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The effect of three rates of cyantraniliprole on the transmission of tomato spotted wilt virus by Frankliniella occidentalis and Frankliniella fusca (Thysanoptera: Thripidae) to Capsicum annuum
Crop Protection 30 (2011) 512e515
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Short communication
The effect of three rates of cyantraniliprole on the transmission of tomato spotted wilt virus by Frankliniella occidentalis and Frankliniella fusca (Thysanoptera: Thripidae) to Capsicum annuum A.L. Jacobson*, G.G. Kennedy* Department of Entomology, North Carolina State University, Research Annex West, Ligon Rd., Box 7630, NCSU Raleigh, NC 27695, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 August 2010 Received in revised form 1 December 2010 Accepted 9 December 2010
Tomato spotted wilt virus (TSWV) is a thrips-transmitted virus that causes major losses in many crops worldwide. Management of TSWV is complex, requiring multiple preventive measures. Currently, there are few chemical options that control thrips populations before they feed upon and transmit TSWV to crop plants. Cyantraniliprole (CyazypyrÔ) is an anthranilic diamide insecticide currently under development that exhibits anti-feedant properties. Transmission of TSWV by Frankliniella fusca (Hinds) to Capsicum annuum L. seedlings was reduced in plants treated with CyazypyrÔ applied to the soil at the rates of 1.45, 2.90 and 4.41 mg ai/plant. Mortality of F. fusca at 3 days post treatment did not differ significantly on excised foliage of CyazypyrÔ treated and control plants, but feeding injury was significantly less on treated foliage. Transmission of TSWV by Frankliniella occidentalis (Pergande) was not reduced in plants treated with 4.41 mg ai/plant. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Anthranilic diamide Cyantraniliprole Antifeedant Thrips Vector
1. Introduction Tomato spotted wilt virus (TSWV) is a thrips-transmitted plant virus capable of causing losses of up to 100% in susceptible crops, which include Capsicum annuum L., Lypersicon esculentum L., Nicotiana tabacum L. and Arachis hypogaea L. (Kucharek et al., 2000; Rosello et al., 1996). At least eight species of thrips have been reported to transmit TSWV, however, in the Southeastern U.S. the primary vectors are Frankliniella fusca (Hinds) and Frankliniella occidentalis (Pergande) (Eckel et al., 1996; McPherson et al., 1999; Whitfield et al., 2005). Thrips vectors transmit TSWV in a persistent, propagative manner, and can infect a plant in as little as 5e10 min of feeding (Chatzivassiliou, 2005; Wijkamp et al., 1996). The short inoculation access period makes management of TSWV difficult because most insecticides do not kill or debilitate the thrips vector before virus transmission occurs. The only chemical management options that currently exist include the use of imidacloprid, a neonicotinoid insecticide that alters the feeding behavior of thrips (Groves et al., 2001; Joost and Riley, 2005), acibenzolar-S-methyl, a plant protectant that induces systemic acquired resistance in the plant * Corresponding authors. Tel.: þ1 919 515 1657; fax: þ1 919 515 3748. E-mail addresses: [email protected] (A.L. Jacobson), george_kennedy@ncsu. edu (G.G. Kennedy). 0261-2194/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2010.12.004
(Mandal et al., 2008), or phorate, which is believed to induce a plant defense response (Csinos et al., 2001; Groves et al., 2001; Herbert et al., 2007; Riley and Pappu, 2004). These chemicals, however, reduce virus incidence only when applications are timed properly, and their effectiveness is not equal in all TSWV susceptible crops. Additional chemistries for TSWV management are desirable because so few options currently exist. The anthranilic diamide insecticides are a new class of insecticides with a novel mode of action targeting the ryanodine receptors in insect muscle cells (IRAC mode of action classification, group 28) (IRAC, 2007; Sattelle et al., 2008). This group of insecticides possesses anti-feedant properties that differ between chemicals and insects (Gonzales-Coloma et al., 1999). Chlorantraniliprole (RynaxypyrÔ), the first commercially available anthranilic diamide, registered for use in the USA in 2008, exhibits anti-feedant activity against chewing insects (DuPont, 2007). Cyantraniliprole (CyazypyrÔ), a new anthranilic diamide currently under development to control lepidopteran and sucking insects, is reported to be active against a broader spectrum of insects than chlorantraniliprole (Burt and Karr, 2008; PAN, 2008; Sattelle et al., 2008). If CyazypyrÔ acts like other insecticides in this class and causes rapid cessation of feeding in affected organisms, it may decrease transmission of insect-transmitted viruses, including TSWV. In this study the incidence of TSWV transmission by F. fusca and F. occidentalis to C. annuum seedlings treated with CyazypyrÔ was evaluated. Additionally, anti-feedant
A.L. Jacobson, G.G. Kennedy / Crop Protection 30 (2011) 512e515
properties were investigated by comparing feeding injury and mortality of F. fusca on C. annuum leaves excised from plants treated with CyazypyrÔ and from water-treated plants. 2. Materials and methods 2.1. Laboratory maintained insect colonies and TSWV isolates Colonies of F. fusca and F. occidentalis were maintained on Phaseolus vulgaris L. bean pods in controlled environments at 24 C with ca. 60% RH and continuous light (Loomans and Murai, 1997). Viruliferous adults of both species were obtained by placing newly emerged (0e6 h old), first instars onto TSWV-infected Emilia sonchifolia L. leaves. After 72 h, the larvae were transferred to 474 ml clear plastic cups (Reynolds Food Packaging, Richmond, VA) covered with thrips-proof screen (Midwest Filter Corporation, Elkhart, IN), and were reared to adults on uninfected bean pods (Wijkamp et al., 1995). The TSWV isolate used in these experiments was collected from L. esculentum in Montgomery County, NC in 2006 and maintained in the greenhouse in E. sonchifolia by transmission with F. fusca. 2.2. Effect of CyazypyrÔ on the transmission of TSWV C. annuum seeds were germinated in a thrips and TSWV-free greenhouse. Four true-leaf stage plants were transplanted to individual 296 ml plastic cups (Solo Cup Company, Lake Forest, IL) with a 25 mm diameter, round, fine mesh screen on the bottom. After transplant, 50 ml of insecticide solution or water was applied to the soil of each cup. CyazypyrÔ SE was tested at three rates: 1.45, 2.90 and 4.41 mg ai per plant; the latter two rates are within a range tested previously in field trials targeting other insect pests of pepper. Water was used as the control. Each rate of CyazypyrÔ was tested against a control in separate experiments. The highest rate was tested first against both F. fusca and F. occidentalis in separate experiments. Because transmission by F. occidentalis was not affected at the highest rate, the lower rates were tested only on F. fusca. Each experiment was conducted as a randomized complete block design with 4 replications and 1018 plants per treatment per replicate, depending on availability of adult thrips. Forty-eight hours after the plants had been treated, 5 randomly selected, potentially viruliferous adult thrips were released onto each C. annuum seedling (Wijkamp et al., 1995). Adults were transferred by placing 5 adults into a 1.5 ml FisherbrandÒ microcentrifuge tube (Fisher Scientific, Pittsburgh, PA) with a paintbrush, and then opening the tube and placing it at the base of the plant. Thrips were contained on the seedling by inverting a plastic cup with screened bottom over the seedling, and sealing it to the cup containing the plant using parafilmÒ. Plants were maintained
513
under constant lights at room temperature (25e30 C). Foliar applications of SpintorÒ, SC (Dow AgroSciences LLC, Indianapolis, IN) were made at a rate of 0.022 ml per plant 4 days later to kill the viruliferous adults, and subsequent applications were made 4 and 8 days after the first application to kill any larvae that may have hatched from eggs laid by the adults. On day 8, C. annuum seedlings were transplanted to 102 mm clay pots and maintained in the greenhouse at 18.9e36 C (average low and high temperatures) until they were tested for TSWV. TSWV infection was confirmed by DAS-ELISA using antisera to the nucleocapsid protein (Agdia Inc., Elkhart, IN), according to manufacturer’s instructions. The first ELISA was conducted 14 days after adults were placed onto plants to confirm early developing infections before early infected seedlings died. At 21e28 days, a second ELISA was conducted on all plants not testing positive in the first ELISA. Optical density readings were made with a THERMOmaxÒ microtiter plate reader (Molecular Devices Corp., Menlo Park, CA, USA) at 405 nm and were considered positive if the optical density reading was greater than the mean þ4 standard deviations of the optical density readings of the non-infected controls on each plate. Each experiment was analyzed separately using the logistic procedure in SAS (SAS Institute Inc., 2005) to compare transmission to CyazypyrÔ-treated plants with that to untreated control plants. Hosmer and Lemeshow goodness-of-fit values calculated for the logistic regression models were used to determine how well the data fit the calculated model. Odds ratios comparing the likelihood of TSWV transmission in the control versus the CyazypyrÔ treatment were tested using Wald Chi Square to determine if the two treatments differed significantly (i.e. odds ratio differed from one) (Davies et al., 1998). Because the different rates of CyazypyrÔ were tested against water-treated controls in separate experiments, a combined analysis of all data was conducted as an incomplete block design using the logistic procedure in SAS to test for significant treatment effects after accounting for effects of block and experiment on transmission rates. Hosmer and Lemeshow goodness-of-fit values were calculated to test significance of the logistic regression model. Contrast statements were used to conduct pair-wise comparisons between treatments using Wald Chi Square tests of odds ratios. 2.3. Feeding damage and mortality assessment An additional experiment was conducted to assess whether F. fusca produced fewer feeding scars and sustained higher mortality on foliage of plants treated with CyazypyrÔ than on foliage of control plants. Forty-eight hours after treatment, whole leaves of approximately the same size were removed from 5 watertreated and 5 C. annuum seedlings treated with 4.41 mg ai CyazypyrÔ/plant and placed individually into 50 9 mm BD
Table 1 Final incidence of TSWV transmission by Frankliniella fusca and Frankliniella occidentalis to water-treated and CyazypyrÔ-treated Capsicum annuum seedlings. Different rates of CyazypyrÔ and their respective controls were evaluated in separate experiments; F. fusca and F. occidentalis were evaluated in separate experiments. Frankliniella fusca Controlb Ratea 1.45 0.31 (0.47) 2.90 0.25 (0.44) 4.41 0.50 (0.50) Frankliniella occidentalis 4.41 0.72 (0.45) a b c d e
Nc 71 72 72
CyazypyrÔb 0.17 (0.38) 0.13 (0.33) 0.06 (0.23)
N 72 72 72
Odds Ratiod 2.70 2.50 18.98
95% CL 1.118e6.527e 1.002e6.238e 6.102e59.059e
c2 (df) 4.871 (1) 3.856 (1) 25.845 (1)
P Value 0.0273e 0.0496e <0.0001e
64
0.60 (0.49)
63
1.78
0.810e3.918
2.066 (1)
0.1506
Rate in mg ai/plant. Proportion of plants infected; mean (standard deviation). N is the total number of plants tested. Ratio of odds of transmission to the control listed to the odds of transmission in the CyazypyrÔ treatment listed in a given row. Indicates the CyazypyrÔ treatment is significantly different from the water-treated control.
514
A.L. Jacobson, G.G. Kennedy / Crop Protection 30 (2011) 512e515
Table 2 Comparison of the final incidence of TSWV transmission by Frankliniella fusca among water-treated and Capsicum annuum seedlings treated with three different rates of CyazypyrÔ. Contrast of CyazypyrÔ Treatments (mg ai/plant) 0b versus 1.45 0 versus 2.90 0 versus 4.41 2.90 versus 1.45 1.45 versus 4.41 2.90 versus 4.41
Odds Ratioa
95% CL
c2 (df)
2.24 2.47 20.83 1.02 8.55 8.45
1.059e5.567c 0.996e6.103 6.659e65.12c 0.298e3.46 2.105e34.48c 1.976e36.07c
4.391 3.812 27.240 0.0006 8.992 8.290
P Value (1) (1) (1) (1) (1) (1)
0.0361c 0.0509 <0.0001c 0.9804 0.0027c 0.0040c
a Ratio of odds of transmission to the first treatment listed to the odds of transmission in the second treatment listed in a given row. b Pooled control data from the separate experiments. c Indicates significant differences exist between the two treatments.
FalconÔ tight fit Petri dish (BD Biosciences, San Jose, CA) lined with moist filter paper. Ten adult F. fusca were placed in each Petri dish. Mortality was assessed after three days by counting dead and living adults in each Petri dish. Antifeedant effects were assessed by visually examining the abaxial and adaxial surfaces of each leaf under a dissecting microscope and estimating the percentage of total leaf surface area that was scarred by thrips feeding. Each experiment contained five replicates of each treatment, and experiments were repeated four times. Feeding damage and mortality were compared using SAS one-sided t-test procedures (SAS Institute Inc., Cary, NC) to test the hypotheses that feeding injury was less and mortality greater on CyazypyrÔ-treated plants. 3. Results and discussion Soil applications of CyazypyrÔ applied at 1.45, 2.90 and 4.41 mg ai per plant to C. annuum seedlings significantly reduced the transmission of TSWV by F. fusca when compared to the water-treated control (Table 1). The odds of TSWV transmission to water-treated pepper plants were ca. 19 times greater than to plants treated with CyazypyrÔ at 4.41 mg ai per plant. In experiments testing lower rates of CyazypyrÔ, TSWV transmission to control plants was 2.5 and 2.7 times more likely than to plants treated with CyazypyrÔ at 2.90 and 1.45 mg ai per plant, respectively. Transmission of TSWV by F. occidentalis was not significantly reduced in the plants treated with CyazypyrÔ at 4.41 mg ai per plant compared to the control (Table 1). Therefore, transmission of TSWV by this vector was not tested at the lower doses of CyazypyrÔ. In the combined analysis that compared the three rates of CyazypyrÔ and the water-treated control to each other in the experiments with F. fusca (Table 2), odds of transmission to the control plants were 2.43 and 20.82 times greater than for plants treated with CyazypyrÔ at the 1.45 and 4.41 mg ai per plant rates (P ¼ 0.0361 and P < 0001, respectively). The odds ratio for transmission to control plants vs. the 2.90 mg ai per plant rate of CyazypyrÔ was 2.46 and approached statistical significance (P ¼ 0.0509). Odds of transmission to plants treated with the 1.45 and 2.90 mg ai per plant rates of CyazypyrÔ were ca. 8.5 times greater than for plants treated with the high rate (P < 0.004). Odds of transmission to plants treated at the 1.45 and 2.90 mg ai per plant rates did not differ significantly. A comparison of the estimated percentage of feeding injury caused by F. fusca on leaves from treated and control plants showed significantly more feeding injury on leaves from control plants (x ¼ 10.3, SE ¼ 3.0) than on leaves from plants treated with 4.41 mg ai per plant of CyazypyrÔ (x ¼ 2.28; SE ¼ 0.5) (t ¼ 2.54; df ¼ 1, 19; P ¼ 0.02). However, the proportion of dead F. fusca did not differ between foliage from CyazypyrÔ-treated plants (x ¼ 0.41; SE ¼ 0.07) or control plants (x ¼ 0.37; SE ¼ 0.06) (t ¼ 0.79; df ¼ 1,19; P ¼ 0.4367).
Because CyazypyrÔ caused a significant reduction in feeding injury without a corresponding increase in mortality of F. fusca adults, it is likely that anti-feedant effects are responsible for the decreased transmission of TSWV by F. fusca observed in this study. It is possible that mortality effects may have been observed with a different experimental design, i.e. using a longer time window to observe mortality (>5 d) or waiting longer before exposing treated plants to thrips (>48 h). Mortality effects on thrips and mobility of CyazypyrÔ through C. annuum are unknown. Future studies should also be conducted utilizing techniques such as electrical penetration graph (EPG) methods (e.g. Harrewijn et al., 1996; Kindt et al., 2003, 2006) that are capable of making precise characterizations of thrips feeding behavior to better understand the responses of both F. fusca and F. occidentalis to CyazypyrÔ-treated foliage. EPG methods have previously been used to study the effects of imidacloprid on thrips feeding behavior (Groves et al., 2001; Joost and Riley, 2005). The results presented suggest that CyazypyrÔ has potential to reduce transmission of TSWV by F. fusca, with greater reduction at 4.41 than at 2.90 and 1.45 mg ai per plant, but is not effective in reducing transmission by F. occidentalis at 4.41 mg ai per plant. Further studies are needed to determine if this holds true for other crop plants affected by TSWV, and whether or not it is effective under field conditions. Reduced virus incidence was not observed in the experiments with F. occidentalis at the rate of 4.41 mg ai/ plant, however, higher rates may prove to be more effective at reducing transmission of TSWV by this vector. Disclosure DuPont has sponsored field evaluations of CyazypyrÔ for insect control by G.G. Kennedy during 2008, 2009 and 2010 and is providing support for additional research on anti-feedant effects of CyazypyrÔ. Acknowledgements We would like to thank Carol Berger for her instruction and assistance with rearing and maintaining TSWV isolates, and Robert Williams of DuPont for providing the CyazypyrÔ used in these experiments. We would also like to thank Chris Franck from the Department of Statistics at North Carolina State University for his assistance with our statistical analysis. References Burt, A., Karr, D., 2008. DuPont and Syngenta Enter into Crop Protection Technology Exchange. http://vocuspr.vocus.com/VocusPR30/Newsroom/Query. aspx?SiteName¼DuPontNew&Entity¼PRAsset&SF_PRAsset_PRAssetID_EQ¼ 110201&XSL¼PressRelease&;Cache¼. Chatzivassiliou, E.K., 2005. Thrips tabaci: an ambiguous vector of TSWV in perspective. In: Proceedings of the 7th International Symposium on Thysanoptera. CSIRO, Australia, pp. 69e75. Csinos, A.S., Pappu, H.R., McPherson, R.M., Stephenson, M.G., 2001. Management of Tomato spotted wilt virus in flue-cured tobacco with Acibenzolar-S-Methyl and Imidacloprid. Plant Dis. 85, 292e296. Davies, H.T.O., Crombie, I.K., Tavakoli, M., 1998. When can odds ratios mislead? BMJ 316, 989e991. DuPont, 2007. RynaxypyrÔ Technical Bulletin. http://www2.dupont.com/Production_ Agriculture/en_US/assets/downloads/pdfs/Rynaxypyr_Tech_Bulletin.pdf. Eckel, C.S., Cho, K., Walgenbach, J.F., Kennedy, G.G., Moyer, J.W., 1996. Variation in thrips species composition in field crops and implications for tomato spotted wilt epidemiology in North Carolina. Entomol. Exp. Appl. 78, 19e29. Gonzales-Coloma, A., Gutierrez, C., Hubner, H., Achenbach, H., Terrero, D., Fraga, B.M., 1999. Selective insect antifeedant and toxic action of ryanoid diterpenes. J. Agric. Food Chem. 47, 4419e4424. Groves, R.L., Sorenson, C.E., Walgenbach, J.F., Kennedy, G.G., 2001. Effects of imidacloprid on transmission of tomato spotted wilt tospovirus to pepper, tomato and tobacco by Frankliniella fusca Hinds (Thysanoptera: Thripidae). Crop Prot. 20, 439e445. Harrewijn, P., Tjallingii, W.F., Mollema, C., 1996. Electrical recording of plant penetration by western flower thrips. Entomol. Exp. App. 79, 345e353.
A.L. Jacobson, G.G. Kennedy / Crop Protection 30 (2011) 512e515 Herbert Jr., D.A., Malone, S., Aref, S., Brandenburg, R.L., Jordan, D.L., Royals, B.M., Johnson, P.D., 2007. Role of insecticides in reducing thrips injury to plants and incidence of tomato spotted wilt virus in Virginia market-type peanut. J. Econ. Entomol. 100, 1241e1247. Insecticide Resistance Action Committee(IRAC), 2007. IRAC Mode of Action Classification Version 5.3. http://www.irac-online.org/documents/IRAC%20MoA %20Classification%20v5_3.pdf. Joost, P.H., Riley, D.G., 2005. Imidacloprid effects on probing and settling behavior of Frankliniella fusca and Frankliniella occidentalis (Thysanoptera: Thripidae) in tomato. J. Econ. Entomol. 98, 1622e1629. Kindt, F., Joosten, N.N., Peters, D., Tjallingii, W.F., 2003. Characterisation of the feeding behavior of western flower thrips in terms of electrical penetration graph (EPG) waveforms. J. Ins. Physiol. 49, 183e191. Kindt, F., Joosten, N.N., Tjallingii, W.F., 2006. Electrical penetration graphs of thrips revised: combining DC- and AC-EPG signals. J. Ins. Physiol. 52, 1e10. Kucharek, T., Brown, L., Johnson, F., Funderburk, J., 2000. Tomato Spotted Wilt Virus of Agronomic, Vegetable, and Ornamental Crops. Plant Pathology Fact Sheet Circ-914. Florida Cooperative Extension Service. http://plantpath.ifas.ufl.edu/ takextpub/FactSheets/circ0914.pdf. Loomans, A.J.M., Murai, T., 1997. Culturing thrips and parasitoids. In: Lewis, T. (Ed.), Thrips as Crop Pests. CAB International, New York, pp. 484e485. Mandal, B., Mandal, S., Csinos, A.S., Martinez, N., Culbreath, A.K., Pappu, H.R., 2008. Biological and molecular analysis of the Acibenzolar-S-Methyl-induced systemic acquired resistance in flue-cured tobacco against Tomato spotted wilt virus. Phytopathology 98, 196e204.
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McPherson, R.M., Pappu, H.R., Jones, D.C., 1999. Occurrence of five thrips species on flue-cured tobacco and impact on spotted wilt disease incidence in Georgia. Plant Dis. 83, 765e767. Pesticide Action Network (PAN), 2008. DuPont/Syngenta Enter Product Sharing Agreement. The Pesticide Monitor. 8. http://www.panap.net/uploads/media/ Pesticide_Monitor-_Vol_8_No_3.pdf. Riley, D.G., Pappu, H.R., 2004. Tactics for management of thrips (Thysanoptera: Thripidae) and tomato spotted wilt virus in tomato. J. Econ. Entomol. 97, 1648e1658. Rosello, S., Díez, M.J., Nuez, F., 1996. Viral diseases causing the greatest economic losses to the tomato crop. I. The Tomato spotted wilt virus d a review. Scientia Horticulturae 67, 117e150. SAS Institute Inc., 2005. SASÒ 9.1.3 Intelligence Platform: Single-User Installation Guide Cary, North Carolina, USA. Sattelle, D.B., Cordova, D., Cheek, T.R., 2008. Insect ryanodine receptors: molecular targets for novel control chemicals. Invert. Neurosci. 8, 107e119. doi:10.1007/ s10158-008-0076-4. Whitfield, A.E., Ullman, D.E., German, T.L., 2005. Tospovirus-thrips interactions. Annu. Rev. Phytopathology 43, 459e489. Wijkamp, I., Almarza, N., Peters, D., 1995. Median latent period and transmission of tospoviruses vectored by thrips. In: Parker, B.L. (Ed.), Thrips Biology and Management. Plenum Press, New York, pp. 153e156. Wijkamp, I., Van De Wettering, F., Goldbach, R., Peters, D., 1996. Transmission of tomato spotted wilt virus by Frankliniella occidentalis median acquisition and inoculation access period. Ann. App. Biol. 129, 303e313.
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Short communication
The effect of three rates of cyantraniliprole on the transmission of tomato spotted wilt virus by Frankliniella occidentalis and Frankliniella fusca (Thysanoptera: Thripidae) to Capsicum annuum A.L. Jacobson*, G.G. Kennedy* Department of Entomology, North Carolina State University, Research Annex West, Ligon Rd., Box 7630, NCSU Raleigh, NC 27695, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 August 2010 Received in revised form 1 December 2010 Accepted 9 December 2010
Tomato spotted wilt virus (TSWV) is a thrips-transmitted virus that causes major losses in many crops worldwide. Management of TSWV is complex, requiring multiple preventive measures. Currently, there are few chemical options that control thrips populations before they feed upon and transmit TSWV to crop plants. Cyantraniliprole (CyazypyrÔ) is an anthranilic diamide insecticide currently under development that exhibits anti-feedant properties. Transmission of TSWV by Frankliniella fusca (Hinds) to Capsicum annuum L. seedlings was reduced in plants treated with CyazypyrÔ applied to the soil at the rates of 1.45, 2.90 and 4.41 mg ai/plant. Mortality of F. fusca at 3 days post treatment did not differ significantly on excised foliage of CyazypyrÔ treated and control plants, but feeding injury was significantly less on treated foliage. Transmission of TSWV by Frankliniella occidentalis (Pergande) was not reduced in plants treated with 4.41 mg ai/plant. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Anthranilic diamide Cyantraniliprole Antifeedant Thrips Vector
1. Introduction Tomato spotted wilt virus (TSWV) is a thrips-transmitted plant virus capable of causing losses of up to 100% in susceptible crops, which include Capsicum annuum L., Lypersicon esculentum L., Nicotiana tabacum L. and Arachis hypogaea L. (Kucharek et al., 2000; Rosello et al., 1996). At least eight species of thrips have been reported to transmit TSWV, however, in the Southeastern U.S. the primary vectors are Frankliniella fusca (Hinds) and Frankliniella occidentalis (Pergande) (Eckel et al., 1996; McPherson et al., 1999; Whitfield et al., 2005). Thrips vectors transmit TSWV in a persistent, propagative manner, and can infect a plant in as little as 5e10 min of feeding (Chatzivassiliou, 2005; Wijkamp et al., 1996). The short inoculation access period makes management of TSWV difficult because most insecticides do not kill or debilitate the thrips vector before virus transmission occurs. The only chemical management options that currently exist include the use of imidacloprid, a neonicotinoid insecticide that alters the feeding behavior of thrips (Groves et al., 2001; Joost and Riley, 2005), acibenzolar-S-methyl, a plant protectant that induces systemic acquired resistance in the plant * Corresponding authors. Tel.: þ1 919 515 1657; fax: þ1 919 515 3748. E-mail addresses: [email protected] (A.L. Jacobson), george_kennedy@ncsu. edu (G.G. Kennedy). 0261-2194/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2010.12.004
(Mandal et al., 2008), or phorate, which is believed to induce a plant defense response (Csinos et al., 2001; Groves et al., 2001; Herbert et al., 2007; Riley and Pappu, 2004). These chemicals, however, reduce virus incidence only when applications are timed properly, and their effectiveness is not equal in all TSWV susceptible crops. Additional chemistries for TSWV management are desirable because so few options currently exist. The anthranilic diamide insecticides are a new class of insecticides with a novel mode of action targeting the ryanodine receptors in insect muscle cells (IRAC mode of action classification, group 28) (IRAC, 2007; Sattelle et al., 2008). This group of insecticides possesses anti-feedant properties that differ between chemicals and insects (Gonzales-Coloma et al., 1999). Chlorantraniliprole (RynaxypyrÔ), the first commercially available anthranilic diamide, registered for use in the USA in 2008, exhibits anti-feedant activity against chewing insects (DuPont, 2007). Cyantraniliprole (CyazypyrÔ), a new anthranilic diamide currently under development to control lepidopteran and sucking insects, is reported to be active against a broader spectrum of insects than chlorantraniliprole (Burt and Karr, 2008; PAN, 2008; Sattelle et al., 2008). If CyazypyrÔ acts like other insecticides in this class and causes rapid cessation of feeding in affected organisms, it may decrease transmission of insect-transmitted viruses, including TSWV. In this study the incidence of TSWV transmission by F. fusca and F. occidentalis to C. annuum seedlings treated with CyazypyrÔ was evaluated. Additionally, anti-feedant
A.L. Jacobson, G.G. Kennedy / Crop Protection 30 (2011) 512e515
properties were investigated by comparing feeding injury and mortality of F. fusca on C. annuum leaves excised from plants treated with CyazypyrÔ and from water-treated plants. 2. Materials and methods 2.1. Laboratory maintained insect colonies and TSWV isolates Colonies of F. fusca and F. occidentalis were maintained on Phaseolus vulgaris L. bean pods in controlled environments at 24 C with ca. 60% RH and continuous light (Loomans and Murai, 1997). Viruliferous adults of both species were obtained by placing newly emerged (0e6 h old), first instars onto TSWV-infected Emilia sonchifolia L. leaves. After 72 h, the larvae were transferred to 474 ml clear plastic cups (Reynolds Food Packaging, Richmond, VA) covered with thrips-proof screen (Midwest Filter Corporation, Elkhart, IN), and were reared to adults on uninfected bean pods (Wijkamp et al., 1995). The TSWV isolate used in these experiments was collected from L. esculentum in Montgomery County, NC in 2006 and maintained in the greenhouse in E. sonchifolia by transmission with F. fusca. 2.2. Effect of CyazypyrÔ on the transmission of TSWV C. annuum seeds were germinated in a thrips and TSWV-free greenhouse. Four true-leaf stage plants were transplanted to individual 296 ml plastic cups (Solo Cup Company, Lake Forest, IL) with a 25 mm diameter, round, fine mesh screen on the bottom. After transplant, 50 ml of insecticide solution or water was applied to the soil of each cup. CyazypyrÔ SE was tested at three rates: 1.45, 2.90 and 4.41 mg ai per plant; the latter two rates are within a range tested previously in field trials targeting other insect pests of pepper. Water was used as the control. Each rate of CyazypyrÔ was tested against a control in separate experiments. The highest rate was tested first against both F. fusca and F. occidentalis in separate experiments. Because transmission by F. occidentalis was not affected at the highest rate, the lower rates were tested only on F. fusca. Each experiment was conducted as a randomized complete block design with 4 replications and 1018 plants per treatment per replicate, depending on availability of adult thrips. Forty-eight hours after the plants had been treated, 5 randomly selected, potentially viruliferous adult thrips were released onto each C. annuum seedling (Wijkamp et al., 1995). Adults were transferred by placing 5 adults into a 1.5 ml FisherbrandÒ microcentrifuge tube (Fisher Scientific, Pittsburgh, PA) with a paintbrush, and then opening the tube and placing it at the base of the plant. Thrips were contained on the seedling by inverting a plastic cup with screened bottom over the seedling, and sealing it to the cup containing the plant using parafilmÒ. Plants were maintained
513
under constant lights at room temperature (25e30 C). Foliar applications of SpintorÒ, SC (Dow AgroSciences LLC, Indianapolis, IN) were made at a rate of 0.022 ml per plant 4 days later to kill the viruliferous adults, and subsequent applications were made 4 and 8 days after the first application to kill any larvae that may have hatched from eggs laid by the adults. On day 8, C. annuum seedlings were transplanted to 102 mm clay pots and maintained in the greenhouse at 18.9e36 C (average low and high temperatures) until they were tested for TSWV. TSWV infection was confirmed by DAS-ELISA using antisera to the nucleocapsid protein (Agdia Inc., Elkhart, IN), according to manufacturer’s instructions. The first ELISA was conducted 14 days after adults were placed onto plants to confirm early developing infections before early infected seedlings died. At 21e28 days, a second ELISA was conducted on all plants not testing positive in the first ELISA. Optical density readings were made with a THERMOmaxÒ microtiter plate reader (Molecular Devices Corp., Menlo Park, CA, USA) at 405 nm and were considered positive if the optical density reading was greater than the mean þ4 standard deviations of the optical density readings of the non-infected controls on each plate. Each experiment was analyzed separately using the logistic procedure in SAS (SAS Institute Inc., 2005) to compare transmission to CyazypyrÔ-treated plants with that to untreated control plants. Hosmer and Lemeshow goodness-of-fit values calculated for the logistic regression models were used to determine how well the data fit the calculated model. Odds ratios comparing the likelihood of TSWV transmission in the control versus the CyazypyrÔ treatment were tested using Wald Chi Square to determine if the two treatments differed significantly (i.e. odds ratio differed from one) (Davies et al., 1998). Because the different rates of CyazypyrÔ were tested against water-treated controls in separate experiments, a combined analysis of all data was conducted as an incomplete block design using the logistic procedure in SAS to test for significant treatment effects after accounting for effects of block and experiment on transmission rates. Hosmer and Lemeshow goodness-of-fit values were calculated to test significance of the logistic regression model. Contrast statements were used to conduct pair-wise comparisons between treatments using Wald Chi Square tests of odds ratios. 2.3. Feeding damage and mortality assessment An additional experiment was conducted to assess whether F. fusca produced fewer feeding scars and sustained higher mortality on foliage of plants treated with CyazypyrÔ than on foliage of control plants. Forty-eight hours after treatment, whole leaves of approximately the same size were removed from 5 watertreated and 5 C. annuum seedlings treated with 4.41 mg ai CyazypyrÔ/plant and placed individually into 50 9 mm BD
Table 1 Final incidence of TSWV transmission by Frankliniella fusca and Frankliniella occidentalis to water-treated and CyazypyrÔ-treated Capsicum annuum seedlings. Different rates of CyazypyrÔ and their respective controls were evaluated in separate experiments; F. fusca and F. occidentalis were evaluated in separate experiments. Frankliniella fusca Controlb Ratea 1.45 0.31 (0.47) 2.90 0.25 (0.44) 4.41 0.50 (0.50) Frankliniella occidentalis 4.41 0.72 (0.45) a b c d e
Nc 71 72 72
CyazypyrÔb 0.17 (0.38) 0.13 (0.33) 0.06 (0.23)
N 72 72 72
Odds Ratiod 2.70 2.50 18.98
95% CL 1.118e6.527e 1.002e6.238e 6.102e59.059e
c2 (df) 4.871 (1) 3.856 (1) 25.845 (1)
P Value 0.0273e 0.0496e <0.0001e
64
0.60 (0.49)
63
1.78
0.810e3.918
2.066 (1)
0.1506
Rate in mg ai/plant. Proportion of plants infected; mean (standard deviation). N is the total number of plants tested. Ratio of odds of transmission to the control listed to the odds of transmission in the CyazypyrÔ treatment listed in a given row. Indicates the CyazypyrÔ treatment is significantly different from the water-treated control.
514
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Table 2 Comparison of the final incidence of TSWV transmission by Frankliniella fusca among water-treated and Capsicum annuum seedlings treated with three different rates of CyazypyrÔ. Contrast of CyazypyrÔ Treatments (mg ai/plant) 0b versus 1.45 0 versus 2.90 0 versus 4.41 2.90 versus 1.45 1.45 versus 4.41 2.90 versus 4.41
Odds Ratioa
95% CL
c2 (df)
2.24 2.47 20.83 1.02 8.55 8.45
1.059e5.567c 0.996e6.103 6.659e65.12c 0.298e3.46 2.105e34.48c 1.976e36.07c
4.391 3.812 27.240 0.0006 8.992 8.290
P Value (1) (1) (1) (1) (1) (1)
0.0361c 0.0509 <0.0001c 0.9804 0.0027c 0.0040c
a Ratio of odds of transmission to the first treatment listed to the odds of transmission in the second treatment listed in a given row. b Pooled control data from the separate experiments. c Indicates significant differences exist between the two treatments.
FalconÔ tight fit Petri dish (BD Biosciences, San Jose, CA) lined with moist filter paper. Ten adult F. fusca were placed in each Petri dish. Mortality was assessed after three days by counting dead and living adults in each Petri dish. Antifeedant effects were assessed by visually examining the abaxial and adaxial surfaces of each leaf under a dissecting microscope and estimating the percentage of total leaf surface area that was scarred by thrips feeding. Each experiment contained five replicates of each treatment, and experiments were repeated four times. Feeding damage and mortality were compared using SAS one-sided t-test procedures (SAS Institute Inc., Cary, NC) to test the hypotheses that feeding injury was less and mortality greater on CyazypyrÔ-treated plants. 3. Results and discussion Soil applications of CyazypyrÔ applied at 1.45, 2.90 and 4.41 mg ai per plant to C. annuum seedlings significantly reduced the transmission of TSWV by F. fusca when compared to the water-treated control (Table 1). The odds of TSWV transmission to water-treated pepper plants were ca. 19 times greater than to plants treated with CyazypyrÔ at 4.41 mg ai per plant. In experiments testing lower rates of CyazypyrÔ, TSWV transmission to control plants was 2.5 and 2.7 times more likely than to plants treated with CyazypyrÔ at 2.90 and 1.45 mg ai per plant, respectively. Transmission of TSWV by F. occidentalis was not significantly reduced in the plants treated with CyazypyrÔ at 4.41 mg ai per plant compared to the control (Table 1). Therefore, transmission of TSWV by this vector was not tested at the lower doses of CyazypyrÔ. In the combined analysis that compared the three rates of CyazypyrÔ and the water-treated control to each other in the experiments with F. fusca (Table 2), odds of transmission to the control plants were 2.43 and 20.82 times greater than for plants treated with CyazypyrÔ at the 1.45 and 4.41 mg ai per plant rates (P ¼ 0.0361 and P < 0001, respectively). The odds ratio for transmission to control plants vs. the 2.90 mg ai per plant rate of CyazypyrÔ was 2.46 and approached statistical significance (P ¼ 0.0509). Odds of transmission to plants treated with the 1.45 and 2.90 mg ai per plant rates of CyazypyrÔ were ca. 8.5 times greater than for plants treated with the high rate (P < 0.004). Odds of transmission to plants treated at the 1.45 and 2.90 mg ai per plant rates did not differ significantly. A comparison of the estimated percentage of feeding injury caused by F. fusca on leaves from treated and control plants showed significantly more feeding injury on leaves from control plants (x ¼ 10.3, SE ¼ 3.0) than on leaves from plants treated with 4.41 mg ai per plant of CyazypyrÔ (x ¼ 2.28; SE ¼ 0.5) (t ¼ 2.54; df ¼ 1, 19; P ¼ 0.02). However, the proportion of dead F. fusca did not differ between foliage from CyazypyrÔ-treated plants (x ¼ 0.41; SE ¼ 0.07) or control plants (x ¼ 0.37; SE ¼ 0.06) (t ¼ 0.79; df ¼ 1,19; P ¼ 0.4367).
Because CyazypyrÔ caused a significant reduction in feeding injury without a corresponding increase in mortality of F. fusca adults, it is likely that anti-feedant effects are responsible for the decreased transmission of TSWV by F. fusca observed in this study. It is possible that mortality effects may have been observed with a different experimental design, i.e. using a longer time window to observe mortality (>5 d) or waiting longer before exposing treated plants to thrips (>48 h). Mortality effects on thrips and mobility of CyazypyrÔ through C. annuum are unknown. Future studies should also be conducted utilizing techniques such as electrical penetration graph (EPG) methods (e.g. Harrewijn et al., 1996; Kindt et al., 2003, 2006) that are capable of making precise characterizations of thrips feeding behavior to better understand the responses of both F. fusca and F. occidentalis to CyazypyrÔ-treated foliage. EPG methods have previously been used to study the effects of imidacloprid on thrips feeding behavior (Groves et al., 2001; Joost and Riley, 2005). The results presented suggest that CyazypyrÔ has potential to reduce transmission of TSWV by F. fusca, with greater reduction at 4.41 than at 2.90 and 1.45 mg ai per plant, but is not effective in reducing transmission by F. occidentalis at 4.41 mg ai per plant. Further studies are needed to determine if this holds true for other crop plants affected by TSWV, and whether or not it is effective under field conditions. Reduced virus incidence was not observed in the experiments with F. occidentalis at the rate of 4.41 mg ai/ plant, however, higher rates may prove to be more effective at reducing transmission of TSWV by this vector. Disclosure DuPont has sponsored field evaluations of CyazypyrÔ for insect control by G.G. Kennedy during 2008, 2009 and 2010 and is providing support for additional research on anti-feedant effects of CyazypyrÔ. Acknowledgements We would like to thank Carol Berger for her instruction and assistance with rearing and maintaining TSWV isolates, and Robert Williams of DuPont for providing the CyazypyrÔ used in these experiments. We would also like to thank Chris Franck from the Department of Statistics at North Carolina State University for his assistance with our statistical analysis. References Burt, A., Karr, D., 2008. DuPont and Syngenta Enter into Crop Protection Technology Exchange. http://vocuspr.vocus.com/VocusPR30/Newsroom/Query. aspx?SiteName¼DuPontNew&Entity¼PRAsset&SF_PRAsset_PRAssetID_EQ¼ 110201&XSL¼PressRelease&;Cache¼. Chatzivassiliou, E.K., 2005. Thrips tabaci: an ambiguous vector of TSWV in perspective. In: Proceedings of the 7th International Symposium on Thysanoptera. CSIRO, Australia, pp. 69e75. Csinos, A.S., Pappu, H.R., McPherson, R.M., Stephenson, M.G., 2001. Management of Tomato spotted wilt virus in flue-cured tobacco with Acibenzolar-S-Methyl and Imidacloprid. Plant Dis. 85, 292e296. Davies, H.T.O., Crombie, I.K., Tavakoli, M., 1998. When can odds ratios mislead? BMJ 316, 989e991. DuPont, 2007. RynaxypyrÔ Technical Bulletin. http://www2.dupont.com/Production_ Agriculture/en_US/assets/downloads/pdfs/Rynaxypyr_Tech_Bulletin.pdf. Eckel, C.S., Cho, K., Walgenbach, J.F., Kennedy, G.G., Moyer, J.W., 1996. Variation in thrips species composition in field crops and implications for tomato spotted wilt epidemiology in North Carolina. Entomol. Exp. Appl. 78, 19e29. Gonzales-Coloma, A., Gutierrez, C., Hubner, H., Achenbach, H., Terrero, D., Fraga, B.M., 1999. Selective insect antifeedant and toxic action of ryanoid diterpenes. J. Agric. Food Chem. 47, 4419e4424. Groves, R.L., Sorenson, C.E., Walgenbach, J.F., Kennedy, G.G., 2001. Effects of imidacloprid on transmission of tomato spotted wilt tospovirus to pepper, tomato and tobacco by Frankliniella fusca Hinds (Thysanoptera: Thripidae). Crop Prot. 20, 439e445. Harrewijn, P., Tjallingii, W.F., Mollema, C., 1996. Electrical recording of plant penetration by western flower thrips. Entomol. Exp. App. 79, 345e353.
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