persistent viruses: cucumis melo amalgavirus 1 (CmAV1), cucumis melo cryptic virus (CmCV), and cucumis melo endonavirus (CmEV). The large number of sRNA reads aligning to the genome (Table 2) and covering the entire length of their genome (Figure 3) added confidence to their identification.…”

“…Viruses 2022, 14, 1310 2 of 13 virus (CuLCrV) (Genus Begomovirus, family Geminiviridae) [2], the cucurbit yellow stunting disorder virus (CYSDV) [3], and the cucurbit chlorotic yellows virus (CCYV) (Genus Crinivirus, family Closteroviridae) [4] are reported on major cucurbits, including cantaloupe, cucumber, squash, watermelon, and zucchini in Georgia [3,5]. Recently, the presence of watermelon crinkle leaf-associated virus 1 (WCLaV-1) was reported in watermelons in Georgia [6]. Furthermore, whitefly-transmitted squash vein yellowing virus (SqVYV) [7] and watermelon crinkle leaf-associated virus 2 (WCLaV-2) [8,9] are a concern to watermelon production in the state due to their recent reports in the neighboring state of Florida.…”

First report of Watermelon crinkle leaf‐associated virus 1 (WCLaV‐1) on Cucurbita pepo in the United States

“…Both the samples (Z272 and Z273) had similar results, therefore only sequences from sample Z272 were submitted to GenBank. The results were further confirmed by SYBR ® ‐based real‐time RT‐PCR using the WCLaV‐1 MP gene‐specific primer pairs (WCLaV‐1FP/WCLaV‐1RP) (Adeleke et al., 2022).…”