First Report of Cucurbit Chlorotic Virus Infecting Melon and Luffa in China

Cucurbit chlorotic virus (CuCV) is an unclassified species in the genus Crinivirus, family Closteroviridae, with a bipartite single-stranded positive-sense RNA genome (RNA1 and RNA2). The virus was first reported infecting cucurbit crops, including Benincasa hispida Cogn. var. chieh-qua How, watermelon (Citrullus lanatus), pumpkin (Cucurbita moschata), and cucumber (Cucumis sativus), in Hainan Province, China, causing typical chlorotic symptoms. (Che et al. 2023) Genome sequences of CuCV isolates from cucumber in Guangdong Province and Taiwan, China, are also available in the GenBank database. In the autumn of 2024, typical virus symptoms were observed on melon (Cucumis melo) in a ~0.75-hectare plastic greenhouse in Xiangcheng Town, Gao’an City, Jiangxi Province. The symptoms were of three main types: 1) leaf curling and dwarfing, 2) mosaic and mottling, and 3) chlorosis and yellowing. To identify the viral pathogen, we collected one leaf each from three individual plants, with each plant exhibiting a distinct symptom type (leaf curling and dwarfing, mosaic and mottling, or chlorosis and yellowing). These three leaves were pooled to create a single composite sample for ribosomal RNA–depleted RNA sequencing and subsequent bioinformatic virus identification, following methods described by (Peng et al. 2019). Briefly, total RNA was extracted from the composite sample, and its quality and integrity were verified. Ribosomal RNA was removed using a rRNA depletion kit, and a cDNA library was constructed and sequenced on an Illumina NovaSeq 6000 platform (Sangon BioTech, Shanghai, China). After removing adapter and low-quality reads, the clean data were subjected to virus identification and annotation using VirusDetect (Zheng et al. 2017). Five viruses were identified in the composite sample: melon necrotic spot virus, watermelon silver mottle virus, tomato leaf curl New Delhi virus, Cucumis melo alphaendornavirus, and CuCV. To improve the completeness of the viral genomes, the clean data were de novo assembled using SPAdes (Prjibelski et al. 2020), yielding the near-full-length genome of a CuCV isolate designated CuCV-GAmelon. The assembled RNA1 and RNA2 sequences were 9,148 nt and 7,780 nt in length, respectively, lacking partial terminal untranslated region sequences. A primer pair (S1-4082F & S1-5271R showed Supplemental Table 1) was designed based on the RNA1 sequence of CuCV-GAmelon. RT-PCR using this primer pair was then performed on the three individual samples used to create the composite pool. This assay yielded negative results for the two samples with leaf curling/dwarfing and mosaic/mottling, but produced the expected ~1.2 kb amplicon for the sample showing chlorosis and yellowing. Sanger sequencing of this amplicon confirmed 100% identity with the corresponding region of CuCV-GAmelon RNA1 obtained by HTS. Furthermore, RT-PCR testing of four additional chlorotic/yellowing samples from the same field also produced the same target amplicon. BLASTn analysis of RNA1 against the core nucleotide database showed the top three matching CuCV isolates were XZ-C-1 from Taiwan, China (PQ618563; 99.87% identity), chieh-qua-CM from Hainan, China (OP976048; 99.83% identity), and watermelon-LS from Hainan, China (OP976050; 99.4% identity). For RNA2, the top three matches were chieh-qua-CM (OP976049; 99.88% identity), watermelon-LS (OP976051; 99.81% identity) from Hainan, China, and XZ-C-1 (PQ618564; 99.78% identity) from Taiwan, China. A comparison of the obtained near-full-length sequences of isolate GAmelon with the complete genome of isolate chieh-qua-CM (RNA1: OP976048, 9157 nt; RNA2: OP976049, 7788 nt) revealed that the GAmelon sequences were missed at the termini: the RNA1 sequence lacked 5 nt at the 5′ end and 4 nt at the 3′ end, while the RNA2 sequence lacked 8 nt at the 5′ end. Given the conserved terminal sequences among crinivirus genomes, four primer pairs were designed based on the Hainan isolate chieh-qua-CM (RNA1: OP976048, RNA2: OP976049) to amplify the 5′ and 3′ terminal regions: S1-5utr & S1-389R and S1-8701F & S1-3utr for RNA1, and S2-5utr & S2-358R and S2-7158F & S2-3utr for RNA2 (Supplemental Table 1). The complete genome sequences of CuCV-GAmelon (RNA1 and RNA2) were obtained and deposited in GenBank under accession numbers PX501711 and PX501712, respectively. In the spring of 2025, 90 symptomatic cucurbit samples, comprising 41 watermelon, 36 melon, 4 pumpkin, 3 cucumber, and 6 luffa (Luffa aegyptiaca) samples, were collected from major cucurbits production areas in Jiangxi Province. RT-PCR screening using primers S1-4082F & S1-5271R detected CuCV in seven out of 90 samples: one melon, one cucumber, one pumpkin, and three luffa samples from Xinfeng County, and one watermelon sample from Xingan County. Using the HTS and full-length amplification strategies described above, the complete genome sequences of three additional CuCV isolates were obtained: XFluffa from luffa in Xinfeng (RNA1: PX501713, RNA2: PX501714), XFmelon from melon in Xinfeng (RNA1: PX501715, RNA2: PX501716), and XGwatermelon from watermelon in Xingan (RNA1: PX501717, RNA2: PX501718). Pairwise sequence comparisons using SDT v1.3(Muhire et al. 2014) among the nucleotide sequences of the four Jiangxi CuCV isolates and eight other isolates from GenBank showed identities ranging from 98.4% to 99.9% for RNA1 among the Jiangxi isolates and 98.3% to 99.9% between Jiangxi and other isolates. All CuCV RNA1 sequences shared 67.9–68.3% identity with those of Crinivirus cucurbitae (cucurbit yellow stunting disorder virus, CYSDV). For RNA2, identities were 99.8–99.9% among Jiangxi isolates and 99.3–99.9% between Jiangxi and other isolates. All CuCV RNA2 sequences shared 68.4–68.6% identity with CYSDV RNA2. These results indicate low genetic diversity among CuCV isolates. To our knowledge, this is the first report of CuCV infecting melon and luffa in China. It also constitutes the first report of this virus occurring on cucurbit crops in Jiangxi Province, an inland region, confirming its spread from coastal to inland areas. These findings contribute to understanding the ongoing emergence and epidemiology of CuCV in Chinese cucurbit crops.