Grapevine red blotch-associated virus (GRBaV) is a recently identified virus that is proposed to be a member of the family Geminiviridae. The virus is associated with red blotch disease of grapevines (Vitis vinifera) (Al Rwahnih et al. 2013). A survey of the prevalence of GRBaV on grapevines showed that the virus is widespread across the United States (Krenz et al. 2014). Some grapevine viruses, including Blueberry leaf mottle virus, Grapevine fleck virus, Grapevine leafroll-associated viruses 1 (GLRaV-1) and GLRaV-3, and Grapevine pinot gris virus, have been identified in Korea (Kwak et al. 2016), but GRBaV has not yet been reported. In July 2014, 25 grapevine samples exhibiting foliar symptoms of mottling, yellowing, reddening, and malformation, and five grapevine samples having no visible symptoms were collected in Suwon and Gyeongsan, Korea, respectively. For high-throughput RNA sequencing-based detection of viruses, all samples were mixed into one pool from which total RNA was extracted with TRI Reag...

Tomato spotted wilt orthotospovirus (TSWV) was first reported in 2004 from paprika in South Korea (Kim et al., 2004), where it is currently widespread. TSWV infections were reported in chili pepper, tomato, weeds, and ornamental plant species in South Korea (Choi et al., 2014; Choi and Choi, 2015; Yoon et al., 2016; Yoon et al., 2018; Yoon et al., 2019). One of the best strategies for TSWV management is planting resistant cultivars containing the Tsw gene. In 2019 virus-like symptoms were observed in chili pepper (Capsicum annuum) plants bearing the Tsw gene in Anseong-si, South Korea. The infected chili peppers showed mosaic and wilting followed by necrosis on leaves and fruits in the field. To identify the causal virus, symptomatic leaf samples were analyzed using ImmunoStrip kits (Agdia, USA); we detected three pepper-infecting viruses: Pepper mild mottle virus, Cucumber mosaic virus, and TSWV. TSWV was only detected from 40 naturally infected chili pepper plants exhibiting virus-like symptoms. To further confirm the presence of TSWV (named TSWV-P1), we amplified reverse-transcription polymerase chain reaction (RT-PCR) products for L, M, and S RNA segments using tospovirus-specific and TSWV-specific primers (Batuman et al., 2014). Expected fragments of 445, 868, and 777 bp in length were amplified and sequenced. The complete genome sequences of TSWV-P1 from a symptomatic chili pepper plant were also determined using TSWV-specific primers (Choi et al., 2014; Lian et al., 2013). The complete genome sequences of TSWV-P1 were deposited to GenBank (LC549179, LC549180, and LC549181). The sequences of each fragment were identical to a consensus sequence, showing 99.1%, 98.5%, and 98.6% identity with TSWV-L, M, and S RNA (KP008132, AY744492, and KP008134), respectively. These results clearly showed only a single TSWV infection among the naturally infected chili pepper plants, without reassortment between TSWV and another tospovirus. To confirm whether TSWV-P1 is a resistance-breaking (RB) strain, Nicotiana rustica was mechanically inoculated with sap from leaves of the infected pepper samples to propagate TSWV-P1. A non-RB TSWV isolate (TSWV-Kor-lisianthus) from lisianthus was used as a control (Yoon et al., 2017). Two resistant (with Tsw) and two susceptible chili pepper cultivars (20 plants per cultivar) were mechanically inoculated with sap from leaves of the TSWV-infected N. rustica. The incidence rates of disease caused by TSWV-P1 were 90-100% for resistant and 95-100% for susceptible cultivars. In contrast, TSWV-Kor-lisianthus caused symptoms only in the susceptible pepper cultivars (90-100% incidence). TSWV infection in representative plants was confirmed using the TSWV- ImmunoStrip kit and RT-PCR. The NSs gene of TSWV-P1 consists of 1,404 nucleotides (468 amino acids); sequence analysis of the TSWV-P1 NSs gene showed high nucleotide (99.7%) and amino acid identities (99.8%) with the NSs sequences of two TSWV isolates (FR693035, CBX24121). Protein sequence analysis of TSWV-P1 NSs revealed that no amino acid mutation was associated with those of a representative TSWV RB strain, as previously described (Almási et al., 2017), suggesting that TSWV-P1 is a RB strain. Because this TSWV-P1 can overcome resistance conferred by the Tsw gene in commercially grown chili pepper cultivars, it represents a potential threat to pepper production in South Korea.

In August and September 2018, apple trees of “Fuji” and “Hongro” cultivars looked abnormal with poor growth. Suspecting infection by viruses, leaf samples were collected from a total of 22 “Fuji” and “Hongro” apple trees in Uiseong, Yeongcheon and Jangsu in Korea. To perform comparative analysis of virus infections, leaves from a healthy and a diseased tree of Fuji and Hongro apple cultivars were chosen. Total RNA was extracted from each of the four selected samples with HiYieldTM Total RNA Mini Kit (Plant; RBC Bioscience, Taipei, Taiwan), and paired-end RNA sequencing was conducted using an Illumina HiSeq4000 system at Macrogen (Daejeon, Korea). Analysis of the de novo assembled contigs showed that the apple trees were infected with several viruses including apple green crinkle associated virus, apple stem grooving virus, apple stem pitting virus, and apricot latent virus. In addition, the samples were also infected with Apple hammerhead viroid (AHVd) recently identified in apple trees (Szostek et al. 20...

Citrus tristeza virus (CTV) is a highly destructive viral pathogen posing a significant threat to citrus crops worldwide. Disease management and crop protection strategies necessitate the development of rapid and accurate detection methods. In this study, we employed Oxford Nanopore sequencing to detect CTV in Citrus unshiu samples. Subsequently, we developed a specific and sensitive detection assay combining CRISPR/Cas12a with reverse transcription-recombinase polymerase amplification. The CRISPR-Cas12a assay exhibited exceptional specificity for CTV, surpassing conventional RT-PCR by at least 10-fold in sensitivity. Remarkably, the developed assay detected CTV in field samples, with zero false negatives. This diagnostic approach is user-friendly, cost-effective, and offers tremendous potential for rapid onsite detection of CTV. Therefore, the CRISPR-Cas12a assay plays a significant role in managing and preserving citrus trees that are free from viruses in the industry.

In December 2018, virus-like symptoms (yellowing, vein clearing) were observed on 2% of muskmelon (Cucumis melo L.) plants in plastic houses on a farm in Gyeongsang province, Korea Total RNA from two symptomatic and two asymptomatic plants was extracted using RNeasy Plant Mini Kit (Qiagen, Germany) for high throughput sequencing (HTS). After pre-processing and Ribo-Zero rRNA removal, a cDNA library was prepared (Illumina TruSeq Stranded Total RNA kit) and sequenced (Illumina NovaSeq 6000 system: Macrogen Inc. Korea). De novo assembly of 88,222,684 HTS reads with Trinity software (r20140717) yielded 146,269 contigs of 201-28,442 bp, which were screened against the NCBI viral genome database by BLASTn. Contigs from cucumber mosaic virus (CMV), melon necrotic spot virus (MNSV), tobacco mosaic virus (TMV) and watermelon mosaic virus (WMV) were identified, all previously reported in Korea. Two contigs (8,539 and 8,040 bp) with 99.9% sequence identity to distinct cucurbit chlorotic yellows virus (CCYV) isolates (JN641883, RNA1, Taiwan; MH819191, RNA2, China) were also identified. The ten sequences most closely related to each RNA of the Korean isolate (≥99% coverage, ≥99.6% nt identity) were from Japan, China, Taiwan, or Israel. CCYV presence was confirmed by reverse transcription-PCR (RT-PCR) using newly designed specific primers, RdRp-F/RdRp-R (5'-ACCGAACACTTGGCTATCCAA-3'/5'-CTTAATGCCGCGTATGAACTCA-3') span style="font-family:'Times New Roman'; letter-spacing:-0.5pt">and HSP-F/HSP-R (5'-TGAACGACACTGAGTTCATTCCTA-3'/5'-CGCCAAGATCGTACATGAGGAA-3'), against RNA dependent RNA polymerase (RdRp; RNA1) and the heat shock protein 70 homolog (HSP70h; RNA2). Symptomatic samples yielded products of expected sizes (RdRp,450 bp; HSP70h, 510 bp) while asymptomatic samples did not. The amplicons were cloned, and two clones of each were sequenced (BIONEER, Korea; GenBank acc. nos. LC592226 and LC592227) showing 100% and 99.2% nt identity with RdRp and HSP70h genes of Chinese CCYV isolate SD (MH819190 and MH819191, respectively) and other Asian isolates. Primers specific for CMV, WMV, beet pseudo-yellows virus (BPYV) (Okuda et al., 2007), TMV (Kim et al., 2018), MNSV (F/R, 5'-ATCTCGCATTTGGCATTACTC-3'/5'-ATTTGTAGAGATGCCAACGTA-3'), cucurbit yellow stunting disorder virus (CYSDV; Zeng et al., 2011) and cucurbit aphid-borne yellows virus (CABYV; F/R, 5'-CGGTCTATTGTCTGCAGTACCA-3'/5'- GTAGAGGATCTTGAATTGGTCCTCA-3') were also used. None of these viruses were detected in the symptomatic samples, but both asymptomatic plants were positive for CMV and WMV, and one also for MNSV. In June and September 2020, muskmelon and oriental melon (Cucumis melo L. var. makuwa) plants with yellowing disease (incidence 80-90%) and whiteflies were observed in all investigated plastic houses of one muskmelon and one oriental melon farm in Gyeonggi and Jeolla provinces. Symptomatic samples (14 muskmelon; 6 oriental melon) were collected and RT-PCR tested as above; 19/20 samples were positive for CCYV, but none for the other viruses. The oriental melon sequence (LC592895, LC592230) showed 99.7% and 100% nt identity with the RdRp and HSP70h genes of Chinese isolate SD, respectively. CCYV was first reported in Japan (Okuda et al., 2010), Taiwan, and China (Huang et al., 2010; Gu et al., 2011); to our knowledge, this is the first report of CCYV infecting muskmelon and oriental melon in Korea. Whitefly-transmitted CCYV could present a serious threat of yield losses to cucurbit crops in Korea, requiring control of vector populations to prevent spread of CCYV.