Frankliniella occidentalis is the key vector responsible for the emergence of Tomato spotted wilt virus as a global threat to agriculture. Frankliniella bispinosa is a common thrips in Florida, the Bahamas, and Bermuda, but the role of F. bispinosa in the epidemiology of the virus is not known. The purpose of this study was to determine the ability of F. bispinosa to acquire and transmit Tomato spotted wilt virus in pepper. In laboratory experiments, the number of larvae produced per F. bispinosa female was less than the number of larvae produced per F. occidentalis female. The larvae of F. bispinosa successfully acquired Tomato spotted wilt virus, although at a lower percentage than F. occidentalis. Viruliferous adults of both species transmitted the virus to pepper. Our results confirm the competence of F. bispinosa as a vector of Tomato spotted wilt virus.
In addition to damaging plant tissues while feeding, some species of thrips vector Tomato spotted wilt virus (TSWV), a tospovirus transmitted through the saliva of thrips during feeding (Hunter & Ullman 1992; Ullman et al. 1997). Tomato spotted wilt was first observed in 1915 in Australia and described as the “spotted wilt” of tomatoes (Brittlebank 1919) that later was associated with transmission by thrips (Pittman 1927). The viral etiology was reported by Samuel et al. (1930).
TSWV causes economic loss in many agricultural crops. The virus has a broad host range, infecting over 1000 plant species, and causing an estimated crop loss of one billion US dollars per year throughout its host range (Prins & Goldbach 1998). Tomato, tobacco, lettuce, pepper, papaya, eggplant, green beans, artichokes, broad beans, celery, some ornamental plants, and other plants experience severe losses due to the virus (Rosella et al. 1996).
Ullman et al. (1997) reviewed the relevant scientific literature involving the relationship between TSWV and its thrips vectors. Only first and second instars of vector thrips species acquire TSWV during feeding upon an infected host, and the virus survives molting, pupation, and the replacement of tissues during the prepupal and pupal stages of thrips development. Adults are unable to acquire TSWV. Infected adults are responsible for transmission and spread.
Outbreaks of tomato spotted wilt are difficult to manage. Growing seedlings under cover, avoiding sequential plantings, removing acquisition hosts for the larvae, rotating with non-susceptible crops, and use of UV-reflective mulch are sometimes useful as management tactics (Cho et al. 1998; Kucharek 1990; Momol et al. 2004; Rosella et al. 1996). Tomato hybrids were developed with a single-gene dominant resistance trait, but this resistance was overcome by strains of the virus (Rosella et al. 1996). Attempts to regulate vector populations with insecticides have not been successful, and populations of thrips developed resistance to broad-spectrum insecticides (Brodsgaard 1994; Immaraju et al. 1992). Further, primary spread of TSWV is not prevented by insecticides because insecticide-exposed viruliferous adults successfully transmitted the virus before death (Momol et al. 2004).
Thrips species known to transmit TSWV are Thrips tabaci (Lindeman), Thrips setosus (Moulton), Frankliniella occidentalis (Pergande), Frankliniella schultzei (Trybom), Frankliniella fusca (Hinds), and Frankliniella intonsa (Trybom) (Sherwood et al. 2001). Frankliniella occidentalis is the primary vector of TSWV due to its increasingly global distribution (Wijkamp et al. 1995). Frankliniella bispinosa (Morgan), which is distributed in parts of the southeastern US, Bermuda, and the Bahamas (Nakahara 1997), has been suspected, but not proven, as a vector of TSWV (Tsai et al. 1996; Webb et al. 1998).
The plants on which adult thrips can be collected have been cited in the literature as host plants (Mound & Teulon 1995), but adults frequently inhabit flowers that are not reproductive hosts. Adults of F. bispinosa are abundant in the flowers of bell pepper, Capsicum annuum L., in Florida along with adults of other species, including F. occidentalis (Funderburk et al. 2000; Hansen et al. 2003; Reitz et al. 2003). The suitability of pepper as a reproductive host of F. bispinosa has not been determined, and the possible role of F. bispinosa in TSWV epidemics is unknown. The purpose of our research was to determine the competence of F. bispinosa as a vector of TSWV in pepper. An experiment was conducted to determine the ability of F. bispinosa to reproduce and acquire the virus on pepper compared to the key vector, F. occidentalis. Another experiment was conducted to verify that F. bispinosa adults are able to transmit the TSWV to un-infected pepper.
Materials and Methods
Pepper Establishment and Maintenance
Individual ‘Camelot X3R’ bell peppers were transplanted into a 16 × 16 cm pot containing soil mixture (Fafard 3B Mix, Agawam, MA) and about 100 were maintained under greenhouse conditions. Plants were fertilized with Peat-Lite special 15-16-17 fertilizer (Scotts-Sierra Horticultural Products Company, Marysville, OH) and Miracle-Gro Bloom Booster 10-52-10 fertilizer (Miracle-Gro, Marysville, OH). Virus acquisition and transmission experiments were conducted in growth rooms at 23 to 25°C under a photoperiod of 14 h light, 10 h darkness.
Virus Acquisition Experiment
Plants for use in TSWV acquisition trials were mechanically inoculated with TSWV isolates collected from naturally infected pepper plants at the North Florida Research and Education Center. Using a mortar and pestle, we homogenized TSWV infected leaf tissue in 5% sodium sulfite solution containing diatomaceous earth in order to prepare an inoculum. Cheesecloth was used to apply inoculum to 3 or 4 leaves per experimental plant. Seven to 10 days after mechanical inoculum, experimental plants were tested for TSWV infection by a commercially available double antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit (Agdia, Elkhart, IN). Glass tubes and polystyrene balls (Precision Plastic Ball Co., Chicago, IL) were used in place of a microplate. Samples were scored for the presence of a colorimetric reaction indicating TSWV infection.
Individual TSWV infected pepper plants between 8 and 10 weeks of age were covered by a polyethylene cylindrical cage (35 cm × 15 cm) (n = 23 and 25 for F. bispinosa and F. occidentalis, respectively). The opening at the top was covered with fine mesh to prevent thrips escape, and there were two side openings (2.5 cm × 2.5 cm) covered with mesh. Ten females of F. occidentalis or F. bispinosa were introduced into the cage containing one infected plant. The adults were removed after 6 d. The plants were visually inspected for larvae at 6, 8, and 10 d after initial infestation with adult thrips. Each larva when found was transferred to green bean pods. After developing into adult, each was tested with an indirect ELISA to detect for the presence of the NSs protein encoded by TSWV RNA (Bandla et al. 1994). The NSs protein is present in thrips cells as a result of TSWV replication, demonstrating that the thrips is a host for TSWV.
The mean numbers of F. occidentalis larvae and F. bispinosa larvae recovered per cage after 10 days were compared with a two sample t-test [PROC TTEST in SAS System Software (SAS Institute 1999)]. The percent virus acquisition of F. occidentalis and F. bispinosa larvae also was compared by a two-sample t-test.
Virus Transmission Experiment
Transmission trials were conducted to confirm that the adults of F. bispinosa and F. occidentalis adults transmit TSWV to pepper. Cohorts of about 50 F. bispinosa and F. occidentalis larvae were allowed to feed on infected tomato fruit until developing into pupae. After developing into an adult, 5 to 10 of each species from each cohort were tested to verify TSWV acquisition with the indirect ELISA method described previously (Bandla et al. 1994). Twenty putatively viruliferous F. occidentalis or F. bispinosa adults from cohorts that tested positive were introduced into a polyethylene cage (55-cm-long × 30-cm-wide × 48-cm-high) containing 4 healthy pepper plants of 8 weeks old. There were 19 and 8 cages established for F. occidentalis and F. bispinosa, respectively. The peppers were tested after 21 days for TSWV by ELISA as described above. Transmission by F. occidentalis and F. bispinosa was compared by a chi-square test.
Results
Pepper was a suitable reproductive host for F. occidentalis and F. bispinosa in our study. The mean total number (+ SEM) of larvae recovered over 10 d when introducing 10 F. occidentalis or F. bispinosa adult females on individually caged pepper plants infected with TSWV was 47.7 (±7.2) and 15.3 (±2.5), respectively. The difference was significant (t = -4.08, df = 58, P < 0.0001).
A higher percentage of F. occidentalis acquired the virus versus F. bispinosa (t = -2.07, df = 53, P < 0.05).The mean percent acquisition (±SEM) of TSWV by F. occidentalis and F. bispinosa larvae feeding on infected pepper plants, as determined by an indirect ELISA to detect for the presence of the NSs protein encoded by the virus RNA, was 21.9 (±3.1) and 14.6 (±2.9), respectively.
The adults of F. bispinosa and F. occidentalis successfully transmitted TSWV to pepper. In the virus transmission experiments, pepper plants exposed to TSWV-infected adults of F. bispinosa were ELISA positive in 4 out of 8 replicates. Pepper plants exposed to viruliferous adults of F. occidentalis were ELISA positive in 6 out of 19 replicates. The difference in transmission between the two species was not significant (χ2 = 0.8; df = 3).
Discussion
The results from the acquisition experiment indicate that under laboratory conditions F. occidentalis is more likely to acquire the virus, and thus may be a more effective vector in pepper than F. bispinosa. The number of larvae of F. occidentalis produced per female was 3.1-fold greater, indicating that F. occidentalis has a greater intrinsic capacity than F. bispinosa to increase on pepper. The greater the intrinsic capacity of increase of a vector species on a host plant the greater the potential for acquisition and spread of TSWV (Peters et al. 1996). Differences in feeding preferences and host suitability between F. occidentalis and F. bispinosa may result in varying abilities of each species to acquire TSWV depending on the host plant. Webb et al. (1998) observed in laboratory studies higher rates of acquisition of TSWV for F. bispinosa than for F. occidentalis when the larvae fed on Datura stramonium L.
Van de Wetering et al. (1999) analyzed 14 populations of F. occidentalis. Each population acquired and transmitted TSWV, but there were marked differences in their efficiency, expressed as the percentage of transmitting adults. Laboratory experiments also do not account for ecological factors that influence thrips populations under field conditions, such as parasitism or predation that may reduce thrips populations, nor does it account for differences in mobility and other behaviors between the two thrips species that might affect the spread of TSWV in field peppers. Ramachandran et al. (2001) showed that the adults of F. bispinosa moved more rapidly in field peppers than the adults of F. occidentalis. This behavior allowed the adults of F. bispinosa to more frequently escape predation of Orius insidiosus.
The abundance of F. bispinosa and F. occidentalis in certain geographical regions also should be considered when assessing these species as a potential threat as a vector of TSWV in field pepper. Hansen et al. (2003) found F. bispinosa in much greater abundance than F. occidentalis in central Florida, while both species were abundant in northern Florida.
In this study, we have shown that under laboratory conditions F. bispinosa is a competent vector of TSWV in pepper. The species reproduced and acquired TSWV from infected pepper plants. Viruliferous adults of F. bispinosa transmitted TSWV to pepper. Reproduction of F. occidentalis on pepper and virus acquisition by the larvae feeding on pepper was greater than that by F. bispinosa; however, species-specific attributes may play a role in the ability of both vectors to vector TSWV in field conditions. The adults of F. bispinosa are more mobile within pepper fields than the adults of F. occidentalis (Ramachandran et al. 2001). TSWV epidemics occur on field pepper in our region (Gataitis et al. 1998), but the role of each species in disease epidemiology under field conditions is not understood.
Acknowledgments
We thank Hank Dankers, Kristen Bowers, and Steve Olson for technical assistance. Heather McAuslane and Michelle Stuckey made comments on the manuscript. The research was supported by a USDA-CSREES grant in Tropical/Subtropical Agriculture.
