Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02396562 2002-07-05
WO 01/50865 PCT/EP00/13094
SYNERGISTIC INSECT CONTROL
Control of insect pests by chemical means has long been a useful
method to protect crops from damage caused by insect attack and
infestation. More recently, methods to control insect crop damage
have been introduced which are specific to the target insect and
avoid environmental and ecological compromise associated with
traditional pesticide usage. One of these methods employs a gene-
tically modified crop which produces insect-specific toxins,
e.g., the Cry toxin from Bacillus thuringiensis. However, the B.
thuringiensis-Cry-toxin-expressing crop may exhibit varying de-
grees of protection from an array.of lepidopteran pest species.
For example, CryIA(c)expressing cotton varieties are highly resi-
stant to tobacco budworm, Heliothis virescens, but only modera-
tely resistant to cotton bollworm Helicoverpa zea (J. H. Benedict
et a1.,1996, Journal of Economic Entomology, Vol. 89 (1), p.
230) .
Another such method of insect control is the application of bio-
logical agents such as a nucleopolyhedrosis virus (NPV)(U.S.Pa-
tent No. 4,668,511), or recombinant nucleopolyhedrosis virus
(rNPV) (U. S. Patents No. 5,662,897 and U.S. 5,858,353). However,
NPV and rNPV may vary in the level of virulence/potency against
various insect species, depending upon the host range of the vi-
ral vectoring agent and the potency of the toxin encoded by the
inserted gene. For example, the insect species Helicoverpa zea is
highly susceptible to the NPV and rNPV designated HzNPV and
HzAaIT, respectively, but only moderately susceptible to the Au-
tographa californica NPV (AcNPV) or its rNVP, AcAaIT (Treacy et
al., 1999, Proceedings Beltwide Cotton Conf., pp. 1076 - 1083).
Although the combination of applying a recombinant nucleopolyhe-
drosis virus which contains a vector which is moderately virulent
to the target insect species to a transgenic crop line has been
described, (All and Treacy, 1997, Proceedings Beltwide Cotton
Conf. p. 1294), neither the transgenic crop nor the rNPV agent,
alone or in combination, provided the level of insect control
needed to prevent crop loss an a commercial basis.
Therefore, it is an object of this invention to provide a method
of synergistic insect control useful for preventing crop damage
and economic loss caused thereby.
It is another object of this invention to provide a method for
the enhanced protection of a transgenic crop from the devastation
and damage caused by insect attack and infestation.
CA 02396562 2002-07-05
WO 01/50865 PCT/EP00/13094
2
It is a feature of this invention that the synergistic insect
control and crop protection methods provided are specific to the
target insect species and demonstrate enhanced environmental and
ecological compatability, while providing commercially acceptable
levels of insect control and crop protection.
Other objects and features of the invention will be apparent to
those skilled in the art from the following description and the
appended claims.
The present invention provides a method for synergistic insect
control which comprises applying to the locus of a transgenic
crop a synergistically effective amount of a recombinant insect
virus containing a vector which is highly virulent to said in-
sect.
Further provided is a method for the enhanced protection of a
transgenic crop from damage caused by insect attack and infesta-
tion.
Although chemical pest control has been an effective means of
controlling important agronomic insect pests, more target insect-
specific methods of control have been introduced. Among these in-
sectspecific methods are the use of a transgenic crop which has
been genetically altered to produce an insect toxin such as Ba-
cillus thuringiensis (Bt) or the use of a naturally occurring vi-
rus such as the nucleopolyhedrosis virus (NPV) or recombinant NPV
(rNPV). However, the transgenic crop which produces a Bt toxin
may exhibit a less than satisfactory degree of protection from
the targeted insect. Similarly, naturally occurring and recombi-
nant insect viruses often demonstrate varying degrees of efficacy
when used as the sole method of insect control.
Although the use of a combination of an rNPV which contains a
vector which is moderately virulent to the target insect species
and a transgenic crop has been described, the results achieved
were not satisfactory for commercial insect control when said
rNPV was applied alone or when said rNPV was applied in combina-
tion with a transgenic crop genetically altered to produce an in-
sect toxin.
It has now been found that the application of a recombinant in-
sect virus which contains a vector which is highly virulent to
the target insect species to a transgenic crop, preferably a
transgenic crop which has been genetically altered to produce an
insect toxin (insecticide), demonstrates a significant synergi-
stic effect (i.e. the resultant insect control is much greater
CA 02396562 2002-07-05
WO 01/50865 PCT/EP00/13094
3
than that which could be predicted from the insect control of the
virulent recombinant insect virus when used alone or from the in-
sect control of the transgenic crop when used alone). This syner-
gistic effect enables a commercially useful level of insect con-
s trol via a non-chemical biological means. Further, the synergi-
stic insect control method of the invention allows for effective
resistance management compatable with sustainable agriculture
practices which are environmentally and ecologically sound.
In accordance with the method of the invention, the application
of a synergistically effective amount of a recombinant insect vi-
rus, preferably a recombinant nucleopolyhedrosis virus (rNPV),
containing a vector which is highly virulent to the target insect
species to a transgenic crop variety, preferably a transgenic
crop which is genetically altered to produce an insect toxin,
provides synergistic control of the insect pest. That is, the ap-
plication of the virulent recombinant insect virus to the trans-
genic crop results in a combination of insecticidal components
which produces a greater insecticidal effect than that which
would be expectedfrom the individual insecticidal compondnts em-
ployed individually (synergistic effect).
Recombinant insect viruses containing a highly virulent vector
which are suitable for use in the method of invention include
rNPVs such as HzNPV, HzAaIT, EGTdel, or a combination thereof.
Transgenic crops which produce an insect toxin suitable for use
in the method of invention include Bt expressing lines of maize
and cotton(BTK lines), such as NuCotn 33BTM, a transgenic cotton
variety derived from Deltapine DP5415TM by the BollgardTM trans-
formation event, or transgenic maize varieties such as those
which express the MON 810TM transformation event (YieldGardTM,
Monsanto Co.).
In actual practice, the virulent recombinant insect virus may be
applied in the form of a formulated composition, such as a wetta-
ble powder, to the locus, foliage or stems, preferably the fo-
liage, of a transgenic crop, particularly a transgenic crop which
has been genetically altered to produce an insect toxin. A pre-
ferred formulation is that described in co-pending U.S. patent
application Serial No. 09/094,279, filed June 9, 1998, incorpora-
ted herein by reference thereto.
The synergistically effective amount of the virulent recombinant
insect virus may vary according to prevailing conditions such as
the degree of insect resistance of the transgenic crop, the ap-
plication timing, the weather conditions, the mode of applica-
CA 02396562 2002-07-05
WO 01/50865 PCT/EP00/13094
4
tion, the density of the insect population, the target crop spe-
cies, the target insect species, and the like. In general, syner-
gistic insect control may be obtained when the virulent recombi-
nant insect virus is applied to the transgenic crop at rates of
1x101 occlusion bodies per hectare (OB/ha) to 1x1013 OB/ha, pre-
feralbly 5x101 OB/ha to 12x1011 OB/ha.
In order to facilitate a further understanding of the invention,
the following examples are presented primarily for the purpose of
illustrating more specific details thereof. The invention should
not be deemed limited thereby except as defined in the claims.
In the following examples, synergism for two-way insecticidal
combinations is determined by the Colby method (Colby, S.R.,
Weeds, 1967 (15), pp.20-22), i.e. the expected (or predicted) re-
sults (percentage of insects eliminated) of the combination is
calculated by taking the sum of the results for each insecticide
component applied alone and subtracting the product of these two
results divided by 100. This is illustrated mathematically below,
wherein a two-way combination is composed of component X plus
component Y.
XY
(X + Y) - ------ - Expected results
100
If the actual observed results are greater than the expected re-
sults calcualted from the formula, synergy exists.
In the present invention, the percent insect control (no external
insecticide applied) exhibited by a transgenic crop of this in-
vention relative to a closely related control crop could be re-
presented by X; and the percent control of a recombinant insect
virus of the invention when used an the control crop could be re-
presented by Y. The foregoing Colby formula can be used to calcu-
late the expected percent control for the combination of the vi-
rus and the transgenic crop. If the observed results (actual per-
cent control.) of the combination of the transgenic crop treat~d
with the -virus is greater than the calculated expected results,
then the combination is synergistic.
45
CA 02396562 2002-07-05
WO 01/50865 PCT/EP00/13094
EXAMPLE 1
Evaluation of the Syneraistic Insecticidal Effect of A
Virulent Recombinant Insect Virus Applied to A Transgenic
5
Crop
In this evaluation a test system is used which approximates fo-
liar-spray and plant architecture parameters typically encoun-
tered in cotton field scenarios. The insecticidal effect of (a)
the application of a wettable powder (WP) formulation of HzAaIT
at rates of 5x1011 OB/ha and 12x1011 OB/ha and (b) the Bacillus
thuringiensis CryIA(c)-expressing cotton variety, 'NuCotn 33B',
is evaluated and compared to combinations using a conventional
cotton variety, 'Deltapine DP54151'.
Plants are grown from seed in 3.8-liter plastic pots which are
filled with commercial potting soil. For comparison purposes,
conventional Deltapine DP5415 cotton is included in the study.
Viral applications to cotton are initiated about 1.5 months after
the cotton planting date. Potted plants are sprayed in an enclo-
sed chamber which is equipped with an overhead, rotary hydraulic
boom. The boom is fitted with three hollow cone nozzles (TX3,
Spraying Systems, Wheaton, IL); one nozzle is mounted to apply
spray directly over plants and two nozzles are mounted an drop
tubes angled at about 45~ to spray sides of plants. The sprayer is
calibrated to deliver 189 liters/ha at 3.5 kg/cm2; compressed air
is used as the spray propellant. The formulated rNPV insecticide
is suspended in dechlorinated water, along with the gustatory
stimulant, CoaxTM (CCT Corp., Carlsbad, CA), at 3.5 L/ha. Plants
are sprayed three times at 7-day intervals. Potted cotton plants
are arranged in a completely randomized design with four replica-
tions an table-tops which are flooded with water to a depth of
about 2 cm to prevent larval migration between plants. Two plants
per treatment are given replicate doses, with replicate subsam-
ples taken from separate tests. Environmental parameters for the
greenhouse during the course of the study are programmed for an
average daily low temperature of about 27°C and an average daily
high of about 32°C.
The plants are infested with laboratory-reared, neonate H. zea at
about 1 hr after each spray session. With the use of a small
paint brush, larvae are placed an leaves and squares throughout
the upper portion of each cotton plant. A total of 30 freshly
hatched larvae are placed an each plant following each of the
three spray sessions. Artificial placement of larvae an plants is
designed to approximate natural distribution of eggs and small
CA 02396562 2002-07-05
WO 01/50865 PCT/EP00/13094
6
larvae of this pest species an cotton (Farrar & Bradley, 1985,
Environ. Entomol .) . Efficacy of treatments applied to cotton is
determined 7 days after the third application session by recor-
ding numbers of damaged and non-damaged squares per plant. Signi-
ficant differences among treatments in injury to cotton by H. zea
are determined by analysis of variance (ANOVA, SAS Institute,
1989). Treatment means are separated by Duncan's multiple range
test (DMRT; SAS Institute, 1989).
Means followed by a common letter are not significantly different
as determined by Duncan's multiple range test
(P < 0.05; F [df 5, 18] - 16.9); percentile data are arcsine
transformed for analysis.
Numbers of damaged and non-damaged squares affixed to each plant
are assessed 7 days after the final application/infestation ses-
sion (7DA3T = 7 Days After 3rd Treatment application)
RESULTS
In this greenhouse study, weekly infestations of H. zea larvae
caused significantly more injury to untreated DP5415 cotton (su-
sceptible) than to untreated NuCotn 33B (resistant),(53.0 % and
20.80 damaged squares, respectively). Foliar applications of
HzAaIT at rates of 5x1011 OB/ha and 12x1011 OB/ha significantly
reduced insect damage an both varieties of cotton. The suscepti-
ble plant variety DP5415 when treated with HzAaIT at rates of
5x1011 OB/ha and 12x1011 OB/ha, gave an average of 27.60 and 23.9
damaged squares, respectively. The resistant plant variety Nu-
Cotn33B when treated with HzAaIT at rates of 5x1011 OB/ha and
12x1011 OB/ha gave an average of 8.8o and S.Oo damaged squares,
respectively. The data are shown an Table I.
As can be seen from the data an Table I, foliar application of a
virulent recombinant insect virus (HzAaIT) to a transgenic crop
(NuCotn33) at a rate of 12x1011 OB/ha reduces the insect damage by
4.2-fold as compared to the insect damage to the untreated trans-
genic crop, whereas the application of said virulent recombinant
insect virus to a susceptible crop (DP5415) at a rate of 12x1011
OB/ha reduces the insect damage by only 2.2-fold as compared to
the untreated susceptible crop. Therefore, the combination of the
application of a virulent recombinant insect virus to a transge-
nic crop gives approximately 2-fold the reduction of insect da-
mage than that which can be expected from either the application
of the virulent recombinant insect virus alone or from the use of
a transgenic crop alone.
CA 02396562 2002-07-05
WO 01/50865 PCT/EP00/13094
7
TABLE I
Control of Cotton Bollworm, Helicoverpa Zea, an Conventional and
Transgenic Cotton Varieties with Foliar Applications of the Re-
combinant Nucleopolyhedrovirus HzNPV (Egtdel)/DA26-ADK-AaIT
(H2AaIT)
Cotton variety Mean % squares
& foliar treat-damaged (SD) o Control2
ment 7DA3T
Observed Expected
DP5415
HZAaIT 27.6 b 47.9 NA
5x1011 OB/ha ( 7.5)
HZAaIT 23.9 b 54.9 NA
12x1011 OB/ha ( 6.8)
Non-treated 53.0 a NA NA
( 9.4)
NuCotn 33 B
HZAaIT 8.8 c 83.4* 79.6
5x1011 OB/ha ( 5.6)
HZAaIT 5.0 c 90.6* 82.3
12x1011 OB/ha ( 3 . 4 )
Non-sprayed 20.8 b 60.8 NA
( 10.4)
1 Means followed by a common letter are not significantly diffe-
rent as determined by Duncan's multiple range test
(P < 0.05; F (df 5, 18] - 16.9); percentile data were arcsine
transformed for analysis.
2
dam.seq. - ~ dam.seq.
(non-treated) (treated)
control = x 100
dam. seq. (non-treated)
* Synergism = Observed > Expected
