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.     

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.     

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.     

Comparative host reactions and Frankliniella occidentalis transmission of different isolates of tomato spotted wilt tospovirus from Spain

The abilities of different isolates of tomato spotted wilt tospovirus (TSWV) collected from northeastern and eastern Spain to infect 10 host species, and to be acquired and transmitted by the western flower thrips (WFT), Frankliniella occidentalis were compared. Two isolates of TSWV from a single source plant could be separated according to the different type of local lesions they induced in Nicotiana glutinosa. Host ranges of the studied TSWV isolates were very similar, but differences were found in the symptoms induced and in their capacity to infect specific hosts systemically. Lycopersicon esculentumDatura stramonium were evaluated for their potential as virus acquisition host species. The proportion of transmitter adult thrips obtained from WFT larvae fed on L. esculentum was greater than from D. stramonium. No differences were detected between TSWV isolates in their ability to be acquired and transmitted by WFT. No evidence was obtained of alterations in TSWV particles which could affect WFT transmissibility due to the repeated mechanical transfers used to clone the isolates. Our findings do not support the existence of pathological effects of TSWV on WFT.     

The proportion of viruliferous individuals in field populations of Frankliniella occidentalis: Implications for tomato spotted wilt virus epidemics in tomato

Tomato spotted wilt tospovirus (TSWV) infected plants and western flower thrips (Frankliniella occidentalis Perg., WFT) adult population densities were monitored during 1993 and 1994 in field tomatoes in Northeastern Spain. The proportion of viruliferous WFT adults in field populations was quantified. A significant association has been found between early population densities of WFT adult thrips and final TSWV incidence for early transplanted tomato crops. In contrast, for late transplanted tomato crops, whereas similar high final disease incidences of TSWV could be attained, very low WFT adult population densities were always detected. The significantly higher infectious potential of WFT populations collected during the early growth stages of late transplanted tomatoes could be relevant for the TSWV incidences attained in spite of the low thrips numbers detected.     

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.     

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.     

The competence of four thrips species to transmit and replicate four tospoviruses

The tospoviruses Tomato spotted wilt virus (TSWV), Tomato chlorotic spot virus (TCSV), Groundnut ringspot virus (GRSV) and Chrysanthemum stem necrosis virus (CSNV) are well-known pathogens on tomato in Brazil. The thrips species Frankliniella occidentalis , F. schultzei , Thrips tabaci and T. palmi were studied for their competence to transmit these tospoviruses. Frankliniella occidentalis transmitted all four tospoviruses with different efficiencies. Frankliniella schultzei transmitted TCSV, GRSV and CSNV. Although F. schultzei has been reported as a vector of TSWV, the F. schultzei population in the present study did not transmit the TSWV isolate used. A population of T. tabaci known to transmit Iris yellow spot virus (onion isolate) did not transmit any of the studied tospoviruses, and nor did T. palmi . Replication of these tospoviruses could be demonstrated by ELISA, not only in the thrips species that could transmit them, but also in those that could not. The results strongly suggest that competence to transmit is regulated not only by the initial amount of virus acquired and replication, but also by possible barriers to virus circulation inside the thrip's body.     

Thrips Resistance in Pepper and Its Consequences for the Acquisition and Inoculation of Tomato spotted wilt virus by the Western Flower Thrips

ABSTRACT Different levels of thrips resistance were found in seven Capsicum accessions. Based on the level of feeding damage, host preference, and host suitability for reproduction, a thrips susceptible and a resistant accession were selected to study their performance as Tomato spotted wilt virus (TSWV) sources and targets during thrips-mediated virus transmission. Vector resistance did not affect the virus acquisition efficiency in a broad range of acquisition access periods. Inoculation efficiency was also not affected in short inoculation periods, but was significantly lower on plants of the thrips resistant accession during longer inoculation access periods. Under the experimental conditions used, the results obtained show that transmission of TSWV is little affected by vector resistance. However, due to a lower reproduction rate on resistant plants and a lower preference of thrips for these plants, beneficial effects of vector resistance might be expected under field conditions.     

Ceratothripoides claratris, a New Vector of a Capsicum chlorosis virus Isolate Infecting Tomato in Thailand

ABSTRACT Ceratothripoides claratris, the predominant thrips species on tomato in Thailand, was tested for vector competence and efficiency to transmit Capsicum chlorosis virus (CaCV) (isolate AIT) to tomato. The efficiency of adult-stage transmission was influenced by the larval stage at which virus was acquired. Adult C. claratris showed 69% transmission efficiency after acquiring the virus as freshly emerged (<1 h) first-instar larvae. However, when just molted (<1 h) second-instar larvae acquired the virus, the percentage of adult transmitters significantly decreased (48%). Transmission efficiency of up to 47% was detected with second-instar larvae of C. claratris which had acquired the virus as freshly emerged first-instar larvae. Transmission efficiency did not significantly differ between adult males and females, irrespective of the larval stage at which the virus was acquired. Highest transmission efficiency for CaCV was recorded in adult C. claratris derived from second-instar larvae collected from infected tomato plants in a greenhouse. Lowest transmission efficiency was observed in adults directly collected from infected tomato plants in the greenhouse. The spread of CaCV on tomato plants in greenhouses showed a close association with thrips infestations.     

Ornamental plants and thrips populations associated with tomato spotted wilt virus in Greece

A survey was conducted in order to record the ornamental plants that are hosts of tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV) in Greece. Polyclonal antibodies prepared against the N protein of a Greek isolate of TSWV fromGerbera jamesonii (GR-34) were used. Leaf samples were taken from plants showing typical symptoms of tospovirus infection such as chlorotic and necrotic rings on the leaves and malformation and necrosis of the flowers. The samples were tested by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using polyclonal antibodies to the N proteins of TSWV and INSV (NL-07). ELIS A-positive samples were mechanically transmitted to plants ofPetunia hybrida, Nicotiana rustica andN. benthamiana to confirm infection. Although none of the samples was found infected with INSV, TSWV presence was recorded in 42 botanical species that belong to 40 genera in 27 families. Among them the speciesBeloperone guttata, Coleus barbatus, Impatiens petersiana andLilium auratum are reported for the first time as hosts of TSWV, whereasBegonia sp.,Catharanthus roseus Celosia cristata, Dianthus chinensis, Fuchsia hybrida andStephanotis floribunda are found as new hosts of the virus in Greece. Thrips collected from TSWV-infected plants were in most cases identified asFrankliniella occidentalis, except from plants ofDendranthema sp. andDianthus caryophyllus whereThrips tabaci individuals were also identified. Different percentages of transmitters were noticed when the thrips populations collected from TSWV-infected ornamental hosts were tested for transmission of TSWV.     

Characterization of a vector-non-transmissible isolate of Tomato spotted wilt virus

The characteristics of a thrips‐non‐transmissible isolate of Tomato spotted wilt virus (TSWV), designated TSWV‐M, were compared with those of a thrips‐transmissible isolate, designated TSWV‐T. TSWV‐M showed a narrower host range than TSWV‐T. Adult thrips failed to transmit TSWV‐M, although the vector acquired the virus during the larval stages. TSWV‐M was detected by RT‐PCR in adult thrips bodies, but not in thrips heads, suggesting that loss of thrips transmissibility was the result of the absence of virus in adult thrips salivary glands. Whereas N (nucleoprotein), NSs (non‐structural protein) and G_C (the C‐terminal portion of the glycoprotein precursor protein) were present in similar amounts in leaf tissue from TSWV‐M‐ or TSWV‐T‐infected plants, G_N (the N‐terminal portion of the glycoprotein precursor protein) was present at much lower amounts in TSWV‐M‐ than in TSWV‐T‐infected plants. SDS‐PAGE and immunoblotting analysis of TSWV‐M and TSWV‐T virion preparations with G_N‐ and G_C‐specific antibodies revealed similar amounts of the G_N and G_C glycoproteins in TSWV‐T virions, but lower amounts of G_N than G_C in TSWV‐M virions. This resulted in a statistically significant reduction in the G_N/G_C ratio in TSWV‐M virions. In affinoblots, the G_C and G_N glycoproteins of TSWV‐M exhibited weak binding with lectins showing affinity for N‐linked oligosaccharide structures. Sequence analysis of M RNA (medium segment of the TSMV genome) revealed no deletions or frameshift mutations in the G_N/G_C precursor of TSWV‐M. However, five amino acid changes were detected in the G_N/G_C precursor. A single, relatively conservative amino acid substitution (V→I) was observed in the NSm protein. Sequence analysis of S RNA (small portion of the TSMV genome) revealed a large intergenic region with no changes in the N protein and with three amino acid changes in the NSs protein.     

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.     

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.     

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.     

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.     

Transmission of tomato spotted wilt tospovirus by Thripstabaci populations originating from leek

The transmission of tomato spotted wilt tospovirus (TSWV) by Thrips tabaci collected from leek was studied using the petunia local-lesion leaf-disc assay. After an acquisition-access period of 72 h given to newborn larvae up to 8 h old, the efficiency of transmission by adults was determined in three inoculation-access periods of 48 h. This efficiency varied for six T. tabaci populations from 0.7 to 11.6% in experiments using the Greek TSWV isolate GR-04. Males were more efficient transmitters than females (19 out of 176 versus five out of 494). Frankliniella occidentalis transmitted the same virus with a higher efficiency (34.8%). The transmission rate differed also among TSWV isolates, as shown in tests with four T. tabaci using two isolates. The virus was more efficiently acquired from infected leaf material of Datura stramonium than from that of Emilia sonchifolia . Plants of the latter species were more susceptible than Nicotiana tabacum in thrips transmission tests.     

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.     

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.