United States
Environmental Protection
Agency
Prevention, Pesticides
And Toxic Substances
(7511W)
EPA 739-S-98-001
May 1998
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TABLE OF CONTENTS
Preface .'.''• . "•. ' : • ' ', ., .,'.--', . .-• •. • . •• i
Bt Plant-Pesticide Resistance Management White Paper Team. ii
Introduction: Background and Purpose of the Paper 1
I. General Discussion of Public Hearing Issues 4
A. The Requirement of Resistance Management Plans 5
B. Scientific Information Needed for Resistance Management Plans 8
C. Use of "Public Good" as a Criterion for the Requirement of 10
Resistance Management Plans
p. Performance of Bt Cotton 12
II. Bt Potato Resistance Management 14
III. Bt Corn Resistance Management 20
IV. Bt Cotton Resistance Management : 51
... • '• •.-..'' ' - • • ' ;- ': , v:' ". ;. .•••/,
V. Document Summary 77
Appendix 1: Table of Pest Acronyms 80
Appendix 2: Table of Registered Bt Plant-Pesticides 81
References v 82
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i , -. ' ':.-•.'-• • , • Preface ' • >. ,.
The,Environmental Protection Agency's White paper on Bt Plant-pesticide Resistance
Management was originally prepared as a support document for the February 9-10, 1998 - ', '
Office of Pesticide Programs (OPP) Federal Insecticide, Fungicide and Rodenticide (FIFRA)
Science Advisory Panel Meeting (SAP) on Bacillus thuringiensis (Bt) plant-pesticides
resistance management., Written statements and other background documents for this SAP
meeting, can be obtained from the OPP Docket Office, 703-305-5805, Docket Number, OPP-
00231. The EPA White Paper can also be obtained electronically from the EPA Home Page
at: Federal Register-Environmental pOeuments~"Law and Regulations"
(http://wwwlepa.gov/fedrgstr/). " •'•'.'•'"
Hard copies of this publication can be obtained by writing to: . - '
Sharlene R. Marten, Ph.D. ' .
Biopesticides and Pollution Prevention Division (7511W) ,
Office of Pesticide Programs
Office of Pesticides, Prevention, and Toxic Substances
U.S-Environmental Protection Agency '
401 M Street, SW : , : ' \
Washington, DC 20460 '
matten.sharlene@epamail.epa.gov , . ' . .
Additional copies can be obtained by contacting the National Center for Environmental
Publications and Information at NCEPI; 11029 Kenwood Road; Building 5; Cincinnati, OH
45242, or by phone (5-13-489-8190) or fax (513-489-8695). ,
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Bt Plant-Pesticide Resistance Management White Paper Team
Office of Pesticide Programs:
Team Leader and Primary Author:
4 i ,,' i , , ..'',. -
Sharlene Matten, Ph. D. Biopesticides and Pollution Prevention Division
Biologist
Science and Regulatory Review:
Alan Reynolds, Entomologist Biopesticides and Pollution Prevention Division
Robyn Rose, Entomologist Biopesticides and Pollution Prevention Division
Douglas Sutherland, Ph.D., B.C.E., Biological and Economic Analysis Division
Entomologist
Phil Hutton, Chief, Biopesticides and Pollution Prevention Division
Microbial Pesticides Branch
Janet Andersen, Ph.D., Director Biopesticides and Pollution Prevention Division
Office of Pesticides, Prevention, and Toxic Substances
f '
Policy Review:
Elizabeth Milewski, Ph.D. Office of Pesticides, Prevention, .and Toxic
Special Assistant for ' Substances
Biotechnology
Cover graphic design layout:
Creavery Lloyd Office of Pesticides, Prevention, and Toxic
Substances
Office of General Counsel
Legal Review:
Mary Beth Gleaves, Attorney Office of General Counsel
n
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Introduction: Background and Purpose of the Paper
The Environmental Protection Agency (EPA) has considered the development of pesticide
resistance and pesticide resistance management in its "regulatory decisions (see reviews Matten
et al, 1996 and updated in Matten, 1997). In general, pesticide resistance management is
likely to benefit the American public by reducing the total pesticide burden on the
environment, and by reducing the overall human and environmental exposure to pesticides.,
Although EPA does not yet have a published policy or standard data requirements in place for
pesticide resistance management, it has required the submission of such data on a case-by-case
basis. EPA supports the efforts of all stakeholders to promote pesticide resistance management
through the development and use of pesticide resistance management plans, appropriate
pesticide labeling and education programs. It is the desire of EPA that this focus on pesticide
resistance management not overly burden the regulated community, jeopardize the registration
of reduced risk pesticides, or exclude conventional pesticides or other control practices which
can contribute to the further adoption of integrated pest management (IPM). EPA believes,that
appropriate resistance management.can further these goals. EPA is continuing to evaluate and
refine the role pesticide resistance management has in the Agency's regulatory decisions.
With a greater focus on pollution prevention and pesticide risk reduction, the EPA believes
that it is important to implement effective resistance management strategies for pesticides such
as Bacillus thuringiensis (Bt) plant-pesticides. A great deal of Agency attention has focused on
the potential development of resistance to the delta-endotoxins of Bt genetically-engineered into
plants (Bt plant-pesticides). This is because Bt plant-pesticides produce the pesticidal active
ingredient, the Cry delta endotoxin, throughout the growing season. Long-term exposure to a
pesticide is one of the factors increasing the potential selection pressure upon both the target
pests and any other susceptible insects feeding on the transformed crop. EPA recognizes the
value of Bt plant-pesticides as effective and safer pest management tools and has determined it
is appropriate to conserve this resource by requiring resistance management plans for certain
transformed crops. The Agency has reviewed the initial strategies from registrants for
managing resistance to Bt delta endotqxins produced in potato (Bt potato), field corn (Bt corn),
and cotton (Bt cotton) and, when necessary, made certain recommendations and requirements
for the development of data to develop and implement long-term resistance management
strategies as part of the registration decisions. EPA has worked and is working with
stakeholders (industry, university, and USDA extension entomologists, individual growers,, user
groups, trade organization, public interest groups, and government agencies) to address
resistance management to Bt plant-pesticides.
The purpose of this paper is to analyze data generated in the 1996 growing season for current
resistance management plans for Bacillus thuringiensis plant-pesticides for Bt potato, Bt corn
and Bt cotton, identify technical modifications that might improve approaches to resistance
management, identify areas of ongoing research, and determine what might be required in the
future for successful implementation of long-term (sustainable) resistance management for Bt
plant-pesticides in these and other crops. This analysis will include information presented in
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the two public hearings hosted by EPA in March and May of 1997, 1996 growing season
reports on resistance management activities and 1997 research efforts for Bt potato, Bt corn,
and Bt cotton, published literature, information from public meetings and discussions with
academic or extension entomologists, EPA reviews of the initial resistance management
strategies, and EPA FACT sheets.
This paper will analyze progress made in resolving issues related to the appropriate resistance
management plans for Bt crops. Good resistance management is dependent on multiple tactics
to decrease the selection pressure on the target pest(s) and employment of different mortality
sources. For Bt plant-pesticides, as for conventional pesticides, an overall IPM program
should include pest resistance management. The characteristics of plant-pesticides (i.e.,
production and use in the plant) allow the implementation of unique pest management
strategies. An example for Bt plant-pesticides is the use of a high dose expression strategy
coupled to the use of an effective refuge as important resistance management tools. For all
pesticides, an effective resistance management plan is likely to include appropriate predictive
tactics, scouting, sampling, and monitoring for changes in pest susceptibility, and evaluation
measures to determine the success of the plan. Perhaps, most critical to the success of a
resistance management strategy, is communication and education efforts targeting growers to
understand and implement the resistance management strategy.
High dosage expression of genes encoding pesticidal proteins will theoretically eliminate all
but rare homozygous resistant individuals. The expectation is that 100 percent of the
susceptible genotypes will be killed by the high dose of the pesticide. Homozygous recessive
(i.e., resistant) individuals are assumed to be so rare as to be insignificant. Effective refuges
allow survival of sufficient numbers of susceptible homozygous individuals to maximize the
probability that resistant homozygotes will mate with susceptible homozygotes, producing
heterozygous progeny that cannot survive on the Bt crop. While the theory of high dose
expression coupled to effective structured refuge is relatively straightforward, its
implementation has been controversial. That is, there is disagreement as to what is the
necessary arrangement and relative size of Bt and refuge field plots, the nature and objective of
performance-monitoring activities, and appropriate incentives to foster grower education and
acceptance. The Agency has and continues to foster efforts to resolve these disagreements to
the satisfaction of all stakeholders and has offered opportunities for public comment and
participation.
No field resistance to Bt plant-pesticides has occurred since the first registrations were issued
in 1995. Field resistance to Bt microbial sprays 'exists for a number of geographically-isolated
diamondback moth populations worldwide: U.S. (Hawaii, Florida, New York), Asia (China,
Japan, Malaysia, Thailand, the Philippines), and Central American (Costa Rica, Guatemala,
Honduras, and Nicaragua) (reviewed in Liu and Tabashnik, 1997; Perez and Shelton, 1997).
Resistance to Bt has also been detected for Plodia interpunctella (Hvibner) in stored grain
(McGaughey and Beeman, 1988). Bt resistance in several insects has been reviewed by
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Tabashnik (1994a) and Bauer (1995). Reports of diamondback moth resistance to Bt microbial
pesticide's have increased everyone's awareness that insect resistance management is important
not just for conventional pesticides, but for biologically-based pesticides, including Bt microbial
pesticides and Bt plant-pesticides.
At the tune of writing this paper, the EPA has registered six Bt plant-pesticide active
ingredients and the genetic material needed for their production in potato, field corn, and
cotton (seven registered products) with certain resistance management recommendations and
requirements. Four of the six plant-pesticides registered for full-scale commercial use have
been for the CryI(A)b or .CryI(A)c Bt delta endotoxin and its respective genetic material
necessary for its production in field corn to control European corn borer (ECB). The six
registered Bt plant-pesticides are as follows: (1) Cry IHA. delta endotoxin and the -genetic
material necessary for its production hi potato (Bt potato) to control Colorado potato beetle
(CPB) (registered May 1995); (2) Cry l(A)b delta endotoxin (and pCIB4431) and the genetic
material necessary for its production in corn (Event 176-derived hybrids, Bt corn) to control
European corn borer (ECB) (2 products registered August 1995); (3) CrylA(c) delta endotoxin
and the genetic material necessary for its production in cotton (Bt cotton) to primarily control
tobacco budworm and pink bollworm, but also to control cotton boll worm (registered October
1995); (4) Cryl(A)b delta endotoxin and the genetic material necessary for its production in
corn (and pZ01502; BTll-derived hybrids) to control ECB (registered August 1996); (5)
Cry l(A)b delta endotoxin (and pV-ZMCTOl; MON 801- and MON 810-derived hybrids) and
the genetic material necessary for its production in corn to control ECB (registered December
1996); and (6) Cryl(A)c delta endotoxin (plus three different plasmids) and the genetic
material necessary for its production in com (DBT418-derived hybrids) to control ECB
(registered March 1997). ^
The Agency mandated specific resistance management data requirements and mitigation
measures with resistance management strategy for all of the Bt corn and Bt cotton registrations.
These registrations were conditional registrations to allow for completion of the studies related
to resistance management. Collection of various data, e.g., target pest biology and behavior,
secondary pest biology and behavior, population dynamics, cross-resistance potential, refuge
strategies, dose deployment adequacy, discriminating concentration, monitoring, and reporting
were made conditions of registration for the Bt corn and Bt cotton registrations. Refuge
requirements were mandatory for Bt cotton. Development of a draft refuge strategy by
August 1998 and a final refuge strategy by January 1999 was required of Bt corn registrations.
No requirements related to resistance management were imposed on the registration of CrylllA
delta endotoxin hi potatoes based on the Agency's analysis and comments received from the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Science Advisory Panel (SAP)
Subpanel on Plant-Pesticides which met on March 1, 1995 (see Office of Pesticide Program
(OPP) docket, OPP-00401). Voluntary interaction between the registrant and EPA was
recommended by the SAP and certain areas of research and monitoring were suggested.
However, Monsanto/Naturemark requires a mandatory refuge through their Grower's
Agreement for each of its growers to follow and the overall Bt potato resistance management
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strategy Is being refined as more data become available.
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The registration for CryffiA 3-endotoxin in potato is not time-limited (no expiration date).
Registrations for the CryI(A)b d-endotoxin in Event 176-derived corn hybrids, MON 810-
derived corn hybrids, BT 11-derived corn hybrids, and CryI(A)c d-endotoxin in DBT 418-
deriyed corn hybrids expire on April 1, 2001. Registration for the CryI(A)c d-endotoxin'
expressed in cotton expires January 1, 2001. EPA will reevaluate the effectiveness of each
registrant's resistance management plan for Bt corn and Bt cotton as more data become
available.
This paper will be organized into five basic sections: (I.) General discussion of public hearing
issues, (II.) Bt potato resistance management, (EL) Bt corn resistance management, and (TV.)
Bt cotton resistance management, and (V.) Conclusions.
I. General Discussion of Public Hearing Issues
EPA continues to monitor and participate hi development of resistance management plans for
Bt potato, Bt corn, and Bt cotton. Because of the high degree of public interest in effective
resistance management for Bt plant-pesticides, the Agency held two public hearings, March
21, 1997 in Washington D. C. and May 21, 1997 in College Station, TX to collect information
on the resistance management plans for plant-pesticides. EPA requested comment on four
issues: (1) The requirement for resistance management plans; (2) Scientific needs for
resistance management plans; (3) The use of "Public Good" as a criterion for the requirement
of resistance management plans; and (4) Performance of Bt cotton.
Approximately 100 individuals/organizations submitted written comments and/or delivered
presentations regarding the subject of Bt plant-pesticide resistance management and the four
issues open for comment. Approximately 30 presenters provided comments at the March 21,
1997 public hearing and approximately 10 presenters provided comments at the May 21, 1997
public hearing. The total number of comments can be classified as follows: industry and
seed companies (7), industry-related groups and trade organizations (3), national/state grower
organizations (6), growers (9), entomologists from academic institutions and the U.S.
Department of Agriculture (extension, research, and forest service) (24), private citizens (31),
public advocacy groups (12), Entomological Society of America (ESA) (1), U.S.,Congress (1),
Canadian Pest Management Regulatory Agency (1), and State FIFRA Issues Research and ,
Evaluation Group (SFIREG) Working Committee on Pesticide Operations and Management
(1). A general analysis of the comments will be provided hi this section. It should be noted
that individuals did not necessarily comment on all four issues. Copies of the written
comments are available in the Office of Pesticide Programs public docket, OPP-00470. Part I
of this paper presents information EPA received at the hearings. Bt cotton performance will be
specifically examined hi Part IV of this paper. Specific scientific data needs for development
of long-term resistance management strategies will be discussed within the context of specific
issues related to Bt potato (Part II), Bt corn (Part HI), and Bt cotton (Part IV).
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A. The requirement for resistance management plans
The Agency sought comment on whether resistance management plans should be mandatory
(i.e., required as a term and condition of registration) or voluntary. EPA also sought
hiformation on the criteria that would indicate a need to require a resistance management plan:
All individuals in one fashion or another indicate that the fundamental basis for resistance ,
management is responsible product stewardship. Based on the verbal presentations and written
comments, there was a split response as to whether the Agency should "allow companies to
voluntarily institute resistance management plans or mandatorily impose such plans as 'a
condition of registration as a means for ensuring successful implementation of Bt plant-
pesticide resistance management. In terms of the numbers of actual individuals, .there was
' about a 75:25 split in favor of mandatorily requiring resistance management plans.
Individuals and organizations representing environmental groups, state grower associations,
private citizens, organic farmers, USDA Forestry Service, ESA, SFIREG, National Cotton
Council, Texas Corn Producers Board, USDA extension and research, and academia (with one
exception) all supported EPA mandatorily requiring resistance management plans to ensure the
long-term success of the resistance management for Bt plant-pesticides. In fact, all of the
private citizens, the organic farmers, organic growers associations, and environmental groups
urged EPA to specifically: 1) Suspend current registrations and forego future approvals of Bt •
crops until workable resistance management plans are available. 2) Convene a meeting of the
Scientific Advisory Panel to evaluate the current management plans. 3) Make-resistance
management plans mandatory. The Union of Concerned Scientists modified the first point and
recommended that EPA: "require 40 to 50% non-sprayed Bt-cotton refuges in Bt-cotton fields
in the 1997 growing season or suspend the current registration of Bt cotton until workable
resistance management plans are available." These individuals all stated, with some urgency,
the desire to maintain the efficacy of Bt products and thus maintain the environmental benefits
of Bt plant-pesticides and Bt foliar pesticide products. The basic sentiment expressed was
that durability of Bt plant-pesticides is too important to be left to depend on voluntary
programs.
1 • * "
Particularly noteworthy in recommending a mandatory role for the Agency was Dr. Mark
Whalon, entomologist, Michigan State University, for the protection of "susceptibility genes"-
through pest resistance management. He. made three recommendations. First, the EPA
should require resistance management plans for all newly registered conventional or biological
pesticides "which will be sold into markets where target or non-target insects, mites,
nematodes or pathogens have developed resistance hi the past." In particular, he noted that
resistance management plans are important to have in markets where chronic resistance
problems exist. Second/the Agency should be required to have "independent scrutiny of the
enforcement of susceptibility management (refuge) hi the case of cotton and corn Bt-transgenic
plants." He believes that the corn situation poses a greater scientific risk than the cotton
situation. Third, EPA should convene a Science Advisory Panel to advise the Agency of "the
development of a comprehensive Susceptibility Management Assessment process hi the
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Agency's pesticide registration responsibility."
1 ''•! ",'' «„',',,' ' '"'•,, •, . ' ' ' • '
SFIREG commented that it shares a common goal with EPA in wanting resistance management
strategies that prevent resistance from developing. "Both the states and EPA understand that
overuse of a single pesticide will likely lead to the development of resistant pests and eliminate
the utility of that tool." A unified product stewardship/education program by registrants for
both the Bt plant-pesticides and Bt microbial pesticides does not exist. In light of that,
SFIREG advised EPA to take a "cautious" regulatory approach and mandate resistance
management plans. SFIREG urged EPA to use requirements on the product registration itself
to manage resistance. The states do not believe that use of labels as an alternative approach to
resistance management would be effective because currently there are many unresolved issues
with plant-pesticide labeling, especially as related to enforceability.
Researchers from Texas A & M commented that "the use of refugia of a minimal size relative
to the acreage of transgenic plants hi the area, in our opinion, should be mandatory." They
further comment that as more scientific information becomes available the size of the refuge
may be increased or decreased, as appropriate. Other comments from this same group
indicated that resistance monitoring is critical and should be done by non-company sources
such as Departments of Entomology in collaboration with USDA or other government
agencies. EPA should be a funding agency for basic research, grower education, and
monitoring programs, but not make these areas a requirement for registration because of the
difficulties in enforcing these requirements.
Conversely, individuals from industry (except for Praxis), seed companies, the American Seed
Trade Association, Insecticide Resistance Action Committee, two members of Congress .from
Idaho [Mike Crapo (U.S. House of Representatives) and Larry Craig (U.S. Senate)] and
individual cotton fapners indicated that EPA should not establish "additional hurdles" to the
development and implementation of these new Bt plant-pesticides and that it is industry's
(including seed company's) responsibility to ensure the successful development and
implementation of resistance management strategies. That is, resistance management to Bt
plant-pesticides should be handled on a voluntary basis. The individual cotton farmers (2) who
commented indicated that Monsanto's actions with regard to developing and implementing a
resistance management program were very useful to help preserve the durability of Bt cotton.
"These actions not only made good business sense for Monsanto, but for all cotton growers as
well." In general, opuiions expressed indicated that resistance management for Bt plant7
pesticides was industry's responsibility and that EPA's role should be to evaluate the safety of
pesticidal products and to participate in discussions with growers, academicians, government
scientists (especially USDA extension and research scientists) and industry to work together to
make resistance management work. EPA should not make specific resistance management
requirements nor require additional data to support particular resistance management strategies
development because of the dynamic nature of pest management, cropping practices, and other
factors in which a great deal of flexibility is required for their implementation. Market forces
should dictate resistance management strategies. EPA's role should not be one of enforcement
6
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of resistance management plans.
A number of individuals, in particular, from industry, Biotechnology Industry Organization
(BIO), American Crop Protection Association (ACPA), and seed companies stated that there
should be a "level playing field" for resistance management of Bt plant-pesticides, These
comments can be summarized by Monsanto ?s statement regarding implementation of Best
Management Practices (BMPs) that there should be "a collaborative stewardship process which
should not require, direction by .the Agency, but one in which all stakeholders, including the
EPA participate and take ownership." Monsanto described a,multiple stakeholder
collaboration that would result in the development of the elements of BMPs for specific Bt
plant-pesticides. Monsanto's current view of appropriate BMPs include the following:
"binding growers to follow insect resistance management BMPs (e.g. grower agreements),
immediate implementation of refuge, extensive grower education programs, surveillance for
and grower reporting of suspected resistance (e.g. 1-800 telephone numbers), and mitigation
planning to address resistance development." Unlike Monsanto, other industry individuals
(Novartis, Mycogen, Pioneer Hibred International, and Holden Foundation Seeds) did not
endorse nor describe specific BAMPs in their comments. BIO supported a collaborative multi-
stakeholder effort. Flexibility in the development and implementation of specific resistance
management strategies was cited as the primary reason for a non-mandatory role for EPA by a
number of individuals. These individuals argued that agroecosystems are dynamic and
flexibility is necessary to respond to dynamic conditions. BIO specifically noted that currently
each individual company must develop its own resistance management strategy and research
priorities and this is not necessarily cost-effective. Several individuals from industry.and
academia noted, as an example of a multi-stakeholder approach to developing resistance
management strategies, the coordinating efforts,of the USD A NC-205 (the regional project
entitled "Ecology and Management of the European Corn Borer and Other Stalk-Boring '
Lepidoptera") that brings together representatives from academia, government, industry
(developers of the gene technology and seed producers), and consultants to discuss and develop
. European corn borer (Ostrinia nubilalis Huebner, ECB) resistance management strategies for
Bt corn and grower education materials.
Some individuals from industry and academia/USDA indicated that EPA should not single out
Bt plant-pesticides for resistance management requirements. As the statement from Pioneer
Hibred International notes "EPA must be equitable in imposing data requirements; it should
not single but plant-pesticides for requirements that have not been, and are hot being, imposed
on numerous other registrants of Bt pesticides." Other individuals noted that the Agency did
not impose resistance management requirements on conventional chemicals that posed a high
degree of selection pressure for resistance on the same target insects as Bt plant-pesticides,
. e.g., imidacloprid, spinosad, fipronil, and insect growth regulators. Pioneer Hibred
International suggested that if a mandatory role was necessary for implementation of resistance
.management strategies it should be USDA rather than EPA that should have the lead.
The USDA Forest Service expressed cpncern about the potential for development of insect
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resistance to Bt used to control forestry pests as a result of registration and use of Bt plant-
pesticides. Currently no Bt-plant-pesticides are registered for forestry use. The Forest Service
recommends that EPA fully assess and document the potential effect of Bt plant-pesticides hi
accelerating forest insect resistance, collect the necessary scientific information to develop
resistance management plans and require these plans to be implemented to manage forest insect
resistance to Bt plant-pesticides.
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B. Scientific information needed for resistance management plans
EPA sought comment on the scientific information needed to develop effective resistance
management plans. That is, what kinds of data are necessary to assess the potential for pest
resistance and/or adequately evaluate proposed resistance management plans. Most specific
comments came from university or USD A entomologists and industry. However, all
individuals indicated a need to collect more scientific information related to resistance
management.
Representatives from industry (in particular, Novartis Seeds, Mycogen, Monsanto, and
Piqneer) believed that areas where more research is needed have been clearly identified and
that research projects were underway to address these areas. Academic and USD A
researchers are active participants hi a number of these research projects. There were
comments received on the research needed for development of sustainable resistance
management strategies for Bt potato, Bt corn, and Bt cotton. The areas identified were: pest
biology (including movement (larval and adult, mating behavior)), ecology (including host
range of target and secondary pests especially in the Bt corn and Bt cotton agroecosystems),
behavioral responses such as emergence differences between resistant and susceptible pests
(including examination of relative fitness differences), susceptibility of the pest(s) to the "
insecticide in question, differences hi population dynamics, baseline susceptibility and
development of discriminating dose assays for the target pests, species-specific resistance
models (development and validation), monitoring for the development of insect resistance
including the development of molecular probes for early detection of the evolution of resistance
in the field, ensuring an adequate high dose, estimates of initial resistance gene frequencies,
impact on beneficial insects, use of alternate hosts as refuges, effective refuge strategies
(development and validation), and development of hybrid varieties that utilize native resistance
genes or other novel resistance genes that can be stacked or pyramided with existing Bt,genes.
Research projects in these areas will be discussed more specifically in the context of particular
resistance management strategies for Bt potato (Section II), Bt corn (Section III), and Bt cotton
(Section IV) below.
Drs. John Witkowski and Blair Siegfried (corn entomologists), University of Nebraska-
Lincoln, hi then: comments to EPA, identified specific research questions and their importance
in developing resistance management plans. For example, these researchers commented that
quantifying ECB local movements and gene flow is critical to determining effective refuge
sizes that maximize the probability that susceptible individuals from a structured refuge will
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find and mate with the few resistant individuals anticipated to survive exposure to the Bt plant.
Additionally, they,stated that it is desirable to understand the biochemical and physiological
nature of resistance in the target pest(s), cross-resistance patterns, arid inheritance of
resistance. Such questions can be addressed by selecting for Bt-resistant ECB populations in
the laboratory. Dr. Liebe Cavalieri (State University of New York at Purchase) commented
on the value of predictive models in understanding the insect pest-pathogen interaction as a
basis for the design of experiments that can be carried out in laboratory microcosms.
Several cotton eritoniologists, as noted below, also provided comments regarding research
efforts and strategies to develop long-term resistance management strategies. These same
basic research areas identified above for fit corn exist for the target lepidopteran pests (tobacco
budworm, pink bollworm, and cotton bollworm) in the Bt cotton agroecosystem. Several
researchers stressed the importance of using Bt cotton in the context of IPM, especially "the use
of crop rotation. That is, the use of the Bt plant-pesticide technology is just one element in the
total mix of pest control strategies in an IPM prograni.
Texas A & M researchers provided extensive comment on 5 research areas, primarily for Bt
cotton, but also for other Bt crops:
1. Assess area wide resistance monitoring needs, [e.g., collect baseline susceptibility data for
major and minor/secondary pests; set up monitoring programs, determine patterns of cross-
resistance in field populations (including use of simulations)] «,
2. Assess the number, of users of Bacillus, thuringiensis (Bt) foliar pesticide products vs. plant-
pesticides using an area wide survey approach. / :
3. Seek information on toxin expression in transgenic plants, [e.g., public disclosure of Bt
toxin expression patterns throughout the plant for the full growing season; ensure high dose
expression (i.e., ensure killing of most heterozygotes, assume that resistance is partially
recessive and due to a single gene)] ,
4. Seek inforination on dispersal from'refuge to/transgenic crops and vice-versa, [e.g., adult
movement, larval movement]
5. Assess the effect of synthetic pyrethroid insecticide treatments against cotton bollworm on
Bt cotton and on the effectiveness of the refuge.
Texas A & M researchers commented that evaluation of any proposed resistance management
plan should include the following two points: L) EPA could assess compliance with refuge
adoption, preference and management plan and 2) EPA could request data to determine the
relationship between the level of insect survival on a Bt crop and the size of refuge needed.
They also indicated that requesting resistance management plans only for Bt plant-pesticides
and not other pesticides is "scientifically and logically flawed".
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Dr. William Meredith (cotton entomologist), USDA/ARS/CP&G in Stoneville, Mississippi
recommends the following to investigate in-field success of Bt cotton performance:
"'!"; ' • • ' , .''•/•.' ..
1. Investigate in the field various management strategies, not just one strategy. Data from
many grower groups in the Mississippi Delta could provide a valuable data base to determine
which resistance management parameters are important and do further studies.
2. Establish a public monitoring system that follows the various management strategies.
3. Establish a public data base collected from the various strategies.
4. Evaluate the effectiveness of various strategies by a team of researchers. Final data
evaluation should be made public.
Possible management parameters would include: appropriate use of chemical and biological
insecticides, crop rotation, rotation of non-Bt and Bt cotton varieties from year to year,
develop and evaluate new resistance genes (natural and transgenic), and all of the above in
combination for a good IPM program.
Dr. Tim, Dennehy (entomology), University of Arizona, provided comments about resistance
management of pink bollworm and deployment of Bt cotton in Arizona including: 1) baseline
susceptibility evaluations for pink bollworm (Pectinophora gossypiella Saunders, PBW)
populations in Arizona, 2) impact of Bt cotton on biological control, and 3) refuge strategy
development, validation, and implementation.
Dr. D. D. Hardee (research cotton entomologist), USDA-ARS at Stoneville, MS commented
on the 1996 and 1997 resistance monitoring strategy work for cotton bollworm and tobacco
budworm hi Bt cotton in 4 cotton-growing states. Preliminary efforts have shown that there
were no shifts hi the baseline susceptibility to CryI(A)c for either cotton bollworm
(Helicoverpa zea Boddie, CBWj or tobacco budworm (Heliothis virescens Fabricius, TBW).
1 ilii " ' • , , • - " '"• •',
C. The use of "Public Good" as a criterion for the requirement of resistance management
plans
The Agency sought coniment on whether "public good" should be used as a criterion
triggering the requirement of resistance management. EPA also sought comment on how
"public good" should be defined. The "public good" criterion was first proposed by the
Pesticide Program Dialogue Committee (PPDC) in July 1996. However, the PPDC were
unable to define what constituted a "public good" except that they agreed that Bt microbial and
Bt plant-pesticides were hi the "public good" and should be protected. The PPDC stated that
EPA was correct to require resistance management plans as part of the registration for Bt
plant-pesticides.
10
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Most individuals did hot specifically address the issue. Commenters from industry indicated,
in general, that the "public good" criterion was not a useful criterion as a basis for requiring
resistance management plans as a condition of registration. Pioneer Hybrid International's
comments areparticularly illustrative of industry's viewpoint, they indicated that "public
good" was an ill-defined concept, not based in FIFRA,1 and singles out Bt plant-pesticides for
additional requirements. .Comments from all stakeholders touched on the "societal" nature of
this question. There were comments from academia and industry on the fact that FIFRA
requires the Agency to make a risk/benefit decision to register a pesticide, and, therefore, if a
pesticide is registered, it is in the "public good". Some felt that mandating resistance
management plans only for Bt plant-pesticides and not other pesticides' is, as some researchers
from Texas A & M noted, "scientifically and logically flawed."
Dr. Kimberly Stoner (entomologist), Connecticut Agricultural Experiment Station in New
Haven stated that "any pesticide discovered and developed with public funds or even grants
from the state or federal governments should be considered a public good." Dr. John Van
Duyh (entomologist), North Carolina State University wrote that "this concept is potentially so
encompassing that it will cause fear in most Americans, from the common and .deeply rooted
suspicion in the government," ,
Howeyer, Dr. Mark Whalon from Michigan State University and a set of Texas A & M
researchers provided an extensive evaluation of "public good." The Texas A & M group
outlined three major criteria to determine public good: 1) Impact on product output, costs of
production and product price. 2) Impacts on the level of pesticide use weighted by the
potential positive and adverse impacts on health and the environment. 3) Impacts oh the
research agenda and the level of support/effort. Public good would be defined by these criteria
which consider the short-run and long-run effects expressed on a present value basis of the
various impact groups.
Dr. Mark Whalon stated that "susceptibility genes in pests are natural resources" which are in
the public good and should be protected. He comments that "the loss of susceptibility genes
through the overuse of pesticides constitutes a tragedy of the commons no less significant than
polluted'air, water or. contaminated food. All mankind suffers from the consequences of this
genetic over-exploitation which can be and should be prevented. It is particularly tragic in that
resistance can be delayed even ameliorated indefinitely with proper EPA-mandated Resistance
Management Plans (RMP) which could be required as EPA has already done for some Bt-
transgenic plants." Dr. Whalon believes that RMPs should be in place at the time a new
pesticide is introduced into the market, particularly where the target is a pest that has already
exhibited a history of resistance. He states that RMPs should be based on five principles: 1)
diversify mortality mechanisms in pest populations (IPM), 2) manage susceptible genes by
providing refuge for untreated pests to survive or manage immigration of susceptible insects in
treated populations, 3) establish base-line susceptibility of target pest populations and, where
possible, monitor these populations as a regular course of operations, 4) when resistance
develops, investigate the mechanisms and inheritance pattern to aid the development of better
: . . ii. . ' ' ' , "' - ,
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RMPs in other areas, 5) develop communication and education programs jthat aid the
introduction, adoption, and maintenance of RMPs.
The Union of Concerned Scientists indicated that "Bt is a public good that should not be
squandered." This viewpoint was echoed by comments from private citizens, organic farmers
and grower groups, and public interest groups.
D. Performance of Bt cotton
EPA sought comment on the performance of Bt cotton in the field. The Agency sought
information regarding reported control failures for Bt cotton hi 1996, possible evaluation tools
concerning these failures, and implications on future resistance management efforts.
Comments are summarized below. Discussion of the resistance management impacts of cotton
bollworm control hi Bt cotton fields will be discussed in the section IV below.
Comments received from private citizens, organic farmers and grower organizations, and
public-interest groups stated a belief that the inability of Bt cotton to control cotton bollworm
during the 1996 growing season showed that the "high dose strategy" was flawed. Therefore,
they felt that the Bt cotton resistance management plan should be reevaluated before the 1997
growing season. Most urged EPA to suspend the registrations of all Bt plant-pesticides and to
hold a FIFRA Science Advisory Panel (SAP) meeting to reevaluate the resistance management
plans for Bt cotton and Bt corn.
Comments received from Monsanto, the National Cotton Council, academic/USDA scientists,
and cotton farmers indicated that Bt cotton performance in 1996 was excellent. These
corftments stated that there was, no breakdown hi the Bt gene technology. Individuals indicated
that there was an unusually high infestation of cotton bollworm in the Cotton belt (south
Texas, mid-south arid southeastern growing regions). Some of these infestations on Bt cotton
required supplemental insecticide treatment.
Monsanto and a number of entomologists noted that cotton bollworm is not as sensitive to the
Bt toxin as tobacco budworm and this information was published in the scientific literature
before commercialization of Bt cotton (Bradley, 1995; Mahaffey ei'dl., 1994). Dr. Van Duyn,
an entomologist from North Carolina State University, stated that his research showed the lack
of very high efficacy in Bt cotton for cotton bollworm and that Bt cotton was not a stand-alone
technology when high populations of cotton bollworm were encountered.
, ii • , ' " ' ' ' .• ''• „: if, ,',,,• -it ,••,''
Monsanto commented that they undertook a number of studies following reports of "Bt cotton
failure in 1996" and tested for cotton bollworm susceptibility and Bt expression hi Bt cotton
areas affected by high cotton bollworm infestations. They found no change in cotton bollworm
susceptibility to the CryI(A)c delta endotoxin and hi Bt expression levels hi the plants as
compared to the baseline susceptibility levels for these locations, these studies showed no
detectable level of resistance hi these populations. Growers realized a $34/acre benefit
" ' « ' , '
, •'• 12
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growing Bt cotton versus non-Bt cotton.. ' ,
Academics and the National Cotton Council all noted that there could be improvements in
communication on cotton bollworm control in Bt cotton with the public, growers, and
consultants. The .National Cotton Council indicated that scouting practices had previously
focused on the top six inches of the plant. As a result of the 1996 Bt cotton growing season,
modified scouting practices hi Bt cotton will now be needed to examine further down hi the
plant canopy especially during peak bloom periods (cottpn bollworm eggs were found on
flowers or blooms on Bt cotton plants), the most critical tune for cotton bollworm co'ntrol in Bt
cotton. Dr. Blake Laytori,. extension entomologist from Mississippi pointed out that producers
and consultants in Mississippi were cautioned via the 1997 Cotton Insect Control Guide, the
weekly Cotton Insect Situation Newsletter, the Cotton Insect Telephone Hotline, and Extension
Publication 2108, "Insect Scouting and Management in Bt-transgenic Cotton" that Bt cotton
may require treatment in cases where high populations of cottpn bollworm occur.
Two cotton farmers from Alabama and Mississippi, both of whom planted Bt cotton in 1996,
indicated that Bt cotton had excellent control of tobacco budworm and significantly reduced
cotton bollworm populations as well. The Mississippi farmer noted that 1300 growers on
nearly 450,000 acres planted Bt cotton in 1996. This grower indicated that less than 10% of
his acres were treated for cotton bollworm. The Alabama farmer noted that 77% of all of the
cotton acres planted in Alabama were Bt cotton. He did not spray one acre of his 6000 acre
farm for cotton bollworms. The;National Cotton Council and Dr. Blake Layton (extension
entomologist from Mississippi State University) pointed out that applications for cotton
bollworm control went from 3.3 applications for non-Bt cotton to 0.3 for Bt cotton in
Mississippi. This type of reduction in insecticide applications for cotton bollworm control was
also noted for, other states employing Bt cotton.
Several entomologists discussed the subject of effective refuge size in then- written comments.
These experts commented that the current refuge options may not be large enough to produce a
relatively high number of susceptible cotton bollworms to mate with any resistant insects that
may develop on Bt cotton with only a moderate dose for cotton bollworm. The presence of a
refuge can still mitigate the effects of resistance development even in the absence of a high
dose. Dr. Mike Caprio from Mississippi State University commented that "while it is true that
a high dose makes the refuge strategy much more effective, we have shown hi simulations of
foliar applications of Bt that even survivorship rates as high as 20% could still delay resistance .
5-fold compared to the rate of resistance evaluation hi the absence of the refuges. "He
encouraged the pyramiding of multiple genes in cotton if such genes increase mortality of
cotton bollworm to delay resistance development in this pest. Dr. Fred Gould (entomologist),
North Carolina State University, indicated in his written comments that there wasn't a high
dose strategy for cotton bollworm and pink bollworm, but rather a moderate dose approach for
cotton bollworm and that the actual effective refuge size should be greater than the current 4%
in Bt cotton. Gould recommended that the effective refuge size for Bt cotton should be
increased to be at least 30% rion-Bt cotton. Texas A & M entomologists commented that if the
. " :• •••"•••' 13 ."'..
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acreage of alternate hosts such as corn, sorghum and soybean is greater than the Bt cotton
acreage, then a 20% or greater sprayed non-Bt cotton refuge may be adequate for managing
resistance to tobacco budworm and cotton bollworm. They recommend that of the two EPA
mandated refuge options for Bt cotton, the 4% non-sprayed refuge option should be replaced
with just the 20% sprayed refuge option. Comments from the National Cotton Council and
University of Arizona indicate that pink bollworm control was excellent.
U. Bt potato resistance management
This section will discuss resistance management activities and results for 1996 and discuss any
lessons learned regarding resistance management since Cry fflA delta endotoxin was registered
in potatoes in 1995. Based on EPA's analysis of the registrant's voluntary resistance
management strategy and comments received from the Science Advisory Panel Subpanel on
plant-pesticides that the registrant's voluntary resistance management strategy was scientifically
adequate (March 1, 1995, see OPP docket, OPP-00401), no requirements related to resistance
management were imposed on the product registration of Cry Hi A delta endotoxin in potatoes.
Voluntary interaction between the registrant and EPA was recommended and certain areas of
research and monitoring were suggested. However, Monsanto/Naturemark requires a
mandatory refuge for each of its growers to follow and the overall Bt potato resistance
management strategy is being refined as more data become available. An annual report on the
status of resistance management activities was not required as part of the registration
agreement, but a 1996-97 status report of resistance management activities was provided to
EPA voluntarily by Monsanto/NatureMark on July 2, 1997. Material provided during the
public hearings, scientific publications, personal communications, EPA's Fact Sheet (U.S.
EPA, 1995a), Agency Reviews (December 23, 1994 and May 2, 1995 regarding pesticide
resistance management) and Monsanto/NatureMark's 1996-97 status report (July 2, 1997) will
be included in this analysis.
Background
The Agency granted a conditional registration without an expiration date for the Cry IIIA delta
endotoxin from Bacillus thuringiensis subspecies tenebrionis in potato to control Colorado
potato beetle (Leptinotarsa decemlineata Say, CPB) in May 1995. This was the first
registration of a plant-pesticide. Bt microbial pesticides with specific activity for CPB have
been registered for a number of years (e.g. M-trak, Foil, Novodor, Raven). The major
difference between the Bt microbial pesticides and the CryJUA delta endotoxin genetically-
engineered into potatoes (Bt potato) is the level of control throughout the growing season. Bt
microbial insecticides have a short persistence and are only effective against young larvae,
necessitating exact timing and several applications to achieve control. The expression of the
CryJJIA delta endotoxin hi the leaves of Bt potato is at a high enough level to be effective
against all stages of the beetle and protection is sustained throughout the season.
Both the Agency and the Science Advisory Panel Subpanel on plant-pesticides (meeting held
14
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, March 1, 1995, see OPP docket, OPP-00401) reviewed Monsanto's resistance management
plan for Bt potato. Review of the resistance management plan was part of the Agency's
risk/benefit decision-making process for registering the Cry HIA delta endotoxin hi potato.
The Agency and the SAP determined that the resistance management plan for Bt potato was
scientifically sound and workable. The SAP stated that the resistance management plan is a
"scientifically credible Colorado potato beetle resistance management protocol." For the Bt
, potato, the SAP recommended that Monsanto provide the Agency with a specific resistance
monitoring plan and requested that specific recommendations be developed on what course of
action should be taken if resistance should be discovered. It was the opinion of the SAP that
Monsanto should work with EPA on developing a long-term resistance management plan, but
that such plans should not be a formal condition of registration. EPA agreed with this
assessment for Bt potato as the pesticide was only for the control of the CPB, the CrylllA delta
endotoxin was at a high dose, and existing Bt microbial products had only limited residual
activity and only worked for early instars of this pest. In addition, the CPB has a limited host
range and limited mobility. EPA recommended further information be collected on
reproductive strategies for CPB with respect to gene flow, optimization of refuge strategies,
continued,development of monitoring plans, development of a discriminating dose assay,
continued development of grower education materials, continued refinement of IPM
recommendations, and continued development of novel CPB control mechanisms involving
different modes of action. -
The SAP further agreed with the seven elements, described by OPP, that need to be addressed
to develop an adequate resistance management plan for Bt plant-pesticides. These elements
are: (1) Knowledge of pest biology arid ecology, (2) Appropriate gene deployment strategy,
(3) Appropriate refuge, (4) Monitoring, and reporting of incidents of pesticide resistance
development, (5) Employment of IPM, (6) Communication and educational strategies for the
use of the product, and (7) Development of alternative modes of action.
Bt potato acreage in 1996 and 1997
Although no specific reporting requirements were required as part of the registration hi 1995,
EPA recommended the continued development of a. data base to monitor the use of the
genetically modified potatoes and correlate possible resistant reports with the use sites. Based
on three-year averages, about .1 million acres of fall potatoes-are planted hi the U.S. annually.
According to information provided in the 1996-97 status report, about 10,000 A (or 1% of the
total) in 1996 and 25,000 A (or 2.5 % of the total) hi 1997 were planted in Bt potatoes
(marketed as NewLeaf® Russet Burbank and NewLeaf® Superior and NewLeaf® Atlantic
varieties) in the U.S. Acreage information was gathered from 94 of 112 total customers hi
1996: The proportion of NewLeaf® potatoes on these farms ranged from 0.1 % to 69% of total
potato acreage. Farm size ranged from less than 500 A to 5000 A.
Analysis of Resistance Management Strategy
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High Dose is Adequate
All available evidence supports the Agency's original finding (December 23, 1994 and May 2,
1995) that NewLeaf® potatoes constitutively express the CrylllA delta endotoxin at sufficiently
high doses to kill all susceptible individuals including resistant heterozygotes. NewLeaf®
potato hybrids are maintaining a "high dose" expression of CrylllA throughout the plant to
provide a "high dose strategy" for resistance management. This conclusion was supported by
the March 1, 1995 SAP (published report, see OPP docket, OPP-00401) and public comments
from entomologists. According to NatureMark's 1996-97 status report, the levels of CrylllA
delta endotoxin expression appear to be "approximately 10-50 times higher than the LQ9 for
CPB larvae."
CPB resistance to Bt Endotoxins
EPA did not require as part of the registration more information on the biology of CPB
resistance and the potential for cross-resistance. However, EPA and the SAP recommended
that this information would be useful to further refine the long-term resistance management
strategy. Field resistance to microbial Bt or to Bt potatoes expressing the CrylllA delta
endotoxin has not been reported to date. However beginning in 1987, Whalon's group selected
field and laboratory colonies with M-One, a commercial foliar Bt spray containing the CrylllA
delta endotoxin, to study the selection and the inheritance of resistance (Whalon et al., 1993).
Whalon's group developed resistant strains that were 59 times more resistant than the
unselected strain. No cross-resistance was observed between prganophosphate, carbamate or
synthetic pyrethroid resistance and the CrylHA-resistant strain. However, this Bt resistant
strain feeding on transgenic potato petioles producing the CrylllA delta endotoxin showed
stage-specific mortality (Wierenga et al., 1996). Results from this study indicated that the
third instars were typically less sensitive than the first and second instars to the Bt delta
endotoxin. The older instars had a significantly slower rate of development than those feeding
on conventional non-Bt foliage. Resistant second instar larvae experienced less than 50%
mortality after 96 h of feeding on a Bt potato plant. After two weeks of feeding on a Bt potato
plant, resistant adult beetles experienced only a 25 % mortality in comparison to susceptible
adults which did not survive.
These studies provide important information about the limitation of a seed-mix refuge strategy
for CPB. Larger larvae surviving on non-Bt plants could move onto Bt plants and receive a
sublethal dose of the CrylUA delta endotoxin and thus speed up the selection process for
resistance. Modeling studies performed by Mallet and Porter (1992) indicate that seed mixes
could enhance selection if there was significant larval movement within a field. Further
selection studies examined the inheritance of resistance. Research by Rahardja and Whalon
(1995) indicated that the genetic inheritance of CPB resistance to the Bt CryDIA delta-
endotoxin was conferred by incomplete dominance genes. After 35 generations of intense
selection to the CrylllA delta endotoxin, the resistance ratio was > 700-fold for Bt-resistant
CPB compared to the susceptible strain and resistance development resulted hi significant
16
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fitness costs, i.e., prolonged larval development, reduced larval weight, shortened oviposition
period, reduced egg-mass size, and reduced fecundity (Trisyono and Whalon, 1997). Further
experiments indicated that these Bt-resistant CPB could survive on transgenic Bt potato plants
expressing the CrylllA protein for a short period of time, although none of the eggs produced
by these adults were viable (DiCosty and Whalon, 1997). Collectively, these studies provide
information^about the nature of CPB resistance under intensive-selection to the CryillA delta
endotoxin in the laboratory and suggest that incorporation of an effective structure refuge into
a resistance management strategy is desirable to slow the potential for resistance to develop.
Further field research is needed to validate the resistant management models, to study the CPB
adult and larval movement, mating behvior, and to study reproductive effects of CPB feeding
on Bt potatoes;
' .'''•"•'' - • . . • ' • • • «.'']"".' •'.''•
Refuge
All available evidence continues to support EPA's original conclusion that a "structured"
refuge is necessary and that the success of the high dose strategy will be compromised if there
is ho effective refuge. Seed mixes were eliminated as a realistic alternative for CPB resistance
management because of the potential for sublethal exposure to the CrylllA delta endotoxin by
later instar larvae surviving on a non-Bt plant and men moving to a Bt plant. However, except
for the elimination of a refuge based on a seed mix, neither the Agency nor' the SAP "
recommended a specific "structured" refuge arrangement and relative size of Bt potato and
refuge plots prior to registration of CrylllA delta endotoxin in potatoes in May 1995. That is,
EPA did not include a refuge requirement as a part of the registration.
Prior to 1997, the refuge concept was included in Monsanto/NatureMark's resistance
management plan as a recommendation rather than a requirement as part of the Technology
Agreement. Growers were instructed by NatureMark to maintain at least 20% of farm potato
acres as non-Bt expressing potatoes that could be treated with conventional insecticides for
CPB. As noted above, compliance with this recommendation was high. The proportion of
NewLeaf® on these farms ranges from 0.1 % to 69% ,of total potato acreage of 94/112 growers-
surveyed by NatureMark. ''..,-
Beginning hi the 1997 growing season, commercial growers were required by NatureMark's
Technology Agreement to maintain at least 20% of their potato acreage as non-Bt expressing
varieties. The recommended spatial arrangement and treatment of these refuge acres is rioted
in NatureMark's Resistance Management Guide. Refuge acres should be growing in close
proximity to NewLeaf® fields. Refuge acres may be treated for CPB, but treatments should be
with foliar (non-Bt insecticides), rather than systemic insecticides to allow enough susceptible
insects to survive. It is recommended that NewLeaf® potatoes should be vine-killed prior to .
non Bt-expressing potatoes. If there are resistant insects surviving on NewLeaf® hybrids, they
should move to the non-Bt expressing potatoes and riiate with susceptible CPB. NatureMark
reports that compliance with this refuge .requirement and other requirements of the Technology
Agreement will be monitored.
.'•'•• . 17 . . • ' '
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A refuge requirement is recommended, but not required by NatureMark for seed growers
because they maintain numerous varieties on their farms. As a result, there is an ample supply
of non-Bt expressing refuge as a natural component of seed production. Historically, CPB
resistance has not been a problem in seed producing areas.
Surveillance and Tracking (Monitoring)
In 1995, the SAP and EPA recommended that a detailed monitoring program and a remedial
action plan should be developed and instituted. The monitoring plan should include sampling
sites, timetable for development, education of growers on sampling for resistance, collecting
specimens to evaluate for resistance, and providing specific recommendations on how to
eradicate resistant individuals to prevent survival of a resistant population. EPA also
recommended the development of a discriminating dose assay. NatureMark has provided a
summary of the baseline susceptibility work, development of a discriminating dose assay, and
a detailed monitoring/surveillance program description including an appropriate remedial
program. NatureMark has a 1-800 number for growers to report unusual CPB survival or for
other technical information.
NatureMark reports that the baseline susceptibility work for CPB populations to the CrylllA
protein has been completed. During a four-year period beginning in 1992, a total of 79
geographically distinct populations were collected from commercial potato farms in 15 states
and two provinces of Canada and were assayed for susceptibility (work completed by Dr.
Galen Dively, Dept. Of Entomology, University of Maryland). Results from this study
indicated a seven-fold difference in CPB baseline susceptibility to the CrylllA protein between
the various populations. A discriminating concentration of 25 jug protein/ml of diet was
selected to test for shifts in susceptibility in suspect CPB populations that may be identified in
the future. This concentration killed 26% of the Whalon resistant strain (Whalon et at., 1993)
compared to 100% of the standard New Jersey susceptible population.
, ill .',,',,, ' . ' • i ', , '
NatureMark has developed user guidelines explaining the deployment of refuges and
monitoring requirements and has put into place an outreach program hi cooperation with seed
suppliers and extension entomologists to look for unexpected levels of CPB survival.
NatureMark has developed a rapid serological test that can be used to identify plants containing
the Bt protein in one hour. If plants are confirmed to be NewLeaf, then "suspect" larvae will
be shipped to the University of Maryland for bioassay to determine actual susceptibility to Bt.
The University of Maryland has a two level testing program that would be implemented if
there is a "suspect" CPB population. This program is designed to detect localized shifts in
susceptibility level rather than shifts hi resistance gene frequency. Monitoring shifts in
resistance gene frequency would be cost-prohibitive. Level 1 testing will have the University
of Maryland bioassay laboratory expose a large number of first instars to the discriminating
concentration and determine if the mortality response is statistically different from the baseline
response. The level 1 testing will take approximately one week. The level 2 testing will
further quantify the concentration-mortality response and provide information on follow-up
18
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actions such as intensified field surveillance in and around the collection site or fact-gathering
to assess reasons for the unusual response, If-CPB are found to be resistant to Bt, they can be'
treated immediately with a conventional insecticide to prevent further reproduction and
movement. , ,: -
NatureMark reports in its 1996-1997 status report of resistant management activities that there
were two situations in which growers alerted NatureMark of CPB larvae surviving on
NewLeaf® plants. In both cases, the plantings were non Bt-expressing plants rather than
NewLeaf® plants due to planting errors. NatureMark indicates that they confirmed that the
plants did not contain the Bt gene.
'• / ; - " • • ' * .
Communication and Training
NatureMark mandates certain activities under its Technology Agreements with growers and
charges a $32 per acre technology usage fee in addition to the cost of the seed potatoes.
NatureMark contacted each NewLeaf® grower personally,by telephone and visited each farm to
discuss resistance management and IPM. This effort helped to ensure that all enlisted growers
had all of the technical information needed to use the product appropriately and successfully.
Monsanto/NatureMark has consulted with research and extension entomologists in the
development of its resistance management program. NatureMark has prepared a simple one-
page summary of its Resistance Management Guide for growers. Additional documents
describe regional pest management recommendations, including non-target pest scouting and
choices for economical and selective pesticides to use in combination with NewLeaf® potatoes'.
Each grower receives all of the technical information includuig resistance management
requirements and recommendations prior to signing NewLeaf®'s Technology Agreement and
again at the completion of signing. These materials are available at trade shows, grower
meetings, and through direct mailings, NatureMark has developed an alliance with three major
chemical distributors, Wilbur Ellis Co., United Agri Products, and Simplot Soilbuilders to
provide crop service and support to NewLeaf® growers. Field representative,s from these
. companies are trained on NewLeaf® Best Management Practices including insect resistance
management. These "Service Partners" are another mechanism to ensure compliance with the
resistance management plan.
Public comments
. Of the 100 comments received as a result of the two public hearings held on Bt plant-pesticide
resistance management this year, only four were specifically related in some fashion to Bt
potato resistance management strategies.
Dr. David Ferro (entomologist), University of Massachusetts and a member of the SAP
Subpanel in 1995 on plant-pesticides, who originally reviewed Monsanto's/NatureMark's
resistance management plan, commented that NatureMark should modify instructions on how
.. " • . -.-:• '• ' 19 •'•'. ".'.:. . , -.
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the 20% non-Bt expressing acreage should be managed. "The 20% refuge may be too far
away from the NewLeaf® plantings to insure mating between RR and SS individuals." He
recommends that no more than 80% of the area in any single 'field be planted to a NewLeaf®
cultivar and that growers must manage the 20% refuge using IPM guidelines. He also
expresses some concern that the use of foliar and soil-applied imidacloprid, if not managed
properly, might eliminate susceptible insects in the refuge.
Praxis, an integrated biological cybernetics company based in southwest Michigan, commented
that CPB resistance management plan for Cry IIIA-expressing potatoes is flawed because of the-
potential impact on beneficial insect populations. However, written comments provided by the
Wisconsin Potato and Vegetable Growers Association arid the Department of Entomology,
University of Wisconsin-Madison, states that populations of beneficial insects are significantly
higher in Bt potatoes than hi those managed with conventional insecticides where "reductions
of 50-75% are common." In 1996 field experiments, beneficial arthropod populations were .
higher than those treated with any other insecticides. However, the predator populations were
not high enough to control late-season aphid populations. Their comments support
NatureMark's 1997 resistance management guidelines: 20% non-Bt refuge, use of alternative
controls, and a monitoring program for Bt susceptibility.
The Canadian Pest Management Regulatory Agency provided a copy of the report of the
October 1996 meeting on integrated management of CPB. This report, while good, does not
include a discussion of Bt potato expressing the CrylUA delta endotoxin.
Summary
NewLeaf potato hybrids are maintaining a " high dose" expression of CrylllA throughout the
plant to provide a "high dose strategy" for resistance management. NatureMark, through its
1997 Technology Agreement with NewLeaf® growers, mandates a 20% non-Bt expressing
refugia. It appears that compliance with the conditions in the Technology Agreement was
excellent. NatureMark has developed a discriminating dose assay, a surveillance and remedial
action plan, and an extensive grower education communication and training program to convey
appropriate resistance management tactics. IPM and scouting are discussed in the technical
material provided by NatureMark. NatureMark has adopted all of EPA's recommendations
regarding resistance management although no specific requirements were mandated as
conditions of registration. Evidence suggests that the resistance management strategy adopted
by NatureMark is a good one, although there is some concern over treatment of the 20% non-
Bt expressing refuge and level of beneficial insects hi the field. Adoption of NewLeaf®
technology may be slowed by the rapid acceptance and implementation of imidacloprid
insecticide as a new chemical control of CPB.
III. Bt corn resistance management
This section will discuss resistance management activities and results for 1996 and discuss any
20
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lessons learned regarding resistance management since the first CryI(A)b delta endotoxin was
registered in corn in 1995. '
The following materials were used in preparing this section of the paper: material provided
during the public hearings, scientific publications, EPA's Fact Sheets (U.S. EPA 1995b; U.S.
EPA, 1996a, b; U.S. EPA, 1997), Agency. Reviews regarding pesticide resistance
management, Research and Extension Entomologists of the-USDA North Central Regional
Research Project (NC-205), Ecology and Management of European Corn Borer and Other
Stalk-Boring Lepidoptera, North Central Regional Extension Publication, NCR 602 entitled
"Bt-corn & European Corn Borer - Long-Term Success Through Resistance Management"
(Ostlie et al. t 1997), Ciba Seeds/Novartis Seeds and Mycogen Plant Sciences 1996-97 status
reports, Letters from Ciba Seeds/Novartis Seeds (November 20, 1996) and Mycogen
(December 9, 1996) regarding silk and kernel expression in Event 176 hybrids, Research Data
on Corn Earworm Relative to Resistance Development and Monsanto's Plans for Producing :
Resistance Predictive Models (MRID 442094-01), and a discussion of 1997 research plans.
Background . ' \
The Agency granted the first two conditional registrations of the Bacillus thuringiensis
subspecies kurstaki CryI(A)b delta endotoxin and the genetic material necessary for its
production in field corn to control European corn borer (Ostrinia nubilalisHuebner, ECB) in
May 1995 to Ciba Seeds (now part of Novartis) and Mycogen Plant Sciences. The first
commercial plantings of Event 176 hybrid Bt corn were in 1996. At the tune this paper was
prepared, the EPA had registered five Bt plant-pesticides for commercial use in field corn: 176
(Ciba/Novartis Seeds and Mycogen Corp.), BT11 (Northrup-King/Novartis Seeds), MON8011
and MON810 (Monsanto) and DBT418 (DEKALB Genetics Corp.). Each of these
transformation events is trademarked'and various seed companies license each event.
Understanding these events and how they effect perfonriance is crucial to the wise selection of
corn hybrids and to appropriate resistance management. ;
Resistance management strategies for the Bacillus thuringiensis subspecies kurstaki CryI(A)b
or CryI(A)c delta endotoxhi and the genetic material necessary for, its production hi field corn
have been reviewed by the Agency. . Summaries of'these analyses and the terms and
conditions of the registration including the resistance management requirements required are
provided in the FACT sheets for the individual registrations (U.S. EPA, 1995b; U.S. EPA,
1996a, b; U.S'. EPA, 1997). Each of these conditional registrations will automatically expire
on midnight April 1, 2001. EPA will reevaluate the effectiveness of each registrant's
resistance management plan before April 1^ 2001 to decide whether to convert the registration
to a registration without an expiration date. Experimental Use Permits (EUPs) have been
^•MON 810 is the major commercialized event and is the only event discussed in detail in this paper. MON801
was registered, but not commercialized. . , '
'••'. 21 . ••" : • •.-;-...•.' ' '
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granted and amendments to registrations are pending for use of a CryI(A)b delta endotoxin in
sweet corn (Rogers Seeds/Novartis Seeds) and in popcorn (Novartis Seeds). An application is
pending for the registration for a Cry 9(c) delta endotoxin in field corn (Plant Genetic
Systems/AgrEvo).
At the time of registration, there were some scientific questions related to long-term resistance
management of the CryI(A)b and CryI(A)c delta endotoxins expressed in corn. Because of
these questions, the Agency imposed specific resistance management requirements. EPA
required as part of the registration, development of a monitoring program (surveillance and
remediation), grower education, and maintenance of a confidential sales database. Each
registrant is required to submit annual progress reports on results and conclusions from
research (including scientific literature) as they become available in the following areas: 1)
information on ECB pest biology and behavior, 2) feasibility of refuge options, 3) development
of discriminating dose concentration assay, 4) effect of corn producing the GryI(A)b or
CryI(A)c delta endotoxin on pests other than ECB including CEW, and 5) the biology of ECB
resistance and cross-resistance. Both Novartis Seeds (Ciba Seeds) and Mycogen Plant
Sciences have submitted their 1996 progress reports (no other delta endotoxins were registered
in corn for full-scale commercial release prior to the 1996 growing season). Progress reports
for 1997 for all Bt plant-pesticides registered for use hi corn will be submitted to EPA by
January 31, 1998.
It is recognized that structured refugia coupled to a high dose expression strategy are two of
the. key resistance management factors for managing pest resistance to Cry delta endotoxins in
corn. However, successful grower education and training are essential to implementation of
any resistance management plan. In addition, it is recognized that long-term resistance
management will involve other IPM practices in addition to the use of Bt corn. In all cases, all
of the general elements of a resistance management plan were addressed by the registrants and
reviewed by EPA. These elements include pest biology, Bt dose deployment, refugia,
monitoring for ECB resistance, susceptible nontarget lepidopteran pests, potential for cross-
resistance development, integrated into an EPM program, grower education and
communication, remedial action plan, and development of alternative pesticides with different
modes of action.
There are two major resistance concerns: 1) development of resistance in the primary target
pest, ECB and 2) development of resistance in secondary target pest'resistance in corn
earworm (CEW) [also known as the cotton bollworm, sorghum headworm, tomato fruitworm,
and soybean pod borer (Helicoverpa zea (Boddie))]. EPA concluded that to manage resistance
effectively and to develop an effective, long-term resistance management strategy, specific data
needs (including target and secondary pest biology and ecology, population dynamics
(modeling), refugia, cross-resistance, baseline susceptibility and discriminating dose
determination), mitigation measures (remedial strategies and limitation of Bt corn acreage hi
the South for Bt corn hybrids producing Bt protein in the silks and kernels), reporting, and
monitoring were required as part of the registration. No specific structured refuge
•" ;' ' • 22'
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requirements were mandated at the time of registration, but thfe registrants were required to
collect research data regarding different refuge strategies in order to determine the necessary
arrangement and relative size of Bt and refuge plots for a long-term resistance management
plan. However, seed mixes were eliminated as an effective refuge strategy because of
relatively high ECB larval movement. Because of this movement^ there is a threat that ECB
larvae would be exposed to sublethal doses of the Bt toxin in Bt plants and then move to non-Bt
plants allowing selection for resistance to occur. By August 9, 1998, a draft refuge strategy
must be submitted to the Agency and a final refuge strategy submitted by January 31, 1999.
The registrant must implement an EPA approved "structured" refuge plan or an EPA approved
alternative resistance management plan no later than April 1, 20.01. Registrants are required to
discuss the development and implementation of the refuge plan and alternative resistance
management practices .with EPA throughout the development and the implementation.
Monsanto Co. and Dekalb Genetics mandated certain activities under their Technology
Agreements with growers and charged a $32 per acre technology usage fee in addition to the
cost of the seed. The Technology Agreements requires that growers who use MON810- or
DBT418^derived hybrids must unplement either a 5% unsprayed non-Bt refuge or a 20%
sprayed non-Bt refuge. Noyartis Seeds and Mycogen Seeds do not require any specific refuge
option to be implemented, but do recommend the use of a refuge.
EPA, a number of entomologists, environmental groups, and other stakeholders have '
expressed concern regarding the impact of CryI(A)b- and CryI(A)c- expressing corn on CEW
in those areas where CryI(A)c-expressing cotton is grown. EPA imposed restrictions on the
, number of acres allowed in the South on Bt corn hybrids expressing the Bt delta endotoxin in
silks and kernels, at present this would include events MON810-, BT11-, DBT418-derived
hybrids, but not Event 176-derived hybrids. A total of 200,000 A was. allowed in the South:
100,000 A each for MON810- and BT 11-derived hybrids and none for DBT418-derived
hybrids. Dekalb's Bt plant-pesticide expressing the CryI(A)c delta endotoxin, DBT418, was
not registered for use hi parts of the South because .the Agency determined that the resistance '
risk'was too high to allow additional Bt corn plant-pesticides mat express Cry delta endotoxins
in silk and kernels to be used there. Based on receptor binding experiments, CryIA(b) or
,CryIA(c) delta endotoxins have similar binding characteristics, indicating that there might be a
high degree of cross-resistance to these two toxins. In addition, the CryI(A)c delta eridotoxin
is also registered in cotton increasing the potential for Bt resistance in pests exposed to this
toxin in both corn and cotton. Event 176 CryI(A)b-expressing corn was not restricted in the
South because, it has only trace (< 8 ppb) levels of the delta endotoxin in silks and kernels and
is nqt expected to select for second generation CEW resistance. In addition to sales
restrictions, research data and model development were required on all the Bt hybrids
registered to evaluate the potential .impact of Bt corn on Bt resistance management programs in
areas growing corn and cotton.
Silk and kernel expression in Bt corn hybrids will likely increase the selection for CEW
resistance especially in cotton-growing areas. If there is silk expression of the
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CryI(A)b/CryI(A)c delta endotoxin at sufficient levels to select for resistant CEW, then
resistant CEW could move from Bt corn to cotton/Bt cotton posing potentially significant
problems in cotton or Bt cotton or potentially in other crops affected by CEW. Where corn
and cotton acres are hi close proximity, there will be migration of second generation CEW
from silk-stage Bt corn to cotton (including Bt cotton) and other crops. In the southeastern
U.S., virtually all second generation CEW funnel through corn where they complete
development on the ear of this preferred host. Selection for CEW/CBW resistance could be
accelerated if Bt corn hybrids became widely adopted in the South if adequate resistance
management was not adopted. In the South, there are 3 to 6 CEW generations and in the
North, there are 1 to 2 generations. Thus, CEW in the South are potentially subject to higher
levels of exposure to the Bt delta endotoxin than CEW in the North. CEW only overwinter in
the South. However, the development of CEW resistance to Bt in the North is also a concern.
The major source of CEW in the northern corn belt is adults flying or being carried on the
prevailing winds from the southern states each year. Should CEW resistance to Bt toxins
develop in the South, it could be equally damaging in the northern states growing Bt corn each
season. In the South, mere would be a higher selection pressure in areas in which Bt corn and
Bt cotton are in close proximity arid in areas in which Bt microbial pesticide products are used.
Resistant CEW could lead to the failure of Bt microbial pesticides used on cotton and other
crops or to the failure of Bt cotton and Bt corn, and other crops both in the South and hi the
North for control of CEW. Although the risk of loss of Bt and increased use of chemical
insecticides cannot be quantified, EPA believes this risk is real. There could also be negative
impacts on organic farmers from the loss of Bt. EPA addressed these risk hi issuing the Bt
corn registrations.
While the theory of high dose expression coupled to effective structured refugia is relatively
straightforward, its implementation and enforcement have been controversial. Long-term Bt
corn resistance management is complicated by the following circumstances: (1) there are
multiple competitors for the technology (there is only a single registrant involved in both Bt
potato and Bt cotton), (2) there are different Bt delta endotoxins and transformation events
being put forward by the various companies with differences in levels of expression through
the plant, (3) research efforts are not consolidated, and (4) there are a large number of states,
large number of growers, and tremendous corn acreage throughout the U.S. involved. With
all of these factors in mind, there is disagreement among stakeholders as to what is (1) the
appropriate arrangement and relative size of Bt corn and refuge plots, (2) the nature and
objective of performance-monitoring activities, (3) research coordination, and (4) appropriate
incentives to foster grower education and acceptance. The Agency has fostered and
participated in efforts to resolve these disagreements to the satisfaction of all stakeholders and
has requested public comment. One such effort to date to bring all stakeholders together has
been by the USDA NC-205 group (research and extension entomologists of the North Central
Regional Research Project (NC-205), "Ecology and Management of European Corn Borer and
Other Stalk-Boring Lepidoptera") (Ostlie etal., 1997). The NC-205 efforts will be discussed
in more detail. This White Paper will analyze where EPA is hi resolving these critical issues
and provide recommendations on improvements for development of a long-term resistance
24
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management strategy for Bt field corn.
Bt corn acreage in 1996 and 1997 ,
EPA required each registrant to submit to EPA annual sales data for each state indicating the
number of units of Bt corn hybrids each registrant sells. Because FIFRA section 10 restricts
the release of certain confidential business information, exact sales data may not be provided.
However, the following information provided by the registrants is publically releasable.
Approximately 400,000 acres (about 0.5 % of the total corn acreage) of Bt corn were planted in
30 states in the U.S. hi 1996 and approximately 3.5 to 4 million acres (about 5% of the total
corn acreage) is expected to have been planted in 1997. Based on a three-year average, there
ar,e approximately 70 to 80 million acres of corn planted annually in the U.S. If the total Bt
corn acreage is in the 4 million range, then the adoption of Bt corn will have grown by about
10-fold between the 1996 and 1997 growing seasons. Part of the reason for this expected
growth is the registration of new CryI(A)b or CryI(A)c delta endotoxins and the necessary
material for their production (BT-11-, MON-810-, and DBT 418-derived Bt corn hybrids) in
late 1996 and early 1997. Sales information for the 1997 growing season will be provided to
EPA by January 31, 1998 as required by the requirements of registration.
Analysis of Resistance Management Strategy
Expression of CryI(A)b/CryI{A)c,and impact on European corn borer '(Ostrinia nubilalis, ECB)
- primary target pest .
The primary target of Bt plant-pesticides used in field corn is the European corn borer.
All of the registered Cry toxins in corn express a dose sufficiently high to control first-
generation ECB in whorl-stage corn. However, the level of control against late-season
ECB generations differs between Bt plant-pesticides. That is, not all of the Bt plant-
pesticides provide a "high dose" to effectively control second (or later) generation
ECB. Events BT11, MON 810 and DBT418 maintain a " high dose ".expression
throughout the growing season in corn plants expressing the CryI(A)b or CryI(A)c for
ECB control. Under heavy ECB pressure in silking corn, BT 11-, MON 810-, and ,
DBT418-derived hybrids provide a higher level of late-season control than Event 176.
Measured in terms of reduction in tunneling damage, the level of control for BT11-,
MON810-, and DBT418-derived hybrids is greater than 95 % for full-season control of
ECB; whereas, the level of control for Event 176rderived hybrids is greater than 95%
.' for first generation ECB and about 70-75% control of second generation ECB. There
is a difference in full season ECB control when CryI(A)b or CryI(A)c is expressed only
in green tissue and pollen in Event 176-derived hybrids as compared to when Cry 1 A(b)
or CrylA(c) is expressed hi all plant tissues including silks and kernels as found hi
BTll-derived hybrids, and MON810- derived hybrids and DBT418-derived hybrids.
Mycogen Plant Sciences, Novartis Seeds, Monsanto, and Dekalb Genetics are -
• ' • ' - " 25 .-."•••'•• . . '• . ' '
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continuing to research the expression patterns hi Event 176-, BT11-, MON810, and
DBT 418-derived hybrids to determine the level of control on first and second
generation ECB. These efforts will be summarized in the forthcoming registrant annual
reports due to be submitted to the Agency by January 31, 1998.
Event 176-derived hybrids - Silk and kernel expression and effect on ECB control
Event 176 Bt corn hybrids express the Cry l(A)b delta endotoxin in silk and kernels at
less than 8 ppb and 5 ppb, respectively (Ciba Seeds/Novartis Seeds letter to EPA dated
November 20, 1996). An ELISA of Mycogen Plant Science's Event 176 Bt hybrids
indicated there was no detectable CryI(A)b expression in silks (Mycogen Corp. Letter
to EPA dated December 9, 1996). There may be trace levels of Bt toxin in the silks
and kernels, but they are below the level of detection and are not at levels thought to
selection for Bt resistance by second generation CEW. Because some hatching larvae
initially colonize ears to feed on silks and developing kernels, these larvae may survive
on Event 176 and may tunnel later in stalks and ear shanks. The presence of second
generation ECB in ears hi Event 176 corn is a topic of resistance management
discussions. Nonetheless, control with Event 176 corn (>95% control of first
generation ECB and about 70-75% control in second generation) is better than
conventional insecticide options [1996 Progress Reports from Ciba Seeds/Novartis
Seeds and Mycogen Plant Sciences]. Insecticides provide 80% and 67% control of first
and second generation ECB, respectively (Ostlie et al., 1997).
Field research performed by Dr. Fred Gould, North Carolina State University in 1996,
indicated that ECB may be surviving on Event 176 Bt corn silks, but the results are
difficult to interpret. Gould concluded that "176 line of Bt corn does not produce a
high enough dose to be considered as part of a resistance management program for
ECB that requires a high dose in the plant during the period when second generation
larvae are present" (from Appendix VI, 1996 Growing Season Report, Ciba
Seeds/Novartis Seeds, January 30, 1997).
Expression of CryI(A)b and CryI(A)c and effects on other pests including CEW and
Southwestern corn borer
While the primary target pest for these Bt corn hybrids is ECB, the CryI(A)b and
CryI(A)c delta endotoxins might suppress or control, to some degree, other
lepidopteran pests. Three non-ECB pests somewhat susceptible to the CryI(A)b or
CryI(A)c delta endotoxins are: southwestern corn borer, Diatraea grandiosella (Dyar)
(SWCB); CEW; fall armyworm, Spodoptera frugiperda (J. E. Smith); and Diatraea
cratnbidoides (Grote), southern cornstalk borer (SCSB). Selection for resistance may
also occur hi these other pest species susceptible to the Bt delta endotoxins and the
Agency is concerned about this risk. Therefore in the conditional registrations, the
Agency asked for data regarding the potential effects and the development of resistance
26
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in SWCB, CEW fall annywofm, SCSB, and other secondary pests, and took other steps
to manage the risks. '<• •
Novartis Seeds and Mycogen Plant Sciences have made no control or suppression
claims on their labels for the control of secondary target pests by Event 176-derived
hybrids expressing CryI(A)b. Dekalb Genetics Corporation has also made no control
or suppression claims on its label for the control of secondary target pests by.DB'T 418-
derived hybrids expressing CryI(A)c. Novartis Seeds has made control or suppression
claims on its label for the control of southwestern corn borer, corn earworm, and fall
armyworm by BT 11-derived hybrids expressing CryI(A)b. Monsanto has made
control or suppression claims for the control on its label of southwestern corn borer and
corn earworm by MON 810-derived hybrids (i.e. Yieldgard™) expressing CryI(A)b.
Research on the effects of CryI(A)b and CryI(A)c on other Lepidopteran pests,
prmiarily conducted in 1996, will be discussed below.
(1). Event 176 efficacy data on multiple secondary Lepidopteran pests (work sponsored
in part by Novartis Seeds): Pilcher et al. (1997) describe field and laboratory
evaluations of Event 176 Bt corn on secondary Lepidopteran pests. Based on these
experiments conducted hi 1994 and 1995, no Bt corn effects were observed on larval
survival, pupal weight,.or days to adult emergence for Agrotis ipsiloh (Hufnagel) (black
cutworm) or Papaipema nebris (Guenee) (stalkborer); however, Pseudaletia unipuncta
(Hawoth) (armyworm) and CEW were affected by Bt (Bt treated diets). Under field
conditions, there were no differences between Bt and non-Bt corn damage caused by
black cutworm, stalkborer,, CEW, and armyworm. CEW survived on and caused
damage to Bt corn ears, and even though there were fewer ears damaged than the non-
Bt controls, there was no difference in the number of live larvae per plant on Bt corn
compared to non-Bt corn. Conversely, there were fewer .feeding scars and no larval
survivors on whorl-stage Event 176 corn; whereas, there were numerous feeding scars
and larval survivors on non-Bt corn.
(2) Southwestern corn borer efficacy data. Field trials conducted at Kansas State
(Bushman and Higgins) and Texas A & M (Archer) (1994-1996) [Work sponsored by
Novartis Seeds, Mycogen Plant Sciences, and Monsanto Co.] showed that BT11- and
MON810 events provided excellent full-season control (> 90%) of southwestern corn
borer, while Event 176 afforded only about 20-50% control. .In this situation, full-
season Bt expression (constitutive expression) provided by the BT11/MON810 events
was necessary to control southwestern corn borer. First brood control was excellent
for all hybrids tested. The greatest difference was seen hi second brood control because.
of the difference hi expression patterns between Event 176 and BT11/MON810
hybrids. Work is continuing by Buschmari and Higgins at Kansas State University to
investigate the efficacy of BT11/MON 810-derived hybrids and Archer at Texas A & M
to investigate the efficacy of Event 176-derived hybrids against SWCB. ,
27
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(3) Southern cornstalk borer efficacy data. Field trials conducted at North Carolina
State University by Dr. John Van Duyn in 1996 (Mycogen Plant Sciences and Novartis
Seeds sponsored work) indicated that there was about 65 % control of southern corn
stalk borer in Event 176 Bt corn. Further efficacy testing will be done in 1997.
Corn Eanvorm (CEW) - Effect ofBt corn on CEW survival: Field Trial Results
Numerous field trials examined CEW susceptibility to the CryI(A)b delta toxin in Event
176 and BT11/MON810 corn hybrids. Only BT-11 and MON 810 labels make claims
for control or supression of CEW. The results of these field trials are discussed below.
Monsanto-sponsored work (MON 810-derived hybrids)
* ., . ,, , ;•'••.•
Monsanto has made control or suppression claims for the control of southwestern corn
borer and CEW by MON 810-derived hybrids (i.e. Yieldgard™) expressing CryI(A)b.
Monsanto was required as a condition of registration to submit available research data
on CEW relative to resistance development and its plans for producing resistance
predictive models by January 31, 1997. The Agency has received Monsanto's available
research data on CEW relative to resistance development and plans for producing
resistance predictive models (MRlD 442094-01, dated January 30, 1997). This
information is summarized below.
(1) CEW densities and development hi MON810 were evaluated in 1995 by Df. Fred •
Gould (North Carolina State University) in North Carolina. CEW growth was slowed
by Bt in MON 810. This work indicates that only a third as many larvae were able to
complete development hi MON810 compared with the non-Bt larvae control. Dr.
Gould concludes that MON810 could change the phenology of adult CEW moth flights
from corn to cotton leading to a need for alteration of scouting procedures in cotton.
In places and years with low CEW densities, delays hi adult emergence could lead to
protracted moth flights which would decrease peak earworm densities in cotton by
spreading out larval hatch over tune. However, protracted moth flights could also
select for resistance in areas such as North Carolina where a large percentage of the
CEW population is found hi ear stage corn.
(2) Three field trials were conducted by Dr. Randall Higgins (Kansas St. University) in
Kansas in 1996. The objective of these trials was to determine the extent hi which
CEW oviposition, larval densities, and larval development differ among MON810,
BT11, and Event 176 corn hybrids compared to non-Bt corn hybrids, Results suggest
that there are no consistent differences hi CEW densities within ears between Bt and
non-Bt corn hybrids. Findings also suggest that CEW development was delayed by the
presence of Bt, although the magnitude of these differences varied among Bt hybrids
and across tune (early- vs. late-planted corn). CEW development was delayed for both
MON810 and BT 11 corn hybrids hi early-planted corn, but not for Event 176 hybrids.
28
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In late-planted cprn, CEW development was delayed for MON810, BTll, and Event
176 corn hybrids, but was greatest for MON810 and BT11 corn hybrids.
(3) In a 1996 Kentucky field trial conducted by Dr. Ricardo Bessih (University of
Kentucky), there was a reduction in CEW larval weight for all Bt hybrids tested (i.e.,
BTll, MON810, Event 176). the greatest reduction in weight was seen in BTll and
MON810 hybrids (82-90%) versus Event 176 (41-64%).
(4) A series of experiments was conducted by Dr. John Van Duyn (North Carolina
State University) in North Carolina in 1996 to evaluate CEW whorl and ear stage
infestation levels, insect development, survival, and fecundity. BTll and MON810
corn hybrids had significantly lower levels of feeding damage from CEW compared
with non-Bt controls. The number of infested ears was not consistently affected by Bt
in the plant. In early-planted corn,, delayed development and reduced larval weights,
were evident for BTll and Event 176 corn hybrids compared with the non-Bt control
(Pioneer 3394). CEW infestation in late-planted corn was more difficult to interpret.
because of overlapping second and third CEW generations, but larval weight reduction
was observed m MON810 hybrids similar to findings in other field trials. Pupal
weights, pupation time, and adult eclosion were delayed in BTll and Event 176 corn
lines compared with the non-Bt control (Pioneer 3394).
(5) In 1996 field trials conducted jointly by Dr. Galen Dively (University of Maryland)
and Dr. Ames Herbert (Virginia Polytechnic University) in Maryland, Virginia and
North Carolina, on CEW survival, development and feeding injury in MON810 and
non-Bt hybrids was evaluated. In addition, ovipositional rates, moth survival, and egg
hatching were evaluated in the laboratory for CEW. Relative to the non-Bt hybrid,
insects feeding on MON810 hybrids were delayed in development and had reduced
fecundity. Evaluation of overwintering survival and construction of life tables are in,
progress.
Dekalb Genetics sponsored work (DBT418-derived hybrids) ,
Dekalb efficacy data indicated that the CryI(A)c protein is much less toxic to CEW than
it is to ECB, Dekalb has made no label claims of control or suppression of CEW on its
DBT 418 (CryI(A)c delta endotoxin and the genetic material necessary for its
production in corn). Results of laboratory and field efficacy studies conducted on CEW
are summarized below. .
(1) The effect of the CryI(A)c protein on the growth of CEW larvae was investigated by
weighing insects following exposure to a series of toxin concentrations. Results were
plbtted as the percentage of control insect weight vs. the concentration of CryI(A)c.
CryI(A)c levels in DBT418 silk and ear leaf tissue were sufficient to slow larval growth
of CEW larvae ,(> 80%),, but not adequate to kill CEW directly. CEW damage to the
29 ' • -'-.'; :''',' I
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ear on DBT418 plants does not appear to be significantly reduced. However, it is
likely the growth inhibition will result in increased CEW field mortality because of
increased predation and parasitism, disease, and losses from exposure to adverse
environmental conditions over a longer period of time.
(2) A field study performed by Dekalb in 1995 indicated a trend towards reduction in
CEW larval weight gain on DBT418 as compared to non-Bt corn plants. A field study
conducted by Dr. Bob Lynch and Dr. Billy Wiseman, USDA-ARS, Tifton, GA also
indicated a reduction in neonate and 3-day larval weight after feeding on leaf and
DBT418 silks incorporated into the diet, but 3-day old larvae were not affected by fresh
DBT418 silks. Six-day old larvae were unaffected by DBT418 silks, fresh or
incorporated into diet. The number of larvae per ear and ear damage following CEW
infestation of a DBT418 hybrid and a non-Bt control were not significantly different.
(3) In 1996-1997, Dekalb is continuing its sponsorship of work on the efficacy of
DBT418 against ear feeding by CEW. Dr. Ricardo Bessin, University of Kentucky,
Lexington is conducting a study to determine the efficacy of DBT 418-expressing
hybrids against ear feeding by CEW. Dr. Billy Wiseman, University of Georgia,
Tifton is conducting two studies involving DBT 418-expressing hybrids. In the first
study, the objectives are to determine the efficacy of DBT 418 corn hybrids against ear
feeding by the CEW and to measure the fitness costs for CEW found on DBT corn
hybrids. In the second study, the objective is to determine the efficacy of
conventionally-bred multiple borer resistant corn lines crossed with DBT 418 hybrids
against ear feeding CEW. Dekalb is also conducting internal studies regarding the
efficacy of DBT 418-expressing hybrids to CEW whorl and ear feeding.
Mycogen Plant Sciences sponsored work (Event 176-derived hybrids)
Mycogen Plant Sciences has made no control or suppression claims on the label for
CEW or other secondary target pests by Event 176-derived hybrids expressing
CryI(A)b. Effects of Event 176 corn on CEW was studied by Galen Dively, University
of Maryland; Randy Luttrell and D. Porter, Mississippi State University; and John Van
Duyn, North Carolina State University. , ' •
(1) The North Carolina field trials conducted by Dr. John Van Duyn, North Carolina
State University in 1996 indicated that there were no reductions in CEW ear feeding
with Event 176 Event com. Ear and silk feeding larvae had slightly longer
developmental tunes on Event 176 Bt corn than on conventional corn. This delay in
development on Event 176 corn will likely increase CEW larval susceptibility to
pathogens, parasites, and predators.
". , , i
(2) A preliminary report in 1996 from the first of a two-year study conducted by
Mississippi State University trials (MSU) indicated that in silking stage corn, there
30
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were no differences in CEW growth and development (including pupal production)
between GEW on Event 116 Corn and non-Bt corn. The MSU work is being continued
in 1997.
"• .'-""'' ,' " - ''"•• ' "
(3) Results from the 1996-1997 University of Maryland studies on whorl feeding CEW
with Event 176 Bt corn are not yet available.
Novartis Seeds sponsored work (BT11 and Event 176-derived hybrids)
Novartis Seeds has made no control or suppression claims on the label for the control
of secondary target pests including CEW by Event 176-derived hybrids expressing
CryI(A)b on its label. Novartis Seeds has made control or suppression claims on the
label for the control of southwestern corn borer, CEW, and fall armyworm by BT11-
derived hybrids expressing CryI(A)b. Research on the effect of Event 176- and BT 11-
derived hybrids on CEW is diseussed'below.
(1) Novartis Seeds reports that CEW survival on silking corn was no different on
Event 176 corn hybrids arid non-Bt corn hybrids. However, there may be a subtle
effect on growth for CEW feeding on Event 176 corn (Novartis letter to EPA dated
November 20, 1996).
(2) In 1995, Ciba Seeds conducted a field evaluation of CEW on whorl-stage Event
176 corn and non-Bt corn. Event 176 plants showed minimal leaf damage by CEW and
non-Bt plants had "shot-hole" injury and elongated feeding lesions on several leaves.
Such data suggest that whorl-stage Event 176 corn shows high efficacy against first
generation CEW. ,
(3) Research performed by Dr. Fred Gould, North Carolina State University, also
showed that CEW survival was no different on silking Event 176 Bt corn hybrids and
non-Bt corn hybrids. There were no statistically-significant differences in number of
live larvae, larval weight, or larval development between larvae that had fed upon ears
from Event 176 corn hybrids or non-Bt corn hybrids. In silking Event 176 corn, if
there is selection for CEW resistance, it is subtle. Thus, Gould suggests, if there is a
high dose of Bt toxin in whorl-stage corn, then Event 176 corn could act as a refuge for
second generation CEW in the South. Gould indicates that Event 176-derived Bt
hybrids are not appearing to exert a significant selection pressure on second generation
CEW populations and may actually provide a refuge from selection for second
generation CEW. However, there is still some concern about the level of selection
pressure on first generation CEW feeding on whorl-stage Event 176 field corn.
The likelihood that second or later generation CEW will develop resistance to CrylA(b)
as expressed in Event 176 Bt corn is considered to be substantially less than for other
corn hybrids that produce Cry proteins in the silks and kernels, the primary feeding
. .• ...,'• '••'•' ' '. ' ;. , si - > . '. '
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tissues for later generations of CEW. Second generation CEW typically oviposit
directly on silks or ears and emerging larvae feed primarily on these tissues. Data
supplied by Mycogen and Novartis indicate that sepond or later generations of CEW are
not exposed to the CrylA(b) toxin in either the silks or the ears and therefore these
generations are not undergoing a selection pressure for the development of CrylA(b)
resistance. However, there is still some concern about the level of selection pressure on
first generation CEW feeding on whorl-stage Event 176 field corn.
(4) Novartis Seeds is also sponsoring work in 1997 by Dr. Galen Dively (University of
Maryland) to examine the performance of Bill-derived hybrids on CEW.
Importance ofBt corn and its impact on Bt resistance management programs in areas growing
corn and cotton
Research data and model development were required on all the Bt hybrids registered to
evaluate the potential impact of Bt corn on Bt resistance management programs in areas
growing corn and cotton. Monsanto and Novartis were required by January 31, 1997 to
submit available research data on CEW relative to resistance development and plans for
producing resistance predictive models to cover regional management zones in the cotton belt
based on CEW/CBW biology and cotton, corn, soybeans, and other host plants. These models
must be field tested. Monsanto and Novartis submitted the available research data on CEW
and plans for predictive models. Novartis, Mycogen, Dekalb, and Monsanto are sponsoring
research activities regarding the development of predictive models on CEW resistance
development and these are summarized below.
1. Modification of a spatially explicit computer simulation model for predicting
resistance development in CEW to Bt corn and effects ofBt corn on parameters of CEW
biology and concomitant effects on population dynamics. Monsanto, Dekalb Genetics,
and investigators from North Carolina State University (Dr. John Van Duyn, Dr. Fred
Gould, Dr. J.R. Bradley, and Dr. George Kennedy), Virginia Polytechnic Institute and
State University (Dr. Ames Herbert), and the University of Maryland (Dr. Galen
Dively) are devising strategies for developing computer simulation models which
predict the evolution of resistance to CryI(A)b/CryI(A)c proteins by CEW within the
corn/cotton system. Additionally, research protocols are being developed for validating
model assumptions and output. Research areas include: (1) Assessing the impact of Bt
corn on CEW adult emergence and oviposition hi cotton; (2) Contribution of alternate
hosts as refuges for CEW; and (3) Impact of Bt on CEW overwintering survival and
fecundity. Information on the effects of CryI(A)b/CryI(A)c proteins on key CEW life
history parameters will be integrated into the computer-based simulation model for
prediction of the resistance development probability and other population biology
events under different Bt crop use scenarios. EPA has limited Bt corn sales in the
South due in part to a lack of biological data and simulations of population dynamics,
gene flow, and resistance development estimates for CEW, a pest which moves freely
32
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from corn to cotton in the south. This research will be critical hi determining ,
appropriate long-term refuge strategies in southern areas: where corn and .cotton are
grown in close proximity.
• ... Data regarding CEW populations on other plant hosts (cotton, sorghum, soybean, a
number of vegetables, ornamentals, and wild hosts) still remain to be gathered and
analyzed by researchers.
> 2. Selection on CEW by 'Event 176 CryI(A)b-expressing corn (sponsored by Novartis •
Seeds and Mycogen Corp.). A multi-year research program is being developed by Dr,
Randy Luttrell at Mississippi State University-Stoneyille to examine the hnpact of
Event 176 CryI(A)b-expressing corn on CEW in those areas where CryI(A)c-expressing
cotton is grown. Results from Luttrell's 1996 field work, as others have shown in
other field tests involving Event 176, indicate that Event 176 Bt corn demonstrates
. high efficacy towards first generation CEW and there are no measurable effects on
second generation GEW.
3.- Modeling the Evolution of Resistance - population dynamics of CEW movement in
' : corn, cotton, and other hosts involving Event 176- and BTll-derived hybrids by
Novartis Seeds). Dr. Randy Luttrell, Mississippi State University-Stoneville is
examining a number of parameters that effect the evolution of CEW resistance in the
, corn-cotton ecosystem. The purpose of this work is four-fold: (1) Estimate impact of
Bt corn on resistance evolution in corn-cotton ecosystem; vary Bt com acreage from 5
, to 100% and vary total refuge from 5 to 20% to determine the most effective strategy.
(2) Simulate impact of cross-resistance between loci that confer resistance to two
different toxins. (3) Simulate expression of Bt protein hi whorl stage corn only, and
expression in whorl and kernel to study hnpact of size of refuge. (4) Simulate hnpact
of wild hosts (density and temporal availability). The field data from research by^
Luttrell noted above in #2 will contribute toward demographic objectives. Validation
of the genetics of the model is not possible within the two year scope of this research
, project. Field validation of the model would require the deliberate generation or
, release of a resistance population which is probably unacceptable.
< .-'''' ' . • • '
European corn borer biology and behavior :
' • ' ' ' ' >' •. . \ , . . «,
A key to developing a long-term resistance management strategy of ECB on Bt corn is based
on the detailed understanding of ECB pest biology and behavior. EPA required as a
requirement of registration additional information regarding ECB pest biology and behavior. ,
Key data regarding adult movement, mating behavior, gene flow, and alternate, hosts of ECB
, are sparse. This information is valuable in designing an effective resistance management" '
strategy that maximizes the probability that susceptible individuals arising from a refuge will
find and mate with the few resistant individuals that survive exposure to the delta endotoxin
produced hi the Bt plant. Research efforts to. assess adult movement, mating, gene flow, and
•~ ' •-. ' -•'"•'•'' ". •'•• 33 " '" •'.•..•,.• :..-'" V .
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survival of ECB on non-corn hosts are ongoing and are summarized below. Results from the
1997 growing season will be discussed in each registrant's annual report required to be
submitted to EPA by January 31, 1998.
1. Long range movement of adult ECB (sponsored by Novartis Seeds and Mycogen
Corp.). The purpose of this research (1996) being conducted by Drs. D. Alstad and
D. Andow, University of Minnesota, is to identify the genetic structure of regional
North American populations of ECB and estimate the intermating (gene flow) between
these populations using isozyme electrophoretic analyses. Electrophoretic and
statistical analyses of the data to estimate gene flow have not been completed.
Preliminary findings indicate that little gene mixing occurred across ECB populations
collected at about 250 kilometer intervals along three transects across U.S. corn
production areas. The final report to be provided with the 1997 annual report, due by
January 31, 1998, may indicate some useful information related to the long range
movement capabilities and gene flow of ECB. '
2. Short range movement of adult ECB (sponsored by Novartis Seeds, Mycogen Plant
Sciences, Dekalb Genetics, Monsanto Co.). The purpose of this research (1996-1997)
being conducted by Drs. John Witkowski and T. Hunt, University of Nebraska, is to
evaluate the short-range movement of adult ECB using a mark-release-recapture
technique. The objectives of this work are: (1) determine how far initial movement is
aw.ay from and around a corn field, (2) determine how dispersion to and distribution
among action sites change with tune, (3) determine how dispersal and/or distribution
differs by sex, and (4) determine how movement changes by generation. Data from
1996 field studies (first year) indicate that the gene flow between adjacent corn fields
does occur. Adult movement indicated that refuge may need to be within 1500-2000
feet of Bt corn fields. Adult movement is influenced by growth stage of the corn, most
adults stay close to where they emerged, and some adults can move at least one mile
within two days of emergence. These preliminary results indicate that the conventional
corii refuge may need to be relatively close, but not necessarily adjacent to Bt corn.
3. Movement of late instar ECB into Bt corn hybrids (sponsored by Dekalb Genetics,
Monsanto Co., Novartis Seeds). The purpose of this two year study (1996-1997)
conducted by Dr. Rick Hellmich (Iowa State University) is to compare movement and
survival of fourth instars on Bt corn hybrids expressing different levels of CryI(A)b or
CryI(A)c. Late instar movement to plants expressing lower levels of Bt and survival on
such plants could cause damage to the plants and reduce the effectiveness of the high
dose strategy.
4. ECB lifetable work (sponsored by DeKalb Genetics, Monsanto Co.). The purpose
of this 1996-1997 work conducted by Dr. Kevin Steffey (University of Illinois -
Urbana-Champaign) is to develop a lifetable for ECB hi Illinois. Information from
lifetables will be used to better understand factors influencing ECB population
'; ' -. , • ' : 34' • -
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fluctuations as well as assist in estimating parameters used in insect resistance
management modeling efforts.
5. 'Overwintering survival (sponsored by Novartis seeds and Mycogen Plant Sciences)
The purpose of this 1996-1997 research conducted by Dr, Blair Siegfried, University of
Nebraska, is to examine the overwintering survival and CryI(A)b tolerance of ECB
larvae derived from Event 176 corn. Mycogen is also conducting internal research in
this area. Results from 1996 and 1997 will be pooled and discussed in the 1997 annual
report due to EPA by January 31, 1998. The number of surviving larvae and the in
vitro CryI(A)b sensitivity of populations derived from these larvae will be established
and well as fitness characteristics assessed. This information will be useful in
determining the significance for resistance management of ECB larvae found surviving
late in the season on Event 176 corn hybrids. -
Laboratory-selection for ECB resistance to Bt Endotoxins '
EPA required as part of registration more information on the biology of ECB resistance and
the potential for cross-resistance. .Information on the nature of resistance to Bt in target pests
such as the ECB are necessary to measure the effectiveness of resistance management for Bt
corn. Such information is useful in evaluating which Bt endotoxins could be used in a
rotational or pyramiding scheme! Currently this work is limited to working with laboratory-
selected populations since resistance in the field has not been detected. Laboratory studies
selecting for ECB-tolerant strains provide information on the genetic potential of ECB to
develop resistance, but are not conclusive on whether.resistance will develop in ECB ,
populations under field conditions. Bt com .and ECB in the field may pose a different situation
than larvae feeding on Bt insecticides in a laboratory diet under controlled conditions.
Laboratory-selected tolerant colonies, nonetheless, will be useful in experiments concerning
; the mechanism of resistance and the genetic basis for resistance, and provide information on
the potential for cross-resistance between Bt toxins. Research (1996-1997) on ECB colonies
. selected for resistance in'the laboratory to CryI(A)c and CryI(A)b are described below.
1. Effect of CryI(A)c resistance on ECB fitness (research by Dr. William Hutchison,
University of Minnesota and sponsored by Novartis Seeds and Mycogen Plant
Sciences). Multiple CryI(A)c-resistant ECB colonies were developed in the laboratory.
These colonies were selected for tolerance to a non-viable formulated Pseudomonas
fluorescens engineered to express the CryI(A)c endotoxin (formulated MVP (Mycogen
Corp.)) under acute exposure conditions. Selection pressure was removed from these
colonies and fitness costs were estimated. Results show that after nine generations of
no exposure to CryI(A)c, there was no difference in susceptibility between the selected
and non-selected colonies. Thus, Hutchison.concluded that there was a significant
fitness cost associated with the development of CryI(A)c tolerance hi these laboratory
colonies. Additional studies on other fitness parameters including fecundity,
developmental rate, survival, and larval weights are being conducted in 1997.
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2. Susceptibility of Cry I (A) c resistant ECB to other Cry proteins (Cross-resistance)
(research by Dr. William Hutchison, University of Minnesota and sponsored by
Novartis Seeds and Mycogen Seeds). In laboratory binding studies, CryI(A)b and
CryI(A)c delta endotoxin are structurally similar and appear to bind to the same midgut
epithelial receptor in ECB (Denolf et al., 1993). The structural and target site
similarities of the two delta endotoxins suggest that insects developing resistance to one
delta endotoxin would likely develop resistance to the other. Research by Dr. Blair
Siegfried tested the estimated LCgg derived from the dose-response curve to CryI(A)b
against the CryI(A)c resistant ECB strains developed at the University of Minnesota.
These strains do not appear to be resistant to CryI(A)b. Resistance to CryI(A)c is not
conferred to the CryI(A)b delta endotoxin. The CryI(A)c resistant strains were tested
for cross-resistance to other Cry proteins. The results of single dose diet surface
bioassays for 17 different single Cry protein preparations suggest that cross resistance
is not present for several toxins, i.e., CryI(A)b, Cry7(A)b, and PS28(C), and is present
for others, i.e., CryI(B), CryI(F)a. These CryI(A)c resistant ECB strains show a very
narrow range of cross-resistance. Additional dose response determinations on
formulated products are continuing in 1997.
3. Laboratory selection experiments for ECB CryI(A)b resistant colonies.
(a) Dr. Keil, University of Delaware (Novartis Seeds sponsored work), selected for a
CryI(A)b-resistant ECB colony under acute exposure conditions in the laboratory. Keil
notes there are fitness costs associated with the development of CryI(A)b tolerance,
including temporal changes in development, reduced pupal weight, as well as reduced
egg deposition in the selected colony. CryI(A)b-tolerant larvae failed to cause any leaf
feeding damage on Event 176-derived corn expressing the CryI(A)b toxin. There was
no difference in feeding damage between the CryI(A)b-tolerant larvae and non-selected
CryI(A)b larvae on Event 176-derived corn. These results indicate that the gene(s)
involved hi tolerance to CryI(A)b hi the laboratory-selected colonies do not confer any
increased tolerance to Event 176-derived corn plants that express the CryI(A)b toxin.
(b) Pioneer Hybrid International has selected for two CryI(A)b-tolerant ECB colonies ;
under chronic exposure conditions hi the laboratory (Lang et at., 1996). Chronic
exposure more closely mimics field exposure conditions for ECB larvae. Following 13
generations of selection pressure, neither colony exhibited any increase in feeding
damage on CryI(A)b-expressing corn plants compared to non-selected larvae. Neither
selected colony was able to survive exposure to Bt concentrations (in an artificial diet)
that approached those concentrations that would be encountered in a Bt corn plant.
(c) Dr. Blair Siegfried (University of Nebraska) and Dr. Randy Higgins (Kansas St.
University) have created two additional CryI(A)b-selected ECB colonies for study.
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Novartis Seeds established a research collaboration with Siefried's laboratory in 1997
to evaluate these colonies for cross-resistance to other Cry toxins, fitness parameters,
and leaf feeding behavior on CryI(A)b-expressing corn plants. Dr. William Hutchison
(University of Minnesota) and Mycogen Plant Sciences are both initiating efforts to
select for CryI(A)b resistance in ECB to facilitate further research on the biology of .
resistance.
1
4. Midgut binding studies (research by Dr. William Hutchinson,University of
Minnesota and sponsored by Mycogen Seeds). Midgut binding studies were expected
to be initiated in 1997. The Cry delta endotoxin binds to a specific receptor,on the
midgut lining and the cells rupture. These studies will examine whether modification in
ECB midgut binding may be the mechanism of resistance.
Effective Refuges
All available evidence supports the conclusion that a "structured" refuge is a necessary
component of a successful long-term resistance management strategy and that the success of the
high dose strategy will be compromised if there is no effective refugia. Stakeholders have
hotly debated the size and deployment of a refuge for Bt coin. Prior to the August 1995
registration of the first CryI(A)b delta endbtxin in corn, a few entomologists recommended a 5
to 50% structured refuge, but no consensus had been reached with industry, EPA, USDA
research and extension scientists, growers, and other stakeholders at that time. In 1995, it
was thought that based on market penetration estimates there would be enough non-Bt crop
acreage to serve as-a viable refuge in the first five years following full-scale
commercialization. A "structured"^ refuge requirement on the registrations thus would not be
necessary. However, the Agency mandated as a condition of registration that research data be
collected to develop an effective refuge with both temporal and spatial refuges to be
investigated. In addition, a draft refuge strategy must.be submitted to the Agency by August
9, 1998 and a final refuge strategy submitted by January 31, 1999. The registrant must
implement an EPA approved "structured" refuge planar an EPA approved alternative
resistance management plan no later than April 1, 2001. Registrants are required to discuss
the development and implementation of the refuge plan and alternative resistance management
practices with EPA throughout the development and the implementation. EPA also required as
a part of the registration for all Bt corn products specific monitoring and remedial action if any
resistance occurs. The registrations indicate that if remedial efforts are not effective in
mitigating resistance, the registrant will voluntarily cease sale of all corn hybrids that contain
the Bt corn plant-pesticides in the county experiencing loss,of product efficacy and the
bordering counties until an effective local management plan approved by EPA has been
implemented. EPA can also halt the future sale of Bt com plant-pesticides if resistance occurs.
, / • r , . i -- • ' - • .
In 1996 and 1997, less than 5 % of the total corn acreage was planted with Bt corn hybrids and
of this acreage, the density in any county or state was low. However, there may be pockets of
contiguous Bt corn acreage with the refuge acreage too far away to provide an adequate supply
'. • ••.•-." • 37 . ' . . . ': . • . '
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of susceptible insects to mate with any potentially Bt-resistant insects that emerge. This is the
contention offered by a review article of Alstad and Andow (1996). They conclude that
structured refuges make as much biological sense at the outset as they do three years following
the initial full-scale commercialization even if there is a lack of scientific information to
describe the optimum size of a refuge. '. ' . .
In 1997, as a result of multi-stakeholder discussions at the USDA Bt Resistance Management
Forum (April 1996) and the USDA NC-205-led Consortium meetings held in 1995-1997, a
more consensus viewpoint on refuge is beginning to emerge. This emerging consensus
focuses on what constitutes an effective refuge size, i.e., how many mating stage adults are
produced in the right place at the flight time to mate with adults emerging from Bt producing
varieties rather than whether a "structured" refuge should be required. The recommendations
of the USDA NC-205 led consortium have been recently published in a report (NCR 602)
entitled "Bt Corn & European Corn Borer - Long-Term Success Through Resistance
Management" (Ostlie et al., 1997). This report recommends having a "structured" refuge
which is 20 to 30% non-Bt corn to prevent Bt delta endotoxin exposure to 20 to 30% of the
larval population. In continuous corn acreage sprayed with insecticides, the refuge size would
be increased to perhaps 40% to compensate for larval mortality. Where there are many
alternate hosts that do not contain Bt proteins, a smaller refuge may be suitable. This
reduction in refuge size assumes that ECB from alternative hosts emerge at similar times as
ECB from corn. At present, the knowledge base is still limited as to what proportion of the
local ECB population flows through non-Bt hosts. More information is needed on the
synchronous emergence from refuges and Bt crops. Therefore, as a baseline, the USDA NC-
205 publication recommended that a 20 to 30% non-Bt corn refuge may be the simplest and
best way to insure delayed resistance. This publication indicated that the actual amount of
refuge required will vary among regions, farms, and corn production system. Therefore,
growers should contact local extension entomologists for specific refuge recommendations.
At present, Monsanto and Dekalb Genetics are the only two registrants that mandate a
particular structured refuge through their Technology Management (Grower) Agreements. A
grower purchasing from Monsanto or Dekalb has two options: a 5% unsprayed non-Bt refuge
or a 20% sprayed non-Bt refuge. These two refuge options are also stated in their respective
grower guides. In 1996 and 1997, Mycogen Plant Sciences's and Novartis Seeds's Grower
Guides/Technical Bulletins indicated their commitment to development of long-term resistance
management strategies through the support of research efforts, but neither mandate nor
recommend a particular refuge option. Mycogen's 1996 and 1997 technical bulletins for
Event-176 corn hybrids indicate a commitment to develop a long-term resistance management
strategy, provide general resistance management guidance, and recommend that not all corn
acres be planted in Bt corn. Novartis Seeds's 1996 and 1997 technical bulletins for Event-176
corn hybrids indicate a commitment to develop a long-term resistance management strategy,
provide general resistance management guidance, and indicate that part of a long-term
resistance management strategy may be "the maintenance of a refuge where susceptible
populations of ECB can escape exposure to the insect control protein in Maximizer hybrids."
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Novartis Seeds for its BT 11 hybrids "encourages" growers to: (1) plant Bt hybrids in large
block, (2) scout for non-target pests and use IPM strategies, (3) maintain a refuge of non-Bt
corn, and (4) monitor for unexpected levels of insect damage in Bt corn. On November 13,
1997, Novartis Seeds announced that in 1998, they would recommend that growers follow the
guidelines outlined in the North Central Regional Publication 602 (Ostlie et al., 1997). As
stated earlier, the NCR 602 report recommends having a "structured" refuge which is 20 to
30% non-Bt corn to prevent Bt delta endotoxin exposure to 20 to 30% of the larval population.
In continuous corn acreage sprayed with insecticides, the refuge size would be increased to
perhaps 40% to compensate for larval mortality. Where there are many alternate hosts that do
not contain Bt proteins, a smaller refuge may be suitable. Again, the report indicates that the
actual amount of refuge may vary between regions, farms, and corn production systems.
Mycogen has indicated in its 1996 year-end report that in some of the verbal presentations to
growers they are supporting the recommendations from the USDA NC-205-led consortium on
Bt corn resistance management. They recommend planting at least 10 to 40% of a grower's
total corn acres with a conventional corn hybrid and to use the high end of this range as a
minimum if insecticides are used to control ECB infestations and the low end if no insecticides
are used to control ECB infestations. Other ECB-susceptible crops may be used such as oats,
peppers, popcorn, potatoes,' snap beans, sorghum, and sweetcorn. The ECB-susceptible crop
should be planted close to the Bt corn to reduce the chances for an isolated population of Bt-
resistant borers to develop. The recommended arrangements for the Bt corn and ECB-
susceptible crops are to: (1) Plant in two blocks of Bt and non-Bt corn in the same field; (2)
Plant Bt and non-Bt corn in adjacent fields, or; (3) Rotate Bt corn fields to a ECB-susceptible
crop in the next growing season. Mycogen indicates to growers that mixing conventional and
Bt corn hybrids in the planter box or splitting the planter to sow alternating strips in the field is
noif recommended. ,
Several research efforts begun hi 1996 and continuing in 1997 are aimed at determining the ',
size, and deployment of an effective refuge .strategy. Research progress and a summary of the
results from the 1997 studies discussed below will be submitted by the registrants to EPA by
January 31, 1998. Efforts to date and 1997 research plans are summarized below,
1. Structured early/middle/late planting time strategy: In 1996, Dr. Marlon Rice and .
collaborators at Iowa State University (Novartis Seeds and Mycogen Corp. jointly-
sponsored research) conducted field research on a block-design planting strategy of Bt
and non-Bt corn, with a fixed amount of refuge (33%). In addition, this strategy
involved a temporal planting .scheme so that non-Bt corn was interspersed between an
early and late-season planting of Bt corn. It is thought that the non-Bt corn could
escape the most dense moth flights yet still support sufficient ECB populations to act as
mating partners to any resistant ECB that may be selected in the Bt corn plots. The
advantages of this strategy to growers, if ,it is effective, is that they will have to
purchase less higher-priced Bt corn seed and they will encounter less"yield loss hi the
non-Bt corn plot. Results from this type of study may help farmers .decide on where
.'• '•• ' ' ' 39- , " : '• ;
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and when to place Bt hybrids hi fields at greatest risk to ECB infestations. This type of
refuge strategy might be an effective option for resistance management. Alstad and
Andpw (1995) discussed the advantages of this approach and modeled potential
outcomes in a recent review.
In 1996, the first year of this two year study, the results indicate that adult ECB
females laid the same number of e,ggs on Bt corn as on conventional hybrids, regardless
of the planting date or ECB brood. Stalk tunneling information indicates that ECB
survived on conventional corn planted in close proximity to Bt hybrids. More
definitive information will be available after the completion of the second year of the
study in 1997.
Additional 1997 studies funded by Novartis Seeds on temporal/spatial refugia are being
conducted by Dr. Ken Ostlie (University of Minnesota), Dr. Murdick McLeod (South
Dakota State University), and Dr. Dennis Calvin (Pennsylvania State University; refer
to #4 below)]
2. Magnitude and mechanism of "Halo Effect" near Bt corn (sponsored by Novartis
Seeds, Mycogen Plants Sciences, Dekalb Genetics, Monsanto Co.). Research
conducted by Drs. D. Andow and D. Alstad, University of Minnesota, indicate that
ECB infestations are reduced in conventional corn fields within 150 ft. blocks of
commercial size plantings of Bt fields. This research supports the finding above that
conventional corn planted in close proximity to Bt corn will support susceptible ECB
populations, although perhaps at a somewhat reduced level than if conventional corn
were planted further away from Bt corn. It also suggests that ECB movement is limited
between Bt and non-Bt fields so that the likelihood of mixing of susceptible with
resistant adults is greater if conventional corn is planted hi close proximity to Bt corn
hybrids. Mycogen Plant Sciences, Dekalb Genetics, Monsanto Co. and Novartis Seeds
are continuing to support research on the halo effect in 1997 to: (1) determine the
benefits for conventional corn within the halo, (2) determine how halo dynamics are
affected by the size and layout of the refuge and Bt units, and (3) determine the
mechanism of the halo effect.
3. Survival of ECB on non-corn hosts (sponsored by Novartis Seeds, Mycogen Plant
Sciences, Dekalb Genetics, Monsanto). The purpose of this research (1996-1997) being
conducted by Dr. Rick Helhnich, USDA/ARS, Iowa State University is to examine the
survival of ECB on non-corn and weed hosts. Preliminary results indicate that weed
hosts produce less than 1 $ of ECB and thus weeds will not be a sufficient ECB refuge
to be useful hi resistance management. Sorghum, some small grams, and some other
non-corn crops may serve as effective refuges, but there isn't sufficient information to
quantify the refuge value of the non-corn crops and make specific recommendations at
this tune. The value of non-corn crops as refuges is particularly important in crop
rotations that include non-corn ECB host crops.
40
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4. Sequential plantings of field corn as temporal refugia for ECB (sponsored by -
Dekalb Genetics, Monsanto Co., Novartis Seeds). The purpose of this two year study
(1996-1997) conducted by Dr. Dennis Calvin, Pennsylvania State University, is to '
evaluate ,the value of temporal refuges as a resistance management strategy as well as
the value of non-transgenic corn patches in a landscape design. The objectives include:
(1) quantifying egg mass recruitment, larval survival, and population stage structure in
sequentially planted field corn, and (2) evaluating the relative attractiveness of various
plant development stages to ECB females.
5. Optimization model for ECB incorporating resistance management factors -,'•.,
including refuge (sponsored by Dekalb Genetics, Monsanto Co., U.S. EPA (Contract
CR822045-01-4). The purpose of this 1997 research to be conducted by Dr. Rick
Hellmich, Dr. Terrance Hurley, and Dr. Bruce Babcock (Iowa State University) is to
develop an economic model which examines the profitability of using a refuge. If
alternate crops are good ECB producers, insect resistance management using crop
combinations could go hand-in-hand with net profits. These researchers developed an
economic model of pest management with pest resistance to estimate the constant
proportion of refuge that maximizes farm income over a fixed planning horizon (Hurley
et al., 1997). Results indicate that there is a clear economic tradeoff between pest
control and population management benefits afforded by a Bt corn variety and the
resistance management benefits and savings in production costs afforded by refuge:
From this model, the researchers concluded that, under certain circumstances, a 20 to
40% refuge is economically sensible. This model could also be used to factor in
cultural practices such as discing that reduce ECB numbers.
6. Evaluation of resistance management strategies for Bt-corn (sponsored by Dekalb
Genetics, Monsanto Co., Novartis Seeds). The purpose of this 1997 research to be
conducted by Drs. Bushman, Higgins, and Sloderbeck, Kansas State University, is to
evaluate ECB production and practical utilization of several refuge planting options and
to evaluate the practical utilization of neighborhood suppression effects of ECB as
suggested by the "halo effect" associated with planting Bt corn hybrids near
conventional hybrids. ; ,
7. Impact of Yieldgard (Bt-11 andMONSW) Bt corn hybrids on management of.ECB
and other corn insects (Dekalb Genetics, Monsanto Co., Novartis Seeds) The purpose
of this 1997 research to be conducted by Mason and Keil, University of Delaware,
Whalon, Michigan St. University, and other researchers is three-fold: (-1) evaluate
effectiveness of other commonly grown host crops in the Mid-Atlantic region as refuges
for non-Bt selected ECB; (2) assess the association between behavioral and
physiological effects on a Delaware population of ECB; and (3) evaluate the
effectiveness of Bt corn hybrids in controlling other corn insect pests including CEW,
fall army worm, true army worm, and southern cornstalk borer. This research will
.••-'""' ' : .- 41 ' ; . . '.• •' •
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provide information related to appropriate refuge sizes for the Mid-Atlantic region as a
result of better quantifying the contribution of non-corn refuges.
8. Grower Surveys - Attitude toward refuges (sponsored by Novartis Seeds and
Mycogen Plant Sciences). Two grower surveys were conducted by entomologists, Dr.
John Witkowski, University of Nebraska and Dr. Marlon Rice, Iowa State University,
in 1996. Based on these first surveys, growers would prefer a rotation option over
planting Bt and conventional hybrids in a specified ratio in field to field patchwork or
in-field refuges. The results of these surveys are important in determining what grower
acceptance is likely to be hi adoption, implementation, and effectiveness of a refuge.
This type of survey also provides insight as to possible research priorities for refuge
research. , /
These type of grower surveys was to be expanded from two to six in 1997 to cover
additional corn growing areas especially hi the Midwestern corn belt. The following
researchers will be conducting grower surveys: Dr. Marlon Rice, Iowa State
University, Dr. John Witkowski, University of Nebraska, Dr. Randy Higgins, Kansas
State University, Dr. Kevin Steffey, University of Illinois, Dr. Dennis Calvin,
Pennsylvania State University, Dr. Ken Ostlie, University of, Minnesota.
9. Impact ofBt corn events on CEW and its implications to resistance and population
suppression in soybeans (Dekalb Genetics and Monsanto Co.) The purpose of this
1997 research to be conducted by Dr. Galen Dively and collaborators, University of
Maryland, and Dr. Ames Herbert, Virginia Polytechnic Institute and State University,
is to quantify the mortality and behavior of CEW feeding on Bt corn hybrids and
investigate the reproductive performance of surviving moths and their role in resistance
development and colonization of nearby soybean fields. This work will improve our
understanding of whether soybean fields are impacted by CEW moving from Bt corn
hybrids to soybeans.
Surveillance and Tracking (Monitoring)
EPA required as part of registration a monitoring plan including the development of ECB
baseline susceptibility responses, development of a discriminating concentration (i.e.,
bioassays that use one optimal or nearly optimal dose, i.e., a discriminating or diagnostic dose
concentration, to distinguish between susceptible and resistant individuals) to detect changes in
ECB sensitivity, routine surveillance, and remedial action if there was suspected resistance.
The purpose of monitoring is to learn whether a field control failure resulted from resistance or
other factors, e.g., factor that might inhibit expression of the Bt Cry delta endotoxin by the
plant. The extent and distribution of resistant populations can be mapped and alternative
control strategies implemented in areas in which resistance has been documented. It may also
be possible to detect resistance before it happens and control failures occur, if monitoring
techniques are sensitive enough to discriminate between resistant and susceptible individuals.
';: : 42
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Surveillance by growers is essential. ,The NC-205 publication NCR 602, "Bt-Corn &
European Corn Borer: Long-Term Success Through Resistance Management" (Ostlie et al,
,1997), discusses how Bt corn can be used to manage ECB and how to use this technology for
long-term profitability. This publication provides specific resistance management
recommendations including monitoring for first and second generation ECB. Current
monitoring aptivities are summarized below. ,
1. Baseline Susceptibility and Development of Diagnostic Bt Concentrations for
Monitoring (Dr. Blair Siegfried, University of Nebraska, sponsored by Mycogen Plant
Sciences,, Novartis Seeds, Northrup-King/Novartis Seeds, Monsanto Co., Dekalb
Genetics Corporation). This work is designed to develop techniques for monitoring Bt
resistance in field populations of ECB. Baseline responses to Cry l(A)b have been
determined for a number of ECB populations collected from across the corn belt over
the last 3 years. Results of baseline susceptibility studies conducted on Nebraska ECB
populations (Siegfried et al., 1997) indicated that distinct geographic populations
showed a greater than 5-fold, based on LC^ variability in their responses to the
CryI(A)b toxin. The toxin was obtained from a fermentation run of Bacillus
thuringiensis, Berliner Subsp. kurstaki (HD1-9 strain) that produces only the CryI(A)b
protein. Baseline susceptibility of ECB using a commercial formulation of Bacillus
thuringiensis Berliner^ Subsp. kurstaki (DipelES) was also conducted by Kansas
researchers using Kansan and Iqwan field populations (Huang et al., 1997). These
researchers found regional differences in susceptibility to Dipel. Siefried et al (1997)
and Huang et al. (1997) indicate that the level of susceptibility reported in their studies
may not accurately reflect me susceptibility of field ECB populations because the
number of insects used to start the colonies was small and may not be representative of
field populations.
Studies in 1996 attempted to develop a diagnostic concentration based on baseline
susceptibility studies conducted in 1995. Experiments were also designed to validate
the LC99 using field populations of ECB. Baseline responses to CryI(A)c are also
being conducted. ,
Larvae were collected from a total of eight sites in seven states (i.e.,Nebraska, Indiana,
'Illinois, Pennsylvania,, North Carolina, Iowa, Minnesota) where Bt corn hybrids had
been planted. Dose-response bioassays were performed using neonate larvae on
artificial diet containing purified CryI(A)b protein.' Results indicated that the difference
in the LC50 values between the ECB populations were similar to those observed during
1995 and represent natural variability. An estimated diagnostic CryI(A)b •
concentration, LC99, was estimated from these baseline data. This work was being
continued in 1997. Mycogen also conducted standard b'ioassays of field collected ECB
in-house in 1997.
The estimated diagnostic LC^ concentration was used to detect changes in the
r "*
' •"• : •' • - :' . " : : "•• ' . 43 '_ '"'• ' ' •' . -; • '••''• •'.
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sensitivity of ECB populations that have been exposed to Bt in 1996. When the eight
ECB populations were exposed to the estimated LQ,9 concentration, 99-100% mortality
was observed. There were differences among the populations tested, but these results
support the conclusion that there was no change in susceptibility of ECB to the
CryI(A)b toxin. The LC99 mortality value is a more useful diagnostic concentration for
estimatingJECB susceptibility levels rather than the ECg9 growth inhibition value.
However, there are significantly higher levels of Bt toxins expressed in Bt corn
meaning that the estimated LCgg is probably several fold lower than what the ECB will
be challenged with (i.e., overall concentrations hi the field will be higher) in Bt corn
fields. Thus, the estimated LC^ will distinguish between resistant and susceptible
individuals and can be used in a diagnostic bioassay for Bt resistance detection.
( • • • •
To further validate the diagnostic concentration, it will be necessary to develop a
CryI(A)b resistant ECB strain. The estimated LCc,9 has been tested against the
CryI(A)c resistant ECB strains developed at the University of Minnesota. However,
these strains do not appear to be resistant to CryI(A)b. Additional studies with
CryI(A)b resistant ECB will be required to confirm the efficacy of the estimated
CryI(A)b LCgg as a discriminating dose. These studies will be conducted when a
resistant CryI(A)b strain becomes available.
2. Establishing CEW baseline susceptibility to CryI(A)b. Dekalb Genetics/Monsanto
Co. are sponsoring the research by Ricerca to establish CEW baseline susceptibility to
CryI(A)b and to determine a diagnostic dose. This work is important in assessing
changes in CEW susceptibility as a result of exposure to CryI(A)b. The project will be
done in cooperation with the current monitoring program for CryI(A)c coordinated by
Dr. Hardee, USDA/ARS, Stoneville, MS (see'description under Section IV. Bt cotton
resistance management).
3. Surveillance and Remedial Action. As part of the requirements of each Bt corn
plant-pesticide registration, each registrant is required to carry out surveillance and
implement a remedial action plan if there are incidents of confirmed resistance. Each
company has instructed its customers to have regular surveillance programs and report
any unexpected levels of ECB and CEW damage via a toll-free customer service
number. Each company will investigate and identify the cause for this damage by local
field sampling of the plant tissue and suspect insect populations followed by appropriate
in vitro and inplanta assays. Confirmed incidences of resistance are required to be
reported to the Agency within 30 days and appropriate remedial action is required to
mitigate ECB and/or CEW resistance. Within 90 days of a confirmed instance of ECB
and/or CEW resistance, the registrant will: (1) notify the Agency of the immediate
mitigation measures that were implemented, and (2) submit to the Agency a proposed
long-term resistance management action plan for the affected area, (3) work closely
with the Agency in assuring that an appropriate long-term reistance management plan
for the affected area is implemented, and (4) implement an action plan that is approved
• ' -
44 -
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by EPA and that consists of, some or all of the following elements: (a) informing
customers and extension agents in the affected areas of ECB and/or CEW resistance,
(b) increasing monitoring in the affected area, and ensuring that local ECB or CEW
populations are sampled on an annual basis, (c) recommending arid implementing
alternative means to reduce or control ECB or CEW populations in the affected areas,
and (d) implementing a structured .refuge in the affected areas based on the latest !
. research results. The implementation of a refuge strategy will be coordinated by the
Agency with other registrants. If the above elements are not effective in mitigating <
January 12, 1998 resistance, the registrant will voluntarily cease sale of all corn hybrids
that contain the particular Bt corn plant-pesticide in the county experiencing loss of
product efficacy and the bordering counties until an effective local management plan
approved by EPA has been implemented.- During the voluntary suspension period, the
registrant may sell and distribute in these counties only by obtaining EPA approval to
study resistance management in those counties. The implementation of such a strategy
will be coordinated by the Agency with other registrants. Seed lot purity wilf
influence the precision required to detect ECB resistance, mdustry. cooperation with
extension entomologists is considered important in coinmunicating specific information
on definitions of "unexpected damage." this topic has been discussed in multi-
stakeholder meetings such as the USDA/ARS NC-205-led meetings on Bt corn
resistance management and the USDA Bt resistance management forum.
Mycogen investigated mree customer calls in 1996 related to incidents of unexpected
levels of ECB and determined that none of these was related to CryI(A)b resistant ECB.
Two of the calls; were from growers who forgot where the Event 176 hybrid corn had
been planted and one came from a crop consultant who misidentified common stalk
- borer feeding for ECB.
Grower Education ,
All registrants are required, as a part of registration, to implement a grower education program
and develop Grower Guides which will include current information regarding insect resistance
management and integrated pest management. Amongst all stakeholders, there is universal
agreement that grower acceptance and adoption of insect resistance management strategies are -
critical to the success of Bt corn. In a 1996 grower survey underwritten by Novartis Seeds
and Mycogen Seeds, the majority of growers who responded indicated that they rely on an
industry representative for their product information. Therefore, industry has a primary
responsibility to provide accurate and effective information regarding insect resistance
management to the grower. Other stakeholders, such as academic and extension
entomologists, county agents, crop consultants, USDA and EPA share this responsibility.
Both Novartis Seeds and Mycogen Plant Sciences reported in their 1996 annual reports that an
extensive grcfwer education program has been implemented. Grower Guides are mailed to
each grower and, information tags are affixed to each bag of seed describing appropriate insect
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resistance management information. Both companies participate in the NC-205-led consortium
discussion on Bt corn resistance management. Mycogen Plant Sciences and Novartis Seeds
noted that they incorporated the specific recommendations for growers on resistance :
management developed by the NC-205-led.consortium and put them in their written materials
and slide presentations. These recommendations will now be part of Novartis Seeds 1998
Grower Guides for BT-11 and Event 176-derived hybrids. Both companies report they give
numerous presentations to growers, agriculture media, university extension and crop
consultants on product performance and insect resistance management and participate in multi-
stakeholder forums to discuss resistance management. Novartis regulatory affairs group hosted
seminars on resistance management issues with Novartis Seeds sales, marketing, and
agronomic staff.
Development of products with alternative modes of action
Industry is developing other corn lines that involve the expression of novel Bt genes acting by
mechanisms different from currently registered Bt genes. These novel genes could be
combined with currently registered Bt genes, insecticidal genes with mechanisms of action
different from Bt, and inherent host plant resistance traits as a means for combating the
development of ECB resistance to either CryI(A)b or Cry(A)c delta endotoxin expressed hi
corn. Pyramiding or stacking genes with different modes of action is advocated by
entomologists as a powerful tool to mitigating the development of resistance. Roush (1994)
has modeled the effects of pyramiding and results indicate that resistance may be delayed by
greater than 1000-fold.
Bt corn impact on mycotoxins and plant diseases
Mycotoxins are secondary metabolites produced by some fungi that contaminate food or animal
feed. Monsanto is funding research to examine mycotoxin reduction hi Yieldgard ears and
determine to what extent Yieldgard hybrids affect mycotoxin levels hi grain as a result of
reduced insect injury in ears. This work is being performed by two research groups: 1)
Munkvold, Hellmich, Showers, and Rice from Iowa State University and 2) Herbert (Virginia
Polytechnic Institute) and Dively (University of Maryland). Reduced mycotoxin levels would
be an additional benefit of the use of Bt corn to humans and animals. Reduction in mycotoxin
levels is also an important IPM consideration for the use of Bt corn hybrids.
Novartis Seeds is also funding internal research to examine to what extent Event 176 and BT11
hybrids affect mycotoxin levels in gram.
Public Comments
Of the 100 comments received as a result of the two public hearings held on Bt plant-pesticide
resistance management this year, about 15 specifically focused on Bt corn resistance
management issues. A discussion of the public hearings is found above in Section I of this
46
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paper.
Comments came primarily from two groups: academic and extension entomologists and
industry/seed companies utilizing the Bt corn hybrid technology. The comments focused on
whether resistance management plans should.be mandatory or voluntary and on the scientific
needs for resistance management plans. Industry/seed companies believe that resistance
management plans should be voluntary and are the responsibility of'product stewardship efforts
by industry. Academic and extension entomologists, with one exception, believe the resistance
management plans should be mandatory for Bt plant-pesticides produced in corn. All parties
agree that additional data need to be gathered to develop a long-term resistance management
strategy. Research in the following areas was generally described as necessary: pest biology,-
genetics, behavior, and ecology-population dynamics, gene flow, refiige strategies, biology of
resistance and cross-resistance, high dose effectiveness on primary and secondary pests, and
importance of Bt corn and its impact on Bt resistance management programs in areas growing
born and cotton. Monitoring (surveillance and tracking) and grower education efforts are
essential. , •
"' ' , • s • .'••'.
However, there were three comments from the University of Missouri, Texas Corn Growers
Association, and Northrup-King/Novartis Seeds who felt that the blanket sales restriction in the
South on Bt corn hybrids with Bt expression in silks and kernels (i.e., MON810, BT11,
DBT418) in parts of Missouri and Texas should be removed because it is economically 'more
desirable to plant Bt corn than Bt cotton. In Texas, the desire is to plant Bt corn hybrids with
silk and kernel expression in counties north and west of Lubbock, i.e. in the Texas Panhandle.
In Missouri, the desire is to plant Bt corn hybrids with Bt expression in silks and kernels in the''
Bootheel region of Missouri. The comments from both states report that it is unlikely that Bt
cotton would be grown in these regions. However, according to the registration for Bt cotton
(issued prior to the registrations for BT 11 and MON 810 corn registrations), Bt cotton can be
grown in the Texas Panhandle and the Missouri Bootheel. In addition, non-Bt cotton could
still be grown in some of these counties. Based on the discussion of Cry effects on CEW
movement above, there would be some selection on CEW/CBW occurring if Bt corn hybrids
with silk and kernel expression were planted in these areas. .Any Bt cotton and Bt microbial
sprays used on cotton or vegetable crops in close proximity to Bt corn hybrids would add
further selection pressure toward the development of Bt resistance,, iff no cotton/ Bt cotton or
other vegetable crops using Bt microbial sprays were grown in these selected counties in the
; Missouri Bootheel or Texas Panhandle, then the selection pressure would be very much
reduced for the development of resistance to later generations of CEW/CBW.
Summary ,
. ' ' , ' '• '*••',- - ' - ' * * /
Prior to the registration of the first Cry delta endotoxin produced, in corn hi August 1995, there
was no consensus on the size and structure of an effective refuge and the incentives needed for
grower adoption. Figures ranged from 50% non-Bt structured refuge needed to 0 to 5% refuge
needed. EPA determined that more information was needed to support specific refuge
-..,''••':''' ..-.-.. 47 •':'•• : •'• , : -,'•'.."'• •'
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options and required as part of registration research to develop the size, structure, and
deployment of a refuge and implementation of a refuge. However, EPA believed that during
the first five years following commercialization (approximate time-limit of the conditional
registrations for Bt corn), there would not be enough Bt corn acreage to provide substantial Bt
selection pressure for the development of ECB resistance. Consequently, EPA did not
mandate any specific refuge requirements. However, EPA identified several research areas in
which data were needed to develop a long-term resistance management strategy: (1)
information on ECB pest biology and behavior, (2) feasibility of refuge options, (3)
development of discriminating dose concentration assay, (4) effect of corn producing the
CryI(A)b or CryI(A)c delta endotoxin on pests other than ECB including CEW, (5) the biology
of ECB resistance and cross-resistance. Research progress in all of these areas will be
reported to the Agency hi each registrant's annual report due January 31, 1998.
EPA required annual reporting of sales information for Bt corn as a requirement of
registration. Approximately 400,000 acres (about 0.5% of the total corn acreage) of Bt corn
were planted in 30 states in the U.S. hi 1996 and approximately 3.5 to 4 million acres (about
5% of the total corn acreage) is expected to have been planted in 1997. Based on a three-year
average, there are approximately 70 to 80 million acres of corn planted annually in the U.S.
Based on efficacy research submitted to the Agency, Bt corn hybrids constitutively expressing
the CryI(A)b and CryI(A)c delta endotoxins throughout the plant, including silks and kernels,
provide an effective high dose for both first and second generation ECB. The registered plant-
pesticides in this category are from MON810, BT11, and DBT418 transformation events.
Event 176 Bt corn hybrids, express the CryI(A)b delta endotoxin hi green tissue and pollen,
and provide an effective high dose in whorl-stage corn for first generation ECB control, but
provide only about 70 to 75% control of second generation ECB in silk-stage corn. Research
and commercial plantings to date indicate that both types of Bt corn hybrids control ECB better
than conventional insecticides. Additional ECB efficacy research is supported by all Bt corn
registrants.
Research results indicate that there is significant short-range ECB adult movement indicating
that it might be more effective to construct the non-Bt refuge in close proximity to Bt acreage.
Alternative crops may serve as effective refuges, but more information is needed. More
information on weedy hosts is needed to determine whether they can serve as effective refuges.
Based on Alstad and Andow's simulation model (Alstad and Andow, 1996), an unsprayed 5%
non-Bt refuge may not be adequate for ECB resistance management. Refuge size may vary
depending on whether insecticides will be used and whether there are alternative hosts in close
proximity to the Bt corn acreage planted. Early research results indicate that a structured
early/middle/late planting time strategy may also be a practical and effective refuge strategy.
More definitive information will be available after the 1997 growing season to assist in
developing a long-term resistance management strategy for Bt corn.
Research on the biology of resistance has led to the development of CryI(A)b and CryI(A)c-
48
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tolerant colonies of ECB, Insects resistant to one of these Cry proteins show no cross-
resistance to the other Cry protein. However, laboratory-selective conditions are not often
representative of field-selective conditions. Laboratory studies selecting for ECB-tolerant
strains provide information on the mechanism of resistance in ECB, but do not predict whether
resistance will develop under field conditions.
EPA required routine monitoring and an annual monitoring report as part of registration for Bt
corn. In 1996 and 1997, the EPA received no reports of putative ECB or CEW resistance to
CryI(A)b or CryI(A)c. Baseline susceptibility studies have indicated that there is a range of
variability in geographically distinct populations, but these studies have not indicated any
significant changes in ECB susceptibility over time. A discriminating concentration, LQ9, has
been developed to detect changes in ECB sensitivity and its efficacy has been tested.
Preliminary results indicate that it can be used for routine surveillance and as a trigger for
remedial action. Researchis continuing on baseline susceptibility and testing the
discriminating dose concentration. ;
Although the primary focus is on ECB, there is also a concern about the potential development
of Bt resistance in CEW. The development of CEW resistance .to Bt produced in crops could
negatively affect the utility of Bt cotton and Bt microbial sprays on vegetables and other crops.
Research efforts to date indicate that silk and kernel expression in Bt corn hybrids will likely
increase the selection for CEW resistance especially in cotton-growing areas. Silk and kernel
expression is found in constitutively expressing Bt corn hybrids, i.e., events MON810,
DBT418, and BT11. In general, laboratory and field studies indicate that CEW development
was delayed and larval weight was reduced by the presence of Bt hi the plants, although the
magnitude of the difference varied among Bt hybrids and across time for the three events
; tested, MON810, BT11, and Event 176. The greatest developmental delays and weight losses
were observed in BT11 and MON810 corn hybrids. There appears to be no selection for Bt
resistance in CEW in Event 176 Bt corn hybrids,and these types of hybrids may actually
provide a refuge from selection for second generation CEW in the South. There is a
sufficiently high dose expressed in whorl-stage Event 176 corn to control first generation
CEW. Additional research efforts are underway to study the impact of Bt corn on CEW. This
research will provide information useful in refining long-term resistance management
strategies. . ,
EPA imposed mitigation measures, in the form of sales and distribution restrictions, as part of
the requirements of registration on Bt corn hybrids that had Bt expression in silks and kernels
to reduce CEW selection pressure in the South. There is a concern that CEW resistance may
develop in insect populations that feed on bom Bt corn and Bt cotton where Bt corn and Bt
cotton/cotton acreage is in close proximity. Even though CEW only overwinter in the South,
resistance would also be found in the northern states carried by migrating or wind blown adults
that had emerged each spring in the South and from Mexico, Sufficient data are not yet
available to indicate whether the selection pressure on CEW/CBW will be increased by large
amounts of Bt corn in the South.
' . . - - • - < -
' •' ' 49''.' ' ' •'•.'.. • • - '
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Grower surveys have indicated the critical need for continuing grower education before
growers accept and implement of a refuge as a permanent part of a resistance management
plan. Based on these surveys, growers would prefer a rotation option in which Bt crops were
alternatively planted with non-Bt crops over planting Bt and conventional hybrids in a specified
ratio in field to field patchworks or in-field refuges. Additional grower surveys were
conducted in 1997 and these results should be reported to the Agency in the annual reports due
January 31, 1998.
Even though use of a structured refuge is accepted by all stakeholders as necessary for a long-'
term resistance management strategy, experts have not reached an agreement as to the size,
structure, and deployment of Bt and non-Bt plots and the nature and objective of performance-
monitoring activities. Agency scientists have participated hi the cooperative efforts of the NC-
205-led consortium. This consortium has identified critical needs for a long-term insect
resistance management strategy, helped set research priorities where uncertainties existed,
encouraged the adoption of national monitoring strategies and developed and disseminated
educational materials to growers and the public. The NC-205 recommendations on ECB
resistance management are described in NCR-602 publication, entitled "Bt-corn & European
Corn Borer: Long-Term Success Through Resistance Management" (Ostlie et al., 1997). The
NC-205 recommendation is to have a structured refuge which is 20-30% non-Bt corn to
prevent Bt delta endotoxin exposure to 20-30% of the larval populations. They also
recommend that in continuous com acreage sprayed with insecticides, the refuge size would be
increased to perhaps 40% to compensate for larval mortality. In addition, a smaller refuge size
may also be suitable if there are many alternate hosts providing adequate numbers of
susceptible ECB. Temporal schemes involving early- and late-plantings of Bt corn
interspersed with non-Bt corn may also be a viable option, but additional data are needed.
Further validation of a structured block design and temporal/spatial structural design options
should be encouraged.
Significant progress has been made to generate the necessary data to develop long-term
resistance management strategies. Coordination of research priorities and combining of
resources would more effectively lead to the development of a long-term resistance
management strategy for Bt corn. EPA has fostered and participated in efforts to provide all
stakeholders with opportunities for public comment on the development and implementation of
long-term resistance management strategies for Bt plant-pesticides. The Agency has held
Science Advisory Panel meetings (e.g., March 1, 1995 SAP Subpanel on Plant-pesticides
regarding the Bt potato risk assessment and resistance management) and two public hearings
(i.e., March and May 1997) on the subject of Bt plant-pesticides resistance management. The
Agency plans to hold a SAP meeting in February 1998 to examine the information the Agency
has gathered hi efforts to develop long-term resistance management strategies for Bt plant-
pesticides. In addition, other SAPs are planned to continue the Agency's dialogue with experts
and stakeholders interested in developing long-term resistance management strategies.
50
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IV. Bt cotton resistance management
This section will.discuss resistance management activities and results for 1996 and discuss any
lessons learned regarding resistance management since CryI(A)c delta endotoxin expressed in
cotton was first registered in 1995. '
The following materials were used in preparing this section of the paper: material provided
during the public hearings, scientific publications, personal communications, 1997 Proceedings
of the Beltwide Cotton Conference, EPA's Fact Sheet (U.S. EPA, 1995c), Agency review
regarding pesticide resistance management, Monsanto's 1996 status reports (November 5
1996 and February 28, 1997), literature review of the biology of the major lepidopteran p'ests
of cotton (MRID 4404225-01), and literature review of cross-resistance potential (D227579
submission dated May 22, 1996) and USDA 1996 cotton statistics. '
Background :
.The Agency granted the conditional registration of the CryI(A)c delta endotoxin from Bacillus
thuringiensis subspecies kurstaki and the genetic material necessary for its production in cotton
to control TBW, CBW, and PfiW in October 1995. The resistance management strategy for
the CryI(A)c delta endotoxin expressed in cotton has been reviewed by the Agency prior to -
registration (U.S. EPA, 1995c). The conditional registration will automatically expire at
midnight January 1, 2001. EPA will reevaluate the effectiveness of the registrant's resistance
. management plan before January 1, 2001 ,to determine whether to convert the registration to a
registration without an expiration date. An Experimental Use Permit has been granted to
Monsanto Co, and their registration submission is pending for use of a CryHA delta endotoxin
in cotton. l .
There are two primary resistance concerns for the registered Bt cotton: 1) development of
resistance in the primary target pests, TBW, CBW, and PBW and 2) cross-resistance to the
CryI(A)c and other Cry delta endotoxins expressed in other Bt plant-pesticides or Bt microbial
products. The Agency concluded that to manage resistance in the long-term and to develop a
long-term resistance management strategy, specific data needed to be collected on all three
target pests and required such data be generated. A multi-factor resistance management plan
was required to be implemented as a condition of the registration for CryI(A)c in cotton. The
Bt cotton registration required a structured refugia and included grower education and training.
The initial resistance management plan addressed all of the general elements of a resistance
management plan. These elements included pest biology, Bt dose deployment, refugia,
monitoring, effects on other susceptible nontarget lepidopteran pests, cross-resistance,'
integration into an IPM-program, grower education and communication, and development of
alternative pesticides with different modes of action: Bt cotton will not control all lepidopteran
and other insect pests. For example, the Bt delta endotoxins do not control boll weevil, fall
armyworm, beet armyworm, silverleaf whitefly, aphids, stink bugs, or plant (Lygus) bugs, all
, 51 ' .'" • .
' •, f
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important pests of cotton. The Cry protein dose expression in the plant is considered to be a
high dose for TBW, but not for CBW and perhaps not for PBW. However, the dosage in the
plant is high enough to kill > 80% of the CBW larvae. Therefore, the use of Bt cotton hi
conjunction with other IPM practices should effectively control high CBW populations and
other important insect pests of cotton. These IPM practices were addressed by Monsanto in its
Bollgard® cotton Grower Guide.
Because of the degree of uncertainty associated with season-long exposure of the target insect
complexes to the CryI(A)c delta endotoxin, the Agency believed that before a conclusion can
be made about the potential long-term success of a resistance management strategy, additional
research data, a specific monitoring plan including the development of discriminating doses for
TBW, PBW, and CBW, field validation of the resistance management strategy, and annual
reporting of use information and monitoring results are required. EPA required as part of
registration for Monsanto: (1) to submit literature and research data on target pest biology and
ecology including the data on the effectiveness of non-cotton hosts as refugia (literature review
due June 1, 1996 and research data due January 31, 1998), (2) to develop a protocol for
determining the likelihood of cross-resistance to other Bt endotoxins (due April 1, 1996), (3) to
evaluate the potential for cross resistance (due January 31, 1998), (4) to submit a plan for a
workable monitoring program (surveillance, tracking and remediation elements) (due March 1,
1996), (5) to submit an annual report of monitoring data (annually November 1 each year for
preliminary results and January 31 each year for the final report for the duration of the
registration), (6) to submit annual use reports (annually November 1 each year for the duration
of the registration, (7) to continue development and distribution of grower education materials,
(8) to continue to investigate the influence of Bt cotton on secondary lepidopteran pests, and
(9) to continue to provide CryI(A)c expression information relevant to susceptibility and
control of the target lepidopteran pests (due January 1, 1998). Two structured refuge options
were mandated as mitigation measures. The registrant is required to submit an annual
monitoring report on results and conclusions from resistance management research. Monsanto
has submitted its progress report for 1996. The due dates for these data submissions are .
indicated in the FACT sheet (U.S. EPA, 1995c).
Two specific refuge options were mandated as requirements of registration to mitigate the
development of resistance. "Option A: For every 100 acres of cotton with the Bollgard® gene
planted, plant 25 acres of cotton without the Bollgard® that CAN be treated with insecticides .
(other than foliar B.t.k2 products) that control the tobacco budworm, cotton bollworm and pink
bollworm. Option B: For every 100 acres of cotton with the Bollgard® gene planted, 4 acres
of cotton without the Bollgard® gene that CANNOT be treated with acephate, amitraz,
endosulfan, methomyl, profenofos, sulprofos, synthetic pyrethroids, and/or B.t.k. insecticides
labelled for the control of tobacco budworm, cotton bollworm and pink bollworm. The refuge
acreage must be managed similarly to the Bollgard® cotton." In addition, if cotton with the
2B.t.k. = Bacillus thuringiensis subsp. kurstaki
52
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.,•.•".. • ' ! '..-./• . ' . •
Bollgard® gene exceeds 75% of the total amount of the cotton planted in any single country or
Parish in aiiy year, growers in that county or Parish choosing to use the 4% untreated refuge
option the following year will be required to plant the 4% refuge within one mile of the
respective Bollgard® cotton field. Similarly, if EPA grants a registration for cotton containing
the B.t.k insect control protein to another company, EPA will determine whether the combined,
acreage of cotton containing the B.t.k. insect control protein exceeds 75% of the total amount '
of the cotton planted ma single county or Parish and inform the registrants that the 4% refuge
must.be planted within one mile of the respective Bollgard® cotton or other B t k cotton
fields. •"..'•' ' ' ' ' -
EPA sought comment on the performance of Bt cotton in the field at two public hearings held
in March and May 1997. The Agency sought information regarding reported control failures ,
for Bt cotton in 1996, possible evaluation tools concerning these failures, and the implications
on future resistance management efforts. Public comments were summarized earlier in this
paper in Section I. To summarize, comments received from private citizens, organic farmers
and grower organizations, environmental groups, and public-interest groups indicated that the
Bt cotton resistance management plan should be reevaluated. Most urged EPA to suspend the
registrations of aJLBt plant-pesticides and to hold a SAP meeting to reevaluate the resistance
management plans for Bt cotton and Bt cofn. Comments received from Monsanto, National
Cotton Council, most academic/USDA scientists, and cotton farmers indicated that Bt cotton
performance in 1996 was excellent They agreed that there was no breakdown in the Bt gene
technology. In general, these commenters indicated that reports of Bt cotton failure were due
to an unusually high infestation of CBW in parts of the Cotton belt (south Texas, Mid-South
and Southeast growing regions). Some of these infestations on Bt cotton required supplemental
insecticide treatment.
As noted earlier, it is recognized that long-term resistance management will involve other IPM
practices in addition to the use of Bt cotton. The three target p^ests, TBW, CBW, and PBW
show a differential susceptibility to the CryI(A)c delta endotoxin expressed in Bt cotton.
Tobacco budworm is the most sensitive of the three species to the CryI(A)c delta endotoxiri. A
high dose .strategy exists for the TBW, but is less certain for PBW, and does not exist for
CBW. The effectiveness of the high dose and refuge strategy along with other element's of the
Bt cotton resistance management strategy such as adaptation to an IPM plan, scouting, and ,
grower education, will be discussed below. Research is underway to address the area's of
uncertainty in the development of a long-term resistance management strategy for Bt cotton.
This paper will discuss the progress made to date to develop a long-term resistance strategy.
Bt cotton acreage in 1996 and 1997
EPA required Monsanto Co. to submit annual sales data for each state indicating the number of
acres of Bt cotton planted per county as a condition of registration. Two Bt cotton varieties '
were available in 1996: NuCotn 33 B and NuCotn 35 B. About 6000 growers planted
approximately 1.8 million acres of Bt cotton. Based on a three year-average, there are about
•••••-• . * ' • ' •"•'•• 53 '•.'•''••: . _• •"• • - ,
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13 to 14 million cotton acres planted annually in 16 states in the U.S. Bt cotton acreage
represented about 13 % of the total 1996 cotton acreage planted. Mosr of the Bt cotton was
grown in the Southeast (779,000 A) and in the Mid-South (728,000 A). Georgia (26%),
Alabama (77%), and Mississippi (42%) planted the greatest number of Bt cotton acres
(> 300,OOOA/state). Fourteen Alabaman counties were planted in > 75 % Bt cotton acreage.
Eight other counties in Florida, Georgia (2), Louisiana, Mississippi (2), North Carolina, and
Texas were planted in greater than 75 % Bt cotton acreage. These counties exceed the 75 % Bt
cotton acreage per county trigger in the registration agreement. As a consequence, in 1997, if
the 4% unsprayed non-Bt refuge was employed, it should be placed within one mile of the Bt
cotton in those counties exceeding the 75 % Bt cotton acreage limit.
Reports indicate that production of 1.8 million acres of Bt cotton in 1996 is thought to be
responsible, in part, for reducing total insecticide applications on cotton for lepidopteran pests
by 250 thousand gallons of formulated conventional insecticides. Seventy-seven percent of the
total U.S. cotton acreage was infested with cotton bollworm/tobacco budworm in 1996,
requiring 1.3 applications of insecticide per acre, down considerably from previous years in
which 5 to 12 spray applications were used on conventional cotton to control these pests.
Nine Bt cotton varieties were available in 1997. Preliminary 1997 reports from Monsanto
indicate that Bt cotton acreage has risen to about 2.2 to 2.4 million acres.
Analysis of Resistance Management Strategy
Dose adequacy
The three target pests, TBW, CBW, and PBW, have a differential susceptibility to the
CryI(A)c delta endotoxin. EPA recognized this fact prior to the registration of the CryI(A)c
delta endotoxin produced in cotton. The tobacco budworm is the most sensitive of the three
species to the CryI(A)c delta endotoxin. The levels of the CryI(A)c delta endotoxin in
Bollgard® cotton are high enough in all plant parts to provide a high dose for control of TBW
through the growing season. Levels of the CryI(A)c delta endotoxin do not appear high
enough to support a high dose to control PBW and CBW, but PBW is more sensitive to the
CryI(A)c toxin than CBW. The Agency has required further research on the effectiveness of
the CryI(A)c on all three target pests. A discussion of the 1996 performance of Bt cotton
follows below for all three target pests.
1) Tobacco budworm control
All reports indicate that TBW control was excellent in 1996, although, generally, there were
unusually low populations of this pest. TBW is a major economic pest of cotton. It has
developed resistance to most insecticides. Bt cotton can play a critical role in the control of
TBW and should lead to a reduction in the use of conventional insecticides used previously to
control this pest. Bt cotton expresses the CryI(A)c at sufficiently high enough levels to be
54
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considered a high dose for control of TBW.
s. - - - ' ' f
2) Cotton bollworm control
While the CryI(A)c expression levels are high enough to kill about 80 % of all susceptible
CBW, during years of high CBW infestations, the dose would not be adequate to control
CBW populations below economic threshold levels during peak periods of ovipositibn. The
Agency utilized previous reported information by Mahaffey et al. {1994) and Bradley (1995)
•that under high CBW infestation pressure, Bt cotton will not provide economic control of
CBW and supplemental insecticidal sprays may be necessary. These reports show that CBW
larval feeding resulted in boll damage levels as high as 32%. and caused significant yield-
reductions when field experiments were conducted under very high CBW larval infestations.
In essence, Bt cotton is not a stand-alone technology when very high cotton bollworm
populations were encountered. CBW is the least susceptible to the CryI(A)c delta endptoxin of
the three target pests. All of the laboratory studies have shown that CryI(A)c, at the levels
expressed in Bt cotton plants is'only moderately toxic to CBW. Current estimates indicate that
Bollgard®3 cotton varieties, NuCotn33B and NuCotn35B, kill between 80 and 95% of the
cotton bollwprm larvae that feed on them. "Technically, this is called a "moderate dose",
although this level provides good practical control of the pest at lower population levels. It
was not expected that Bt cotton would provide a high dose to control CBW. Warnings in
Monsanto's grower guides and from extension entomologists to this effect were provided to
growers. For example, Dr. Blake Layton, extension entomologist from Mississippi pointed
out that producers and consultants in Mississippi were cautioned via the 1996 and 1997 Cotton
Insect Control Guides, the weekly Cotton Insect Situation Newsletter, the Cotton Insect
Telephone Hotline, and extension publication 2108, ".Insect Scouting and Management in Bt-
transgenic Cotton" that'Bt cotton may require treatment in cases where high populations of
CBW occurred. As a result of EPA's analysis of the potential for resistance to develop in
CBW, as well as in TBW and PBW, EPA required monitoring of the target pest populations
for resistance. EPA's analysis indicated that, in some cases, additional control measures
would be necessary to reduce CBW populations below .economic threshold levels. The level of
CBW control in Bt cotton, the availability of other best management practices, and the pest
biology were all considered as part of the development of the initial resistance management
strategy both by Monsanto in its initial resistance management plan and by EPA in it
evaluation of resistance risk.
In 1996, cotton bollworm populations ,were the highest seen in ten years in parts of the Cotton
belt (i.e., Brazos Valley,Texas, Mid-South and Southeast growing regions). The lack of
economic control of CBW by Bt cotton was observed by independent crop consultants,
Monsanto, and others who advised affected growers ,'to use alternative chemical controls, if
necessary, as part of an overall IPM cotton growing program. In some cases, growers failed-
Bollgard® cotton is Monsanto's trademark for Bt cotton varieties containing the CryI(A)c gene.
. - "' •'•' -'• ' ''55 ' - •'•'• ' • , •
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to understand the Bt cotton technology and rushed to apply insecticides when economic
thresholds had not been exceeded. Nonetheless, in 1996, applications for CBW control were
about 3.3 applications for non-Bt cotton and about 0.3 for Bt cotton in Mississippi. That is, it
was expected that under unusually high CBW infestation pressure that supplemental insecticide
treatments might be necessary and that scouting and monitoring practices and other IPM
practices would have to be tailored to most effectively use Bt cotton to control insect pests. A
number of other best management practices can be employed as part of a resistance
management plan when using Bt cotton to control CBW. These include use of: crop rotation,
structured non-Bt cotton refuges, non-Bt alternate hosts as refuge (corn, sorghum, and
soybeans), alternative insecticides — synthetic pyrethroids and new foliar spray technologies to
control CBW, stalk removal, and novel Cry insecticidal genes (e.g., CryllA). However, more
documentation is needed as to their effectiveness as part of a long-term resistance management
strategy.
Monsanto reported to the Agency the potential Bt cotton control failures as early as July 1996
and followed up with a full analysis of these incidents in the Fall of 1996. Monsanto
performed studies at all Bt cotton areas affected by high cotton infestations to determine
whether cotton susceptibility to the CryI(A)c toxin had changed and whether the Bt cotton was
expressing the CryI(A)c and whether the CryI(A)c expression levels and patterns had changed.
Monsanto also provided the results of these studies in its 1996 annual report on resistance
monitoring activities. Results of these studies indicate that there was no change in cotton
bollworm susceptibility and no change in Bt expression in the Bt cotton areas affected by high
cotton bollworm infestations. These studies indicated no detectable level of resistance in these
populations. Unusually high infestation levels of CBW may have, in part, resulted from the
dramatic increase in corn acreage in the South.
3) Pink bollworm control ,
Reports submitted to the Agency prior to registration indicated that the CryI(A)c expression
levels may not be at high dose levels for the control of pink bollworm. The Agency
recognized at the time of registration that there are existing best management practices that can
be used in conjunction with Bt cotton to more effectively control of PBW while making the
need for a high dose requirement less critical. These best management practices include:
shredding and/or plow down requirements for PBW to destroy overwintering larvae and
employing late planting dates that result hi early "suicide" emergence of many PBW adults.
These practices plus the use of pheromone trapping and release of sterile PBW moths under a
USD A/APHIS quarantine program, together with the use of Bt cotton are likely to be effective
in controlling the development of resistance. However, more information is needed on
whether any putative resistant adults will be temporal synchrony with susceptible insects
produced in the refuge or through the use of other best management practices.
There are essentially no alternative hosts for PBW in cotton growing areas. Okra is the only
known alternate host for PBW in the U.S. Research is being conducted on an effective refuge
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size and structure for PBW resistance management at the University of Arizona (and its
collaborators). Comments provided to the Agency at the two public hearings from the
National Cotton Council and University of Arizona indicate in their opinion that pink
bollworm control was excellent by Bt cotton in 1996.
, • ' ' * "• . "" -. . . ' „
• ' ' ' "" ',.••"*•"'•."
4) Other control issues
Traditionally, TBW has been a greater concern for cotton growers than CBW because CBW
resistance to conventional pesticides is limited and the cost to control CBW is relatively low
compared to the cost to control TBW. In 1996, farmers in South Carolina experienced failure
controlling CBW larvae with synthetic pyrethroid applications (cypermethrin resistance)
(Brown et al, 1997). There is a concern that CBW resistance will develop to the CryI(A)c
delta endotoxin which is expressed at high levels in Bt cotton over the course of full season.
The major concern is that there will be added selection pressure for the- development of Bt«
resistance as CEW moves from Bt corn to Bt cotton increasing the exposure to similar Bt delta
endotoxins expressed in both crops. This selection pressure will be especially intense if there
is wide scale planting of Bt corn across the south and southeastern U.S. where Bt cotton is
used and Bt microbial pesticide products are applied on other host crops of CEW/CBW.
Other generations of CEW are partitioned amongst a wider number of alternate wild hosts and
crops such as cotton, soybean, peanut, and vegetables as well as corn.and thus the selection
pressure is not as intense, However, we do not really know what proportion of CEW/CBW
feed on non-cotton hosts. Planting and growing of Bt corn hybrids, expressing the CryIi(A)b
and CryI(A)c delta endotoxin in silks and kernels,- were restricted by EPA as a mitigation
measure against the development of CEW/CBW resistance (see earlier discussion under
Sectionlll).
Rapid pest adaptation resistance to an insecticide is highly dependent on the initial frequency of
resistance alleles in field populations. A high dose strategy is based on the assumption that
resistant alleles in field, populations will be rare and occur at a gene frequency that is less than
1 X 10'3. Gould et al. (1997) were able to directly estimate the field frequency of alleles for
resistance in TBW as 1.5 X 1CT3 by individually mating over 2,000 male TBW collected in four
states to females of a Bt toxin-resistant laboratory strain, YHD2, and then screening Fj and F2
offspring for tolerance to the toxin protein. The direct estimate of the initial resistance allele .
frequency of 1.5 X 10'3 in TBW supports the 1(T3 preliminary estimate of the YHD2 resistance
allele (Gould et al., 1995). Genetic models indicate that a recessive allele at this frequency
could lead to rapid evolution of resistant populations if Bt cotton is grown without adequate
refuges (Gould, 1986; Mallet and Porter, 1992; Roush, 1994; Alstad and Andow,, 1995;
Tabashnik, 1994b, Liu and Tabashnik, 1997), Gould et al. (1997) wrote "assuming that
resistance alleles are at least partially, recessive and at approximately 10"3 and the
Environmental Protection Agency mandate of a 4% refuge to maintain SS moths is followed, it
should be at least 10 years before Bt resistance becomes a problem in H. virescens
populations." This evidence for a high initial frequency of resistance alleles in TBW "
'•••.-•. '. ' : . " . 57 • : '•-''.' .' "• •"
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emphasizes the need for caution and effective resistance management at. the outset of deploying
Bt cotton to control TBW. Using the measured resistance allele frequency of 1.5 X 10~3 for
TBW, predictive models can be refined and used to establish useful parameters for field
experiments. Tests of resistance management tactics, such as a refuge, in the field are still
needed even where there are good predictive models.
Gould et al. in their 1997 paper indicate that resistance could be a problem for CBW in far less
time than for TBW because the CryI(A)c toxin currently deployed in cotton is not as toxic to
CBW as it is to TBW. Gould et al. (1997) indicates that fewer than 90% of the natural,
undetected larvae of CBW and ECB are killed by commercially available Bt crops. These
authors indicate that if one assumes the initial frequency of a partially recessive resistance
allele is 10"3 for CBW as it has been measured for TBW, then genetic models predict that
CBW populations could become resistant to the Bt cotton in 3 to 4 years even with the 4%
refuge currently in use (Gould et al., 1997). However, no estimates of the initial resistance
allele frequency currently exist for CBW or ECB and it cannot be assumed that CEW/CBW
will have the same initial resistance allele frequency as TBW. The Agency has required
appropriate research on the effect of the CryI(A)c on CEW/CBW, refuge strategies, and
monitoring for changes in baseline susceptibilities to all three target pests. To date, no
evidence of resistance exists for any of the three target species.
Pest Biology
A long-term resistance management strategy for Bt cotton is linked to understanding TBW,
CBW, and PBW biology and ecology. EPA required as part of registration a literature review
and additional information regarding TBW, CBW, and PBW biology and ecology. Key
literature information regarding pest biology, adult movement, mating behavior, gene flow,
and alternate Hosts for these target pests has been reviewed and evaluated from the perspective
of resistance management options for Bt cotton by Dr. Michael Caprio (Mississippi St.
University) and Dr. John Benedict (Texas A & M University) for Monsanto (MRID 2204225-
01, Biology of the Major Lepidopteran Pests of Cotton (June 24, 1996)). Some of this
information has been useful hi evaluating the effect of CryI(A)b and CryI(A)c delta endotoxins
on CEW in Bt corn and its movement into Bt cotton where it is called CBW. The Agency has
reviewed this information and its contents are summarized below.
Biology
larval development time is affected by a number of factors including temperature,
humidity, predators and parasites. The number of generations CEW/CBW and TBW
populations complete in a year varies by location. PBW prefers to oviposit directly on
bolls; whereas TBW and CEW normally oviposit in the upper third of the cotton plant.
Timing and temporal synchrony in adult behaviors are clearly important. Mating
disruption has been an effective method for control of PBW and should be compatible
with resistance management strategies for Bt cotton. The physical structure of the leaf
58 .
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surface (e.g., trichome density) may alter larval movement. Heliothine larvae (e.g.,
CEW/CBW and TBW) have the genetic potential to alter behavior in response to plant
defensive compounds which may limit survival and development. Cotton with high
gossypol levels hinders the development of heliothine larvae. Production of plant,
defensive compounds are another factor to consider with other resistance management
strategies for Bt cotton. The pupal stage in heliothines is important because it is the
overwintering stage that can be disrupted using cultural practices, e.g., plowing.
Overwintering stage disruption should, be compatible with resistance management
strategies for Bt cotton. This practice is particularly useful in controlling PBW, more
so than for TBW and CBW.
Host plant preference :
Host plant preference is useful in determining whether alternate hosts may be used as
refuge sources. Both CBW and TBW have a number of alternate hosts and emerge
prior to planting of their preferred hosts. The first generation in both cases utilize a
variety of wild host plants. The most comprehensive review of host plant use by the
CBW/TBW complex is Stadelbacher et al. (1986).
There are 9 different early season wild hosts of differing quality for TBW in May:
Geranium,dissectum (wild geranium), Trifolium incarnatum (crimson clover), T.
resupinatum (Persian clover), G. carolidnum (Carolina cranesbill), Viciavillosa
(winter vetch), Medicagosativa (alfalfa), Lathyrus hirsutus (Caley pea), Abutilon
theorphrasti (velvetleaf), and Sicy.os dngulatus (bur-cucumber). The preferred hosts for
TBW are tobacco and toadflax. Corn is not a host for TBW. TBW moves to cotton in
the summer and the density is about ten times less than oh tobacco. Soybeans,
especially late-planted soybeans, may become attractive to TBW in August, but are a
less desired host than tobacco or cotton. The final generation of TBW either enters
diapause in cotton fields or .moves into wild hosts (e.g., beggarweed, Desmodium
tortuosum), alfalfa,-or soybeans. , '
As with TBW, first generation CEW/CBW primarily feeds on wild hosts during the
early spring. There are five to'eight wild hosts that have been noted as early season
hosts: Amaeranthus sp., Desmodium spp., Jussiaes decurrens, Linaria canadensis yar.
Texana,, Solanum carolinense, G. dissectum, T. incarnatum, and A. iheophrasti.
Alfalfa and early season wheat can also be hosts for CEW/CBW. Cultivated hosts are,
more attractive to second generation CEW/CBW. These hosts include peanuts, sweet
pepper, strawberries, gladiolus, soybeans, cotton, okra, sweet potato, tomato, alfalfa,
tobacco, geranium, lima beans, wax beans, snap beans, sorghum, and corn. The
primary hosts for the second generation .include tobacco, toadflax, whorl stage com and
cotton. There is a broader cultivated host range for CEW/CBW than for TBW. As
corn begins^to silk, it becomes very attractive to CEW/CBW. Populations hi silk stage
corn can reach very-high densities. By the third generation of CEW/CBW, corn has
' " • .• • v ; ,' "
" •"' ' ' -' ' " ''•' ' ;. •' '•' ;59 • -'; ., • •" - '
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begun to dry up and is not as attractive so alternate hosts including cotton, sorghum,
peanuts, tomatoes, and late-planted soybeans become more important. Millet and
cowpeas may also be important hosts at this time. After the third and fourth
generations have survived primarily cultivated hosts, the final generation or two may
move back onto wild hosts such as purselane (Portulaca oleraced) or alfalfa.
Although late-planted soybeans may be considered as a suitable refuge crop for cotton,
they are not the primary host for either CEW/CBW or TBW. Cotton is preferred by
both CEW/CBW and TBW over soybeans. Soybeans can support heliothine
populations, but they will only do so when the more attractive cotton has already started
to senesce. Soybean may be a better host for CEW/CBW in some areas, like North
Carolina, but more research on the cotton/soybean interface is warranted. It seems
unlikely that soybeans are a significant refuge crop while cotton is still blooming.
It is clear from the literature review that there are many possible alternate hosts for
CEW/CBW and TBW during the season. However, the exact utilization patterns vary
with climate and cultivation practices. The complexity of movement of CEW/CBW
and TBW amongst various possible hosts requires more study before it is possible to
determine which alternate hosts may serve as a refuge. Therefore, for now, we must
rely on non-Bt cotton as the refuge.
PBW, in contrast to either CEW/CBW or TBW, is more restricted to cotton in the U.S.
Populations of PBW don't increase until there are bolls. There are 36 possible
alternative host plants, but the extent to which these alternative hosts sustain PBW has
not been addressed. In Arizona, only okra and wild cotton act as possible alternative
hosts for PBW, but these areas where okra and wild cotton grow are very small and
isolated from the cotton growing areas.
Movement
, ii, i. , , :
Research has shown that CBW and TBW are highly mobile moths, both from
mark/recapture studies and from studies of genetic structure. Data suggest that
CEW/CBW is a more mobile moth than TBW . It is possible to see long-distance
dispersal in CEW/CBW of more than 160 km, particularly as it moves out of corn.
Estimates of the genetic variability between populations predict the gene flow over long
periods of time (long term movement rates). It is important to measure the distance
moved before mating. Another indication of mobility is the appearance of moths hi an
area prior to local emergence. This is common for CEW/CBW, but not for TBW. On
the other hand, ultra field movement is more limited for PBW. Most flights are
nocturnal and are short, less than 200 m. Genetic studies have indicated that there are
low levels of population differentiation suggesting high rates of genetic exchange
between populations. In contrast to CEW/CBW and TBW, PBW is much less mobile.
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Because the use of refuges in resistance management depends upon the assumption of
random mating between moths from refuge and treated sites and random oviposition, it
is important to improve estimates of the likelihood of matings to occur at a localized
sites versus those between moths from long distance and gene flow throughout the
growing season.
Based on the published data, additional research efforts are needed to address larval and adult
movement, mating behavior, ovipositional preferences, population dynamics, gene flow,
survival and fecundity, fitness costs, and the use of alternate cultivated or wild hosts as '
refuges. The varie'd cropping systems for cotton, including local and regional differences,
should be considered. This information is valuable in designing an effective refuge strategy
that maximizes the probability that susceptible individuals arising from a structured refuge will
find and mate with the few resistant individuals that survive exposure to the delta endotoxin
produced in the ,Bt plant. Research data regarding these efforts must be submitted by
Monsanto to the Agency by January 31, 1998. ...
Biology of Resistance
EPA required as a condition of registration a protocol for determining the likelihood of cross
resistance for the CryI(A)c delta endotoxin to other Bt endotoxins. Monsanto provided a
number of published articles regarding the potential for cross-resistance and the results of a
midgut binding assays to examine receptor binding properties for CryI(A)c and CryllA in
TBW, CBW, and ECB (D227579, dated May 22, 1996). Monsantp concluded that the
potential for cross resistance to develop among Bt proteins exists and that additional laboratory
or field studies will not add significantly to the current body of knowledge concerning the
development of cross resistance between the CryI(A)c and CryllA delta endotoxins. Instead,
Monsanto has proposed three criteria to determine the suitability of insect control proteins for
insertion into plants for a multiple gene strategy: (1) use of a non-Bt protein with a distinct
mode of action (when available) (2) the similarity (degree of amino acid homology) between
the two protein sequences if derived from Bt; and (3) differences in the mode of action or in
binding parameters if derived/rom Bt. Monsanto does not intend to send additional data
regarding the potential development of cross resistance between, the CryI(A)c and CryllA delta
endotoxins. EPA is currently reviewing Monsanto's proposal. Currently, one of the
conditions of registration is for data evaluating the potential for cross resistance to develop.
These data are due to EPA by January 31, 1998.
Information ori the nature of resistance to Bt in TBW, CBW, and PBW are necessary to
develop effective long-term resistance management for Bt cotton. Such information is useful
in evaluating the potential for Bt endotoxins to be used in rotational or pyramiding schemes.
Currently this work is limited to working with laboratory-selected insect populations since
resistance in the field has not been detected; Laboratory studies selecting for TBW, CBW, or
PBW-tolerant strains provide information on the genetic potential of each of these three target
pests to develop resistance, but do not provide information on whether resistance will develop
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under field conditions. Bt cotton and TBW, CBW, or PBW in the field may pose a different
situation than larvae feeding on Bt insecticides in a laboratory diet. Laboratory selected
TBW and PBW colonies tolerant to Cry proteins exist. The CBW-tolerant colony selected
against CryI(A)c at Mississippi State University no longer exists. Laboratory-selected Bt-
tolerant TBW, CBW, and PBW colonies will allow experiments concerning the mode of
resistance, the genetic basis for resistance, and the potential for cross resistance to other Bt
toxins to be conducted. These laboratory-selected Bt-tolerant insect colonies can also be used
to estimate the initial resistant allele frequencies in field populations and be used to confirm the
efficacy of the estimated LC^, as a discriminating dose.
1. Laboratory selection for TBW Cry-tolerant colonies and Cross-resistance.
There is a limited knowledge on the genetics and mechanisms of resistance to CryI(A)c
toxin in TBW, CBW, and PBW. Several laboratory strains of TBW have been selected
for resistance to the Bt delta endotoxins (Stone et at, 1989; Sims and Stone, 1991;
Gould et at., 1992; Gould et al, 1995).
The YHD2 strain of TBW developed a very high level of resistance to CryI(A)c and
related Cryl endotoxins (Gould et al., 1995). This colony was developed before Bt
cotton was commercially deployed in 1996 and was selected on an artificial diet with an
acute exposure to high amounts of purified CryI(A)c. The LQo for YHD2 was 2,OOOX
more resistant to the Bt endotoxin than the LQ0 for the susceptible TBW colony.
Recent experiments have shown that YHD2 can survive on Bt cotton that expresses the
CryI(A)c toxin. A major portion of the resistance in the YHD2 strain is encoded by a
single gene with mostly recessive inheritance. YHD2 confers resistance to CrylA
toxins (a, b, and c) as well as CrylF. Research using genetic markers have indicated
the location of this major resistance gene is on linkage group 9 with some evidence that
there is a minor resistance gene located on linkage group 11. Biochemical analyses
indicate that resistance in the YHD2 strain is associated with decreased toxin binding to
the membrane of larval midgut cells, the toxin's apparent binding site and site of action.
Other decreases in Bt toxin binding have also been found hi Bt-resistant strains of other
lepidopteran species and inheritance of the Bt resistance was at least partially or
completely recessive (Van Rie et al., 1990; Ferr6 et at., 1991).
, ' , /- ' .„,- ; , , . i
Gould et al. (1992) reported broad cross-resistance in another laboratory strain of TBW
that was not as highly resistant as YHD2 that did not appear to be related to binding site
modifications. Laboratory selections of a TBW population using CryI(A)c resulted hi
varying levels of cross-resistance to CryI(A)a, CryI(A)b, CrylB and CrylC insect
control proteins. These laboratory data indicate that there is the potential for cross-
resistance by TBW to develop to a number of Cry proteins. It is unlikely that
laboratory selective procedures provide the identical selective conditions as exist in the
field. The ability to select for tolerance to Cry proteins hi the laboratory in different
insect pests indicates that it is prudent to use appropriate resistance management
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strategies.
Midgut binding assays performed by Monsanto (D227579, dated May 22, 1996)
examining the mode of action of CryllA have shown that, unlike Cryl and CrylllA,
. neither saturable binding nor a saturable binding component was found for CryllA on
the midgut brush border, of CEW/CBW, TBW, or ECB larvae.' CryllA did not dilute
and block CryI(A)c binding; however, CryI(A)c effectively diluted CryHA and stopped
the initial binding of CryllA to the brush border. These observations indicate that
CryI(A)c and CryllA share a common component for binding on the midgut brush
border. These same.results were reported for CEW/CBW alone by English et al.
• (1994). These authors went further to conclude that CryllA is significantlyless soluble
, in the digestive fluids of.CEW/CBW than CryI(A)c." Third instar CEW/CBW feeding
was arrested by both proteins, but acute morbidity was delayed for CryllA. CryllA
formed voltage-dependent and not highly cation-selective channels in planar lipid
bilayers unlike CryI(A)c and CrylllA. These authors conclude, that CryllA is less
bioactive against CEW/CBW than CryI(A)c, but represents a unique mode of action
among the delta endotoxins. CEW/CBW is the least sensitive of three target species
(i.e., TBW, CEW/CBW, and PBW) to me CryI(A)c delta endotoxin expressed in
cotton. The results seem to indicate that CryllA may be less effective than CryI(A)c
' • . • in controlling CEW/CBW. However, the CryHA delta endotoxin has a different target
binding site on the midgut membrane than CryI(A)b/CryI(A)c and .thus may be useful in
pyramiding or stacking with other Cry genes to combat insect pest resistance.
2. Predicting the Evolution of CEW/CBW resistance - Computer simulation models.
Monsanto, Dekalb, Novartis Seeds and investigators from North Carolina State
University (Dr. John Van Duyn, Dr. Fred Gould, Dr. J.R. Bradley, and Dr. George
Kennedy), Virginia Polytechnic Institute and State University (Dr. Ames Herbert) and
from the University of Maryland (Dr. Galen Dively) are developing strategies for
developing computer simulation models which predict the evolution of resistance to
CryI(A)b/CryI(A)c proteins by CEW/CBW within the corn/cotton system.
Additionally, research protocols are being developed for validating model assumptions
and output. Research areas include: (1) Assessing the impact of Bit corn on CEW/CBW
adult emergence and oviposition in cotton; (2) Contribution of alternate hosts as refuges
for CEW/CBW; and (3) Impact of Bt on CEW/CBW overwintering survival and
fecundity; and (4) Assessing survivorship of CEW/CBW and TBW on pollen.
Effective Refuges . ,
All available evidence supports the conclusion that a "structured" refuge is necessary to the
success of a long-term resistance management strategy. Two refuge options were mandated as
requirements of the Bt cotton registration to mitigate the development of resistance: 20%
sprayed refuge (Option A) and 4% unsprayed refuge (Option B):
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Monsanto's 1996 year-end report indicates that compliance with the refuge requirements was
excellent, >98% of those growers surveyed. Monsanto visited 2,346 of 6000 Bollgard®
growers (about 40% of the total) and found that 60% used Option A (the 20% sprayed non-Bt
cotton refuge) and 38% used Option B (the 4% unsprayed non-Bt cotton refuge) and 1 % used
both. The remaining 1 % did not have an adequate refuge in place due to flooding or other
circumstances. Between 82-88% of the growers surveyed had their refuges within 1 mile of
the Bollgard® cotton. This means that 12-18% of the growers had refuges more than 1 mile
from Bt cotton, this could be a problem.
Several entomologists discussed the subject of effective refuge size and structure in their
written comments to EPA and hi the published literature. Experts including Dr. Mike Caprio,
Dr. Fred Gould, Dr. Rick Roush, and Dr. John Benedict have provided written comments to
the Agency during the two public hearings in March and May 1997 and stated that the 4%
unsprayed refuge may not be large enough to produce a relatively high number of susceptible
cotton bollworm adults to mate with any resistant insects that may develop on Bt cotton with a
low dose for CBW. Some growers employing this Option sustained large yield losses in the
refuge due to fall army worm and CBW infestations. Resistance management analyses by
EPA (September 27, 1995) indicated that the 4% unsprayed refuge was not the preferred
option because of the potential for its failure, under high pest population pressure, to provide
enough susceptible individuals to mate with any resistant individuals throughout the growing
season. Economic considerations may also contribute to its potential lack of success.
Fanners may be unwilling because of the cost involved to treat the unsprayed refuge areas hi
an identical fashion, agronomically, e.g., weed control, irrigation, fertilization etc. They may
choose agronomically undesirable land or place the refuge too far away from the Bollgard®
cotton acreage to be of any use hi resistance management.
Reports from extension entomologists in the 1997 Proceedings of the Beltwide Cotton
Conferences indicated that in some cotton-growing areas, 50 to 80% yield losses were
incurred in the 4% unsprayed refuge areas. Some indicate that they would not be
recommending the use of the 4% unsprayed refuge in 1997 (Smith, 1997). A number of
entomologists have indicated hi their written comments to EPA that the lack of high dose for
CBW control should be considered hi designing an effective refuge strategy and the existing
refuge requirements should be modified. These experts are recommending that Option B/the
4% unsprayed refuge should be discontinued or drastically expanded to perhaps, 30 to50%.
Option A/the 20% sprayed refuge should be continued or expanded.
Dr. Fred Gould (entomologist), North Carolina State University, commented that Bt cotton
does not provide a high dose strategy for control of CBW and PBW. Furthermore, the current
refuge options associated with Bt cotton amounted to an effective refuge size of 4% and in his
estimation the effective refuge size would need to be increased to counterbalance the lack of a
high dose in CBW. The 20% sprayed refuge was considered to be a 4% effective refuge.
Gould indicated the effective refuge size for Bt cotton, under the current dose situation, would
have to be at least 30% non-Bt cotton.
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A panel of Texas A & M entomologists stated in their written comments to EPA that a 30 %
unsprayed refuge may be impractical or too costly to the grower. They recommend that a 20%
non-Bollgard cotton sprayed refuge be planted and managed in a manner to coincide with the
'Bollgard®, cotton so the refuge is attractive to ovipositioning CBW adults and will produce
moths at.the same time and in the same geographical vicinity as the Bollgard® cotton, i.e.,
within 1 mile of the Bollgard® cotton. They recommend that the 4% unsprayed refuge should
be replaced with a 20% sprayed refuge. They explained that a 20% sprayed refuge should be
effective, as long as the combined corn, sorghum and soybean acreage is planted in relatively
close proximity to the Bt corn to act as a refuge, is greater, than the Bollgard® cotton acreage
in these Bollgard® cotton producing counties.
Dr. Mike Caprio (entomologist) from Mississippi State University commented that a delay in
emergence of resistance could still exist in the absence of a high dose strategy, but the
emergence would occur sooner than it would in the presence of a high dose strategy. He
states that "while it is true that a high dose makes the refuge strategy much more effective, we
have shown in simulations of" foliar applications of Bt that even survivorship rates as high as
20% could still delay resistance 5 fold compared to the rate of resistance evaluation in the
absence of the refuges." He encourages me:pyramiding of multiple genes in cotton if such
genes increase mortality of cotton bollworm to delay resistance development in this pest.
,- . • - ' • Ł "I--''
^Gould et ql. (1997) measured an initial resistance alMe frequency of 1.5 X 10 "3 for TBW.
Using this measured frequency, these authors caution that an effective refuge is needed at the ,
outset to manage the development of resistance to the CryI(A)c produced in Bt cotton. They
concluded that the current refuge options mandated by EPA should be effective for at least 10 '
years before a Bt resistance .problem might be seen in TBW populations. They commented that
Bt resistance could be a problem for CBW in far less time than for TBW because the CryI(A)c
toxin currently deployed hi cotton is not as toxic to CBW as it is to TBW. These authors
indicate that if one assumes the initial frequency of a partially recessive resistance allele is 10"3
for CBW as it has been measured for TBW, then genetic models predict that CBW populations
could become resistant to the Bt cotton in 3 to 4 years even with the 4% refuge currently in use
(Gould et al., 1997). However, no estimates of the initial resistance allele frequency currently
exist for CBW or ECB and it cannot be assumed that CEW/CBW will have the same initial
resistance allele frequency as TBW.
Tabashnik (1997) commented on Gould et al. 's 1997 paper. He also suggests that the odds
for, delaying resistance could be improved by requiring larger refuges than the 4 % unsprayed
or 20% sprayed refuges required for Bt cotton. The 4% unsprayed refuge may not produce
enough susceptible insects throughout the growing season and the 20% sprayed refuge may
suppress susceptible insects because of the effectiveness of the conventional insecticide
treatments. In both cases, the refuges would be ineffective^
Recent experiments by Liu and Tabashnik (1997) suggest that a 10% structured refuge helped
to maintain susceptibility of a diamondback moth to B. thuringiensis subsp. aizawai. These
'. . • ' '.' • -65 '." - ' '' *- ' :; : "•
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data provide experimental evidence to support the mathematical models that refuges can delay
insect adaptation to Bt (Gould, 1988; Mallet and Porter, 1992; McGaughey and Whalon, 1992;
Tabashnik 1994 a, b; Alstad and Andow, 1995).
Experts have argued that a mixed seed refuge will be ineffective for managing resistance of
TBW and CBW to the CryI(A)c delta endotoxin expressed in cotton because of short-range
larval movement between plants. There is a threat that TBW and CBW larvae would be
exposed to sublethal doses of the Bt toxin on Bt plants and then move to non-Bt producing
plants allowing selection for resistance to occur. However, PBW has very restricted larval
movement and a seed mix refuge strategy is more likely to work for this pest. Furthermore,
PBW has a very limited host range, essentially it feeds only on cultivated cotton in the U.S.
Okra and wild cotton are alternative hosts, but these plantings are extremely small and isolated
from cotton acreage in Arizona.
,l ' i!li . , , .. ' • ' , ' '
Monsanto is wholly or partially funding a number of research activities along with other
sponsors, including USD A, to determine what constitutes effective refuges for several pests.
These are summarized below. *
1. "Managing Resistance to Bt-Transgenic Plants: Greenhouse and Field Tests" (A. M.
Shelton, J. D. Tang, andE. D Earle, Cornell University, Geneva, NY and R. Roush,
University of Adelaide, Australia) Using resistant diambndback moth populations,
small-scale field tests are being conducted in Bt broccoli to examine the effects on
refuge size, proximity, and effects of conventional insecticide treatment on the refuge.
Results from greenhouse and larval movement data, and field data showed that the
number of larvae per plant hi the mixed refuge was less than the number of larvae per
plant in the separate refuge. This suggests that more larvae were exposed to toxin
which should have intensified selection in the mixed refuge. The level of resistance,
however, was low for all refuge treatments. Immigration of native susceptibles were
sufficient to dilute the resistance. Both mixed and separate refuge treatments were
effective in managing resistance, but it is riot known what will happen if the field
studies were extended to 8 or 9 insect generations. However, these researchers
recommend that a separate refuge should be used over a mixed refuge. Further field
experiments on refuges will be done to study the release of greater numbers of larvae
earlier in the field season and release at higher resistant allele frequencies.
2. Evaluate resistance management strategies for PBW in Bt cotton infield tests (Tim
Dennehy, University of Arizona, Tuscan, AZ; Western Cotton Research Laboratory-
USDA; USDA-APHIS-PPQP, Arizona Cotton Growers Association, Cotton
Incorporated, and Monsanto Co.) A three-year, 200 acre study was begun hi 1997 in
Eloy, Arizona to contrast the outcomes of resistance development of PBW subjected to
different Bt use strategies. Five different treatments are being studied: 1) 80% Bt
cotton/20% non-Bt cotton, 2) Rotations of Bt cotton (one year) with non-Bt cotton (next
year); 3) In-field refuges, i.e., mixed seed, 80% Bt c6tton/20% non-Bt cotton; 4)
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Biologically-intensive strategy (multiple control measures, Bt cotton + pheromones +
nematodes + sterile male releases); arid 5) Control group, non-Bt cotton with PBW
controlled with conventional insecticides. PBW populations will be collected from each
• treatment annually and will be placed in culture and bioassayed for susceptibility to the
CryI(A)c delta endotoxin. ,
Monitoring for Resistance-Surveillance/tracking
As a requirement of registration, EPA required that Monsanto submit a plan for a workable
monitoring program, and submit the existing data for baseline susceptibility data for TBW,
PBW, and CBW by March 1, 1996. Where these data do not exist, data must be submitted
which provided baseline susceptibility and discriminating doses for these pests. The
monitoring plans should establish specific locations in selected states that will be monitored
annually at a central laboratory location, with duplicate sample collections sent to a second lab
for confirmation, Monsanto will also follow up on grower, extension specialists, or consultant
reports of less than expected results or control failures for TBW, CBW, and PBW as well as
for cabbage looper, soybean looper, saltmarsh caterpillar, cotton leafperforator and EGB.
Monsanto will ,also indicate in the monitoring plans how resistance management strategies
would be altered should resistance be detected. A preliminary report on monitoring must by
submitted to EPA annually by November 1 each year and a final report Submitted to EPA
annually by January 31 each year for the duration of the condition registration. Monitoring .
and remedial action plans were submitted in March 1996, reviewed by the Agency in April
1996, and found to be acceptable. The purpose of monitoring is to learn whether a field
control failure resulted from resistance or other factors, other than resistance, that might
inhibit the expression" of the CryI(A)c delta endotoxin. It may be possible to develop
monitoring techniques sensitive enough to detect early changes in resistance in pest populations
before it becomes widespread. Regular surveillance by growers is essential to early detection
of resistance. Monitoring activities and> the monitoring and remedial action plans are
summarized below.
. 1. Baseline susceptibility and development of diagnostic Bt concentrations for
monitoring for TBW and CBW (Dr. Hardee and Dr. L.C. Adams, USDA-ARS-SIML,
Stoneville, Mississippi, Monsanto sponsorship). Diagnostic doses for CEW and TEW
have been developed over several years hi insect control labs at Monsanto (Suns et al.,
1996). The LC <& estimates for the full-length CryI(A)c protein are 6.6 |j,g/mL for
. TBW and 13322 |j,g/mL for CBW. Clearly, the differences in diagnostic dose
concentration indicate that CBW is significantly less sensitive to the CryI(A)c protein
than TBW. Sims.er at. (1996) evaluated the growth inhibition response using the full-
length CryI(A)c protein for TBW and CBW. The EQ, was 0.058 jag/mL for TBW and
28.8 |o,g/mL for CBW. These ECgg estimates are considerably lower, 114-fold less for
TBW and 463-fold less for CBW, than the corresponding LC <# estimates for the full-
length CryI(A)c protein. That is, the larval growth inhibition response is more
sensitive than the diagnostic dose response arid is a reasonable starting point for
•'.'••'-•• • 67 • • . ', .' :..•'•• '
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detecting changes in CBW and TBW susceptibility to the Bt CryI(A)c protein. Sims et
al. (1996) validated the concept of a diagnostic dose in combination with a larval
growth inhibition assay to unambiguously separate resistant from susceptible insects
using a CryI(A)c protein resistant strain of TBW and Fj hybrids derived by crossing the
resistant strain to a susceptible TBW strain. These data indicate tht it may be hard to
detect resistance with a simple LC50 test or to develop a simple diagnostic mortality
dose. The weight gain data are much more quantitative. A combination of the
diagnostic dose and larval growth inhibition assay seems to be the most efficient means
of tracking population susceptibility, especially when the assay can detect the decreased
susceptibility present hi resistant heterozygotes.
Monitoring efforts for CBW and TBW resistance were initiated in 1996. Twenty-three
different populations of these insects were collected hi Arkansas, Mississippi,
Oklahoma, and Texas and subjected to field doses of MVP II Bt foliar insecticide. The
CryI(A)c delta endotoxin in this foliar insecticide is the most lexicologically analogous
to the CryI(A)c protein expressed hi Bollgard® cotton. Monitoring results showed no
shifts in baseline susceptibility levels to the CryI(A)c protein. However, there were
exceptionally low populations of TBW across the Cotton Belt in 1996; therefore, data
from only 3 colonies of this insect were tested. This sample size is very small and is
not adequate to accurately predict whether resistance is occurring in the field.
Resistance monitoring efforts were expanded and continued in 1997. Determination,of
threshold levels of initiating remedial action need to be developed as well as the specific
programs for appropriate remedial actions. The 1997 monitoring report should be
submitted to the Agency by January 31, 1998.
2. Baseline susceptibility and development of diagnostic Bt concentration for
monitoring for PBW. (Dr. Alan C. Bartlett, Western Cotton Research Laboratory,
USDA/ARS, Phoenix, AZ; Dr. Tim Dennehy, Dept. of Entomology, University of
Arizona, Tuscan, AZ; Dr. Larry Antilla, Arizona cotton Research and Protection
Copuncil, Tempe, AZ, Monsanto sponsorship). Twenty-five percent of Arizona's
cotton acreage was planted with Bt cotton hi 1996. The key target of Bt cotton
expressing the CryI(A)c delta endotoxin in Arizona is PBW. Baseline susceptibility to
the CryI(A)c delta endotoxin was determined from five PBW populations in five
Arizona counties (i.e., Stanfield, Yuma, Buckeye, Parker, and Marana). Newly
hatched larvae from these populations were subjected to artificial diets containing doses
of a purified solution of Bt CryI(A)c delta endotoxin provided by Monsanto. None of
the neonate larvae were able to reach maturity when the CryI(A)c dose exceeded 0.005
Hg/mL of diet (Bartlett, et al., 1997). Baseline monitoring studies were expanded in
1997. Preliminary results will be available hi 1998.
3. Surveillance and remedial action.
As noted earlier, Monsanto is required as part of the Bt cotton registration to have a
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workable monitoring plan and a remedial action plan. Monsanto is required to instruct
customers to contact them (e.g., toll-free customer service number) if incidents of
unexpected levels of TBW, CBW, and PBW damage occur as well as unexpected
damage by cabbage looper, soybean looper, saltmarsh caterpillar, cotton leafperforator
and ECB. Monsanto is required to report to EPA suspected incidents of resistance to
these three pests. Monsanto will investigate and identify the cause for this damage by
local field sampling of the plant tissue and suspect insect populations, followed by
appropriate in vitro and inplanta assay. Any confirmed incidents of resistance are
required to be reported to EPA. If resistance is confirmed then appropriate remedial
action is required to mitigate resistance. Remedial actions include: informing
customers and extension agents in the affected areas of resistance problems,
implementing alternative means to reduce or control the resistant populations,
increasing monitoring in the affected areas, modifying refuges in the affected areas, and
ceasing of sales in the affected and bordering counties. In its Bollgard® Grower
Guide, Monsanto has instructed its customers to have regular surveillance programs
and report any unexpected levels of TBW, CBW, and PBW damage to them and to
their local extension agents. EPA considers that industry cooperation; with extension
and academics entomologists and consultants to be important in communicating specific
information of definitions of "unexpected damage" and appropriate remedial action.
l)TBWandCBW Y ,
In 1996, Monsanto investigated claims of Bt cotton failure in the Brazos River bottoms
in East Texas and reported this information to the EPA immediately in July 1996.
Monsanto investigated these "failures" at the affected sites. CBW and Bt cotton tissue
were collected from high infestation areas. CBW susjceptiblity and Bt expression in Bt
cotton areas affected by high cotton infestations were determined. There was no
change in cotton bollworm susceptibility or in Bt expression in these areas. These
studies showed no detectable level of resistance in CBW populations collected in the
affected areas. Experts agree that the Bt cotton performed as expected under high
infestation conditions of CBW. Reports indicate that CBW populations were at the
highest level measured in a decade. Bollgard® cotton killed greater than 80% of these
hatching CBW, but survivors exceeded the economic threshold for control.
The situation unfolded as follows. As corn began to senesce after producing two
generations of CEW, nearby cotton acreage experienced extremely heavy CBW
infestations, especially in areas with high corn acreage. In many cases, these CBW
larvae were able to survive, by feeding on pollen-material and then moving to bolls
lower in the plant canopy where expression of the CryI(A)c prptehi is lowest. Dr,
Blake Layton, extension entomologist from Mississippi, reported that the percent of
CBW damaged bolls was considerably lower in the Bt due to larvae feeding on pollen
in blooms and then moving to bolls rather than in the terminal region of the plant.
These CBW larvae escaped detection because scouting techniques for conventional
'• '•'''."'.' •••-••:• 69 •• - " • .. ' ' - •
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cotton normally are for the top 6" of the plant canopy. Coupled with the natural
tolerance of CBW to the CryI(A)c protein compared to TBW, it is likely that a
proportion of the population survived on pollen and grew large enough to tolerate
higher levels of the CryI(A)c protein in other tissues. Supplemental insecticide sprays
to control CBW on Bt cotton were used in some instances, but not all Bt cotton acreage
was treated or needed to be treated. The results of these investigations were presented
verbally to EPA at a meeting held on September 11, 1996. The formal results of the
investigations were presented to EPA in written form in Monsanto's preliminary
monitoring report dated November 5, 1996 and its final annual monitoring report dated
February 28, 1997. EPA agrees with the findings as presented by Monsanto in these
reports. ,
2) PBW
While the focus has been on the control of the TBW/CBW complex in the majority of
cotton growing areas located from Texas eastward, PBW is the major target insect of Bt
cotton in Arizona, California, and New Mexico cotton-growing areas. In addition to
Monsanto's required efforts to respond to putative reports of resistance, a multi-agency
Rapid Response Team consisting of the University of Arizona, Cotton Research and
Protection Council, and headed by the Arizona Cotton Growers Association has been
organized to promptly and rigorously investigate growers' claims of failure of Bt cotton
to control PBW in Arizona. Putatively resistant populations will be put into culture and
tested for susceptibility to Bt toxin.
Development of products with alternative modes of action
Industry is developing other cotton lines that involve the expression of novel Bt genes acting
by mechanisms different from currently registered Bt genes. These novel genes could be
combined with currently registered Bt genes that could be combined with existing Bt genes,
insecticidal genes with mechanisms of action different from Bt, and inherent host plant
resistance traits as a means for combating the development of TBW, CBW, or PBW resistance
to the Cry(A)c delta endotoxin expressed in cotton, An EUP has already been granted for the
Cry HA delta endotoxin expressed in cotton and the registration submission is under review.
This delta endotoxin operates by a voltage-dependent mechanism. Pyramiding or stacking
genes is advocated by entomologists as a powerful tool to mitigate the development of
resistance. Roush (1994) has modeled the effects of pyramiding and results indicate that
resistance may be delayed by greater than 1000-fold. Dr. Dave Ferro (University of
Massachusetts) and Dr. Fred Gould (North Carolina State University) both provided comments
encouraging the pyramiding of multiple genes in cotton if such genes increase mortality of
cotton bollworm to delay resistance development hi this pest. Monsanto is conducting
research on the effects of CryI(A)c and CryriA gene combinations on efficacy and resistance
management. They also have ongoing efforts to discover non-Bt genes to control TBW, CBW,,
and PBW among other insect pests. There are also many novel non-Bt conventional insecticide
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products with different modes of action available to cotton growers to control the TBW/CBW
complex, e.g., Tracer, Pirate, Intrepid, and Proclaim. The availability of many control
options with different modes of action to control the TBW/CBW complex will help to reduce,
the reliance on any one control option. , . '-
Grower Education
As a requirement of registration, Monsanto was required to continue the development and
distribution of grower education materials including: instructions on the appropriate use of
Bollgard® cotton in a resistance management program, monitoring, and reporting of resistance,
Monsanto has extensive grower activities, e.g., educational seminars, brochures, video tapes,
cassettes etc. Monsanto supports a number of research activities on resistance management
(discussed above), Monsanto has had .involved discussions with growers, crop consultants and
USDA researchers and extension specialists to develop information on resistance management
and integrated pest management. This information was included in the Bollgard® cotton
Grower Guide. :
Monsanto reported to EPA that it made personal grower surveys in 1996 and 1997 to quantify
grower experience with Bollgard® cotton. The 1996 sample included 89 growers. Bollgard®
cotton growers reported an average yield improvement of 6% to 16% depending on the cotton-
growing region. Taking into account total insecticide system control costs and yield, they saw
an economic advantage of $33 per acre from using Bollgard® cotton even after paying the $32
technology fee. Planting of Bollgard® cotton resulted in the elimination of the equivalent of a
250 thousand gallons of formulated insecticide products in the U.S.
Based on Bt cotton performance in 1996, academics, Monsanto,'and the National Cotton
Council noted that there could be improvements made in communication on CBW control in Bt
cotton with the growers, general public, and consultants. The National Cotton Council
indicated that scouting practices had previously focused on the top six inches of the plant. As
,a result of observations, made during the 1996 Bt cotton growing season, including the outbreak
of CBW in the Brazos Valley in Texas, modified scouting practices in Bt cotton will be
employed to examine the whole plants especially during peak bloom periods. It is currently
recommended that growers continue to scout their crop on a regular basis by inspecting the
entire plant, including blooms and bloom tags, to obtain an accurate larval count and damage
assessment. Monsanto, growers, crop consultants, USDA researchers and extension
specialists are working together to continue to develop information on how to best scout
Bollgard® cotton. Their collective scouting recommendations are reflected in the 1997
Grower Guide. The 1997 Grower Guide states that "scoutuig at least twice per week is
recommended during periods of heavy or sustained egg lay, especially during peak bloom.
Scout the entire plant, including blooms'and bloom tags. Larvae greater than 1/4 inch (2- 4
days old) are generally recognized as survivors that will be difficult to control with Bollgard®
alone. Apply remedial insecticides if the frequency of advanced stage larvae or plant damage
warrants treatment. Consult your local university and extension service for advice on
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.' 'I!'1' ' , , • i . . , ' ' '
thresholds appropriate for your area. Changes to these recommendations may be required
under unique circumstances; consult your local crop advisor."
,;, ' , " • " "I "',,,"
Reports from extension entomologists and consultants hi the 1997 Proceedings of the Beltwide
Cotton Conferences indicate that effective and timely scouting is extremely important for
Bollgard® cotton. Based on the 1996 Bt cotton performance, there would be an extra cost for
scouting Bollgard® cotton because of the higher monitoring frequency, the more exacting and
different monitoring requirements (not overreacting to egg or to the tiny first-stage larvae,
judging what constitutes a second-stage larva, monitoring for stink bugs, fall armyworms etc.)
compared with conventional non-Bt cotton. These higher costs may cause some growers not
to use Bollgard® cotton. Additional training and labor requirements in effectively monitoring
Bollgard® cotton are needed in the short term until Bt cotton scouting and treatment practices
become more routine. Pest surveys should be modified and economic thresholds may be more
difficult to determine hi Bollgard® cotton.
Consultants and entomologists report in the 1997 Proceedings of the Beltwide Cotton
Conferences that Bollgard® cotton is more valuable to the grower after boll weevil eradication
programs because the CryI(A)c delta endotoxin is not effective against boll weevil.
Insecticidal treatment for boll weevils will be required hi areas in which they have not been
eradicated. However, treating for boll weevil will also suppress the beneficial insect
populations allowing other secondary pest populations to surge which then may need
insecticidal treatment. Therefore, if Bollgard® cotton is used in areas that have not
undergone boll weevil eradication, then strategies to ensure the preservation of beneficial
insects especially later in the season will be important to help manage non-lepidopteran pests,
because beneficial insects will be destroyed during early season treatment for boll weevil.
Reports indicate the 1.8 million acres of Bollgard® cotton grown in 1996 is thought to be
responsible, in part, for reducing total formulated chemical insecticide applications by 250
thousand gallons. Seventy-seven percent of the U.S. cotton acreage was infested with
CEW/CBW complex hi 1996. On average,"there were 1.3 applications of insecticide sprays
per acre where Bollgard® cotton was grown. This represents a significant reduction from
previous years in which 5 to 12 applications were used on conventional cotton to control
CEW/CBW populations.
Cotton consultants had little practical experience with Bt cotton prior to its wide-scale
commercialization in 1996. Experiences in 1996 were shared via the Consultants Instant
Information Network (GEN). Lack of experience may have caused the costs of pest control
to be higher in some instances. If so, greater familiarity with specific scouting and monitoring
practices for Bollgard® cotton should lower these costs. Some consultants in the Mid-South
have raised concerns about whether Bollgard® cotton is well suited for the Mid-South cotton
production areas (Farr et al., 1997). They are interested in several issues: whether new Bt
cotton varieties will perform better agronomically than the standard non-Bt cotton lines, will
they perform better hi controlling CBW than the standard non-Bt cotton lines, will the Mid-
• • ' • " 72 , ', , , ':
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South generate more boll weevil adults to; overwinter out of Bt cotton fields, will resistance
increase more rapidly in Bt cotton due to full season production of the toxin in the plant, and
will plant bugs increase in Bt cotton.
The most important consideration for growers is whether the Bollgard® cotton'will provide a
greater economic value than use of conventional cotton. Comments by Texas A & M
entomologist, Dr. John Benedict, and other entomologists have noted that Bollgard® cotton is
not useful to all cotton producers, but is of value, to those spending more than $40 per acre for
control of TBW, PBW or mixed populations of CBW, TBW or PBW on their cotton. It allows
those farmers to use their equipment for other farm functions, reduces hazards of pesticide
exposure and poisoning to the grower, farm employees, farm families, and consumers. Bt
cotton will also be useful to improve the grower's ability to fully use IPM, to allow beneficial
arthropods to be more effective, and to provide greater likelihood of relief from past crop
losses, and costs of unsuccessful attempts to control insecticide-resistant TBW.
Public Comments .
Of the 100 comments received as a result of the two public hearing held on Bt plant-pesticide
resistance management this year, about 15 specifically focused on Bt cotton resistance
management issues. These comments came from academic and extension entomologists,
grower groups, trade organizations, environmental groups, and industry/seed companies/trade
organizations. The Agency sought information regarding reported control failures for Bt
cotton in 1996, suggested evaluation tools concerning these failures, and, implications on future
resistance management efforts at two public hearing held in March and May 1997. These
comments are summarized above in Section I of this paper. ,
In general, comments received from private citizens, organic farmers and grower
organizations, environmental groups, and public-interest groups indicated that the Bt cotton
resistance management plan should be reevaluated and that a SAP should be held. Experts
from industry, academia (almost all), and USDA noted in their 'comments that Bt cotton
performance was excellent for control of TBW, CBW, and PBW. In 1996 .following reports
of Bt cotton failures in the Brazos Valley area of Texas and some other areas in the Mid-South,
Monsanto tested for cotton bollworm susceptibility and Bt expression in Bt cotton areas
affected by high cotton bollworm infestations. They found no change in CBW susceptibility to
the CryI(A)c delta eridotoxin and in Bt expression levels in the plants as compared to the
baseline susceptibility levels for these locations. That is, these studies showed no detectable
level of resistance in the CBW populations. Comments indicated that additional data need to
be gathered to develop a long-term resistance management strategy. Progress in these
research areas was summarized above.
Summary , •
It is* recognized that long-term resistance management will involve other IPM practices hi
. • • ' 73 ' ••• ' . '- . " .
-------
addition to the use of Bt cotton. The number of insecticide applications and the type of
insecticides used vary widely in Bt cotton and non-Bt cotton. Based on the 1996 Bt cotton
performance, there would be an extra cost for scouting Bt cotton because of the higher
monitoring frequency, the more exacting and different monitoring requirements (not
overreacting to egg or to the tiny first-stage larvae, judging what constitutes a second-stage
larva, monitoring for stink bugs, fall armyworms etc.) compared with conventional non-Bt
cotton. Additional training and labor requirements in effectively monitoring Bt cotton appear
to be needed in the short term until these practices become more routine. Pest surveys will
have to be modified and economic thresholds may be more difficult to determine in Bt cotton.
Best management practices should be tailored specifically for Bt cotton. A unified, multi-
stakeholder effort to determine research priorities and develop a long-term resistance
management strategy is essential.
The three target pests, TBW, CBW, and PBW show a differential susceptibility to the
CryI(A)c delta endotoxin expressed in Bt cotton. Tobacco budworm is the most sensitive of the
three species to the CryI(A)c delta endotoxin. A high dose strategy exists in Bt cotton for the
TBW, but the existence of a high dose strategy is less certain for PBW, ancl does not exist for
CBW. In 1996, there was an unusually high infestation of CBW in the Cotton belt.
Monsanto's researcli to determine cotton susceptibility and Bt expression in the Bt cotton areas
affected by high cotton bollworm infestation showed no detectable level of resistance in these
populations. That is, there was no change in cotton bollworm susceptibility or Bt expression
levels in the affected areas as compared to previous levels. Reports in the published literature,
from Monsanto, and EPA's analysis recognized the fact that Bt cotton would not produce a
high dose to control CBW and that supplemental insecticide treatment might be necessary.
Supplemental insecticide treatment was necessary in some cases in 1996 because of high CBW
populations, but not on all Bt cotton acreage. In 1996, overall insecticide use dropped on
cotton by about 250 thousand gallons, hi part, because of the introduction of Bt cotton.
EPA concluded that to develop a long-term resistance management strategy and manage
resistance effectively, specific data needs were made requirements of the Bt cotton registration:
(1) target and secondary pest biology and ecology, (2) cross-resistance potential, (3)
monitoring data (baseline susceptibility and discriminating dose determination), (4) effect of Bt
cotton on secondary lepidopteran pest (e.g., cabbage looper, soybean looper, saltmarsh
caterpillar, cotton leaf perforator, and ECB), and (5) expression. EPA also required annual
monitoring and annual use reports. EPA mandated specific refugia requirements and grower
compliance has been high. EPA required continued development and distribution of grower
education materials.
An extensive literature review regarding pest biology, host-preference, and movement were
provided by Monsanto and reviewed by EPA. However to develop a long-term resistance
management strategy, research efforts appear to be needed to address larval and adult
movement, mating behavior, ovipositional preferences, population dynamics, gene flow,
survival and fecundity, fitness costs, and the use of alternate cultivated or wild hosts as
74
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refuges. Currently, one of the requirements of the registration agreement is for further
evaluation of the target pest biology. These data must be submitted to EPA by January 31
1998.
Literature regarding the potential for cross-resistance to develop between CryI(A)c and CryllA.
and a protocol for evaluating the cross-resistance between CryI(A)c, CryllA and other Bt
toxins were submitted by. Monsanto. Monsanto concluded that the potential for cross-
resistance amongst Cry proteins exists and that additional data and field studies will not add
significantly to the current body of knowledge concerning the development of cross-resistance
between the CryI(A)c and CryllA delta endotoxins. Data presented indicated that CryI(A)c
and CryllA operate by a different biochemical mechanism of action. Monsanto does not intend
to send additional data regarding the potential development of cross resistance between the
CryI(A)c and CryllA delta endotoxins. EPA is currently reviewing Monsanto's proposal.
Current registration requirements require additional data evaluating the cross-resistance
potential to be submitted by January 31, 1998.
Data on the genetics and mechanism of resistance to Bt in TBW, CBW, and PBW are useful to
measure the effectiveness of a long-term resistance management strategy for Bt cotton. Such
information is useful in evaluating which Bt endotoxins could be used effectively in a rotational
or pyramiding scheme. In the absence of field resistance in any of the three target pests,
laboratory-selected tolerant colonies are needed to examine the mechanism of resistance, the
genetic basis for resistance, and the potential for cross-resistance. These laboratory-selected
colonies can also be used to estimate the initial resistant allele frequencies in field populations
and be used to confirm the efficacy of the estimated LC,9 as a discriminating dose. A number
of registrants are collaborating with academic entomologists to develop computer simulation
models which predict the evolution of resistance to CryI(A)b/CryI(A)c proteins by CEW/CBW
within the corn/cotton system. ,
As a requirement of registration, Monsanto must provide annual reports on its monitoring
activities. Monsanto submitted a monitoring and remedial action plan hi 1996. EPA accepted
these plans. The 1996 results of its monitoring program include the development of baseline
susceptibility data and discriminating dose concentrations for each of the three primary target
- pests. Baseline susceptibility and development of diagnostic Bt concentration for monitoring
have been developed for TBW and PBW and are under development for CBW. Validation of
these diagnostic doses for detecting susceptibility changes in Bt cotton fields is needed.
Baseline monitoring studies for TBW/CBW and PBW were expanded hi 1997. No changes hi
susceptibility to the CryI(A)c delta endotoxin were detected hi the tested populations of TBW,
CBW, and PBW. The monitoring programs for TBW, CBW and PBW were expanded hi
1997. Threshold levels for remedial action still need to be developed as well as the remedial
action themselves. . . •
Two structured refuge options were mandated by EPA as mitigation measures: Option A, a
20% sprayed refuge and Option B, a 4% unsprayed refuge; Monsanto's 1996 annual report
. - '. ••'.--•• .-•••' 75 . • ••'•'.. •'•:", .
-------
indicates that there was a high compliance with the refuge requirements amongst the growers
surveyed. Sixty percent used Option A and 38% used Option B. A number of entomologists
have indicated that the lack of a high dose for CBW control was not considered hi models
predicting the effectiveness of the refuge strategy and that the existing refuge requirements
should be modified for Bt cotton. One option discussed by these experts was to discontinue the
4% unsprayed non-Bt cotton refuge because it may not produce enough susceptible insects to
mate with putative resistant insects that emerge from Bt cotton fields throughout the growing
season. These experts believe that the 20% sprayed non-Bt cotton refuge should be continued "
or expanded. They indicate that the sprayed refuge should be planted within one mile of the •
Bt cotton and that it should be managed hi the same way as the Bt cotton. The refuge must be
temporally and agronomically equal to the Bt cotton so that it produces susceptible insects at
the appropriate tune and place to mate with resistant insects produced on the Bt cotton. One
recommendation was that the size of the refuge be expanded to 30% unsprayed non-Bt cotton
acreage. However, such an increase in size of untreated acreage may not be economically
attractive for growers. Yield considerations and cost of maintenance of the refuge acreage are
important considerations for the adoption of a particular refuge option.
,:, • " i if ' , •
Entomologists suggest that a seed mix refuge strategy may work for PBW because of its
limited larval movement. A seed mix refuge strategy to control PBW resistance development
is being field tested over the next 3 years in Arizona.
In conclusion, a great deal of scientific data have been gathered during the first two years of
commercialization of Bt cotton. Progress has been made toward the development of a long-
term resistance management strategy. Additional scientific data are still required as part of the
Bt cotton registration: (1) target and secondary pest biology and ecology, (2) cross-resistance
potential, (3) monitoring data (baseline susceptibility and discriminating dose determination),
(4) effect of Bt cotton on secondary lepidopteran pest (e.g., cabbage looper, soybean looper,
saltmarsh caterpillar, cotton leaf perforator, and ECB), and (5) expression. EPA also required
annual monitoring and annual use reports. EPA mandated specific refugia requirements and
grower compliance has been high. EPA required continued development and distribution of
grower education materials. Grower and consultant education is vital to the success of any
long-term resistance management strategy. The high dose and refuge strategy should be
integrated into a set of best management practices specific for Bt cotton. These practices
include: use of biological control, appropriate use of conventional insecticides to control
CBW, application of pheromone for mating disruption, sterile insect release, and stalk
destruction programs for PBW; application of new selective foliar sprays that kill Bt resistant
and susceptible pests while preserving beneficial,insects to clean up target pests that escape the
insecticide, deployment of effective refuges to manage resistance, and crop rotation.
76
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V. Document Summary .
This paper has provided a review of the public hearing comments and an update on the status
of the resistance management plans in Bt potato, Bt corn, and Bt cotton. EPA is holding a
Science Advisory Panel meeting in February 1998 to obtain input from resistance management
experts regarding this information and the development of long-term resistance management
. strategies for Bt potato, Bt corn, Bt cotton and other Bt crops. The Agency plans to host
additional SAP meetings to continue this evaluation process.
Since Bt plant-pesticides became commercially available in 199(5, growers have adopted this
technology as part of their IPM practices to control pests in potato, corn, and cotton. Based
on industry reports sent to EPA, the greatest adoption of Bt crop technology has been by
cotton growers, especially in the southeastern United States in 1996, with about 13% of the
, cotton acreage, 1.8 million acres, and an estimated 2.2 to 2.4 million acres, in 1997 planted in
Bt cotton. Corn growers planted in Bt corn about 400,000 acres in 30 states in 1996, but are
expected to have planted an estimated 4 million acres in 1997. Potato growers planted about
10,000 acres of Bt potato in 1996 and an estimated 25,000 acres in 1997. The differences in
the rate of adoption of Bt potato, Bt corn, and Bt cotton are likely due,,inpart, to the
availability of effective alternatives, the cost of the biotechnology crop, extent of regional pest
problems, and familiarity and acceptance of the technology by growers. For example, there
are several insecticide alternatives for Colorado potato beetle control. The cost of the
technology and familiarity with the technology .may have discouraged a wider adoption by corn
growers. The adoption rate for Bt cotton was especially high for a new technology because
few, if any, effective alternatives existed to control tobacco budworm in cotton especially
where resistance to registered conventional pesticides was extremely high in states such as
Mississippi and Alabama. . ,
No evidence exists that resistance to Bt Cry proteins produced in transgenic potato, corn, or
cotton has developed in the 1996 or 1997 growing .season. Monitoring for susceptibility
changes to the different registered Cry proteins, GryI(A)b, Cryl(A)c, and CrylHA, has been"
conducted for Colorado potato beetle, European corn borer, tobacco budworm, cotton
bollworm, and pink bollworm. Baseline susceptibility studies show a wide-range of
variability, so it is important to look at susceptibility changes in the context of the baseline
range for a particular geographic location of the pest (i.e., different portions of a State). No
changes in baseline susceptibility have been detected for any of the target insects exposed to
the Cry proteins expressed in Bt potato, Bt corn, and Bt cotton. This information indicates that
there has been no measured increase in tolerance to date to the Cry proteins expressed in Bt
crops. However, current monitoring methods may not be sensitive enough to detect changes in
susceptibility to the toxins.
Laboratory-tolerant colonies of Colorado potato beetle, European corn borer, tobacco
.budworm, and pink bollworm have been created through selection against purified Cry
proteins or mixtures of Cry proteins using Bt microbial pesticides. Laboratory colonies
• . • ' • ." - , - ' 77 '. .'• " ' . '•
-------
tolerant to high levels of Cry proteins do not exist for all target pests, e.g., CEW/CBW. The
ability of insects to develop high levels of tolerance to Bt in the laboratory indicates that these
insects possess the genetic potential to develop resistance to Cry delta endotoxins expressed as
Bt plant-pesticides. It is unlikely that laboratory selective procedures provide the identical
selective conditions as exist hi the field. The ability to select for tolerance to Cry proteins hi
the laboratory in different insect pests indicates that it is prudent to use appropriate resistance
management strategies.
The CBW outbreak beginning in East Texas in 1996 has not been shown to be due to CBW
resistance to the CryI(A)c delta endotoxin expressed in Bt cotton, but due to extremely high
infestation levels of the insect and the lower sensitivity this insect has to the CryI(A)c delta
endotoxin relative to TBW and PBW. Scouting detected the CBW lower in the plant canopy of
Bt cotton than expected and, hi some cases, supplemental chemical insecticides were used to
control CBW. The fact that supplemental insecticides might be necessary to control unusually
high CBW infestations was not unexpected and was considered hi the Agency's review of the
initial resistance management strategy for Bt cotton. Modifications to the CBW scouting
program for Bt cotton were made for the 1997 season to improve detection of the CBW larvae
which might escape the Bt delta endotoxin by feeding on blooms and bloom tags that are lower
in the cotton plant.
Most cotton growers complied with the structured refuge requirements. Cotton growers seem
to prefer the 20% sprayed refuge option (Option B) which allows them to treat the refuge with
chemical insecticides normally used to control TBW, CBW, and PBW (except for Bt microbial
pesticides). This option appears to more reliably provide a higher yield in the refuge acreage
than the 4% unsprayed refuge option (Option A) which often had higher management costs and
lower yields. Most cotton researchers who commented at the two public hearings favored the
20% structure refuge as a better strategy for Bt cotton resistance management because this
refuge is more likely to provide a greater percentage of susceptible .insects throughout the
growing season to mate with any rare resistant individuals that might survive in the Bt cotton
fields. EPA received comments that the 4% unsprayed refuge was decimated early hi the
growing season so there were few, if any, adult moths surviving to mate with any resistant
insects that survived hi the Bt cotton fields.
EPA believed that during the first five years following commercialization (approximate tirne-
limit of the conditional registrations for Bt corn, there would not be enough Bt corn acreage to
provide substantial Bt selection pressure for the development of ECB resistance. Consequently,
EPA did not mandate specific refuge requirements for Bt corn, but EPA has required research
data on the size, structure, and deployment of a structured refuge. A combination of temporal
and structured refuges are being studied. A draft refuge strategy must be submitted to the
Agency by August 1998 and a final refuge strategy is required to be submitted by January
1999. Implementation of an EPA-approved structured refuge plan or an EPA-approved
alternative resistance plan is required no later than April 1, 2001. Monsanto and Dekalb are
requiring structured refuges as part of grower agreements. Beginning in the 1998 growing
, , , ,, . j «,,_,
:• i , ' •' , i"1 ,- /
78
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season, Novartis Seeds has adopted the NC-205 consortium's recommendations published in
NCR-602 publication entitled "Bt Corn & European Corn Borer - Long Term Success Through
Resistance Management" -(Ostlie et al., 1997). The NC-205 recommendation is to have a
structured refuge which is 20-30% non-Bt corn to prevent Bt delta endotoxin exposure to 20-
30% of the larval populations. They also recommended that in continuous corn acreage
sprayed with insecticides, the refuge size would be increased to perhaps 40% to compensate
for larval mortality. In addition, a smaller refuge size may also be suitable if there are many
alternate hosts providing adequate numbers of susceptible ECB. Mycogen has not made any
specific refuge recommendations in its Grower Guide, but is supportive of the use of refuges
.and supportive of the NC-205 recommendations.
Monsanto/Naturemark requires a structured refuge as part of grower agreements for use of Bt
potato. EPA has required that Monsanto mandate specific refuge requirements as a condition
of registration for Bt cotton. Monsantd has implemented these refuge requirements through a
grower agreement. Research is underway to study whether the seed mix option is viable for
PBW resistance management. EPA is encouraged by reports of the high level of compliance
with structured refuge in Bt cotton and Bt potato and by the tremendous reduction in the use of
conventional insecticides that has resulted from adoption of Bt cotton. .
!-""•'" ." - . |
A great deal of research is underway to study the elements that are necessary for long-term
resistance management strategies for Bt potato, Bt corn, and Bt cotton. Specific research data
were required as part of the Bt corn and Bt cotton conditional registrations and was
recommended for the Bt potato registration. These data included: the dosage effectiveness on
the target pest(s), monitoring data including baseline susceptibility and validation of the
diagnostic dose concentration, pest biology and ecology, influence of the Bt crop oh secondary
lepidopteran pests, the impact of CryI(A)b/CryI(A)c produced hi Bt corn on the selection of
CEW/CBW resistance hi Bt corn and Bt cotton, impact of Bt on CEW overwintering survival
and fecundity, effective refuges, alternate hosts as refuges, and cross-resistance potential.
Additionally, alternative pest control strategies and integration into existing IPM programs are
being examined for each of the Bt plant-pesticides. All of these data will provide the basis for
specific improvements to the existing resistance management strategies. Future information is
especially important for understanding the selection of CEW/CBW resistance hi Bt corn and Bt
cotton especially in the southern United -States because CEW/CBW usually moves from silking
corn to cotton, has multiple generations per'year, and overwinters in the South., Exposure to
Cry delta endotoxins produced in both Bt corn and Bt cotton hi two or more generations a year
could rapidly accelerate development of resistance to Cry delta endotoxins. Research results
and predictive models studying this situation are expected to be submitted to the Agency in
1998.
79
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Appendix 1: Table of Pest Acronyms
Acronym
CBW
CEW
CPB
ECB
FAW
PBW
>
SCSB
SWCB
TBW
Common Name
Cotton Bollworm
Corn Ear Worm
Colorado Potato
Beetle
European Corn Borer
Fall Army worm
Pink Bollworm
Southern Corn Stalk
Borer
Southwestern Corn
Borer
Tobacco Budworm
Scientific Name
Helicoverpa zea
(Boddie)
Helicoverpa zea
(Boddie)
Leptinotarsa
decemlineata (Say)
Ostrinia nubilalis
(Huebner)
Spodoptera
frugiperda (J. E.
Smith)
Pectinophora
gossypiella
(Sounders)
Diatraea
crambidoides (Grote)
Diatraea
grandiosella (Dyar)
Heliothis virescens
(Fabricius)
Crop
cotton
corn
potato
corn
corn
cotton
corn
corn
cotton
80
,1 ,, ' „!' iji., ',•'
-------
Appendix 2: Table of Registered Bt Plant-Pesticides
Events/Products
NewLeaf®
176 .
'.
BT11
. MON801
MON810 .
DBT418
Bollgard®
Toxin :
• . Cry IIIA
CryI(A)b.
, CryI(A)b
CryI(A)b
CryI(A)b
Cry I(A)c
CryI(A)c
Crop
Potato
Corn
Co.rn
Corn
Corn
, Corn
Cotton
Company(s)
NatureMark/
Monsanto
Ciba/Novartis Seeds,
Mycogen Corp.
Northrup-
King/Novartis Seeds
Monsanto
Monsanto
DEKALB Genetics
Corp.
Monsanto
81
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