2-Plants: First field system to test Bt resistance management plans developed (2)
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TITLE: Field tests on managing resistance to Bt-engineered plants
SOURCE: Nature Biotechnology, Volume 18 Number 3
by A. M. Shelton et al.
DATE: March 2000
-------------------- archive: http://www.gene.ch/ --------------------
Field tests on managing resistance to Bt-engineered plants
Anthony M. Shelton1, Juliet D. Tang1, 2, Richard T. Roush3, 4,
Metz5, 6 & Elizabeth D. Earle5
[see also Cornell University Press Release at
1. Department of Entomology, Cornell University, New York State
Agricultural Experiment Station, Geneva, NY 14456.
2. Current address: Department of Entomology and Plant Pathology,
Mississippi State University, MS 39762.
3. Department of Entomology, Cornell University, Ithaca, NY 14853.
4. Current address: Department of Crop Protection, Waite Institute,
PMB 1, Glen Osmond, SouthAustralia 5064 Australia.
5. Department of Plant Breeding, Cornell University, Ithaca, NY 14853.
6. Current address: Department of Biological Sciences,Campbell
University, Buies Creek, NC 27506.
Correspondence should be addressed to A M Shelton. e-mail:
Several important crops have been engineered to express toxins of
Bacillus thuringiensis (Bt) for insect control. In 1999, US farmers
planted nearly 8 million hectares (nearly 20 million acres) of
transgenic Bt crops approved by the EPA. Bt-transgenic plants can
greatly reduce the use of broader spectrum insecticides, but insect
resistance may hinder this technology. Present resistance management
strategies rely on a "refuge"composed of non-Bt plants to conserve
susceptible alleles. We have used Bt-transgenic broccoli plants and
the diamondback moth as a model system to examine resistance
management strategies. The higher number of larvae on refuge plants
in our field tests indicate that a "separate refuge" will be more
effective at conserving susceptible larvae than a "mixed refuge" and
would thereby reduce the number of homozygous resistant (RR)
offspring. Our field tests also examined the strategy of spraying the
refuge to prevent economic loss to the crop while maintaining
susceptible alleles in the population. Results indicate that great
care must be taken to ensure that refuges, particularly those sprayed
with efficacious insecticides, produce adequate numbers of
susceptible alleles. Each insect/Bt crop system may have unique
management requirements because of the biology of the insect, but our
studies validate the need for a refuge. As we learn more about how to
refine our present resistance management strategies, it is important
to also develop the next generation of technology and implementation
Keywords: Insecta, Plutella xylostella, Bacillus thuringiensis,
resistance, transgenic plants
Expression of proteins produced by a common bacterium, Bacillus
thuringiensis (Bt), in transgenic plants to protect them from insect
attack is revolutionizing agriculture1. The insecticidal proteins
produced by Bt are toxic to major pests of many of the world's most
important crops such as cotton, rice, and corn. Of the $US 8.1
billion spent annually on insecticides worldwide, it is estimated
that nearly $2.7 billion could be substituted with Bt biotechnology
applications2. At least 16 companies are presently developing
transgenic crops with Bt genes, and at least 18 Bt-transgenic crops
have been approved by the US Department of Agriculture (USDA) for
When incorporated into plants, Bt proteins are made much more
persistent and effective, even against insects that feed at sites
difficult or impossible to reach with sprays4. Bt cotton was one of
the first insecticidal plants to be approved for commercial use in
1995, and since then the adoption of this technology has been rapid
not only in the United States but also in Australia and China. The
reasons for the rapid adoption of this new technology are compelling.
For example, Bt cotton required three or fewer insecticide
treatments, compared with historical averages of 5-12insecticide
sprays per year for cotton in the United States5. Despite the
considerable advantages of Bt-transgenic crops, both to the
environment and to farm worker safety, concern is widespread that
these gains will be short-lived because of evolution of resistance in
Various deployment strategies have been proposed to delay the onset
of resistance6, and modeling studies have examined the effect of
different deployment strategies7-10; however, few empirical data
exist. The only commercially available strategy is use of a high dose
of a single gene (>LC90 of heterozygous RS insects) in combination
with a refuge. The refuge is composed of nontransgenic plants that
will generate enough SS (homozygous susceptible) individuals to
outnumber RR (homozygous resistant) individuals during mating, so
that the majority of the population will remain either RS or SS. For
cotton in the United States, "expert opinion" in 1994 and a marketing
strategy have resulted in the nontransgenic plants being deployed as
either a separate refuge in which 20% of the field is planted in
nontransgenic plants that can be treated with a non-Bt foliar
insecticide, or a 4% refuge of nontransgenic plants that are left
untreated. The concept is that the refuge will generate enough
susceptible insects to dilute resistant alleles while at the same
time allowing the nontransgenic plants to generate high yields.
Recently the debate on the appropriate strategies for controlling
insects through the use of Bt plants has focused on the size of the
refuge needed11, or indeed whether refuges that are large enough can
be economically acceptable to the users or sellers of Bt crops. In
cotton, for example, some workers have called for a dramatic increase
in refuge sizes over the Environmental Protection Agency (EPA)
requirements, such as refuges as large as 50%, if farmers are allowed
to spray them12.The use of current transgenic cultivars thus faces
the following dilemma. The maximum benefits to crop production, 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. Careful modeling studies and empirical data are
needed to address this question.
Testing a resistance management strategy is inherently difficult
because it requires both a Bt-expressing plant and an insect that has
developed resistance to the Bt toxin expressed in the plant. For this
study,we have used the diamondback moth, Plutella xylostella, the
only insect that has developed resistance to Bt toxins in the
field13, in combination with crucifers engineered to express a
Cry1A(c) toxin14 to study factors that influence the development of
resistance. Resistance in this population of diamondback moth was due
to a single autosomal recessive gene15, and the plants expressed high
levels of the toxin14.
In green house trials16 we introduced diamondback moths that had an
initially low Cry1A(c) resistance gene frequency into cages with
various ratios of Bt broccoli and non-Bt broccoli plants. The insect
populations were allowed to cycle and, after a set number of
generations, the larvae were tested for resistance. We found that
pure stands of Bt-expressing plants (0% refuge) resulted in rapid
development of highly resistant diamondback moth 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 an
overall decline in the number of susceptible alleles. This resulted
in a more rapid development of resistance than when plants were
separated by a distance that limited the movement of larvae.
Additional greenhouse and laboratory data demonstrated that resistant
diamondback moths display similar levels of weight gain, growth, and
survival on Bt plants as they do on non-Bt plants17.
These studies have documented that Bt-resistant insects can survive
on Bt plants and that different management strategies will influence
the durability of resistance. Although these studies provided some
insight into variables that could be manipulated to delay the onset
of resistance, the present field study was performed to provide
further data to help identify variables that may influence resistance
management in the field.
Results and discussion
Our 1996 field experiment 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
diamondback moths 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 (Table 1). This finding
indicates that, as in our previous greenhouse experiments, a separate
refuge is more effective at conserving the number of susceptible
alleles 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 finding provides
evidence to support the use of a separate refuge for Bt-transgenic
crops that are attacked by insects that can move between plants as
larvae. On the Bt-expressing plants over the season, an average of
0.3 larva was found in any of the treatments, indicating that the
diamondback moth population was being controlled by the Bt-expressing
plants (Table 1). This was also confirmed by the absence of any
larvae on the Bt-expressing plants at the end of the season. In leaf-
dip assays taken through the season, no differences in susceptibility
were detected between diamondback moths taken from any of the
treatments (Table2). Furthermore, comparing the level of resistance
at the beginning of the test to the level at the end, it appears that
the insects actually became more susceptible. This was the result of
immigration of native susceptible diamondback moths into our field
plots, which diluted the frequency of resistant alleles of the
released insects and prevented the establishment of resistance even
when R allele frequencies of released larvae were as high as 0.12.
This result was not seen in our previous greenhouse studies in which
we had a closed system prohibiting immigration. Despite the
differences in the number of larvae on refuge plants in the mixed and
separate refuges in this field study (Table 1), we were not able to
document differences in mortality (Table 2) over the relatively short
period of thisexperiment. However, the differences in larval
populations on the refuge plants in these treatments do lay the
groundwork for differences in susceptibility to occur given a longer
Our results from this field study might be taken as justification for
not needing any refuge within a planting because of the presence of
immigrating susceptible alleles. However, such an approach would only
be justified if immigration patterns of susceptible insects were well
known and had been shown to be consistent. Usually one does not know
a priori whether such immigration of susceptible alleles will occur.
Under conditions in which there is no such immigration, high levels
of resistance and crop damage can occur16.
Growers may be unwilling to sacrifice large numbers of refuge plants
to delay the onset of resistance. Thus, current recommendations allow
the management of insects on these refuge plants through the use of
insecticides with a different mode of action than the Bt-transgenic
plants. The critical question in such a strategy is whether enough
susceptible insects will survive in the refuge to provide an
effective source of susceptible alleles.
Our 1997 field experiments examined the results of spraying the
plants in the 20% separate refuge. Because there is no documented
cross-resistance between Cry1C and Cry 1A Bt toxins18, 19, we
examined how spraying the refuge with M-C (Mycogen, encapsulated
Cry1C) affected DBM larval density and resistance on Cry1Ac broccoli.
Our results indicate that in both 100% refuge treatments (where
insects were released or where insects were not released),
susceptibility increased significantly over time (Fig. 1).With a
discriminating dose of 10 p.p.m., the population had a rate of 27%
mortality before release into the treatments, but in both 100% refuge
treatments the mortality at 10 p.p.m. increased to >70% by the third
count. The similar increase in susceptibility in both treatments is
indicative of immigration of susceptible insects into those plots, as
was also seen in the 1996 field studies. However, despite high rates
of immigration of susceptible insects, when resistance allele
frequencies in the plot were high, spraying the refuge resulted in
progressively higher levels of resistance over the course of the
season than when the refuge was not sprayed (Fig. 1).In both the
second and third counts, the insect population in the sprayed refuge
had a significant and >15% lower average mortality at the diagnostic
dose for resistance (10 p.p.m.), compared with the insects in the
unsprayed refuge. Insects collected from the Bt plants would have a
RR genotype for Bt var. kurstaki resistance, and we consistently
found significantly higher numbers of Bt var. kurstaki-resistant
larvae on the Bt plants when the refuge was sprayed than when it was
not sprayed (Fig. 2). This is the opposite of what should occur if
resistant alleles are to be maintained in the refuge for an effective
resistance management strategy.
To illustrate this further, we examined the overall diamondback moth
population within our experimental plots of 300 broccoli plants.
Because each 20% refuge plot had 240 Bt plants and 60 refuge plants,
a higher number of larvae per Bt plant translated to a significantly
higher overall populationin the plot in the second and third counts
when the refuge was sprayed than when not sprayed (Fig. 3). The
important point demonstrated here is that spraying the refuge reduces
its potential to dilute resistance. By leaving the refuge unsprayed
and giving more susceptible insects a chance to survive, short-term
sacrifices of relatively more insects in the refuge may translate to
seasonal reductions in resistance and reductions in the total number
of larvae per plot. The critical question is whether such populations
would result in unacceptable crop losses.
The high-dose/refuge strategy is the current foundation for managing
pest resistance to Bt plants. Whereas the consensus is that the
efficiency of this strategy depends on early implementation before
the frequency of resistance alleles is high, evaluation under field
conditions with this criterion is inherently difficult. We can
approach such an evaluation by increasing the R allele frequency, as
we did with multiple releases, and then assess changes in
susceptibility and effectiveness of the refuge in conserving
susceptible alleles within a field. Our results indicate that the use
of refuges can be a sound strategy. However, this strategy will also
depend on our ability to effectively monitor and manage susceptible
alleles on an individual field or farm basis as well as on an
areawide 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 is rendered
ineffective 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. An
alternative to the strategy of having a 20% refuge that can be
sprayed (the requirement for cotton) is the EPA-approved strategy
(also in cotton) of having a 4% refuge that remains unsprayed.
Critical experiments need to be performed to assess which approach,
as well as which refuge size, would be more effective in conserving
susceptible alleles while providing acceptable crop yields, and such
tests need to be performed in the specific insect/Bt crop system.
As we refine resistance management strategies for the currently
available Bt crops, it is also imperative that other strategies for
managing overall resistance to Bt be developed and implemented in the
near future. Having Bt expressed in plants so that the insect
population is subjected to selection pressure for particular periods
of time (e.g., through an inducible promoter) or in particular plant
parts (e.g., through tissue-specific promoters) may provide larger
refuges for susceptible alleles both within the field and within a
region while at the same time minimizing crop loss10. Although this
appears to be technically difficult at present, it may be an approach
that merits further development. Other options may be more feasible
in the near future. 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 temporally10, 20, and may require
smaller refuges. Other non-Bt genes may also aid in managing
resistance to Bt crops. Currently the most promising ones being
evaluated in transgenic plants include vegetative insecticidal
proteins (vips), as well as various genes from other insects,
animals, plants,and bacteria that act as inhibitors of insect
digestive enzymes (e.g., protease inhibitors, -amylase inhibitors,
and cholesterol oxidase21).
The development and implementation of engineered insecticidal plants
is currently in its infancy and the only available technology is that
of Bt-transgenic plants. Using the diamondback moth/Bt broccoli
system as a model, we have investigated aspects important to the long-
term deployment of this novel technology. Although the diamondback
moth/Bt broccoli system may not exactly duplicate the currently
available insect/Btcrop systems such as cotton, corn, and potatoes,
it can help identify areas for further work. Concurrently with more
field studies conducted to refine the presently utilized
recommendations, industry, public sector scientists, and farmers must
work together to develop a second generation of technology and
implementation strategies to ensure the even longer term durability
of Bt-transgenic plants.
The diamondback moth. Using field-collected diamondback moth
populations and our laboratory susceptible population (G88) we were
able to make synthetic populations of diamondback moth with the
desired resistance allele frequency for Cry1A toxins. Field-collected
diamondback moth populations that showed resistance to Cry1A were
collected in 1996 and 1997. The year the population was collected it
was mated with G88 to create the synthetic population, then reared on
plant material and re-released in the field. Although these synthetic
populations were manipulated for field studies in isolated research
plots, there was no carryover through the winter, since the
diamondback moth does not overwinter in upstate New York where these
tests were conducted22.
The transgenic plants. Using an Agrobacterium tumefaciens-mediated
transformation system and a synthetic cry1A(c) gene provided by
Monsanto, we produced diamondback moth-resistant broccoli plants23.
The plants allowed survival of about 90% of neonate larvae from the
resistant strain but caused 100% mortality of the F1 neonates
heterozygous for resistance14. Mortality assays with susceptible
larvae using leaves taken from different locations within the plant
showed that toxin expression is fairly uniform throughout the plant
when the plant is in its vegetative stage17. We used cytoplasmic male
sterile transgenic plants hemizygous for the cry1A(c) gene for field
Field tests, 1996. Four treatments with three replicates were
arranged in a completely randomized design: 0% refuge, 20% separate
refuge, 20% mixed refuge, and 100% refuge (plots were located at the
New York State Agricultural Experiment Station in Geneva, NY, three
plot replicates per treatment, 300 plants per plot, and 60 m minimum
distance separating plots), into which we released second-instar
diamondback moth larvae (312 larvae per plot per release) at seven
periods over the course of the season, at an approximate R allele
frequency of 0.12. Plants were spaced at 46 cm between plants and 90
cm between rows. Plots with a separate refuge had two border rows on
one side of the field, and the border rows were separated from the Bt
plants by one blank row of bare ground. Mixed refuges had non-Bt
plants randomly assigned within the plot. Releases were initiated on
16 June and continued at one- to three-week intervals until 26
August. We inoculated the plots with insects at various times to
ensure high diamondback moth populations, with multiple and
overlapping generations and with R alleles. We were simulating what
would happen when insect populations immigrate into and cycle within
a field, and are challenged by Bt plants. Additional treatments were
included for reference to reflect native diamondback moth populations
and were 0% refuge and 100% refuge with no insect release in either
(one replicate each). Counts of larvae (all stages) were taken at
five periods over the season beginning on 1 July and ending on 12
September. Leaf-dip bioassays with JavelinWG (Bt var. kurstaki;
Novartis) to evaluate resistance were done with progeny of the
released larvae (time zero) and with progeny of larvae counted in the
Field studies, 1997. Four treatments with three replicates were
arranged in a completely randomized design: 20% refuge, 20% sprayed
refuge (sprays were applied five times at 8- to 13-day intervals
throughout the growing season), and 100% refuge into which we
released diamonback moth pupae at six periods early in the season at
approximate R allele frequencies of 0.8 based on 10 p.p.m. Javelin.
The fourth treatment was a 100% refuge with no release. Releases were
initiated on 15 May and continued at one- to two-week intervals until
25 June for the same purposes as noted in 1996. Plant and plot
spacing were as in 1996. Larval counts (eight plants sampled
destructively per plot) were taken at three periods over the season
beginning on 22 July and ending on 11 August. In each sample, all
larvae counted were taken back to the lab and reared. In each of the
20% refuge plots, larvae were collected from equal numbers of refuge
and Bt plants and then combined. Progeny of larval collections and
time zero larvae (i.e., representative of what was released into the
plots) were used in leaf-dip bioassays with Javelin to evaluate
Data in Table 1 on the number of larvae per plant were analyzed by
ANOVA with the means weighted inversely by their variances. Data in
Figures 2 and 3 were analyzed using ANOVA, and non overlap of the
standard errors indicates significant differences. Because the plots
were inoculated with equal numbers of insects, less than normal
variation within a treatment probably occurred. The bioassays for
insects were conducted using our normal protocol24, and the
concentration or dose/mortality relationships contained in Table 1and
Figure 1 were estimated assuming a probit model by using POLO25.
Received 15 July 1999; Accepted 2 January 2000.
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We thank Melissa Birkett, Hilda Collins, Jennifer Cooley, Bryna
Mitchell, Sabrina Siebert, Powell Smith, and Joe Zhao for their
assistance in this project. The research was funded by USDA NRI
grants 91-37302-6199, 93-01977 and 95-37302-1783.
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