Comparison of Frankliniella fusca and Frankliniella occidentalis (Thysanoptera: Thripidae) as Vectors for a Peanut Strain of Tomato Spotted Wilt Orthotospovirus

Comparison of Frankliniella fusca and Frankliniella occidentalis (Thysanoptera: Thripidae) as... Abstract Tomato spotted wilt orthotospovirus (TSWV) is a major disease in peanut, Arachis hypogaea L., across peanut producing regions of the United States and elsewhere. Two thrips, Frankliniella fusca Hinds and Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), are considered important vectors of TSWV in peanut in the Southeast. We compared the efficiency of acquisition (by larvae) and transmission (adults) of both thrips species for TSWV (Texas peanut-strain) to leaf disks of peanut (Florunner), as well as to Impatiens walleriana Hook. f. (Dwarf White Baby) and Petunia hybrida Juss. ‘Fire Chief’ using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Both species were competent TSWV vectors in peanut and Impatiens, although F. fusca was the more efficient vector overall, i.e., virus acquisition and transmission rates for F. fusca averaged over several bioassays were 51.7 and 26.6%, respectively, compared with 20.0 and 15.3% for F. occidentalis. Neither species effectively transmitted this TSWV strain to Petunia (i.e., ≤3.6% transmission). We found statistically similar virus acquisition and transmission rates between both sexes for each species. We also detected no differences in TSWV-acquisition and transmission frequency between macropterous and brachypterous (short-wing) forms of F. fusca collected from a field population in south Texas. DAS-ELISA failed to detect low levels of TSWV in a few thrips that subsequently proved to be competent vectors. tomato spotted wilt orthotospovirus, brachyptery, western flower thrips, tobacco thrips, peanut Orthotospovirus, the only plant-infecting genus in the Order Bunyavirales, are exclusively transmitted by thrips in the family Thripidae (Riley et al. 2011). To date, 11 species of orthotospoviruses are recognized by the International Committee on Taxonomy of Viruses (ICTV 2017), but many more putative viruses are awaiting classification (Rotenberg et al. 2015). The tomato spotted wilt orthotospovirus (TSWV), the type member of the genus, is among the most economically important plant viruses worldwide, known to infect more than 1000 vegetable and ornamental plant species (Parrella et al. 2003, Scholthof et al. 2011). TSWV is a major disease of peanut, Arachis hypogaea L., in the southern United States (Srinivasan et al. 2017). The first U.S. report of TSWV in peanut was from Texas in 1971 (Halliwell and Philley 1974). By the late 1980s, TSWV became one of the most serious diseases for peanut production in the southeastern United States, especially in Alabama, Florida, and Georgia (Culbreath and Srinivasan 2011, Culbreath et al. 2003); in 2013, these three states alone accounted for over 70% of the U.S. peanut production (USDA NASS 2015). The potential impact for growers across the United States includes millions of dollars annually in lost production and pest control costs. For example, the severity of TSWV in the Georgia peanut crop in 2015 was estimated at 3% yield loss from a production value of $685 million (Little 2017). These losses prompted the development of integrated thrips management programs using a combination of chemical and cultural management practices and varieties more tolerant to TSWV (Srinivasan et al. 2017). Quantifying the primary routes of virus transmission is needed to develop grower-acceptable integrated disease management strategy for TSWV (Pappu et al. 2009). Since neither mechanical inoculation (Mandal et al. 2001) nor seed-transmission (Pappu et al. 1999) of TSWV readily occurs in peanut, thrips are the putative vectors in the TSWV-peanut pathosystem. Both tobacco thrips, Frankliniella fusca Hinds (Thysanoptera: Thripidae) and western flower thrips, Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), are found on peanut throughout the southeastern United States (Srinivasan et al. 2014). Moreover, both thrips species are known to be competent vectors for TSWV (Riley et al. 2011). While F. fusca was reported to be more numerous in peanut when compared with F. occidentalis (Todd et al. 1995, 1996), the relative extent to which each species acquires and vectors TSWV in different growing regions is not clear. Biological aspects of different thrips species in peanut may also influence their role as TSWV vectors. In the case of F. fusca, the presence of both macropterous (full wing) and brachypterous (short-wing) forms has implications for the transmissibility of TSWV. Since brachypterous F. fusca are unable to move large distances, viruliferous forms implies a within-field source of virus acquisition and secondary spread (Chamberlin et al. 1992). Since the severity of TSWV varies among locations and years (Culbreath et al. 2010, Culbreath and Srinivasan 2011, Srinivasan et al. 2017), predicting the relative severity of TSWV in a given year may assist growers evaluating management options. Understanding the importance of different thrips vectors is useful for making such decisions. In the current study, we compared the effect of TSWV on the development of F. fusca and F. occidentalis, and their ability to acquire and transmit this virus in peanut and additional ornamental plants. We also investigated the proportions of macropterous and brachypterous F. fusca acquiring TSWV in peanut fields associated with a virus outbreak in south central Texas. Materials and Methods Thrips Colonies F. occidentalis and F. fusca, originating from field populations collected in Frio County Texas, were reared on fresh green bean pods in 30-ml plastic cups closed with a paper lid (Frontier Agricultural Sciences, Newark DE). Bean pods were sterilized in 5% v/v sodium hypochlorite and cut into 25–35-mm long sections with the ends sealed in paraffin. New colonies were set up weekly. First instars for experiments were generated by incubating female thrips on peanut ‘Florunner’ leaf discs (13-mm diameter) inside microcentrifuge vials (1.5 ml) for a 24-h oviposition period. The microcentrifuge vials were incubated at 25°C, 76% RH, and 14:10 L:D, which generated a uniform cohort of larvae (<24 h old) after 4 d. Virus Source A strain of TSWV obtained from peanut ‘Florunner’ (Frio Co, TX) was used for host acquisition and transmission experiments. Virus levels remain stable in leaves stored for up to 10 d at 4°C (Kresta et. al. 1995). To expand the scope of the study, the peanut strain of TSWV was mechanically inoculated onto Impatiens ‘Dwarf White Baby’ using methods outlined previously (Mandal et al. 2001). All plants were confirmed with DAS-ELISA to be infected prior to use in acquisition experiments. For the ELISAs, reagents and antisera were purchased commercially (Agdia Inc., Elkhart, IN) and used according to manufacturer’s instructions. Development and TSWV-Acquisition of F. fusca and F. occidentalis on Peanut and Impatiens First instar thrips were confined in small groups on both symptomatic and asymptomatic peanut leaf tissue using Tashiro-Munger cells (Tashiro 1967) and maintained at 25°C, 76% RH, and 14:10 L:D photoperiod. Asymptomatic plants came from uninoculated control plants maintained in a separate location. Leaf tissues remained viable for thrips larval development, i.e., within 7 d under these conditions (Lowry et al. 1992). Mortality was noted and propupae were transferred to individual 1.5-ml microfuge tubes. One 13-mm leaf disc from healthy peanut was added when adults emerged. Two-day-old adult thrips were ground in 50-μl buffer (Cho et al. 1988) and subjected to DAS-ELISA and analysis as per Kresta et al. (1995). Because TSWV also impacts ornamental plants (Adkins et al. 2005), the efficiency with which F. fusca and F. occidentalis larvae acquired TSWV was assessed on preflowering Impatiens using the same procedures. The infection status of all symptomatic leaf tissues was confirmed by grinding in 100-μl extraction buffer (approximately 1:10 w/v) and testing via DAS-ELISA, as per Kresta et. al. (1995). Only symptomatic samples testing positive were used in the analysis, with a minimum of 30 thrips tested for TSWV and controls per host plant treatment. Thrips were similarly tested in 50-μl buffer and subjected to ELISA at the end of the experiment. TSWV-Transmission by F. fusca and F. occidentalis on Peanut and Impatiens First instar thrips were confined individually in 1.5-ml microcentrifuge vials containing a fresh 13-mm leaf disc from symptomatic plants for a 24-h acquisition access period. Leaf tissues were again tested for TSWV, while those from healthy plants served as controls. A small piece of absorbent tissue paper at the bottom of the vial served to control humidity. Leaf discs were replaced daily with healthy plant tissues until thrips pupation. Two-day-old adult thrips (from these treatments) were provided with leaf discs from healthy plants for virus transmission assessments to the same corresponding plant species. Leaf discs were replaced daily after a 24-h inoculation access period. Thrips-exposed leaf discs were floated on distilled water in 24-well tissue culture plates at 25°C, 14:10 photoperiod for 2 wk for multiplication and lesion development (chlorotic and mosaic ring spots). Wijkamp and Peters (1993) evaluated the leaf disc method with Petunia × hybrida and reported no difference in virus transmission rates when compared with whole Petunia plants. The leaf disks and thrips were subjected to DAS-ELISA as noted above. Studies were repeated with at least 75 thrips of each species tested per host plant with tests run sequentially as the tissue/thrips were available. Transmission of TSWV by F. fusca and F. occidentalis to Additional Plants The following plants were tested; peanut ‘Florunner’, Impatiens ‘Dwarf White Baby’, and Petunia ‘Fire Chief’. Impatiens is a known systemic host, while petunia is a local lesion host and a nonsystemic infection would appear as a lesion (Wijkamp and Peters 1993). Plants were reared in growth chambers at 25°C and 14:10 (L:D) h photoperiod. Plants were grown from seed in Sunshine Mix #1 (Fisons, Vancouver, British Columbia, Canada), watered twice weekly, and fertilized weekly with 10-15-10 liquid fertilizer (Schultz All Purpose, Knox Fertilizer Co., Knox, IN). Two-day old-adult thrips that were prior inoculated with TSWV as first instars on peanut as noted above were sequentially exposed to leaf discs of each test plant for 24-h inoculation periods. Tests were done in 1.5-ml microcentrifuge vials, as noted above. Approximately 100 adult thrips of each species (F. fusca and F. occidentalis) were exposed sequentially to each of the three plant species; i.e., each thrips was exposed to each test plant provided in a random sequence. There were two sequential 24-h inoculation access periods per test plant per thrips. Effect of F. fusca Wing Form on TSWV Acquisition and Transmission (Field Population) Adult F. fusca were collected from a peanut field in Frio County, TX, that was experiencing a TSWV epidemic, with disease prevalence approaching 70% of plants. Thrips were hand collected from the foliage of plants in several fields showing yellowing and ring spot symptoms. For transmission experiments, thrips were individually transferred into 1.5-ml microfuge tubes containing a 13-mm peanut leaf disk from healthy plants, as noted above. Transmission of TSWV by each field-collected adult was assessed over 5-d, by conducting a new inoculation access period with a noninfected leaf disc every 24 h. Exposed leaf discs and thrips were individually assessed via DAS-ELISA as noted above. In total, 150 thrips were used in the study; data were compared separately for macropterous and brachypterous wing forms of either sex. Statistical Analysis Among thrips exposed to TSWV as larvae, the proportion acquiring virus (i.e., testing positive in DAS-ELISA as adults) and becoming virus transmitters (i.e., inoculating leaf discs with TSWV) were compared among treatments with Pearson χ2 tests for independence. Where appropriate, means were separated using adjusted residual (z-score) post-hoc analysis (Garcia-Perez and Nunez-Anton 2003). Since sex of the vector can influence transmission of TSWV (Van de Wetering et al. 1998, Ogada and Poehling 2015) male and female thrips were compared separately. For the last study with field-collected thrips, the frequency of virus transmission over time was analyzed using a one-way ANOVA with arcsine-transformed data. All analysis was performed with SPSS (v.24; IBM, Inc., Armonk, NY). Results Development and TSWV-Acquisition of F. fusca and F. occidentalis on Peanut and Impatiens F. fusca larval development (survival to adults reared in Tashiro-Munger cells) was affected by plant species (χ2 = 25.94 =, df = 1, P < 0.001) and whether plants were TSWV-infected (χ2 = 25.18, df = 1, P < 0.001). Survival rates were higher on peanut and noninfected leaf tissues (Table 1). However, virus acquisition rates (i.e., ELISA positive thrips) were statistically similar between those reared on TSWV-infected peanut and impatiens plants for both male (χ2 = 0.23, df = 1, P =0.63) and female thrips (χ2 = 0.93, df = 1, P = 0.34). No evidence for TSWV-acquisition was observed among virus-free control plants. In the case of F. occidentalis, successful development to adult was lower overall (≤35% survival) by comparison to F. fusca, and neither affected by plant species (χ2 = 1.97, df = 1, P = 0.16) nor the infection status of plants (χ2 = 0.65, df = 1, P = 0.42) (Table 2). Virus acquisition rates for F. occidentalis were statistically similar among plant species for both male (χ2 = 2.62, df = 1, P = 0.11) and female thrips (χ2 = 3.24, df = 1, P = 0.07). Table 1. Effect of host plant and TSWV-status on development and virus acquisition by F. fusca larvae Host plant TSWV infection N % survivala % ELISA positiveb ♂ ♀ Peanut + 54 68.5bc 82.4a 80.0a − 85 82.4a 0.0b 0.0b Impatiens + 305 47.9c 76.9a 69.1a − 103 65.7b 0.0b 0.0b Host plant TSWV infection N % survivala % ELISA positiveb ♂ ♀ Peanut + 54 68.5bc 82.4a 80.0a − 85 82.4a 0.0b 0.0b Impatiens + 305 47.9c 76.9a 69.1a − 103 65.7b 0.0b 0.0b aOf larvae through adult emergence reared in Tashiro-Munger cells. bIn adult thrips reared on test plants. cPost hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Table 1. Effect of host plant and TSWV-status on development and virus acquisition by F. fusca larvae Host plant TSWV infection N % survivala % ELISA positiveb ♂ ♀ Peanut + 54 68.5bc 82.4a 80.0a − 85 82.4a 0.0b 0.0b Impatiens + 305 47.9c 76.9a 69.1a − 103 65.7b 0.0b 0.0b Host plant TSWV infection N % survivala % ELISA positiveb ♂ ♀ Peanut + 54 68.5bc 82.4a 80.0a − 85 82.4a 0.0b 0.0b Impatiens + 305 47.9c 76.9a 69.1a − 103 65.7b 0.0b 0.0b aOf larvae through adult emergence reared in Tashiro-Munger cells. bIn adult thrips reared on test plants. cPost hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Table 2. Effect of host plant and TSWV-status on development and virus acquisition by F. occidentalis larvae Host TSWV N % Survivala % ELISA positiveb ♂ ♀ Peanut + 142 25.4ac 40.0a 38.5a − 30 16.7a 0.0b 0.0b Impatiens + 90 26.7a 25.0a 31.3a − 80 35.0a 0.0b 0.0b Host TSWV N % Survivala % ELISA positiveb ♂ ♀ Peanut + 142 25.4ac 40.0a 38.5a − 30 16.7a 0.0b 0.0b Impatiens + 90 26.7a 25.0a 31.3a − 80 35.0a 0.0b 0.0b aOf larvae through adult emergence reared in Tashiro-Munger cells. bIn adult thrips reared on test plants. cPost-hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Table 2. Effect of host plant and TSWV-status on development and virus acquisition by F. occidentalis larvae Host TSWV N % Survivala % ELISA positiveb ♂ ♀ Peanut + 142 25.4ac 40.0a 38.5a − 30 16.7a 0.0b 0.0b Impatiens + 90 26.7a 25.0a 31.3a − 80 35.0a 0.0b 0.0b Host TSWV N % Survivala % ELISA positiveb ♂ ♀ Peanut + 142 25.4ac 40.0a 38.5a − 30 16.7a 0.0b 0.0b Impatiens + 90 26.7a 25.0a 31.3a − 80 35.0a 0.0b 0.0b aOf larvae through adult emergence reared in Tashiro-Munger cells. bIn adult thrips reared on test plants. cPost-hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large TSWV-Transmission by F. fusca and F. occidentalis on Peanut and Impatiens Survival of F. fusca reared individually on test plants was neither affected by the TSWV-infection status of the plant (χ2 = 0.53, df = 1, P = 0.47) nor plant species (χ2 = 1.13, df = 1, P = 0.72) (Table 3). Virus acquisition rates from symptomatic peanut and Impatiens plants were again similar for both male (χ2 = 0.92, df = 1, P = 0.34) and female thrips (χ2 = 1.12, df = 1, P = 0.29). The proportion of adult F. fusca transmitting TSWV to leaf discs was statistically similar between peanut and Impatiens test plants, both for male (χ2 = 1.31, df = 1, P = 0.25) and female thrips (χ2 = 1.84, df = 1, P = 0.18). No evidence for TSWV-acquisition was observed among thrips reared on healthy control plants. Table 3. Host plant and TSWV-status effects on survival, acquisition and transmission by F. fusca Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 80 68.8ac 43.3a 12.0a 36.7a 24.0a − 30 86.7a 0.0b 0.0b 0.0b 0.0b Impatiens + 50 80.0a 30.0a 20.0a 20.0a 36.7a − 25 68.0a 0.0b 0.0b 0.0b 0.0b Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 80 68.8ac 43.3a 12.0a 36.7a 24.0a − 30 86.7a 0.0b 0.0b 0.0b 0.0b Impatiens + 50 80.0a 30.0a 20.0a 20.0a 36.7a − 25 68.0a 0.0b 0.0b 0.0b 0.0b aReared individually in microcentrifuge vials. bAmong all virus-exposed thrips surviving until adults. cPost hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Table 3. Host plant and TSWV-status effects on survival, acquisition and transmission by F. fusca Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 80 68.8ac 43.3a 12.0a 36.7a 24.0a − 30 86.7a 0.0b 0.0b 0.0b 0.0b Impatiens + 50 80.0a 30.0a 20.0a 20.0a 36.7a − 25 68.0a 0.0b 0.0b 0.0b 0.0b Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 80 68.8ac 43.3a 12.0a 36.7a 24.0a − 30 86.7a 0.0b 0.0b 0.0b 0.0b Impatiens + 50 80.0a 30.0a 20.0a 20.0a 36.7a − 25 68.0a 0.0b 0.0b 0.0b 0.0b aReared individually in microcentrifuge vials. bAmong all virus-exposed thrips surviving until adults. cPost hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large In the case of F. occidentalis, survival to adults was not affected by plant infection status (χ2 = 0.24, df = 1, P = 0.63) but was affected by plant species (χ2 = 18.50, df = 1, P < 0.001) (Table 4). Virus acquisition rates (based on ELISA) were low overall, and statistically similar among test plant for both male (χ2 = 0.46, df = 1, P = 0.48) and female thrips (χ2 = 0.79, df = 1, P = 0.38). The proportion of F. occidentalis exposed to TSWV as larvae and transmitting as adults was statistically similar between plant species, for both male (χ2 = 1.78, df = 1, P = 0.18) and female thrips (χ2 = 0.45, df = 1, P = 0.51). Virus acquisition rates of both thrips species were lower overall when compared with larvae reared in Tashiro-Munger cells (Tables 1 and 2). Table 4. Host plant and TSWV-status effects on survival, acquisition and transmission by F. occidentalis Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 95 34.7b 10.0ac 0.0a 10.0ac 21.7a − 25 76.0a 0.0b 0.0a 0.0b 0.0b Impatiens + 85 78.8a 12.5a 2.3a 20.8a 20.9a − 10 20.0b 0.0b 0.0a 0.0b 0.0b Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 95 34.7b 10.0ac 0.0a 10.0ac 21.7a − 25 76.0a 0.0b 0.0a 0.0b 0.0b Impatiens + 85 78.8a 12.5a 2.3a 20.8a 20.9a − 10 20.0b 0.0b 0.0a 0.0b 0.0b aReared individually in microcentrifuge vials. bAmong all virus-exposed thrips surviving until adults. cPost hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Table 4. Host plant and TSWV-status effects on survival, acquisition and transmission by F. occidentalis Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 95 34.7b 10.0ac 0.0a 10.0ac 21.7a − 25 76.0a 0.0b 0.0a 0.0b 0.0b Impatiens + 85 78.8a 12.5a 2.3a 20.8a 20.9a − 10 20.0b 0.0b 0.0a 0.0b 0.0b Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 95 34.7b 10.0ac 0.0a 10.0ac 21.7a − 25 76.0a 0.0b 0.0a 0.0b 0.0b Impatiens + 85 78.8a 12.5a 2.3a 20.8a 20.9a − 10 20.0b 0.0b 0.0a 0.0b 0.0b aReared individually in microcentrifuge vials. bAmong all virus-exposed thrips surviving until adults. cPost hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Virus Transmission of F. fusca and F. occidentalis to Additional Host Plants Overall, TSWV transmission among virus exposed thrips was highest on Impatiens (average 31.4% infected leaf discs) but was relatively low on both peanut and petunia (4.9 and 1.5% infected leaf discs, respectively) (Table 5). Overall, F. fusca was the more efficient vector with this isolate of TSWV; i.e., thrips species was a significant variable for both Impatiens (χ2 = 6.55, df = 1, P = 0.01) and petunia (χ2 = 6.38, df = 1, P = 0.01) and approached significance for peanut (χ2 = 3.01, df = 1, P = 0.08). However, the sex of thrips was not a significant factor in virus transmission rates to any host plant (χ2 ≤ 1.15, df = 1, P ≥ 0.28). Table 5. Effect of thrips species and sex on TSWV-transmission to three host plants Thrips Sex N Host planta Peanut Impatiens Petunia F. fusca ♂ 40 9.5ab 42.5a 2.5ab ♀ 55 5.5a 38.2a 3.6a F. occidentalis ♂ 34 2.9a 20.6b 0.0b ♀ 66 1.5a 24.2b 0.0b Thrips Sex N Host planta Peanut Impatiens Petunia F. fusca ♂ 40 9.5ab 42.5a 2.5ab ♀ 55 5.5a 38.2a 3.6a F. occidentalis ♂ 34 2.9a 20.6b 0.0b ♀ 66 1.5a 24.2b 0.0b aTransmission during either of two 24-h inoculation access intervals. bPost-hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Table 5. Effect of thrips species and sex on TSWV-transmission to three host plants Thrips Sex N Host planta Peanut Impatiens Petunia F. fusca ♂ 40 9.5ab 42.5a 2.5ab ♀ 55 5.5a 38.2a 3.6a F. occidentalis ♂ 34 2.9a 20.6b 0.0b ♀ 66 1.5a 24.2b 0.0b Thrips Sex N Host planta Peanut Impatiens Petunia F. fusca ♂ 40 9.5ab 42.5a 2.5ab ♀ 55 5.5a 38.2a 3.6a F. occidentalis ♂ 34 2.9a 20.6b 0.0b ♀ 66 1.5a 24.2b 0.0b aTransmission during either of two 24-h inoculation access intervals. bPost-hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Effect of F. fusca Wing-Form on Virus Acquisition and Transmission (Field Population) The proportion of ELISA-positive field collected adult F. fusca was neither significantly affected by wing-form nor sex (χ2 ≤ 1.19, df = 1, P ≥ 0.28; Table 6). The proportion of all thrips transmitting TSWV was also not affected by wing form nor sex (χ2 ≤ 0.86, df = 1, P ≥ 0.35). Among viruliferous thrips, the frequency of virus transmission was not affected by wing form or sex treatments (F3,145 = 1.38, P = 0.25). Table 6. Effect of wing-form and sex on TSWV-transmission by F. fusca collected from diseased peanut (Frio County, TX) Wingform Sex N % ELISA positive % TSWV transmission All thripsa Frequencyb Macropterous ♂ 41 56.1ac 36.6ac 49.3ad ♀ 56 69.6a 26.8a 36.0a Brachypterous ♂ 33 66.7a 36.4a 48.3a ♀ 19 68.4a 42.1a 45.0a Wingform Sex N % ELISA positive % TSWV transmission All thripsa Frequencyb Macropterous ♂ 41 56.1ac 36.6ac 49.3ad ♀ 56 69.6a 26.8a 36.0a Brachypterous ♂ 33 66.7a 36.4a 48.3a ♀ 19 68.4a 42.1a 45.0a aTransmitted TSWV to leaf disc at least once (tested daily over 5 d). bFrequency of transmission among viruliferous thrips (tested daily over 5 d). cPost-hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). dPost-hoc tests (in columns) based on one-way ANOVA with arcsine-transformed data (P < 0.05). View Large Table 6. Effect of wing-form and sex on TSWV-transmission by F. fusca collected from diseased peanut (Frio County, TX) Wingform Sex N % ELISA positive % TSWV transmission All thripsa Frequencyb Macropterous ♂ 41 56.1ac 36.6ac 49.3ad ♀ 56 69.6a 26.8a 36.0a Brachypterous ♂ 33 66.7a 36.4a 48.3a ♀ 19 68.4a 42.1a 45.0a Wingform Sex N % ELISA positive % TSWV transmission All thripsa Frequencyb Macropterous ♂ 41 56.1ac 36.6ac 49.3ad ♀ 56 69.6a 26.8a 36.0a Brachypterous ♂ 33 66.7a 36.4a 48.3a ♀ 19 68.4a 42.1a 45.0a aTransmitted TSWV to leaf disc at least once (tested daily over 5 d). bFrequency of transmission among viruliferous thrips (tested daily over 5 d). cPost-hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). dPost-hoc tests (in columns) based on one-way ANOVA with arcsine-transformed data (P < 0.05). View Large Discussion The number of different orthotospovirus–plant–vector interactions now recognized (Riley et al. 2011, Rotenberg et al. 2015) shows that this relationship can be specific and intricate. Thus far, few studies have directly compared vector competency of difference thrips species for TSWV to different host plants. Our data confirm the ability of F. fusca and F. occidentalis to vector field strains of TSWV in peanut, as well as Impatiens. Overall F. fusca was the more competent vector of the peanut-TSWV strain, based on both acquisition and transmission studies. In the southeastern United States, F. fusca is the predominant thrips species that reproduces on peanut, and therefore considered the most important vector (Culbreath and Srinivasan 2011). Additionally, F. fusca start to colonize peanut plants early in the season at the seedling stage; by contrast F. occidentalis is mostly found later in the season when plants are less susceptible to TSWV infection (Shrestha et al. 2015). Different feeding behaviors of these thrips species may also influence virus transmission in ‘choice’ situations. Chitturi et al. (2006) showed that the addition of pine pollen increased the selection and settling behavior (attractancy) of peanut to F. occidentalis (more of a generalist feeder) but not to F. fusca. We investigated several factors influencing the epidemiology of TSWV in peanut. We found little statistical evidence for differences in the vector competency between male and female thrips. Ogada and Poehling (2015) reported that transmission efficiency of TSWV on Capsicum annum by F. occidentalis was significantly higher in males, although females had a higher percentage of transmitting individuals. Van de Wetering et al. (1998) also reported higher rates of TSWV transmission among male F. occidentalis, which they attributed to the higher mobility and probing frequency of males while feeding. Stafford et al. (2011) also reported that male TSWV-infected F. occidentalis increased their probing frequency relative to uninfected males, thus increasing the probability of virus inoculation. It is possible that our method of confining thrips on small leaf discs would partially mask effects of higher mobility and probing on transmission efficiency. We also detected no differences in vector competency between field-collected F. fusca based on wing form, confirming that either form can acquire and transmit TSWV to field peanut. Wells et al. (2002) reported no significant difference between brachypterous and macropterous F. fusca in their ability to transmit TSWV in the laboratory, although suggested that macropterous individuals are required to colonize (and hence transmit TSWV to) newly emerged crops. By contrast, brachypterous individuals, which are more common in the late fall, may help maintain TSWV between seasons by harboring inoculum in the population until the development and dispersal of macropterous thrips in the spring (Chamberlin et al. 1992, Wells et al. 2002). We observed reduced survival rates from F. fusca developing on TSWV-infected peanut, although the same trend was not observed with F. occidentalis. An earlier laboratory study by Arthurs and Heinz (2003) also detected no differences in development rates or longevity of viruliferous and nonviruliferous F. occidentalis. These results are in broad agreement with previous laboratory studies demonstrating that F. fusca is more sensitive to the direct effects of TSWV than F. occidentalis. Stumpf and Kennedy (2005) reported that TSWV-infected F. fusca developing on TSWV-infected Datura stramonium L. and Emilia sonchifolia L. plants developed more slowly and were smaller compared with virus-free adults reared on the same TSWV-infected foliage, indicating a direct, negative effect of TSWV infection on F. fusca. Comparable studies conducted later (Stumpf and Kennedy 2007) also indicated that F. occidentalis reared on TSWV-infected D. stramonium and E. sonchifolia exhibited significantly improved survival and decreased development time. In both cases, host survival and transmission of TSWV was affected by an interaction between host plant and temperature. The reason for differential development response among thrips species to TSWV-infected host plants in our tests unknown; however, we note that F. occidentalis exhibits much higher polyphagy, which may make it more tolerant of nutritional or other biochemical changes associated with tospovirus-infected hosts. We used DAS-ELISA to test for viruliferous thrips based on earlier observations (Ullman et al. 1992, Cho et al. 1988) that thrips feeding on contaminated material as larvae showed a positive result when tested with DAS-ELISA as adults. However, we observed TSWV-transmission among ELISA-negative thrips on at least one occasion, suggesting that this test may not be completely reliable for detecting low titers in thrips. By contrast, relatively higher virus-detection rates were observed among field collected thrips. An increased proportion of field-collected adult thrips testing positive (when compared with transmission rates) might be explained by false positives, i.e., virus detected in the gut of nonviruliferous adult thrips recently feeding on infected plant material. An epithelial midgut barrier prevents systemic virus acquisition by adult thrips (Ullman et al. 1992, van de Wetering et al. 1996). An alternative ELISA method to overcome this problem involves detecting a small nonstructural protein encoded by the small RNA of TSWV that is only present if the virus has replicated within the host (Wells et al. 2002, Arthurs et al. 2003). TSWV was a major problem in peanut in the 1980s and 1990s, with crop losses over 50% reported in some states (Culbreath and Srinivasan 2011). The incidence of TSWV has declined in the southern United States since the 2000s, due to the adoption of various risk reduction practices, including second- and third-generation resistant peanut cultivars; although there has been an uptick in recent years, possibly due to new resistance breaking strains of the virus (Srinivasan et al. 2017). Currently, the mechanisms influencing field resistance of TSWV in peanut are poorly understood, but include suppressed thrips feeding and development as well as constitutive and induced plant defense proteins in TSWV resistant cultivars. Currently, host resistance remains the most important management option available to peanut growers. Our experiments were done with Florunner, which is considered a susceptible variety. It would be informative to compare these data with modern cultivars (such as Georgia 10T, Georgia 12Y, Georgia 13M, Georgia 14N, and Florun 107), which exhibit several fold increase in field resistance to TSWV compared with Florunner. Acknowledgments The efforts of SPA and KMH were supported by a grant from the Texas A&M AgriLife Research Insect Vector Diseases Seed Grant Program. We thank Kenya Kresta for assistance in laboratory studies, and Deborah Groth-Helms and Elizabeth Kmieciak (Agdia Inc.) for technical support. This work was also supported by the Texas Peanut Producers Board. References Cited Adkins , S. , T. Zitter , and T. Momol . 2005 . Tospoviruses (Family Bunyaviridae, Genus Tospovirus) . 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Walker , and D. E. Ullman . 2011 . Infection with a plant virus modifies vector feeding behavior . P. Natl. Acad. Sci . 108 : 9350 – 9355 . Google Scholar CrossRef Search ADS Stumpf , C. F. , and G. G. Kennedy . 2005 . Effects of tomato spotted wilt virus (TSWV) isolates, host plants, and temperature on survival, size, and development time of Frankliniella fusca . Entomol. Exp. Appl . 114 : 215 – 225 . Google Scholar CrossRef Search ADS Stumpf , C. F. , and G. G. Kennedy . 2007 . Effects of tomato spotted wilt virus isolates, host plants, and temperature on survival, size, and development time of Frankliniella occidentalis . Entomol. Exp. Appl . 123 : 139 – 147 . Google Scholar CrossRef Search ADS Tashiro , H . 1967 . Self-watering acrylic cages for confining insects and mites on detached leaves . J. Econ. Entomol . 60 : 354 – 356 . Google Scholar CrossRef Search ADS Todd , J. W. , A. K. Culbreath , J. R. Chamberlin , R. J. Beshear , and B. G. Mullinix . 1995 . 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Todd , A. S. Csinos , B. Mandal , and R. M. McPherson . 2002 . Dynamics of spring tobacco thrips (Thysanoptera: Thripidae) populations: implications for tomato spotted wilt virus management . Environ. Entomol . 31 : 1282 – 1290 . Google Scholar CrossRef Search ADS van de Wetering , F. , R. Goldbach , and D. Peters . 1996 . Tomato spotted wilt tospovirus ingestion by first instar larvae of Frankliniella occidentalis is a prerequisite for transmission . Phytopathology . 86 : 900 – 905 . Google Scholar CrossRef Search ADS van de Wetering , D. , Hulshof , J. , Posthuma , K. , Harrewijn , P. , Goldbach , R. , and D. Peters . 1998 . Distinct feeding behavior between sexes of Frankliniella occidentalis results in higher scar production and lower tospovirus transmission by females . Entomol. Exp. Appl . 88 : 9 – 15 . Google Scholar CrossRef Search ADS Wijkamp , I. , and D. Peters . 1993 . Determination of the median latent period of two tospoviruses in Frankliniella occidentalis using a novel leaf disk assay . Phytopathology . 83 : 986 – 991 . Google Scholar CrossRef Search ADS © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Environmental Entomology Oxford University Press

Comparison of Frankliniella fusca and Frankliniella occidentalis (Thysanoptera: Thripidae) as Vectors for a Peanut Strain of Tomato Spotted Wilt Orthotospovirus

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Abstract

Abstract Tomato spotted wilt orthotospovirus (TSWV) is a major disease in peanut, Arachis hypogaea L., across peanut producing regions of the United States and elsewhere. Two thrips, Frankliniella fusca Hinds and Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), are considered important vectors of TSWV in peanut in the Southeast. We compared the efficiency of acquisition (by larvae) and transmission (adults) of both thrips species for TSWV (Texas peanut-strain) to leaf disks of peanut (Florunner), as well as to Impatiens walleriana Hook. f. (Dwarf White Baby) and Petunia hybrida Juss. ‘Fire Chief’ using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Both species were competent TSWV vectors in peanut and Impatiens, although F. fusca was the more efficient vector overall, i.e., virus acquisition and transmission rates for F. fusca averaged over several bioassays were 51.7 and 26.6%, respectively, compared with 20.0 and 15.3% for F. occidentalis. Neither species effectively transmitted this TSWV strain to Petunia (i.e., ≤3.6% transmission). We found statistically similar virus acquisition and transmission rates between both sexes for each species. We also detected no differences in TSWV-acquisition and transmission frequency between macropterous and brachypterous (short-wing) forms of F. fusca collected from a field population in south Texas. DAS-ELISA failed to detect low levels of TSWV in a few thrips that subsequently proved to be competent vectors. tomato spotted wilt orthotospovirus, brachyptery, western flower thrips, tobacco thrips, peanut Orthotospovirus, the only plant-infecting genus in the Order Bunyavirales, are exclusively transmitted by thrips in the family Thripidae (Riley et al. 2011). To date, 11 species of orthotospoviruses are recognized by the International Committee on Taxonomy of Viruses (ICTV 2017), but many more putative viruses are awaiting classification (Rotenberg et al. 2015). The tomato spotted wilt orthotospovirus (TSWV), the type member of the genus, is among the most economically important plant viruses worldwide, known to infect more than 1000 vegetable and ornamental plant species (Parrella et al. 2003, Scholthof et al. 2011). TSWV is a major disease of peanut, Arachis hypogaea L., in the southern United States (Srinivasan et al. 2017). The first U.S. report of TSWV in peanut was from Texas in 1971 (Halliwell and Philley 1974). By the late 1980s, TSWV became one of the most serious diseases for peanut production in the southeastern United States, especially in Alabama, Florida, and Georgia (Culbreath and Srinivasan 2011, Culbreath et al. 2003); in 2013, these three states alone accounted for over 70% of the U.S. peanut production (USDA NASS 2015). The potential impact for growers across the United States includes millions of dollars annually in lost production and pest control costs. For example, the severity of TSWV in the Georgia peanut crop in 2015 was estimated at 3% yield loss from a production value of $685 million (Little 2017). These losses prompted the development of integrated thrips management programs using a combination of chemical and cultural management practices and varieties more tolerant to TSWV (Srinivasan et al. 2017). Quantifying the primary routes of virus transmission is needed to develop grower-acceptable integrated disease management strategy for TSWV (Pappu et al. 2009). Since neither mechanical inoculation (Mandal et al. 2001) nor seed-transmission (Pappu et al. 1999) of TSWV readily occurs in peanut, thrips are the putative vectors in the TSWV-peanut pathosystem. Both tobacco thrips, Frankliniella fusca Hinds (Thysanoptera: Thripidae) and western flower thrips, Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), are found on peanut throughout the southeastern United States (Srinivasan et al. 2014). Moreover, both thrips species are known to be competent vectors for TSWV (Riley et al. 2011). While F. fusca was reported to be more numerous in peanut when compared with F. occidentalis (Todd et al. 1995, 1996), the relative extent to which each species acquires and vectors TSWV in different growing regions is not clear. Biological aspects of different thrips species in peanut may also influence their role as TSWV vectors. In the case of F. fusca, the presence of both macropterous (full wing) and brachypterous (short-wing) forms has implications for the transmissibility of TSWV. Since brachypterous F. fusca are unable to move large distances, viruliferous forms implies a within-field source of virus acquisition and secondary spread (Chamberlin et al. 1992). Since the severity of TSWV varies among locations and years (Culbreath et al. 2010, Culbreath and Srinivasan 2011, Srinivasan et al. 2017), predicting the relative severity of TSWV in a given year may assist growers evaluating management options. Understanding the importance of different thrips vectors is useful for making such decisions. In the current study, we compared the effect of TSWV on the development of F. fusca and F. occidentalis, and their ability to acquire and transmit this virus in peanut and additional ornamental plants. We also investigated the proportions of macropterous and brachypterous F. fusca acquiring TSWV in peanut fields associated with a virus outbreak in south central Texas. Materials and Methods Thrips Colonies F. occidentalis and F. fusca, originating from field populations collected in Frio County Texas, were reared on fresh green bean pods in 30-ml plastic cups closed with a paper lid (Frontier Agricultural Sciences, Newark DE). Bean pods were sterilized in 5% v/v sodium hypochlorite and cut into 25–35-mm long sections with the ends sealed in paraffin. New colonies were set up weekly. First instars for experiments were generated by incubating female thrips on peanut ‘Florunner’ leaf discs (13-mm diameter) inside microcentrifuge vials (1.5 ml) for a 24-h oviposition period. The microcentrifuge vials were incubated at 25°C, 76% RH, and 14:10 L:D, which generated a uniform cohort of larvae (<24 h old) after 4 d. Virus Source A strain of TSWV obtained from peanut ‘Florunner’ (Frio Co, TX) was used for host acquisition and transmission experiments. Virus levels remain stable in leaves stored for up to 10 d at 4°C (Kresta et. al. 1995). To expand the scope of the study, the peanut strain of TSWV was mechanically inoculated onto Impatiens ‘Dwarf White Baby’ using methods outlined previously (Mandal et al. 2001). All plants were confirmed with DAS-ELISA to be infected prior to use in acquisition experiments. For the ELISAs, reagents and antisera were purchased commercially (Agdia Inc., Elkhart, IN) and used according to manufacturer’s instructions. Development and TSWV-Acquisition of F. fusca and F. occidentalis on Peanut and Impatiens First instar thrips were confined in small groups on both symptomatic and asymptomatic peanut leaf tissue using Tashiro-Munger cells (Tashiro 1967) and maintained at 25°C, 76% RH, and 14:10 L:D photoperiod. Asymptomatic plants came from uninoculated control plants maintained in a separate location. Leaf tissues remained viable for thrips larval development, i.e., within 7 d under these conditions (Lowry et al. 1992). Mortality was noted and propupae were transferred to individual 1.5-ml microfuge tubes. One 13-mm leaf disc from healthy peanut was added when adults emerged. Two-day-old adult thrips were ground in 50-μl buffer (Cho et al. 1988) and subjected to DAS-ELISA and analysis as per Kresta et al. (1995). Because TSWV also impacts ornamental plants (Adkins et al. 2005), the efficiency with which F. fusca and F. occidentalis larvae acquired TSWV was assessed on preflowering Impatiens using the same procedures. The infection status of all symptomatic leaf tissues was confirmed by grinding in 100-μl extraction buffer (approximately 1:10 w/v) and testing via DAS-ELISA, as per Kresta et. al. (1995). Only symptomatic samples testing positive were used in the analysis, with a minimum of 30 thrips tested for TSWV and controls per host plant treatment. Thrips were similarly tested in 50-μl buffer and subjected to ELISA at the end of the experiment. TSWV-Transmission by F. fusca and F. occidentalis on Peanut and Impatiens First instar thrips were confined individually in 1.5-ml microcentrifuge vials containing a fresh 13-mm leaf disc from symptomatic plants for a 24-h acquisition access period. Leaf tissues were again tested for TSWV, while those from healthy plants served as controls. A small piece of absorbent tissue paper at the bottom of the vial served to control humidity. Leaf discs were replaced daily with healthy plant tissues until thrips pupation. Two-day-old adult thrips (from these treatments) were provided with leaf discs from healthy plants for virus transmission assessments to the same corresponding plant species. Leaf discs were replaced daily after a 24-h inoculation access period. Thrips-exposed leaf discs were floated on distilled water in 24-well tissue culture plates at 25°C, 14:10 photoperiod for 2 wk for multiplication and lesion development (chlorotic and mosaic ring spots). Wijkamp and Peters (1993) evaluated the leaf disc method with Petunia × hybrida and reported no difference in virus transmission rates when compared with whole Petunia plants. The leaf disks and thrips were subjected to DAS-ELISA as noted above. Studies were repeated with at least 75 thrips of each species tested per host plant with tests run sequentially as the tissue/thrips were available. Transmission of TSWV by F. fusca and F. occidentalis to Additional Plants The following plants were tested; peanut ‘Florunner’, Impatiens ‘Dwarf White Baby’, and Petunia ‘Fire Chief’. Impatiens is a known systemic host, while petunia is a local lesion host and a nonsystemic infection would appear as a lesion (Wijkamp and Peters 1993). Plants were reared in growth chambers at 25°C and 14:10 (L:D) h photoperiod. Plants were grown from seed in Sunshine Mix #1 (Fisons, Vancouver, British Columbia, Canada), watered twice weekly, and fertilized weekly with 10-15-10 liquid fertilizer (Schultz All Purpose, Knox Fertilizer Co., Knox, IN). Two-day old-adult thrips that were prior inoculated with TSWV as first instars on peanut as noted above were sequentially exposed to leaf discs of each test plant for 24-h inoculation periods. Tests were done in 1.5-ml microcentrifuge vials, as noted above. Approximately 100 adult thrips of each species (F. fusca and F. occidentalis) were exposed sequentially to each of the three plant species; i.e., each thrips was exposed to each test plant provided in a random sequence. There were two sequential 24-h inoculation access periods per test plant per thrips. Effect of F. fusca Wing Form on TSWV Acquisition and Transmission (Field Population) Adult F. fusca were collected from a peanut field in Frio County, TX, that was experiencing a TSWV epidemic, with disease prevalence approaching 70% of plants. Thrips were hand collected from the foliage of plants in several fields showing yellowing and ring spot symptoms. For transmission experiments, thrips were individually transferred into 1.5-ml microfuge tubes containing a 13-mm peanut leaf disk from healthy plants, as noted above. Transmission of TSWV by each field-collected adult was assessed over 5-d, by conducting a new inoculation access period with a noninfected leaf disc every 24 h. Exposed leaf discs and thrips were individually assessed via DAS-ELISA as noted above. In total, 150 thrips were used in the study; data were compared separately for macropterous and brachypterous wing forms of either sex. Statistical Analysis Among thrips exposed to TSWV as larvae, the proportion acquiring virus (i.e., testing positive in DAS-ELISA as adults) and becoming virus transmitters (i.e., inoculating leaf discs with TSWV) were compared among treatments with Pearson χ2 tests for independence. Where appropriate, means were separated using adjusted residual (z-score) post-hoc analysis (Garcia-Perez and Nunez-Anton 2003). Since sex of the vector can influence transmission of TSWV (Van de Wetering et al. 1998, Ogada and Poehling 2015) male and female thrips were compared separately. For the last study with field-collected thrips, the frequency of virus transmission over time was analyzed using a one-way ANOVA with arcsine-transformed data. All analysis was performed with SPSS (v.24; IBM, Inc., Armonk, NY). Results Development and TSWV-Acquisition of F. fusca and F. occidentalis on Peanut and Impatiens F. fusca larval development (survival to adults reared in Tashiro-Munger cells) was affected by plant species (χ2 = 25.94 =, df = 1, P < 0.001) and whether plants were TSWV-infected (χ2 = 25.18, df = 1, P < 0.001). Survival rates were higher on peanut and noninfected leaf tissues (Table 1). However, virus acquisition rates (i.e., ELISA positive thrips) were statistically similar between those reared on TSWV-infected peanut and impatiens plants for both male (χ2 = 0.23, df = 1, P =0.63) and female thrips (χ2 = 0.93, df = 1, P = 0.34). No evidence for TSWV-acquisition was observed among virus-free control plants. In the case of F. occidentalis, successful development to adult was lower overall (≤35% survival) by comparison to F. fusca, and neither affected by plant species (χ2 = 1.97, df = 1, P = 0.16) nor the infection status of plants (χ2 = 0.65, df = 1, P = 0.42) (Table 2). Virus acquisition rates for F. occidentalis were statistically similar among plant species for both male (χ2 = 2.62, df = 1, P = 0.11) and female thrips (χ2 = 3.24, df = 1, P = 0.07). Table 1. Effect of host plant and TSWV-status on development and virus acquisition by F. fusca larvae Host plant TSWV infection N % survivala % ELISA positiveb ♂ ♀ Peanut + 54 68.5bc 82.4a 80.0a − 85 82.4a 0.0b 0.0b Impatiens + 305 47.9c 76.9a 69.1a − 103 65.7b 0.0b 0.0b Host plant TSWV infection N % survivala % ELISA positiveb ♂ ♀ Peanut + 54 68.5bc 82.4a 80.0a − 85 82.4a 0.0b 0.0b Impatiens + 305 47.9c 76.9a 69.1a − 103 65.7b 0.0b 0.0b aOf larvae through adult emergence reared in Tashiro-Munger cells. bIn adult thrips reared on test plants. cPost hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Table 1. Effect of host plant and TSWV-status on development and virus acquisition by F. fusca larvae Host plant TSWV infection N % survivala % ELISA positiveb ♂ ♀ Peanut + 54 68.5bc 82.4a 80.0a − 85 82.4a 0.0b 0.0b Impatiens + 305 47.9c 76.9a 69.1a − 103 65.7b 0.0b 0.0b Host plant TSWV infection N % survivala % ELISA positiveb ♂ ♀ Peanut + 54 68.5bc 82.4a 80.0a − 85 82.4a 0.0b 0.0b Impatiens + 305 47.9c 76.9a 69.1a − 103 65.7b 0.0b 0.0b aOf larvae through adult emergence reared in Tashiro-Munger cells. bIn adult thrips reared on test plants. cPost hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Table 2. Effect of host plant and TSWV-status on development and virus acquisition by F. occidentalis larvae Host TSWV N % Survivala % ELISA positiveb ♂ ♀ Peanut + 142 25.4ac 40.0a 38.5a − 30 16.7a 0.0b 0.0b Impatiens + 90 26.7a 25.0a 31.3a − 80 35.0a 0.0b 0.0b Host TSWV N % Survivala % ELISA positiveb ♂ ♀ Peanut + 142 25.4ac 40.0a 38.5a − 30 16.7a 0.0b 0.0b Impatiens + 90 26.7a 25.0a 31.3a − 80 35.0a 0.0b 0.0b aOf larvae through adult emergence reared in Tashiro-Munger cells. bIn adult thrips reared on test plants. cPost-hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Table 2. Effect of host plant and TSWV-status on development and virus acquisition by F. occidentalis larvae Host TSWV N % Survivala % ELISA positiveb ♂ ♀ Peanut + 142 25.4ac 40.0a 38.5a − 30 16.7a 0.0b 0.0b Impatiens + 90 26.7a 25.0a 31.3a − 80 35.0a 0.0b 0.0b Host TSWV N % Survivala % ELISA positiveb ♂ ♀ Peanut + 142 25.4ac 40.0a 38.5a − 30 16.7a 0.0b 0.0b Impatiens + 90 26.7a 25.0a 31.3a − 80 35.0a 0.0b 0.0b aOf larvae through adult emergence reared in Tashiro-Munger cells. bIn adult thrips reared on test plants. cPost-hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large TSWV-Transmission by F. fusca and F. occidentalis on Peanut and Impatiens Survival of F. fusca reared individually on test plants was neither affected by the TSWV-infection status of the plant (χ2 = 0.53, df = 1, P = 0.47) nor plant species (χ2 = 1.13, df = 1, P = 0.72) (Table 3). Virus acquisition rates from symptomatic peanut and Impatiens plants were again similar for both male (χ2 = 0.92, df = 1, P = 0.34) and female thrips (χ2 = 1.12, df = 1, P = 0.29). The proportion of adult F. fusca transmitting TSWV to leaf discs was statistically similar between peanut and Impatiens test plants, both for male (χ2 = 1.31, df = 1, P = 0.25) and female thrips (χ2 = 1.84, df = 1, P = 0.18). No evidence for TSWV-acquisition was observed among thrips reared on healthy control plants. Table 3. Host plant and TSWV-status effects on survival, acquisition and transmission by F. fusca Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 80 68.8ac 43.3a 12.0a 36.7a 24.0a − 30 86.7a 0.0b 0.0b 0.0b 0.0b Impatiens + 50 80.0a 30.0a 20.0a 20.0a 36.7a − 25 68.0a 0.0b 0.0b 0.0b 0.0b Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 80 68.8ac 43.3a 12.0a 36.7a 24.0a − 30 86.7a 0.0b 0.0b 0.0b 0.0b Impatiens + 50 80.0a 30.0a 20.0a 20.0a 36.7a − 25 68.0a 0.0b 0.0b 0.0b 0.0b aReared individually in microcentrifuge vials. bAmong all virus-exposed thrips surviving until adults. cPost hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Table 3. Host plant and TSWV-status effects on survival, acquisition and transmission by F. fusca Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 80 68.8ac 43.3a 12.0a 36.7a 24.0a − 30 86.7a 0.0b 0.0b 0.0b 0.0b Impatiens + 50 80.0a 30.0a 20.0a 20.0a 36.7a − 25 68.0a 0.0b 0.0b 0.0b 0.0b Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 80 68.8ac 43.3a 12.0a 36.7a 24.0a − 30 86.7a 0.0b 0.0b 0.0b 0.0b Impatiens + 50 80.0a 30.0a 20.0a 20.0a 36.7a − 25 68.0a 0.0b 0.0b 0.0b 0.0b aReared individually in microcentrifuge vials. bAmong all virus-exposed thrips surviving until adults. cPost hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large In the case of F. occidentalis, survival to adults was not affected by plant infection status (χ2 = 0.24, df = 1, P = 0.63) but was affected by plant species (χ2 = 18.50, df = 1, P < 0.001) (Table 4). Virus acquisition rates (based on ELISA) were low overall, and statistically similar among test plant for both male (χ2 = 0.46, df = 1, P = 0.48) and female thrips (χ2 = 0.79, df = 1, P = 0.38). The proportion of F. occidentalis exposed to TSWV as larvae and transmitting as adults was statistically similar between plant species, for both male (χ2 = 1.78, df = 1, P = 0.18) and female thrips (χ2 = 0.45, df = 1, P = 0.51). Virus acquisition rates of both thrips species were lower overall when compared with larvae reared in Tashiro-Munger cells (Tables 1 and 2). Table 4. Host plant and TSWV-status effects on survival, acquisition and transmission by F. occidentalis Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 95 34.7b 10.0ac 0.0a 10.0ac 21.7a − 25 76.0a 0.0b 0.0a 0.0b 0.0b Impatiens + 85 78.8a 12.5a 2.3a 20.8a 20.9a − 10 20.0b 0.0b 0.0a 0.0b 0.0b Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 95 34.7b 10.0ac 0.0a 10.0ac 21.7a − 25 76.0a 0.0b 0.0a 0.0b 0.0b Impatiens + 85 78.8a 12.5a 2.3a 20.8a 20.9a − 10 20.0b 0.0b 0.0a 0.0b 0.0b aReared individually in microcentrifuge vials. bAmong all virus-exposed thrips surviving until adults. cPost hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Table 4. Host plant and TSWV-status effects on survival, acquisition and transmission by F. occidentalis Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 95 34.7b 10.0ac 0.0a 10.0ac 21.7a − 25 76.0a 0.0b 0.0a 0.0b 0.0b Impatiens + 85 78.8a 12.5a 2.3a 20.8a 20.9a − 10 20.0b 0.0b 0.0a 0.0b 0.0b Host TSWV N % Survivala % ELISA positiveb % Transmittingb ♂ ♀ ♂ ♀ Peanut + 95 34.7b 10.0ac 0.0a 10.0ac 21.7a − 25 76.0a 0.0b 0.0a 0.0b 0.0b Impatiens + 85 78.8a 12.5a 2.3a 20.8a 20.9a − 10 20.0b 0.0b 0.0a 0.0b 0.0b aReared individually in microcentrifuge vials. bAmong all virus-exposed thrips surviving until adults. cPost hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Virus Transmission of F. fusca and F. occidentalis to Additional Host Plants Overall, TSWV transmission among virus exposed thrips was highest on Impatiens (average 31.4% infected leaf discs) but was relatively low on both peanut and petunia (4.9 and 1.5% infected leaf discs, respectively) (Table 5). Overall, F. fusca was the more efficient vector with this isolate of TSWV; i.e., thrips species was a significant variable for both Impatiens (χ2 = 6.55, df = 1, P = 0.01) and petunia (χ2 = 6.38, df = 1, P = 0.01) and approached significance for peanut (χ2 = 3.01, df = 1, P = 0.08). However, the sex of thrips was not a significant factor in virus transmission rates to any host plant (χ2 ≤ 1.15, df = 1, P ≥ 0.28). Table 5. Effect of thrips species and sex on TSWV-transmission to three host plants Thrips Sex N Host planta Peanut Impatiens Petunia F. fusca ♂ 40 9.5ab 42.5a 2.5ab ♀ 55 5.5a 38.2a 3.6a F. occidentalis ♂ 34 2.9a 20.6b 0.0b ♀ 66 1.5a 24.2b 0.0b Thrips Sex N Host planta Peanut Impatiens Petunia F. fusca ♂ 40 9.5ab 42.5a 2.5ab ♀ 55 5.5a 38.2a 3.6a F. occidentalis ♂ 34 2.9a 20.6b 0.0b ♀ 66 1.5a 24.2b 0.0b aTransmission during either of two 24-h inoculation access intervals. bPost-hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Table 5. Effect of thrips species and sex on TSWV-transmission to three host plants Thrips Sex N Host planta Peanut Impatiens Petunia F. fusca ♂ 40 9.5ab 42.5a 2.5ab ♀ 55 5.5a 38.2a 3.6a F. occidentalis ♂ 34 2.9a 20.6b 0.0b ♀ 66 1.5a 24.2b 0.0b Thrips Sex N Host planta Peanut Impatiens Petunia F. fusca ♂ 40 9.5ab 42.5a 2.5ab ♀ 55 5.5a 38.2a 3.6a F. occidentalis ♂ 34 2.9a 20.6b 0.0b ♀ 66 1.5a 24.2b 0.0b aTransmission during either of two 24-h inoculation access intervals. bPost-hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). View Large Effect of F. fusca Wing-Form on Virus Acquisition and Transmission (Field Population) The proportion of ELISA-positive field collected adult F. fusca was neither significantly affected by wing-form nor sex (χ2 ≤ 1.19, df = 1, P ≥ 0.28; Table 6). The proportion of all thrips transmitting TSWV was also not affected by wing form nor sex (χ2 ≤ 0.86, df = 1, P ≥ 0.35). Among viruliferous thrips, the frequency of virus transmission was not affected by wing form or sex treatments (F3,145 = 1.38, P = 0.25). Table 6. Effect of wing-form and sex on TSWV-transmission by F. fusca collected from diseased peanut (Frio County, TX) Wingform Sex N % ELISA positive % TSWV transmission All thripsa Frequencyb Macropterous ♂ 41 56.1ac 36.6ac 49.3ad ♀ 56 69.6a 26.8a 36.0a Brachypterous ♂ 33 66.7a 36.4a 48.3a ♀ 19 68.4a 42.1a 45.0a Wingform Sex N % ELISA positive % TSWV transmission All thripsa Frequencyb Macropterous ♂ 41 56.1ac 36.6ac 49.3ad ♀ 56 69.6a 26.8a 36.0a Brachypterous ♂ 33 66.7a 36.4a 48.3a ♀ 19 68.4a 42.1a 45.0a aTransmitted TSWV to leaf disc at least once (tested daily over 5 d). bFrequency of transmission among viruliferous thrips (tested daily over 5 d). cPost-hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). dPost-hoc tests (in columns) based on one-way ANOVA with arcsine-transformed data (P < 0.05). View Large Table 6. Effect of wing-form and sex on TSWV-transmission by F. fusca collected from diseased peanut (Frio County, TX) Wingform Sex N % ELISA positive % TSWV transmission All thripsa Frequencyb Macropterous ♂ 41 56.1ac 36.6ac 49.3ad ♀ 56 69.6a 26.8a 36.0a Brachypterous ♂ 33 66.7a 36.4a 48.3a ♀ 19 68.4a 42.1a 45.0a Wingform Sex N % ELISA positive % TSWV transmission All thripsa Frequencyb Macropterous ♂ 41 56.1ac 36.6ac 49.3ad ♀ 56 69.6a 26.8a 36.0a Brachypterous ♂ 33 66.7a 36.4a 48.3a ♀ 19 68.4a 42.1a 45.0a aTransmitted TSWV to leaf disc at least once (tested daily over 5 d). bFrequency of transmission among viruliferous thrips (tested daily over 5 d). cPost-hoc tests (in columns) based on adjusted residual comparisons (z-score) tests (P < 0.05). dPost-hoc tests (in columns) based on one-way ANOVA with arcsine-transformed data (P < 0.05). View Large Discussion The number of different orthotospovirus–plant–vector interactions now recognized (Riley et al. 2011, Rotenberg et al. 2015) shows that this relationship can be specific and intricate. Thus far, few studies have directly compared vector competency of difference thrips species for TSWV to different host plants. Our data confirm the ability of F. fusca and F. occidentalis to vector field strains of TSWV in peanut, as well as Impatiens. Overall F. fusca was the more competent vector of the peanut-TSWV strain, based on both acquisition and transmission studies. In the southeastern United States, F. fusca is the predominant thrips species that reproduces on peanut, and therefore considered the most important vector (Culbreath and Srinivasan 2011). Additionally, F. fusca start to colonize peanut plants early in the season at the seedling stage; by contrast F. occidentalis is mostly found later in the season when plants are less susceptible to TSWV infection (Shrestha et al. 2015). Different feeding behaviors of these thrips species may also influence virus transmission in ‘choice’ situations. Chitturi et al. (2006) showed that the addition of pine pollen increased the selection and settling behavior (attractancy) of peanut to F. occidentalis (more of a generalist feeder) but not to F. fusca. We investigated several factors influencing the epidemiology of TSWV in peanut. We found little statistical evidence for differences in the vector competency between male and female thrips. Ogada and Poehling (2015) reported that transmission efficiency of TSWV on Capsicum annum by F. occidentalis was significantly higher in males, although females had a higher percentage of transmitting individuals. Van de Wetering et al. (1998) also reported higher rates of TSWV transmission among male F. occidentalis, which they attributed to the higher mobility and probing frequency of males while feeding. Stafford et al. (2011) also reported that male TSWV-infected F. occidentalis increased their probing frequency relative to uninfected males, thus increasing the probability of virus inoculation. It is possible that our method of confining thrips on small leaf discs would partially mask effects of higher mobility and probing on transmission efficiency. We also detected no differences in vector competency between field-collected F. fusca based on wing form, confirming that either form can acquire and transmit TSWV to field peanut. Wells et al. (2002) reported no significant difference between brachypterous and macropterous F. fusca in their ability to transmit TSWV in the laboratory, although suggested that macropterous individuals are required to colonize (and hence transmit TSWV to) newly emerged crops. By contrast, brachypterous individuals, which are more common in the late fall, may help maintain TSWV between seasons by harboring inoculum in the population until the development and dispersal of macropterous thrips in the spring (Chamberlin et al. 1992, Wells et al. 2002). We observed reduced survival rates from F. fusca developing on TSWV-infected peanut, although the same trend was not observed with F. occidentalis. An earlier laboratory study by Arthurs and Heinz (2003) also detected no differences in development rates or longevity of viruliferous and nonviruliferous F. occidentalis. These results are in broad agreement with previous laboratory studies demonstrating that F. fusca is more sensitive to the direct effects of TSWV than F. occidentalis. Stumpf and Kennedy (2005) reported that TSWV-infected F. fusca developing on TSWV-infected Datura stramonium L. and Emilia sonchifolia L. plants developed more slowly and were smaller compared with virus-free adults reared on the same TSWV-infected foliage, indicating a direct, negative effect of TSWV infection on F. fusca. Comparable studies conducted later (Stumpf and Kennedy 2007) also indicated that F. occidentalis reared on TSWV-infected D. stramonium and E. sonchifolia exhibited significantly improved survival and decreased development time. In both cases, host survival and transmission of TSWV was affected by an interaction between host plant and temperature. The reason for differential development response among thrips species to TSWV-infected host plants in our tests unknown; however, we note that F. occidentalis exhibits much higher polyphagy, which may make it more tolerant of nutritional or other biochemical changes associated with tospovirus-infected hosts. We used DAS-ELISA to test for viruliferous thrips based on earlier observations (Ullman et al. 1992, Cho et al. 1988) that thrips feeding on contaminated material as larvae showed a positive result when tested with DAS-ELISA as adults. However, we observed TSWV-transmission among ELISA-negative thrips on at least one occasion, suggesting that this test may not be completely reliable for detecting low titers in thrips. By contrast, relatively higher virus-detection rates were observed among field collected thrips. An increased proportion of field-collected adult thrips testing positive (when compared with transmission rates) might be explained by false positives, i.e., virus detected in the gut of nonviruliferous adult thrips recently feeding on infected plant material. An epithelial midgut barrier prevents systemic virus acquisition by adult thrips (Ullman et al. 1992, van de Wetering et al. 1996). An alternative ELISA method to overcome this problem involves detecting a small nonstructural protein encoded by the small RNA of TSWV that is only present if the virus has replicated within the host (Wells et al. 2002, Arthurs et al. 2003). TSWV was a major problem in peanut in the 1980s and 1990s, with crop losses over 50% reported in some states (Culbreath and Srinivasan 2011). The incidence of TSWV has declined in the southern United States since the 2000s, due to the adoption of various risk reduction practices, including second- and third-generation resistant peanut cultivars; although there has been an uptick in recent years, possibly due to new resistance breaking strains of the virus (Srinivasan et al. 2017). Currently, the mechanisms influencing field resistance of TSWV in peanut are poorly understood, but include suppressed thrips feeding and development as well as constitutive and induced plant defense proteins in TSWV resistant cultivars. 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Journal

Environmental EntomologyOxford University Press

Published: Mar 27, 2018

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