Pest resistance to insecticides is a worldwide phenomenon and current estimates indicate that over 500 arthropod species have developed strains resistant to one or more pesticides. These cases of resistance are not limited to synthetic insecticides, but also include a wide range of `natural products' including pathogens and insect growth regulators. Unfortunately, this also includes resistance to toxins from strains of the common bacterium, Bacillus thuringiensis (Bt). Bt products had been used for more than 40 years as insecticidal sprays without any evidence of resistance in field situations until a report from the Philippines indicated control failures of the diamondback moth (DBM), Plutella xylostella. Subsequent studies in Hawaii and Florida documented the genetic basis of resistance of Bt and further reports have documented control failures of Bt against DBM in other parts of the US, Japan, Central America and China. Laboratory populations of at least 10 species of moths, two species of beetles and four species of flies have been exposed to selection against Bt toxins and ten-fold increases in tolerance have occurred in nine of the 16 species (1). These findings warn of the possibility of insects developing resistance to transgenic insecticidal plants containing Bt toxins. Although there are no cases of insects developing resistance to Bt transgenic plants in the field, laboratory populations of Cry1A-resistant DBM have been shown to be able to survive on transgenic crucifers expressing high levels of Cry1Ac (2).

The development of resistance to Bt transgenic plants would negate the benefits of this new technology, now grown on ca. 11.8 million hectares worldwide. Some of the benefits of Bt plants can be illustrated with Bt cotton in the US. Since the commercialization of Bt cotton in 1996, insecticide spays on cotton have been reduced by approximately 3.8 million liters of formulated product per year in the US and this has led to a significant reduction in the use of more hazardous organophosphate and pyrethroid insecticides (3).

Can Bt plants be deployed so that resistance will be delayed or avoided? It has been suggested that Bt plants may, in fact, be more effective at managing resistance than Bt foliar sprays because one is able to regulate the dose more effectively in the plant than with a spray. While this may bode well for Bt plants, the question still remains about how to deploy them to reduce the likelihood of resistance developing. Various strategies have been proposed but the only commercially available approach is the use of a high dose of a single gene, producing 25 times the toxin concentration needed to kill susceptible insects, in combination with a refuge. The refuge is composed of non-transgenic plants and is intended to generate sufficient numbers of susceptible insects to dilute resistant alleles, while at the same time allowing the non-transgenic plants to generate high yields. While this sounds like a good idea, there is considerable debate on the required size of a refuge. Presently, a 20% refuge is recommended for cotton and corn but some workers have called for refuges as large as 50%, if farmers are allowed to spray them. This allotment size presents a dilemma since farm profitability and reduction of pesticide use may come from larger proportions of transgenic crops, but long-term enjoyment of these benefits may be feasible only by limiting the percentage of the crops that are transgenic.

We have used DBM in combination with crucifers engineered to express a Cry1Ac toxin to study factors that influence the development of resistance (4). In greenhouse trials we introduced DBM that had an initially low Cry1Ac resistance gene (R) frequency into cages with various ratios of Bt broccoli and non-Bt broccoli plants. The insect populations were allowed to cycle for several generations and then the larvae were tested for resistance. Pure stands of Bt-expressing plants (0% refuge) resulted in rapid development of highly resistant DBM populations, and increasing the size of the refuge delayed the development of resistance. Furthermore, the placement of the refuge plants significantly affected the development of resistance. When both plant types were mixed in a random spatial arrangement (`mixed seedling model') larvae were able to move between plant types. As they moved from refuge plants to Bt-expressing plants, they died and caused a decline in the number of susceptible alleles (S) in the overall population. This resulted in a more rapid development of resistance than when plants were separated by a distance that limited the movement of larvae.

We then took our studies into the field. For the first year of tests, we examined the effect of refuge size and refuge placement (mixed vs. separate refuges) on the distribution of the larvae within the plots as well as the level of resistance in DBM at the end of the season. Our results demonstrated that the cumulative number of larvae per plant on refuge plants through the season in the 20% mixed refuge was significantly lower (6.4 vs. 14.6) than the 20% separate refuge. This finding indicates that, as in our previous greenhouse experiments, a separate refuge is more effective at conserving the number of susceptible alleles. This is because larvae on these refuge plants will be more likely to survive to adults (either SS or RS) that can mate with RR individuals and thereby reduce the number of RR offspring. This evidence supports the use of a separate refuge for Bt-transgenic crops susceptible to insects that can move between plants as larvae.

We then examined the effects of spraying plants in the 20% separate refuge and, as expected, spraying reduced the capacity of the refuge to dilute resistance in the larger field. Our results indicated that if the refuge were left unsprayed, it would give a larger number of susceptible insects a chance to survive. The short-term sacrifices of having relatively more insects in the unsprayed refuge would translate to seasonal reductions in resistance and in the total number of larvae per plot. However, the critical question is whether such populations would result in unacceptable crop losses, and the answer to this will depend on the particular crop/insect system and the techniques used to manage the insects in the refuge.

Our studies provided the first empirical data demonstrating the usefulness of the refuge strategy for Bt plants. However, they also indicate the need to effectively monitor and manage susceptible alleles on an individual field or farm basis as well as on an area-wide basis. Within an individual field or farm, treating the refuge with a highly effective insecticide may dilute the abundance of susceptible alleles to such an extent that the refuge becomes less effective unless there is substantial immigration of susceptible alleles from wild hosts or from surrounding non-Bt crops. On the other hand, growers may be reluctant to sacrifice a large number of refuge plants to insects just to maintain susceptible alleles. Critical experiments need to be performed in the specific insect/Bt crop system to determine the correct balance between conserving susceptible alleles while providing acceptable crop yields.

The theory of resistance management has the potential to work in the field for the first generation of insecticidal plants. New technologies under development for the second generation of plants include Bt expression modes that subject insects to selection pressure for specified periods of time, and in particular, plant parts by using inducible and/or tissue specific promoters. These techniques may allow for larger refuges for susceptible alleles both within the field and within a region while at the same time minimizing crop loss. Other options are also possible. Theoretical models suggest that pyramiding two dissimilar toxin genes in the same plant has the potential to delay the onset of resistance much more effectively than single-toxin plants released spatially or temporally and may require smaller refuges (5). Other non-Bt genes may also aid in managing resistance to Bt crops.

The development and implementation of engineered insecticidal plants is currently in its infancy but is providing substantial benefits for growers and the environment. It is important that industry, public-sector scientists, and farmers work together to develop a second generation of technology and implementation strategies to ensure that insects do not rapidly develop resistance to Bt crops.

Sources

1. Tabashnik BE. 1994. Evolution of resistance to Bacillus thuringiensis. Annual Review of Entomology 39:47-79.

2. Metz TD, Roush RT, Tang JD, Shelton AM, and Earle ED. 1995. Transgenic broccoli expressing a Bacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies. Molecular Breeding 1:309-317.

3. US Environmental Protection Agency. 27 May 1999, revisions 12 July 1999. [Online.] EPA and USDA position on insect resistance management in Bt crops. http://www.epa.gov/oppbppd1/biopesticides/otherdocs/bt_position_paper_618.htm.

4. Shelton AM, Tang JD, Roush RT, Metz TD, and Earle ED. 1999. Field tests on managing resistance to Bt-engineered plants. NatureBiotechnology 18: 339-342.

5. Roush RT. 1998. Two-toxin strategies for management of insecticidal transgenic crops: Can pyramiding succeed where pesticide mixture have not? Philosophical Transactions Royal Society of London B 353: 1777-1786.

A. M. Shelton
J. Z. Zhao
Cornell University
mailto:ams5@cornell.edu

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