spatially-explicit test of the refuge strategy for delaying insecticide resistance

The refuge strategy is used worldwide to delay the evolution of pest resistance to insecticides that are either sprayed or produced by transgenic Bacillus thuringiensis (Bt) crops. This strategy is based on the idea that refuges of host plants where pests are not exposed to an insecticide promote survival of susceptible pests. Despite widespread adoption of this approach, large-scale tests of the refuge strategy have been problematic. Here we tested the refuge strategy with 8 y of data on refuges and resistance to the insecticide pyriproxyfen in 84 populations of the sweetpotato whitefly (Bemisia tabaci) from cotton fields in central Arizona. We found that spatial variation in resistance to pyriproxyfen within each year was not affected by refuges of melons or alfalfa near cotton fields. However, resistance was negatively associated with the area of cotton refuges and positively associated with the area of cotton treated with pyriproxyfen. A statistical model based on the first 4 y of data, incorporating the spatial distribution of cotton treated and not treated with pyriproxyfen, adequately predicted the spatial variation in resistance observed in the last 4 y of the study, confirming that cotton refuges delayed resistance and treated cotton fields accelerated resistance. By providing a systematic assessment of the effectiveness of refuges and the scale of their effects, the spatially explicit approach applied here could be useful for testing and improving the refuge strategy in other crop-pest systems. pesticide resistance | predictive evolutionary models | pest management | resistance management P opulation growth will continue to favor agricultural intensification for decades. Because agricultural intensification is associated with increased pest pressure, pesticides generally help to increase yield (1-3). Although significant progress has been made to reduce reliance on pesticides (4, 5), an increasing number of insects and mites exhibit field-evolved resistance to synthetic pesticides, Bacillus thuringiensis (Bt) sprays, and transgenic Bt crops (6, 7). Negative consequences of resistance include increased pesticide use, disruption of food webs and ecosystem services, increased risk to human health, and loss of profits for farmers and industry (1, 3). One of the main strategies for delaying resistance promotes survival of susceptible pests by providing refuges, which are areas of host plants where pests are not exposed to an insecticide. Theory predicts that refuges will slow the evolution of resistance by reducing the fitness advantage of resistant individuals (7-9). Refuges can also reduce the heritability of resistance when susceptible individuals mate with resistant individuals surviving exposure to an insecticide (7). Empirical support for the refuge strategy was provided by short-term laboratory and greenhouse experiments (10, 11). Although these experiments test the hypothesis that mating between susceptible and resistant individuals delays the evolution of resistance, they do not consider several factors that affect resistance in the field (7-9), and thus only provide partial support for effectiveness of the refuge strategy in the field. Retrospective analyses of variation in resistance evolution in the field also suggest that refuges have been effective, but these previous tests have been based primarily on comparisons among species, or qualitative comparisons within species based on a limited number of widely separated geographic areas (12, 13). In such tests, factors that vary among species or geographic areas can confound the effects of refuges. Accordingly, large-scale field tests of the refuge strategy for a single species within a geographic area where factors affecting resistance are similar are needed to test the refuge strategy more rigorously. Moreover, tests of predictive refuge strategy models are required to determine if the refuge strategy can delay resistance (14). Furthermore, to improve our ability to develop efficient refuge strategies, empirical approaches are necessary to characterize effects of refuges on resistance evolution (7, 15). Here we tested the refuge strategy using 8 y of data on refuges and resistance to the insecticide pyriproxyfen in 84 populations of the sweetpotato whitefly (Bemisia tabaci) sampled in cotton fields of central Arizona. We studied the B biotype of B. tabaci, also known as the Asia Minor-Middle East 1 species, which is a key pest of cotton and other crops in Arizona and worldwide (16). The insect growth regulators pyriproxyfen (a juvenile hormone analog) and buprofezin (a chitin synthesis inhibitor) are selective insecticides that have been used for whitefly control in Arizona cotton (Gossypium spp.) since 1996 (17, 18). A single application of either insecticide on cotton when B. tabaci populations start to increase has substantially reduced sprays of broad-spectrum insecticides, helped to conserve natural enemies, and restored farmers ' profits (18, 19). To deter rapid evolution of resistance, farmers in Arizona generally have not used pyriproxyfen to control B. tabaci on crops other than cotton Although B. tabaci is polyphagous, few whitefly crops other than cotton are available in central Arizona from June to September, when pyriproxyfen is sprayed on cotton. In principle, crops that could act as refuges include spring melons (Citrullus lanatus and Cucumis melo), alfalfa (Medicago sativa) and cotton not treated with pyriproxyfen (referred to hereafter as untreated cotton). B. tabac

Assessment of Knack Field Performance Through Precision Field and Laboratory Bioassays in Cotton

When a product performs better or worse than expectations, there are many biological, ecological, and operational factors that must be examined. Genetic resistance to the pesticide itself is often a concern. The control interval depends on the ecological impact of biotic (e.g., presence and function of natural enemies) and abiotic (e.g., frequency and severity of storms) factors. Timing, rates, and application methods used are also key factors affecting product performance. A four-year study to evaluate pyriproxyfen (Knack®) field performance in Arizona was initiated in 2004, after levels of whitefly susceptibility in statewide surveys were observed to be decreasing. Grower sites in Maricopa, Buckeye, Wellton, and Marana were used. We controlled for major operational factors by using a common timing, rate, and aerial application for each Knack spray. Resistance bio-assays were conducted on progeny of field-collected adults, pre- and post-spray. New eggs were marked in-field prior to spraying and examined in the field and lab in order to isolate Knack-associated mortality caused by direct toxicity as well as by ecological factors (e.g., bioresidual). Nymphal bioassays were used to evaluate metamorphosis inhibition. Population trends were estimated using standard sampling methods. Appropriate check plots were compared to the Knack treatment. Study results suggest Knack field performance and pyriproxyfen resistance has not changed significantly among the years or locations examined to date. In 2005, many struggled to gain control over whitefly populations. This work indicated that Knack performance and resistance parameters were within the range expected for the last several years. However, operational and ecological barriers to the performance of Knack and other chemistry were in play. Late planted conditions, lush winter vegetation capable of hosting whiteflies, poor growing conditions, and an extended period of extreme immigration pressure were all factors that diminished the impact of Knack and other products in 2005. In contrast, the winter preceding 2006 was among the driest on record followed by a very active monsoon season in central Arizona. High winds and dust movement, and a very active natural enemy community helped to continually lower whitefly populations. The result was a whitefly season characterized as light, with overall foliar insecticide usage setting a 28-yr record low for Arizona cotton. Barring all other operational and ecological factors at work, control intervals should have been similar each year. Yet, observed intervals have been different (e.g., 2005 vs. 2006) and point to the importance of these external factors in assessing product performance. Work will continue in 2007 to identify factors that contribute to whitefly outbreak conditions. These data will be key to understanding any performance changes, either due to operational or ecological factors mentioned above or due to innate changes in whitefly susceptibility. This will be important in advising growers about the risk factors associated with whitefly outbreaks and should lead to recommendations for minimizing these risks

Six Years of Successful Management of Whitefly Resistance in Arizona Cotton

Arizona cotton experienced a severe crisis in 1995 stemming from resistance of whiteflies to synergized pyrethroid insecticides. The insect growth regulators (IGRs) Knack® (pyriproxyfen) and Applaud® (buprofezin) served a pivotal role in resolving this problem. Statewide monitoring of whitefly resistance is conducted annually in Arizona to assess the status of resistance in this important pest. In this paper we provide an update on results from whitefly collections made from 19 cotton fields located throughout Arizona. Overall, whitefly control in Arizona cotton remained excellent in the 2001 season and there were no reported field failures. However, we detected major decreases in susceptibility to Knack of whiteflies collected from cotton. Whereas it was extremely rare to have any whiteflies surviving bioassays of 0.1 μg/ml from 1996 to 1998, this changed in 1999, and by the 2001 season over 60% of Arizona sites evaluated had •2% pyriproxyfen-resistant whiteflies. One collection from Eloy, Arizona, in 2000 had >50% of whiteflies surviving Knack bioassays of 0.1 μg/ml. Whiteflies throughout Arizona continued to be moderately less susceptible to Applaud, relative to susceptibility levels in 1996, when the IGRs were first introduced. In contrast to our findings with Knack, changes in susceptibility to Applaud have been only moderate and quantitative. Arizona whiteflies continued a six year trend of reduced resistance to synergized pyrethroid insecticides, as indicated by bioassays with mixtures of Danitol and Orthene. Problematic frequencies of whiteflies resistance to synergized pyrethroids were found at only two of 19 locations sampled. Steps should be taken now to prepare for the onset of more severe resistance to IGRs in Arizona cotton. Factors that could undermine the current success of whitefly resistance management in Arizona are discussed. Education efforts should reinforce the importance of limiting IGR use in cotton to a maximum of one treatment each per season and rotating conventional insecticides as recommended in the three-stage resistance management strategy implemented in 1996. Because Knack and Applaud have received registrations for use in Arizona vegetable and melon crops grown in proximity to cotton, it is now especially critical that Extension education efforts focus on cross-commodity coordination of IGR use recommendations to preserve the activity of these important insecticides

Multi-Toxin Resistance Enables Pink Bollworm Survival on Pyramided Bt Cotton

Transgenic crops producing Bacillus thuringiensis (Bt) proteins kill key insect pests, providing economic and environmental benefits. However, the evolution of pest resistance threatens the continued success of such Bt crops. To delay or counter resistance, transgenic plant "pyramids" producing two or more Bt proteins that kill the same pest have been adopted extensively. Field populations of the pink bollworm (Pectinophora gossypiella) in the United States have remained susceptible to Bt toxins Cry1Ac and Cry2Ab, but field-evolved practical resistance to Bt cotton producing Cry1Ac has occurred widely in India. Here we used two rounds of laboratory selection to achieve 18,000- to 150,000-fold resistance to Cry2Ab in pink bollworm. Inheritance of resistance to Cry2Ab was recessive, autosomal, conferred primarily by one locus, and independent of Cry1Ac resistance. We created a strain with high resistance to both toxins by crossing the Cry2Ab-resistant strain with a Cry1Ac-resistant strain, followed by one selection with Cry2Ab. This multi-toxin resistant strain survived on field-collected Bt cotton bolls producing both toxins. The results here demonstrate the risk of evolution of resistance to pyramided Bt plants, particularly when toxins are deployed sequentially and refuges are scarce, as seen with Bt cotton and pink bollworm in India.Peer reviewed: YesNRC publication: Ye

Large-scale, spatially-explicit test of the refuge strategy for delaying insecticide resistance

The refuge strategy is used worldwide to delay the evolution of pest resistance to insecticides that are either sprayed or produced by transgenic Bacillus thuringiensis (Bt) crops. This strategy is based on the idea that refuges of host plants where pests are not exposed to an insecticide promote survival of susceptible pests. Despite widespread adoption of this approach, large-scale tests of the refuge strategy have been problematic. Here we tested the refuge strategy with 8 y of data on refuges and resistance to the insecticide pyriproxyfen in 84 populations of the sweetpotato whitefly ( Bemisia tabaci ) from cotton fields in central Arizona. We found that spatial variation in resistance to pyriproxyfen within each year was not affected by refuges of melons or alfalfa near cotton fields. However, resistance was negatively associated with the area of cotton refuges and positively associated with the area of cotton treated with pyriproxyfen. A statistical model based on the first 4 y of data, incorporating the spatial distribution of cotton treated and not treated with pyriproxyfen, adequately predicted the spatial variation in resistance observed in the last 4 y of the study, confirming that cotton refuges delayed resistance and treated cotton fields accelerated resistance. By providing a systematic assessment of the effectiveness of refuges and the scale of their effects, the spatially explicit approach applied here could be useful for testing and improving the refuge strategy in other crop–pest systems