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Since their introduction in 1996, the area grown to transgenic plants
expressing insecticidal proteins from the bacterium Bacillus thuringiensis
(Bt) has grown rapidly. In 2004, Bt plants were grown on over 13 million ha
in the U.S. and 22.4 million ha worldwide1,2. Bt corn and Bt cotton
comprised about one-third and one-half, respectively, of the U.S. corn and
cotton markets in 2004. Worldwide, approximately 15 million ha of Bt corn
and 7.4 million ha of Bt cotton were grown in 2004. The economic and
environmental benefits of Bt crops, compared to other technologies, have
been well-documented3. Reports from China have also shown that fewer
insecticide poisonings occur when Bt cotton is used4, September 2005 by
Anthony Shelton.

Despite the extensive use of Bt crops over an 8-year period, there have
been no reports of product failure or increased resistance in insect pests5.
To put this in perspective, Bt crops have already exceeded the length of
time that typically passes in the field before resistance is first
documented with most conventional neurotoxic pesticides, despite undergoing
what has been hailed as one of the world?s largest selection for
resistance6. However, because of the demonstrated ability of two insect
species (the diamondback moth and the cabbage looper) to evolve resistance
to sprays of Bt proteins in commercial settings, there is concern that some
insects may also evolve resistance in the field to Bt plants. To prevent or
delay resistance to Bt crops, various insecticide resistance management
(IRM) strategies have been proposed. These strategies include manipulation
of the transgenic elements of the plants, such as the genes and promoters,
and the manner in which plants are deployed in the landscape. The most
widely employed tactic to delay resistance is the use of plants expressing a
high dose of a single Bt protein throughout the life of the plant, combined
with a "refuge" of non-Bt plants nearby that can maintain an adequate supply
of susceptible alleles within the insect population. We believe this "high
dose/refuge" strategy has played a major role in the lack of resistance to
date for Bt plants, but recognize that other strategies must be developed as
the use of Bt plants continues to grow and selection for resistance
intensifies.

Over the past 15 years, our cooperative program has tested various IRM
strategies using a unique system. The system is composed of different
populations of diamondback moth that have evolved resistance to one or two
Bt proteins (Cry1A and/or Cry1C) and broccoli plants that express either one
or both of these proteins. Our greenhouse and field tests are described in a
recent paper6 in a historical perspective of IRM and Bt plants. In that
paper, we describe the use of this unique system to demonstrate the
effectiveness of the "high dose/refuge" strategy and the use of an inducible
promoter as "proof of concept" by using the inducible promoter so that the
Bt protein is expressed only at a specific period of time, thus reducing the
selection pressure for resistance. Such inducible promoters allow Bt
proteins to be expressed only during specific periods of time, and could be
used in situations in which plants could withstand some insect defoliation
early in their growth, but the marketable part of the plant must be kept
clean during the later stages of growth (e.g., tomatoes).

An IRM strategy outlined in a 1998 paper by Roush7 suggested that pyramiding
two dissimilar Bt proteins in the same plant could delay resistance
development compared to plants that expressed only one Bt protein. The
models contained in the paper were evaluated using our diamondback moth/Bt
broccoli system, and we found that such Bt pyramided plants significantly
delayed the evolution of resistance8.

Pyramided cotton plants ("Bollgard II") with two genes derived from Bt
(Cry1Ac and Cry2Ab2) were approved for commercial use in Australia and the
U.S. in 2002, and several companies are developing new cotton and corn
varieties with pyramided Bt genes. However, there is concern that the
optimal benefits of pyramided Bt genes for resistance management may be lost
if one-gene plants sharing similar Bt toxins continue to be deployed. Newly
developed pyramided varieties of Bt cotton and corn currently contain the
same or similar genes as one-gene (Cry1Ac for Bt cotton, Cry1Ab for Bt corn)
Bt plants already marketed. If market forces result in a complicated
landscape mix of one- and two-gene Bt plants, the benefits of pyramided Bt
plants for slowing resistance evolution could be undermined. For example, a
modeling study9 suggested that Cry2A resistance evolution in a cotton pest
was maximized when Bt cotton varieties expressing one- and two-genes were
both available, and that the overall durability of two-gene plants would be
greater if they were deployed alone, compared to a sequential or mosaic
deployment with Bollgard (Cry1Ac alone). However, the risk of pest
adaptation to pyramided Bt plants used in conjunction with one-gene plants
had not been quantified empirically.

To assess the risk of resistance evolution to pyramided plants when they
were simultaneously deployed with single gene plants containing one of the
Bt genes in the pyramided Bt plants, we used broccoli plants transformed to
express different Cry toxins (Cry1Ac, Cry1C, or both) and populations of
diamondback moth carrying resistance to each of the Bt Cry toxins8. Three
treatments were tested in separate large greenhouse cages: (1) 45% Cry1Ac
and 45% two-gene plants plus 10% refuge; (2) 45% Cry1C and 45% two-gene
plants plus 10% refuge; (3) 90% two-gene plants plus 10% refuge. Diamondback
moths with a known frequency of resistance alleles to Cry1Ac and Cry1C were
then introduced into each cage. We allowed the insect populations in each
cage to reproduce normally over time. Every few generations, we counted the
number of insects produced in each cage and determined the frequency of
resistance alleles in the insect population. The results were rather
dramatic. After 24 ? 26 generations of selection in the greenhouse, the
concurrent use of one- and two-gene plants resulted in control failure of
both types of Bt plants. When only two-gene plants were used in the cage, no
or few insects survived in subsequent tests on one- or two-gene Bt plants.
Overall, this clearly indicated that the concurrent use of transgenic plants
expressing a single and two Bt genes will select for resistance to two-gene
plants more rapidly than the use of two-gene plants alone. The results of
this experiment agree with the predictions of a Mendelian deterministic
simulation model.

What does this mean for the commercial use of Bt plants? Simply put, the
concurrent use of single and two-gene Bt plants can offer exposed insect
populations a "stepping stone" to develop resistance to both toxins. Thus,
from a resistance management perspective, it appears that using pyramided Bt
plants simultaneously with single-gene plants, if they share similar Bt
toxins, will negate some of the benefits of the two-gene plants. In
Australia, pyramided Bt cotton (Bollgard II) has been commercially available
since 2002. The use of both one- and two-gene varieties was permitted for
the first two years following the introduction of Bollgard II, but now only
two-gene varieties are allowed. The rapid phaseout of one-gene varieties was
intended to minimize pest exposure to the single Bt toxin and thus to reduce
the risk of resistance to pyramided plants. In the U.S., plants with a
single Bt gene remain the dominant Bt varieties. Our results indicate that
the introduction of pyramided plants with currently deployed single gene
plants should be examined carefully by regulatory agencies. Our data are
consistent with models and suggest that, from an IRM standpoint, it could be
advantageous for regulatory agencies to consider deregulating single gene
plants as soon as pyramided plants are available.

We recognize that economic considerations must be factored into decisions
regarding the deployment of single and pyramided gene plants. From an
industry standpoint, however, there could be distinct economic and marketing
advantages for promoting pyramided plants rather than single gene plants. In
addition to providing superior resistance management, pyramided plants may
provide improved control of some harder-to-kill insects, and require a
smaller area set aside for the refuge. The smaller refuge size has
particular relevance for IRM; models have indicated that a 30?40% refuge for
single gene plants is equivalent to a 5?10% refuge for pyramided plants7.
Thus, the two-gene strategy is especially suitable for developing countries
such as China, where farms on average are only 0.5 ha, and the practice of
setting aside land for a refuge is highly impractical.

References

1. U.S. Department of Agriculture?NASS (2004)

2. James C. (2004) ISAAA Briefs No. 32. Ithaca, NY: ISAAA. 43 pp

3. Shelton AM, Zhao J-Z & Roush RT. (2002) Ann. Rev. Entomol. 47, 845-881

4. Pray CE, Ma D, Huang J & Qiao F. (2001) Impact of Bt cotton in China.
World Dev. 29, 813-825

5. Tabashnik BE, Carri?re Y, Dennehy TJ, Morin S, Sisterson MS, Roush RT,
Shelton AM & Zhao J-Z. (2003) J. Econ. Entomol. 96, 1031-1038

6. Bates SL, Zhao J-Z, Roush RT & Shelton AM. (2005) Nature Biotechnol. 22,
57-62

7. Roush RT. (1998) Phil. Trans. R. Soc. Lond. B 353, 1777-1786

8. Zhao J-Z, Cao J, Li YX, Collins HL, Roush RT, Earle ED & Shelton AM.
(2003) Nature Biotechnol. 21, 1493-1497

9. U.S. Environmental Protection Agency (2002)

Anthony M. Shelton*, J-Z Zhao*, J Cao?, HL Collins*, SL Bates*, RT Roush§, &
ED Earle?

*Dept. of Entomology, Cornell Univ. / NYSAES, Geneva, NY
?Dept. of Plant Breeding and Genetics, Cornell Univ., Ithaca, NY
§Statewide IPM Program, University of California, Davis, CA

[www.isb.vt.edu]

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