3. DEVELOPING A ROBUST IRM PLAN

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1 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops

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3 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops Table of Contents ACKNOWLEDGEMENTS ABBREVIATIONS TERMINOLOGY EXECUTIVE SUMMARY INTRODUCTION Stewardship Insect protected biotech-derived crops Insect resistance management DEVELOPING A ROBUST IRM PLAN Biology and ecology of major pests Product deployment strategies Pyramiding versus single insecticidal traits Local cropping systems Planting choices Alternative pest management options INSECT RESISTANCE MANAGEMENT TOOLS Structured refuges Scouting and applying insecticides as needed Limiting the use of multiple crops with the same insect control proteins Capping sales to limit market penetration Cultivation and destruction of crop residues Variety resistance and good crop management practices Using multiple traits targeting the same pests BASELINE SUSCEPTIBILITY AND MONITORING DAMAGE INTEGRATED PEST MANAGEMENT ENGAGEMENT, EDUCATION AND COMMUNICATION EXAMPLES OF IRM PLANS Case study: Indian IRM requirements for insect protected cotton IMPLEMENTING STRUCTURED REFUGES Flexibility Seed distribution Grower education and communication Refuge management Planning Planting Recording Monitoring the implementation of refuges Reporting REMEDIAL ACTION PLANS DISCUSSION AND CONCLUSIONS REFERENCES Appendix 1. SUMMARY OF KEY POINTS Appendix 2. RECORD OF REFUGE PLANTING Appendix 3. RECORD OF MONITORING Appendix 4. EXAMPLES OF IRM LOCAL AND REGIONAL PROGRAMMES

4 Acknowledgements This training manual was developed from the paper Managing the Risk of Insect Resistance to Transgenic Insect Control Traits: Practical Approaches in Local Environments written by Susan C. MacIntosh, MacIntosh & Associates, Incorporated, 1203 Hartford Avenue, Saint Paul, MN for CropLife International. Additional information was obtained from academics and the slide set How to Develop an Insect Resistance Management Plan: Practical Approaches for Local Environments developed by the Insect Resistance Action Committee of CropLife International. The Insect Resistance Action Committee (IRAC) is supported by the member companies of the IRAC Plant Biotechnology Working Group: Bayer CropScience, Dow AgroSciences LLC, E.I. Dupont De Nemours and Company, Monsanto Company and Syngenta Plant Sciences. Abbreviations ABSTC Bt CICR GEAC IRM IRAC IPM OECD QC/QA US-EPA Agriculture Biotechnology Stewardship Technical Committee Bacillus thuringiensis Central Institute of Cotton Research Genetic Engineering Approval Committee Insect Resistance Management Insect Resistance Action Committee Integrated Pest Management Organisation for Economic Co-operation and Development Quality Control and Quality Assurance United States Environmental Protection Agency 2

5 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops Terminology Biotech-derived: refers to crops improved through molecular biology techniques that alter the crop genetics. Bt: is an insecticidal protein obtained from the bacterium, Bacillus thuringiensis, or Bt. It is a commonly used source of insect protection in biotech-derived crops. Diapause: refers to a period of inactivity or rest in response to adverse environmental conditions, especially winter, during which insect development is suspended. Grower: refers to a farmer who purchases insect protected, biotech-derived planting material. Grower agreement: refers to an agreement between the grower and the technology provider that is established at purchase of the planting material and which may stipulate insect resistance management requirements for the particular crop-trait combination and growing area among other stewardship practices. Growing area: refers to the region where the crop is grown. Insect resistance management requirements may vary depending on factors present in various growing areas. High dose: refers to trait-insect combinations where the trait is sufficiently effective, and the target insect pest sufficiently sensitive, so that very few if any of the exposed target pest insects survive. It has been defined as the dose necessary to control target insects that are heterozygous for resistance alleles. Insect protected: refers to crops that have been developed to withstand damage from specific insect pests. IRM: refers to insect resistance management and details the measures taken to delay the development of resistance to pest control measures in target pest populations. Key target pests: refers to those pests in a cropping system, that are the most economically damaging and are targeted by an insect protection trait. Primary and secondary pests: refers to the dominant pests and the less important pests on a crop, based on typical population sizes and the levels of crop injury they cause. Pyramiding genes: refers to a special case of gene stacking where two of more transgenes are combined in one crop that each provides protection from the same target pest(s) so that there are at least two modes of action. Refuge: refers to an area of the same crop, or natural vegetation, that does not contain the biotech-derived, insect protection control mechanism. Refuge calculator: refers to a table of formulas provided to assist growers in the calculation of acceptable refuge area measurements. Regulatory authority: refers to any national regulatory authority which might stipulate insect resistance management conditions for the production of specific biotech-derived crops. Trait: refers to a genetically determined characteristic. Trait provider: refers to a company or public centre that develops new traits for biotech-derived crops and makes these available for improved seed. Transgene: refers to a gene or genetic material that has been transferred by a genetic engineering technique from one organism to another. Seed distributor: refers to the local company or organisation that distributes seed to growers for crop production. Stacking genes: refers to inserting two or more transgenes into one crop which may be for different traits. Sustainable agriculture: refers to a combination of farming methods with the common goals of providing more farm profits, achieving greater environmental stewardship, and benefiting families and communities today without compromising the ability of future generations to meet the same goals. Threshold levels: refers to levels of pest damage that will affect the yield of the crop. Threshold levels are determined for specific pests on specific crops in specific growing areas and are available from extension officers and seed suppliers. 3

6 1. Executive Summary Growers have rapidly adopted biotech-derived crops that have been improved to express proteins for insect control, because these crops provide excellent protection from key damaging insect pests around the world. Insect protected crops also offer superior environmental and health benefits while increasing grower income. However, insect resistance development is an important concern for all stakeholders, including growers, technology providers, and the seed companies that develop biotech-derived crops. Given the benefits associated with insect protected seed, insect resistance management (IRM) must be a consideration when cultivating these crops. It is important that concerns about resistance should not be allowed to prevent the use of biotech crops; rather, these concerns should result in stewardship and management programmes that effectively delay resistance while enabling the benefits of the technology to the environment and to agriculture to be realised. The technical data and practical experience accumulated by developers, researchers and growers with insect protected crops in many global regions can inform different aspects of resistance management leading to robust, science-based IRM plans. A range of elements should be considered in assembling any IRM strategy, including: In addition, insect susceptibility monitoring measures any changes in pest susceptibility to the insect protected crop, while stakeholder and grower communication and education inform the end-users of any IRM stewardship guidelines and requirements. Infrastructure should be developed such that a remedial action plan can be designed and implemented should resistance develop. Each of these elements is described in more detail, with specific examples of how they can be combined and tailored to local/regional environments and grower practices. Insect resistance management plans need to be suitable for the given production situation. What works for large monoculture production systems in North and South America is unlikely to be appropriate for the small, more diverse agriculture of Southeast Asia or Africa. Although it is clear that insect protected crops impart considerable value to growers, it is also clear that it is in the best interest of all stakeholders to preserve insect protected crops for the long-term benefits they provide. key target pest biology/ecology; efficacy of and target pest sensitivity to the insectprotection traits; pyramided versus single insecticidal traits; product deployment patterns; local cropping systems; and availability of alternative pest management options, including biotech, chemical, biological and cultural control options. 4

7 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops 2. Introduction 2.1 STEWARDSHIP Stewardship is defined as the responsible management of a product from its inception through to its use and ultimate discontinuation (ETS, 2009). In plant biotechnology, stewardship includes the responsible introduction and use of biotech-derived products across the entire plant product life cycle, from idea, through development and launch, to discontinuation. Stewardship is a shared responsibility of the entire value chain including technology developers, seed producers, seed dealers/distributors, grower advisors, growers, and consumers. Insect resistance management, one of the first industry-wide stewardship programmes, was introduced in conjunction with the launch of insect protected, biotech-derived crops in the mid-1990s. For the first generation of insect protected traits (Bt corn and Bt cotton) in the United States, the traits are considered to be high dose against several key target pests and the Environmental Protection Agency (EPA) worked with the technology providers, university scientists, and grower groups to develop high-dose/ refuge-based IRM plans. In developing the IRM plans, it was important to keep the refuge requirements simple and flexible. For their part, the agricultural biotechnology companies agreed to implement communication programmes to help growers understand the needs and benefits of refuge areas and to implement programmes to monitor adherence. Local, regional, and international organisations have been formed that bring together the trait providers, grower organisations, and academic researchers to address scientific issues central to the responsible stewardship of biotech products in modern agriculture. For example, in the United States the Agriculture Biotechnology Stewardship Technical Committee (ABSTC) is a coalition of biotech companies that plays a key role in the grower-industry interaction on IRM. Similar organisations exist in all countries where Bt crops are commercially cultivated. The Insect Resistance Action Committee (IRAC), a specialist technical group of the CropLife International industry association, has undertaken to provide additional guidance for the development of IRM plans that address these stakeholder concerns. 2.2 INSECT PROTECTED BIOTECH- DERIVED CROPS An assortment of crops expressing different Bacillus thuringiensis proteins (Bt) has been commercialised for insect control and additional products are under development. Growers have embraced crops (i.e., maize, cotton, potato and rice) genetically improved to express different insect control proteins (i.e., Cry1Ab, Cry1Ac, Cry1F, Cry1A.105, Cry2Ab, mcry3aa, Cry3Bb, Cry3Aa, Cry34/35, Vip3A), as they provide excellent protection from key damaging insect pests in global regions (James, 2008). There is a history of safe use of these proteins, both for the environment and human health (Betz et al., 2000; OECD, 2007; US-EPA, 2001). Recent evaluations have shown that these traits provide economic value to adopting countries through increased grower income (Brookes and Barfoot, 2006; Gianessi, et al., 2002). The continuing value of this technology can be enhanced through appropriate stewardship, such as IRM plans, that can prolong trait efficacy against the target insect pests. 2.3 INSECT RESISTANCE MANAGEMENT Insect control traits introduced into plants using modern biotechnology methods have shown high economic value across the globe. Over the past century, hundreds of insect species have developed resistance to one or more control measures, severely impacting the economics of crop production. Most cases of insect resistance to date involve synthetic chemical insecticides (Yu, 2008), but resistance has also developed to microbial agents, such as sprayed formulations of Bt (Ferré and Van Rie, 2002). The evolution of insect resistance is an ongoing concern to crop protection users, including those who apply insecticide applications, cultural practices and host plant resistance. The goal of resistance management is to delay the evolution of resistance in pest populations exposed to a pest management tool. Resistance can and has evolved to all forms of pest management, including chemical, biological, and cultural tools, and is not a unique concern for biotech-derived crops. However, the benefits of biotech-derived insect protection traits are considered so valuable that the technology providers and other stakeholders have placed huge emphasis on prolonging their durability by slowing the rate of resistance development in target pests. Multiple tactics are available to preserve the durability of insect management technologies, including using the technology only against the most economically damaging pest populations, alternating among different control tactics, or integrating multiple tactics into a pest management programme. 5

8 3. Developing a robust IRM plan By using a risk management approach, developers and growers implement practices that will delay the development of resistance in insect pests to these new crops. This proactive approach of devising and implementing resistance management strategies will help ensure that the new technology is effective for many years so that growers, consumers and the environment can benefit from its effectiveness. Durable, science-based IRM plans have been based on an extensive array of research data that was collected before and since the initial introduction of insect protected crops. Many important factors, such as pest biology, pest/crop interactions and resistance genetics have been investigated. Computer simulation models have enabled researchers to evaluate the relative effectiveness of different resistance management options. However, from the beginning it was understood that no matter how detailed the results, science alone will not result in a robust IRM plan in the absence of practical field experience, information on growing environments and grower practices. Insect resistance management plans need to be suitable for the given production situation. While practical experience has accumulated with insect protected crops in the U.S., Canada, Australia, Argentina, Philippines, South Africa, Spain, and China (Fit, 2003; Wu and Guo, 2005; Matten, et al., 2008), and can inform certain elements of IRM plans, other aspects must take into account the unique pest population spectrums and distinctive agricultural practices found in local growing environments. For example, what works for large monoculture production systems in North America is unlikely to be appropriate for the small, more diverse agriculture of Southeast Asia or Africa. Uncertainties, such as those inherent in biological processes and changes in growing environments, trigger a regulatory temptation to be overly conservative in setting IRM measures, potentially unnecessarily limiting the use and availability of No matter how detailed the results, science alone will not result in a robust IRM plan in the absence of practical field experience, information on growing environments and grower practices. the technology. By contrast, there is a natural tendency for growers to be less cautious, driven by short term needs to produce a crop in a costeffective manner. Practical IRM needs to strike a balance between these competing perspectives. The goal should be to enable growers to have access to the technology, while providing effective stewardship that will provide an acceptable level of protection against resistance. Insect resistance management plans need to be suitable for the given production situation. A range of factors needs to be considered when developing an IRM plan for specific crops in specific growing areas BIOLOGY AND ECOLOGY OF MAJOR PESTS The core of the IRM plan is based on the biology of key target pests and on pest-crop interaction. Insect resistance management plans developed for different countries or regions should be tailored to the specific local needs, and while all the elements outlined in this manual should be considered, some may not be locally appropriate or feasible. Most crops have a complex of primary and secondary pests. Many secondary pests can be controlled via other integrated pest management (IPM) tools if the key target pest is controlled by the trait. The primary target pests of the insect protected crop should be identified for the region and the efficacy of the insect protected crop characterised for each of these. The information on the biology of the target pest should include the history of control measures, such as classes of insecticides sprayed in the area and the combination of IPM approaches adopted, to assess the potential for resistance development. The life cycle of the insect pest, including the number of generations of the pest in different growing regions and seasons, annual migrations and the movement of both larval and adult stages of each target pest on the crop and other host plants, should be investigated with the help of local entomology and pathology experts. The location, timing and distribution of feeding damage on the crop should be understood. If refuge is an aspect of the IRM plan, insect movement has a direct impact on the design of a refuge for producing susceptible insects. For polyphagous pests, that eat many different plant species, the availability and suitability of alternative host plants, such as other crops, natural vegetation and weeds, or a combination of these, can be considered as refuge so long as sufficient susceptible 6

9 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops Table 1: Examples of generalist and specialist insect pests (developed from Bernays and Minkenberg, 1997). Generalist pests Cotton bollworm, Helicoverpa zea Old world bollworm, Helicoverpa armigera Tobacco budworm, Heliothis virescens Cabbage looper, Trichoplusia ni Specialist pests European corn borer, Ostrinia nubilalis Western corn rootworm, Diabrotica virgifera virgifera Plants eaten Wide variety of cultivated and uncultivated species Primarily maize insects are reliably produced at the same time and in the same areas as the Bt crop is grown. This is commonly referred to as a natural refuge and can dominate the life system of a target pest species in a particular release area such that the resistance risk is acceptable. Some examples of generalist and specialist insect pests are given in Table PRODUCT DEPLOYMENT STRATEGIES Characterisation of the insect protected crop as it relates to the IRM plan should include the effectiveness of the trait at protecting the crop and controlling the insect pests in different plant parts and across the growing season. This characterisation of the insect protected crop will help identify possible control gaps where additional control measures might be necessary. For example, under heavy insect pressure, it is not unusual for certain types of insect protected crop to require supplemental insecticide sprays to protect yield. Such sprays are expected to reduce the selection pressure for resistance to the insect protected crop. A high dose trait-insect combination means that the trait is sufficiently effective, and the target insect pest sufficiently sensitive to it, that very few if any of the exposed target pest insects survive. In this situation, the high-dose/refuge approach may be appropriate, whereby provision of a small refuge, consisting of a host crop that does not have traits for controlling the pest, allows the production of large numbers of susceptible insects. These susceptible insects are then available to mate with any resistant insects surviving in the insect protected crop and to pass susceptibility on to the offspring. In some crops, the refuge can be provided as a seed blend of insect protected and non-insect protected seeds; in others, and more commonly today, the refuge should be planted separately from the insect protected crop, as a block or as strips within the insect protected field, or in a nearby field. The high-dose/refuge The goal approach is not should be to enable always applicable growers to have to all situations access to the technology, as it relies on while providing effective high sensitivity stewardship that will of the pest population to the provide an acceptable insect protected level of protection crop and a farming against resistance. infrastructure that is amenable to managing structured refuges. In many situations, one or more of the target pests is not highly sensitive. In these cases, while a refuge helps in reducing selection pressure for resistance, other considerations are also important such as: the availability of alternative host plants (including varieties of the same crop that do not have insect protection traits) that provide a natural refuge; long-range dispersal of the insect population across different cropping regions, and the use of supplemental control measures that provide additional pest management. There are multiple techniques to reduce insect selection pressure, incorporating methods based on IPM wherever possible. These include, but are not limited to: the use of refuge; scouting and applying insecticides as needed; rotating different modes of action; restricting the use of a single insect control protein across multiple crops that form a natural refuge complex; or limiting the use of multiple crops with the same insect control proteins; 7

10 3. Developing a robust IRM plan capping sales to limit market penetration; destruction of crop residues; using locally adapted crop varieties with native resistance; and combining multiple traits targeting the same pests within a plant. The best IRM plan will match the available products with the local environment and pest pressure conditions in an IPM manner. The ideal level of expression of insect protection proteins in insect protected crops provides: adequate control of the target pest(s) at below the economic threshold for damage; consistent expression throughout the growing season to ensure control as pest populations increase; and control of partially resistant insects. been the most frequently identified resistance mechanism associated with high levels of resistance to microbial Bt pesticides (Van Rie et al., 1990; Ferré et al., 1991; Tabashnik, et al., 1996). Pyramiding of different insect protection proteins into the same plant controls not only a broader spectrum of insects, but may provide superior IRM properties if the insect protection proteins act by unique mechanisms of action on the same target pest. This improved IRM strategy (called pyramiding ) is based on the concept that resistance to two different control proteins present in the same plant is far less likely to develop than resistance to a single control protein (Roush, 1994; Roush, 1997; Gould, 1988). The decrease in likelihood of resistance development is based on the very low potential for two random mutations to occur in a single insect pest in one generation. Insects that have one such mutation giving resistance to one trait are still controlled by the second trait. Knowing the potential for cross-resistance patterns between the proteins is useful. Insecticidal proteins, which bind to different receptors in the insect midgut, such as Cry1 and Cry2 Bt proteins in tobacco budworm, are examples of such a strategy. The level of target pest control will determine the amount of non-insect protected plant refuge needed to support sufficient susceptible populations. In some cases, the refuge is a structured refuge, i.e. a planting of the non-insect protected crop, matched by maturity level and genetics to the insect protected crop, planted in close proximity. The refuge can also be unstructured, consisting of alternative host plants, such as other crops, natural vegetation and weeds or a combination of these so long as sufficient susceptible insects are produced. The practice of saving a portion of the crop seed harvest for planting in the next growing season can have important negative impacts on resistance management for insect protected crops. Where pyramiding strategies for insect control traits consist of the simultaneous use of two insecticidal agents with different modes of action against the same target pests, these can be used to reduce the development of insect resistance. In using this approach, knowing the potential for cross-resistance patterns between the proteins is useful, and seed suppliers of pyramided traits need to ensure that the IRM requirements are clearly laid out. Insect protection proteins found in U.S. maize events that were available in 2011 are provided in Table 2. Alternating insect protected crops with different modes of action can be used as part of a resistance management strategy to prevent the survival of partially resistant pests that may survive from one season to the next. To use this strategy, growers must alternate the planting of two insect protected crops with different modes of action (see next section) Pyramiding versus single insecticidal traits Insect control Bt proteins act through a receptormediated mechanism. A change in the receptor has Researchers have highlighted the IRM advantages of pyramiding insect control proteins in the same crop variety indicating that the refuge size could be greatly reduced or a natural refuge may be sufficient without increasing the risk of insect resistance (Roush, 1997; Zhao et al., 2005). This has been implemented in the US for cotton with pyramided insect control traits (US-EPA, 2001) and in South Africa for smallholder cotton production. It is important to note that where stacked genes target different pests, there is no IRM advantage and the full refuge requirements are still needed. 8

11 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops Table 2. Available maize traits in the U.S., their spectrum of control, refuge amounts and distances for the Midwest (April 2011). (Adapted from: DiFonzo and Cullen, MSU Field Crops Entomology Program, CDD #288) Current Bt Protein(s) Insects controlled (bold) Herbicide Refuge %, distance or suppressed (italics) tolerance in the MIDWEST Above ground In soil Agrisure products Agrisure CB/LL Cry1Ab ECB CEW FAW SB LL 20% 1 /2 mile Agrisure GT/CB/LL Cry1Ab ECB CEW FAW SB _ GT LL 20% 1 /2 mile Agrisure RW mcry3a CRW 20% adjacent Agrisure GT/RW mcry3a CRW GT 20% adjacent Agrisure CB/LL/RW Cry1Ab mcry3a ECB CEW FAW SB CRW LL 20% adjacent Agrisure 3000GT Cry1Ab mcry3a ECB CEW FAW SB CRW GT LL 20% adjacent Agrisure Viptera Cry1Ab Vip3A BCW CEW GT LL 20% 1 /2 mile 3110 ECB FAW WBC SB Agrisure Viptera Cry1Ab mcry3a BCW CEW CRW GT LL 20% adjacent 3111 Vip3A ECB FAW WBC SB Agrisure Viptera Cry1Ab Cry1F BCW CEW GT LL 5% 1 /2 mile 3220 Vip3A ECB FAW WBC SB Herculex products Herculex 1 (HX1) Cry1F BCW ECB FAW WBC LL 20% 1 /2 mile CEW RR2 (some) Herculex RW (HXRW) Cry34/35Ab1 CRW LL 20% adjacent Herculex XTRA (HXX) Cry1F BCW ECB FAW WBC CRW LL 20% adjacent Cry34/35Ab1 CEW RR2 (some) Optimum products Optimum Intrasect Cry1F Cry1Ab BCW ECB FAW WBC LL RR2 5% 1 /2 mile CEW Optimum AcreMaxRW Cry34/35Ab1 CRW RR2 10% in the bag Optimum AcreMax1 Cry1F BCW ECB FAW WBC CRW LL RR2 10% in the bag (CRW) (AM1) Cry34/35Ab1 CEW & 20% 1 /2 mile (ECB) YieldGard products YGCB Cry1Ab ECB CEW FAW SB RR2 (some) 20% 1 /2 mile YGRW Cry3Bb1 CRW RR2 (some) 20% adjacent YieldGard Plus Cry1Ab Cry3Bb1 ECB CEW FAW SB CRW RR2 (some) 20% adjacent YieldGard VTRW Cry3Bb1 CRW RR2 20% adjacent YieldGard VT Triple Cry1Ab Cry3Bb1 ECB CEW FAW SB CRW RR2 20% adjacent Genuity/SmartStax products Genuity VT Cry1A.105 CEW ECB FAW RR2 5% 1 /2 mile Double Pro (VT2P) Cry2Ab2 Genuity VT Cry1A.105 CEW ECB FAW CRW RR2 20% adjacent Triple Pro (VT3P) Cry2Ab2 Cry3Bb1 SmartStax (Dow) or Cry1A.105 BCW CEW CRW LL RR2 5% adjacent Genuity Smartstax Cry2Ab2 Cry1F ECB FAW WBC (Monsanto) (SSX) Cry3Bb1 Cry34/35Ab1 Genuity SSX Same as SSX Same as SSX LL RR2 For 2012 RIB Complete (Mon) 5% in the bag REFUGE ADVANCED Same as SSX Same as SSX LL RR2 For 2012 Powered by 5% in the bag SSX (Dow) BCW black cutworm; CEW corn earworm; CRW corn rootworm; ECB European corn borer; FAW fall armyworm; SB stalk borer; WBC western bean cutworm; GT glyphosate tolerant; LL Liberty Link, glufosinate tolerant; RR2 Roundup Ready 2, glyphosate tolerant. 9

12 3. Developing a robust IRM plan Local cropping systems Agricultural cropping systems vary by crops, country and culture. High production agriculture may favour crop monocultures across very large areas with few wild or uncultivated areas. Other environments may support a wide diversity of crops on small plots, or crops intermixed with wild vegetation within the landscape. The risk of resistance development is affected by these patterns, with increased risk often associated with monoculture cropping and higher market penetration. The practice of saving a portion of the crop seed harvest for planting in the next growing season can have important negative impacts on resistance management for insect protected crops. The practice would undermine IRM efforts by: creating a mixture of plants with different genotypes, including some plants without genes for insect protection; removing quality control and quality assurance (QC/QA) efforts in the production of the seed resulting in variable trait expression; limiting the accuracy of market penetration assessments, if saved seed is not registered when planted; weakening insect monitoring plans by circumventing the record keeping of volumes and areas where specific varieties are grown; preventing effective grower communication and education efforts; and separating pyramided traits that were combined during crop breeding. Without proper QC/QA by a professional seed company, uniform high dose deployment of the trait and even trait purity can be compromised. selection for resistance is relaxed, and resistant insects may be at a fitness disadvantage. In other cases, there may be year-round production of the crop, perhaps with sequential plantings, in which case selection pressure for resistance may be continuous. The geographic fit of a particular insect protected crop may be limited, perhaps only being preferred in areas of repeatedly high pest pressure; in other areas the pest population may remain at low levels. Such a situation would reduce populationwide selection pressure for resistance. Alternating insect protected crops with different modes of action can be used as part of an IRM strategy to prevent the survival of partially resistant pests that may survive from one season to the next. To use this strategy, growers must alternate the planting of two insect protected crops with different modes of action. Availability of competing insect protection traits in the crop against the same pests can also reduce selection pressure. If different traits are available in one crop or in the market, the selection pressure for resistance to any one is reduced compared with the situation where a single trait dominates the market. The latter case is the situation under which IRM plans were first developed in North America, as at that time only Cry1Ab was available to control European corn borer (ECB) in corn and only Cry1Ac was available to control tobacco budworm in cotton. Monitoring the adoption rate of insect protected crops on a regular basis is important for identifying the highest risk areas. Knowledge of market penetration together with regional market triggers provides useful risk management tools for refuge deployment and insect susceptibility monitoring. Without proper QC/QA by a professional seed company, uniform high dose deployment of the trait and even trait purity can be compromised. If the market penetration numbers are inaccurate, market triggers and caps become impractical and insect monitoring for insect susceptibility will be hampered because the available sampling locations are not all identified. In order to share critical education information, technology providers, distributors and dealers must be able to identify growers who will benefit from this information Planting choices In some cases, the crop may only be grown during certain times of the year, while the pest population persists throughout the year, surviving on other host plants. This can create a temporal refuge whereby Alternative pest management options Only rarely will insect-protected biotech products represent the only option for managing a pest population. Usually crop damage by insects is managed using a combination of pest management tools, such as manipulation of planting dates to avoid peak pest populations at times when the crop is vulnerable. Using native tolerance or resistant crop varieties can limit the impact of pest populations. Crop rotation is a very effective tool against relatively sedentary pests. Synthetic or biological insecticides are frequently available to reduce populations below economically damaging levels. Other biotech traits may also be available. Use of these alternatives means that selection pressures for resistance to a newly introduced biotech trait can be much lower than 10

13 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops anticipated. Similarly, availability of these tools means that pest populations that are evolving resistance can still be managed, so that the impact of resistance on crop production can be lower than anticipated. When pest pressure levels trigger the need for additional control measures, growers should consult local guidelines and chose a control option, or combination of treatments, that cause the least impact on beneficial organisms. Beneficial organisms are important components in IPM and should be protected as much as possible. When selecting chemical control measures growers should follow the label requirements on the chosen When pest pressure levels trigger the need for additional control measures, growers should consult local guidelines and chose a control option, or combination of treatments, that cause the least impact on beneficial organisms. product and the guidelines for the insect protected crop. Importantly, no pest control products containing Bt should be used on fields that contain biotech-derived insect protected plants with in-plant Bt control mechanisms. Some refuges may not be treated with chemical controls at certain growth stages of the target organism. For example, in some U.S. growing areas, insecticides labelled for adult pest control should not be used in the refuge during the emergence of the adult pests. In some cases, the crop may have inplant weed control mechanisms not present in the refuge, or vice versa. The grower needsto select appropriate weed control mechanisms for the crop and the refuge based on the genetics of the varieties. 11

14 4. Insect resistance management tools There are multiple techniques that can reduce the resistance selection pressure on insect pest populations. The best IRM plan will match the available products with the local environment and growing conditions in an integrated manner. 4.1 Structured refuges Refuges contain crop plants without a biotech trait for protection against the target insects and provide an area where susceptible insects can thrive. This area serves to dilute selection pressure for resistance. In the case of a high dose trait, the susceptible insects produced by the refuge area additionally serve to mate with any rare resistant insects surviving in the insect protected crop area. This prevents inheritance by the progeny of resistance to the control protein. The size of the structured refuge area must take into account the factors that affect the selection pressure for resistance discussed above, as well as grower acceptance. Refuge areas typically yield less than their biotech counterparts because they must sustain some level of insect damage to produce susceptible insects. The value of the crop and the level of industrialisation of the agricultural system (versus subsistence farming) must be taken into account when determining appropriate refuge sizes. In many situations, it is most desirable for the refuge to be planted as a separate block, or in a separate field from the insect protected crop. This isolation prevents wandering insects from sampling insect protected and non-insect protected plants thereby receiving less than the full insecticidal dose. Cross over of insects from protected to refuge crops or vice versa, can reduce the effective refuge size and can favour the survival of partially-resistant insects. With a separate refuge, the grower is responsible for ensuring the refuge is planted alongside the insect protected variety. Such a separate refuge can be provided by supplying a small package of refuge seed along with the larger amount of biotech-derived seed, or by encouraging the grower to separately purchase refuge seed. In other situations, it can be more desirable for the refuge to be provided as a seed blend. This simplifies the grower s operations and shifts the onus of compliance with refuge requirements to the seed provider. In cases where insect movement among plants is limited, or where the movement doesn t favour survival of partially-resistant insects, seed blends may be highly effective. 4.2 Scouting and applying insecticides as needed No biotech-derived insect protected crop should be regarded as a complete solution to all pest problems. Any trait is only effective against a subset of pest species, and the level of control of the target species is often imperfect. This means that growers of insect protected crop varieties must continue to scout their fields for damaging insect populations and use insecticides when economic damage thresholds are reached. Application of insecticides to a target pest population will help control any portion of that population that may be developing resistance to the insect control proteins in the crop. 4.3 Limiting the use of multiple crops with the same insect control proteins In situations where a natural refuge is considered an important factor in reducing selection pressure for resistance and where a large proportion of that natural refuge is also a crop, it may be helpful to limit the use of the same or similar insect control proteins in the natural refuge crop. This could be accomplished by limiting the areas in which insect protected varieties of the second crop are grown, or by requiring the use of a structured refuge for the refuge crop. This would require planning that includes an economic and social analysis to balance the relative benefits of using insect protection traits in the different crops. For example, in the United States, it is recognised that host crops for Helicoverpa zea, such as maize and soybean, represent important components of the natural refuge for insect protected cotton. No insect protection traits are currently commercially used in soybean, and insect protected hybrids of maize require a structured refuge. Therefore both conventional corn and soybeans act as refuge for insect protected cotton. 4.4 Capping sales to limit market penetration As market adoption of insect protection traits increases, so does the selection pressure for resistance. One tool to limit selection pressure is to cap sales of insect-protected varieties at some level. Monitoring the adoption rate of insect protected crops on a regular basis is important for identifying the highest risk areas and monitoring data can be used to trigger refuge deployment and insect susceptibility monitoring. For example, in the Philippines, when a specific market penetration level is reached (i.e., 80%), then all single gene insect protected maize crops must deploy a structured refuge. Until then, the prevalence of non-insect protected host plants can serve as the refuge. 12

15 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops Specific market caps can limit the planting of insect protected crops or favour insect control proteins in one crop but not another as a method of reducing selection pressure. Caps could be applied to specific geographic regions. However, a decision to implement such a cap, and the limits placed on planting would have to take into account the economic and social implications of restricting availability of a beneficial crop protection tool. It can also be logistically complex to enforce such a cap. For example, a market cap of 30% was put in place for the first insect protected cotton grown in Australia, which contained a single Cry protein. In addition, the growers were required to: plant refuge; avoid late planting to prevent high pest pressure; cultivate to destroy pupae in crop residue; work within defined spray thresholds; and monitor moth populations for resistance to the Cry protein (Davidson, 2003). The subsequent introduction of biotech-derived cotton with two different insect control genes for the targeted pests helped to reduce these IRM requirements. 4.5 Cultivation and destruction of crop residues For insects that over-winter in the soil, tillage can reduce survival of any resistant insects by exposing them to adverse environmental conditions, resulting in mortality through natural processes such desiccation, freezing, or over-heating. Pupae busting with cultivation is a central component of the insect protected cotton resistance management programme in Australia. For insects that over-winter within crop residues, such as corn borers that diapause as larvae inside tunnels at the bottom of corn stalks, destruction of the crop residues after harvest will kill large numbers of these pests. This action serves to reduce pest presence in the following growing cycle. Destruction of crop residues can also be effective for insects that feed within the harvested portion of the crop, such as in fresh produce fruits and potato tubers. In some cases, harvesting the insect protected crop before insects complete their development will reduce overall survival of any resistant insects, while elimination and destruction of infected harvested material will lower incidence rate for the pest. 4.6 Variety resistance & good crop management practices For several reasons, healthy locally adapted crops will provide resistance management advantages. Locally adapted varieties often possess native insect resistance or tolerance traits, having evolved or been developed to withstand some level of pest pressure. Healthy crops, produced with good crop management practices, will produce the expected levels of the insect protection proteins, ensuring that the dose and efficacy are matched with the IRM plan. Healthy refuge crops will produce more susceptible insects and better yields than inferior varieties or poorly managed crops. 4.7 Using multiple traits targeting the same pests As with any pest control technology, over-reliance on a single mode of action can rapidly lead to resistance development. As discussed above, pyramiding insect traits that provide high levels of protection from specific pest species are far more robust than are single traits. A pyramiding strategy for insect control traits consists of the simultaneous use of two insecticidal agents with different modes of action. In using this approach, knowing the potential for cross-resistance patterns between the proteins is useful. Insecticidal proteins, which bind to different receptors in the insect midgut, such as Cry1 and Cry2 Bt proteins against tobacco budworm, are examples of such a strategy. Pyramiding of insect control proteins has reduced refuge requirements in the U.S. for cotton (US-EPA, 2001) and maize (Table 2). In addition to pyramiding, multiple traits can be used in a mosaic application, where a patchwork of crops with different insect protection traits is planted on a farm. This mosaic of different traits will reduce selection pressure for resistance against any one control protein, and help preserve the durability of the technology. It can be important therefore that development and introduction of new traits is encouraged as part of the IRM programme. In the U.S., the first insect protected maize and cotton varieties only produced Cry1Ab or Cry1Ac Bt proteins (which are closely related) for protection from corn borers and other lepidopteran pests. However, in subsequent years, new lepidopteran-protection traits have been introduced, such as Cry1F, Cry2Ab, Cry1A.105, and Vip3A, which all add to the diversity of modes of action encountered by the target pests and help extend the durability of these crops. 13

16 5. Baseline susceptibility and monitoring damage Insect monitoring encompasses two basic approaches: monitoring the insects for changes in susceptibility to the control protein and monitoring fields for signs of unexpected levels of damage due to a key target pest. Routine insect monitoring can provide important information about the effectiveness of IRM programmes and detect shifts in pest susceptibility before widespread resistance occurs at the field level. This enables implementation of actions to mitigate the crop damage and manage resistant insect pest populations. Possible resistant insect populations are indicated when insect damage surpasses levels that are normally expected based on the trait and the pest population patterns. It may be possible to contain local or isolated hotspots of resistance with appropriate mitigation measures. Once widespread field failures occur, due to resistance in the key target pest, it may be too late to rescue or sustain the insect protection efficacy against the resistant species in the affected region; however, if the crop remains effective against other primary pests, the overall utility of the insect protected trait may be maintained. Insect monitoring encompasses two basic approaches: monitoring the insects for changes in susceptibility to the control protein and monitoring fields for signs of unexpected levels of damage due to a key target pest. As a first step, measuring the baseline susceptibility of key target pest populations to the trait across the growing area should be completed prior to widespread planting of insect protected crops. Subsequent periodic monitoring (e.g., annual or biennial, depending on the pest and crop) may be warranted to compare insect susceptibility to the baseline data or known susceptible populations. Sampling should focus on regions with highest anticipated risk of resistance development, i.e., areas with high levels of market penetration (adoption) and those areas where insecticide treatment of non-insect protected crops is highest. Insect sampling should be done in areas nearby, but not within or adjacent to the insect protected crop in order to collect sufficient numbers for testing and to ensure that the sample collected is representative of the local pest population. To represent a location, it is recommended that at least 100 larvae, 100 adults, 50 mated females or 50 egg masses are collected, but if populations in the field are small, one half of these numbers will also provide a valid sample. These collections are used to establish laboratory populations for testing using standardized bioassay techniques, where laboratory cultures are feasible. Tests should be performed on the earliest lab generation possible (ideally the first generation progeny if collected as larvae or adults, or the larvae hatching if collected as egg masses). Test systems should be adapted to the species and trait of interest, and usually consist of purified insecticidal proteins overlaid or incorporated into artificial diet. Endpoints can be taken as mortality, growth inhibition, or moult inhibition. Bioassays can be conducted at a set protein concentration with a known response in susceptible populations (e.g., a discriminating concentration that kills or prevents development of at least 99% of susceptible insects, Marçon et al., 2000), or concentration-response curves to estimate parameters such as the LC 50 (concentration required to cause 50% mortality). Grower monitoring of fields for damage due to the key target pests is probably the most important component of resistance monitoring. Grower monitoring of fields for damage due to the key target pests is probably the most important component of resistance monitoring. Growers are likely to be the first to identify a relevant change in the level of field efficacy of a insecticidal trait. Therefore, information should be readily available for growers so they can report any findings of damage in the insect protected crop to seed company or technology provider representatives. Contact information should be provided to distributors, dealers and customers in various forms of product literature, i.e., grower guides and product labels. Once a report is made, one-on-one communication with the grower should be initiated to investigate the source of the damage. Once it is verified that the damage was in a field planted with insect protected crop and a key target pest was involved, a technical representative should visit the grower to: investigate the level of crop damage; assist the grower in mitigating the problem to preserve the current season crop; and report the results for further follow up, if justified. 14

17 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops authorities, i.e. when reporting is required under the conditions of the regulatory approval for commercial production, or providing regulators with safety information in a timely manner. The protocols for these activities are generally included in company standard operating procedures and in stewardship guidelines. If further follow up confirms that this is a case of suspected resistance, additional investigations should be conducted to confirm the level of sensitivity to the insect protection protein compared with baseline measures. If necessary, remedial actions that are in line with the severity of the incident should be initiated. These may include reporting to regulatory 15

18 6. Integrated pest management When pest pressure levels trigger the need for additional control measures, growers should consult local integrated pest management (IPM) guidelines and chose a control option, or combination of treatments, that cause the least impact on beneficial organisms. Beneficial organisms are important components in IPM and should be protected as much as possible. Biotech-derived insect protected crops themselves are known to be extremely benign to non-target arthropods. The refuge approach to IRM is well suited to sustainable agriculture and complements IPM activities, such as the appropriate use of pest thresholds and sampling to inform spray regimes. Cultural and biological pest control practices are also compatible with IPM and are encouraged to improve the management of biotechderived crops. However, Bt pesticides should not be used on Bt-containing biotech-derived crops, or on refuges serving these crops. When selecting chemical control measures growers should follow the label requirements on the chosen product and the guidelines for the biotech-derived crop. Importantly, in addition to not using Bt insect control products on refuges, some refuges may not be treated with chemical controls at certain growth stages of the target organism. For example, in some U.S. growing areas, insecticides labelled for adult pest control should not be used in the refuge during the Bt pesticides should not be used on biotech-derived, insect protected crops or on refuges serving these crops if they contain in-plant protection from a Bt protein. emergence of the adult pests. The reduction of general pesticide use may increase the potential for the emergence of secondary pests and an IPM approach can help to manage these secondary pests. Infestations of pests not controlled by the in-plant insect protection in biotech-derived crops are possible during any growing season. In some cases even target pest infestations will rise above economic thresholds for damage and growers will need to apply additional control measures. Growers are encouraged to use local IPM guides to identify appropriate monitoring schedules, the pests to be monitored, critical economic thresholds for damage, and the recommended alternative control measures. There are IPM checklists to assist growers. These include actions such as: selecting appropriate cultivars for the growing areas; using cultural control measures to reduce pest loads; using recommended scouting and monitoring programmes; and when needed, choosing additional control measures that will have the least impact on beneficial organisms. 16

19 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops 7. Engagement, education and communication Resistance management is the responsibility of all stakeholders. The use of insect protected crops provides a novel insect control option for the marketplace. Although it is clear that the availability of insect protected crops imparts considerable value to growers, it is also clear that it is in the best interest of all stakeholders to preserve insect protection proteins for the long-term benefits they will provide (Gianessi et al, 2002). In fact, the proactive effort for grower education on the responsible use of insect protected crops in the U.S., for example, as well as the speed at which these products have been adopted, has been greater than for any other single insecticidal product in history (James, 2010). Adoption of IRM plans for insect protected crops has been successful to date in part because they were developed with broad stakeholder participation. Experts representing trait providers, academic institutions, regulatory authorities, grower and commodity groups, and other local and regional support groups have played a role during the development and maintenance of IRM strategies. At commercialisation, grower education on the proper use of the crops and associated IRM plans is a key component of how the product is marketed and sold. In line with best practices and product launch policy, seed companies, distributors and It is important that growers are trained through familiar venues that are relevant to their situation, taking into account the culture, language, education levels, avenues of access to information and how products are procured. Resistance management is the responsibility of all stakeholders. growers should be informed about correct product use, including the consequences of resistance development. For example, grower education meetings could be held for purchasers of the product. Other mechanisms for education could include technical bulletins, product brochures, sales meetings, articles in trade journals, presentations by local experts, and grower guides that accompany the commercial product. All of these can be implemented in addition to the bag tag that accompanies the insect protected crop planting material, and which outlines the contents of the product and directions for use. Successful resistance management should begin before introduction of insect protected crops through: The establishment of a local infrastructure of experts who can provide input on key target pest biology and pest/crop interactions that can help to tailor IRM recommendations; Evaluation by scientific experts of the available data to determine if additional research is needed to support implementation of an initial IRM plan and to refine the deployment of an IRM strategy as new research data and experience with the technology becomes available; Provision by grower groups and farm advisor organisations of specific crop production practices; evaluation of how IRM components can be practically implemented; and how information on IRM can be effectively disseminated; Provision of technical information from commodity groups and technology developers, to enable the development of educational materials for stakeholders and users; While this is the ideal approach, it must be realised that not all situations will meet this model. For example, in developing countries, the infrastructure might not exist for widely educating growers on insect resistance management. In these cases, it will be necessary to explore options for implementation. 17

20 7. Engagement, education and communication It is important that growers are trained through familiar venues that are relevant to their situation, taking into account the culture, language, education levels, avenues of access to information and how products are procured. Complex IRM plans that require growers to perform additional tasks beyond their normal planting and cultivation activities could counter good stewardship, so it is better to keep both the plan and the message simple and direct for growers. Education programmes for growers should cover the following areas relating to resistance management: The characteristics of the trait and how it provides insect protection; Guidance on scouting for target pest damage and thresholds for insecticide applications or other pest management tools; Guidance on how to implement refuge requirements, if applicable; Potential consequences of not following best crop management practices, including lost yield potential, resistance development, and loss of the technology; Instructions for communicating with crop advisors, seed dealers and/or technology providers if there are questions about the product performance or management. Expected efficacy against primary and secondary target insects; Guidelines for planting, management, and harvest for optimal productivity, including application of fertilisers, weed management, and other IPM techniques; 18

21 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops 8. Examples of IRM plans United States. The introduction of insect protected crops in the United States brought powerful new pest control agents to growers, which are so effective and safe that the EPA made the unprecedented demand for resistance management plans prior to market introduction. Specifically, these early IRM plans were based on: presence in the plant tissues of a high dose of the insect control protein; the use of refuges with no insect control protein control measures, where susceptible insects could thrive; and the expectation that genes for resistance to the insect control proteins initially occur only rarely in insect pest populations. The first IRM plans in the U.S. required an area of plants without insect control proteins, a refuge, that was close to the insect protected crop so that any rare resistant insects that emerge from the insect protected fields could easily find and mate with susceptible insects. In this scenario, the genetics of resistance is diluted out to keep offspring susceptible to the control protein expressed in the crop. In theory, this IRM strategy should delay resistance development as long as the refuge produces sufficient numbers of susceptible insects so that mating between resistant and susceptible insects is significantly more likely to occur than mating between insects that both have some resistance. The refuge requirements set by the US-EPA in 2006 for insect protected crops are described in Table 3 for maize and Table 4 for cotton. Table 3. Refuge requirement for Bt maize set by the US-EPA (US-EPA, 2006) Trait target Refuge size Deployment Proximity Corn borer 20% (maize regions) Discrete corn borer refuge Internal or external blocks 50% (cotton regions) within ½ mile or in-field strips (at least 4 rows wide) Rootworm 20% Discrete rootworm refuge Internal or external blocks adjacent or in-field strips (at least 4 rows wide) Corn borer + 20% (maize regions) 2 options: Internal or external blocks Rootworm 50% (cotton regions) 1. Common rootworm/corn adjacent or in-field strips borer refuge (at least 4 rows wide) 2. Discrete rootworm/corn Separate fields should be borer refuges used within ½ mile Table 4. Refuge requirement for Bt cotton set by the US-EPA (US-EPA, 2006) Gene # Region Refuge size Deployment Proximity Single All 1. 5% external unsprayed 1. At least 50m wide 1. ½ mile (¼ mile preferred) Dual Arizona, 2. 5% embedded 2. At least 50m wide 2. Embedded in field California, 3. 20% external sprayed 3. N/A 3. 1 mile New Mexico, (½ mile preferred) West Texas Dual Southeast US Natural refuge no structured refuge requirement Single / dual For PBW a only N/A At least one row for Embedded in field Arizona and every 6 to 10 rows California of Bt cotton a pink bollworm 19

22 8. Examples of IRM plans Refuges can be economically practical for growers especially if chemical insecticide treatments (non-bt) are allowed in the refuge to protect the yield while allowing sufficient insects to survive that are susceptible to the control proteins in the insect protected crop. Other elements of these initial IRM plans included: annual monitoring of target pests for susceptibility to the control protein expressed in the crop in areas with high adoption rates; communication and education for growers so that they fully understand and carry out resistance measures; monitoring growers for adherence with the IRM plan (i.e., ensuring that the refuge plots are large enough and at the appropriate distance from the insect protected field); enforcement of adherence through removal of technology from growers found repeatedly out of compliance; and a remedial action plan should resistance be detected. A structured non-insect protected crop refuge provides one source of susceptible insects but it may not be the only source because many insect pests are generalists (Table 1) which feed and develop on a variety of different host plants, e.g., cotton bollworm, Helicoverpa zea; tobacco budworm, Heliothis virescens; cabbage looper, Trichoplusia ni (Bernays and Minkenberg, 1997). These pests can develop on other cultivated crops as well as non-crop alternative hosts, such as those in uncultivated areas and weedy field borders. Other insects are classified as specialists which feed on only one or a few different species (i.e., European corn borer, Ostrinia nubilalis; western corn rootworm, Diabrotica virgifera virgifera). For the generalists, an alternative host refuge may produce equal to or larger quantities of insects than the structured non-insect protected crop refuge. The US-EPA acknowledged this when they removed the need for a structured non-insect protected cotton refuge for biotech-derived cotton varieties with pyramided insect protection genes, relying instead on the abundance of non-cotton plants to provide a natural refuge for Noctuidae species (US-EPA, 2001). In other areas of the world, a variety of IRM tactics have been utilised for insect protected crops taking into account local cropping practices. Australia. Australian authorities and growers worked together to establish IRM plans with the introduction of single-gene insect control cotton in 1996 by limiting each grower to a cap of 30% of the total cotton grown per farm, thereby ensuring a large noninsect protected cotton refuge. This cap was removed when pyramided insect protected cotton events (containing more than one different insect protection protein) were introduced seven years later. The revised regulatory requirement reflects the reduced potential for resistance development associated with varieties that have pyramided control genes. Some other continuing elements of the IRM plan in Australia include restricted planting times, limited insecticide use on refuges and required cultivation after harvest ( pupa-busting ), all in an effort to minimise resistance risk (Davidson, 2003). China. Because farm production in China is typically a mixture of small plots of cotton, maize, soybean, wheat and peanut (<5 ha in total) that serve as natural hosts for the major pest (old world bollworm (OWB), Helicoverpa armigera), there is no requirement for a separate structured non-insect protected cotton refuge for insect protected cotton in China (Wu and Guo, 2005). To date, insect protected maize has not been approved in China, thereby limiting selection pressure on OWB which also feeds on maize. India. In India 1, each bag of insect protected cotton seed includes a second bag containing an additional 20% of non-insect protected cottonseed to plant a refuge. Information is shared on the proper deployment of the non-insect protected refuge. Philippines. In the Philippines 2, where farm size is also small, adopted IRM requirements for insect protected maize have based the need for a structured IRM plan on market penetration, similar to the Australian plan but with different metrics. Until growers in a region plant above 80% of their maize crop as hybrids containing an insect protected trait, they are not required to plant non-insect protected maize as a refuge for the Asian corn borer, Ostrinia furnacalis. All the plans described above include either annual insect susceptibility monitoring and/or routine monitoring for insect damage and follow up testing if populations of the primary target insects are found damaging an insect protected crop. South Africa. The IRM requirements for insect protected cotton production at commercial level are a compulsory 5% refuge of conventional cotton that is never sprayed. A local study has demonstrated that

23 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops sufficient natural refuge exists in the surrounding vegetation in cotton growing areas (Green et al., 2003). The combination of the 5% refuge and the natural refuge provides a reduced potential for resistance development to the Cry1Ac protein by the local cotton pests: African bollworm, Helicoverpa armigera (Hübner); red bollworm Diparopsis castanea (Hampson); and two spiny bollworm species, Earias biplaga (Walker) and E. insulana (Boisduval). Cotton events with pyramided insect protection genes have recently been approved in South Africa CASE STUDY: INDIAN IRM REQUIREMENTS FOR INSECT PROTECTED COTTON The Genetic Engineering Approval Committee (GEAC) is the official body in charge of commercial release of transgenic crops in India, including strategies for insect resistance management. This is advocated so as to increase the durability of the technology and reduce chances for resistance development. Accordingly, biotech-derived insect protected cotton has to be planted in the centre of the plot and non-transgenic cotton has to be planted as refuge crop surrounding the central plot, such that it covers at least five rows or 20% of the total sown area, whichever is more (Figure 1). Figure 1. Required refuge planting pattern in India for insect protected cotton Refuge: Non-Bt cotton 5 rows or 20% Crop: Bt cotton Mixing insect protected (the crop) and non-insect protected (refuge) seeds together before planting is not recommended, because larvae move easily from one plant to the next. The guidelines indicate that each pack of insect protected cottonseed, when sold, should also contain, in a separate bag, the required quantity of non-transgenic seeds to meet the requirements of planting the refuge crop. The planting layout has to be indicated in product literature included in the seed pack. Since the standard pack size of cottonseeds in India is 450 g, each pack of insect protected cottonseed sold contains one pouch of 450 g of insect protected cottonseeds and an additional pouch of 120 g of non-insect protected cottonseeds. For the few years after insect protected cotton was first released for commercial cultivation in India, the non-insect protected counterpart hybrid seeds were used for refuge planting. More recently, the GEAC has allowed the flexibility of any conventional non-insect protected hybrid to be used in refuge planting. However, there is no legal basis for the grower to follow the requirement of refuge planting and adherence enforcement has been a challenge for seed companies. Additionally, it has been suggested by the scientific community that considering the abundant, diverse alternate host availability for the target pest and the cropping patterns followed by Indian growers, the requirement for planting refuge areas may be unnecessary in some parts of India (Singla et al., 2010). The many insect host plants in natural vegetation and the small cotton field sizes, interspersed with vegetable crops, should ensure that resistant moths emerging from cotton fields will easily mate with susceptible moths from other crops and natural vegetation. Structured refuges remain necessary for areas where P. gossypiella and Eraias spp. are primary pests. The requirement for baseline susceptibility data generation by the applicant prior to commercial release of insect protected cotton, followed by regular resistance monitoring after commercial release, has been implemented as a part of the IRM strategy. The Central Institute of Cotton Research 3 (CICR), India s premier public cotton research institute, has been entrusted with the responsibility for the regular monitoring of insect susceptibility

24 9. Implementing structured refuges Where a separate structured refuge is indicated as being important for delaying the development of resistance, special consideration needs to be given to promote grower implementation and appropriate management of the refuge FLEXIBILITY Providing a choice of appropriate systems is essential as it enables growers to choose an acceptable system that best fits their growing conditions, resources and preferences. For example, growers with appropriate planting equipment might prefer to plant strips of refuge within the insect protected crop, while others may prefer to plant separate blocks of the refuge and insect protected varieties. Appropriate refuge guidelines or requirements clearly define the planting configuration options and the crop protection treatments required for each option. The refuge can be embedded in the biotech-derived fields, adjacent to these fields or, in some growing areas, shared between fields for some crops. Refuge can be sprayed or unsprayed, depending on the nature of the biotech-derived crop and the size of the refuge area. In general, refuge that will be sprayed for insect control needs to be larger than unsprayed refuge. For some insect protected crops, where the natural vegetation in the growing area has sufficient host plants to support a population of target pests, the use of natural vegetation as refuge has been approved for these growing environments. When insect protected crops have combinations of different protection for different pests, each of these control measures may need to have its own refuge. For example, maize with two different insect protection proteins that control European corn borer and corn rootworm, will need to have refuges appropriate for both of these pests. In some instances it is possible for certain control agents to share refuge, based on the biology of the pests, the growing environment and the control mechanisms. The size and distance of these common refuge areas is determined by the traits being served by the shared refuge. Grower guides provide planting configuration options for specific crops in specific growing areas (Figure 2). The refuge area should be planted and managed in the same way as the crop. For example, the refuge and insect protected crops should be planted close in time; planted with varieties that have similar maturation times; be given the same inputs and management (soil preparation, irrigation, weeding, fertiliser, pesticide treatments, etc.); and be planted at a similar density. These measures are to ensure that the refuge remains as attractive as the crop to local pests throughout the growing season. Figure 2. Examples of refuge configurations allowed for two crops in certain growing areas. (Adapted from industry IRM guidelines.) Within or Adjacent or 800m option Block Block Adjacent <800m ( 1 /2 mile) Perimeter 800m option valid in some growing areas only Strips Within adjacent field Road, path ditch, etc. Common refuge of non-biotech corn. Minimum four rows for each component Biotech corn with protection against two different pests A. 20% corn refuge option for a cotton growing area 22

25 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops Biotech cotton with one insect protection trait <1.6km (1 mile) Refuge of nonbiotech cotton <1.6km (1 mile) Refuge requirements May be treated with any insecticides, except Bt products B. 20 % sprayed cotton refuge option 9.2. SEED DISTRIBUTION Information for growers about feasible IRM strategies and, the legal instruments to enforce IRM systems, can be linked to the procurement of seed for insect protected varieties (Figure 3). Figure 3. Example of a seed label that indicates IRM requirements for purchased seed (NCGA). Before opening a bag of seed, be sure to read and understand the stewardship requirements, including applicable refuge requirements for insect resistance management, for the biotechnology traits expressed in the seed set forth in the technology agreement that you sign. By opening and using a bag of seed, you are reaffirming your obligation to comply with those stewardship requirements. In the U.S. and Canada growers who purchase or obtain biotech-derived seed are required to sign grower agreements that bind them to the use of one or more IRM systems during the production of the insect protected crop. Information on the IRM options for the crop are distributed with the seed and it is up to the growers to choose which system best suits their growing conditions, cultivation practices and resources. However, legal instruments such as these may not be acceptable or effective in more diverse or less industrialised agricultural systems GROWER EDUCATION AND COMMUNICATION Grower education should cover not just what the refuge requirements are, but why they exist and why they benefit the grower and the farming community in the long run. Frequent, localised communication may be needed to help growers fully understand their responsibilities and how refuge requirements or guidelines can be met in their particular circumstances REFUGE MANAGEMENT If the chosen IRM strategy includes the planting of refuge areas, the layout, the seed needs and the input requirements must be fully understood and prepared for before planting. Similarly, if the IRM strategy requires insecticide treatment, the treatment schedule and the availability of the identified pesticides both need to be verified before planting. In some cases, the crop may have in-plant weed control mechanisms not present in the refuge, or vice versa. The grower needs to select appropriate weed control mechanisms for the crop and the refuge based on the genetics of the varieties. 23

26 9. Implementing structured refuges Figure 4. Example of a field map showing the relative positions of insect protected crops and refuge areas. Creekside Farm Summer 2010 N Hay Hay Hay R24 Soybean RR 5.1 Ha Maize 43 Bt1 + Bt2 1.7 Ha Maize 87 sprayed Maize 87 Maize 43 sprayed 1.2 Ha Bt1 + Bt2 0.3 Ha Maize 43 Bt1 + Bt2 1.8 Ha 0.9 Ha Maize 43 Bt1 + Bt2 2.1 Ha Maize 43 Bt1 + Bt2 2.9 Ha building 50m Planning Growers who chose a refuge option for IRM may wish to map out the planting areas and ensure that the volume and proximity of the refuge are in compliance with the growers agreement for IRM requirements for each insect protected variety. Field maps help to define the planting strategy for each season, as well as guide the management of the crops and the refuge areas through the growing period and during harvest (Figure 4). Refuge planning, planting and crop management may be facilitated if fields and refuge areas are mapped. Refuge areas may have different pesticide application requirements and field maps help ensure that pesticide applications are applied to the correct fields. Where common refuge areas are allowed for certain insect protected products, growers should plan these before seed purchase and planting to ensure that they meet the IRM requirements. If maps are not feasible, other records of where refuge and insect protected fields are located should be maintained. Refuge calculators are developed to help determine the amount of refuge needed for a specific insect protected product or the amount of communal refuge needed for two or more insect protected products. Table 5 gives examples of refuge calculators for two different refuge area requirements. 24

27 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops Table 5. Examples of refuge calculators supplied with grower IRM requirements when biotech-derived insect protected seed is purchased. A: For 5% refuge requirement Examples Your field Field size (hectares) = Maximum insect-protected hectares: Field size x 0.95 = Minimum 5% refuge hectares: Field size x 0.05 = B: For 20% refuge requirement Examples Your field Field size (hectares) = Maximum insect protected hectares: Field size x 0.80 = Minimum 20% refuge hectares: Field size x 0.20 = Planting Refuges for many insect protected crop events entail accurate planting of specific seed in specific areas on a farm. Growers should plant and manage both the refuge areas and the insect protected crop with the same protocols to ensure that the plants grow and mature at comparable rates. In some cases, insect protected plants that are interspersed in a refuge area can lessen the effectiveness of the IRM strategy and the productivity of the refuge area. As such, growers should ensure that planting machinery is cleaned thoroughly prior to planting a refuge area. Refuge can be planted within or adjacent to the insect protected crop and must not contain the same insect protection that is present in the crop. When perimeter or in-field strip refuge is planted, a minimum width of the strips may be determined for each crop-traitenvironment combination and should be described in information material that is provided to the grower. When planting strip refuge within the field, the required volume of refuge seed can be loaded into the specific hoppers on the planter that will ensure planting of the required refuge strip width. When the refuge seed is finished, the hoppers can be filled with the insect protected seed for the rest of the planting. In some cases refuge areas should be under the control of the same grower who is managing the insect protected crop, especially if farm or field sizes are large. It may be appropriate for a group of growers to cooperate in managing refuge areas across multiple farms, especially if farm size is small Recording At planting, the growers are encouraged to record the placement and dimensions of any refuge with respect to the insect protected crop. The refuge areas should receive the same inputs and management protocols applied the insect protected events. To provide a record of adherence, the grower is encouraged to record the soil preparation protocols that were used and all inputs for the crop and the refuge during the growing season. In addition, it is useful to record all scouting activities and to document any insect damage identified in the insect protected crop and in the refuge. Recording is an important tool for managing the development of insect resistance in insect protected crops, however it can also be time consuming and is not completed by many growers. In some cases growers file the labels on planting material packaging and write the planting date on these. Together with a map of the planting areas, this would provide some record of the crops on the farm for that season. This manual contains examples of forms designed to facilitate recording of the information that is relevant to insect resistance management. Growers can adapt these forms to their requirements. 25

28 9. Implementing structured refuges 9.5 MONITORING THE IMPLEMENTATION OF REFUGES It is standard practice for technology developers to assess implementation of the IRM strategies recommended for their seed. Some regulatory authorities require compliance monitoring and reporting as part of the authorisation conditions for commercial use of insect protected plants. Most developers or seed companies work with their customer during the growing season, discussing their crop management programmes, challenges encountered, and checking for insect damage. Growers It is standard practice for technology developers to assess implementation of the IRM strategies recommended for their seed. who have not complied with the IRM requirements for insect protected biotech-derived seed should be provided with additional education and assistance. In some cases, growers who repeatedly ignore refuge requirements can be denied access to the biotech seed in subsequent seasons. Information from refuge implementation assessments should be used to refine education programmes around the need for refuges and how best to implement them. This information can also be used to review the IRM requirements themselves to see if they can be made more flexible or more practical without significantly compromising their effectiveness REPORTING Growers are encouraged to report unexpected insect activity and damage during the growing season. Contact details for submitting these reports should be part of the seed package label. The seed provider will discuss the damage with the grower and together determine how best to protect the crop and what follow-up actions are needed to investigate and manage any potential insect resistance development in the local pest populations. 26

29 Practical Approaches to Insect Resistance Management for Biotech-Derived Crops 10. Remedial action plans of the insect population(s) for susceptibility to the biotech-derived insect protection protein. This testing will be coordinated by the technology developer and should be done in the specific area of crop damage and in surrounding regions, to delineate the scope of resistance and compare the bioassay results to precommercial baseline data. The implementation of a remedial action plan should occur if field resistance to an insect protected crop is confirmed in a key target pest. Depending on the situation, remedial action plans could have multiple components, such as planting a structured refuge, applying insecticides or other pest management tools, and/or temporarily halting sales of the affected insect protected crop in the affected area. The immediate remedial action goals should be to: For a worst-case scenario, where insect resistance is confirmed and appears to be spreading, a long-term remedial action plan should be developed. This may consist of the integration of additional pest control technologies. Other options for controlling the key target pests should be identified for growers, including soil cultivation, insecticide applications and use of alternative biotech-derived insect control traits. If no effective alternatives are available and the insect protected crop is no longer providing economic levels of control of key target pests, sales of varieties expressing this insect protection protein could be suspended or restricted in the affected region in future growing seasons. protect the grower s crop investment; characterise the resistant insects to enable the development of case-specific management options; and contain the spread of these insects utilising the best available control methods. Furthermore, once it has been determined that the damage is caused by a targeted insect and that the plants contain the insect protection protein, the investigation should move to collection and testing 27