| In the ongoing battle
against Helicoverpa armigera, Bollgard II cotton varieties are about
to replace Ingard varieties. Bollgard II produces two proteins from
Bt (Cry1Ac and Cry2Ab) which are toxic to the moth’s larvae (Ingard
contains only Cry1Ac).
It is hoped that this double attack by Bollgard II will reduce opportunities
for H. armigera to develop resistance to Bt cottons, and so prolong
their ‘life’.
H. armigera has proved itself to be very effective at evolving resistance
to insecticides. There has always been a concern that it would also
become resistant to the toxin in Ingard. Indeed, researchers at CSIRO
have selected for a laboratory strain of the moth that is highly resistant
to Cry1Ac. But, as yet, there is no evidence of resistance in the field.
In a CRDC funded project, we have now begun looking at the possibility
of H. armigera becoming resistant to the toxins in Bollgard II. Early
results from this research show that cotton growers need to remain vigilant,
and that adherence to a Resistance Management Strategy (RMS) will be
important in retaining the value of Bt cotton varieties.
The Transgenic and Insect Management Strategy Committee (TIMS) of the
Australian Cotton Growers Research Association (ACGRA) has asked for
the results of this preliminary research and planned future research
to be made available to cotton growers. TIMS’ purpose in doing
this is to keep the industry informed on issues that may need to be
reviewed prior to the final endorsement of the 2004–05 Resistance
Management Plan (RMP) for Bollgard II. At present a draft 2004–05
RMP for Bollgard II has been supported by TIMS and submitted to the
Australia Pesticides and Veterinary Medicines Authority (formerly the
NRA) for approval.
What the research found
One group of larvae, established from parents collected as eggs on a
maize crop near Griffith, exhibited marked resistance to Cry2Ab. The
results indicate a much higher frequency of resistant alleles (see ‘Background’
box) than expected, particularly as H. armigera populations have never
been exposed to cotton-related Cry2Ab.
What it means
The expectation has been that Bollgard II, with its two toxins, will
be much more resilient to the evolution of resistance than varieties
like Ingard which carry only a single toxin. This would be true unless
there is a gene that confers resistance to both Cry1Ac and Cry2Ab simultaneously.
This is considered unlikely, and there is no evidence of such cross-resistance
to the two quite different toxins in other species of moths. But models
indicate that the longevity of Bollgard II (the period before resistance
evolves), is sensitive to the frequency of resistance alleles at the
time it is introduced. The more common the resistance alleles, the shorter
the time until resistance develops.
To date, the aim of resistance management in Bt cotton (Ingard) has
been to maintain Cry1Ac resistance at low levels until two-gene cotton
(such as Bollgard II) became available. This seems to have been successful,
as there is no evidence of resistance to Cry1Ac in the field. But the
background frequency of resistance alleles for the second toxin, Cry2Ab
appears to be unexpectedly high, and it is possible this could impact
on the longevity of Bollgard II.
While Cry2Ab resistance may be more common than expected, it must be
emphasised that the level of threat, if any, to the longevity of Bollgard
II posed by the resistance gene is not yet known. The resistant colony
derived from field collected eggs and a quite independent colony selected
by low doses of Cry2Ab in the laboratory (see below) are under intensive
investigation right now to assess that risk.
Importantly, the research performed to date on insects from the field
resistant colony (as yet, almost nothing is known about the laboratory
selected colony) has shown that they show no cross-resistance to Cry1Ac,
implying that larvae carrying this resistance will still be susceptible
to the Cry1Ac in Bollgard II. Equally importantly, the resistance appears
to be largely recessive — that is it must be inherited from both
parents before it is expressed, which will also hinder the development
of resistance.
What could have caused it?
Forms of genes that confer resistance will be generated through naturally
occurring mutations. Importantly, these are spontaneous and selection
is not involved in the generation of mutations but is important later
in causing such mutations to increase in frequency.
Insects with mutant versions of genes that confer resistance often suffer
from poor fitness. This means they are unable to thrive or survive and
so these gene versions (alleles) are likely to remain rare unless there
is a selective force — such as a toxin expressed in a cotton plant
which gives individuals carrying the mutant an advantage over non-mutant
carrying individuals.
One explanation for the unexpectedly high frequency of alleles that
confer resistance to Cry2Ab in H. armigera, is that there may be sufficient
Bacillus thuringiensis (Bt) expressing this toxin in Australian soils
to ‘pre-select’ populations of insects.
While superficially an attractive theory, there are problems with it.
Soil-borne Bt in Australia frequently produce Cry1Ac, Cry2Ab or other
Cry proteins. But Cry1Ac is by far the most commonly produced protein,
so if the ‘pre-selection’ theory is true, why is resistance
to Cry2Ab apparently more common than resistance to Cry1Ac?
An alternative theory is that the mutation rate at the gene or genes
that confer resistance to Cry2Ab may be high, and/or, such mutations
may not cause deleterious effects. Under high mutation rates and low
fitness costs, mutant alleles would accumulate in the population.
The research — in more detail
When monitoring for resistance to Cry1Ac, the standard practice has
been to ‘challenge’ larvae reared from field-collected eggs
by feeding them on a diet containing a discriminating dose (see ‘Background’)
of toxin. Any larvae which survive and grow on the diet could be resistant.
To date, the resistance monitoring program (presently carried out by
CSIRO, but until the 2002–03 season by NSW Agriculture) has detected
no evidence of increasing levels of resistance to Cry1Ac despite Ingard
cottons being grown in Australia for seven years. So there appears to
be no cause for concern that H. armigera is developing field resistance
to Cry1Ac.
This favourable situation has been the objective of the RMS that has
been in place since Ingard was first planted — including the deployment
of refuges to ‘dilute’ resistant genes, the 30 per cent
Ingard area cap and ‘pupae busting’ to reduce the population
of possibly resistant H. armigera diapausing under Ingard.
The Bt monitoring program provides an overall assessment of changes
in resistance levels in H. armigera populations throughout the industry
and most cotton growing areas are included in the survey. But the program
does not provide information on the frequency of resistance alleles,
and this information is invaluable for improving models used to develop
the RMS.
It is possible to obtain such information through the use of an ‘F2
screen’ that involves exposing the descendants (the human analogy
would be the grand children) of two moths (one male, one female) to
discriminating doses of toxin (Figure 1).
We were interested in looking at the potential for F2 screens to supplement
discriminating dose assays as a more sensitive way of detecting resistance
in its early stages. It was expected to be more sensitive as (in other
species of moth and therefore probably in H. armigera) Bt resistance
is normally ‘recessive’ (see ‘Background’).
However in the early stages of the evolution of resistance, such individuals
are extremely rare. More common are individuals carrying one mutant
and one normal form of the gene and these are likely to be still susceptible
to Bt and therefore ‘missed’ in discriminating dose assays.
During the 2002–03 season we conducted a preliminary survey to
evaluate this methodology by testing the descendants of 33 single pairs
of moths. In separate assays, groups of larvae were challenged with
either Cry1Ac or Cry2Ab. Only minor, low-level resistance was detected
for Cry1Ac but one pair of moths from Griffith produced descendants
with unexpectedly high resistance to Cry2Ab.
As each parent of a ‘pair’ contributed two copies of each
gene, the survey examined 33 (pairs) x 2 (female) x 2 (male) = 132 alleles.
Thus the calculated frequency of the resistant form of the gene is 1/132
or 0.008. Resistance alleles were expected, but to find one among the
first 132 examined was unexpected.
In related research, CSIRO’s Dr Ray Akhurst and colleagues exposed
a different colony of H. armigera established from eggs collected in
various locations to low levels of Cry2Ab toxin. This colony now also
exhibits resistance to Cry2Ab, implying that at least one of the individuals
that were incorporated into the colony carried a resistance gene.
Ongoing research
Following discussions on the research reported above, TIMS has resolved
that there is a need to learn more about background levels of resistance
to Cry2Ab toxins in H. armigera populations and opportunities for survival
of resistant genotypes on Bollgard II.
While the available information on the newly discovered Cry2Ab resistance
(no cross-resistance and recessive nature) is particularly favourable,
further research during the coming field season (2003– 2004) will
allow a better evaluation of the threat posed by it.
We will be looking at several issues. Firstly, additional work will
improve the accuracy of the assessment of the frequency of resistance
genes. If an additional 100 or 200 F2 analyses are performed and no
further resistance alleles are isolated, then there would be cause for
celebration. On the other hand, isolation of further resistance genes
would require additional research including a very careful re-evaluation
of the resistance management model upon which the future management
plan for Bollgard II is based.
Secondly, we will determine if resistant individuals can survive on
Bollgard II. The season-end decline of Cry1Ac may provide an opportunity
for Cry2Ab resistant but Cry1Ac susceptible individuals to exploit.
Lastly, the fitness of resistant individuals on non-Bt hosts needs to
be examined.
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