Burdock (Arctium lappa L., belongs to the family Asteraceae), is an edible plant and an oriental medicinal herb in Korea (Han and Koo, 1993). In July 2023, burdocks showing chlorotic ringspots and yellowing on the leaves were observed in nine of approximately 4,000 plants in a greenhouse in Daegu, South Korea. To determine the causal virus species, nine symptomatic leaves from each individual plant were collected and tested using commercially available immunostrips (Agdia, Elkhart, USA) for cucumber mosaic virus (CMV) and tomato spotted wilt virus (TSWV). Seven out of nine samples tested positive for TSWV only. TSWV in South Korea was first reported on sweet pepper from Yesan in 2004 (Kim et al., 2004) and has since spread to various crops. The first report of TSWV infecting burdock plants in the world was from Hawaii in 1995 (Bautista et al., 1995), but TSWV-infected burdock has not been reported in Korea. To further confirm the presence of TSWV, total RNA was extracted from TSWV-positive burdock leaves using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and used in reverse transcription-polymerase chain reaction (RT-PCR) assays with a specific primer set that amplifies 777 bp of nucleocapsid gene (N gene) of TSWV (Yoon et al., 2014). To obtain the complete genome sequence of this TSWV in the burdock plant, named TSWV-DG, fragments of L, M, and S segments were amplified and sequenced. The complete genome sequences of the L (8914 nt), M (4773 nt), and S (2946 nt) segments were obtained by overlapping RT-PCR amplicons. RT-PCR products were cloned into the pGEM-T Easy vector, and selected DNA clones were sequenced using Sanger method (Bioneer, Korea). The complete genome sequences were deposited to GenBank (LC790665, LC790666, and LC790667, respectively). BLASTn analysis showed that sequences of each TSWV-DG segment had maximum nucleotide identities of 99.5%, 99.5%, and 99.5% with TSWV-L, TSWV-M, and TSWV-S (OM154971, OM154970, and OM154969, respectively), which were isolated from water dropwort (Oenanthe crocata) in China (Qiu et al., 2023). To assess the biological activity of TSWV-DG, A. lappa and Nicotiana benthamiana were inoculated mechanically with sap from infected burdock leaves and maintained for visual inspection of virus symptoms at 25 ℃ at 3 weeks. TSWV-DG produced symptoms on the systemic leaves of A. lappa, that included chlorotic spots and yellowing, and on the leaves of N. benthamiana, that included chlorotic spots and mosaic patterns from 14 days-post-inoculation. Meanwhile, mock-inoculated A.lappa and N.benthamiana remained symptomless. The presence of TSWV on the inoculated leaves was subsequently confirmed through Immunostrip and RT-PCR analyses. TSWV may pose a significant threat to the production of A. lappa, which is cultivated as both leafy greens and root vegetables in Korea. Furthermore, A. lappa may not only be at risk of damage from TSWV infection but also act as a potential source of TSWV infection, thereby posing a risk of transmission to other key crops in Korea, such as pepper or potato (Yoon et al., 2014). This is the first report TSWV infecting burdock in South Korea.

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Several studies have showed that the nucleotide and dinucleotide composition of viruses possibly follows their host species or protein coding region. Nevertheless, the influence of viral segment on viral nucleotide and dinucleotide composition is still unknown. Here, we explored through tomato spotted wilt virus (TSWV), a segmented virus that seriously threatens the production of tomatoes all over the world. Through nucleotide composition analysis, we found the same over-representation of A across all viral segments at the first and second codon position, but it exhibited distinct in segments at the third codon position. Interestingly, the protein coding regions which encoded by the same or different segments exhibit obvious distinct nucleotide preference. Then, we found that the dinucleotides UpG and CpU were overrepresented and the dinucleotides UpA, CpG and GpU were underrepresented, not only in the complete genomic sequences, but also in different segments, protein coding regions and host species. Notably, 100% of the data investigated here were predicted to the correct viral segment and protein coding region, despite the fact that only 67% of the data analyzed here were predicted to the correct viral host species. In conclusion, in case study of TSWV, nucleotide composition and dinucleotide preference of segment viruses are more strongly dependent on segment and protein coding region than on host species. This research provides a novel perspective on the molecular evolutionary mechanisms of TSWV and provides reference for future research on genetic diversity of segmented viruses.

The tomato spotted wilt virus (TSWV) is a member of the Tospoviridae family and has an negative/ambisense single-stranded RNA genome. Frankliniella occidentalis and F. intonsa are known to be dominant pests in Capsicum annuum (hot pepper) and can cause damage to the plant either directly by feeding, or indirectly by transmitting TSWV in a persistent and propagative manner, resulting in serious economic damage. This study compared the immune responses of two different thrips species against TSWV infection by transcriptome analysis, which then allowed the assessment of antiviral responses using RNA interference (RNAi). Both adult thrips shared about 90 % of the transcripts in non-viruliferous conditions. Most signal components of the immune pathways were shared by these two thrips species, and their expression levels fluctuated differentially in response to TSWV infection at early immature stages. The functional assays using RNAi treatments indicated that the Toll and JAK/STAT pathways were associated with the antiviral responses, but the IMD pathway was not. The upregulation of dorsal switch protein one supported its physiological role in recognizing TSWV infection and triggering the eicosanoid biosynthetic pathway, which mediates melanization and apoptosis in thrips. In addition, the signal components of the RNAi pathways fluctuated highly after TSWV infection. Individual RNAi treatments specific to the antiviral signalling and response components led to significant increases in the TSWV amount in the thrips, causing virus-induced mortality. These findings suggest that immune signalling pathways leading to antiviral responses are operating in the thrips to regulate TSWV litres to prevent a fatal viral overload. This study also indicates the differential antiviral responses between the TSWV-transmitting F. occidentalis and F. intonsa.

Tomato spotted wilt virus (TSWV) and related thrips-borne orthotospoviruses are a threat to food and ornamental crops. Orthotospoviruses have the capacity for rapid genetic change by genome segment reassortment and mutation. Genetic resistance is one of the most effective strategies for managing orthotospoviruses, but there are multiple examples of resistance gene breakdown. Our goal was to develop effective multigenic, broad-spectrum resistance to TSWV and other orthotospoviruses. The most conserved sequences for each open reading frame (ORF) of the TSWV genome were identified, and comparison with other orthotospoviruses revealed sequence conservation within virus clades; some overlapped with domains with well-documented biological functions. We made six hairpin constructs, each of which incorporated sequences matching portions of all five ORFs. Tomato plants expressing the hairpin transgene were challenged with TSWV by thrips and leaf-rub inoculation, and four constructs provided strong protection against TSWV in foliage and fruit. To determine if the hairpin constructs provided protection against other emerging orthotospoviruses, we challenged the plants with tomato chlorotic spot virus and resistance-breaking TSWV and found that the same constructs also provided resistance to these related viruses. Antiviral hairpin constructs are an effective way to protect plants from multiple orthotospoviruses and are an important strategy in the fight against resistance-breaking TSWV and emerging viruses. Targeting of all five viral ORFs is expected to increase the durability of resistance, and combining them with other resistance genes could further extend the utility of this disease control strategy. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

An in-house tomato inbred line, YNAU335, was planted in a greenhouse in spring from 2014 to 2017, and showed immunity to tomato spotted wilt virus (TSWV). YNAU335 was infected with TSWV in the spring from 2018 to 2020, and disease was observed on the leaves, sepals, and fruits. In 2021 and 2022, YNAU335 was planted in spring in the same greenhouse, which was suspected of being infected with TSWV, and visible disease symptoms were observed on the fruits. Transmission electron microscopy, deep sequencing of small RNAs, and molecular mutation diagnosis were used to analyze the pathological features and genetic polymorphism of TSWV infecting tomato fruit. Typical TSWV virions were observed in the infected fruits, but not leaves from YNAU335 grown between 2021 and 2022, and cross-infection was very rarely observed. The number of mitochondria and chloroplasts increased, but the damage to the mitochondria was greater than that seen in the chloroplasts. Small RNA deep sequencing revealed the presence of multiple viral species in TSWV-infected and non-infected tomato samples grown between 2014-2022. Many virus species, including TSWV, which accounted for the largest proportion, were detected in the TSWV-infected tomato leaves and fruit. However, a variety of viruses other than TSWV were also detected in the non-infected tissues. The amino acids of TSWV nucleocapsid proteins (NPs) and movement proteins (MPs) from diseased fruits of YNAU335 picked in 2021-2022 were found to be very diverse. Compared with previously identified NPs and MPs from TSWV isolates, those found in this study could be divided into three types: non-resistance-breaking, resistance-breaking, and other isolates. The number of positive clones and a comparison with previously identified amino acid mutations suggested that mutation F at AA118 of the MP (GenBank OL310707) is likely the key to breaking the resistance to TSWV, and this mutation developed only in the infected fruit of YNAU335 grown in 2021 and 2022.

Capsicum annuum (CA) is grown outdoors across fields in Jeollabuk-do, South Korea. The weeds surrounding these fields were investigated regarding the infection of 11 viruses infecting CA during the year 2014-2018. In the reverse transcription polymerase chain reaction diagnosis, 546 out of 821 CA samples (66.5%) were infected by nine viruses, and 190 out of 918 weed samples (20.7%) were infected by eight viruses. Correlation analysis of the mutual influence of the viruses infecting CA and weeds during these 5 years showed that five viruses had significant positive correlations with the infection in both CA and weeds. Over the study period, the weeds infected by cucumber mosaic virus (CMV) in the previous year were positively correlated with the incidence of CMV infection in CA in the current year, although the correlation was lower for tomato spotted wilt virus (TSWV) compared to CMV. The CMV infection percent was 14.0% in summer annuals, 11.4% in perennials, and 7.8% in winter annuals. However, considering the overwintering period without CA, the infection percent was 5.2% higher in winter annuals and perennials than that in summer annuals, indicating that winter annual and perennial weeds served as the main habitats for insect vectors. The TSWV infection percent in weeds was 10.4% in summer annuals, 6.4% in winter annuals, and 6.2% in perennials. The weeds surrounding CA fields, acting as the intermediate hosts, were found to be the potent sources of infection, influencing the spread and diversity of CA-infecting viruses. The results of this study can contribute to prevent viral infection in agricultural fields.

Tomato spotted wilt virus (TSWV, family Tospoviridae, genus Orthotospovirus) is a thrips-vectored pathogen that infects lettuce (Lactuca sativa) and many vegetable crops (Kuo et al. 2014, Hasegawa et al. 2022). Another thrips-borne pathogen of lettuce, impatiens necrotic spot virus (INSV, Tospoviridae, Orthotospovirus), was first reported in 2021 in Yuma, Arizona (Hasegawa et al. 2022). Symptoms of both viruses in lettuce are similar and include necrotic spotting, leaf chlorosis and plant stunting (Kuo et al. 2014). Beginning February through April of 2022, lettuce displaying symptoms of orthotospovirus infection was collected from romaine lettuce (var. longifolia) fields in three regions of Yuma County. A total of 96 plants were collected (5 from Tacna on 2/21, 5 from Wellton on 2/21, 15 from Wellton on 3/23, 30 from Tacna on 4/4, 20 from Wellton on 4/4, and 21 from Yuma Valley on 4/4). The area of the fields ranged from 10 to 18 acres, and the percent disease incidence ranged from 0.8% (Tacna on 4/4) to 2.75% (Tacna on 2/21). Thrips vector were present in all fields were symptomatic plants were observed. One leaf disk per plant (8 mm in diameter) was sampled with a cork borer and grounded individually with a micro pestle in a 1.7 ml microcentrifuge tube with 150 ul of Tri-reagent (Molecular Research Center). Total RNA was extracted from each sample using the Zymo Direct-zol-96 kit (Zymo Research). Samples were diluted with water to a ratio of 1:10 after RNA extraction. RT-qPCR was performed in 20 ul reactions with 5 ul of input RNA using the PCR Biosystems qPCRBIO Probe 1-Step Go No-ROX for the cDNA/qPCR master mix. RT-qPCR assays were carried out in multiplex reactions using primers specific for TSWV and INSV, in addition to a lettuce internal control gene (LOC111918243), along with negative controls. Primer and probe sequence details are reported in supplemental Table 1. We used a cycle threshold (ct) < 40 to indicate a positive result for both INSV and TSWV (Chen et al. 2013; Boonham et al. 2002). RT-qPCR successfully amplified INSV in 90 out of 96 samples and TSWV in 8 out of 96 samples. These 8 samples tested positive for both TSWV and INSV, showing that INSV and TSWV co-infected lettuce plants. Thus overall, ∼ 95% of symptomatic plants were infected with INSV alone, and ∼ 8% were co-infected with TSWV and INSV. Amplicons of 4 samples testing positive for TSWV were sent for Sanger sequencing (Eurofins Genomics, Louisville, KY). All were identified as TSWV. One amplicon with TSWV was sequenced for INSV and double infection was confirmed. BLAST results from the NCBI nt database show 100% (138 bp) identity to TWSV (MW519211) for the 4 TWSV amplicons and 99.22% (137 bp) identity to INSV (KX790323) for the INSV amplicon. Sanger sequence data are in the GenBank (accession: OQ685940-OQ685944). Based on RT-qPCR results, all TSWV infected plants were also infected with INSV. INSV may have been introduced to Yuma by infected plants or thrips from lettuce transplants produced in California (Hasegawa et al. 2022). TSWV could have been introduced similarly. To our knowledge, this is the first report of TSWV infecting lettuce in Yuma and the first report of INSV and TSWV co-infecting lettuce. TSWV and INSV infections have remained low since their discovery in Yuma, in part due to effective cultural and chemical management by lettuce growers (Palumbo, 2022). However, an increase in disease incidence and severity in the future could have a significant negative impact on production of romaine lettuce in the region.

The nonstructural protein NSm of tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant of the tomato single dominant Sw-5 resistance gene. Although Sw-5 effectiveness has been shown for most TSWV isolates, the emergence of resistance-breaking (RB) isolates has been observed. It is strongly associated with two point mutations (C118Y or T120N) in the NSm viral protein. TSWV-like symptoms were observed in tomato crop cultivars (+Sw-5) in the Baja California peninsula, Mexico, and molecular methods confirmed the presence of TSWV. Sequence analysis of the NSm 118-120 motif and three-dimensional protein modelling exhibited a noncanonical C118F substitution in seven isolates, suggesting that this substitution could emulate the C118Y-related RB phenotype. Furthermore, phylogenetic and molecular analysis of the full-length genome (TSWV-MX) revealed its reassortment-related evolution and confirmed that putative RB-related features are restricted to the NSm protein. Biological and mutational NSm 118 residue assays in tomato (+Sw-5) confirmed the RB nature of TSWV-MX isolate, and the F118 residue plays a critical role in the RB phenotype. The discovery of a novel TSWV-RB Mexican isolate with the presence of C118F substitution highlights a not previously described viral adaptation in the genus Orthotospovirus, and hence, the necessity of further crop monitoring to alert the establishment of novel RB isolates in cultivated tomatoes.

Tomato spotted wilt virus (TSWV) poses a serious threat to tomato (Solanum lycopersicum) production. In this study, tomato inbred line YNAU335 was developed without the Sw-5 locus, which confers resistance or immunity to TSWV (absence of infection). Genetic analysis demonstrated that immunity to TSWV was controlled by a dominant nuclear gene. The candidate genes were mapped into a 20-kb region in the terminal of the long arm of chromosome 9 using bulk segregant analysis and linkage analysis. In this candidate region, a chalcone synthase-encoding gene (SlCHS3) was identified as a strong candidate gene for TSWV resistance. Silencing SlCHS3 reduced flavonoid synthesis, and SlCHS3 overexpression increased flavonoid content. The increase in flavonoids improved TSWV resistance in tomato. These findings indicate that SlCHS3 is indeed involved in the regulation of flavonoid synthesis and plays a significant role in TSWV resistance of YNAU335. This could provide new insights and lay the foundation for analyzing TSWV resistance mechanisms.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s11032-022-01325-5.