Repellency and Repulsiveness of Selected Agrichemicals to the Two-Spotted Spider Mite (Tetranychus urticae) on Grape Foliage

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1 2005 Plant Management Network. Accepted for publication 7 November Published. Repellency and Repulsiveness of Selected Agrichemicals to the Two-Spotted Spider Mite (Tetranychus urticae) on Grape Foliage Douglas B. Walsh, Associate Entomologist, and Gary G. Grove, Associate Plant Pathologist, Irrigated Agriculture Research and Extension Center, Washington State University, N. Bunn Road, Prosser Corresponding author: G. G. Grove grove@wsu.edu Walsh, D. B., and Grove, G. G Repellency and repulsiveness of selected agrichemicals to the two-spotted spider mite (Tetranychus urticae) on grape foliage. Online. Plant Health Progress doi: /php rs. Abstract Leaf disk and whole leaf bioassays were conducted to evaluate the repellency and repulsiveness of selected fungicides, insecticides, spray adjuvants, and petroleum-derived spray oils (PDSO) to the two-spotted spider mite, Tetranychus Urticae Koch (Acari: Tetranychidae). Compounds were evaluated for their ability to stimulate the movement of T. urticae from treated to nontreated grape tissue (repellency) and to inhibit their movement from untreated to treated tissue (repulsiveness). All insecticides other than Bacillus thuringiensis and some fungicides were highly repellent. Several insecticides and fungicides, and all spray adjuvants, were highly repulsive. We conclude that many agrichemicals are irritating to two-spotted spider mites and may impact their spatial dynamics within plant canopies. Introduction Concern about the sub-lethal effects of pesticides on non-target organisms has increased during the last decade. Once the efficacy of a pesticide has been determined against the target pests, subsequent research efforts by ecotoxicologists typically focus on the detrimental effects of pesticide residues on beneficial arthropods, such as susceptible indicator species or key climax/regulated species. Little consideration has been directed towards the understanding of the effects of pesticides on the spatial distribution of specific non-target pests as a consequence of pesticide exposure. The spatial distribution of arthropod pests within host plant canopies is important in determining their potential for reaching damaging population densities. The two-spotted spider mite is a phytophagous and secondaryinduced pest in many cropping systems (7) (Fig. 1). Population outbreaks of T. urticae are often attributed to the application of pesticides, which can negatively impact the arthropod predators that naturally regulate T. urticae populations (8,9), or which may trigger an increase in reproduction or fecundity (hormolygosis) in the mites (1,4). Other researchers have documented shifts in the age structure, fecundity, and sex ratio of mite populations based on their location on the host plant and its nutritive value (11). Pesticide use in vineyards in eastern Washington is common. During recent years the effect of some pesticides on insect populations in vineyards has been documented (5), and there is growing concern about the negative impact of fungicides and spray adjuvants on non-target organisms and overall grape integrated pest management (IPM). The purpose of this study was to investigate the effect of selected fungicides, insecticides, petroleum-derived spray oils (PDSO), and spray adjuvants for their potential effects on the spatial dynamics of T. urticae populations within grape canopies.

2 Fig. 1. Two-spotted mites browsing on the surface of a leaf. Investigating Behavioral Response of T. urticae to Selected Agrichemicals Vinifera grape (Vitis vinifera L. cv. Merlot ) was used as a host species in this study because T. urticae is occasionally an economically significant pest on this host in eastern Washington (10). All of the T. urticae used in these experiments originated from a greenhouse colony maintained on beans (Phaseolus lunatus L. dwarf cv. Henderson bush ) grown in vermiculite. Colony foundress females were obtained from a commercial vineyard (cv. Merlot ) located near Paterson, WA on 20 June Types, classes, active ingredients, trade names, common names, and manufacturers of pesticides evaluated in this study are presented in Table 1. Two series of bioassays (using leaf disks and two-leaf vine sections) were conducted to evaluate the repellency and repulsiveness of selected agrichemicals to T. urticae and their affect on spatial dynamics of T. urticae populations within plant canopies. We define repellency in T. urticae as the consistent movement of mites from pesticide-treated surfaces to similar areas free of pesticide residue. We define repulsiveness as a response in T. urticae when mites placed on plant materials free of pesticide residues avoid moving onto similar available pesticide-treated plant surfaces. Leaf disk bioassays included the evaluation of 18 synthetic fungicides, 8 insecticides, 4 spray adjuvants, and 3 PDSO. Pesticide concentration was based on recommended rates of products per hectare specified for wine grapes in eastern Washington. In this study we did not attempt to differentiate the pesticidal effects caused by the active or inert ingredients that comprise each product. Leaf disks (1.5 cm 2 in diameter) were cut using a cork borer from mature leaves of Merlot grape plants. Plants were maintained in a lath house at the Washington State University Irrigated Agriculture Research and Extension Center (IAREC) in Prosser, WA. One-half of each leaf disk was immersed for 5 s in a suspension of pesticide and allowed to dry under a fume hood for a minimum of 30 min. Control disks were similarly immersed in distilled water. When dry, leaf disks were positioned with the abaxial surface facing down on a moist 4.5-cm cm cotton square positioned in a 9-cm-diameter Petri dish. This incubation system provided mites with the ability to move freely on leaf disks while inhibiting their movement off of them. To measure repellency, ten adult female T. urticae were transferred to the treated side of the disk. To measure repulsiveness, ten adult female T. urticae were transferred to the untreated side of the leaf disk. The synthetic pyrethroid permethrin (Table 1) at a rate 12.4 µl/liter solution served as the treated control for these studies. The strong repellency of pyrethroid insecticides to some predaceous mites has been documented previously (11). Untreated controls were established by placing ten T. urticae randomly on leaf disks treated with water. This is a high population density for fecund adult female mites on leaf disks this diameter and the mites will exhibit irritable behavior with a tendency to disperse (Walsh, unpublished). The disks were incubated at 24 C for 24 h. Repellency or repulsiveness was assessed microscopically at 10 by determining the number of mites present on non-treated side of the leaf disks 24 h after inoculation. Treatments were replicated in separate Petri dishes 10 times for each material.

3 Table 1. List of pesticides and spray adjuvants tested for repellency and repulsiveness to Tetranychus urticae. Type Fungicide (continued) Class anilinopyrimidine Active ingredient Trade name and formulation cyprodinil Vangard 75 WG cyprodinil + fludioxinil (phenylpyrrole) Switch 62.5WG biofungicide Bacillus subtilis Serenade 15WP Manufacturer Syngenta Syngenta Agraquest benzanilide boscalid Endura 30W BASF chloronitrile chlorothalonil Bravo Weather Stik54F dithiocarbamate thiram hydroxyanilide fenhexamid DMI (imidazole) Thiram 65WSB Elevate 50WDG Syngenta UCB Tomen Agro triflumizole Procure 50WS Chemtura (Triazole) myclobutanil Rally 40W DowAgro (pyrimidine) fenarimol Rubigan 1E Gowan QOI trifloxystrobin Flint 50WG Bayer azoxystrobin Abound 2.08F Syngenta kresoxim-methyl Sovran 50WG BASF pyraclostrobin Cabrio EG BASF pyraclostrobin + boscalide (benzanilide) Pristine 38WG quinoline Quinoxyfen Quintec 2.08SC BASF DowAgro SAR chitinase Elexa 4F Safe Science sulfur Sulfur Thiolux 80DF Syngenta Insecticides pyrethroid permethrin Pounce 3.2 EC FMC organosphospate fenopropathrin Danitol 2.4 EC Valent USA chlorpyrifos Lorsban 4E DowAgro carbamate methomyl Lannate 90W DuPont Ag IGR tebunfenozide Confirm 2F DowAgro methoxyfenozide Intreprid 2F DowAgro biological spinosad Success 2 SC DowAgro

4 Table 1. (continued) Type Petroleum derived spray oils Spray adjuvants Class Active ingredient Trade name and formulation paraffinic oil JMS Stylet Oil non-inonic non-ionic emulsified paraffin base petroleum oil + non-ionic organosilicone phthalic/glycerol aklky resin + butyl alcohol alkylphenol ethoxylate, butyl alcohol, dimethylpolysiloxane silicone-polyether copolymer Omni Supreme Oil Volck Supreme Oil Latron 1956B Manufacturer JMS Flower Farms Helena Valent DowAgro R-11 Wilbur-Ellis Mor-Act Silwet L-77 Wilbur-Ellis Setre Chemical Company Two-leaf bioassays comprised the second series of experiments. The pesticides chosen for this series of experiments were selected based on results of the leaf-disk bioassay and are presented in Table 3. Bioassay arenas were created from potted cv. Merlot maintained as described above. Grape cuttings were taken and transported to the laboratory and used to construct two-leaf arenas. Arenas were comprised of two mature leaves (with less than 3 cm between the leaf petioles) and 6 cm of vine below the bottom leaf. Insect adhesive (Tanglefoot, Grand Rapids, MI) was applied around the vine 1 cm below the lower petiole to prevent movement of mites off the arenas. The base of the vine was then placed in a 2-ml glass test tube filled with water and the test arenas were subsequently maintained at 24 C in a 12-h photoperiod. One leaf was dipped into a pesticide suspension (Table 3) and then air-dried. Non-treated controls were established by dipping leaves in distilled water. For repulsiveness tests, 20 adult female T. urticae were placed on the non-treated leaf and after 96 h the numbers of mites remaining on the leaf were determined as described above. For repellency tests, 20 adult female T. urticae were placed on treated leaves and the numbers moving to untreated leaves were counted after 96 h. Treatments were replicated five times. These bioassays were conducted over a period of 96 h to permit mites to traverse the additional distances associated with the 2 leaf bioassay arenas. Bioassays were conducted by chemical type on 12 separate dates. Analysis of variance among the non-treated controls for these 12 dates determined that there were no significant differences among respective sample dates over different bioassay dates (F = 0.29). This permitted pooling sample dates among all respective treatments for analysis of variance among all chemical treatments tested (6). Significant (P < 0.05) and highly significant (P < 0.01) repellency and repulsiveness effects were observed for fungicide, insecticide, PDSO, and spray adjuvant products in these bioassays. Pairwise t-tests were performed between the non-treated control and the chemical treatments to determine if individual agrichemicals were repellent or repulsive to spider mites. All statistical analyses were performed using the Statistical Analysis System (SAS). All experiments were conducted twice.

5 Effect of Selected Agrichemicals on Behavior of T. urticae Summary. The repellency (F = 4.7) and repulsiveness (F = 10.8) of various pesticides and adjuvants on leaf disks were significant at P < The repellency (F = 2.9) and repulsiveness (F = 6.2) of various pesticides and adjuvants on two-leaf bioassays were also significant at P < Fungicides (Tables 1-4). Fungicides comprised the largest group of agricultural chemicals evaluated in this study and their effects on T. urticae are summarized in Table 4. Several fungicides (e.g., quinoxyfen, azoxystrobin, trifloxystrobin, kresoxim-methyl, and fenarimol) widely used in regional grape powdery mildew management programs (3) had no apparent effect on T. urticae. Among the formulated fungicides tested, Bacillus subtilis and thiram were repellent, while triflumizole, pyraclostrobin + boscalid, cyprodinil, and myclobutanil were highly repellent (P < 0.01). Triflumizole, pyraclostrobin/boscalid, fludoxinil/cyprodinil (Switch), and mylobutanil were repulsive (P < 0.05), or highly repulsive (P < 0.01), respectively. All of the latter are among compounds in widespread local use for managing powdery mildew or Botrytis bunch rot (3). Table 2. Repellency and repulsiveness of agrichemicals to Tetranychus urticae on leaf disks. All products were diluted at the quantities listed into liters of distilled water. Pesticide classes, trade names, and formulations are presented in Table 1. Common Type name Amount 1 Repellency 2 Repulsiveness 3 Water control 5.2 ± ± 0.2 Fungicides azoxystrobin 455 ml ns ns Bacillus subtilis 908g a ns pyraclostrobin 5.5 ± 1.0 ns 5.1 ± 0.8 ns boscalid + pyraclostrobin 6.8 ± 0.7 b 6.3 ± 0.6 z chitosan 0.2% v/v ns ns chlorothalonil 5678 ml b ns cyprodinil 283 g b ns cyprodinil + fludioxinil 340 g b y fenarimol 177 ml ns ns fenhexamid 454 g ns ns kresoxim-methyl 90.8g ns ns myclobutanil 148 ml b y quinoxyfen 207 ml ns ns sulfur (flowable) 2268 g ns ns thiram 1814 g a y trifloxystrobin 59 ml ns ns triflumizole 226 g a z (continued)

6 Table 2. (continued) Type Common name Amount 1 Repellency 2 Repulsiveness 3 Insecticides permethrin ml b y fenopropathrin ml n/a y Bacillus thuringiensis kg n/a y Petroleum derived spray oils Spray adjuvants chlorpyrifos 946 ml n/a y methomyl kg n/a y methoxyfenozide 473 ml n/a y spinosad 298 ml n/a y Paraffinnic oil (JMS Stylet Oil) Paraffinnic oil (Omni Supreme) Paraffinnic oil (Volck Supreme) Non-ionic (Phthalic/glycerol aklky resin + butyl alcohol; Latron 1956B) Non-ionic (alkylphenol ethoxylate, butyl alcohol, dimethylpolysiloxane; R- 11) Emulsified paraffin base petroleum oil + non-ionic (Mor- Act) organosilicone (Silwet L-77) 1% v/v ns y 1% v/v b y 1% v/v a y 236 ml b y 473 ml b y 473 ml a y 473 ml b y ns not significant a Significantly repellent compared to untreated control by pairwise t-test at P < 0.05 b Significantly repellent compared to untreated control by pairwise t-test at P < 0.01 z Significantly repulsive compared to untreated control by pairwise t-test at P < 0.05 y Significantly repellent compared to untreated control by pairwise t-test at P < Amount corresponds to maximum labeled rate recommended for grapes in Washington State, USA. 2 Repellency is mean number of mites out of 10 per leaf disk bioassay that moved off treated surface 24 h after placement on treated surface. 3 Repulsiveness is the number of mites out of 10 remaining 24 h after placement on non-treated surface. 4 Rate of permethrin was 0.1 of a typical field application rate.

7 Table 3. Repellency and repulsiveness of agrichemicals to Tetranychus urticae on 2-leaf bioassay. All products were diluted at the quantities listed into liters of distilled water. Common Type name Amount 1 Repellency 2 Repulsiveness 3 Untreated control Fungicides myclobutanil 148 ml b z ziram 1814 g b y sulfur 2268 g b y Insecticides fenpropathrin ml b z methoxyfenozide 473 ml a y Petroleum derived spray oils paraffinnic oil (Omni Supreme) 1% v/v b y Spray adjuvants Phthalic/glycerol aklky resin + butyl alcohol (Latron B-1956) 236 ml a y a Significantly repellent compared to untreated control by pairwise t-test at P < 0.05 b Significantly repellent compared to untreated control by pairwise t-test at P < 0.01 z Significantly repulsive compared to untreated control by pairwise t-test at P < 0.05 y Significantly repellent compared to untreated control by pairwise t-test at P < Amount corresponds to maximum labeled rate recommended for grapes in Washington State, USA. 2 Repellency is mean number of mites out of 10 per leaf disk bioassay that moved off treated surface 24 h after placement on treated surface. 3 Repulsiveness is the number of mites out of 10 that remained 24 h after placement on non-treated surface.

8 Table 4. Summary of repellency and repulsiveness characteristics of selected grape fungicides on the two-spotted spider mite, Tetranychus urticae. Class Active ingredient Repellent Repulsive Anilopyrimidine + phenylpyrrole cyrodinil + fludioxinil yes yes anilopyrimidine cyprodinil yes no carboximide boscalid no no chloronitrile chlorothalonil yes no DMI (imidazole) triflumizole yes yes DMI (triazole) myclobutanil yes yes DMI (pyrimidine) fenarimol no no EBDC thiram yes no hydroxyanilide fenhexamid no no Paraffinic oil paraffinic oil no yes Paraffinic oil paraffinic oil yes yes Paraffinic oil paraffinic oil yes yes QOI azoxystrobin no no QOI + carboximide Pyraclostrobin + boscalid yes yes QOI trifloxystrobin no no QOI kresoxim-methyl no no quinoline quinoxyfen no no biological Bacillus subtilis yes no Insecticides (Tables 1-3). All of the insecticides tested except the biological insecticide Bacillus thuringiensis were highly repellent. However, when mites were placed directly on fenpropathrin or chlorpyrifos for the repellency test, approximately 40% died from pesticide exposure. Mite mortality had not been observed in any of the other types of agrichemicals tested. This biased our results and for this reason we eliminated all of the insecticides from our overall repellency analysis of variance. Bacillus thuringiensis was not repellent. However the insecticides methomyl, spinosad, methoxyfenozide, fenpropathrin, tebufenozide, and chlorpyrifos were all highly repulsive (P < 0.01). PDSO fungicides/miticides Volck Supreme and Omni Supreme Spray were highly repellent (P < 0.01) and JMS Stylet Oil, Volck Supreme, and Omni Supreme Spray were highly repulsive (P < 0.01). JMS Stylet Oil was not repellent. Spray adjuvants (Table 1). Mor-Act was repellent (P < 0.05), and Latron B-1956, R-11, and Silwet L-77 were highly repellent (P < 0.01). All spray adjuvants were highly repulsive (P < 0.01). Conclusions We were unable to draw further conclusions about the general effects of fungicide classes on T. urticae (Table 4) and conclude that the effects of fungicides on nontarget organisms should be determined by evaluating each fungicide. For example, fungicides comprised of only QOI active ingredients were neither repellent nor repulsive but a formulation comprised of a combination of QOI (pyraclostrobin) and carboximide (boscalid) active ingredients were both repellent and repulsive. Triazole and imidazole DMI fungicides were both repellent and repulsive but the pyrimidine DMI fenarimol was neither. More definitive information about the effects of DMI fungicides could result from testing several members within each subclass. PDSO Volck and Omni Supreme Oils were repellent and repulsive but JMS Stylet Oil was only repulsive. Our experiments did not differentiate sub-lethal effects caused by

9 active ingredients from those caused by inert ingredients. Perhaps some of the variability observed within and across fungicide classes could be attributed to the effects of the latter on T. urticae. Spider mites under natural conditions are pseudocolonial; groups tend to aggregate near their foundress female. Dispersal typically takes place after ambient conditions become unfavorable through resource depletion, overpopulation, or when environmental cues promote a dispersal response (e.g., decreasing photoperiod or temperature). Mites irritated by exposure to many of the agrichemicals tested in these trials will likely migrate to areas of the canopy where chemical contact is reduced. Changes in the distribution of mites in the canopy could complicate both mite population abundance sampling and biological control (2). Female predatory mites will typically lay their eggs near groups of spider mites to ensure that recently hatched offspring will have ready access to prey (7). Examples of chemically irritated mites reproducing more rapidly have been documented for certain organophosphate and pyrethroid insecticides (1). Agrichemicals affecting spider mite populations and their distribution are likely to also have direct and/or indirect effects on predatory mites and other beneficial arthropods. Our results suggest that applications of some pesticides commonly used in Washington grape production may be having significant non-lethal effects on the biology and ecology of non-target mite predators within grape canopies through direct effects on their host. Acknowledgments We sincerely appreciate the support of this project by the Washington Wine Advisory Committee; Washington Liquor Control Board; Washington State University Agricultural Research Center (ARC), Pullman, WA; and Washington State University Irrigated Agriculture Research and Extension Center (IAREC), Prosser, WA. Literature Cited 1. Ayyappath, R., Witkowski, J. F., and Higley, L. G Ovipositional responses of two species of spider mites (Acari: Tetranychidae) to sublethal concentrations of permethrin and methyl parathion on corn. Environ. Entomol. 26: Huffaker, C. B., Van de Vrie, M., and McMurtry, J. A Ecology of the Tetranychid mites and their natural enemies: A review II. Tetranychid populations and their possible control by predators: An evaluation. Hilgardia 40: Grove, G., Lunden, J., and Spayd, S Use of petroleum-derived spray oils in Washington grapevine powdery mildew management programs. Plant Health Progress doi: /php rs. 4. James, D. G., and Price, T. S Fecundity in twospotted spider mite (Acari: Tetranychidae) is increased by direct and systemic exposure to imidacloprid. J. Econ. Entomol. 4: James, D. G., Price, T. S., Wright, L. C., and Perez, J Abundance and phenology of mites, leafhoppers, and thrips on pesticide-treated and untreated wine grapes in Southcentral Washington. J. Agric. Urban Entomol. 19: Ott, L An Introduction to Statistical Methods and Data Analysis. PWS, Kent Publishing Co., Boston, MA. 7. Rabbigne, R Damage and Control. Pages in: Spider Mites, Their Biology, Natural Enemy, and Control. W. Helle, ed. Vol. I. Elsevier, Amsterdam, The Netherlands. 8. Sabelis, M. W Predation on spider mites. Pages in: Spider Mites, Their Biology, Natural Enemy, and Control. Vol. I. W. Helle and M. W. Sabelis, eds. Amsterdam, Elsevier. 9. Van de Vrie, M., McMurtry, J. A., and Huffaker, C. B Ecology of tetranychid mites and their natural enemies: a review. iii. Biology, ecology, and pest status, and host-plant relations of tetranychids. Hilgardia 41: Walsh, D Spider mites: Secondary pests of Washington State wine grapes. Online. Proc. of the Annu. Meeting, Wash. State Grape Soc. 11. Walsh, D. B., Zalom, F. G. and Stimmann, M Effects of pyrethroid insecticide residues on almond leaves on the biology of the western orchard predator mite Galendromus occidentalis (Nesbitt) (Acari: Phytoseiidae). Acta Hortic. 470:

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