Tomato Spotted Wilt Virus Infection Research Articles

Virus-vectoring thrips regulate the excessive multiplication of tomato spotted wilt virus using their antiviral immune responses.

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.

Comprehensive phytohormone metabolomic and transcriptomic analysis of tobacco (Nicotiana tabacum) infected by tomato spotted wilt virus (TSWV)

Tomato spotted wilt virus (TSWV) is ranked among the top 10 most destructive viruses globally. It results in abnormal leaf growth, stunting, and even death, significantly affecting crop yield and quality. Phytohormones play a crucial role in regulating plant-virus interactions. However, there is still limited research on the effect of TSWV on phytohormone levels, particularly growth hormones and genes involved in the phytohormone pathway. In our study, we combined phytohormone metabolomics and transcriptomics to examine the impact of TSWV infection on phytohormone content and gene expression profile. Metabolomic results showed that 41 metabolites, including major phytohormones and their precursors and derivatives were significantly altered after 14 days of TSWV inoculation tobacco plants cvK326, with 31 being significantly increased and 10 significantly reduced. Specifically, the levels of abscisic acid (ABA) and jasmonoyl-isoleucine (JA-Ile) were significantly reduced. The levels of indole-3-acetic acid (IAA) have remained unchanged. However, the levels of cytokinin isopentenyladenine (iP) and salicylic acid (SA) significantly increased. The transcriptome analysis revealed 2,746 genes with significant changes in expression. Out of these, 1,072 genes were significantly downregulated, while 1,674 genes were significantly upregulated. Among them, genes involved in ABA synthesis and signaling pathways, such as 9-cis-epoxycarotenoid dioxygenase (NCED), protein phosphatase 2C (PP2C), serine/threonine-protein kinase (SnRK2), and abscisic acid responsive element binding factor (ABF), exhibited significant downregulation. Additionally, expression of the lipoxygenase gene LOX, Jasmonate ZIM domain-containing protein gene JAZ, and transcription factor gene MYC were significantly down-regulated. In the cytokinin pathway, while there were no significant changes in the expression of the cytokinin synthesis genes, a significant downregulation of transcriptionally active factor type-B response regulators (type-B RRs) was observed. In terms of SA synthesis and signaling pathways, the isochorismate synthase gene ICS1 and the pathogenesis-related gene PR1 were significantly upregulated. These results can strengthen the theoretical foundation for understanding the interaction between TSWV and tobacco and provide new insights for the future prevention and control of TSWV.

Genome-wide characterization of the tomato UDP-glycosyltransferase gene family and functional identification of SlUDPGT52 in drought tolerance

Abiotic stresses are major factors constraining the growth, development and productivity of tomato (Solanum lycopersicum), the most cultivated vegetable crop worldwide. Uridine diphosphate glycosyltransferases (UDPGTs or UGTs) are essential enzymes that utilize 5-uridine diphosphate as a glycosyl donor molecule to facilitate the catalysis of glycosylation reactions across diverse substrates, thereby playing a pivotal role in conferring abiotic stress tolerance. Currently, there is a limited understanding of the structure and functions of the UDPGT gene family in tomato. In this work, 106 members of the SlUDPGT gene family were identified through in silico analysis, besides, their protein sequence properties, phylogenetic relationships, gene structure, chromosomal distribution, cis-acting elements, tissue expression and hormone- and stress-induced expression were comprehensively investigated. The expression of representative SlUDPGTs under abiotic stress and exogenous hormone treatments, including salt, polyethylene glycol, methyl viologen, gibberellic acid, jasmonic acid, abscisic acid and brassinolide, was investigated through qRT‒PCR analysis. Numerous cis-acting elements linked to stress and hormone signaling were present in the promoter regions of SlUDPGTs. According to microarray data, most SlUDPGT genes were responsive to hormones and abiotic stresses, while certain SlUDPGTs were specifically differentially expressed under Botrytis cinerea and tomato spotted wilt virus infection. Additionally, diverse expression profiles of SlUDPGTs were observed in various tissues and developmental stages. Furthermore, CRISPR/Cas9-mediated knockout of SlUDPGT52 led to enhanced drought tolerance due to enhanced reactive oxygen species (ROS) scavenging. These findings lay the foundations for the future functional characterization of specific UDPGT gene family members, assisting the biotechnology-mediated improvement of tomato and other horticultural crops.

First Report of Tomato spotted wilt virus infecting Helichrysum bracteatum in China.

Strawflower (Helichrysum bracteatum, Asteraceae) , an annual or biennial herb, is one of the most popular flowers in the world because of the colorful flowers and the long flowering period. However, the ornamental plants belonging to Asteraceae are susceptible to numerous viruses such as cucumber mosaic virus (CMV) (Cucumovirus, Bromoviridae) , potato virus Y (Potyvirus, Potyviridae), tomato mosaic virus (ToMV) (Tobamovirus, Virgaviridae), tobacco mosaic virus (TMV) (Tobamovirus, Virgaviridae), chrysanthemum virus B (CVB) (Carlavirus, Betaflexiviridae), tomato aspermy virus (TAV) (Cucumovirus, Bromoviridae), tomato spotted wilt virus (TSWV) (Orthotospovirus tomatomaculae, Tospoviridae), and impatiens necrotic spot virus (INSV) (Orthotospovirus impatiensnecromaculae, Orthotospovirus) resulting in severe yield loss (Verma et al. 2003; Raj et al. 2007; Kondo et al. 2011; Liu et al. 2014; Marys et al. 2014; Min et al. 2020; Gautam et al. 2021; Read et al. 2022; Supakitthanakorn et al. 2022). Among these viruses, the TSWV, a thrips-transmitted negative-stranded RNA virus, is well known to cause viral disease in several plant species while is less reported in Helichrysum, especially in China. In April 2021, viral attack symptoms were detected on the leaves of H. bracteatum during our routine checks in the greenhouse located at Shunyi District, Beijing, China, such as wilting, shrinking, chlorotic blotches, chlorotic ring spots. To investigate the virus infecting H. bracteatum, in total of 25 symptomatic and 5 asymptomatic leaves were sampled and tested by the effective double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using antisera against CMV, PVY/PVX, ToMV, TMV, CVB, TAV, TSWV, INSV, separately (Agdia, USA). Only the TSWV showed positive in symptomatic samples, and asymptomatic samples were all negative, which implied TSWV infection. To further confirm the virus type of TSWV isolated from H. bracteatum samples, the genomic RNA of the virus was isolated using reverse transcription and polymerase chain reaction (RT-PCR), and then was cloned, sequenced and analyzed. Total RNA of five symptomatic leaves (ELISA-positive) were extracted using the FastPure Plant Total RNA Isolation Kit (Vazyme, China), and then were reverse transcribed by HiScript II Reverse Transcriptase (Vazyme, China). Each genome segments were amplified using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China) with TSWV-specific primers newly designed and listed in Table S1. The PCR setup was as follow: 95°C for 30 s, followed by 35 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 1.5 min, with a final extension at 72°C for 10 min. All PCR products were cloned into the TA/Blunt-Zero vector (Vazyme, China) and sequenced (GENEWIZ, Inc.). We assembled and then analyzed the evolutionary relationship of three genomic fragments, that is, TSWV-BJFC-Hb S (2923 bp), M (4785 bp) and L (8971 bp) using the BLAST tools. The results showed high similarity with TSWV-henan isolated from pepper in China (99.6% to TSWV-S (MT799179.1), 99.8 % to TSWV-M (MT799178.1) and 99.8 % TSWV-L (MT799177.1)). These sequences have been submitted to the GenBank (OM982910, OM982911 and OM937131). Taking all of these evidences together, the viral disease observed in H. bracteatum was closely associated with TSWV. TSWV is currently widespread in China, infecting Nasturtium, Chrysanthemum and cowpea (Xiao et al. 2015; Hu et al. 2018; Yu et al. 2021). Epidemics of TSWV have also been reported in several other countries such as Korea, North Carolina, Turkey and India (Renukadevi et al. 2015; Koehler et al. 2016; Kwak et al. 2021; Erilmez, S. 2022). This is the first report of TSWV infection on H. bracteatum in China. Due to the fast spread and serious economic losses of TSWV, the rapid detection may be the essential way to prevent this viral disease among crops (Macharia et al. 2014).

Evaluation of the Weeds around Capsicum annuum (CA) Cultivation Fields as Potential Habitats of CA-Infecting Viruses.

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.

HMG-like DSP1 is a damage signal to mediate the western flower thrips, Frankliniella occidentalis, immune responses to tomato spotted wilt virus infection

Tomato spotted wilt virus (TSWV) causes a serious plant disease and is transmitted by specific thrips including the western flower thrips, Frankliniella occidentalis. The persistent and circulative virus transmission suggests an induction of immune defenses in the thrips. We investigated the immune responses of F. occidentalis to TSWV infection. Immunofluorescence assay demonstrated viral infection in the larval midguts at early stage and subsequent propagation to the salivary gland in adults. In the larval midgut, TSWV infection led to the release of DSP1, a damage-associated molecular pattern, from the gut epithelium into the hemolymph. DSP1 up-regulated PLA2 activity, which would lead to biosynthesis of eicosanoids that activate cellular and humoral immune responses. Phenoloxidase (PO) activity was enhanced following induction of PO and its activating protease gene expressions. Antimicrobial peptide genes and dual oxidase, which produces reactive oxygen species, were induced by the viral infection. Expression of four caspase genes increased and TUNEL assay confirmed apoptosis in the larval midgut after the virus infection. These immune responses to viral infection were significantly suppressed by the inhibition of DSP1 release. We infer that TSWV infection induces F. occidentalis immune responses, which are activated by the release of DSP1 from the infection foci within midguts.

Two Alimentary Canal Proteins, Fo-GN and Fo-Cyp1, Act in Western Flower Thrips, Frankliniella occidentalis TSWV Infection.

Tomato spotted wilt virus (TSWV) is a plant virus that causes massive economic damage to high-valued crops. This virus is transmitted by specific thrips, including the western flower thrips, Frankliniella occidentalis. TSWV is acquired by the young larvae during feeding on infected host plants. TSWV infects the gut epithelium through hypothetical receptor(s) and multiplies within the cells for subsequent horizontal transmission to other plant hosts via the salivary glands during feeding. Two alimentary canal proteins, glycoprotein (Fo-GN) and cyclophilin (Fo-Cyp1), are thought to be associated with the TSWV entry into the gut epithelium of F. occidentalis. Fo-GN possesses a chitin-binding domain, and its transcript was localized on the larval gut epithelium by fluorescence in situ hybridization (FISH) analysis. Phylogenetic analysis indicated that F. occidentalis encodes six cyclophilins, in which Fo-Cyp1 is closely related to a human cyclophilin A, an immune modulator. The Fo-Cyp1 transcript was also detected in the larval gut epithelium. Expression of these two genes was suppressed by feeding their cognate RNA interference (RNAi) to young larvae. The RNAi efficiencies were confirmed by the disappearance of the target gene transcripts from the gut epithelium by FISH analyses. The RNAi treatments directed to Fo-GN or Fo-Cyp1 prevented the typical TSWV titer increase after the virus feeding, compared to control RNAi treatment. Our immunofluorescence assay using a specific antibody to TSWV documented the reduction of TSWV in the larval gut and adult salivary gland after the RNAi treatments. These results support our hypothesis that the candidate proteins Fo-GN and Fo-Cyp1 act in TSWV entry and multiplication in F. occidentalis.

First report of tomato spotted wilt virus infecting water dropwort in China.

Water dropwort (Oenanthe javanica) is an aquatic perennial plant that has been cultivated in many regions in Asia for thousands of years. In China, it is an economically important vegetable that has been consumed as food, while also being used as a folk remedy to alleviate diseases (Liu et al., 2021). In 2021, during a disease survey of a greenhouse in Beijing, China, chlorotic spots were detected on many water dropwort plants (Fig. S1A). Twenty-seven water dropwort samples were collected for the extraction of total RNA using the TRIzol reagent (Invitrogen, USA). High-quality RNA samples from three water dropwort plants were combined and used as the template for constructing a single small RNA library (BGI-Shenzhen Company, China). The Velvet 1.0.5 software was used to assemble the clean reads (18 to 28 nt) into larger contigs, which were then compared with the nucleotide sequences in the National Center database using the BLASTn algorithm. Thirty-eight contigs matched sequences in the tomato spotted wilt virus (TSWV) genome. No other viruses were detected. Twenty-seven leaf samples were analyzed in an enzyme-linked immunosorbent assay (ELISA) with anti-TSWV antibody (Agdia, USA), which revealed 17 positive reaction. Two sets of primer pairs targeting different parts of the S RNA (Table S1) was used to verify the TSWV infection on water dropwort by reverse transcription (RT)-PCR followed by Sanger sequencing (BGI-Shenzhen, China). The TSWV target sequences were amplified from 17 samples, which was consistent with the ELISA results. The sequenced 861-bp PCR product shared 99.8% nucleotide sequence identity with TSWV isolate MR-01 (MG593199), while the 441-bp amplicon shared a 99.2% nucleotide sequence identity with MR-01 (MG593199). To obtain the whole genome sequence of TSWV (S, M, and L RNA sequences), specific RT-PCR primers were designed (Table S1) and used to amplify their respective fragments from one representative sample (TSWV-water dropwort). The amplified products were inserted into PCE2TA/Blunt-Zero vector (Vazyme Biotech Co., Ltd, China) and then sequenced (BGI-Shenzhen, China). The S, M, and L RNA sequences were determined to be 2,952 nt (accession no. OM154969), 4,776 nt (accession no. OM154970), and 8,914 nt (accession no. OM154971), respectively. BLASTn analysis demonstrated that the whole genome sequence was highly conserved. The nucleotide identities between this isolate and other TSWV isolates ranged from 98.6% to 99.6% (S RNA), 98.9% to 99.2% (M RNA), and 97.3% to 98.7% (L RNA). Using MEGA 7.0, the phylogenetic relationships of TSWV were determined on the basis of the S, M, and L RNA full-length sequences (Kumar et al., 2016). In the S RNA derived phylogenetic tree, the water dropwort isolate was closely related to the MR-01 isolate from the USA (MG593199). In the M RNA and L RNA derived phylogenetic trees, the water dropwort isolate formed a branch with only a TSWV isolate from eggplant. Additionally, the M and L RNA sequences were most similar to sequences in TSWV isolates from China and Korea, respectively (Fig. S1B). To the best of our knowledge, this is the first report of water dropwort as a natural host for TSWV in China and the second report worldwide since the first finding in the Korea (Kil et al. 2020). TSWV has caused serious problems on many crops in the world, and the infection of TSWV on water dropwort in a greenhouse should not be looked lightly. Firstly, the virus can be passed on from generation to generation in infected water dropwort due to the vegetative propagation mode of the plant in production, thus threaten the production of this vegetable crop. In addition, infected water dropwort may serve as a reservoir for the virus, thus potentially posing a threat for causing TSWV spread in the affected greenhouses. The author(s) declare no conflict of interest. Funding: This research was supported by the Beijing Academy of Agriculture and Forestry Foundation, China (QNJJ202131, KJCX20200212, and KJCX20200113).

Determination of Some Virus Diseases on Gerbera Plants in Antalya Province

Gerbera spp., which is in the Asteraceae family, is the most widely cultivated cut flower ornamental plant in Türkiye after carnation. Approximately 74% of Türkiye's production is met from Antalya province. Virus diseases are a problem in the cultivation of Gerbera as they significantly reduce the yield and quality. In this study, which was carried out to detect some virus diseases seen in Gerbera production areas in Antalya province, surveys were carried out in commercial production areas in the 2021-2022 production season. For this purpose, a total of 112 leaf samples, which are thought to be infected with viruses, were collected from 100 Gerbera plants showing symptoms similar to virus disease symptoms and 12 selected weeds regardless of the presence of symptoms. These leaf samples, which were later brought to the laboratory, then were tested by the Double Antibody Sandwich Enzyme-linked Immunosorbent Assay (DAS-ELISA) method to detect infections of Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Tobacco streak virus (TSV) and Impatiens necrotic spot virus (INSV). As a result of the tests, TSWV (41.9%), CMV (1.7%) and TSV (37.5%) infections were detected in Gerbera plant samples. In addition, TSWV (25%) infections were most common in weed samples, followed by TSV (16%) and CMV (8.3%) infections. The presence of INSV virus was not detected in any of the leaf samples tested by DAS-ELISA. With this study, TSWV, TSV and CMV infections in Gerbera production areas in Türkiye were revealed for the first time.

First Report of Tomato Spotted Wilt Virus in Oxypetalum coeruleum in Korea

<i>Oxypetalum coeruleum</i>, commonly known as Tweedia, is a perennial herbaceous plant of the <i>Apocynaceae</i> family native to southern Brazil and Uruguay. Tweedia plants are grown as one of the most popular ornamental flowers for floral arrangement in Korea. In May 2021, several tweedia plants in a single greenhouse in Gimje, Jeollabuk-do were found to show virus-like symptoms including necrotic rings, vein-clearing, chlorotic mottle, and mosaic on the leaves, and necrosis on the stems. Here, we have identified tomato spotted wilt virus (TSWV) in symptomatic tweedia leaves by applying high-throughput RNA sequencing. In the result, a single infection by TSWV was verified without mixed infections of different virus species. To confirm the presence of TSWV, a reverse transcription polymerase chain reaction was performed with a specific primer set to the N gene of TSWV. The complete genomic sequence of L, M, and S segments of TSWV ’Oxy’ isolate were determined and deposited in GenBank under accession numbers LC671525, LC671638, and LC671639, respectively. In the phylogenetic tree analysis by maximum likelihood method, ’Oxy’ isolate showed a high relationship with TSWV ’Gumi’ isolate from <i>Gerbera jamesonii</i> in Gyeongsangbuk-do, Korea; for all three RNA segments. To our knowledge, this is the first report of TSWV infection of <i>O. coeruleum</i> in Korea.

Three-dimensional analysis of membrane structures associated with tomato spotted wilt virus infection.

To study viral infection, the direct structural visualization of the viral life cycle consisting of virus attachment, entry, replication, assemblyand transport is essential. Although conventional electron microscopy (EM) has been extremely helpful in the investigation of virus-host cell interactions, three-dimensional (3D) EM not only provides important information at the nanometer resolution, but can also create 3D maps of large volumes, even entire virus-infected cells. Here, we determined the ultrastructural details of tomato spotted wilt virus (TSWV)-infected plant cells using focused ion beam scanning EM (FIB-SEM). The viral morphogenesis and dynamic transformation of paired parallel membranes (PPMs) were analyzed. The endoplasmic reticulum (ER) membrane network consisting of tubules and sheets was related to viral intracellular trafficking and virion storage. Abundant lipid-like bodies, clustering mitochondria, cell membrane tubules, and myelin-like bodies were likely associated with viral infection. Additionally, connecting structures between neighboring cells were found only in infected plant tissues and showed the characteristics of tubular structure. These novel connections that formed continuously in the cell wall or were wrapped by the cell membranes of neighboring cells appeared frequently in the large-scale 3D model, suggesting additional strategies for viral trafficking that were difficult to distinguish using conventional EM.

Sex-biased proteomic response to tomato spotted wilt virus infection of the salivary glands of Frankliniella occidentalis, the western flower thrips

Successful transmission of tomato spotted wilt virus (TSWV) by Frankliniella occidentalis requires robust infection of the salivary glands (SGs) and virus delivery to plants during salivation. Feeding behavior and transmission efficiency are sexually-dimorphic traits of this thrips vector species. Proteins secreted from male and female SG tissues, and the effect of TSWV infection on the thrips SG proteome are unknown. To begin to discern thrips factors that facilitate virus infection of SGs and transmission by F. occidentalis, we used gel- and label-free quantitative and qualitative proteomics to address two hypotheses: (i) TSWV infection modifies the composition and/or abundance of SG-expressed proteins in adults; and (ii) TSWV has a differential effect on the male and female SG proteome and secreted saliva. Our study revealed a sex-biased SG proteome for F. occidentalis, and TSWV infection modulated the SG proteome in a sex-dependent manner as evident by the number, differential abundance, identities and generalized roles of the proteins. Male SGs exhibited a larger proteomic response to the virus than female SGs. Intracellular processes modulated by TSWV in males indicated perturbation of SG cytoskeletal networks and cell-cell interactions, i.e., basement membrane (BM) and extracellular matrix (ECM) proteins, and subcellular processes consistent with a metabolic slow-down under infection. Several differentially-abundant proteins in infected male SGs play critical roles in viral life cycles of other host-virus pathosystems. In females, TSWV modulated processes consistent with tissue integrity and active translational and transcriptional regulation. A core set of proteins known for their roles in plant cell-wall degradation and protein metabolism were identified in saliva of both sexes, regardless of virus infection status. Saliva proteins secreted by TSWV-infected adults indicated energy generation, consumption and protein turnover, with an enrichment of cytoskeletal/BM/ECM proteins and tricarboxylic acid cycle proteins in male and female saliva, respectively. The nonstructural TSWV protein NSs - a multifunctional viral effector protein reported to target plant defenses against TSWV and thrips - was identified in female saliva. This study represents the first description of the SG proteome and secretome of a thysanopteran and provides many candidate proteins to further unravel the complex interplay between the virus, insect vector, and plant host.

Genotypic, Temporal, and Tissue-type Effects on the Incidence of Tomato spotted wilt virus in Peanut (Arachis hypogaea L.)

Spotted wilt, caused by Tomato spotted wilt virus (TSWV), is one of the major diseases affecting peanut production in the southeastern United States. Growers use integrated disease management to control spotted wilt; however, disease resistance of a cultivar is the most important factor for managing this disease. This research evaluated genotypic, temporal, and tissue-type effects on the incidence of TSWV on each of four peanut cultivars. Four different types of tissues (leaf, root crown, old leaf and young leaf) were collected from each of four peanut cultivars (Florida-EPTM ‘113’, Florida-07, Georgia Green and Georgia Valencia) at five time points during the growing season. Immunoassays were conducted to detect viral presence. The incidence of TSWV infection increased over time during the growing season. Root crown tissue had higher infection than other types of tissue. Florida-EPTM ‘113’ had the lowest infection incidence. Compared to Florida-EPTM ‘113’, Florida-07 was seven times more likely to be infected by TSWV, while Georgia Green and Georgia Valencia possessed even higher probability, 18 times and 21 times higher, respectively. Florida-EPTM ‘113’ had significantly reduced TSWV infection likely due to slowing movement of the virus throughout the plant.

Incidence and possible sources of Tomato spotted wilt virus in tobacco grown in Denizli Province, Turkey

Tomato spotted wilt virus (TSWV) is economically prominent disease for its impact on tobacco (Nicotiana tabacum L.) production worldwide. An increase of the incidence of symptoms typical of TSWV has been observed in tobacco production areas in Denizli province of Turkey where tobacco is significantly grown. Surveys were conducted to determine the prevalence status of TSWV in tobacco cultivars and its possible sources of infections in four tobacco growing districts of Denizli province. A total of 501 plant samples from field-grown tobaccos, weeds, potential intermediate hosts, seedlings and seeds were collected during 2019 and tested by DAS-ELISA. Of these plants, 243 belong to 55 different weed species from 26 different families with intermediate host potential. Throughout the study, 40 crop plant samples which could be intermediate hosts and 39 tobacco seed samples were also taken for testing. Adult thrips specimens were picked up from the fields and brought to the laboratory for preparations. Four vector virus species were detected when adult thrips individuals were diagnosed: Thrips tabaci Lindeman (Thysanoptera: Thripidae), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), Aeolothrips intermedius Bagnall (Thysanoptera: Aeolothripidae) and Thrips major Uzel (Thysanoptera: Thripidae). Of the 179 tobaccos sampled, 31.2% was positive; besides, of 243 weeds tested 10 were found to be infected. Echinochloa crus-galli and Tordylium apulum were determined to be new host recordings for TSWV infection. Only one tomato plant from the crop plants as intermediate hosts was infected. Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV) and Potato virus Y (PVY) was also confirmed in tobacco fields.

Konya İli Marul Ekim Alanlarında Sorun Olan Virüs Hastalıklarının Belirlenmesi

Lettuce (Lactuca sativa L.) is a member of the family Compositae (Asteraceae). This plant, which is grown for its leaves, is grown in open field and greenhouse conditions almost everywhere in the world and in Turkey. With the present study virus diseases of lettuce and their prevalence in Konya province was revealed for the first time. For this purpose, leaf samples were taken from lettuce plants showing virus diseases symptoms with surveys carried out in Konya province lettuce planting areas from May to August in 2020. Then the collected lettuce leaf samples were tested in laboratory conditions by Double Antibody Sandwich Enzyme-linked Immunosorbent Assay (DAS-ELISA) method to reveal infections of Cucumber mosaic virus (CMV), Miraflori lettuce big vein virus (MiLBVV), Lettuce mosaic virus (LMV), and Tomato spotted wilt virus (TSWV). According to the information obtained from the results of the study, it was determined that 40 out of 97 (41.23%) lettuce plant samples and all (6) weed samples were infected with at least one of the viruses. In lettuce leaf samples; TSWV (27.83%), LMV (12.37%), CMV (10.31%) and MiLBVV (5.15%) infections have been detected. In weed samples; infections of CMV (83.33%), LMV (66.66%), and TSWV (50%) have been revealed. The infections of TSWV, LMV, CMV, and MiLBVV on lettuce plants in Konya province were reported firstly with the study.

Population analysis on tomato spotted wilt virus isolates inducing various symptoms on tomato, pepper, and Chenopodium album in Turkey

Molecular identification of tomato spotted wilt virus (TSWV) has been conducted in several surveys in Turkey, but the population structure of the virus in the country remains unknown. During 2019–2020, 227 viral symptomatic tomato (Solanum lycopersicum), pepper (Capsicum annuum), and weeds leaf samples were collected from Ankara, Bartın, Eskisehir, Konya, and Zonguldak provinces. RT-PCR tests confirmed TSWV infection in 99 tomato, pepper, and Chenopodium album samples (43.6%). Phylogenetic analysis based on complete nucleotide sequences of N gene clustered the 23 newly sequenced Turkish isolates and other 281 isolates in NCBI GenBank into three major clades: p202/3WT, Tarquinia, and p105. All tested Turkish isolates were included in either p202/3WT or p105 clade. Global isolates retained high nucleotide and amino acids identity for N gene according to percentage identity analysis. N gene of TSWV was under very strong negative selection pressures, as shown by the very low ω values (<0.15) for all analyzed populations. Three neutrality tests also suggested that these populations are undergoing balancing selection by assigning negative values to most of them. The genetic differentiation and gene flow analysis among three clades demonstrated that they are genetically divergent from each other, but their medium FST values of around 0.5 showed that gene flows among clades are rare. This is the first detailed study on the genetic diversity and population structure of TSWV in Turkey.

A sequel study on the occurrence of Tomato spotted wilt virus (TSWV) in cut-chrysanthemum by DAS-ELISA using recombinant nucleocapsid protein to produce polyclonal antiserum

The tomato spotted wilt virus (TSWV) belonging to the genus Orthotospovirus, family Tospoviridae, causes severe necrotic disease in field crops and horticultural crops, resulting in considerable yield loss worldwide. The development of protein-based diagnostics is essential to track the virus transmission and prevent its spread in vegetatively propagated crops such as ornamentals. In this study, nucleocapsid (N) gene of TSWV was cloned in pET 28 a (+) expression vector. Expression of the 32 kDa recombinant TSWV-N protein was induced in BL21 (DE3) cells using 1 mM of Isopropyl β-d-1-thiogalactopyranoside (IPTG), and was confirmed through SDS-PAGE and Western blot by fluorescent-labeled secondary antibody. The bacterial cells expressed recombinant TSWV-N protein up to a concentration of 9.48 μg/mL. The purified protein was used for immunization of a rabbit to produce specific polyclonal antiserum. The TSWV antiserum was conjugated with the enzyme alkaline phosphatase (ALP). Double Antibody Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA) was developed and validated against TSWV infected hosts. This antiserum specifically reacted with recombinant N protein as well as TSWV infected hosts, but not with groundnut bud necrosis orthotospovirus (GBNV) as well as capsicum chlorosis orthotospovirus (CaCV) infecting tomato and chilli plants. The coating antibody at 1 μg/mL concentration and 1:500 dilution of enzyme conjugate were found to be effective and economical in the detection of recombinant N protein of TSWV and the virus present naturally in the infected hosts. Using standardized DAS-ELISA protocol, the TSWV titer also was quantified in artificially inoculated assay hosts. Among 11 hosts tested, higher virus titer was recorded in Nicotiana tabacum (0.270 μg/100 μL), followed by Impatiens balsamiana (0.185 μg/100 μL) and Dahlia pinnata at a low virus tire of 0.083 μg/100 μL. The diagnostic reagents and protocol (DAS-ELISA) are further validated by detecting the infection of TSWV in chrysanthemum stem cuttings from six different nurseries in the hill stations of Tamil Nadu, India. The DAS-ELISA assay experimented on six varieties from four different nurseries revealed that the Mum Yellow variety had a higher percentage of TSWV infection (36 %), which was followed by the Mum White variety (33 %); both collected from Kotagiri Nursery. The same variety exhibited a higher virus titer by DAS-ELISA, an A405 value range of 0.733 (̴ 0.115 μg) and 0.711 (̴ 0.111 μg) respectively, and a total of 27 % of TSWV infection was confirmed by screening 800 stem cuttings by DAS-ELISA. The presence of TSWV was also detected in 54 (6.75 %) asymptomatic stem cuttings from different locations, and the A405 value ranged from 0.325 to 0.468.(̴ 0.044−0.069 μg/100 μL); this is the first reported development of immune-based diagnostics for TSWV in India. This protocol and diagnostics will be highly useful for quarantine purposes while trading large quantities of planting materials.

Auxin drives tomato spotted wilt virus (TSWV) resistance through epigenetic regulation of auxin response factor ARF8 expression in tomato.

Tomato spotted wilt virus (TSWV) causes severe losses of tomato crops worldwide. To cope dynamically with such a threat, plants deploy strategies acting at the molecular and the epigenetic levels. We found that tomato symptoms progress in a specific-genotype-manner upon TSWV infection. Susceptible genotypes showed within the Auxin Response Factor (ARF8) promoter coupled to enhanced expression of miRNA167a, reduced ARF8 gene and decreased levels of the hormone auxin. This constitutes a deliberate attempt of TSWV to disrupt plant growth to promote spread in sensitive cultivars. Epigenetic regulation through the level of cytosine methylation and the miR167a-ARF8 module are part of a complex network modulating auxin-triggered synthesis and shaping tomato responses to TSWV. Furthermore, modulation of miR167a-ARF8 regulatory module could be applied in tomato-resistance breeding programs.

Integration of transcriptomics and network analysis reveals co-expressed genes in Frankliniella occidentalis larval guts that respond to tomato spotted wilt virus infection

BackgroundThe gut is the first barrier to infection by viruses that are internally borne and transmitted persistently by arthropod vectors to plant and animal hosts. Tomato spotted wilt virus (TSWV), a plant-pathogenic virus, is transmitted exclusively by thrips vectors in a circulative-propagative manner. Frankliniella occidentalis (western flower thrips), the principal thrips vector of TSWV, is transmission-competent only if the virus is acquired by young larvae. To begin to understand the larval gut response to TSWV infection and accumulation, a genome-assisted, transcriptomic analysis of F. occidentalis gut tissues of first (early L1) and second (early L2 and late L2) instar larvae was conducted using RNA-Seq to identify differentially-expressed transcripts (DETs) in response to TSWV compared to non-exposed cohorts.ResultsThe larval gut responded in a developmental stage-dependent manner, with the majority of DETs (71%) associated with the early L1 stage at a time when virus infection is limited to the midgut epithelium. Provisional annotations of these DETs inferred roles in digestion and absorption, insect innate immunity, and detoxification. Weighted gene co-expression network analysis using all assembled transcripts of the gut transcriptome revealed eight gene modules that distinguish larval development. Intra-module interaction network analysis of the three most DET-enriched modules revealed ten central hub genes. Droplet digital PCR-expression analyses of select network hub and connecting genes revealed temporal changes in gut expression during and post exposure to TSWV.ConclusionsThese findings expand our understanding of the developmentally-mediated interaction between thrips vectors and orthotospoviruses, and provide opportunities for probing pathways for biomarkers of thrips vector competence.

First Report of Tomato Spotted Wilt Virus in Angelica acutiloba

In June 2019, Angelica acutiloba plants showing virus-like symptoms such as chlorotic local lesion and mosaic on the leaves were found in a greenhouse in Nonsan, South Korea. To identify the causal virus, we collected 6 symptomatic A. acutiloba leaf samples and performed reverse transcription polymerase chain reaction (RT-PCR) analysis using specific detection primers for three reported viruses including tomato spotted wilt virus (TSWV). RT-PCR results showed that five symptomatic samples were positive for TSWV. Mechanical sap inoculation of one of the collected TSWV isolate (TSWV-NS-AG28) induced yellowing, chlorosis and mosaic symptoms in A. acutiloba and necrotic local lesions and mosaic in Solanaceae species. Phylogenetic analysis based on the complete genome sequences showed that TSWV-NS-AG28 had a maximum nucleotide identity with TSWV-NS-BB20 isolated from butterbur in Nonsan, South Korea. To our knowledge, this is the first report of TSWV infection in A. acutiloba.