Tomato spotted wilt virus (TSWV) infection of Physcomitrella patens gametophores

Following mechanical inoculation of the moss Physcomitrella patens (Hedw.) B.S.G. with Tomato spotted wilt virus (TSWV), the virus encoded N nucleocapsid protein was detected in gametophores harvested 11 and 29 dpi and the non-structural NSm movement protein was observed 29 dpi. The detection of both viral proteins presumes that P. patens could serve as a new lab–host for TSWV, allowing reverse genetics by gene targeting to elucidate the role of specified molecular virus–host interactions.     

The molecular population genetics of the Tomato spotted wilt virus (TSWV) genome

RNA viruses are characterized by high genetic variability resulting in rapid adaptation to new or resistant hosts. Research for plant RNA virus genetic structure and its variability has been relatively scarce compared to abundant research done for human and animal RNA viruses. Here, we utilized a molecular population genetic framework to characterize the evolution of a highly pathogenic plant RNA virus [Tomato spotted wilt virus (TSWV), Tospovirus, Bunyaviridae]. Data from genes encoding five viral proteins were used for phylogenetic analysis, and for estimation of population parameters, subpopulation differentiation, recombination, divergence between Tospovirus species, and selective constraints on the TSWV genome. Our analysis has defined the geographical structure of TSWV, attributed possibly to founder effects. Also, we identify positive selection favouring divergence between Tospovirus species. At the species level, purifying selection has acted to preserve protein function, although certain amino acids appear to be under positive selection. This analysis provides demonstration of population structuring and species-wide population expansions in a multisegmented plant RNA virus, using sequence-based molecular population genetic analyses. It also identifies specific amino acid sites subject to selection within Bunyaviridae and estimates the level of genetic heterogeneity of a highly pathogenic plant RNA virus. The study of the variability of TSWV populations lays the foundation in the development of strategies for the control of other viral diseases in floral crops.     

Screening Capsicum germplasm for resistance to tomato spotted wilt virus (TSWV)

Resistance conferred by the Tsw locus from Capsicum chinense against Tomato spotted wilt virus (TSWV) has been widely used in breeding programmes. Nevertheless, this resistance depends on inoculation conditions, and isolates able to overcome it have already been detected. In this work 29 accessions of several Capsicum species have been mechanically inoculated with TSWV to identify new sources of resistance. Five accessions showed variable percentages of resistant plants, two of which did not show local lesions on inoculated leaves, suggesting that the response was not mediated through hypersensitivity. Two of these accessions also had a remarkable reduced viral accumulation compared to susceptible control. ECU‐973., a C. chinense accession, showed the best performance against TSWV, with 100% resistant plants. This response was confirmed after mechanical inoculation with three different TSWV isolates. The resistance was maintained when the accession was inoculated with TSWV using a high pressure of viruliferous thrips. These results open new possibilities in the development of a durable resistance to TSWV in pepper.     

New RAPD markers of tomato spotted wilt virus (TSWV) resistance in Lycopersicon esculentum Mill

The selection of TSWV resistant individuals can be facilitated by molecular markers. RAPD analysis was carried out on three forms (Stevens × Rodade — resistant; Rey de los Tempranos — moderately tolerant; Potentat — susceptible) with the use of 271 primers. Out of 271 primers 28 generated stable polymorphism and so they were tested for linkage to resistance gene. Bulk segregant analysis (BSA) was applied to F₂ segregating progeny developed from resistant × susceptible parents. As a result, 5 primers enabling us to distinguish between resistant and susceptible forms were detected. Only one of them had previously been reported by Chague et al. (1996). The analysis should be repeated on a larger population to confirm the results obtained.     

A CAPS marker to assist selection of tomato spotted wilt virus (TSWV) resistance in pepper.

The hypersensitive resistance to tomato spotted wilt virus (TSWV) in pepper is determined by a single dominant gene (resistant allele: Tsw) in several Capsicum chinense genotypes. In order to facilitate the selection for this resistance, four RAPD (among 250 10-mer primers tested) were found linked to the Tsw locus using the bulked segregant analysis and 153 F2 individuals. A close RAPD marker was converted into a codominant cleaved amplified polymorphic sequence (CAPS) using specific PCR primers and restriction enzymes. This CAPS marker is tightly linked to Tsw (0.9 +/- 0.6 cM) and is helpful for marker-assisted selection in a wide range of genetic intercrosses.     

Selection of tomato plants resistant to a local Polish isolate of tomato spotted wilt virus (TSWV)

We found that the Sw-5 gene confers resistance to one of the Polish isolates of tomato spotted wilt virus (TSWV). A series of tomato breeding accessions was analysed along with standards of resistance and susceptibility to TSWV. The presence of the Sw-5 gene was determined using the available PCR marker. Subsequently plants from these accessions were grown in the presence of the TSWV isolate from Poland. Some of them developed severe symptoms of the TSWV disease. Expression of the virus proteins was also assayed in tissues of the investigated plants. We found general agreement between either lack or presence of the disease symptoms, virus proteins and resistance gene. Some observed discrepancies of these data are also discussed. Our results indicate that marker-assisted selection can be used for breeding of the TSWV-resistant tomato in Poland.     

Prospects of engineered forms of resistance against tomato spotted wilt virus

Tomato spotted with virus (TSWV) is deviant from most other plant viruses in having (mainly) a negative-strand RNA genome and a lipid envelope, and in being propagatively transmitted by thrips. Due to increasing resistance of thrips to insecticides as well as to a relative shortage of natural resistance genes suitable for breeding, genetically engineered forms of resistance to TSWV infections are urgently needed. This review will discuss the recently approved strategy of N gene-mediated protection, which has been shown to hold even against thrips-mediated virus inoculation, as well as a number of potential strategies that are likely to be developed in the near future.     

Tsw gene-based resistance is triggered by a functional RNA silencing suppressor protein of the Tomato spotted wilt virus

As a result of contradictory reports, the avirulence (Avr) determinant that triggers Tsw gene-based resistance in Capsicum annuum against the Tomato spotted wilt virus (TSWV) is still unresolved. Here, the N and NSs genes of resistance-inducing (RI) and resistance-breaking (RB) isolates were cloned and transiently expressed in resistant Capsicum plants to determine the identity of the Avr protein. It was shown that the NSs^RI protein triggered a hypersensitive response (HR) in Tsw-containing Capsicum plants, but not in susceptible Capsicum, whereas no HR was discerned after expression of the N^RI/RB protein, or when NSs^RB was expressed. Although NSs^RI was able to suppress the silencing of a functional green fluorescence protein (GFP) construct during Agrobacterium tumefaciens transient assays on Nicotiana benthamiana, NSs^RB had lost this capacity. The observation that RB isolates suppressed local GFP silencing during an infection indicated a recovery of RNA silencing suppressor activity for the NSs protein or the presence of another RNA interference (RNAi) suppressor. The role of NSs as RNA silencing suppressor and Avr determinant is discussed in the light of a putative interplay between RNAi and the natural Tsw resistance gene.     

Use of Pseudomonas fluorescens-based formulations for management of tomato spotted wilt virus (TSWV) and enhanced yield in tomato

Strains of Pseudomonas fluorescens were investigated for biocontrol efficacy against tomato spotted wilt virus (TSWV) in tomato both alone and in mixtures. P. fluorescens strains applied to seed, soil and foliage or as a seedling dip significantly reduced TSWV, with a concomitant increase in growth promotion in both the glasshouse and field. Two native strains (CoP-1 and CoT-1) and one foreign strain (CHAO) reduced TSWV. In P. fluorescens-treated tomato plants, increased activity of polyphenol oxidase, β-1,3-glucanase and chitinase was observed, and induction of chitinase was confirmed by western blot analysis. Induction of new protein (18 kDa) detected by SDS-PAGE in P. fluorescens-treated tomato plants was not found in healthy and P. fluorescens-untreated virus inoculated control plants. Indirect ELISA clearly showed a reduction in viral antigen concentration in P. fluorescens-treated tomato plants corresponding to reduced disease ratings. All the P. fluorescens-treated tomato plants also showed enhanced growth and yield compared to control plants. Hence, plant growth promoting rhizobacteria (PGPR) could play a major role in reducing TSWV and increasing yield in tomato plants.     

Tomato spotted wilt virus (TSWV) in Greece: its incidence following the expansion of Frankliniella occidentalis, and characterisation of isolates collected from various hosts

Leaf samples were collected from plants with tospovirus‐like symptoms from various hosts in different regions of Greece where Thrips tabaci, Frankliniella occidentalis or both vectors occur. The viruses infecting these plants were identified with polyclonal antibodies raised against the N proteins of Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) by ELISA. All samples tested positive for TSWV, but not for INSV. ELISA of thirty three isolates, using monoclonal antibodies against the N protein of TSWV, revealed the existence of five epitopes on the N protein. RT‐PCR tests on a few randomly‐selected isolates, using a pair of universal primers, a pair of primers specific for the L gene and a pair of primers specific for the N gene, as well as sequence analysis of a part of the S gene of one isolate, confirmed the authenticity of the virus isolated as TSWV. Host range studies showed differences in susceptibility, especially among species belonging to the Leguminosae and Cucurbitaceae. The species Beloporone guttata and Coleus sp. are reported for the first time as hosts of the virus, whereas Solanum melongena, Celosia cristata, Dianthus chinensis, Stephanotis floribunda and Catharanthus roseus were identified as new hosts of TSWV in Greece.     

Agrobacterium-mediated transformation of cultivated tomato with construct carrying the nucleoprotein (N) gene from tomato spotted wilt virus (TSWV)

Significant yield losses in commercial tomato production caused by tomato spotted wilt virus (TSWV) are the reason why we have undertaken studies on resistance to this pathogen. One of the possible sources of resistance can be the incorporation of the nucleoprotein N viral gene by Agrobacterium transformation. The N gene was introduced into three Lycopersicon esculentum forms. Out of the total of 3044 cotyledon explants 14.7% regenerated shoots, but only a few were rooted on medium containing kanamycin. The preliminary analysis indicated that 18 plants are putative transformants.     

Specificity of accumulation and transmission of tomato spotted wilt virus (TSWV) in two genera, Frankliniella and Thrips (Thysanoptera: Thripidae)

The accumulation and transmission of tomato spotted wilt virus (TSWV) was examined in second instar larvae and adults of two thrips genera, Frankliniella and Thrips. The species tested were F. occidentalis (Pergande), F. intonsa (Trybom), T. tabaciLindeman, T. setosus Moulton, T. palmi Karny and T. hawaiiensis (Morgan). In a standard petunia leaf disc assay, the efficiencies of TSWV transmission by two species of Frankliniella were higher than those of any Thrips species in the adult stage. A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) showed that large amounts of the TSWV-nucleocapsid (N) protein were present in the ELISA-positive larvae of each species, with the exception of T. palmi. The ELISA titre of and the proportion of virus-infected individuals of the two Frankliniella species increased or did not significantly change from the larval to the adult stages, whereas those of the four Thrips species decreased significantly. These results show that the specificity of virus transmission by adult thrips is probably affected by the amount of viral N protein accumulation in the adults and that the accumulation pattern from the larval to the adult stages is in between the two genera tested in the present study.     

番茄斑萎病毒运动蛋白NSm在植物细胞和昆虫细胞内的定位

[目的]研究番茄斑萎病毒(Tomato spotted wilt virus,TSWV)运动蛋白NSm的细胞定位.[方法]将NSm基因融合GFP后构建到植物双元表达载体pCHF3中,农杆菌介导浸润本氏烟叶片,同时将融合GFP的NSm基因利用Bac-to-Bac杆状病毒表达体系转染昆虫Tn细胞.在激光共聚焦显微镜下观察NSm-GFP在烟草表皮细胞和昆虫Tn细胞中的定位情况.[结果]观察发现与单独表达GFP在细胞壁周围和细胞核处均匀分布不同,NSm-GFP融合蛋白会在植物细胞内扩散,能够在细胞壁边缘定位,并且在胞间连丝处呈不连续的绿色荧光小点,偶尔成对出现在相邻的两个细胞之间;NSm蛋白在昆虫Tn细胞表面产生数量众多的管状结构并向外延伸.[结论]研究结果表明NSm能够特异性定位在植物细胞的胞间连丝处,并能在昆虫Tn细胞内表达,在细胞表面产生运动蛋白小管.     

Symptom development and distribution of Tomato spotted wilt virus in flue-cured tobacco

Tomato spotted wilt virus (TSWV) is an economically important viral pathogen of flue‐cured tobacco, Nicotiana tabacum. Disease development and in planta distribution of TSWV were studied following mechanical inoculation of cv. K326 at various stages of growth. The effect of plant age on the disease development, distribution of symptoms and TSWV were studied by inoculating plants in five age groups, 40, 60, 75, 95 and 100 days after sowing (DAS). The plant age at the time of infection had no significant influence on the incidence of localised infection; however, it had a significant effect on the development of systemic symptoms and distribution of TSWV in the plant. In a higher proportion of plants (89.2%), no systemic symptoms developed when plants were inoculated at 60–100 DAS. However, 90% of plants became systemically infected when plants were inoculated at 40 DAS. The systemic symptom expression was severe and distributed in all the leaves in 40‐DAS plants, whereas in 60‐ to 100‐DAS plants, it was erratic and restricted only to a few upper leaves. Results show that plant age is an important factor for TSWV infection of tobacco and mature tobacco plants significantly reduced the systemic development of the disease.     

Monoclonal Antibodies against the Recombinant Nucleocapsid Protein of Tomato spotted wilt virus and its Application in Virus Detection

Tomato spotted wilt virus (TSWV) is the type member of the tospovirus genus and causes significant losses in a wide range of economically important ornamental and vegetable crops worldwide. The nucleocapsid gene, located on the ambisense S RNA segment of TSWV was expressed in Escherichia coli using pET-32a as vector and correct expression of recombinant protein was confirmed by Western blot using an anti-TSWV monoclonal antibody (MAb). The recombinant protein was purified using Ni-NTA agarose and the purified protein was used for the production of MAbs. Three murine MAbs against the recombinant nucleocapsid protein were produced. Triple antibody sandwich enzyme-linked immunosorbent assay and immunocapture RT-PCR methods were then established for reliable and efficient detection of TSWV using the produced MAbs.     

Suppressing spread of Tomato spotted wilt virus by drenching infected source or healthy recipient plants with neonicotinoid insecticides to control thrips vectors

Field experiments were done to determine whether drenching plants with two systemically active neonicotinoid insecticides, thiamethoxam and imidacloprid, suppresses spread of Tomato spotted wilt virus (TSWV) by thrips vectors. Separate treatments to TSWV ‘infector’ tomato (source) and healthy lettuce (recipient) plants provided information on the relative importance of targeting control at virus acquisition by nymphs versus virus transmission to healthy plants by adults. Drenches were applied either to seedlings just before transplanting or to soil around plants. The thrips vectors recorded were Frankliniella occidentalis, F. schultzei and Thrips tabaci, but F. schultzei and T. tabaci predominated. Overall ratios of external to internal TSWV spread into and within plots without insecticide ranged from 1 : 2.3 to 1 : 2.8 between field experiments. Applying thiamethoxam as a soil drench to both young source plants and recipient seedling transplants suppressed TSWV incidence by 86%, while such application to either young source or recipient seedlings diminished incidence by 67–70%. When thiamethoxam was applied either as a soil drench to old source plants and concurrently as a seedling drench to recipient plants or as a seedling drench to recipient plants alone, incidence was suppressed by 65–73% and 54–73%, respectively. Thiamethoxam applied as a soil drench to old source plants diminished incidence by only 33% or not significantly. Imidacloprid applied either as a soil drench to old source plants and concurently as a seedling drench or as a seedling drench alone, suppressed TSWV incidence by 90–92% and 80% respectively. Although adult vector thrips and nymph numbers were low, fewer adults and/or nymphs were sometimes recorded due to insecticide application. Drenching healthy seedlings with neonicotinoid insecticides just before transplanting can be an effective chemical control measure to include in integrated disease management strategies to suppress TSWV epidemics in short‐duration crops like lettuce.     

Transmission of Tomato spotted wilt virus isolates able and unable to overcome tomato or pepper resistance by its vector Frankliniella occidentalis

Tomato spotted wilt virus (TSWV) causes serious diseases of many economically important crops. Disease control has been achieved by breeding tomato and pepper cultivars with the resistance genes Sw‐5 and Tsw, respectively. However, TSWV isolates overcoming these genetic resistances have appeared in several countries. To evaluate the risk of spread of these resistance‐breaking isolates, we tested their ability of transmission by the main vector of TSWV, the thrips Frankliniella occidentalis. We compared the transmission rate by thrips of six TSWV isolates of different biotype (able or unable to overcome this resistance in pepper and tomato), and with divergent genotype (A and B). Our results indicate that the transmission rate was related to the amount of virus accumulated in thrips but not to virus accumulation in the source plants on which thrips acquired the virus. No correlation was found between transmission efficiency by thrips and the genotype or between transmission efficiency and the ability of overcoming both resistances. This result suggests that resistance‐breaking isolates have the same potential to be transmitted as the isolates unable to infect resistant tomato and pepper cultivars.     

Overcoming host- and pathogen-mediated resistance in tomato and tobacco maps to the M RNA of Tomato spotted wilt virus

A viral genetic system was used to map the determinants of the ability of Tomato spotted wilt virus (TSWV) to overcome the R gene (Sw-5) in tomato and the resistance conferred by the nucleocapsid gene of TSWV (N gene) in tobacco. A complete set of reassortant genotypes was generated from TSWV isolates A and D. TSWV-A was able to overcome the Sw-5 gene in tomato and the TSWV N gene in tobacco, whereas TSWV-D was repressed by both forms of resistance. The ability to overcome both forms of resistance was associated with the M RNA segment of TSWV-A (M(A)). Overcoming the Sw-5 gene was linked solely to the presence of M(A), and the ability of M(A) to overcome the TSWV N gene was modified by the L RNA and the S RNA of TSWV-A, which is consistent with previous reports that suggest that the nucleocapsid gene is not the primary determinant for overcoming the nucleocapsid-mediated resistance. Sequence analysis of the M RNA segment of TSWV-A, -D, and the type isolate BR-01 revealed multiple differences in the coding and noncoding regions, which prevented identification of the resistance-breaking nucleotide sequences.