90 L. Pathak et al.
ceptible to Bt. Recommended amounts of refuge
to delay resistance is 20 % or more located within
one-half mile of the Bt corn.
If transgenic plants can express a cry gene at
doses high enough to kill even homozygous re-
sistant insects, that crop will become a nonhost.
While such an ultrahigh dose might be imprac-
tical with a sprayable product due to high cost,
incomplete coverage, toxin breakdown, and plant
growth, it may be possible with toxin-engineered
plants, taking into account the currently attain-
able levels of Cry expression in planta (Jansens
et al. 1997 ). For example, a Colorado potato
beetle population 100-fold resistant to a Cry3A-
containing B. thuringiensis spray could not sur-
vive on potato plants expressing the same pro-
tein (Altre et al. 1996 ). It remains to be seen if
a combination of toxins with ultrahigh expres-
sion can overcome all homozygous resistance
alleles, changing the crops into nonhost plants.
Metz et al. ( 1995 ) demonstrated that F1 larvae
from a cross between a susceptible laboratory
P. xylostella colony and a field-resistant colony
did not survive on transgenic broccoli expressing
Cry1Ac. It has been reported that the inclusion
of refuge plants in cages with transgenic broccoli
plants resulted in slower evolution of resistance in
populations of P. xylostella. Supporting evidence
also comes from selection experiments using B.
thuringiensis subsp. aizawai and a diamondback
moth population that had evolved resistance to
Cry1Ab and Cry1Ca in the field. In these stud-
ies, a 10 % refuge delayed resistance over a nine-
generation test (Luiand Tabashnik 1997 ).
A specific planting strategy that has been
recommended to reduce selection is the use of
seed mixtures of toxin-expressing and toxin-free
plants to provide prepackaged refugia. The seed
mix strategy, still controversial, would probably
only be effective for insect species whose larvae
move very little between plants (Mallet and Por-
ter 1992 ). Another valuable option for resistance
management, in combination with the use of re-
fugia, is the expression of multiple Cry proteins
in crops or incorporation of multiple proteins in
B. thuringiensis sprays, provided these toxins
have different modes of action with respect to
the insect’s mechanism of resistance. Cry tox-
ins that recognize different receptors in the same
target species could be deployed in this strategy,
since they are less prone to cross-resistance. As
noted above, diamondback moth populations re-
sistant to field applications of Cry1A-containing
B. thuringiensis formulations showed minimal
cross-resistance to other crystal proteins such
as Cry1Ba, Cry1Bb, Cry1Ca, Cry1Da, Cry1Ia,
Cry2A, and Cry9Ca, while they were cross-resis-
tant to Cry1Fa and Cry1Ja (Lambert et al. 1996 ).
For many insect species, multiple Cry1A proteins
would not be an appropriate choice, since some
of these proteins share binding sites with one
another and even with other toxins of the Cry1
class. Yet for other insects, Cry1A proteins have
been shown, at least on ligand blots, to recog-
nize different binding proteins. Additionally, B.
thuringiensis Cry toxins could be combined with
other insecticidal proteins.
The multiple-attack strategy assumes that
within a population, if insects homozygous for
one resistance gene are rare, then insects homo-
zygous for multiple resistance genes are extreme-
ly rare. Crops or sprays deploying multiple toxins
would still control even insects homozygous for
one or two resistance genes yet heterozygous for
another gene. A critical condition for the success
of this strategy is that each of the insecticides on
its own should have high mortality for suscep-
tible homozygote. An example is O. nubilalis, in
which Cry1Ab and Cry1Ba, both highly active,
bind to different receptors. A strong argument for
the utility of multiple-gene pyramiding is found
in the recent results of Georghiou and Wirth
( 1997 ). Their field-collected C. quinquefascia-
tus populations readily developed resistance in
the laboratory to a single B. thuringiensis subsp.
israelensis toxin (Cry11A) but remained remark-
ably sensitive when selection was with the full
complement of toxins from this variety.
Due to the urgent need for a more complete
understanding of the parameters of effective re-
sistance management, companies developing B.
thuringiensis biopesticidal sprays and transgenic
plants formed the B. thuringiensis Management
Working Group to promote research on the judi-
cious use of B. thuringiensis products. It is hoped
that an increased understanding of the complex