Acquisition of Tomato spotted wilt virus by Adults of Two Thrips Species

ABSTRACT Only larval thrips that acquire Tomato spotted wilt virus (TSWV), or adults derived from such larvae, transmit the virus. Nonviruliferous adults can ingest virus particles while feeding on TSWV-infected plants, but such adult thrips have not been shown to transmit TSWV. Immunofluorescence microscopy was used to show that thrips 1, 5, 10, and 20 days after adult emergence (DAE) fed on TSWV-infected plants acquired TSWV with virus replication and accumulation occurring in both epithelial and muscle cells of Frankliniella fusca (tobacco thrips [TT]) and F. occidentalis (western flower thrips [WFT]), as indicated by immunodetection of the nonstructural (NSs) protein encoded by the small RNA and the nucleocapsid (N) protein, respectively. Adult WFT acquired TSWV more efficiently than TT. There was no significant effect of insect age on TSWV acquisition by TT. In contrast, acquisition by adult WFT at 1 and 5 DAE was higher than acquisition at 10 and 20 DAE. Subsequent transmission competence of adult cohorts was studied by vector transmission assays. All adult thrips tested that had an acquisition access period as an adult were unable to transmit the virus. These results indicate the susceptibility of adult TT and WFT to infection of midgut cells by TSWV and subsequent virus replication and confirm earlier studies that adult thrips that feed on virus-infected plants do not transmit the virus. The role of a tissue barrier in TSWV movement and infection from midgut muscle cells to the salivary glands is discussed.     

The Efficiency by Which Thrips tabaci Populations Transmit Tomato spotted wilt virus Depends on Their Host Preference and Reproductive Strategy

ABSTRACT Arrhenotokous and thelytokous populations of Thrips tabaci from tobacco or leek plants were evaluated for their ability to transmit Tomato spotted wilt virus (TSWV) and for their host preference. Transmission efficiencies were comparatively studied using leaf disks of Petunia hybrida, Datura stramonium, and Nicotiana tabacum cv. Basmas. Adults of arrhenotokous populations collected on infected tobacco plants in the field were efficient transmitters (up to 48.5% transmission) and remained so when maintained on tobacco for several generations. Arrhenotokous T. tabacipopulations from leek plants were poor transmitters (up to 3.1% transmission), whereas no transmission was obtained with thelytokous populations from leek. All populations could infest leek, however none of the arrhenotokous and thelytokous populations from leek plants was able to infest tobacco. TSWV could be acquired by both first and second larval instars of a T. tabacipopulation from tobacco. However, the transmission by adults decreased with the age at which the virus was acquired by larvae. The highest efficiencies (61% of males and 51% of females transmitted) were obtained when newborn (0- to 24-h old) larvae acquired the virus. The majority of thrips started to transmit after becoming adult and rates were positively correlated with the temperature at which the thrips were kept. The median latent period values found for adults decreased with increasing temperature. The median acquisition access period (AAP50) of the population was 41 min, whereas the AAP(50) was 65 min for males and 35 min for females. The median inoculation access period of males was 246 and 365 min on tobacco and petunia, respectively, and 96 and 345 min for females. The results show that T. tabaci forms a complex in terms of host preference, reproductive strategy, and ability to transmit TSWV. The transmission parameters show that the thrips of arrhenotokous populations infesting tobacco are highly efficient vectors.     

Restricted Spread of Tomato spotted wilt virus in Thrips-Resistant Pepper

ABSTRACT Spread of Tomato spotted wilt virus (TSWV) and population development of its vector Frankliniella occidentalis were studied on the pepper accessions CPRO-1 and Pikante Reuzen, which are resistant and susceptible to thrips, respectively. Viruliferous thrips were released on plants of each accession (nonchoice tests) or on plants in a 1:1 mixture of both accessions (choice tests) in small cages containing 8 or 16 plants. Significantly fewer CPRO-1 plants became infected in the primary infection phase in both tests. In the nonchoice test, virus infection of the resistant plants did not increase after the initial infection, but all plants eventually became infected when mixtures of both cultivars were challenged in the secondary infection phase. Secondary spread of TSWV from an infected resistant or susceptible source plant was significantly slower to resistant plants than to susceptible plants, independent of source plant phenotype. The restricted introduction and spread of TSWV in the thrips-resistant cultivar was confirmed in a large-scale greenhouse experiment. The restricted and delayed TSWV spread to plants of the resistant accession in both the cage and the greenhouse experiment was explained by impeded thrips population development. The results obtained indicate that thrips resistance may provide a significant protection to TSWV infection, even when the crop is fully susceptible to the virus.     

Assessing the susceptibility of chrysanthemum cultivars to tomato spotted wilt virus

Three methods were compared to assess the susceptibility of vegetatively propagated chrysanthemum to tomato spotted wilt tospovirus (TSWV): mechanical and thrips-mediated inoculation of whole plants, and a leaf-disc assay. As symptom expression was often poor or even absent, TSWV infections and subsequent susceptibility to TSWV were determined by ELISA. All 15 chrysanthemum cultivars tested were susceptible to TSWV, irrespective of their degree of vector resistance (based on feeding-scar damage rates). Thrips-mediated inoculation using different numbers of thrips revealed that 100% infection was obtained when plants were challenged by six thrips per plant, whereas 80 and over 50%, respectively, of the plants became infected when inoculated by a single male or female thrips. However, false negatives were scored even after intensive sampling because of erratic, cultivar-specific and time-dependent virus distribution after inoculation in the plants. Labour-intensive samplings and long incubation periods could be overcome by a readily applicable leaf-disc assay. This assay was as reliable as thrips-mediated inoculation of whole plants, and its use is therefore favoured to assess chrysanthemum cultivars for TSWV susceptibility.     

Detection of tomato spotted wilt virus and transmission by Frankliniella occidentalis in France

Tomato spotted wilt virus (TSWV) was isolated from pepper, tomato, eggplant, broad bean, lettuce, basil, chrysanthemum, aster, New Guinea impatiens, anemone and gloxinia plants. Virus identification was based on host range, vector transmission, serology and electron microscopy. TSWV was readily detected by ELISA in naturally or artificially infected cultivated or weed plants. The virus was also detected in individual F. occidentalis thrips. The spread of TSWV in vegetable and ornamental plants in greenhouses and/or in the open is related to the close relationship of the virus with the vector.     

Tomato spotted wilt virus Infection Improves Host Suitability for Its Vector Frankliniella occidentalis

ABSTRACT The effect of Tomato spotted wilt virus (TSWV) infection on plant attractiveness for the western flower thrips (Frankliniella occidentalis) was studied. Significantly more thrips were recovered on infected than were recovered on noninfected pepper (Capsicum annuum) plants in different preference tests. In addition, more offspring were produced on the virus-infected pepper plants, and this effect also was found for TSWV-infected Datura stramonium. Thrips behavior was minimally influenced by TSWV-infection of host plants with only a slight preference for feeding on infected plants. Offspring development was positively affected since larvae hatched earlier from eggs and subsequently pupated faster on TSWV-infected plants. These results show a mutualistic relationship between F. occidentalis and TSWV.     

Effect of temperature, vector life stage, and plant access period on transmission of banana bunchy top virus to banana

The study of the transmission biology of insect-borne plant viruses is important to develop disease control practices. We characterized the transmission of a nanovirus, Banana bunchy top virus (BBTV), by its aphid vector Pentalonia nigronervosa Coquerel (Hemiptera, Aphididae) with respect to temperature, vector life stage, and plant access time. Adult aphids transmitted BBTV more efficiently than third instar nymphs at all temperatures tested. Adult aphids transmitted the virus more efficiently at 25 and 30 degrees C than at 20 degrees C, but temperature had no impact on transmission efficiency by nymphs. By decoupling the relationship between temperature and aphid BBTV acquisition or inoculation, we determined that temperature affected inoculation events more strongly than acquisition. Longer plant access periods increased viral acquisition and inoculation efficiencies in a range of 60 min to 24 h. Both BBTV acquisition and inoculation efficiencies peaked after 18 h of plant access period. We also show that BBTV transmission by P. nigronervosa requires a latent period. Our results demonstrate that vector transmission of BBTV is affected by temperature, vector life stage, and plant access period.     

Binding of Tomato Spotted Wilt Virus to a 94-kDa Thrips Protein

ABSTRACT Using protein blot assays, a 94-kDa thrips protein was identified that exhibited specific binding to tomato spotted wilt virus (TSWV) particles. Renaturation of the 94-kDa protein, which is conserved among the two major vector species of TSWV, Frankliniella occidentalis and Thrips tabaci, was crucial for its virus-binding properties, whereas under the same conditions no specific binding was observed with aphid (Myzus persicae) proteins. The 94-kDa protein species was present in all developmental stages of both vectoring thrips, whereas it was present mainly in the adult stage of a nonvectoring thrips species, Parthenothrips dracenae. Using antibodies against the different TSWV structural proteins, the G2 envelope glycoprotein was identified as the viral determinant involved. Because the virus-binding protein is present throughout the thrips body, but not in the gut, it may represent a receptor protein involved during circulation of the virus through its vector but probably not during viral uptake in the midgut.     

Effects of Latent Infection of Stock Plants and Abundance of Thrips on the Occurrence of <Emphasis Type="Italic">Tomato spotted wilt virus </Emphasis>in Chrysanthemum Fields

DAS-ELISA proved to be reliable enough to detect a latent infection by Tomato spotted wilt virus (TSWV) in asymptomatic stock plants of chrysanthemum. A high density of Frankliniella occidentalis, the predominant vector, in the presence of latently infected stock plants resulted in a high incidence of disease in the chrysanthemum production field. The incidence of disease was low when the vector thrips were not abundant in spite of the presence of latently infected stock plants. These results suggest that an infestation of the vector thrips causes severe secondary spread of TSWV originating from latently infected stock plants in chrysanthemum production fields. Received 27 July 2001/ Accepted in revised form 27 November 2001     

Overwintering of Frankliniella fusca (Thysanoptera: Thripidae) on Winter Annual Weeds Infected with Tomato spotted wilt virus and Patterns of Virus Movement Between Susceptible Weed Hosts

ABSTRACT Overwintering of tobacco thrips, Frankliniella fusca, was investigated on common winter annual host plants infected with Tomato spotted wilt virus (TSWV). Populations of tobacco thrips produced on TSWV-infected plants did not differ from those produced on healthy plants, whereas populations varied greatly among host plant species. The mean per plant populations of F. fusca averaged 401, 162, and 10 thrips per plant on Stellaria media, Scleranthus annuus, and Sonchus asper, respectively, during peak abundance in May. Adult F. fusca collected from plant hosts were predominately brachypterous throughout the winter and early spring, but macropterous forms predominated in late spring. Weed hosts varied in their ability to serve as overwintering sources of TSWV inoculum. Following the initial infection by TSWV in October 1997, 75% of Scleranthus annuus and Stellaria media retained infection over the winter and spring season, whereas only 17% of Sonchus asper plants remained infected throughout the same interval. Mortality of TSWV-infected Sonchus asper plants exceeded 25%, but mortality of infected Stellaria media and Scleranthus annuus did not exceed 8%. TSWV transmission by thrips produced on infected plants was greatest on Stellaria media (18%), intermediate on Scleranthus annuus (6%), and lowest on Sonchus asper (2%). Very few viruliferous F. fusca were recovered from soil samples collected below infected wild host plants. Vegetative growth stages of Stellaria media, Sonchus asper, and Ranunculus sardous were more susceptible to F. fusca transmission of TSWV than flowering growth stages, whereas both growth stages of Scleranthus annuus were equally susceptible. In a field study to monitor the spatial and temporal patterns of virus movement from a central source of TSWV-infected Stellaria media to adjacent plots of R. sardous, the incidence of infection in R. sardous plots increased from <1% in March to >42% in June 1999. Infection levels in the Stellaria media inoculum source remained high throughout the experiment, averaging nearly 80% until June 1999 when all Stellaria media plants had senesced. Dispersal of TSWV from the inoculum source extended to the limits of the experimental plot (>37 m). Significant directional patterns of TSWV spread to the R. sardous plots were detected in April and May but not in June. R. sardous infections were detected as early as March and April, suggesting that overwintering inoculum levels in an area can increase rapidly during the spring in susceptible weed hosts prior to planting of susceptible crops. This increase in the abundance of TSWV inoculum sources occurs at a time when vector populations are increasing rapidly. The spread of TSWV among weeds in the spring serves to bridge the period when overwintered inoculum sources decline and susceptible crops are planted.     

Dynamics of Tomato spotted wilt virus Replication in the Alimentary Canal of Two Thrips Species

ABSTRACT Transmission of Tomato spotted wilt virus (TSWV) is dependent on virus uptake in the midgut prior to virus movement to the salivary glands. Replication of TSWV in the alimentary canal of tobacco thrips (TT, Frankliniella fusca) and western flower thrips (WFT, F. occidentalis) was investigated by immunolocalization of the nonstructural protein (NSs) encoded by the small RNA of TSWV and fluorescence microscopy. Analysis of cohorts during development from larva to adults following virus acquisition by first instar larva indicated that virus replication followed a specific time-course pattern in the foregut, regions of the midgut, salivary glands, and ligaments between the midgut and salivary glands. Initial virus replication occurred only in epithelial cells of midgut-1 but, upon infection of muscle cells, the virus moved to the midgut-2, foregut, midgut-3, and salivary glands. The ligaments between the midgut and salivary glands appeared to be a route for virus to invade the salivary glands. No virus replication was observed in the hindgut, Malpighian tubules, or tubular salivary glands. The dynamics of TSWV replication, as measured by NSs accumulation, were similar in both TT and WFT.     

Development of an assay system using thrips-mediated inoculation to evaluate resistance of Capsicum spp. to Tomato spotted wilt virus

Considerable losses in pepper production by Tomato spotted wilt virus (TSWV) have been reported worldwide. In breeding programs, an assay for resistance that accurately estimates field occurrence of TSWV during pepper production is critical because the virus is vector transmitted. Here, we establish an assay system of TSWV-resistant Capsicum spp. using insect-mediated inoculation within an acrylic chamber in which environmental conditions such as temperature, light intensity, and nutrient supply are controlled. This chamber enables transmission of TSWV from viruliferous plants to plants used in the resistance assay with Frankliniella occidentalis safely, quickly, and precisely.     

Interaction of Tomato Spotted Wilt Tospovirus (TSWV) Glycoproteins with a Thrips Midgut Protein, a Potential Cellular Receptor for TSWV

ABSTRACT Interactions between viral and cellular membrane fusion proteins mediate virus penetration of cells for many arthropod-borne viruses. Electron microscope observations and circumstantial evidence indicate insect acquisition of tomato spotted wilt virus (TSWV) (genus Tospovirus, family Bunyaviridae) is receptor mediated, and TSWV membrane glycoproteins (GP1 and GP2) serve as virus attachment proteins. The tospoviruses are plant-infecting members of the family Bunyaviridae and are transmitted by several thrips species, including Frankliniella occidentalis. Gel overlay assays and immunolabeling were used to investigate the putative role of TSWV GPs as viral attachment proteins and deter mine whether a corresponding cellular receptor may be present in F. occidentalis. A single band in the 50-kDa region was detected with murine monoclonal antibodies (MAbs) to the TSWV-GPs when isolated TSWV or TSWV-GPs were used to overlay separated thrips proteins. This band was not detected when blots were probed with antibody to the non-structural protein encoded by the small RNA of TSWV or the TSWV nucleocapsid protein, nor were proteins from nonvector insects labeled. Anti-idiotype antibodies prepared to murine MAbs against GP1 or GP2 specifically labeled a single band at 50 kDa in Western blots and the plasmalemma of larval thrips midguts. These results support the putative role of the TSWV GPs as viral attachment proteins and identified potential cellular receptor(s) in thrips.     

Transmission Efficiency of 'Candidatus Liberibacter solanacearum' and Potato Zebra Chip Disease Progress in Relation to Pathogen Titer, Vector Numbers, and Feeding Sites

ABSTRACT With diseases caused by vector-borne plant pathogens, acquisition and inoculation are two primary stages of the transmission, which can determine vector efficiency in spreading the pathogen. The present study was initiated to quantify acquisition and inoculation successes of 'Candidatus Liberibacter solanacearum', the etiological agent of zebra chip disease of potato, by its psyllid vector, Bactericera cockerelli (Hemiptera: Triozidae). Acquisition success was evaluated in relation to feeding site on the host plant as well as the acquisition access period. Inoculation success was evaluated in relation to vector number (1 and 4) on the plants. Acquisition success was influenced by the feeding site on the plant. The highest acquisition success occurred when insects had access to the whole plant. The results of the inoculation study indicated that the rate of successfully inoculated plants increased with the vector number. Plants inoculated with multiple psyllids had higher bacterial titer at the point of inoculation. Although disease incubation period was significantly shorter in plants inoculated with multiple psyllids, this effect was heterogeneous across experimental blocks, and was independent of pathogen quantity detected in the leaflets 3 days postinoculation. Disease progress was not affected by bacterial quantity injected or psyllid numbers.     

Transmissibility of four tospoviruses by a thelytokous population ofThrips tabaci from Liguria, Northwestern Italy

Studies were carried out on a population ofThrips tabaci Lindeman (Thysanoptera: Thripi-dae) from Liguria to assess its sex-ratio and its ability to transmit four tospoviruses: tomato spotted wilt (TSWV), impatiens necrotic spot, tomato chlorotic spot and groundnut ringspot. The population was composed of females only (therefore thelytokous). The first instar larvae were allowed to acquire the virus for 48 h on infected leaves of datura, basil or pepper, and then reared on cucumber until emergence, which medially occurred 9.5 days after hatching. Transmission capacity was checked using two inoculation access periods (lAPs) of 48 h each on pepper leaf disks.T. tabaci was able to transmit TSWV isolate P105 with an efficiency of 16.7% and 4.4% in the first and second IAP, respectively, and TSWV isolate BR-01 with an efficiency of 2.0%. The onion thrips did not transmit the three other tospoviruses. During the IAPs, almost all adults fed on the leaf disks, producing evident silvery scars. The presence of tospovirus nucleocapsids in thrips was assayed by Triple Antibody Sandwich (TAS) and cocktail ELISA. Not all adults that had transmitted TSWV were positive in the tests, whereas some non-transmitter individuals proved positive. For each of the other tospoviruses, some thrips were positive in at least one test, although none was able to transmit the virus.     

Role of weed hosts and the western flower thrips, Frankliniella occidentalis, in epidemiology of Tomato spotted wilt virus in the Çukurova region of Turkey

Surveys of thrips and Tomato spotted wilt virus (TSWV) on weeds were conducted in the eastern Mediterranean region of Turkey during the years 2004–2006. Thrips species were collected by vigorously shaking weedy plants into a white container for 15 sec. Plant material collected during field surveys and plants which were used for mechanical inoculation of TSWV, were tested by DAS–ELISA. The weed species Ranunculus muricatus, Melilotus officinalis, Sinapis arvensis and Portulaca oleracea were used for the virus transmission experiments in an enclosed high plastic tunnel and in cage experiments. Western flower thrips, Frankliniella occidentlis (Pergande) (Thysanoptera: Thripidae), was the most common thrips, inhabiting 80 of the 82 weed species sampled. Adults of F. occidentalis and thrips larvae were significantly more abundant on S. arvensis than on the other weed species sampled (P<0.05). Adult and larval thrips showed peak densities on most weeds in April or May. Summer annual weeds were not good hosts for reproduction of the thrips. A total of 90 samples from 17 plant species belonging to 11 plant families were ELISA positive for TSWV. No TSWV was detected on 65 weed species belonging to 26 plant families. High numbers of plant samples infected by TSWV were obtained in P. oleracea (21 samples) and in R. muricatus (15 samples). In field surveys symptoms of TSWV were detected on only R. muricatus. Incidence of the TSWV on weeds ranged between 5% and 25%. Transmission rates of TSWV by F. occidentalis to the weeds ranged from 33% to 83% in the pepper plastic tunnel and cage experiments.     

Impeded Thrips Transmission of Defective Tomato spotted wilt virus Isolates

Two defective RNA-containing isolates (Pe-1 and 16-2) and an envelope-deficient (env ) isolate of Tomato spotted wilt virus (TSWV) were tested for their transmissibility by Frankliniella occidentalis. The Pe-1 isolate contained a truncated L RNA segment that barely interfered with symptom expression and replication of the wild-type (wt) L RNA segment. This isolate was transmitted with an efficiency of 51%, a value comparable to that found for wt TSWV (54%). Isolate 16-2, which contained a genuine defective interfering L RNA as concluded from its ability to suppress wt L RNA synthesis and attenuation of symptom expression, was not transmitted at all. The midguts of all larvae that ingested Pe-1 became infected, whereas limited midgut infections were found in 24% of the larvae that ingested 16-2. This difference in infection could be explained by the presence of a low number of infectious units in the inoculum ingested from plants as demonstrated in infection experiments and verified by northern blot analysis. The env isolate failed to infect the midgut after ingestion and could not be transmitted by any thrips stage. This isolate also cannot infect primary thrips cell cultures. Taken together, these results suggest that the envelope of TSWV contains the determinants required for binding and subsequent infection of thrips cells.     

Replication of Tomato spotted wilt virus After Ingestion by Adult Thrips setosus is Restricted to Midgut Epithelial Cells

ABSTRACT If acquisition access feeding (AAF) is first given after adult eclosion, none of the nine thrips species able to serve as tospovirus vectors can become infective. The previous cellular investigations of this phenomenon, carried out only in Frankliniella occidentalis, suggested that infectivity was prevented because the type member of the tospoviruses, Tomato spotted wilt virus (TSWV), was unable to enter the midgut of adult thrips. The present study extends a cellular view of tospovirus-thrips interactions to a species other than the western flower thrips, F. occidentalis. Our findings show that TSWV enters and replicates within the midgut of adult Thrips setosus, but does not infect cells beyond the midgut epithelia. After AAF as adult, TSWV replicated in T. setosus midgut cells as indicated by significant increases in nucleocapsid (N) protein detected by double-antibody sandwich enzyme-linked immunosorbent assay, and the presence of inclusions containing the S RNA-encoded nonstructural and N proteins revealed by microscopic observations. Electron microscopic observations of adult insects showed that no infection occurred in cells beyond the midgut epithelia, and insects subsampled from the same cohorts could not transmit TSWV. In contrast, electron microscopy observations of larval T. setosus revealed that TSWV infected the midgut and muscle cells, and adult insects developing from these cohorts had infected salivary glands and were able to transmit TSWV. Mature virions were observed only in the salivary glands of adults developing from infected larvae. Our findings suggest that the barrier to infectivity in T. setosus adults differs from that shown for F. occidentalis adults.     

Incidence of weed reservoirs and vectors of tomato spotted wilt tospovirus on southern Tasmanian lettuce farms

Thrips species and tomato spotted wilt virus (TSWV) alternate weed hosts were surveyed on two lettuce farms in southern Tasmania during 1994 and 1995. Only one known vector species, Thrips tabaci, was found at either site, comprising on average 36.8% of the total monthly catch. A major peak of thrips activity in the summer corresponded with an increase of disease in autumn harvested lettuce. Two thrips species new for Tasmania were recorded, Pseudanaphothrips achaetus and Tenothrips frici . Infection patterns within the crop indicated that localized weed infestations were the most likely reservoir of virus. ELISA testing showed that TSWV was present in a range of dicotyledonous weed species, although usually infecting only a low percentage of the plants. Arctotheca calendula appeared to be the single most important reservoir host species at one property, whilst this species and Sonchus oleraceus, Malva sylvestris, Brassica rapa ssp. silvestris, Erodium moschatum and Trifolium sp. were probably the most important reservoirs at the other property. Two new natural TSWV host species were recorded, Erodium moschatum and Brassica rapa ssp. silvestris . The property with the highest incidence of TSWV-infected lettuce had a relatively higher proportion of virus-infected weeds but less thrips activity during the infection period.