Information Systems for Biotechnology/NBIAP News Report

SPECIAL ISSUE ON BT - December, 1995


In This Issue:
BT Insecticidal Crystal Proteins And Their Mode Of Action
The Empirical And Theoretical Basis For BT Resistance
EPA And BT Plant-Pesticide Resistance Management
Development Of A Comprehensive Resistance Management Plan For Newleaf Potatoes
Resistance Management Strategy For Ciba Seeds' Transgenic BT Corn
Developing New Recombinant Strains Of BT
An Industrial Working Group Promotes Responsible Use Of BT Products
The Business Side Of BT Products: A Market Analysis
Organic Farmers And Genetically Engineered BT
Transgene Pyramids For Resistance Management
Engineering Insect Resistant Plants: BT And Beyond

Introduction
This month, the NBIAP News Report is devoted to the subject of managing pest resistance to Bacillus thuringiensis, an effective and safe biological pesticide commonly referred to as Bt. Crops genetically engineered with Bt toxin genes for protection against insect pests have the potential to reduce chemical pesticide use, thereby lowering the environmental and human health costs engendered by toxic compounds, and to lower production costs. At issue is whether widespread planting of these crops will hasten the development of insect populations that are resistant to Bt, resulting in the loss of this valuable and environmentally benign pesticide.

We asked people in academia, industry, government, and farming to comment on the issue of pest resistance to Bt and how best to preserve the utility of Bt as a biological pesticide. Collectively, their articles describe current thinking with respect to the potential risk, the various approaches to risk management, and what constitutes appropriate regulatory oversight.

The News Report begins with three articles that provide background information and define the issue:

The next four articles offer industry views and resistance management plans for three Bt products, and describe an industry- wide consortium formed to promote responsible use of Bt products.

The last four articles look at the future of Bt products from different perspectives - financial, ecological, and technological points of view are presented.

We hope you find this News Report interesting and informative, and as always, your comments are welcomed.

Pat Traynor, Information Systems for Biotechnology
540-231-2620; email: traynor@nbiap.biochem.vt.edu


BT INSECTICIDAL CRYSTAL PROTEINS AND THEIR MODE OF ACTION

Bacillus thuringiensis (Bt) is a gram-positive soil bacterium noted for its abundant production during sporulation of insecticidal proteins in the form of a crystal or crystal- complex. The insecticidal crystal proteins are commonly designated as "Cry" proteins and the genes encoding them as "cry" genes. Cry proteins have been classified according to their insect specificity and nucleotide sequence (see table below; based on Hofte and Whiteley, 1989; Microbiol. Rev. 53:242). Generalizations in terms of their insecticidal activity have been made across classes of Cry proteins but are not strict. For example, members of a protein class can vary significantly in activity against insects within a single insect Order. The same protein isolated from different Bt strains can vary slightly in its amino acid sequence (1 or 2 residues), resulting in dramatic effects on insecticidal activity.

Gene              Crystal Shape    Protein Size (kDa)    Insect Activity
----              -------------    -----------------     ---------------
cryI; A(a),       Bipyramidal      130-138               Lepidopteran larvae
A(b), A(c), B, 
C, D, E, F,G 

cryII; A, B, C    Cuboid or ??     71, 71, 69            Lepidopteran and 
						         Dipteran larvae

cryIII; A, B,     Flat or          73, 74, 74            Coleopteran larvae 
B(b)              Irregular


cryIV; A, B,      Bipyramidal or   134, 128, 78, 72      Dipteran larvae
C, D              Round (?)

cryV - cryIX      Various          129, 73, 35, 38       Various

To date over fifty Cry proteins have been sequenced and the simplified classification shown above is no longer adequate. Classification schemes based solely on sequence data create a different picture of the relationships among the various Bt proteins, and perhaps more accurately reflect the evolutionary relationships between the various Cry proteins (Yamamoto and Powell, 1993. In "Advanced Engineered Pesticides", Leo Kim, ed., Mercel Dekker, Inc.).

The Cry proteins typically require both solubilization and activation steps before they become biologically active toxins. For most, solubilization occurs in the highly alkaline environment of lepidopteran insect midguts. Activation occurs via discrete proteolysis by insect gut enzymes and may occur concomitantly with the solubilization step. The highly acidic nature of most mammalian guts is not a favorable environment for the Cry toxin. The low pH of most mammal guts would solubilize and denature the Cry proteins, making them susceptible to hydrolysis by native gut proteases into inactive small peptides and free amino acids. The Cry I type proteins are typically processed from a 130-kDa protoxin to the active 55 to 65 kDa form. It is generally accepted that the toxin recognizes certain receptors on the surface of insect midgut epithelial cells. A pore-complex forms through the cell membrane, resulting in the loss of potassium ions which affects the insect's ability to regulate osmotic pressure. Eventually the animal dies due to massive water uptake.

Crystallography studies with Cry IIIA protein toxin (Li, et al; 1991. Nature 353:815) indicate three structurally distinct domains. Domain I consists of seven alpha-helices and is believed to be involved with membrane interactions and the insertion of the toxin into the insect's midgut epithelium and pore formation. Domain II appears as a triangular column of three beta-sheets and is reported to be involved in receptor binding. Domain III consists of anti-parallel beta-strands in a "jellyroll" configuration and, like Domain II, is implicated in insect specificity and stability. It appears that several of the reported cases of insect resistance to specific Cry proteins are due to altered receptor binding specificities.

Presently, our knowledge of the various Cry proteins is insufficient to predict how specific protein modifications may affect the efficacy or activity spectrum of a particular protein. However, as our knowledge base expands we can predict that protein chemists will be making alterations or fusions of the various Cry proteins or their domains, to increase both their potency and activity spectra.

Steve DeWald, Northrup King Co.
507-663-7667; email: dewaldsg@mr.net


THE EMPIRICAL AND THEORETICAL BASIS FOR BT RESISTANCE MANAGEMENT

Bacillus thuringiensis (Bt) toxins are specific to a small subset of insects and quickly break down to non-toxic compounds when exposed to ultraviolet light and other environmental factors. These characteristics of Bt have earned it a reputation for being environmentally friendly; the kind of insecticide we would like to be able to use for a long time.

As Bt toxins become more widely used in transgenic crops, the question of whether targeted insect pests will evolve resistance becomes an important issue. To date, only two species have evolved resistance to Bt in the field but over ten species have been shown capable of evolving resistance based on laboratory selection experiments. For example, the tobacco budworm, which is one of the most important pest targets for Bt-expressing cotton, has evolved a number of different types of resistance to Bt in the laboratory. One strain developed resistance to the CryIA(c) toxin by a mechanism that gave it broad resistance to many diverse Bt toxins including CryIIA, which is extremely different from the CryIA(c) toxin used in selection. This finding indicates that although there are many Bt toxins, once resistance evolves to one of them, we may not be able just to substitute a different Bt toxin.

Another strain of the tobacco budworm that was selected to adapt to the CryIA(c) toxin developed a more specific mechanism for resistance. This strain now has over 5,000 fold resistance to CryIA(c), which means it takes 5000 times more toxin to kill the resistant strain than it takes to kill normal strains. The resistant strain also has high resistance to a number of other Bt toxins such as CryIF. Interestingly, this strain is not highly resistant to CryIIA. It has been estimated that one in a thousand tobacco budworms carries a gene for Bt resistance. This is a higher frequency than expected for conventional pesticides and may be related to the fact that Bt toxins are so species specific.

Strategies to Manage Resistance
With the potential for resistance so high, and our need for environmentally safe insecticides so pressing, it is clear that we need to do something to maintain the efficacy of Bt toxins. Entomologists and population geneticists have been working on ways to slow down or "manage" the evolution of pesticide resistance for many years. These scientists have recently focused their attention on Bt toxins produced by transgenic plants. They have come up with a number of strategies for decreasing the rate at which targeted pests will adapt to Bt-expressing transgenic plants.

I will briefly describe five general strategies developed for managing resistance to Bt expressing crops and will then focus on one strategy that is most likely to be implemented. The five strategies are as follows:
(1) Constitutive expression of high levels of single toxins in all plants
(2) Constitutive expression of high levels of two or more toxins in all plants
(3) Spatial or temporal mixtures of plants having high levels of constitutive expression of one or more toxins with other plants having no toxin expression
(4) Low levels of expression of single toxins interacting with the pests' natural enemies
(5) Targeted Bt gene expression.

Strategies 1 and 2 are based on the view that if we can make the toxic barrier facing the pest extremely difficult to circumvent, the pest is unlikely to have the genetic potential to rapidly overcome the barrier. While these strategies have merit, they are risky because we know that pests have the ability to develop very high levels of resistance and that this resistance may impact the efficacy of a number of Bt toxins. Strategy 4 takes the opposite approach of 1 and 2, with the perspective that in many cases a very low level of toxin that only slows the growth of insect pests could offer sufficient control because the partially debilitated pests would be more vulnerable to predators and parasites for a longer period of time. There have been some field tests that have demonstrated the ecological efficacy of this approach, but there are problems with the consistency of control and companies have not embraced this approach. Strategy 5 is based on the idea that in many crops you don't need to protect all parts of the plant all of the time. In some cases, a pest may feed on both early and late stages of a plant but may only cause economic damage to the late stages (fruiting stages). In such cases advanced techniques in molecular biology could turn on the genes for toxin production only in later stages of plant development. This would decrease the exposure of the pest to the toxin, and thus decrease the rate of resistance development.

Strategy 3, in which plants that constitutively express high levels of one or more toxins are spatially or temporally mixed with plants that do not express any toxin, is the strategy most likely to be implemented in the field. This approach combines strategies 1 and 2 with the presence of non-Bt plants that act as a refuge for susceptible pest individuals. The advantage of this strategy is that even when there are some insects that can genetically overcome the effects of high doses of one or more Bt toxins, they will mate with Bt susceptible insects coming from the refuges of non-Bt plants. Studies have shown that the hybrid offspring of resistant and susceptible individuals can not withstand high doses of Bt toxins. The higher the number of susceptible insects produced in refuges, the more likely they are to mate with resistant individuals that develop on the Bt producing plants. If there are enough of these susceptible insects, almost all of the resistant insects will mate with them instead of mating with other resistant individuals. This should result in a dramatic decrease in the rate at which resistant individuals take over the population.

Potential Problems
There are three potential problems with strategy 3. First, if there is a gene for high Bt resistance that is genetically dominant, the offspring of resistant and susceptible insects will be highly resistant and the population of pests will rapidly be taken over by resistant individuals. Fortunately, such a resistance gene has yet to be encountered. The second problem is that strategy 3 requires that the Bt expressing plants always produce a dose of Bt toxin high enough to kill insects with intermediate levels of resistance. Some of the engineered crop varieties that will be planted in 1996 fall far short of this standard and are likely to undermine this strategy. A final problem with strategy 3 is that the refuge plants must be planted close enough to the Bt plants so that adult insects emerging from the refuge can fly over to the Bt fields and mate with the few resistant insects that emerge from the Bt plants. If the refuge plants are too far away, this won't happen and resistant insects will be likely to mate with each other and will produce cohorts of resistant offspring.

Strategy 3 makes a lot of sense from a theoretical level. However it will be up to companies, farmers, extension agents and regulatory agencies to make sure that this strategy is properly implemented. The EPA has taken an important step in this direction by requiring that in order to sell Bt producing cotton, industry must make sure that non-Bt cotton is planted as a refuge. The next steps are to make sure that the refuges are large enough and in the right places, and that the cotton expresses Bt at high enough levels to kill insects that are partially resistant to the toxin. Because many pests move from one crop to another it will be important to make sure that all crop varieties accessible to a pest either contain a high dose of the toxin(s) or no toxin at all.

Fred Gould, Department of Entomology
North Carolina State University
919-515-1647


EPA AND BT PLANT-PESTICIDE RESISTANCE MANAGEMENT

The problem of pest resistance to pesticides is a worldwide concern. The U.S. Environmental Protection Agency (EPA) has historically considered pesticide resistance management important in determining environmentally sound pest management practices. However, EPA does not have an official policy or standard data requirements in place. With a greater focus on use reduction of the higher risk pesticides, the EPA believes that it is very important to implement effective resistance management strategies. This article will focus on how the Agency has considered pesticide resistance management for pesticides and, in particular, Bt plant-pesticides.

Historical Role of Pesticide Resistance in EPA Regulatory Decisions EPA has considered pesticide resistance when making certain regulatory decisions. The Agency has addressed pesticide resistance issues under a number of sections of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) including: Sections 18 (Emergency Exemptions), 6 (Special Review), and 3 (Registration).

With respect to registration, pesticide resistance has not been a consideration upon determining whether a new pesticide should be registered. However, beginning in the late 1980s, in specific cases where pesticide resistance development has been a concern, EPA has worked with some pesticide registrants to develop appropriate pesticide label language to advise pesticide users on ways to avoid or delay the onset of pesticide resistance. Registration labels have included statements related to resistance management that include recommending the use of alternative pesticides if resistance were already a factor. In addition, EPA has reviewed several pesticide resistance management strategies that were voluntarily submitted to the Agency by pesticide registrants.

The November 23, 1994 Federal Register (59 FR 60496) notice of the Plant-Pesticide Proposed Policy also indicated that the Agency was considering how to best encourage development of agricultural practices that will minimize resistance development to plant-pesticides.

Refining the Role EPA Plays in Pesticide Resistance Management
In August 1992, the Assistant Administrator requested that an Office of Pesticide Programs (OPP) workgroup be formed following discussions at OPP's FIFRA Science Advisory Panel meetings and letters from Public Interest Groups regarding potential for development of pesticide resistance to Bacillus thuringiensis (Bt) foliar sprays because of the pending introduction of Bt plant-pesticides. At this time, the Pesticide Resistance Management Workgroup (PRMW) was formed. The PRMW includes scientists from several scientific disciplines, e.g., plant pathologists, microbiologists, entomologists, weed scientists, biologists, and biochemists. The workgroup considers EPA's role concerning the resistance management of conventional, biological, and genetically-engineered plant pesticides.

Registration of Plant-Pesticides
The PRMW has identified seven elements that need to be addressed to develop an adequate resistance management plan. A subpanel of the FIFRA Science Advisory Panel (SAP) approved of these seven factors on March 1, 1995. These elements are: (1) knowledge of pest biology and ecology, (2) appropriate gene deployment strategy, (3) appropriate refugia (primarily for insecticides), (4) monitoring and reporting of incidents of pesticide resistance development, (5) employment of IPM, (6) communication and educational strategies on use of the product and (7) development of alternative modes of action.

The PRMW has reviewed plant-pesticide resistance management strategies which have been voluntarily submitted by the registrants. Reviews of resistance management plans that have been completed by the PRMW are: (1) the Bt CryIIIA delta endotoxin produced in potato to control Colorado potato beetle (registered May of 1995); (2) the Bt CryIA(b) delta endotoxin produced in field corn to control European corn borer (registered in August of 1995), and (3) the CryIA(c) delta endotoxin produced in cotton to control pink bollworm, cotton bollworm, and tobacco budworm (registered in October of 1995).

OPP used the PRMW's reviews of the management plans to make suggestions to registrants to help them improve their management plans, and, when necessary, establish conditions for the registration of plant pesticides. The EPA believes that resistance management is critical to the long-term viability of plant-pesticides. For example, if no resistance management plan is implemented for Bt plant-pesticides, it is expected that widespread pest resistance would develop in less than five years after transgenic crops have been grown uniformly over large areas following registration. Because the pesticidal protein in Bt plant-pesticides, CryI delta endotoxins, are also widely used in a variety of Bt foliar spray products on many crops, resistance development to Bt plant-pesticides would also affect the efficacy of foliar Bt products.

Workgroup Accomplishments and Proposed Bt Plant-Pesticide Registrant Task Force
The following list summarizes the PRMW's accomplishments on regulation and policy for pesticide resistance management:

(1) Established a list of appropriate factors to be considered in developing a pesticide resistance management plan. This list was approved by the March 1, 1995 Subpanel on Plant-Pesticides of the FIFRA Science Advisory Panel.

(2) Recommended reporting requirements for incidents of pesticide resistance development that are included in the revision of the adverse effects reporting rule (FIFRA Section 6(a)2 Rule, in draft at the time).

(3) Recommended revisions to EPA policy to allow emergency exemptions to be granted under certain conditions for two or more unregistered pesticides for the purpose of avoiding or delaying the buildup of pest resistance (when resistance has not yet been documented). State pesticide regulatory agencies have requested these changes.

(4) Recommended revising EPA policy to include resistance management criteria for issuing special local needs (FIFRA section 24(c)) registrations. EPA proposed a change in policy in the draft guidance for special local needs registrations in which EPA would allow a special local needs registration to avoid or delay the buildup of pest resistance under certain conditions. State pesticide regulatory agencies have requested pesticide resistance management be a part of the guidance document.

(5) Developed criteria for when pesticide resistance management plans should be implemented for experimental use permits (FIFRA Section 5) and prior to registration of a new active ingredient (FIFRA Section 3).

(6) Encouraging the development of a Bt plant-pesticide registrant task force to address, more uniformly, resistance management issues for Bt/corn and Bt/cotton.

Proposed Pesticide Resistance Screening Process and Request For Comments
The Agency believes that resistance management should be considered for all pesticides, but the Workgroup is not recommending across-the-board data requirements for resistance management or specific labeling for all pesticides. A screening process is being considered to identify pesticides and pests which pose the greatest concern for the development of pesticide resistance and pesticide resistance management. At this early stage of development, OPP is considering the following criteria to identify pesticides which may require the development of a pesticide resistance management strategy as a condition of registration: 1) classes of pesticides with a known history of pesticide resistance, 2) target pests with a known history of pest resistance, 3) pesticides with new modes of action, 4) reduced risk pesticides which the Agency has determined require pesticide resistance management concerns, and 5) new uses of pesticides which may dramatically increase the use of a pesticide and consequently pose a greater selection pressure on the target pest(s).

We are encouraging comments on these potential criteria. We would like to know which pests and classes of pesticides pose the greatest resistance management concerns in order for the Agency to more clearly focus its resources. Please send your comments to the Office of Pesticide Programs, U.S. Environmental Protection Agency, 401 M St., S.W., Washington D. C. 20460, or by email to one of the addresses provided.

[The views expressed in this article are those of the authors and do not necessarily represent those of the United States government.] Sharlene R. Matten Environmental Fate and Effects Division (7507C) 703-305-7974; email: matten.sharlene@epamail.epa.gov Paul I. Lewis Special Review and Reregistration Division (7508W) 703-308-8018; email: lewis.paul@epamail.epa.gov


DEVELOPMENT OF A COMPREHENSIVE RESISTANCE MANAGEMENT PLAN FOR NEWLEAF POTATOES

In May of 1995, NewLeaf Russet Burbank potatoes became the first genetically modified, insect-resistant crop to receive full federal regulatory approval for commercialization. NewLeaf potato plants express the CryIIIA protein derived from Bacillus thuringiensis subsp. tenebrionis (B.t.t.), which is selectively active against certain Coleopteran insects including the Colorado potato beetle (CPB, Leptinotarsa decimilineata). Three federal agencies evaluated various aspects of NewLeaf potatoes, including their food quality (Food and Drug Administration), potential for becoming a plant pest (United States Department of Agriculture), and the human, environmental, and non-target safety of the B.t.t. protein itself (Environmental Protection Agency, EPA). The EPA was the lead agency in this process, and their evaluation was as rigorous as that conducted for conventional pesticides. However, unlike their reviews of previous microbial or chemical insecticides, the EPA also considered the risk of insect resistance development to NewLeaf potatoes as part of their regulatory assessment.

In 1992, EPA formed the Pesticide Resistance Management Workgroup (PRMW) within the Office of Pesticide Programs, to begin consideration of resistance as one element of environmental risk in the evaluation of herbicides, fungicides, and insecticides. During the regulatory review process from 1993 to 1995, Monsanto Co. and its seed potato division, NatureMark, worked closely with the PRMW to identify research needs and generate the data necessary to devise workable resistance prevention and management strategies for NewLeaf potatoes. NatureMark, Monsanto Co., outside academic experts, and representatives from the potato industry, including potato growers, met with the EPA repeatedly during the three year review period to keep them abreast of progress toward the development of a comprehensive management plan. In July of 1994, NatureMark and Monsanto submitted a written document to the EPA outlining these resistance management plans. These strategies, briefly described below, were based largely on research results from multiple field and laboratory experiments carried out by academic and government cooperators. They continue to form the basis of NatureMark's resistance management strategy for NewLeaf potatoes.

1) Agronomic and other pest management practices that promote multiple tactics for insect control, including cultural, biological, and chemical factors. NewLeaf potatoes should be incorporated into IPM programs as an integral, but not a stand-alone, measure. Growers should continue to utilize existing, accepted pest management practices that are designed to reduce pesticide inputs and delay resistance development. In addition, it is anticipated that non-chemical tactics for CPB management will increase the potential for biological control by allowing beneficial arthropod predators and parasites to increase in the agroecosystem. These natural enemies can also contribute to pest and resistance management of CPB and other potato insects.

2) Monitoring of CPB populations for susceptibility to the B.t.t. protein. The first step in resistance management is to establish an estimate of the target pest's baseline susceptibility to the pesticide. In 1992, NatureMark initiated a program for monitoring the susceptibility of CPB to the B.t.t. protein expressed in NewLeaf potatoes. This program will continue in both the immediate and long term.

3) High dose expression of the B.t.t. protein in potatoes to control CPB heterozygous for resistance alleles. Assuming that resistance to B.t.t. is the result of a single major gene that is inherited as a recessive or co-dominant trait, the "high-dose" hypothesis predicts that all homozygous susceptible and heterozygous resistant individuals will be killed. Therefore, only homozygous resistant beetles will have the ability to survive on NewLeaf plants. These insects will be extremely rare and will most likely mate with susceptible insects giving rise to heterozygous progeny. The results of several studies support the conclusion that the expression of B.t.t. protein in the NewLeaf potatoes represents a high-dose approach to pest and resistance management.

4) Refugia as hosts for B.t.t. susceptible insects provided through non-CPB resistant potatoes. There are several potential strategies under review for incorporating a refuge for B.t.t. susceptible CPB into the cropping system such as seed mixes, trap cropping. etc. In addition, not all potato varieties will contain the B.t.t. protein. A number of factors will dictate the choice and ultimate success of the various strategies. These include, but are not limited to, the extent of CPB movement from plant to plant, mate and host finding behavior, and industry acceptance.

5) Development of novel CPB control proteins with a distinct mode of action from the NewLeaf insect control protein. Multiple gene and alternate gene strategies hold potential for substantially delaying or halting resistance development. Monsanto is actively searching for additional insect active proteins and other mechanisms for transgenic control of the Colorado potato beetle.

On March 1, 1995, following internal review and prior to granting the registration for the B.t.t. protein, the EPA convened a Scientific Advisory Panel (SAP) meeting to facilitate an open discussion and review of the resistance management plan by individuals from academia, industry, regulatory agencies, and the general public. Members of the SAP consisted of several entomologists, plant breeders, toxicologists and other biologists. The panelists found the plan to be detailed, thorough, and appropriate to delay or prevent the widespread development of resistance by the CPB to the B.t.t. protein produced in NewLeaf potatoes (U.S. EPA memorandum, 1995). The SAP also recommended that the Agency approve the registration of the B.t.t. protein so that NewLeaf potatoes could be commercialized.

Shortly after the SAP, on March 2, the USDA granted a determination of nonregulated status for seven lines of NewLeaf Russet Burbank potatoes. The EPA registration of the B.t.t. protein produced in NewLeaf potatoes was finalized on May 5, 1995. This action signified the final federal regulatory approval necessary for the sale of these seed potatoes to growers.

NewLeaf potatoes were commercialized in 1995 and Monsanto recently received registration for the B.t.k. protein in BollGard cotton. Both of these crops will be widely planted in 1996. Monsanto's pest and resistance management research programs continue to generate new data and information, which are broadening our understanding of pest biology and behavior, and the potential of these insects to develop resistance to Bt. Monsanto and NatureMark have been proactive in developing strategies to preserve the long-term durability of these products. The development and implementation of resistance management strategies is an ongoing and dynamic process. Innovation, flexibility and the integrated efforts of industry, academic and government scientists, as well as growers, is required to successfully implement these strategies. Monsanto intends to remain a leader in the development and commercialization of transgenic plants and remains committed to deploying these products so that the efficacy of Bt will be preserved.

Terry Stone, Monsanto Co.
314-537-6547; email: tbston@monsanto.com

Jennifer Feldman, NatureMark
208-344-4491


RESISTANCE MANAGEMENT STRATEGY FOR CIBA SEEDS' TRANSGENIC BT CORN

Ciba Seeds and Mycogen Plant Sciences recently received the first approvals from United States government regulatory agencies to market hybrid field corn that produces an insect control protein representing a truncated form of the CryIA(b) protein that occurs naturally in Bacillus thuringiensis subsp. kurstaki (B.t.k.). The product, commonly referred to as 'Bt Corn', will be marketed by Ciba Seeds as Maximizer hybrid corn with KnockOut built-in corn borer control. Naturally-occurring B.t.k. proteins have been commercially produced and used as insecticides for decades. An extensive body of safety testing and experience supports their lack of toxicity to humans and other mammals, and the absence of adverse effects on nontarget organisms and the environment.

Transgenic Corn Resistant to European Corn Borer B.t.k. proteins are very effective against certain lepidopteran (caterpillar) insects, including European corn borer (ECB), Ostrinia nubilalis (Hubner). This major corn pest reduces yield by disrupting normal plant physiology and causing physical damage to the plant and ear that results in stalk lodging, dropped ears and damaged grain. Economic loss in the United States due to ECB infestation is estimated to approach $1 billion annually, with yield losses as high as 30% in heavily infested fields. Although chemical insecticides (e.g., organophosphates and synthetic pyrethroids) and foliar Bt microbial insecticides can be somewhat effective against ECB, applications must be carefully timed before the insect bores into the stalk, and repeat applications are often necessary to achieve limited control.

Results of small-scale field tests performed from 1991 to 1995 by Ciba Seeds indicate that corn plants producing the CryIA(b) protein are highly effective in controlling ECB, even though only minute quantities of the insect control protein are produced. Under conditions of natural ECB infestation, hybrids expressing the CryIA(b) protein exhibited significantly increased yield and overall superior agronomic performance as compared to non-transformed isogenic control hybrids grown under identical conditions.

Crop plants genetically engineered to resist pests potentially fall under the jurisdictions of three federal agencies: the U.S. Department of Agriculture (USDA), the Food and Drug Administration (FDA), and the Environmental Protection Agency (EPA). The USDA's regulatory oversight extends to the importation, interstate movement and environmental release of any organism that poses a potential threat to U.S. agriculture. For many genetically engineered crops, these activities require a simple notification to USDA in order for an applicant to pursue any of these activities. To commercialize a crop that falls under the USDA's rules, the crop must be exempted from further regulation by a formal petition process. In addition, companies commercializing transgenic crops are consulting with the FDA in compliance with that agency's 1992 policy covering foods developed from new varieties of crops. Finally, EPA has proposed a policy under which plant-produced pesticides could be regulated under existing pesticide statute. Ciba Seeds worked with all three federal agencies in order to clear Maximizer corn for commercialization. This process concluded with EPA's granting of a pesticide registration for marketing and sale of Maximizer hybrid corn with KnockOut built-in corn borer control. During the registration process, EPA decided that a regulatory approach toward managing potential pest resistance to the active endotoxin was appropriate, given the unique status of Bt as a "natural resource."

Occurrence of Insect Resistance to Bt Endotoxins
The only documented instance of insect resistance to Bt endotoxins, resulting from repeated use of Bt products in agriculture, is in the diamondback moth (Plutella xylostella). In laboratory studies, insects derived from fields with a history of repeated, intensive use of foliarly-applied commercial Bt products had significantly higher tolerance to Bt endotoxin than laboratory strains of P. xylostella.

Resistance to Bt endotoxins (commercial formulations or purified endotoxin) in laboratory-reared colonies of several insects has been reported. In each case, insects were acutely exposed to sublethal concentrations of a Bt preparation, with each subsequent generation exposed to incrementally higher concentrations. Researchers have reported the selection of insect colonies that are >150 times more tolerant to endotoxin than unselected laboratory colonies. Several of these endotoxin-tolerant colonies have been bioassayed on appropriate transgenic host plants that express the same endotoxin, or an endotoxin to which the insect has exhibited cross-resistance in vitro. In each case, there have been few if any surviving insects, even in those instances where the plants expressed very low amounts of endotoxin. There is little argument that these colonies are valuable research tools. However, it is not yet clear whether the genotypic properties associated with the observed in vitro tolerance to Bt will bear any resemblance to the mechanism(s) of resistance that may develop under actual field conditions.

Ciba Seeds Resistance Management Strategy for Bt Corn
Ciba Seeds and Mycogen Plant Sciences submitted to the EPA two supplements to the Bt Corn registration package that were directed specifically to the issue of resistance management. One document focused its discussion on European corn borer, and provided an overview of the current state of knowledge and theory of resistance management principles as they applied to ECB and corn. The second supplement focused its attention on lepidopteran pests other than ECB.

Early in 1995, the EPA convened a Science Advisory Panel to address what general elements should be part of resistance management strategies that may be applied to all transgenic plants expressing Bt delta endotoxins. A public meeting with this expert panel was held later that Spring, at which Ciba Seeds addressed specific questions regarding Bt Corn. During this period and in the ensuing months leading up to registration, Ciba and Mycogen maintained an ongoing dialogue with the EPA's Pesticide Resistance Management Workgroup, the group charged with reviewing resistance management plans. Ciba and Mycogen developed a plan for research, monitoring, mitigation and education that will be workable for at least the first five years of commercialization of Bt Corn. The plan presented below details Ciba Seeds' resistance management strategy for its product.

High Dose Strategy & Refugia
One component of this resistance management program is the adoption of a high dose strategy. The corn transformation event that was chosen for commercialization expresses the insecticidal protein in green tissue and pollen at a level exceeding the LC99 for ECB by up to 33-fold. This nearly guarantees that all ECB that are heterozygous for a "resistance" allele will receive a lethal dose of the CryIA(b) protein after exposure to these tissues.

It is the general belief among resistance management researchers that the success of the high dose strategy is contingent upon there being sufficient refuge (non-toxic host plants). This refuge will serve to maintain sufficient susceptible (non-Bt resistant) insect populations to reduce the likelihood of any resistant individuals mating with each other. The refuge would reduce the buildup of any homozygote populations, thus preventing the proliferation of resistance.

Ciba Seeds recognizes that refugia are part of an effective resistance management program. What is not yet apparent, however, is 1) how much non-Bt Corn refuge will be necessary to maintain susceptible populations, and 2) the most effective way to deploy the refuge. Based upon the diversity of researcher's opinions on the size of the refuge (5 - 50%), and deployment strategies (seed mixtures; on-farm; adjacent/independent acreage; 50/50 early-late season planting) Ciba believes it is premature to recommend to growers a specific refugia strategy until there is field-validated data to support it. The refugia strategy must be proven practical, and be able to be integrated into current agronomic practices employed by growers.

Ciba Seeds is committed to the concept of refugia as a component of a viable resistance management strategy. For the first several years of commercialization of Ciba's Bt Corn, adequate amounts of refugia will be available throughout the Bt Corn distribution area due to a number of factors. These factors include considerations of market share, farmer adoption rates and buying practices. Extensive market research data were collected by Strategic Marketing Research & Planning, Inc. (Chesterfield, MO) under contract to Ciba Seeds. In a 1994 survey, 925 corn growers across the major corn growing areas of the U.S. farmers were asked about their hybrid buying practices. On average, corn growers purchase a total of six hybrids from two or three different seed companies. The survey results clearly indicate that growers manage their production risk through the genetic diversity of their chosen corn hybrids, and that this hybrid diversity increases with the size of the grower's production.

Information contained within the market research report clearly supports the conclusion that growers typically plant 4-5 established hybrids for conventional production purposes on the majority of their corn acreage. The remaining 1-2 hybrids, representing new hybrids on the market, are typically planted on smaller plots for evaluation purposes and comparison against the more established hybrids with which the grower has had prior experience. These survey conclusions reinforce the well-known grower conservatism in adopting new hybrids. They also reinforce the commonly recognized risk management practices of using established hybrids from different seed companies on the majority of acres planted, while experimenting with newer hybrids on fewer acres, as a means of maintaining diversity of germplasm that can perform well under a variety of environmental conditions. Based upon these grower buying and adoption practices, more than adequate refugia on non-Bt Corn will already exist and will be widely maintained by market forces for at least the first few years after commercial introduction of Ciba Seeds' Bt Corn.

Monitoring Program & Resistance Mitigation
Since 1993 Ciba Seeds, as a member of the Bt Resistance Management Workgroup (an industry consortium), has been sponsoring research at academic institutions to establish CryIA(b) baseline susceptibility values for ECB populations throughout the major corn growing regions of the United States. As a component of its management plan, Ciba will continue to sponsor these studies. In addition, populations of ECB will be collected from representative distribution areas of Bt Corn, with particular focus on those areas of highest distribution. The in vitro susceptibility of these populations to CryIA(b) protein will be compared to the historical CryIA(b)-susceptibility as determined from the baseline studies. Elevation of in vitro CryIA(b)-tolerance levels may be an indication that selection for resistance is occurring in the field.

Customers will be instructed to contact Ciba Seeds if incidents of unexpected levels of ECB damage occur in fields of Bt Corn. Ciba Seeds will investigate and identify the cause for this damage by local field sampling of plant tissue to confirm that the plants contain CryIA(b) protein, as well as sampling of local ECB populations for in vitro CryIA(b)-susceptibility studies. If the LC50 of the local ECB populations to CryIA(b) exceeds certain levels, as established by the baseline susceptibility studies, and these same ECB populations cause excessive damage to and survive on CryIA(b)-positive leaf tissue, this will be considered a confirmed case of resistance. Ciba Seeds will report any confirmed finding of resistance to the EPA within 30 days, and immediately initiate mitigation measures (customer notification; recommend use of alternative ECB-control measures; recommend that crop residue be soil-incorporated to minimize the overwintering of ECB). Within 90 days of a confirmed incidence of resistance Ciba Seeds will also increase the ECB-monitoring program in the affected area, and implement a structured refuge strategy (coordinated by EPA with other registrants).

If these efforts are not effective in mitigating resistance, Ciba Seeds will voluntarily cease sales of all Bt Corn in the affected county as well as bordering counties until an effective EPA- approved resistance management plan is implemented. The execution of such a strategy will be coordinated by the EPA with other registrants. Ciba Seeds may resume sales in affected areas when the EPA agrees that an appropriate resistance management plan has been implemented.

Grower Communication & Research Program
Ciba Seeds will implement a grower education program directed at increasing grower awareness of resistance management. As specific Bt Corn resistance management recommendations are developed through ongoing research and experience, these will be incorporated into various grower communication and educational media (e.g., technical bulletins, sales and marketing brochures). Ciba Seeds is also developing a Grower Guide that will include current information regarding Bt Corn resistance management and integrated pest management.

Ciba Seeds will expand its current in-house resistance management research program and also its research funding at public institutions. Current target areas of research include ECB movement and mating patterns, field evaluation of refuge options, effects of Bt Corn on pests other than ECB, identification of new ECB control principles, and studies focused on the biology and genetics of ECB resistance to Bt. Ciba Seeds will work closely with the EPA in developing this multifaceted program, and will apply the findings toward development of a long-term resistance management strategy.

Jeffrey Stein and Richard Lotstein, Ciba Seeds
919-541-8683; email: steinj@am.abru.cg.com


DEVELOPING NEW RECOMBINANT STRAINS OF BT

Bacillus thuringiensis (or Bt, as it is commonly called) has been the active ingredient in a wide array of biological insecticides for nearly a half century. These products have been utilized as highly safe alternatives and supplements to chemical insecticides for applications in agriculture and forestry, and for control of disease vectors such as mosquitos and blackflies. In agriculture, most of the products have been based on a single strain of Bt, termed HD-1, that was isolated in 1970 by Dr. H. Dulmage at the USDA Cotton Insects Research Laboratory in Brownsville, Texas.

Since the early 1980's, research on Bt has shown that the insecticidal crystal proteins (ICPs) produced by sporulating cells are encoded on extrachromosomal plasmids. These plasmids are capable of being transferred between strains of Bt by a conjugation-like process. It has also been shown that many strains of Bt harbor multiple ICP genes, thus producing either multiple insecticidal crystals or mixed crystals containing several different but related ICPs. Bioassays of individual purified ICPs have revealed that each one has a unique insecticidal activity spectrum for certain insects in the Lepidoptera (caterpillar), Coleoptera (beetle) or Diptera (fly) groups. These ICPs are classified according to their genetic relatedness, and there have by now been some thirty or more different ICP genes cloned and sequenced. By a process of cloning and expression of individual ICPs in a common Bt strain background, it is now possible to identify those that are particularly active on various target insects, and to use genetic techniques to construct strains carrying several ICPs selected for both optimized activity on the desired insect targets and for the management of the potential for insect resistance.

At Ecogen we have utilized these approaches to construct a number of new Bt strains for different crop and insect applications. The choice of Bt itself as the expression host has several advantages. First is that Bt is naturally capable of stably maintaining several different ICP genes without undergoing loss or gene rearrangement. Second, Bt can express these genes to high levels such that 25-30% of its total protein can be ICP protein. Third, we can take advantage of natural Bt plasmids as cloning vectors for constructing new ICP combinations, as well as a Bt transposon that encodes both a transposase and a site-specific recombinase. These elements greatly facilitate the construction of new ICP combinations that do not contain antibiotic resistance genes or other undesired foreign genes. Thus, the new Bt constructs consist only of Bt DNA, a definite advantage when seeking regulatory approval for conducting large-scale field trials or product registration. In fact, by employing this strategy Ecogen was granted a blanket approval in 1992 by the Environmental Protection Agency for conducting small-scale field trials of any new recombinant Bt without having to obtain separate approvals.

Ecogen's first new product derived by this recombinant technology is called Raven, and was developed as a superior product for control of Colorado potato beetle, as well as caterpillar pests of potato, tomato, and eggplant. This product, the first live Bt derived by recombinant DNA technologies, was approved for registration by the EPA within ten and a half months of submission. The Raven strain contains two different ICP proteins of the beetle-active CryIII group, in addition to two caterpillar-active CryI genes. The two CryIII genes contribute to a much higher productivity in fermentation of this strain as compared to its predecessor strain in the now-discontinued Foil product.

In addition, the particular combination of genes in the Raven strain is designed to minimize the development of resistance to the product by the Colorado potato beetle, which is recognized as perhaps one of the most active of all insect pests in developing resistance to chemical insecticides. This strategy involves two different approaches. First is that the two CryIII proteins expressed in the Raven strain have different binding characteristics on potato beetle midgut cell membranes. In studies conducted with researchers at Michigan State University, laboratory-selected potato beetles that are resistant to one of the CryIII proteins showed only minimal resistance to the second CryIII. Thus, in practice, an individual beetle would have to undergo two independent resistance mutations to become resistant to the Raven product.

Second, it was found that when the beetle strain selected for resistance to the one CryIII protein is exposed to a mixture of that CryIII protein and the CryI protein contained in Raven, the CryIII resistance is strongly reduced. This effect is presumably due to some protein-protein interaction that occurs between the two ICPs at the level of midgut binding. Thus, the Raven strain incorporates two different strategies to minimize the likelihood that the principal insect target would develop resistance to the product.

Currently we have two other products under development using the recombinant system described, one (CryMax) for applications on an array of caterpillar pests of vegetables and horticultural crops, and a second (CryStar) specifically aimed at the control of fall armyworm on sweet corn and other vegetables, an insect for which no Bt product is currently available. In the future we expect to continue to develop novel ICPs that have different properties with respect to their modes of action on important insect pests. These activities will come from a combination of new gene discovery efforts and by employing approaches such as protein engineering of selected genes to alter their activities and other physiological properties. We believe that this combined approach will allow us not only to develop new and improved products, but also to effectively manage the potential for insect resistance development by continuing to exploit the ability of Bt to express multiple ICP genes having a diversity of activities.

Bruce C. Carlton, Ecogen Inc.
215-757-1595 ext. 216


AN INDUSTRIAL WORKING GROUP PROMOTES RESPONSIBLE USE OF BT PRODUCTS

The Bt Management Working Group (BtMWG) is an international group of 13 companies that are using a variety of approaches to develop improved Bt-based products for plant protection. These approaches include traditional strain optimization, genetic improvements resulting in new combinations of insect control proteins, and novel Bt delivery methods such as transgenic plants.

Founded in 1988, the group is committed to fostering the judicious use of Bt-based products, funding research to address both the potential for development of resistance to Bt and to develop strategies that will minimize or prevent resistance. Over the past six years we have provided $370,000 in funding, nearly 100% of our budget, for research projects at twelve university labs.We have disseminated this research information by sponsoring various symposia on insect resistance at both the annual meetings of the Entomological Society of America and the Society of Invertebrate Pathology. At these symposia, scientists we supported presented their research results. A summary document has been produced that highlights more than 30 abstracts from publications that resulted, at least partly, from our research funds.

Due to the diversity of Bt-based products, the BtMWG anticipates wider use of biological pest control tactics and has taken a proactive approach to minimize the threat of insect resistance to these products. Bt products must be wisely used in IPM programs. Technical representatives from all member companies, working as a team, meet regularly to review scientific issues related to the preservation of Bt as a biocontrol agent.

Individually and in collaboration as an industry, companies developing Bt products have devised resistance management plans to ensure the long-term durability of Bt. This effective collaboration grew out of a sense of cooperation fostered by the BtMWG. The most critical challenge of the next few years will be the implementation of sound management strategies. Successful implementation will extend the team based approach to include end users, specifically pest control agents, applicators, growers and product distributors.

The members of the BtMWG recognize the importance of Bt as a valuable pest control resource. Investment in programs to assess and manage resistance risks represents an important component of stewardship of Bt products, to ensure their continued value as safe and effective tools for crop protection. Industrial participants in the BtMWG include: Abbott Laboratories, Calgene Inc., Ciba, DeKalb Genetics, DuPont Agricultural Products, Ecogen Inc., ICI Agrochemicals / ICI Seeds, Monsanto Company, Mycogen Corporation, Novo Nordisk Entotech Inc., Plant Genetic Systems, Pioneer Hi-bred, and Sandoz Agro Inc.

Sue MacIntosh, Plant Genetic Systems
Chairperson, BtMWG
515-276-6642; email: suemac94@aol.com


THE BUSINESS SIDE OF BT PRODUCTS: A MARKET ANALYSIS

Chemical control of insect pests is one of the most costly aspects of crop production, estimated to be $3-5 billion annually worldwide. The costs for pesticide application and from economic losses to pests can be staggering. Recent estimates (TIBTECH 13:362-368, Sept. 1995; Bio/technology 13:434-435, May 1995) include the following: Each year in the U.S. over $400 million is spent just for the control of lepidopteran pests. Coleopteran pests such as Colorado potato beetle (CPB) and corn rootworms cost U.S. farmers more than $1 billion annually. Insect damage to cotton crops costs an estimated $645 million per year; cotton bollworm is responsible for annual losses estimated at $16 million. Potato growers spend between $75 and $100 million to control CPB on 480,000 hectares of potatoes. European corn borer (ECB) causes an estimated $1 billion in crop losses annually in the United States. ECB costs $50 million to the state of Illinois alone; losses in Nebraska are over $48 million

Genetic engineering techniques are offering relief from insect pests through the development of two new insect control measures based on Bacillus thuringiensis (Bt) genes. First is the development of new bioinsecticides based on genetically modified microbial strains. These Bt strains are improved from the wild type strains that have been in use for years. The genetically modified strains are more potent and do not need to be applied as often as non-engineered strains. Some of the new modified strains have an increased range of target pests; other strains target specific pests such as the fall army worm, the number one problem in sweet corn for which no biopesticide currently exists. The costs to the farmer are the same as other for Bt sprays, but, due to a decrease in the number of applications required, the genetically modified products could actually lower expenses.

The second type of insect control based on Bt is the insertion of Bt toxin genes directly into plants. Genetic engineering of plants to make them resistant to specific insect pests has become a reality. The first Bt plant products will soon be hitting the market. The industry has collectively spent hundreds of millions of dollars on Bt biotechnology in plants, and millions more will likely be required for future products. Introduction of insect resistant crop plants, however, should benefit not only farmers by cutting their crop production costs, but also the environment by reducing the amount of pesticides used.

Over 3 million acres of corn are treated with pesticides to control European corn borer (ECB). Ciba Seeds (Greensboro, NC) has just introduced Maximizer hybrid corn, which contains a Bt gene that confers resistance to ECB. Losses due to ECB can be as high as 8% to 10% per borer per stalk, with an average of 3.3 larvae per stalk. In addition to saving on time and costs of pesticide applications, the grower who plants Bt corn does not need to spend as much time in the field scouting for pests. Results from 1994 demonstrated that Maximizer hybrids had a 14.3 bushel yield increase, 0.63 pound heavier test weight and were 75% drier than conventional hybrids.

Mycogen (San Diego, CA) was recently approved to sell its own new corn hybrids, also resistant to ECB. Sold under the name NatureGard, these hybrids were developed with both Bt and native resistance genes in the same plant and will be available for commercial sale in 1996. Additional crops resistant to other insect are under development and will also be sold under the NatureGard umbrella.

Partnerships Pool Technologies and Resources
According to Carl Eibl, executive VP of Mycogen, companies such as Mycogen have neither the time nor the resources to go it alone in developing new engineered plant varieties, and thus seek partnerships to develop the technology. Mycogen is now collaborating with Pioneer Hybrid (Des Moines, IA) for development of insect resistant crops based on Bt. Pioneer is providing $51 million in total funds; $21 million for R&D and $30 million to purchase 3 million shares of Mycogen common stock. In addition, Pioneer will devote staff and other resources to the project. In return, Pioneer will receive non-exclusive rights to all Bt crop protection technology developed by Mycogen during the next 10 years.

Other companies are also forming agreements to share Bt technology. Ecogen (Langhorne, PA) and Monsanto (St. Louis, MO) are pairing up in a $25 million deal in which Monsanto will develop Ecogen's Bt technology for in-plant applications. Ecogen owns one of the world's largest Bt gene libraries with more than 10,000 strains. Monsanto will use the gene library to develop in- plant Bt varieties, while Ecogen will continue development of Bt spray products.

These collaborations, by combining complementary areas of expertise, will allow companies to make more rapid progress in development of new agricultural products. For example, Mycogen will now be able to develop four or five products at a time instead of just one. Mycogen has already received U.S. Patents covering techniques to alter Bt genes to resemble plant gene sequences. Additional patents have been granted, or are pending, for over 30 Bt gene sequences encoding toxins active against numerous pests. In addition to Mycogen's Bt corn hybrid with resistance to ECB, the company has developed a proprietary delivery system for a Bt-based bioinsecticide.

Although licensing agreements have been developed for particular aspects of Bt technology, rivalry between companies is still keen. As is often the case in the biotechnology industry, the courts have become one of the major venues for competition. Mycogen recently filed a declaratory judgement action in Federal District Court in San Diego seeking to invalidate two U.S. patents held by Plant Genetic Systems (PGS-Ghent, Belgium) covering pest resistance technology for plants. This is in response to notification of PGS's intentions to file suit alleging that Mycogen and Ciba Seeds' insect-resistant corn infringed on PGS's patents covering truncated Bt genes in plants (Biotech Reporter 12 (11):1,4; November 1995).

Companies Protect Their Investments
With large investments at stake, companies are also concerned about the stability of the products protected by these patents. The major concern is the potential for increased resistance in insects exposed continually to high levels of Bt, as would be found in transgenic plants. Companies have been developing strategies to combat such occurrences.

Monsanto has been studying the problem for over ten years, and believes they have developed a strategy that will ensure the longevity of their products. The company plans to advise growers of their transgenic cotton to follow specific agronomic practices, such as crop rotation and revised planting and plow down dates that minimize the insect's exposure to Bt. The company is requiring farmers to sign a licensing agreement and attend training and educational meetings in order to purchase their Bt engineered seed. Farmers will have to agree to a resistance management plan, but will have the option to follow different plans that create refuges for sensitive insects. For example, for every 100 acres planted with Bt producing plants, the farmer must also plant 25 acres of non-producing plants of the same crop. These plants are not allowed to be treated with any type of Bt spray or product but can be treated with other types of insecticides. An alternative approach is to plant 4 acres of non-producing plants for every 100 acres of Bt producing plants, but not treat them with any pesticides at all.

Farmers have shown enthusiasm for Bt producing plants. Monsanto is currently marketing their Bt cotton to farmers who spray at least three or more times per growing season. Many farmers spray cotton up to six or seven times while others only require one spray. Approximately 16 million acres of cotton are in production in the U.S.; of these, four to five million acres require at least three sprays. Seed costs will be approximately the same as for non-Bt seeds, but growers will also have to pay a small technology fee, as well as an insect control fee based on the numbers of acres to be planted. The fee is expected to be between $30 and $40 per acre. The farmer, however, will save on not having to buy chemical insect controls, as well as on the expenses incurred to apply those chemicals, such as renting airplanes for spray applications.

Because insect resistance to spray Bt insecticides is a potential problem, Mycogen emphasizes that microbial Bt products should be used as part of on overall pest control program that also includes use of beneficial insects and crop rotation. Problems arise when a single chemical pest control method is relied upon as the exclusive control measure forcing the insect into evolving resistance. Mycogen recommends a management program with several approaches. Planting different fields with different types of resistance in each helps to ensure that corn borers always face different control methods, thus making unlikely that they will develop resistance to any one method. Selection of planting dates to avoid ECB infestations based on local trends can also decrease incidence of the problem. Additional controls include select usage of proper pesticides and cultural controls to remove debris in which ECB overwinters.

Investors Are Becoming Optimistic
As pesticide resistant crops near market, it appears that Wall Street's chill towards some agbiotech stocks may be starting to thaw. While these stocks in general have been ignored in recent years, some of the companies working with Bt genes have seen their stocks rise. Mycogen's stock price as of the end of November continued to hover around its 52-week high of $14.25 per share after having climbed almost 35 percent in September due to the partnership with Pioneer Hybrid. Pioneer's stock has also done well, having reached a 52-week high of $56.5 per share at press time. Monsanto, another major Bt company that has made significant investments in biotechnology, has also seen steady growth of its stock price during 1995, increasing from $72 to over $118 per share since December of 1994.

Sano Shimoda, President of BioScience Securities (Orinda, CA), an institutional research and investment banking firm focusing on agricultural, chemical, and environmental companies, believes that this is only the tip of the iceberg. Shimoda believes that new agricultural products now coming to market, brought about by breakthroughs in genetic engineering and biotechnology, represent a revolutionary change in the agricultural industry. He feels that the increases seen in some of the agbiotech stocks are signs that Wall Street is beginning to pay attention, and that within the next 12 months, market pull from farmers desiring new Bt products will capture investor's full attention. Investments in Bt companies such as Mycogen and Ecogen by larger ag corporations like Pioneer and Monsanto help validate the technology and stimulate interest on Wall Street.

The new Bt products, both plants and sprays, are greeted with enthusiasm by farmers with high hopes of controlling destructive insect pests. While biopesticides currently only represent a small fraction of the multi-billion dollar pesticide industry, the market will likely grow rapidly as an increasing number of genetically engineered pesticides reach the market and pressures mount to replace more traditional toxic chemicals.

William O. Bullock and Cynthia Sollod
Institute for Biotechnology Information
919-544-5111; e-mail: bbullock@nando.net.


ORGANIC FARMERS AND GENETICALLY ENGINEERED BT

Organic farmers have traditionally avoided the use of synthetic materials in crop production because, as a rule, they short circuit, rather than enhance, the ecological balances of nature. Synthetic materials, consequently, often create the problems they purport to solve. For example, R. Hindmarsh has pointed out that annual crop losses to insects doubled during the same period of time that insecticide use increased tenfold (The Ecologist, Sept. 1991, pp.198-199).

Organic farmers predict that genetically engineered organisms in farming systems will have similar, but accelerated effects. Their predictions may be well founded. The past may be a harbinger of the future. The systematic application of synthetic agrochemicals induced resistance in fungi, insects and other disease vectors. We know that this happened because the use of these technologies fostered crop homogenization which, in turn, created the need for more of the same technologies. Mono-cropping leads to more of the same crop pests and therefore the application of more of the same pesticides. Any situation where pest control is homogenized, while the pest naturally evolves, will lead to greater resistance. Genetically engineering Bt into crop plants doesn't alter this pattern, it only accelerates it.

Bt sprays have long been used successfully in diversified cropping systems as a limited-use pesticide, by organic farmers and backyard gardeners. Organic farmers' first line of defense against pests is to increase and manage biodiversity. They only use Bt sprays in emergencies. The combination of maintaining a balanced relationship between pests and plants, and drastically limiting the amount of Bt released into the environment in any given time period, slows or even eliminates the probability of resistance.

By persistently placing Bt into the environment with transgenic crops, pests will likely build up resistance to Bt in five to seven years (some scientists predict three years). The rule is that the more we increase homogenization, taper genetic precision and/or reduce diversity in response to specific pest problems, the more likely we are to increase resistance. As a result, genetic engineering will, in a few years, destroy a tool that organic farmers have used effectively for decades. It is ironic that transgenic crops are being introduced to reduce chemical pesticide use when they are taking away one of the rare pest control tools that farmers now use to avoid synthetic chemicals.

While it is heartening to see that companies developing Bt transgenic plants recognize that resistance will be a problem, their proposals for reducing resistance with unique field management recommendations are ineffectual. Most farmers operate under very intense financial constraints. Consequently, they make field management decisions based on immediate financial returns, not future problems. They are too preoccupied with today's doom to worry much about tomorrow's doom.

In the early 1980's, for example, farmers in North Dakota were warned by everyone from extension agents to seed sales people, that failing to rotate sunflowers would invite sunflower insect and disease disasters. But sunflowers were a good cash crop that produced much needed revenue, so most farmers raised sunflowers in the same fields at least every other year, and in some instances they continuous-cropped them. Within a few years insect and disease problems became so severe that the cost of pest control forced many farmers to get out of sunflower production. It is not that farmers were stupid or unconvinced of the risks. Short term economics simply took precedence over long term economics. Management decisions for transgenic plants will be no different.

But resistance is not the only problem. Equally disturbing to organic farmers is the possible ecological disruptions which transgenic Bt crops may cause. History clearly demonstrates that we can never predict all the effects that the introduction of a novel organism may have on a complex and interconnected ecology. Examples abound. Just recently (New York Times, October 9, 1995) two USDA scientists reported that the infestation of the beet armyworm on Rio Grande Valley and San Angelo, Texas cotton crops may have been caused by heavy applications of malathion designed to eradicate the boll weevil. The malathion, they said, caused "a disruption of the beneficial insect complex that normally suppresses the beet armyworm." Transgenic crops, which introduce instantaneously-created new life forms into the environment, dramatically increase the potential for such disruptions, many of which may be irreversible.

Moving cautiously with respect to introducing transgenic crops is essential. We must ask societal and ecological questions that extend the level of inquiry far beyond whether this will boost the production of cotton, corn or potatoes, or make pest control more convenient next year.

Frederick Kirschenmann, President
Farm Verified Organic, Inc.
701-486-3578; email: 76372.2132@compuserve.com

Carolyn Raffensperger, Executive Director
The Science and Environmental Health Network
701-763-6287; email: 75114.1164@compuserve.com


TRANSGENE PYRAMIDS FOR RESISTANCE MANAGEMENT

There is a strong risk that large-scale deployment of plants genetically engineered with Bacillus thuringiensis (Bt) genes will cause insects to become resistant. Bt has been used as a biological insecticide for over 30 years as a spray. Sprays are not often used by growers, however, because of relatively highcost, critical timing of application, and low efficacy. Thus, although Bt sprays have been used commercially and Bt microorganisms co-exist naturally with herbivores, there has been little opportunity for insects to evolve resistance. This scenario may change rapidly because of massive deployment of Bt transgenic crops such as cotton, corn, and potato that will imminently be commercialized. Plants containing a single insect resistance transgene will be planted commercially by growers for the first time in the USA in the Spring of 1996.

In transgenic plants, Bt insecticidal crystal proteins will be produced continually in plant tissue. This effect will create strong selection pressure on both economically important and unimportant insects. If genes conferring resistance to Bt become fixed in insect populations, both sprays and transgenic plants would become ineffective as insect control agents.

Researchers are formulating strategies to delay the onset of resistance to Bt. Planting strategies typically manipulate transgenic plants in time and space in order to prevent immediate development of resistance. These include using rotations (in which transgenics may be alternated in time with non- transgenics), refugia (in which a portion of a field may be planted with non-transgenics), and mosaics (in which mixtures of transgenic and non-transgenic plants are grown together). There are no empirical data showing that any of the above strategies are universally better than the others, and mathematical models predict various results that depend upon initial assumptions and parameters. A serious drawback to any of the time and space strategies is that they may be difficult for farmers to implement. For instance, if farmers were to deviate from the prescribed planting regime, the strategy would fail.

Use of multiple transgene combinations (gene pyramiding), would arguably be more effective and easier to implement. Instead of directional selection driving insect populations toward resistance to Bt, other genes engineered into plants would give insect pests additional forces to reckon with. They would then have to overcome two or more insecticidal genes to become resistant, a less likely occurrence known as disruptional selection. Besides helping to prevent the development of resistant insect biotypes, combining Bt with another gene or genes for insect resistance also increases the toxic effect to insects. For example, feeding assays have shown that mixtures of Bt and proteinase inhibitors have a synergistic interaction.

Some genes are better candidates than others for pyramiding with Bt genes. For example, combining two Bt genes would not be an effective strategy since it has been shown that insects that are resistant to one are likely resistant to other Bt toxins, a phenomenon known as cross-resistance. Furthermore, genes already resident in a crop plant may not generate enough disruptive selection to appreciably slow down the development of resistance to Bt. The best pyramiding strategy would combine Bt genes with unrelated transgenes whose products have different modes of action. Additionally, these genes should code for proteins that insects have not been heavily exposed to.

The class of insect resistance genes that encode proteinase inhibitors provides some good candidates for pyramiding with Bt genes. Preliminary findings, however, indicate that more than one proteinase inhibitor may be necessary for effective control. A likely reason for this is that many insect species have evolved partial resistance to individual proteinase inhibitors because they have been exposed to these naturally-occurring compounds for a long time. In one study a cabbage proteinase inhibitor that was not very effective on cabbage worms was nonetheless effective in controlling insects that do not typically feed on cabbage (Broadway, 1995. J. Insect Physiology 41:107-116).

Transgenic plants expressing a single plant proteinase inhibitor usually do not control insects as effectively as Bt transgenics. In certain crops, combining two or more proteinase inhibitor genes may be more effective. For example, combining genes from different sources (e.g., pairing proteinase inhibitor genes from a plant and an insect) may be an effective general strategy. Thus, in some insects and plant combinations, the effect of proteinase inhibitors may be increased synergistically. The proteinase inhibitor pair may then be combined with Bt to create strong disruptional selection for resistance in insect populations, significantly delaying resistance to either Bt or the tandem proteinase inhibitor suite.

As crop plants become more easily engineered and more research is performed in gene pyramiding, in ten years it may be unusual for any insecticidal transgenic plants to have fewer than five transgenes. If Bt is to be one of those genes, we must ensure its long-term viability and efficacy.

C. Neal Stewart, Jr., Department of Biology
University of North Carolina at Greensboro
910-334-5391 ext. 52; email: nstewart@goodall.uncg.edu


ENGINEERING INSECT RESISTANT PLANTS: BT AND BEYOND

First generation transgenic crops will be protected from insect feeding by single genes that encode, as the primary gene product, an insecticidal protein. In addition to Bt toxins, there are a number of naturally occurring biologically active proteins that can and will be used to protect plants from insects. Examples include lectins, protease inhibitors, antibodies, wasp and spider toxins, and insect peptide hormones. In the case of Bt toxins, this simple approach has produced crops with surprising levels of resistance to insects. As successful as the primary gene product approach is, however, it represents only the beginning of the many possibilities in store for future transgenic plants.

Advances in our understanding of protein structure and function is allowing researchers, using protein engineering technologies, to begin to design chimeric proteins. These chimeras are constructed by piecing together at the gene level discrete functional parts of the protein, called domains. For example, different Bt toxin domains (discussed in the first article) can be swapped to change or increase their normal species activity spectrum.

Besides Bt toxins, nature has produced a myriad of unique proteins that are very toxic to insects. The long-term goal of protein engineering is construction of modular "smart proteins" that will target specific pests and, like Bt, not harm beneficial animals. In principle, any domain from any protein can be used in this modular system to construct proteins with a given set of attributes. Although still in its infancy, protein engineering will allow us to design proteins for use against our most severe pests -- pests that presently are not controlled by Bt toxins.

Entomologists have long known that plants make use of a wide array of non-protein molecules to kill insects. Some of these compounds, such as the insecticide azadirachtin, isolated from the Neem tree, are widely used by people and are seen as safe biological pesticides. Unfortunately, these non-protein toxins are produced as a result of metabolic pathways that rely on several enzymes to produce the active molecule. Early research avoided work on these potent insecticidal molecules because it was thought that entire pathways would have to be engineered into a plant for production of the compound. However, since even the most complex molecules are derived secondarily from metabolic pathways common to many plants, there is the potential to identify insecticidal molecules that are a mere enzymatic step away from those existing pathways. Identification of the enzyme responsible for the terminal step and transfer of its gene to the crop plant may result in expression of the non-protein compound. Functionally, the engineered enzyme acts as a primary gene product.

A case in point is the non-protein compound limonene. This natural product occurs in many fruits and vegetables. Recent experiments show that transformation of corn with the gene encoding limonene synthase results in enhanced accumulation of limonene (Meyer and Roth, 1994. International patent application No. PCT/US94/03011; International Publication WO 94/22304). This has led to control of some insect pests, and was accomplished without the need for complete reengineering of the plant.

In summary, we are witnessing a truly revolutionary time in agriculture. Transgenic plants expressing Bt toxins provide a level of insect control not seen since the introduction of chemical insecticides decades ago. Unlike the release of new technologies in the past, we in industry and academia are keenly aware of the need to manage resistance in insects to this promising new technology. Accordingly, other non-Bt proteins are being identified that will be used in combination with, or in place of, Bt as one component of resistance management. As exciting as this first generation technology is, it pales in comparison to the second and third generation technologies under development. We can expect to see in the future "smart-proteins" designed and constructed by protein engineering that will specifically target even our most problematic pests. Finally, the ability to engineer plants to produce novel non-protein compounds will provide crop pest managers with an almost limitless number of options for controlling insect pests.

Joseph E. Huesing, Northrup King Co.
507-663-7670; email: huesing@mr.net


The material in this News Report is compiled by NBIAP's Information Systems for Biotechnology, a joint project of USDA/CSREES and the Virginia Polytechnic Institute and State University. It does not necessarily reflect the views of the U.S. Department of Agriculture or of Virginia Tech.

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