Twenty leaf samples of Jack beans (C. ensiformis) plants showing virus-like symptoms were collected from different farms and stored at −80 °C for mechanical inoculation, conventional reverse transcription polymerase chain reaction (RT-PCR), and high-throughput sequencing after quick freezing. Leaf samples of the symptomatic Jack bean plants were homogenized in 10 mM sodium phosphate buffer (pH 7.4) and centrifuged at 10,000 rpm for 3 min. Then, supernatant was subjected to identify a causal virus using TEM. All sample preparations were negatively stained on formvar-coated grids with 2.0% (w/v) sodium phosphotungstic acid solution. Virus particles were observed using a TEM (Carl Zeiss EM LEO 906E; Carl Zeiss, Jena, Germany).

A total of 20 symptomatic leaf samples of Jack beans plants were subjected to DAS-ELISA using Potyvirus-specific antisera (Agdia, Elkhart, IN, USA), according to manufacturer's instruction. In brief, leaf tissues were homogenized in 100 mM sodium phosphate buffer (pH 7.4) containing 0.02% NaN₃, 0.1% Tween 20, and 0.1% skim milk powder at a sample-to-buffer ratio of 1:3 (w:v), and 100 μl of extracted sap was loaded in duplicate onto microtiter plates. The primary antibody specific to potyviruses, purchased from Agdia was diluted to 1:100 in carbonate buffer (50 mM sodium carbonate, pH 9.6) and the diluted antibody solution (1 μg/ml) was used for potyvirus detection in microtiter plates. Subsequently, goat anti-rabbit IgG conjugated alkaline phosphatase was used as a secondary antibody, according to the manufacturer's instructions (Promega, Madison, WI, USA). Substrate, 4-nitrophenyl phosphate (0.6 mg/ml), was allowed to react at room temperature for 1 h (SigmaAldrich, St. Louis, MO, USA). Plates were read with an automated plate reader (Titertek, Huntsville, AL, USA) at 405 nm. A sample was considered positive if the optical density (OD405) was greater than three times the mean of the healthy controls (Yoon et al., 2011).

Leaf tissue exhibiting mosaic symptoms was ground using a mortar and pestle in 50 mM sodium phosphate buffer (pH 7.4). Subsequently, the extract then was used to inoculate on Chenopodium quinoa as a local lesion host. Local lesions were carefully excised from C. quinoa as soon as they became visible, ground as above, and inoculated to C. quinoa. After three times back-inoculation, Nicotiana clevelandii was mechanically inoculated with the sap of infected C. quinoa for virus propagation. A Korean isolate of ClYVV (named ClYVV-Ce-JH) selected was further confirmed by a biological method with some plant species as indicator plants by mechanical inoculation. The host range of ClYVV-Ce-JH was determined by mechanical inoculation onto several plant species using extracts from systemically infected N. clevelandii plants in 10 mM sodium phosphate buffer (pH 7.4). The diagnostic host species were as follows: C. ensiformis, C. quinoa, C. amaranticola, Glycine max, N. benthamiana, N. clevelandii, N. tabacum cv. Samsun NN, Solanum lycopersicum, Vicia faba, and Vigna unguiculate (Table 1). Five plants for each indicator species were inoculated with 50 mM sodium phosphate buffer (pH 7.4) as negative controls. All the inoculated plants were maintained in a greenhouse at 25±3 °C with a 16 hr light period and were observed for symptom production until 21 days after inoculation.

One hundred mg of leaf tissue of infected Jack bean plants was frozen in liquid N₂ and ground to a fine powder. Total RNA was extracted using a RNeasy plant mini-kit, according to the manufacturer's instructions (Qiagen, Hilden, Germany). Briefly, 0.1 g of leaf tissue of the diseased Jack bean plants was ground with lysis buffer from the kit in a 1.5 ml microcentrifuge tube using a bead beater. Subsequently, a contaminated DNA in the eluted total RNA solution was removed from the samples by on column DNase digestion with the RNase-Free DNase Set (Qiagen) according to the manufacturer's protocol. RNA was eluted from the columns with 50 μl nuclease-free water, and the concentration was measured using NanoDrop and a QuantiT RiboGreen RNA assay kit according to the manufacturer's instructions (Qiagen). Total RNA containing genomic RNA of ClYVV-Ce-JH was reverse-transcribed using Super-Script III^® reverse transcriptase (ThermoScientific, Waltham, MA, USA) and the first cDNA was amplified using the oligo-nucleotide primers as shown in Table 2. The thermo-cycling conditions for all PCR amplifications were as follows; 3 min at 95 °C (1 cycle), 94 °C, 30 sec, 55 °C, 30 sec and 72 °C, 2 min (35 cycles), and a final extension at 72 °C for 10 min. The syn-thesized RT-PCR products were purified using Qiaquick PCR purification (Qiagen) and cloned into pGEM-T easy vector (Promega), according to the manufacturer’ instructions. We generated the RNA-seq libraries using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions. Each library was paired-end (150 bp×2) sequenced by Illumina's HiSeq 4000 system (Macrogen, Daejeon, Korea). All raw sequence data were de novo assembled by the Trinity program with default parameters as described previously (Jo et al., 2022). The obtained contigs were subjected to BLASTX search with E-value 1e^-10 as a cutoff against the plant viral database derived from the NCBI. The 5′/3′-terminal sequence of ClYVV-Ce-JH was amplified using SMARTer RACE 5′/3′ kit (TaKaRa Bio., Kyoto, Japan) according to the manufacturer's instructions. The 5′-terminal sequence was amplified by RT-PCR using a generic primer for the sequence ends of ClYVV (Takahashi et al., 1997) and an internal specific primer (5′-GGGTCCAGCCTCGATGTGAGTACTGG-3′). The cDNA synthesis was conducted using SMARTScribe transcriptase according to the manufacturer's instructions (TaKaRa Bio.). PCR was carried out using SeqAmp DNA polymerase (Takara Bio., Shiga, Japan) under the thermal cycling conditions as follows: pre-denaturation at 94 °C for 2 min, 25 cycles of 94 °C for 30 sec, 68 °C for 30 sec, and 72 °C for 3 min, and a final extension of 72 °C for 10 min. The amplicons that were obtained were purified using Qiaquick PCR purification (Qiagen) and cloned into pGEM-T easy vector (Promega), according to the manufacturer’ instructions. The cDNA clones were sequenced by the Sanger method. The assembled complete genome sequences of CSNV-Ce-JH has been deposited to NCBI GenBank (accession no. LC729726).

Analysis of the nt and deduced amino acid (aa) sequences were done using BLAST search and DNASTAR Lasergene Genomics Suite software (DNASTAR Inc., Madison, WI, USA). All ClYVV-associated contigs were selected using the BLASTX results. Of the ClYVV-associated contigs, we selected viral contigs with sizes greater than 1,000 bp to identify ClYVV sequences that covered ORFs using the NCBI ORF-finder. For sequence comparison, 14 genomic sequences of ClYVV isolates available from the GenBank database were added to our data set (Table 3). The sequences from the database that were redundant, or smaller than the size of full-length ClYVV polyprotein were omitted. For phylogenetic analysis of ClYVV, nt and aa sequences of 15 ClYVV isolates including ClYVV-Ce-JH were aligned using MUSCLE implemented in MEGA 11 followed by manual modification (Tamura et al., 2021). The deduced full-length polyprotein or CP sequences of ClYVV were manually adjusted using CLUSTAL W (Tamura et al., 2021) for calculation of sequence identities. Phylogenetic trees were constructed based on the neighbor-joining method and Maximum likelihood method. Bootstrap resampling (1,000 replications) was used to measure the reliability of individual nodes in each phylogenetic tree.