Proposal Category: x Research Outreach Proposal Status: x New Proposal Previously funded by SRSFC; has been previously funded for years Determining the status of twospotted spider mite acaricide resistance in southeast strawberries Rebecca Schmidt-Jeffris 2700 Savannah Hwy Charleston, SC 29414 rschmi3@clemson.edu Hannah Burrack Box 7634, NCSU Raleigh, NC 27695 hannah burrack@ncsu.edu Abstract The purpose of this project was to determine the extent of acaricide resistance in twospotted spider mite (TSSM) populations from strawberry in North (NC) and South Carolina (SC). This was done by collecting populations of TSSM from strawberry fields, rearing the populations in colonies, and then applying acaricides to the mites in laboratory assays. For each population, we used a diagnostic dose of the high field rate divided by 5 (FD/5) to determine if resistance was likely for a particular active ingredient. Any populations with <50% mortality at FD/5 was further screened to determine the lethal dose that kills 50% of the population (LD50). This allowed us to compare LD50 s between the field-collected populations and a laboratory population that was known to be pesticide susceptible. Unfortunately, the rate of mortality in the water check was high the NC populations, so these assays will be repeated. In the SC populations, the pre-screen identified that all populations were likely to be resistant to bifenthrin (Brigade) and fenbutatin-oxide (Vendex). One population was also flagged for potential abamectin (Agri-Mek) resistance. The LD50 calculations determined that the tested populations were 100-60,000 less susceptible to bifenthrin and 25-123 less susceptible to fenbutatin-oxide than the laboratory population. The population tested for abamectin resistance was 25 less susceptible. In the case of bifenthrin and abamectin, the LD50 values were above or near field rate, indicating very likely application failure of these products. Fenbutatin-oxide field failure may also occur due to resistance. Growers should use and rotate between the newer acaricide products for which resistance has not yet been observed in strawberry to delay resistance development. Objectives: 1. Determine presence of acaricide resistance in twospotted spider mite populations from SC and NC strawberries using diagnostic doses 2. Determine the LD50 of any population determined to be resistant 3. Educate growers about resistance occurrence and management
Introduction Tetranychus urticae, commonly known as twospotted spider mite (TSSM), is an extremely polyphagous insect with a worldwide host range making them a serious pest of crops globally. TSSM are considered a pest because they pierce into plant cells and feed on the xylem, which causes leaf spotting and if the infestation because severe enough can result in leaf necrosis. In strawberry, this damage has been reported to cause 10-25% crop loss (Gimenez- Ferrer et al., 1994; Walsh et al., 1998). With warmer, drier years becoming increasingly common twospotted spider mite outbreaks continue to increase, which could result in greater crop damage. In strawberry, it is common for broad-spectrum insecticides to be applied to manage pests. The application of these insecticides causes the natural enemy population to decline, resulting in TSSM outbreaks. Once these outbreaks occur, apply acaricides to control TSSM. First, TSSM is intensively managed in both the nursery and fruit production phases of production, and for mites transferred from nursery to production fields, they are potentially exposed to acaricide residues for 10 to 12 continuous months. Frequent opportunities for resistance development are particularly problematic for TSSM, as this pest is notorious for rapid resistance development. Therefore, it is in the continued interest of growers to screen population to provide updated resistance information to improve management of systems. The goal of this study was to determine the current extent of TSSM resistance to acaricides registered in strawberry. Knowing the status of acaricide resistance will aid growers in decision making and allow for immediate improvements in management of TSSM. Seeing the magnitude of resistance in some populations may lead to increased adoption of chemical rotation and non-chemical management practices, including biological control. These studies will also serve as baseline resistance data, which can be used to track the development of resistance, especially to newer materials (e.g. Nealta). This baseline data is lacking for strawberries and many other crops in the southeast. Materials and Methods Colony Collection and Rearing. TSSM populations were collected from farms across the states of South Carolina and North Carolina (Table 1) and a susceptible population was obtained from Cornell University. The populations were then reared in isolated colonies on lima bean plants. Diagnostic Dose Bioassays. Plastic cups (96.1 ml) were used as arenas for all bioassays. Five replicates Table 1. Collection information for tested TSM populations. Population # City GPS Collection date SC-1 Lake City, SC 33.8717, -79.9097 31 Mar 2018 SC-2 Gable, SC 33.8763, -80.1327 31 Mar 2018 SC-3 Lexington, SC 33.9582, -81.3102 2 Apr 2018 SC-4 York, SC 35.0406, -81.2493 3 Apr 2018 SC-5 Fort Mill, SC 34.9798, -80.9605 3 Apr 2018 SC-6 Mt. Pleasant, SC 32.8544, -79.8227 19 Apr 2018 NC-1 Clinton, NC 35.0252,-78.2764 13 Jun 2018 NC-2 Carthage, NC 35.3550, -79.3454 25 May 2018 NC-3 Indian Trail, NC 35.0873, -80.5969 25 May 2018
per treatment were prepared. Within each replicate arena, a 0.5% solution of agar was poured in the cup and allowed to solidify. A bean leaf disk (2.2 cm) was placed lower surface facing upwards on this solution. For adulticides, 20 female TSSM were placed in the arena. The acaricide concentration used was based on the maximum label rate of the product for strawberry, applied at 100 GPA (935 L/ha) (Table 2). All acaricide solutions were created by mixing the appropriate amount of formulated product to make a 1 L solution. The arenas were sprayed with 2 ml of acaricide or water using a Potter Tower (Burkard Mfg, Rickmansworth, UK). Following application, arenas were held in a growth chamber at 27.29 ± 0.01 C, 75.0 ± 0.3% RH, 16:8 light and 16:8 photoperiod. The assays were evaluated at 24, 48, and 72 h following treatment to determine the number of live, dead, and runoff (drowned in agar) females. This was repeated for all field-collected colonies being screened. Data shown in results are for 24 h only. For ovicides, arenas were prepared in the same manner as previously mentioned and there were five replicates per treatment. Ten adult female TSSM were placed on each arena and held in a growth chamber overnight. The following day, adult female TSSM were removed and 20 eggs were marked in the arena, while all other eggs were removed. Following this, acaricides were applied as indicated above (Table 2). These assays were held until egg hatch in the check was >90%, at which point all treatments were evaluated to determine the number of hatched and unhatched eggs per arena. This was repeated for all field collected colonies that were screened. Dose Response Bioassays. If percent mortality or hatch was <50% for any diagnostic dose bioassay, an additional bioassay was conducted to determine the lethal dose for 50% (LD50) of the population for each acaricide and population tested. Each acaricide was tested using 6 serially diluted rates of product and a water control. These assays were set up as previously described, with five replicates per dose. Assays were evaluated in the same manner as diagnostic bioassays. This was also the only bioassay conducted for the susceptible population. Statistical Analysis. Mortality and hatch data from dose response bioassays were analyzed using POLO-Plus (LeOra Software, Parma, MO) to LD50 of the population for each acaricide and colony tested. A resistance ratio (RR) was calculated for each material and population by taking the field collected population LD50 divided by the susceptible population (RR= LD50 resistant/ld50 susceptible). Table 2. Acaricides used in resistance screening bioassays. Active Ingredient Classification Mode of Action Brand Name/Formulation FD/5 (g AI/ha) Bifenthrin Adulticide 3A Brigade WSB 44.83 Abamectin Adulticide 6 Agri-Mek SC 4.29 Bifenazate Adulticide 20D Acramite 50WS 45.36 Acequinocyl Adulticide 20B Kanemite 15 SC 67.86 Cyflumetofen Adulticide 25 Nealta 40.07 Fenpyroximate Adulticide 21A Portal XLO 9.07 Fenbutatin-oxide Adulticide 12B Vendex 50 WP 90.72 Spiromesifen Ovicide 23 Oberon 2 SC 56.04 Hexythiazox Ovicide 10A Savey 50DF 42.03 Etoxazole Ovicide 10B Zeal Miticide 30.26
Results and Discussion Diagnostic Dose Bioassays. Check mortality was much higher in the NC populations than the SC populations in both ovicide and adulticides bioassays (Table 3). Runoff was higher in these assays, so lids were added to the arenas. It is possible that the increased humidity within the arena was harmful to TSSM. Assays will be repeated for these populations this winter. Based on current results, populations NC-2 and NC-3 are likely to be resistant to bifenthrin and NC-3 is also likely to be resistant to fenbutatin-oxide (Table 3). Additional active ingredients may be flagged for screening after these assays are repeated. All SC populations were marked as potentially resistant to bifenthrin, with percent mortality ranging from 2-22% at FD/5 (Table 3). Similarly, all SC populations were flagged for dose response screening to fenbutatin-oxide. One population (SC-1) was borderline (53% mortality), but the standard error overlapped the <50% mortality criteria (Table 3). Finally, SC-2 was also marked as potentially resistant to abamectin, with FD/5 mortality at 32% (Table 3). Dose Response Bioassays. LC50 values were successfully determined for the susceptible population for all tested acaricides (Table 4). These values can be used in future screening efforts. LC50 values for each population active ingredient combination that was flagged in the diagnostic dose screening were able to be determined (Fig. 1). In the SC populations, bifenthrin resistance was ubiquitous and high (Fig. 1A). RRs varied from 116-59,956. In all but SC-6, the LC50 was above field rate, indicating a strong likelihood of failure of the application to control TSSM. While bifenthrin and other pyrethroids are typically applied for controlling other insect pests (e.g. strawberry clippers, aphids, tarnished plant bugs, and spotted wing drosophila), Brigade (bifenthrin) is labelled for TSSM control. Growers should be aware that this product is not likely to provide any level of TSSM control. Additionally, because pyrethroids have non-target effects on natural enemies (Gerson and Cohen, 1989; Theiling and Croft, 1988) and bifenthrin does not harm TSSM, application of this product for other pests is likely to cause TSSM flare ups. SC TSSM were 25-123 less susceptible to fenbutatin-oxide than the susceptible population. All LC50 values were below field rate, but high enough compared to field rate that application failure of the product is possible. This is an older acaricide and reports of field failure and resistance are common (Arthropod_Pesticide_Resistance_Database, 2018; Liburd et al., 2007; Urbaneja et al., 2008). The RR for abamectin in SC-2 TSSM was 25. This is above the field rate, indicating likely failure of this product to control this population. This is an older active ingredient and reports of TSSM resistance to abamectin are common (Arthropod_Pesticide_Resistance_Database, 2018). Because of the non-target effects of abamectin on the beneficial mites that control TSSM (Abraham et al., 2012; Bostanian and Akalach, 2006; Kim et al., 2005) and known resistance issues, this product should not be a first choice for strawberry growers. Extension efforts. To date, the results from these studies have been presented at one strawberry grower meeting in Lexington Co., SC on August 13, 2018; 12 growers attended this meeting. Additionally, spider mite resistance management was discussed at an additional seven extension meetings in South Carolina, with a total of ~300 attendees receiving the information. The myipm app strawberry mite management section has been updated to indicate that bifenthrin and fenbutatin-oxide resistance occurs in South Carolina. This information will continue to be used in future presentations.
Table 3. Percent mortality (mean ± SE) for all TSSM populations tested. Populations flagged for additional screening (<50% mortality) are highlighted in yellow, borderline cases where mortality overlapped with the cutoff when SE was accounted for are green. Active Ingredient SC-1 SC-2 SC-3 SC-4 SC-5 SC-6 NC-1 NC-2 NC-3 Bifenthrin 10 ± 3.5 22 ± 5.6 7 ± 2.5 7 ± 3.0 14 ± 4.3 2.0 ± 1.2 87.5 ± 4.0 39.4 ± 6.8 52.6 ± 7.7 Abamectin 100 ± 0 32 ± 7.2 61 ± 7.0 97 ± 2.0 100 ± 0 99 ± 1.0 98.0 ± 2.0 96.9 ± 1.2 78.0 ± 4.5 Fenbutatin-oxide 53 ± 8.7 18 ± 5.1 24 ± 5.1 21 ± 5.6 29 ± 13.2 2.0 ± 1.3 80.4 ± 6.3 61.7 ± 11.1 45.5 ± 5.7 Acequinocyl 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 98.9 ± 1.1 98.9 ± 1.1 64.1 ± 9.6 Bifenazate 100 ± 0 100 ± 0 100 ± 0 95 ± 2.2 100 ± 0 99 ± 1.0 96.1 ± 1.8 87.3 ± 5.2 64.8 ± 7.2 Fenpyroximate 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 70.2 ± 13.0 Cyflumetofen 99 ± 1.0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 95 ± 3.9 95.3 ± 1.2 99.0 ± 1.0 72.4 ± 5.5 Check (Adulticide) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 1 ± 1.0 31.2 ± 7.2 44.2 ± 7.1 62.1 ± 7.7 Etoxazole 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 99.0 ± 1.0 99.0 ± 1.0 100 ± 0 Spiromesifen 98 ± 2.0 100 ± 0 100 ± 0 100 ± 0 97 ± 2.0 90 ± 1.6 100 ± 0 100 ± 0 100 ± 0 Hexythiazox 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 98.8 ± 1.3 Check (Ovicide) 8 ± 2.6 4 ± 2.4 1 ± 1.0 1 ± 1 7 ± 2.5 4 ± 2.9 75.0 ± 4.7 37.0 ± 6.4 21.0 ± 5.1
Summary Results from this study indicate that bifenthrin and fenbutatin-oxide are unlikely to control TSSM in grower fields. All SC populations tested indicated some level of resistance to these products. In some cases, growers may also experience field failure of abamectin due to resistance. Fortunately, resistance to other active ingredients was not found. Strawberry growers should rotate use of newer acaricides to prevent resistance development. Many newer active ingredients, for which this study did not find resistance, have the added benefit of minimal nontarget effects on natural enemies. In particular, hexythiazox, cyflumetofen, bifenazate, and acequinocyl should be considered due to their selectivity. References Abraham, C.M., Braman, S.K., Oetting, R.D., Hinkle, N.C., 2012. Pesticide compatibility with natural enemies for pest management in greenhouse gerbera daisies. J. Econ. Entomol. 106, 1590-1601. Arthropod_Pesticide_Resistance_Database, 2018. Available online: https://www.pesticideresistance.org/. Bostanian, N.J., Akalach, M., 2006. The effect of indoxacarb and five other insecticides on Phytoseiulus persimilis (Acari: Phytoseiidae), Amblyseius fallacis (Acari: Phytoseiidae) and nymphs of Orius insidiosus (Hemiptera: Anthocoridae). Pest Manag. Sci. 62, 334-339. Gerson, U., Cohen, E., 1989. Resurgences of spider mites (Acari: Tetranychidae) induced by synthetic pyrethroids. Exp. Appl. Acarol. 6, 29-46. Gimenez-Ferrer, R.M., Erb, W.A., Bishop, B.L., Scheerens, J.C., 1994. Host-pest relationships between the twospotted spider mite (Acari: Tetranychidae) and strawberry cultivars with differing levels of resistance. J. Econ. Entomol. 87, 168-175. Kim, S.S., Seo, S.G., Park, J.D., Kim, S.G., Kim, D.I., 2005. Effects of selected pesticides on the predatory mite Amblyseius cucumeris (Acari: Phytoseiidae). J. Entomol. Sci. 40, 107-114. Liburd, O.E., White, J.C., Rhodes, E.M., Browdy, A.A., 2007. The residual and direct effects of reduced-risk and conventional miticides on twospotted spider mites, Tetranychus urticae (Acari: Tetranychidae) and predatory mites (Acari: Phytoseiidae). Fla. Entomol. 90, 249-257. Theiling, K.M., Croft, B.A., 1988. Pesticide side-effects on arthropod natural enemies: a database summary. Agric. Ecosyst. Environ. 21, 191-218. Urbaneja, A., Pascual-Ruiz, S., Pina, T., Abad-Moyano, R., Vanaclocha, P., Monton, H., Dembilio, O., Catanera, P., Jacas, J.A., 2008. Efficacy of five selected acaricides against Tetranychus urticae (Acari: Tetranychidae) and their side effects on relevant natural enemies occurring in citrus orchards. Pest Manag. Sci. 64, 834-842. Walsh, D.B., Zalom, F.G., Shaw, D.V., 1998. Interaction of the twospotted spider mite (Acari: Tetranychidae) with yield of day-neutral strawberries in California. J. Econ. Entomol. 91, 678-685.
Table 4. Acaricide LD50 values for the susceptible laboratory TSSM colony (LD50 and 95% Confidence Interval, CI). 95% CI Active Ingredient LD50 (ppm) Lower Limit Upper Limit Bifenthrin 0.704 0.379 1.159 Abamectin 0.016 0.013 0.019 Fenbutatin-oxide 1.414 0.858 1.931 Acequinocyl 0.003 0.001 0.008 Bifenazate 1.604 1.29 1.885 Spiromesifen 0.302 0.193 0.44 Cyflumetofen 3.027 2.525 3.53 Fenpyroximate 0.014 0.007 0.026 Hexythiazox 0.002 0.001 0.006 Etoxazole 0.016 0.005 0.033
Fig. 1. LD50 values (± 95% CI) for all populations that underwent dose response bioassays for a) bifenthrin, b) fenbutatin-oxide, and c) abamectin. The dashed line represents PPM of active ingredient for the high field rate.