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Insect Resistance Management for Bt Crops

Bt作物的昆虫抗药性管理Tessa Milofsky, M.S. and Sharlene Matten, Ph.D.泰莎 . 米洛夫斯基 硕士沙利恩 . 马顿 博士U.S. Environmental Protection Agency Office of Pesticide Programs美国环保局 杀虫剂项目办公室

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IRM: what is it?昆虫抗药性管理定义Insect resistance management (IRM) is a strategy to mitigate the development of insects resistant to a pesticide (e.g., resistance to Bacillus thuringiensis or Bt)

昆虫抗药性管理 (IRM) 是为了减轻害虫对杀虫剂 ( 例如对 BT) 产生抗性而采取的一种策略 .

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IRM: why is it important?IRM的重要性 Maintaining the efficacy of Bt, a low-risk pesticide, is beneficial for

the following reasons: 维持低风险杀虫剂 Bt的功效有如下的优点 :

– Bt replaces higher risk pesticides– Bt 杀虫剂可以代替其他高风险性的杀虫剂– Less exposure of nontarget organisms to superficially applied pesticides– 降低非目标生物暴露于杀虫剂的可能性– Use of Bt crops may provide greater profits to farmers, due to reduced use

of insecticides– 由于减少了杀虫剂的使用 ,Bt 作物可以给农民带来更大的收益

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Critical components of IRM planIRM计划的重要组成成分 Pest biology 害虫生物学 Dose 剂量 Cross-resistance 抗性转移 Refuge 避难所 Modeling 建立模型 Resistance monitoring 抗性监测 Remedial action 矫正措施 Compliance assurance 确保规范性 Grower education 种植者教育

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Pest biology害虫生物学 Important factors include:

– Number of insect generations per year– Host range– Insect movement and dispersal patterns– Mating and ovipositional behavior– Population dynamics

要点包括 :– 每年害虫繁殖的代数– 宿主的范围– 害虫移动和分布的模式– 交配和产卵行为– 种群动态

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Pest biology: an example害虫生物学举例 European corn borer (ECB), Ostrinia nubilalis

– 1-4 generations per year– Polyphagous (prefers corn)– Movement:

Larvae - can move from plant-to-plant within a field Adults - mostly local movement, but some long distance migratory movement can

occur 欧洲玉米螟 (ECB)

– 每年繁殖 1-4 代– 杂食性 ( 偏好玉米 )– 移动

幼虫可以在同一田地内不同植株上移动 成虫大部分在本地活动 , 但也有可能进行长距离迁移

Female雌性Male雄性

late instars晚期幼虫ECB

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Dose 剂量 Dose = the amount of toxin expressed over time by a transgenic crop,

relative to the susceptibility of a target pest; can be determined through field efficacy trials or laboratory bioassays

剂量 = 转基因作物在一定时间内表达的毒素的量 , 和目标害虫的敏感性相关 ; 通常由田间功效实验或实验室生物测试所决定 . High dose = at least 25x the amount of toxin needed to kill all

susceptible target insects; preferred over low dose events for refuge-based IRM strategies

高剂量 = 至少为杀死所有敏感目标害虫剂量的 25 倍 ; 在以避难所为基础的昆虫抗性管理策略中，使用高剂量遗传事件体要比低剂量遗传事件体好

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Dose assumptions剂量假设 Three genotypes for resistance/susceptibility

(SS, rS, rr) Resistance (rr) is recessive and rare Refuge supplies SS (homozygous susceptible)

genotype Random mating:

– rr x SS → rS– rS x SS → rS or SS

SS and rS susceptible to high dose 三种抗性 /敏感性基因型 (SS,rS,rr) 抗性基因 (rr)是隐性的 ,少见的 安全基因型 SS(纯合敏感 ) 随机交配 :

– rr x SS → rS– rS x SS → rS or SS

SS 和 rS对高剂量敏感

DoseMortality

SSrSrr

LC50

0

100

低 高

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Dose levels: some examples关于剂量水平的一些例子 Lepidopteran-active corn (Cry1Ab & Cry1F):

– High dose European corn borer (ECB), Ostrinia nubilalis Southwestern corn borer (SWCB) Diatraea crambidoides

– Non-high dose Corn earworm (CEW), Helicoverpa zea

鳞翅目敏感玉米 (Cry1Ab & Cry1F):– 高剂量

欧洲玉米螟 (ECB), Ostrinia nubilalis 西南玉米螟 (SWCB) Diatraea crambidoides

– 非高剂量 玉米棉铃虫 (CEW), Helicoverpa zea WCR

female male

late instars

ECB

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Dose levels: some examples关于剂量水平的一些例子 Rootworm-active corn (Cry3Bb1, Cry34/35, mCry3A)

– Non-high dose Western corn rootworm (WCR), Diabrotica virgifera virgifera Northern corn rootworm (NCR), D. barberi Mexican corn rootworm (MCR), D. virgifera zea

Secondary pests– Non-high dose for some pests

根虫敏感玉米 (Cry3Bb1, Cry34/35, mCry3A)– 非高剂量

西部玉米根虫 (WCR), Diabrotica virgifera virgifera 北部玉米根虫 (NCR), D. barberi 墨西哥玉米根虫 (MCR), D. virgifera zea

次级害虫– 非高剂量 WCR

female male

late instars

ECB

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Cross-resistance抗性转移 When resistance to a single Bt toxin confers pest resistance to a cluster

of related toxins Factors affecting cross-resistance include:

– Similarity/dissimilarity of commercialized toxins– Use of single toxin versus multiple toxins– Level of resistance in an insect population

害虫对单一 Bt 蛋白的抗性使得害虫对其他一系列相关毒素都产生抗性 影响抗性转移的因素包括 :

– 商品用毒素的异同点– 单一毒素和复合毒素的使用– 昆虫种群的抗性水平

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Cross-resistance: an example(抗性转移 :举例说明 )

Binding site diagram for European corn borer( 欧洲玉米螟结合位点简图 )Cry1Ab Cry1FCry1Ac

220 KDaCadherin

154 KDaAPN

145/167 KDa APN

Other

European corn borer (based on Hua, G., Masson, L., Jurat-Fuentes, J. L., Schwab, G., Adang, M. J. 2001. Binding analyses of Bacillus thuringiensis Cry d-endotoxins using brush border membrane vesicles of Ostrinia nubilalis. Appl. Env. Microbiol. 67: 872-879)

( 其他 )

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Refuge 避难所• Any host plant that does not produce Bt toxin and has

not been treated with conventional Bt formulations (may be treated with non-Bt insecticide)

• 任何不产生 Bt 毒素并且也未经常规 Bt 杀虫剂处理过的宿主植物 ( 可能经非 Bt 杀虫剂处理过 )• Enables mating between resistant and susceptible

adults, resulting in production of susceptible offspring• 抗性和敏感性成熟植株杂交可以产生敏感性的后代

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Refuge options 避难所的选择 Structured refuge = specified size, location in relation to Bt

field, and crop/plant composition Community-based refuge = neighboring growers establish

shared structured refuge Unstructured or natural refuge = use of alternative host

plants/crops in place of defined refuge 结构避难所 = 特定大小 , 并且位置和 Bt 植物种植区有一定关系的区域 , 此区域既有农作物 , 又有植被 以社区为基础的避难所 = 相邻的种植者建立的一种共享的结构避难所 非结构或称天然避难所 = 用一种寄主植物和作物互相交替种植的模式来代替人为限定的避难所模式

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Refuge options 避难所的选择 Seed bag mix = Bt seed bags contain Bt seed and non-Bt seed (e.g.,

80% Bt, 20% non-Bt); seed may be bagged separately or mixed together

Regional limit = national/regional government enforced limit on Bt seed sales (e.g., 80% in defined region)

混装种袋 = 在这种含 Bt 种子的袋子中既含有 Bt 种子 , 也含有非 Bt 种子( 例如 :80%Bt 种子 , 20% 非 Bt 种子 ), 种子可以先按小袋分装好再混和 ,也可以直接混在一起 .

区域限制 = 国家或地方政府对 Bt 种子的买卖进行严格限制 ( 例如 : 在一定区域内，只允许 80% 的 Bt 种子的买卖 )

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Seed mix

Block

In-field strips田地内的条带模式

Internal block(s)内部种植模式 Border (block)周边种植模式

Structured refuge options结构避难所的选择

External block外部种植模式

种子混和

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Other refuge options其他避难所选择

*********************Refuge Field**********************

*********************Refuge Field**********************

One mile or less

One mile or less

BollgardBollgard

BollgardBollgardBollgardBollgard

BollgardBollgard

BollgardBollgard

BollgardBollgard

BollgardBollgard

BollgardBollgard

BollgardBollgard

(20%)Sprayed

(20%)Sprayed

Community refuge社区避难所模式80% Bt seed corn玉米种子 20% non-Bt

seed corn玉米种子

Natural refuge天然避难所

Bt cottonBt 棉花 w/ TBW

Soybean大豆 Tobacco烟草

Tobacco budworm, 烟草蚜虫Heliothis virescens

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Bt corn refuge specifications: an exampleBt玉米避难所规范示例 20% refuge required in corn belt; 50% refuge

required in corn/cotton region Structured refuge is required. Refuges may be

planted in one of the following designs:– In-field strips (4-6 rows)– Blocks (internal or external)

Refuge requirements are the same for lepidopteran and coleopteran active products

在 Bt 玉米田中 20% 为避难所 , 在玉米 / 棉花田中则为 50% 结构性避难所是必须的 . 避难所可以按如下的设计来耕作 :

– 田地内条带 (4-6 行 )– 内部或外部田块

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Modeling 模拟 Useful tool for predicting the likelihood of resistance, for evaluating the

durability of IRM strategies, and for helping to focus data needs. Model components include:

– Biological information (pest biology, population dynamics)– Genetics (R-allele dominance/frequency, cross resistance)– Operational aspects (dose, single/stacked toxin(s), refuge)

建立模型是预测抗药可能性 ,评价 IRM 策略持久性和帮助调整数据需求的一种有效方法 . 模型包括以下组成成分 :– 生物信息 ( 害虫生物学 , 种群动态 )– 遗传学 (R-等位基因显性 /频率 , 抗性转移 )– 运行情况 ( 剂量 , 单一的 /叠加的毒剂 , 避难所 )

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Resistance monitoring抗性监测 Can help to prolong the efficacy of an insect resistant crop Designed to detect resistance 2-3 years before field failure Target pest sampling consists of:

– Grower reports of unexpected pest damage in insect-protected crop fields– Random population sampling:

Sampling based on pest prevalence in region Sampling target: number of insects needed to determine resistance at

p<0.01 可以延长抗虫作物的功效 可以在失效前监测抗性 2-3 年 目标害虫取样包括 :

– 种植者在抗虫作物区域内由害虫造成的意外损失报告– 随机种群取样 :

害虫流行区域取样 取样目标 : 用来确定抗性所需的害虫数量 (p<0.01)

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Resistance monitoring (cont.)抗性监测 (续 )

Bioassays are used to detect potential resistance in sampled populations. Assays include:– Pest susceptibility (LC50)– Discriminating Dose (LC99)– F2 screen

Confirmation studies are used to verify resistance. Questions include:– Is there field failure? – Is the insect resistance trait expressed? – Is insect resistance trait inherited?

用生物鉴定来检测样品种群中的潜在抗性 . 生物鉴定包括 :– 害虫敏感性鉴定 (LC50)– 差异剂量 (LC99)– F2筛选

确认研究用来核实抗性 . 包括 :– 区域失效了么 ?– 抗性特征表达了么 ?– 抗性性可遗传么 ?

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Resistance monitoring: positive hits for Bt corn in the US? 抗性监测 : 美国 Bt玉米正采样数 In 2001, mortality for one European corn borer (ECB)

population (Kandiyohi, MN) was < 99% in Cry1Ab discriminating dose assay

Follow-up tests with Bt plant material revealed that population was not field resistant

No field resistance confirmed for any monitored Bt corn pest 2001 年在 Cry1Ab 不同剂量分析中 , 欧洲玉米螟种群的死亡率小于 99% Bt 作物的后续测试表明种群并没有田间抗药性 被监测的 Bt 玉米害虫没有被证实产生田间抗药性

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Remedial action plan矫正措施If resistance is confirmed through resistance monitoring, a remedial action plan is implemented to contain or slow the spread of resistant pest populations

如果抗性监测确认了抗药性 , 矫正措施可以减缓害虫种群抗药性的传播

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Remedial action plan: an example矫正措施举例 EPA’s plan includes:

– Mandatory reporting of suspected resistance– Use of alternate control measures in affected areas– Increased resistance monitoring– Suspension of Bt crop sales in affected areas– Investigation of cause of resistance

美国环保局措施包括 :– 强制要求对有怀疑的抗药性表现上报– 在有影响区域使用其他管理措施– 增强抗性监测– 在被影响区域终止 Bt 玉米销售– 对抗药性原因进行调查

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Compliance assurance确保规范性Compliance measures implemented to ensure that growers comply with approved IRM program

规范操作可以确保种植者按照害虫抗药性管理规则执行耕种操作 .

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Compliance assurance: an example确保规范性举例 EPA requires registrants to implement the following measures:

– Contracts affirming grower agreement to comply with approved IRM strategy– 3rd party grower surveys of IRM implementation– On-farm field visits to verify IRM implementation– Tips and complaints on non-compliers– Suspension of seed access for non-compliers

美国环保局规定登记者必须实施以下方法 :– 合同规定种植者必须遵守 IRM 策略– 第三方对 IRM执行情况进行调查– 田间观察以确定 IRM 的执行情况– 揭发和投诉不遵守规则的种植者– 对不遵守规则的种植者终止种子供应

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Grower education种植者教育Education about the need for IRM, how to implement requirements, and grower responsibility in complying with IRM requirements

对 IRM 策略的必要性 , 怎样完成要求 , 和种植者对完成 IRM 要求所担负的责任进行教育

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Summary of EPA’s IRM requirements for lepidopteran-active Bt corn美国环保局对鳞翅目昆虫敏感的 Bt玉米 IRM要求概述 20% refuge in corn belt; 50% refuge in cotton belt Structured refuge required. Refuges planted in one of the

following designs:– in-field strips (4-6 rows)– blocks (internal or external)

Industry-wide collaboration on compliance surveys 在玉米种植带中 20% 为避难所 ; 在棉花种植带中为 50% 结构性避难所是必需的 . 避难所种植需为如下设计中的一种 :

– 田间条带 (4-6 行 )– 田块 ( 内部或外部 )

广泛合作开展规范性调查

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Summary of EPA’s IRM requirements for lepidopteran-active Bt corn (cont.)对鳞翅目昆虫敏感的 Bt玉米 IRM要求概述(续 )

Industry-wide uniformity of:– Grower agreements– Grower training– Implementation of tips and complaints

以下行为保持高度一致性 :– 种植者协议– 种植者培训– 监测和投诉的实施

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Summary of EPA’s IRM requirements for lepidopteran-active Bt corn (cont.)对鳞翅目昆虫敏感的 Bt玉米 IRM要求概述 (续 )

•Annual reporting requirements: － Compliance assurance (e.g., surveys, grower contracts) － Changes to grower education － Resistance monitoring data － Sales data

年度报告需要 :–规范性确认材料 ( 例如调查 , 种植者合同 )–种植者教育的变化–抗性监测数据–销售数据

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Data sources for EPA’s IRM program美国环保局 IRM项目的数据来源 Published scientific literature Industry submissions Experts (academia, government, industry) Meetings (advisory panels, workshops, ESA, NC205, NC46) Field visits 已出版的科学文献 工业企业的报告 专家 ( 学术界 ,政府 ,工业界 ) 会议 (顾问团 ,研讨会 ,ESA,NC205,NC46) 田间观察

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Keys to success at EPA美国环保局 (EPA)取得成功的关键 EPA is responsive to new scientific information EPA seeks scientific input from state and federal

agencies, academic experts, and public interest groups

EPA 对最新的科技信息反应积极 EPA 从各州和联邦的政府部门 , 学术专家和公众团体中寻求科技力量

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Keys to success at EPA美国环保局 (EPA)取得成功的关键 Conditional (time-limited) registrations enable EPA to adapt

IRM requirements over time EPA negotiates regulatory changes with registrants and

grower organizations

有时间限制的注册可以使 EPA适应 IRM随着时间变化而产生的新要求 EPA 经常与注册者 , 种植者组织就规则调整进行协商

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IRM challengesIRM面临的挑战 Plans are specific to the:

– Pest/crop– Country/region

Bt selection intensity will be affected by:– Adoption/distribution of a Bt crop– Cross-resistance potential with similar products (similar modes of action)

计划往往针对 :– 害虫 / 作物– 国家 / 地区

Bt选择强度会受到以下情况影响 :– Bt 作物的种植 / 分布– 相似产品 ( 相似作用模式 )潜在的抗性转移

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IRM challenges (cont.)IRM面临的挑战 (续 )

Plans must be economically feasible to: – Growers (planting refuge)– Industry (education, compliance, monitoring and evaluation)– Government agencies (program evaluation, policy

negotiation/implementation) 对种植者 ( 种植避难所 ),工业企业 ( 教育 , 规范性 , 监测和评估 ) 以及政府部门 ( 项目评估 ,政策商讨 /执行 ) 来说 ,计划必须在经济上是可行的 .

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IRM challenges (cont.) IRM面临的挑战 (续 )

Plans must be flexible/adaptable enough to consider:– Evolving science/new data– New products– Need for consistency across products

考虑到以下情况 ,计划必须是灵活的 , 可变通的 :– 学科发展 /新数据– 新的农产品– 产品一致性的需要

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Conclusions结论 IRM is based on strong science Plans must be:

– Pest specific– Flexible and adaptable– Easy to implement– Economical

IRM 基于很强的科学背景 计划必须 :

– 对害虫有专一性– 灵活的 , 可变通的– 易于实施– 经济实用