First Report of a New Begomovirus Infecting <i>Crepidiastrum denticulatum</i> in China

Crepidiastrum denticulatum is an annual or biennial herbaceous plant belonging to the family Asteraceae (Compositae). Its extract can reduce pollutant-induced damage and has anti-inflammatory and hepatoprotective effects (Kim et al. 2017; Yoon et al. 2019; Yoo et al. 2014). In April 2024, yellow vein symptoms on leaves were observed in approximately 20% C. denticulatum plants on a hillside in Ningbo City, Zhejiang Province, China. Symptomatic leaves from five plants were collected and pooled. Total RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, USA) and used for high-throughput sequencing. The cDNA library was constructed using a TruSeq RNA Sample Preparation Kit (Illumina) and an Illumina NovaSeq 6000 platform was utilized to yield 150 nt paired-end reads. A total of 47,866,048 paired-end reads were obtained, and 94,589 contigs (65 - 29,688 nt) were generated de novo and compared with sequences in the NCBI nt and nr databases using BLASTn and BLASTx, respectively. A total of 84,542 reads were mapped to the genome of Clerodendrum golden mosaic Jiangsu virus (ClGMJsV-[CN-JsXY2-08]) (Li and Zhou 2010; GenBank accession no. FN396966; Begomovirus clerodendrumjiangsuense) with an average coverage of 4533x. A contig of 2742 nt was aligned to ClGMJsV by BLASTn with the highest nt sequence identity of 88.6% and 94% query coverage, suggesting that the samples were infected with a begomovirus. No other viral sequence was identified from the constructed contigs. Subsequently, to determine the genome sequence of this virus, one plant was selected for genome validation. The full length of DNA-A component was amplified with back-to-back primers ISF (5’-ATACAGTTACTGGTGGTCAGTA-3’)/ ISR (5’-TGTATACGTAAACTTCCGCAATAC-3’) designed based on the assembled viral metagenomic sequencing data and cloned into the pEASY®-Blunt Zero Cloning vector (TransGen Biotech, China). After sequencing, the complete DNA-A sequence was determined to be 2,742 nts and deposited in GenBank (accession no. PX310655). SDT v1.3 was used to compare the sequence with those of other begomoviruses identified in the initial BLASTn analysis (Muhire et al. 2014). The new sequence had the highest identity (88.6%) to ClGMJsV-[CN-JsXY2-08]. According to the current demarcation threshold for begomoviruses (&lt;91 % nt identity) (Brown et al. 2015), this virus is a new species in the genus Begomovirus, which we have named Crepidiastrum yellow vein virus (CrYVV-[CN-Nb-2024]). Phylogenetic analysis was performed based on a multiple alignment of DNA-A sequences using our CrYVV isolate and other related geminiviruses using MEGA11. CrYVV clustered together with ClGMJsV and then senecio yellow mosaic virus (SYMV; Begomovirus senecionis). To confirm the infectivity of CrYVV-[CN-Nb-2024], we constructed its 1.43× infectious clone, and agrobacterium-infiltrated it into Nicotiana benthamiana, tomato, and pepper. At 21 days post-inoculation, compared with the control plants, N. benthamiana plants showed slight downward leaf curling symptoms, while pepper and tomato plants were dwarfed, and with upward curling on the leaves of pepper. Specific bands for DNA-A could be detected from new leaves of all inoculated plants by PCR. To the best of our knowledge, this is the first report of a begomovirus infecting C. denticulatum in China. Given its pathogenicity on tomato and pepper, future research should enhance monitoring of dispersal routes and assessment of potential risks.