AU3727001A - Synergistic insect control - Google Patents

Description

WO 01/50865 PCT/EP00/13094 SYNERGISTIC INSECT CONTROL Control of insect pests by chemical means has long been a useful 5 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 10 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 15 stant to tobacco budworm, Heliothis virescens, but only modera tely resistant to cotton bollworm Helicoverpa zea (J.H. Benedict et al.,1996, Journal of Economic Entomology, Vol. 89 (1), p. 230). 20 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 25 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 30 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 35 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. 40 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 45 the enhanced protection of a transgenic crop from the devastation and damage caused by insect attack and infestation.

WO 01/50865 PCT/EPOO/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 5 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. 10 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 15 sect. Further provided is a method for the enhanced protection of a transgenic crop from damage caused by insect attack and infesta tion. 20 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 25 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 30 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 35 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 40 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 45 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 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 5 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. 10 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 15 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 20 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 25 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 30 variety derived from Deltapine DP5415TM by the BollgardTM trans formation event, or transgenic maize varieties such as those which express the MON 81Om transformation event (YieldGard2m, Monsanto Co.). 35 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 40 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 45 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- WO 01/50865 PCT/EPOO/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 5 1x10 10 occlusion bodies per hectare (OB/ha) to lx10 1 3 OB/ha, pre feralbly 5x10 10 OB/ha to 12x10 11 OB/ha. In order to facilitate a further understanding of the invention, the following examples are presented primarily for the purpose of 10 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., 15 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, 20 wherein a two-way combination is composed of component X plus component Y. XY (X + Y) - ------ = Expected results 100 25 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 30 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 35 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 treated with the -virus is greater than the calculated expected results, then the combination is synergistic. 40 45 WO 01/50865 PCT/EPOO/13094 5 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 10 tered in cotton field scenarios. The insecticidal effect of (a) the application of a wettable powder (WP) formulation of HzAaIT at rates of 5x10 11 OB/ha and 12x10 11 OB/ha and (b) the Bacillus thuringiensis CryIA(c)-expressing cotton variety, 'NuCotn 33B', is evaluated and compared to combinations using a conventional 15 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. 20 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 25 spray directly over plants and two nozzles are mounted an drop tubes angled at about 450 to spray sides of plants. The sprayer is calibrated to deliver 189 liters/ha at 3.5 kg/cm 2 ; compressed air is used as the spray propellant. The formulated rNPV insecticide is suspended in dechlorinated water, along with the gustatory 30 stimulant, CoaxTd (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 35 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 0 C and an average daily high of about 32 0 C. 40 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 45 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 WO 01/50865 PCT/EPOO/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 5 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). 10 Means followed by a common letter are not significantly different as determined by Duncan's multiple range test (P < 0.05; F [df 5, 181 = 16.9); percentile data are arcsine transformed for analysis. 15 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 20 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 20.8% damaged squares, respectively). Foliar applications of 25 HzAaIT at rates of 5x10 11 OB/ha and 12x10 11 OB/ha significantly reduced insect damage an both varieties of cotton. The suscepti ble plant variety DP5415 when treated with HzAaIT at rates of 5x10 11 OB/ha and 12x10 1 1 OB/ha, gave an average of 27.6% and 23.9% damaged squares, respectively. The resistant plant variety Nu 30 Cotn33B when treated with HzAaIT at rates of 5x10 11 OB/ha and 12x10 11 OB/ha gave an average of 8.8% and 5.0% damaged squares, respectively. The data are shown an Table I. As can be seen from the data an Table I, foliar application of a 35 virulent recombinant insect virus (HzAaIT) to a transgenic crop (NuCotn33) at a rate of 12x10 11 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 12x10 1 1 40 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 45 of the virulent recombinant insect virus alone or from the use of a transgenic crop alone.

WO 01/50865 PCT/EPOO/13094 7 TABLE I Control of Cotton Bollworm, Helicoverpa Zea, an Conventional and 5 Transgenic Cotton Varieties with Foliar Applications of the Re combinant Nucleopolyhedrovirus HzNPV (Egtdel) /DA26-ADK-AaIT (HzAaIT) Cotton variety Mean % squares & foliar treat- damaged ( SD) % Control 2 10 ment 7DA3T Observed Expected DP5415 HZAaIT 27.6 b 47.9 NA 15 5x10 1 1 OB/ha (± 7.5) HZAaIT 23.9 b 54.9 NA 12x10 11 OB/ha (± 6.8) Non-treated 53.0 a NA NA 20 ( 9.4) NuCotn 33 B HZAaIT 8.8 c 83.4* 79.6 5x10 11 OB/ha (± 5.6) 25 HZAaIT 5.0 c 90.6* 82.3 12x10 11 OB/ha (± 3.4) Non-sprayed 20.8 b 60.8 NA ( 10.4) 30 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, 181 = 16.9); percentile data were arcsine transformed for analysis. 35 % dam.seq. - % dam.seq. control = (non-treated) (treated) x 100 % dam. seq. (non-treated) 40 * Synergism = Observed > Expected 45

Claims (10)

1. A method for synergistic control of an insect which comprises 5 applying to the locus, foliage or stem of a transgenic crop which produces an insect toxin a synergistically effective amount of a recombinant insect virus containing a vector which is highly virulent to said insect. 10

2. The method according to claim 1 wherein said recombinant virus is a recombinant nucleopolyhedrosis virus.

3. The method according to claim 2 wherein said recombinant virus is HzNPV, HzAIT, EGTdel or a combination thereof. 15

4. The method according to claim 1 wherein said transgenic crop is a crop plant which has been genetically altered to express Bacillus thuringiensis toxin. 20

5. The method according to claim 4 wherein said transgenic crop is maize.

6. The method according to claim 4 wherein said transgenic crop is cotton. 25

7. The method according to claim 6 wherein said crop is NuCotn 33B.

8. The method according to claim 2 where the synergistically ef 30 fective amount of said recombinant insect virus is 1x10 10 OB/ha to 1x10 1 3 OB/ha.

9. The method according to claim 8 wherein the insect is Lepidoptera. 35

10. The method according to claim 9 wherein the insect is Helico verpa zea. 40 45