Expression and characterization of a soluble form of tomato spotted wilt virus glycoprotein GN

Tomato spotted wilt virus (TSWV), a member of the Tospovirus genus within the Bunyaviridae, is an economically important plant pathogen with a worldwide distribution. TSWV is transmitted to plants via thrips (Thysanoptera: Thripidae), which transmit the virus in a persistent propagative manner. The envelope glycoproteins, G(N) and G(C), are critical for the infection of thrips, but they are not required for the initial infection of plants. Thus, it is assumed that the envelope glycoproteins play important roles in the entry of TSWV into the insect midgut, the first site of infection. To directly test the hypothesis that G(N) plays a role in TSWV acquisition by thrips, we expressed and purified a soluble, recombinant form of the G(N) protein (G(N)-S). The expression of G(N)-S allowed us to examine the function of G(N) in the absence of other viral proteins. We detected specific binding to thrips midguts when purified G(N)-S was fed to thrips in an in vivo binding assay. The TSWV nucleocapsid protein and human cytomegalovirus glycoprotein B did not bind to thrips midguts, indicating that the G(N)-S-thrips midgut interaction is specific. TSWV acquisition inhibition assays revealed that thrips that were concomitantly fed purified TSWV and G(N)-S had reduced amounts of virus in their midguts compared to thrips that were fed TSWV only. Our findings that G(N)-S binds to larval thrips guts and decreases TSWV acquisition provide evidence that G(N) may serve as a viral ligand that mediates the attachment of TSWV to receptors displayed on the epithelial cells of the thrips midgut.     

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.     

Midgut infection by tomato spotted wilt virus and vector incompetence of Frankliniella tritici

Abstract:  The mechanism leading to vector competence of thrips species to transmit tomato spotted wilt virus (TSWV) is not well characterized. We investigated the interaction of TSWV and the non-vector species Frankliniella tritici . A monoclonal antibody to the non-structural protein (NSs) of the TSWV was used to detect TSWV replication within the thrips by immunofluorescence microscopy and enzyme-linked immonosorbent assay (ELISA). TSWV was acquired by F. tritici , replicated and moved within the alimentary canal of F. tritici similar to a known vector of TSWV, Frankliniella occidentalis . However, virus was not found in the salivary glands of F. tritici , which is a prerequisite to virus transmission. Thus, movement to the salivary glands may determine vector incompetence of F. tritici .     

Effects of tomato spotted wilt virus isolates, host plants, and temperature on survival, size, and development time of Frankliniella occidentalis

Tomato spotted wilt virus (TSWV) replicates in both its plant hosts and its thrips vectors. Replication of TSWV within thrips suggests the potential for pathological effects that could affect the fitness of its vectors directly, whereas infection of the plant may alter its suitability as a host for thrips development. This study was undertaken to examine the influence of TSWV isolate, host plant, and temperature on potential direct and host-mediated effects of virus infection of the thrips and the plant on Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), an important vector of TSWV. Neonate F. occidentalis were reared to adult eclosion on excised foliage of Datura stramonium (L.) (Solanaceae) or Emilia sonchifolia (L.) (Compositae) infected with either the CFL or RG2 isolate of TSWV, or not infected. Effects of the TSWV isolates and host plants on thrips were measured at 18.3, 23.9, and 29.4 °C. Results demonstrate significantly improved survival and a small but significant decrease in development time of F. occidentalis on TSWV-infected plants. These effects resulted from the combined influence of the direct effects of the virus on infected thrips and plant-mediated effects resulting from virus infection of the thrips’ host plant. Our results extend previous findings and help to explain inconsistencies among previously published reports by demonstrating that the manifestation and magnitude of effects of TSWV on F. occidentalis are dependent on host plant, virus isolate, and temperature.     

Antagonistic plant defense system regulated by phytohormones assists interactions among vector insect, thrips and a tospovirus

The western flower thrips (Frankliniella occidentalis) is a polyphagous herbivore that causes serious damage to many agricultural plants. In addition to causing feeding damage, it is also a vector insect that transmits tospoviruses such as Tomato spotted wilt virus (TSWV). We previously reported that thrips feeding on plants induces a jasmonate (JA)-regulated plant defense, which negatively affects both the performance and preference (i.e. host plant attractiveness) of the thrips. The antagonistic interaction between a JA-regulated plant defense and a salicylic acid (SA)-regulated plant defense is well known. Here we report that TSWV infection allows thrips to feed heavily and multiply on Arabidopsis plants. TSWV infection elevated SA contents and induced SA-regulated gene expression in the plants. On the other hand, TSWV infection decreased the level of JA-regulated gene expression induced by thrips feeding. Importantly, we also demonstrated that thrips significantly preferred TSWV-infected plants to uninfected plants. In JA-insensitive coi1-1 mutants, however, thrips did not show a preference for TSWV-infected plants. In addition, SA application to wild-type plants increased their attractiveness to thrips. Our results suggest the following mechanism: TSWV infection suppresses the anti-herbivore response in plants and attracts its vector, thrips, to virus-infected plants by exploiting the antagonistic SA-JA plant defense systems.     

Tactics for management of thrips (Thysanoptera: Thripidae) and tomato spotted wilt virus in tomato

Four studies were conducted in Georgia during spring 1999, 2000, 2001, and 2002 to evaluate various management tactics for reducing thrips and thrips-vectored tomato spotted wilt virus (TSWV) in tomato and their interactions relative to fruit yield. Populations of thrips vectors of TSWV, Frankliniella occidentalis (Pergande) and Frankliniella fusca (Hinds), were determined using flower and sticky trap samples. The management practices evaluated were host plant resistance, insecticide treatments, and silver or metallic reflective mulch. Averaged over all tests, the TSWV-resistant tomato 'BHN444' on silver mulch treatment had the largest effect in terms of reducing thrips and spotted wilt and increasing marketable yield. Of the insecticide treatments tested, the imidacloprid soil treatment followed by early applications of a thrips-effective foliar insecticide treatment provided significant increase in yield over other treatments. Tomato yield was negatively correlated with the number of F. fusca and percentage of TSWV incidence. F. occidentalis per blossom was positively correlated with percentage of TSWV incidence, but not with yield. No significant interactions were observed between cultivar reflective mulch main plot treatments and insecticide subplot treatments; thus, treatment seemed to be additive in reducing the economic impact of thrips-vectored TSWV. Control tactics that manage thrips early in the growing season significantly increased tomato yield in years when the incidence of TSWV was high (>17%).     

番茄斑萎病毒对多杀菌素抗性西花蓟马发育繁殖和药剂敏感性的影响

【目的】西花蓟马Frankliniella occidentalis (Pergande)是重要的入侵害虫,是番茄斑萎病毒（TSWV）最有效的传播媒介,TSWV对西花蓟马的生长发育有一定的影响。多杀菌素是防治西花蓟马最有效的药剂之一,但已有田间西花蓟马对多杀菌素产生抗药性的报道。TSWV对抗性西花蓟马是否也有影响及程度如何尚不清楚。本研究通过对此问题进行深入研究,以期为进一步了解TSWV对西花蓟马的影响提供依据。【方法】应用特定年龄-龄期及两性生命表的方法,研究用番茄斑萎病毒处理和未处理的多杀菌素抗性和敏感西花蓟马种群的生物学特性;用叶管药膜法测定不同处理种群对3种药剂（多杀菌素、甲氨基阿维菌素苯甲酸盐和虫螨腈）的敏感性变化。【结果】对于抗性品系,TSWV处理后西花蓟马的发育历期缩短,雌成虫寿命和产卵量略高,但与对照组差异不显著(P＞0.05),内禀增长率（r）和净生殖率（R₀）分别为0.0433 d^-1和2.210,显著高于对照组（分别为0.0356 d^-1和1.972）（P ≤ 0.001）。对于敏感品系,TSWV处理后西花蓟马的发育历期缩短,雌雄成虫寿命均显著延长（P ≤ 0.001）,产卵量也略有提高,R₀为4.125,显著高于对照组（3.979）（P ≤ 0.001）。TSWV处理后敏感和抗性西花蓟马对多杀菌素的敏感性没有发生明显变化,对甲氨基阿维菌素苯甲酸盐和虫螨腈的敏感性显著降低。【结论】番茄斑萎病毒对多杀菌素敏感和抗性西花蓟马均有直接有利影响,病毒处理的西花蓟马发育历期缩短,繁殖能力增强,成虫寿命延长,对药剂的敏感性降低。     

Влнянне характера эаселення ноля капустноii совкоii(Barathra brassicae L.) эффективноеть трихограммы (Trichogramma evanescens Westw.) (Lepidoptera,Noctuidae; Hymenoptera,Trichogrammatidae)

TSWV belongs to the genus Tospovirus which was established in the family Bunyaviridae, a family of animal viruses. Besides TSWV, Impatiens necrotic spot virus (INSV) and ground nut bud necrosis virus (GBNV) were established as different Tospovirus species. Tospoviruses have quasispherical particles of 85 nm diametre which are surrounded by a membrane and contain 3 RNA species and 4 structural proteins. In Tospovirus infected plant cells virions were detected in cavaties of the endoplasmatic reticulum and additionally amorphous electron dense material accumulates in infected cells. Defective forms of TSWV lack the ability to form complete virus particles. TSWV is the only plant pathogenic virus that is transmitted by thrips which transmit the virus with different efficiency. The virus has an extensive plant host range of more than 360 different species. The developing symptoms depend on the Tospovirus species, the virulence of the virus strains and the environmental conditions.

Based on the reaction of TSWV isolates with N‐specific polyclonal antisera, 3 serogroups were established. The most frequently used technique for serologically based diagnosis of Tospoviruses is DAS ELISA with N‐specific or preadsorbed antisera against complete virus. For TSWV epidemiology distinct weeds and cultural host plants play an important role for the survival of virus and vector. Breeding for resistance is the most important preventive measure of control.     

Role of insecticides in reducing thrips injury to plants and incidence of tomato spotted wilt virus in Virginia market-type peanut

Tomato spotted wilt virus (family Bunyaviridae, genus Tospovirus, TSWV), transmitted by many thrips species, is a devastating pathogen of peanut, Arachis hypogaea L. TSWV has become a serious problem in the Virginia/Carolina peanut-growing region of the United States. During 2002, TSWV was present in 47% of the North Carolina hectarage and caused a 5% yield reduction in Virginia. Factors influencing levels of TSWV in runner market-type peanut cultivars, which are primarily grown in Alabama, Flordia, Georgia, and Texas, have been integrated into an advisory to help those peanut growers reduce losses. An advisory based on the southeast runner market-type version is currently under development for virginia market-type peanut cultivars that are grown primarily in the Virginia/ Carolina region. A version based on preliminary field experiments was released in 2003. One factor used in both advisories relates to insecticide use to reduce the vector populations and disease incidence. This research elucidated the influence of insecticides on thrips populations, thrips plant injury, incidence of TSWV, and pod yield in virginia market-type peanut. Eight field trials from 2003 to 2005 were conducted at two locations. In-furrow application of aldicarb and phorate resulted in significant levels of thrips control, significant reductions in thrips injury to seedlings, reduced incidence of TSWV, and significant increases in pod yield. Foliar application of acephate after aldicarb or phorate applied in the seed furrow further reduced thrips plant injury and incidence of TSWV and improved yield. These findings will be used to improve the current virginia market-type TSWV advisory.     

Impact of early-season thrips management on reducing the risks of spotted wilt virus and suppressing aphid populations in Flue-cured tobacco

The influence of tray drench (TD) treatments, with and without foliar applications of the plant activator acibenzolar-S-methyl (Actigard), was examined in replicated field plots in 2000--2002. TD treatments of Actigard, imidacloprid (Admire), and these two products combined had little effect on seasonal mean thrips populations; however, thrips densities were lower in the Admire-treated plots at 4 and 5 wk after transplanting. Actigard and Admire TD treatments significantly reduced the seasonal incidence of tomato spotted wilt virus (TSWV) symptomatic plants in 2 yr in the study. The combination of both products was better in reducing TSWV than Actigard alone. Three early-season foliar sprays of Actigard had no effect on thrips population densities, but they did reduce TSWV incidence. The tobacco thrips, Frankliniella fusca (Hinds), comprised 92-95% of the thrips complex each year. Other thrips collected on tobacco foliage at very low densities included Haplothrips spp., Chirothrips spp., Limothrips cerealium (Haliday), other Frankliniella spp. and other unidentified species. Using nonstructural TSWV protein enzyme-linked immunosorbent assay, 1.5-2.3% of the F. fusca tested positive for nonstructural TSWV protein. Cured yields were higher in the TD treatments and the Actigard foliar treatments in the years with high TSWV in the untreated plots. The TD treatments and foliar Actigard had little impact on plant height or grade index; however, TD treatments with Admire had low tobacco aphid, Myzus nicotianae Blackman, populations through 10 wk after transplanting. The early-season Actigard and Admire treatment options are management decisions that can effectively reduce the risks of TSWV incidence in flue-cured tobacco.     

Differential Susceptibilities between Leaf Disks and Plants in the Transmission of Tomato Spotted Wilt Virus by Frankliniella occidentalis to TSWV Hosts and Transgenic Plants

The efficiency by which tomato spotted wilt virus (TSWV) was transmitted to plants and leaf disks by the vector Frankliniella occidentalis , was analysed. The virus was efficiently transmitted to Datura stramonium, Impatiens sp. and tobacco plants, i.e. 60–100% of the plants became infected when 1–3 viruliferous thrips were confined per plant for a period of 3 days. However, lettuceexhibited a lower susceptibility since only 25% of the test plants were infected when challenged by 10 viruliferous thrips per plant for 3 days. In contrast, complete resistance was found when transgenic tobacco plants, expressing the nucleocapsid protein of TSWV, were challenged with up to 10 viruliferous thrips per plant, whereas all non-transgenic control plants were infected when 5 viruliferous thrips per plant were used. To improve and accelerate the tramission studies, the applicability of leaf disks in these studies was tested. Leaf disks of 16 different plant species appeared to be highly susceptible. Infection ratings ranging from 51.6 to 95.0% were obtained when one viruliferous adult was placed singly on these leaf disks for a period for 24 h. The leaf disk assay was also employed to screen resistance of transgenic plants expressing the nucleocapsid protein of TSWV. One transgenic tomato line displayed complete immunity whereas a second line appeared to be susceptible. For the transgenic tobacco line, positive ELISA reactions were found for a few leaf disks (7.5%) suggesting that some virus replication did occur. However, the ELISA readings for these disks were significantly lower than those for leaf disks of non-transgenic controls. Finally, the significance of the use of the leaf disks and test plants in virus-vector studies is discussed.     

Effects of tomato spotted wilt virus (TSWV) isolates, host plants, and temperature on survival, size, and development time of Frankliniella fusca

The effects of different isolates of the tomato spotted wilt tospovirus (TSWV), host plants, and temperatures on Frankliniella fusca (Hinds) (Thysanoptera: Thripidae), the most important vector of TSWV in North Carolina, were measured in the laboratory. Thrips were reared at either 18.3, 23.9, or 29.4 °C until adult eclosion on excised leaves of Datura stramonium L. or Emilia sonchifolia (L.). Plants were either infected with the TSWV isolates CFL or RG2, or left uninfected (control). The results revealed a positive relationship between larval survival and temperature, regardless of host plant or TSWV isolate. Both survival to adult and percentage transmission of TSWV by F. fusca were significantly affected by the interaction between host plant and TSWV isolate. The consequence of this interaction was that the cohort‐based percentage transmission from infected E. sonchifolia plants for CFL was 1.3‐fold greater than that of RG2, whereas the percentage transmission from infected D. stramonium plants for RG2 was twice that of CFL. Both host plant and TSWV isolates showed significant effects on thrips development time to adult and head capsule width of adult thrips, as well as on the incidence of thrips infection with TSWV. The infection status of these thrips was determined by ELISA for the NSs viral protein. Infected thrips reared on infected host foliage took longer to develop to adult and were smaller than non‐infected thrips which had also been reared on infected host foliage, demonstrating a direct effect of the TSWV on thrips. However, non‐infected thrips reared on non‐infected leaves took longer to develop than non‐infected thrips reared on infected leaves, suggesting an effect of the plant tissue on thrips. In addition, adult thrips reared on TSWV‐infected D. stramonium at 29.4 °C developed smaller head capsules than thrips developing on infected foliage at lower temperatures and on non‐infected leaves of D. stramonium or E. sonchifolia. Both TSWV isolates and host plants differentially affected females more than males. In conclusion, both the infection of thrips by TSWV and TSWV‐mediated changes in host plant quality were found to have significant biological effects on F. fusca.     

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.     

Preliminary evidence of recovery from Tomato spotted wilt virus infection in Frankliniella occidentalis individuals

In this study we analysed the ability of individual thrips to transmit Tomato spotted wilt virus (TSWV) in a population of Frankliniella occidentalis over their lifespan as adults (about 10 days). In three experiments a total of 636 thrips were individually tested for their transmission capacity through leaf disc assays using four inoculation access periods (IAPs). Almost half of the transmitting thrips maintained the capacity to infect leaf discs in each of the four IAPs, confirming the persistent propagative nature of the transmission modality. Nevertheless, a relevant number of thrips (9.25% of transmitter thrips) was able to transmit in the early phases of their adult life (for the first two IAPs), but did not transmit the virus for the remainder of their lifetime. We compared the virus titer of these individuals at the end of the fourth IAP with that of individuals that maintained transmission ability in the four IAPs and showed a statistically significant difference. This difference could be evidence for recovery from TSWV infection in individual thrips.     

Specific Insect-Virus Interactions Are Responsible for Variation in Competency of Different Thrips tabaci Isolines to Transmit Different Tomato Spotted Wilt Virus Isolates

Local adaptation between sympatric host and parasite populations driven by vector genetics appears to be a factor that influences dynamics of disease epidemics and evolution of insect-vectored viruses. Although T. tabaci is the primary vector of Tomato spotted wilt virus (TSWV) in some areas of the world, it is not an important vector of this economically important plant virus in many areas where it occurs. Previous studies suggest that genetic variation of thrips populations, virus isolates, or both are important factors underlying the localized importance of this species as a vector of TSWV. This study was undertaken to quantify variation in transmissibility of TSWV isolates by T. tabaci, in the ability of T. tabaci to transmit isolates of TSWV, and to examine the possibility that genetic interactions and local adaptation contribute to the localized nature of this species as a vector of TSWV. Isofemale lines of Thrips tabaci from multiple locations were tested for their ability to transmit multiple TSWV isolates collected at the same and different locations as the thrips. Results revealed that the probability of an isofemale line transmitting TSWV varied among virus isolates, and the probability of an isolate being transmitted varied among isofemale lines. These results indicate that the interaction of T. tabaci and TSWV isolate genetic determinants underlie successful transmission of TSWV by T. tabaci. Further analysis revealed sympatric vector-virus pairing resulted in higher transmission than allopatric pairing, which suggests that local adaptation is occurring between T. tabaci and TSWV isolates.     

Weed species in tomato production and their role as alternate hosts of Tomato spotted wilt virus and its vector Frankliniella occidentalis

Tomato spotted wilt virus (TSWV) is an important plant virus that infects a wide range of hosts including weeds making its management difficult. A survey was undertaken to establish the occurrence of weed species in tomato production systems in Kenya and their role as hosts of TSWV and its vectors. Selected weed species were further evaluated for their reaction to TSWV, transmission efficiency by Frankliniella occidentalis and ability to support thrips reproduction. Of the 43 weed species identified in the field, 29 species had been reported as hosts of TSWV, two were non‐hosts and 11 had no record of their status. Among the more common species, Amaranthus hybridus, Solanum nigrum, Tagetes minuta and Datura stramonium were susceptible to the virus and supported high levels of thrips reproduction. The TSWV could not be transmitted to Galinsoga parviflora and Sonchus oleraceus by F. occidentalis despite them being highly susceptible in mechanical transmission tests. There was a significant correlation between feeding damage and number of larvae of F. occidentalis on different weeds. Occurrence of weeds that support thrips reproduction and are good hosts of TSWV is a clear indicator of their role in epidemiology and the importance of their management for disease control.     

Propagation of tomato spotted wilt virus in Frankliniella occidentalis does neither result in pathological effects nor in transovarial passage of the virus

The effect of tomato spotted wilt virus (TSWV) on Frankliniella occidentalis Pergande (Thysanoptera; Thripidae) following a 6-hour acquisition access period on infected plants was investigated. No statistically significant differences were observed among viruliferous, non-viruliferous and control thrips with respect to developmental time, reproduction rate and survival. Thrips larvae, exposed or non-exposed to TSWV, developed from egg to adult in 13.1 and 13.2 days, respectively. Exposed females produced an average of 28.3 larvae whereas control thrips produced 22.3 larvae and longevity was 13.4 and 12.5 days, respectively. None of these values were significantly different. Population reproductive statistics, net reproductive rate (R ₀), mean generation time (T) and intrinsic rate of increase (r _m) were calculated from the life fertility tables. R ₀ and r _m were higher for viruliferous thrips as compared to non-viruliferous and non-exposed thrips. Virus transmission studies revealed that viruliferous thrips were able to transmit virus until death and that TSWV was not transovarially transmitted.