Tomato Spotted Wilt Virus Isolates Research Articles

A novel tomato spotted wilt virus isolate encoding a noncanonical NSm C118F substitution associated with Sw-5 tomato gene resistance breaking.

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.

First Report of Resistance-Breaking Variants of Tomato Spotted Wilt Virus (TSWV) Infecting Tomatoes with the Sw-5 Tospovirus-Resistance Gene in North Carolina.

Widespread use of tomato cultivars with the Sw-5 resistance gene has led to the emergence of resistance-breaking (RB) strains of tomato spotted wilt virus across the globe. In June of 2022, tomato spotted wilt (TSW) symptoms were observed at two farms (A and B, within 15 miles of each other) in Rowan County, NC on several commercial TSW resistant tomato cultivars (all heterozygous for the Sw-5 gene). At farm A, ~10% of plants had symptomatic foliage with ~30% of fruit with symptoms, while at farm B, up to 50% of plants had symptomatic foliage with ~80% of fruit with symptoms. Visual symptoms included stunting, severe leaf curling and bronzing, necrotic lesions on leaves, petioles and stems, and concentric ring spots on fruit (Supplementary Fig. 1). TSWV ImmunoStrips (AgDia, Elkhart, IN) and reverse-transcription (RT)-PCR with NSm primers (di Rienzo et al 2018) confirmed the presence of TSWV in 12 symptomatic plants sampled across the two farms. Primers designed to detect Impatiens necrotic spot virus, groundnut ringspot virus, tomato chlorotic spot virus, tomato chlorosis virus, alfalfa mosaic virus, and tomato necrotic streak virus (ilarvirus, Badillo et al., 2016) failed to generate amplicons of the expected size from cDNA generated from these field samples. The amplicons from full-length NSm cDNA were sequenced from independent, single-leaflet isolates from the TSWV-positive plants (three from farm A, nine from farm B) with the expectation of finding an amino acid (aa) substitution associated with the Sw-5 RB phenotype identified previously in CA (C118Y, Batuman et al. 2017) or Spain (C118Y and T120N, Lopez et al. 2011). All three nucleotide sequences from farm A contained the NSm C118Y substitution reported in CA. All three sequences were 99% identical (including the C118Y mutation) to NCBI GenBank accession KU179600.1, a TSWV isolate collected from GA in 2014 with no cultivar information reported. The nine nucleotide sequences from farm B contained neither of the two previously reported aa substitutions associated with the RB phenotype. Instead, all contained a D122G substitution within a conserved region of the TSWV NSm protein reported to be involved in direct interaction with the Sw-5 protein (Zhu et al 2017). Likewise, Huang et al (2021) generated a D122A mutation in TSWV-NSm, resulting in failure to elicit a Sw-5 mediated hypersensitive response. Three NSm sequences retrieved from GenBank contained the D122G substitution (AY848921.1, HM015516.1, KU179582.1), however, this mutation was not implicated directly with RB phenotypes (Ciuffo et al., 2005; Lopez et al., 2011; Marshall, 2016). The RB phenotype was confirmed with the NC variants on 'Mountain Merit' (Sw-5) by two means of virus inoculation: mechanical, rub-inoculation with extracted sap from infected plants, and thrips transmission assays with lab colony-maintained, Frankliniella occidentalis, the western flower thrips. Symptomatic leaf tissue obtained from these inoculation assays tested positive for TSWV by DAS-ELISA (AgDia, Elkhart, IN) and RT-PCR with NSm primers, providing definitive evidence of the occurrence of RB-TSWV at both farms, and subsequent sequencing confirmed the C118Y and D122G substitutions. This report warrants further investigation of the putative origins, prevalence and epidemiological implications of RB-TSWV variants in NC tomato production, and the development of new sources of resistance to TSWV.

İzmir ilinde domates ve biber bitkilerini etkileyen tomato spotted wilt virus (TSWV) ve cucumber mosaic virus (CMV) etmenlerinin moleküler karakterizasyonu

Objective: The objective of this study was to investigate tomato spotted wilt virus (TSWV) and cucumber mosaic virus (CMV) infections in tomato and pepper plants showing virus-induced symptoms in vegetable growing districts of İzmir, Turkey. Material and Methods: Surveys were carried out in tomato and pepper plantations in 2019 and 2021, and the incidences of these viruses in the collected leaf samples were determined by RT-PCR. Nucleotide identities and phylogenetic relationships of the TSWV and CMV isolates with other isolates retrieved from the GenBank database were determined. Results: The results of this study showed that tomato plants were infected at the same rate (21.50%) with TSWV and CMV. Out of the tested pepper samples, 64.15% were infected with TSWV and 25.47% with CMV. The results showed that, the identity rate of nucleoprotein region of TSWV isolates from tomato was 99-96% at nucleotide level while the isolates from pepper showed 100-95% identity. On the other hand, the capsid protein gene region of the tomato isolate of CMV had nucleotide identity rate of 98-95% with other isolates in GenBank, while that of its pepper isolates had 100-98% identity. Also, CMV isolates of this study showed close phylogenetic relationship with the CMV isolates of subgroup IB. Conclusion: This study revealed the prevalence of TSWV and CMV in symptomatic tomato and pepper samples in İzmir province and some molecular properties of them.

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).

Molecular Characteristics of Subgenomic RNAs and the Cap-Dependent Translational Advantage Relative to Corresponding Genomic RNAs of Tomato spotted wilt virus

Tomato spotted wilt virus (TSWV) causes severe viral diseases on many economically important plants of Solanaceae. During the infection process of TSWV, a series of 3'-truncated subgenomic RNAs (sgRNAs) relative to corresponding genomic RNAs were synthesized, which were responsible for the expression of some viral proteins. However, corresponding genomic RNAs (gRNAs) seem to possess the basic elements for expression of these viral proteins. In this study, molecular characteristics of sgRNAs superior to genomic RNAs in viral protein expression were identified. The 3' ends of sgRNAs do not cover the entire intergenic region (IGR) of TSWV genomic RNAs and contain the remarkable A-rich characteristics. In addition, the 3' terminal nucleotides of sgRNAs are conserved among different TSWV isolates. Based on the eIF4E recruitment assay and subsequent northern blot, it is suggested that the TSWV sgRNA, but not gRNA, is capped in vivo; this is why sgRNA is competent for protein expression relative to gRNA. In addition, the 5' and 3' untranslated region (UTR) of sgRNA-Ns can synergistically enhance cap-dependent translation. This study further enriched the understanding of sgRNAs of ambisense RNA viruses.

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.

Diagnosis of Important Virus Diseases of Ornamental Anthurium in Screen- Houses in Tehran Province

Introduction: One of the limiting factors for Anthurium production is its infection with various viruses. During the year 2014- 2015, a total of 504 symptomatic ornamental Anthurium leaf samples with virus-like symptoms including leaf and flower blister, dwarfing, mosaic, yellowing, leaf marginal chlorosis, and deformity were collected from Varamin and Pakdasht (Tehran province) Screen-houses. Aim: Samples were tested for infection to Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV) and Dasheen mosaic viruses (DaMV). Method: Biological, serological (ELISA) and molecular methods (RT-PCR) were performed for the detection and analysis of the important viral agents infecting Anthurium ornamental plants. Results: According to ELISA test results the prevalence of CMV were (31.54%) followed by TSWV (25%) and DaMV (7.93%) viruses respectively. Results of mechanical inoculation and host range reaction of CMV, TSWV and DaMV isolates were also recorded (fig 2). Coat protein gene sequence of Iranian CMV isolate showed the highest similarity (97%) with a CMV isolate Cucurbit pepo (Acc. No.GU327368.1) from South Korea. DMV on ornamental Anthurium is reported for the first time in this research from Iran. Conclusion: Despite the detection of CMV, TSWV and DaMV in this study, viral pathogens are one of the most economically damaging agents in the ornamental Anthurium plant. Development of research methods for, the detection and study of different isolates of these agents from different hosts, and study of the characteristics of each of these isolates has always been a scientific need.

Determination of key residues in tospoviral NSm required for Sw-5b recognition, their potential ability to overcome resistance, and the effective resistance provided by improved Sw-5b mutants.

Sw‐5b is an effective resistance gene used widely in tomato to control tomato spotted wilt virus (TSWV), which causes severe losses in crops worldwide. Sw‐5b confers resistance by recognizing a 21‐amino‐acid peptide region of the viral movement protein NSm (NSm21, amino acids 115–135). However, C118Y or T120N mutation within this peptide region of NSm has given rise to field resistance‐breaking (RB) TSWV isolates. To investigate the potential ability of TSWV to break Sw‐5b‐mediated resistance, we mutagenized each amino acid on NSm21 and determined which amino acid mutations would evade Sw‐5b recognition. Among all alanine‐scan mutants, NSmP119A, NSmW121A, NSmD122A, NSmR124A, and NSmQ126A failed to induce a hypersensitive response (HR) when coexpressed with Sw‐5b in Nicotiana benthamiana leaves. TSWV with the NSmP119A, NSmW121A, or NSmQ126A mutation was defective in viral cell‐to‐cell movement and systemic infection, while TSWV carrying the NSmD122A or NSmR124A mutation was not only able to infect wild‐type N. benthamiana plants systemically but also able to break Sw‐5b‐mediated resistance and establish systemic infection on Sw‐5b‐transgenic N. benthamiana plants. Two improved mutants, Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q , which we recently engineered and which provide effective resistance against field RB isolates carrying NSmC118Y or NSmT120N mutations, recognized all NSm21 alanine‐substitution mutants and conferred effective resistance against new experimental RB TSWV with the NSmD122A or NSmR124A mutation. Collectively, we determined the key residues of NSm for Sw‐5b recognition, investigated their potential RB ability, and demonstrated that the improved Sw‐5b mutants could provide effective resistance to both field and potential RB TSWV isolates.

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.

English

The molecular diversity of Tomato spotted wilt virus (TSWV) isolates from Pakistan was investigated based on nucleocapsid protein (N) gene. It revealed that the Pakistani isolates shared 98.2–99.3% (nt) and 95.7–97.4% (aa) identities with each other and 97.2–99.3% (nt) and 92.7–97.8% (aa) with other TSWV isolates retrieved from Genbank. In a neighbour-joining phylogenetic reconstruction, four Pakistani isolates clustered with South Korean and Turkish isolates and one with Australian and South African isolates. All the Pakistani isolates shared maximum genetic diversity (0.024–0.039 and 0.026–0.029) with South African (AJ296600) and Australian (AY879109) isolates and the lowest values of genetic diversity (0.009–0.013) was recorded with Czech Republican isolate (AJ296599). The gene differentiation co-efficient value (Fst) was 0.33 less than standard value noticed as 0.05145, between globally reported and Pakistani TSWV isolates considering it a frequent gene flow. The significant values of statistical tests based on three genetic differentiation analysis; Ks⃰; 4.86231, Z; 34.27778 and Snn; 0.55556 were observed. The negative values were recorded according to statistical test as Fu, &amp; Li’s D ⃰ (-2.55465), Fu, &amp; Li’s F ⃰ (-2.78103) and one positive value from Tajima’s D (2.10697). These results indicate low polymorphism frequency in TSWV population of Pakistan. © 2021 Friends Science Publishers

Current Status and Potential of RNA Interference for the Management of Tomato Spotted Wilt Virus and Thrips Vectors.

Tomato spotted wilt virus (TSWV) is the type member of the genus Orthotospovirus in the family Tospoviridae and order Bunyavirales. TSWV, transmitted by several species of thrips, causes significant disease losses to agronomic and horticultural crops worldwide, impacting both the yield and quality of the produce. Management strategies include growing virus-resistant cultivars, cultural practices, and managing thrips vectors through pesticide application. However, numerous studies have reported that TSWV isolates can overcome host-plant resistance, while thrips are developing resistance to pesticides that were once effective. RNA interference (RNAi) offers a means of host defence by using double-stranded (ds) RNA to initiate gene silencing against invading viruses. However, adoption of this approach requires production and use of transgenic plants and thus limits the practical application of RNAi against TSWV and other viruses. To fully utilize the potential of RNAi for virus management at the field level, new and novel approaches are needed. In this review, we summarize RNAi and highlight the potential of topical or exogenous application of RNAi triggers for managing TSWV and thrips vectors.

Biological and molecular characterization of tomato spotted wilt virus (TSWV) infecting Chrysanthemum in India

Tomato spotted wilt virus (TSWV) infecting chrysanthemum (Dendranthema grandiflora L.) has been identified as a major constraint for chrysanthemum cut-flower industries in Southern India. The disease symptoms include veinal necrosis, necrotic spots on leaves, deformed flowers, and stem necrosis. The presence of TSWV in chrysanthemum plants collected from farms at Kotagiri, Nedugula, and Yercaud in Tamil Nadu State, India, was confirmed by the triple antibody sandwich-enzyme-linked immunosorbent assay (TAS-ELISA). The pathogenicity of TSWV was established for three isolates (TN1KO, TN2NE, and TN3YE) by mechanical inoculation of sap into chrysanthemum cuttings. Host range experiments in different host plants indicated that the isolates expressed symptoms typical of TSWV in all hosts assayed. Furthermore, Nicotiana tabacum L. had maximum virus titre among the tested host plants, and therefore was identified as the best indicator and the propagative host. The nucleocapsid (N) gene of the three studied isolates shared 99.2–100% identity at the amino acid level among themselves and 96.5–99.6% identity with reference TSWV isolates. The non-structural movement protein (NSm) gene of studied isolates shared 99.0% identity among themselves and 96.0%–100% identity at the amino acid level with reference TSWV isolates. The phylogenetic relationship analyzed based on the amino acid sequence identity of the N gene revealed that the studied isolate clustered as a single group with all the TSWV isolates taken for comparison. However, analysis based on the NSm gene indicated that the Indian isolates grouped with isolates from South Korea, the USA, Italy, Spain, and Australia.

First report of Tomato spotted wilt virus in Petasites japonicus in Korea.

Butterbur (Petasites japonicus [Siebold & Zucc.] Maxim.) is a perennial herb of the Asteraceae family that is cultivated for medicinal and nutritional purposes. Due to long-term vegetative propagation of virus-infected native species, the yield and quality of butterbur plants have deteriorated. Five viruses have been reported to infect this species: alfalfa mosaic virus (AMV), arabis mosaic virus (ArMV), butterbur mosaic virus (ButMV), broad bean wilt virus 2 (BBWV-2), and cucumber mosaic virus (CMV) (Ham et al. 2016; Tochihara and Tamura 1976). From 2018 to 2019, butterbur plants in four greenhouses in Nonsan, South Korea (Supplementary Figure S1a, b) were found to show virus-like symptoms such as chlorotic and necrotic ring spots, necrosis, and mild mosaic on the leaves. Disease incidence was greater than 80% in one greenhouse (~1,000 m2). To identify the causal virus, we collected 17 symptomatic butterbur leaf samples from these greenhouses and performed reverse-transcription polymerase chain reaction (RT-PCR) analysis using species-specific detection primers for the five reported viruses and tomato spotted wilt virus (TSWV) (Supplementary Table S2). RT-PCR results showed that 12 samples from three greenhouses showing necrotic ring spots and mosaic symptoms were infected with a mixture of TSWV and ButMV, whereas 5 samples from one greenhouse showing mild mosaic symptoms were infected only with ButMV. TSWV (genus Orthotospovirus, family Tospoviridae) is transmitted by thrips and causes serious damage to a wide range of economically important plants (Pappu et al. 2009). ButMV (genus Carlavirus, family Betaflexiviridae) is transmitted by aphids, as well as infected vegetative propagation material (Hashimoto et al. 2009) and is the most predominant virus in butterbur in Korea (Ham et al. 2016). To isolate TSWV from butterbur, leaf extracts from symptomatic samples were mechanically inoculated on an assay host, Chenopodium quinoa, via three single-lesion passages followed by propagation in Nicotiana tabacum cv. Samsun. Thirty different indicator plant species were used for the bioassay of the TSWV isolate (TSWV-NS-BB20) by mechanical inoculation method (Supplementary Table S3). RT-PCR analysis confirmed that TSWV-NS-BB20 induced necrotic local lesions and mosaic on Nicotiana species and ring spots and mosaic on tomatoes and peppers. Notably, TSWV-NS-BB20 reproduced necrotic local lesions and mild mosaic symptoms on butterbur plants which were infected with ButMV with no obvious symptoms. To characterize TSWV-NS-BB20 genetically, the complete genome sequences of L (8914 nt), M (4751 nt), and S (2917 nt) RNA segments were obtained by RT-PCR using specific primers for TSWV as described previously (Kwak et al., 2020). The obtained sequences were deposited in GenBank under accession nos. MT643236, MT842841, and MN854654, respectively. BLASTn analysis showed that sequences of each segment had maximum nucleotide identities of 99.0, 98.9, and 98.6% to TSWV-L, M, and S (KP008128, FM163373, and KP008129) of TSWV-LL-N.05 isolate from tomato in Spain. Since 2018, TSWV outbreaks on butterbur are observed every year and thus may act as a potential source of TSWV infection for other crops of importance to Korea, such as pepper. Owing to the butterbur vegetative propagation, the identification of TSWV infection in butterbur will be helpful for future virus management to generate virus-free materials. To our knowledge, this is the first report of TSWV infection of butterbur.

Developmentally regulated Arabidopsis thaliana susceptibility totomato spotted wilt virus infection.

Tomato spotted wilt virus (TSWV) is one of the most devastating plant viruses and often causes severe crop losses worldwide. Generally, mature plants become more resistant to pathogens, known as adult plant resistance. In this study, we demonstrated a new phenomenon involving developmentally regulated susceptibility of Arabidopsis thaliana to TSWV. We found that Arabidopsis plants become more susceptible to TSWV as plants mature. Most young 3‐week‐old Arabidopsis were not infected by TSWV. Infection of TSWV in 4‐, 5‐, and 6‐week‐old Arabidopsis increased from 9%, 21%, and 25%, respectively, to 100% in 7‐ to 8‐week‐old Arabidopsis plants. Different isolates of TSWV and different tospoviruses show a low rate of infection in young Arabidopsis but a high rate in mature plants. When Arabidopsis dcl2/3/4 or rdr1/2/6 mutant plants were inoculated with TSWV, similar results as observed for the wild‐type Arabidopsis plants were obtained. A cell‐to‐cell movement assay showed that the intercellular movement efficiency of TSWV NSm:GFP fusion was significantly higher in 8‐week‐old Arabidopsis leaves compared with 4‐week‐old Arabidopsis leaves. Moreover, the expression levels of pectin methylesterase and β‐1,3‐glucanase, which play critical roles in macromolecule cell‐to‐cell trafficking, were significantly up‐regulated in 8‐week‐old Arabidopsis leaves compared with 4‐week‐old Arabidopsis leaves during TSWV infection. To date, this mature plant susceptibility to pathogen infections has rarely been investigated. Thus, the findings presented here should advance our knowledge on the developmentally regulated mature host susceptibility to plant virus infection.

Phylogenetic analysis based on full genome sequencing of Italian tomato spotted wilt virus isolates identified in “Roggianese” sweet pepper and chilli pepper

AbstractA study aimed at defining population structure of Italian tomato spotted wilt virus (TSWV) isolates was performed. Full genome sequencing of six TSWV isolates found in two Italian regions (two from Latium: Lazio 17 and Tarquinia; and four from Calabria: PepCal 10, 12, 22 and 24) were assembled. Identity percentages in nucleotide sequence among these TSWV isolates are here provided. The six full length genome sequences were compared with other two Italian isolates (p105 and p202/3WT) already fully sequenced, as well as full TSWV genomes that could be retrieved from GenBank. Phylogenetic analysis, performed in concatenated sequences and for each gene of each genome segment (L, M and S), confirmed the presence of two clades, namely A‐like and D‐like. In particular, the phylogenetic tree based on segment L grouped all the newly sequenced TSWV isolates in D‐like clade. In the M segment phylogenetic tree, all our TSWV isolates shifted in the A‐like clade. Isolates separation was not correlated to their geographical origin in phylogenetic study of distinct ORFs encoded by the RNA S segment. In fact, in nucleocapsid‐encoding phylogenetic tree, PepCal 10 and 22 grouped in an A‐like clade with p105, PepCal 12 and 24 in a D‐like clade with p202/3WT, whereas Lazio 17 and Tarquinia in a third well distinct group. NSs tree displayed only PepCal 10 with p105 in A‐like clade, whereas PepCal 12, 22, 24 with p202/3WT in D‐like subclade; and isolates from Latium grouped a separated clade adjacent to D‐like isolates. Additional analysis on putative reassortment events showed that TSWV Calabrian isolates likely originated from a reassortment event in M RNA and other in S RNA with p105 as major parent. Recombination events were detected in isolates from Latium in L and S RNAs with Chinese isolates as putative major parent.

Full genome sequence of a chrysanthemum-infecting tomato spotted wilt virus isolate from Zimbabwe obtained by next-generation sequencing.

Tomato spotted wilt virus (TSWV) is an economically important pathogen of many crops worldwide. However, prior to this study, only one complete genome sequence of an African TSWV isolate was available in public databases. This limits genetic diversity and evolutionary studies of the pathogen on the continent. TSWV was detected in symptomatic Zimbabwean chrysanthemum plants using late-ral flow kits. The presence of the pathogen was subsequently confirmed by double antibody sandwich enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction (RT-PCR). Total RNAs for RT-PCR and next-generation sequencing (NGS) were extracted using an RNA extraction kit. NGS performed on an Illumina HiSeq platform was used to recover the full TSWV genome and analyzed by different software packages. The tripartite genome of the Zimbabwe TSWV isolate consisted of L, M and S RNAs of 8914, 4824 and 2968 nucleotides, respectively. This isolate shared highest protein and nucleotide sequence identities with the isolate LK-1 from neighboring South Africa. The Zimbabwe TSWV isolate was found to be a non-recombinant and non-resistance-breaking. This study provides the first full genome of TSWV from Zimbabwe. It also adds useful information towards understanding the evolution of the pathogen. Keywords: Africa; tospovirus; phylogenetic analysis; recombination; virus identification.

Biological and molecular characterization of tomato spotted wilt virus (TSWV) resistance‐breaking isolates from Argentina

Tomato spotted wilt virus (TSWV) has been present in Argentina since 1938 and had limited sweet pepper and tomato production until the introduction of resistant cultivars bearing Tsw and Sw‐5b genes. However, the wide use of TSWV‐resistant pepper plants in La Plata Horticultural Belt (LPHB) triggered the emergence of resistance‐breaking isolates (RB), increasing the economic impact of TSWV in pepper. This work characterized 11 natural RB pepper isolates from LPHB that have overcome the Tsw resistance gene in Capsicum sp. but are unable to break the Sw‐5b‐mediated resistance in tomato. Phylogenetic analysis of the N gene showed that the LPHB isolates are most closely related to isolates from Asia, indicating that Argentine TSWV isolates might have emerged from the Asian continent. The NSs sequence analysis reinforces the hypothesis that the appearance of an RB phenotype is a consequence of a number of different single amino acid substitutions spread along the NSs gene that lead to multiple independent evolutionary events. These results provide information on the current situation of the tospovirus–pepper/tomato pathosystems in LPHB, which represents a fundamental prerequisite to include these RB isolates in future screening programmes in order to select new and durable sources of resistance to TSWV in pepper.

Phenotypic expression and species-specific reaction of Lactuca sativa ‘PI 342444’ to groundnut ringspot virus (GRSV) and tomato spotted wilt virus (TSWV) isolates

Currently, diseases caused by tospovirus species are among the most limiting factors for lettuce (Lactuca sativa L.) production in Brazil, where losses can reach up to 100%. In cultivated lettuce germplasm, the highest levels of resistance to tospoviruses were identified in the accession ‘PI 342444’ (= cultivar ‘Tinto’). We carried out controlled inoculation assays aiming to characterize the phenotypic expression and verify the spectrum of ‘PI 342444’-mediated resistance against a panel of isolates of two tospovirus species: Tomato spotted wilt virus (TSWV) and Groundnut ringspot virus (GRSV). Lettuce seedlings were mechanically inoculated with seven isolates (three TSWV and four GRSV). Plants of ‘PI 342444’ did not exhibit symptoms after inoculation with TSWV isolates, whereas severe symptoms were observed on the susceptible lettuce control (cultivar ‘Vanda’). The absence of TSWV infection in the majority of ‘PI 342444’ plants was also confirmed by serological testing. In contrast, all ‘PI 342444’ plants inoculated with GRSV isolates displayed early onset of severe symptoms, indicating that this resistance is not effective against this viral species. Further studies shall be conducted to characterize the genetic factors associated with a putative TSWV-specific resistance observed in ‘PI 342444’. On the other hand, our assays indicated that additional lettuce germplasm screenings searching for sources of resistance effective against GRSV isolates are among the major breeding priorities under Brazilian conditions, since this ‘PI 342444’ resistance-breaking virus is currently the predominant lettuce-infecting tospovirus species in the country.

Detection and Characterization of Tomato spotted wilt virus and Cucumber mosaic virus on Pepper Growing Areas in Antalya

The most efficient method to control the plant virus diseases is breeding resistant cultivars. However, the resistance could be broken down after using resistant cultivars. This study was aimed to determine the prevalence and also serological and molecular characterization of Tomato spotted wilt virus (TSWV) and Cucumber mosaic virus (CMV) that cause infections, especially, in resistant pepper cultivars. For this reason, samples were collected from pepper growing greenhouses and open fields during vegetation period of 2015 in different parts of Antalya province including Kumluca, Demre, Serik and Aksu districts. Out of 148 pepper samples collected, 53 (35.81%) were infected with TSWV and 11 (7.34%) with CMV as a result of Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DASELISA) test. Some regions on S RNA (nucleocapsid protein gene), M RNA (glycoprotein gene) and L RNA (RNAdependent RNA polymerase gene) of TSWV genome; RNA 1 (helicase/methyltransferase gene) and RNA 3 (coat protein gene) of CMV genome of DAS-ELISA positive samples were amplified by RT-PCR with specific primers. Nucleotide similarity rates of nucleocapsid protein gene, glycoprotein gene and RNA-dependent RNA polymerase gene regions of TSWV isolate varied between 92-98% identity with other isolates in GenBank and CMV isolate varied between 89-96%. TSWV isolate showed nucleotide identity varied between 92-97% with Tsw resistance breaking isolates from Capsicum plants in Turkey, Italy and Spain. Genetic determinant for overcoming pepper Tsw resistance is located in S segment but aminoacid substitutions responsible for TSWV breakdown remain contradictory in several reports.

First Report of Tomato Spotted Wilt Virus Identified in Tulbaghia violacea in Florida

Tulbaghia violacea Harv., also known as society garlic, belongs to the genus Tulbaghia (Amaryllidaceae). It is diversely used for medicinal, culinary, and ornamental purposes. In June 2018, T. violacea plants from Lake County, FL, with typical tospovirus-like symptoms of chlorotic ring patterns, were submitted to the Florida Department of Agriculture and Consumer Services, Division of Plant Industry (Gainesville, FL) for identification. Representative leaf tissue from four plants tested positive in a serological assay and reverse transcription-polymerase chain reaction. Amplicons of the expected size were produced using the tomato spotted wilt virus (TSWV) nucleocapsid (N) gene primers. They were directly sequenced and deposited in GenBank (MK567883). The 777-bp N gene sequence had 98 to 99% nucleotide identity with the corresponding region of previously described Florida TSWV isolates and 96 to 99% nucleotide identity with all TSWV isolates in GenBank. Epidemics of TSWV occur frequently in numerous crops in Florida, chiefly tomato, pepper, tobacco, and ornamentals. The western flower thrip, Frankliniella occidentalis (Pergande), is the most efficient vector of TSWV and has become established in Florida. This is the first report of TSWV infection in T. violacea.