First Report of Pepper Chlorosis-Associated Virus in Burdock () in Korea.

Burdock () is a fiber-rich root vegetable widely used in Asian cuisine with many health benefits. In July 2023, two burdock leaf samples showing viral symptoms, such as yellowing and interveinal chlorosis, were collected from a field in Daegu, South Korea, with an incidence rate of about 10%. The samples were pooled, ground with liquid nitrogen, and total RNA was extracted using a Maxwell® 16 LEV Plant RNA Kit (Promega, Madison, USA). A cDNA library was constructed using TruSeq Standard Total RNA with Ribo-Zero (Illumina, San Diego, USA) and sequenced on an Illumina NovaSeq 6000 (BIOTO, Daejeon, Korea) with 100 bp paired-end reads. Of the approximately 84 million reads generated, about 61 million clean reads were identified using Trimmomatic 0.39 with default parameters. Subsequently, Trinity 2.13.0 was used for assembly, resulting in 179,910 contigs, which, when annotated as previously described (Bak et al., 2024), revealed the presence of tomato spotted wilt virus (TSWV) and pepper chlorosis-associated virus (PepCaV). Three PepCaV-related contigs, representing a near-complete genome, were submitted to NCBI GenBank (PP502423-PP502425). BLASTn analysis showed 95.21% (7275/7641 bp), 95.89% (1680/1752 bp), and 96.18% (1461/1519 bp) identities with the L, M, and S segments of the PepCaV isolate Higashitsuno_2021 (LC719619-LC719621), respectively. RT-PCR was performed to confirm the presence of viruses using specific primers for PepCaV (5'-CACCTTGATTATAGACATGACAG/GTTCTCAAATCTTCCCAATCG-3') targeting the coat protein region, and TSWV (5'-GCAACAACTTGCAAGAAGATG/CATGACCTTCAGAAGGCTTG-3'; Kim et al., 2021). Initial results showed both samples were positive for TSWV, and one was positive for PepCaV. Additionally, 26 symptomatic and 8 asymptomatic samples were collected from the same field. PepCaV was positive in 3 samples, TSWV in 12, and TSWV+PepCaV in 11 by RT-PCR. All asymptomatic samples were negative. Leaves infected with PepCaV, whether solely or co-infected with TSWV, initially exhibited interveinal chlorosis and vein contraction on one side of the main vein (Fig. S1). In plants infected with PepCaV alone, these symptoms were mild or less noticeable in the initial phases. The majority of PepCaV-infected samples were co-infected with TSWV, during which symptoms appeared more severe. Following this, an amplicon, derived from the sample that initially tested positive for PepCaV in the first RT-PCR was cloned using the All in OneTM Cloning Kit (BioFact, Daejeon, Korea) and sequenced by Bionner (Daejeon, Korea). The resulting sequence, submitted to NCBI GenBank (PP928956), demonstrated 96.87% (805/831 bp) identity with the PepCaV isolate Higashitsuno_2021 (LC719600). Moreover, sap inoculation was performed on , , , , , , , and , but no transmission was identified in any case. PepCaV, recently identified and likely belonging to the genus within the family , is known to infect pepper and tobacco (Shimomoto et al., 2023; Wang et al., 2024). This is the first report of PepCaV in Korea and the first identification of burdock as a natural host. Although studies on its vectors and pathogenicity are lacking, PepCaV's presence suggests potential risks. Given the widespread cultivation of burdock in Korea, PepCaV could significantly impact local agriculture by affecting crop yield and health.