BG64408B1 - Mixtures of gentically modified insect viruses with chemical and biological insecticides for enhanced insect control - Google Patents

Abstract

Incecticidal compositions are described for use against insects comprising mixtures of genetically modified insect viruses with chemical and biological insecticides for enhanced insect control. The genetic modification of the virus comprises the insertion of a gene which expresses an insect controlling or modifying substance, for example, a toxin, a neuropeptide or a hormone, or an enzyme. The genetic modification of the virus also comprises a deletion in a gene.

Description

Technical field

The invention relates to insecticidal compositions for use against insects, which compositions contain mixtures of genetically modified insect viruses with chemical and biological insecticides to improve insect control.

BACKGROUND OF THE INVENTION

The control of insect pests that are destroying commercially valuable crops has been the subject of many proposals. Chemical insecticides are widely used. However, there are some objections to their use. Thus, chemical insecticides can damage beneficial insect species along with harmful insect species. Insects tend to become resistant to such chemicals, necessitating the development of new chemicals. Chemicals can remain in the environment for a long time after use.

In an effort to reduce the use of chemical insecticides, insect-specific viruses have been used to attack insects in their larval stages. Insect specific viruses include both DNA viruses and RNA viruses. DNA viruses include entomopox viruses (EPV) and Baculoviridae viruses such as nuclear polynuclear viruses (NPV), granulosa viruses (GV), and non-occluded baculoviruses (NOB) and the like. RNA viruses include togaviruses, flaviviruses, picornaviruses, cytoplasmic polynuclear viruses (CPV), and the like. The subfamily of double-stranded DNA viruses, Eubaculovirinae, includes two genera NPV and GV, which are especially useful for biological control because they produce occlusal bodies (OBs) throughout their life cycle.

Examples of NPVs are Lymantria dispar NPV, Autographa californica MNPV, Syngrapha falcifera NPV, Spodoptera litturalis NPV, Spodoptera frugiperda NPV, Spodoptera exigua NPV, Heliothassa NPestera NPV, Heliothoris NPV , Trichoplusia ni NPV, Helicoverpa zea NPV, etc. Examples of GV viruses are Cydia pomonella GV (apple fruit worm GV), Pieris brassicae GV, Trichoplusia ni GV, etc. Examples of NOB are Orcytes rhinoceros NOB and Heliothis zea NOB. Examples of entomopox viruses are Melolontha melonotha EPV, Amsacta moorei EPV, Locusta migratoria EPV, Melanoplus sanguinipes EPV, Schistocerca gregaria EPV, Aedes aegypti EPV, Chironomu luridus EPV, etc.

More than 400 baculovirus isolates occurring in invertebrates have been described. Autographa californica Multiple Nuclear Polynuclear Virus (AcMNPV) is a virus prototype of the Baculoviridae family and has hosts in a wide range. The AcMNPV virus was initially isolated from Autographa californica (A.cal.), A lepidopteran noctuid (which at night is a butterfly at night), commonly known as the alfalfa caterpillar. This virus infects 12 families and more than 30 species in the Lepidoptera insect order. There is no known productive infection of other species outside this order.

The baculovirus life cycle, as shown by AcMNPV, involves two stages. Each stage of the life cycle is represented by a specific form of the virus: extracellular (extracellular) viral particles (ECV), which are not occluded and occluded viral particles (OV). Extracellular and occluded viral forms have the same genome but exhibit different biological properties. The maturation of each of the two forms of the virus is guided by separate sections of the viral genes, some of which are unique to each form.

In its naturally occurring insect infectious form, multiple virions are located in the para-crystalline protein matrix known as the occlusal body (OB), which is referred to as the polyhedron inclusion body (PIB). Protein viral occlusions are cited as polyhedra (polyhedron is the singular form). A polynuclear protein having a molecular weight of 29 KD is the major viral encoded structural protein of viral occlusions (similar to GV produces OBs composed mainly of granulin rather than polyhedrin).

Viral occlusions are an important part of the natural life cycle of baculoviruses, providing the means for horizontal (from insect to insect) transfer between susceptible insect species. In the environment, a susceptible insect (usually in the larval stage) ingests viral occlusions from an infectious food source, such as a plant. Crystalline occlusions are dispersed inside the susceptible insect, releasing infectious viral particles. These resulting polyhedron viruses (PD V) invade and reproduce in the cells of the tissues of the midgut.

Viral particles are thought to invade cells by endocytosis or fusion, and viral DNA opens to the nuclear pore or nucleus. Viral DNA replication was detected within 6 h. For 10-12 hours after infection (p.i.), secondary infection spreads to other insect tissues by developing extracellular virus (ECV) from the cell surface. The ECV form of the virus is responsible for spreading the virus from cell to cell in a single infected insect, as well as for transmitting the infection to cell culture.

Late in the infection cycle (12 h after infection), the polynuclear protein can be detected in the infected cells. 18-24 hours after infection, the polynuclear protein is collected in the nuclei of the infected cells and the viral particles are anchored in protein-like occlusions. Viral occlusions accumulate in large numbers over a period of 4-5 days, with the cells lysed. These polyhedra do not play an active role in the spread of infection in the larva. ECVs disseminate in infected larvae, causing the larvae to die.

When the larva dies, millions of polyhedra remain in the decaying tissue until the ECV decomposes. When other larvae are exposed to the polyhedra, for example by ingesting infected plants or other nutrient material, the cycle is repeated.

In summary, the occluded form of the virus is responsible for infecting the insect through its insides as well as for the stability of the virus in the environment. PDVs are not essentially infectious when administered by injection, but are highly infectious orally. The non-occluded form of the virus (eg ESS) is responsible for viral viraemia and infection from cell to stool in tissue culture. EC V are highly infectious for cell culture or internal insect tissue when injected, but are not substantially infectious for oral administration.

These insect viruses are not pathogenic to vertebrates or to plants. In addition, baculoviruses generally have a narrow host range. Many strains are restricted to one or more species of insect.

The use of baculoviruses as bioinsecticides is promising. One of the major obstacles to their widespread use in agriculture is the prolongation of the time between the onset of the insect infection and its death.

This time delay can be in the order of several days to several weeks. During this time, the insect larva continues to feed, causing further damage to the plant.

Many researchers have attempted to overcome this deficiency by incorporating a xenogeneic gene into the viral genome so as to express an insect control or modifying substance such as a toxin, neuropeptide, and hormone or enzyme.

However, so far, such genetically modified insect viruses have not been used in combination with chemical insecticides as part of an integrated pest management scheme. Combinations of wild-type insect viruses with chemical insecticides have been reported, but their results are not optimal in view of the limitations of wild-type viruses (Aspirot, J., et al., US 4 668 511, Mohamed, A. 1 , et al., Environ. Entomology, 12,478-481 (1983), Mohamed, A.I., etal., Environ. Entomology, 12,140320 1405 (1983), Velichkova-Kozhukharova, M., et al., Restenievad Nauki, 25.80-86 (1988), Jagues, RP, et al., “Compatibility of Pathogens with Other Methods of Pest Control and with Different Crops”, Chapter 38, pages 695-715). Researchers 25 have made attempts to control insects with other biological control agents such as bacteria (eg Bacillus thuringiensis), fungi, protozoa and nematodes, either alone or in combination with insect viruses or chemical insecticides, but they have not yielded the optimum 30 results (Mohamed, AI, et al., Environ. Entomology, 12,478-481 (1983), (Mohamed, A. 1., et al., Environ. Entomology, 12,1403-1405 (1983), Jagues, RP. et al., “Pathogens Compatibility with Other Pest Control Methods and with Different Crops,” Chapter 38, 35 pages 695-715, Geerviliet, JBF, et al., Med. Fac.

Landbouww. Rijksuniv. Gent, 56,305-311 (1991). Therefore, there is a need to develop combinations of chemical insecticides and genetically modified insect viruses to provide benefits to both components, reducing the amount of chemicals used and reducing the killing time compared to that obtained with natural viruses when used. of genetically engineered insect viruses.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide insecticidal compositions for use against 50 lepidopteran insects containing mixtures of genetically modified insect viruses and chemical and biological insecticides to improve insect control. Genetic modification of the virus involves the inclusion of a gene that expresses an insect controlling or modifying substance, for example a toxin, neuropeptide or hormone or enzyme. Genetic modification of the virus also involves deletion into the gene.

The present invention provides insecticidal compositions comprising:

(a) an effective amount of a chemical insecticide selected from the group consisting of pyrethroids, arylpyrroles, diacylhydrazines and formamidines, and

(b) an effective amount of a genetically modified Autographa califomica nuclear polynuclear virus (AcMNPV) that contains either (i) an inserted gene expressing the insect toxin Androctonus australis (AalT), or (ii) a deletion in the gene encoding transdiferaseT EG (EG) of AcMNPV, these compositions being used against lepidopteran insects, provided that when the insects are Heliothis zea and the chemical insecticide is formamidine, the genetically modified AcMNPV contains an inserted gene that expresses AalT.

According to one embodiment of the present invention, insecticidal compositions are provided for use against Heliothis virescens insects containing:

d) an effective amount of a chemical insecticide selected from the group consisting of pyrethroids and arylpyrroles, and

(b) an effective amount of genetically modified AcMNPV that contains either (i) an inserted gene expressing AalT or (ii) a deletion in the gene encoding the EGT of AcMNPV.

According to another embodiment of the present invention, insecticidal compositions for use against Heliothis zea are provided comprising:

(a) an effective amount of a chemical insecticide selected from the group consisting of arylpyrroles and diacylhydrazines, and

(b) an effective amount of genetically modified AcMNPV that contains either (i) an inserted gene expressing AalT, or (ii) a deletion into the gene encoding the EGT of AcMNPV.

According to another embodiment of the invention, insecticidal compositions are provided for use against Heliothis zea insects comprising:

(a) an effective amount of a chemical insecticide selected from the formamidine group; and

(b) an effective amount of genetically modified AcMNPV that contains an inserted gene expressing AalT.

The present invention further provides a method of controlling the insecticide of the lepidopter, which consists in applying to these insects or to the culture with which these insects are fed, the insecticidal compositions described above.

Short description of the figures

Figure 1 is a graphical representation of the data presented in Table 13 denoting a mortality rate of 1.4 and 10 days for the first three treatments set out in Table 13, except that the data from “untreated controls” in Table 13 are not depicted in FIG. 1.

Figure 2 is a graphical representation of the data presented in Table 14 indicating the mortality rates at 1.4 and 10 days for the first three treatments set out in Table 14, except that the data from the "untreated controls" in Table 14 are not depicted in FIG. The AcMNPV AalT in Table 14 is the same as the rNPV in Figure 2.

Lepodoptera insects undergo a well-characterized sequence of actions during their development from egg to adult insect. After hatching the egg, the insect larva enters a period of extensive feeding. During this time, she changes her shell several times to allow for continuous growth. The steps between successive replacement of the shell are referred to as larval stages. At the end of the larval growth period, the larvae turn into cocoa beans and emerge as an adult insect. It is an object of the present invention to improve the control of harmful insects in the larval stage. The Lepidoptera families, which are known to be major pests of crops, are Noctuidae, Notodontidae, Arctiidae, Pyralidae, Plutellidae, Pieridae and Geometridae.

Two criteria are used to determine whether an insecticide composition provides effective control of insect pests. One is the number of larvae killed over a period of time. This is given as "% mortality". The other is the kill rate. Even if there is no improvement in% mortality after the end of time, but if more larvae are killed in the early stages of the period, this is favorable because there is less time to feed and thus less damage to the crop . If there is an improvement in mortality% or in the rate of killing, the test composition can be considered as improved over existing formulations.

A combination of a genetically modified insect virus with a chemical or biological insecticide is called "synergistic" if the mortality caused by the combination is greater than the mortality caused by the sum of the individual components administered alone, "additive" if the mortality from the combination is equal of mortality from the sum of the individual components administered alone, "subadmissive" if the mortality of the combination is greater than the mortality caused by any individual component administered alone but less than by the mortality of the sum of the individual components administered alone and "antagonistic" if the mortality from the combination is less than the mortality caused by any of the individual components administered alone.

There are advantages when the combination is synergistic by reducing the dose of either of the two components compared to the dose when administered individually, with cost savings as well as environmental benefits such as reducing the amount a chemical insecticide that reduces the durability and development of resistance.

The insecticidal composition is advantageous if it provides improved control over either of the two admissible or semi-admissible insects. An insect tolerant is generally 100-1000 times more susceptible to the insect virus or chemical insecticide than the semi-tolerant insect. For example, H. virescens is tolerant of AcMNPV, whereas H. zea is palutolerant of AcMNPV.

An additional advantage of the present invention is that the combination of a chemical insecticide and an insect virus is such that it can target more insect species than the individual components alone. Both chemical insecticides and insect viruses have a specific host range. The combinations may extend the reach of hosts due to the presence of two components. However, this effect is not due to any interaction between the insecticidal components.

A large number of groups of chemical insecticidal substances are used to control insect pests. Below is a brief overview of many of these groups and describes their course of action.

Pyrethroids are compounds that bind to a protein from the sodium ion channel, thereby causing a change in the action potential across the axonal membrane. This in turn destroys the intrinsic functioning of the insect's nervous system. Examples of pyrethroids are cypermethrin (? -Cyano-3-phenoxybenzyl-cis / trans-3- (2,2-dichlorovinyl) -2,2-dimethylcyclopropanecarboxylate, FMC Corp.). ), PERMETHRIN ™ (3-phenoxybenzyl-cis / trans-3- (2,2-dichlorovinyl) -2,2-dimethylcyclopropanecarboxylate, Coulston International Corp.), fenvalerate (α-cyano-3-phenoxybenzyl3- (2-chloro- 3,3,3-Trifluoroprop-1-enyl) -dimethylcyclopropanecarboxylate.

Formamidines are compounds that have several established modes of action, including binding to the octopamine (neurohormone / neurotransmitter) receptor and acting as an agonist, increasing cAMP production and inducing changes in behavior or inhibition of mixed function or monoamine oxidase. Examples of formamidines are Amirtaz (N '- (2,4-dimethyl phenyl) -N [[(2,4-dimethylphenyl) imino] methyl] -N-methylmethanimidate, NOR-AM, Schering AG) and chlordimeform (N' ( 4-chloro-o-tolyl) -N, N-dimethylformamidine).

Allylpyrroles are mitochondrial toxins that have a lethal effect by interrupting oxidative phosphorylation. Examples of arylpyrroles are 4-bromo-2- (p-chlorophenyl) -1- (ethoxymethyl) -5- (trifluoromethyl) pyrrole-3-carbonitrile (i 8 5 310 938) and the compounds described in US 5 010 098.

Diacylhydrazines are non-steroidal insect growth regulators whose initial action is as ecdysone agonists. Examples of diacylhydrazines are dibenzoyl-tert-butylhydrazine (the preparation of which is described in US 5 300 688) and MIMIC ™ (3,5-dimethylbenzoic acid 1- (1,1-dimethyl ethyl) 2- (4-ethylbenzoyl) hydrazide, Rohm & Haas Co.).

Cyclodienes bind to the receptor subunit of the GABA complex. An example of cyclodiene is endosulfan (6,7,8,9,10,10-hexachloro-1,5,5,6,9,9-hexahydro-6,9-methano-2,4,3-benzodioxathiopin-3-oxide, Hoechst).

Carbamates act as cholinesterase inhibitors. Examples of carbamates are thiodicarb (damethyl-N-thiobis (methylimino) carbonyloxy) bis (ethanimidothioate), Rhone-Poulenc) and methomyl (Smethyl H - [(methylcarbamoyl) oxy] thioacetimidate).

Organophosphates act as cholinesterase inhibitors. Examples of organophosphates are profenofos (O-4-bromo-2-chlorophenyl O-ethyl Spropylphosphorothioate, Ciba-Geigy), malathion (0,0dimethyl phosphorodithioate of diethyl mercaptosuccinate), sulfprophos (O-ethyl O- [4- (methyl) ] S propyl phosphorodithioate and dimethoate (0,0-dimethyl (8-methylcarbamoylmethyl) phosphorodithioate.

Pyrazoles are inhibitors of mitochondrial respiration through specific action on Complex I of the electronic transport system. Examples of pyrazoles are tebufenpyrad (N- (4-tert-butylbenzyl) -4-chloro-3-ethyl-1-methylpyrazole5-carboxamide, Mitsubishi Kasei, American Cyanamid Company) and compounds described in EP 289 879.

Nitroguanidines prevent the binding of acetylcholine to some choline receptors in the postsynaptic membrane, by interrupting neurotransmission by binding to the receptors themselves. Examples of nitroguanidines are imidacloprid (1 - [(6-chloro-3-pyridyl) methyl] -N-nitro-2-imidazolidinimine, Bayer) and derivatives thereof.

Milbemycin initially binds to the site in the GABA complex of the chlorine ion receptor / channel and then causes paralysis and death of the insect by inhibiting signal transmission in the neuromuscular link. An example of melbimycin is abamectin (a mixture of avermectins containing> 80% avermectin B1a and <20% avermectin Bib, Merck, Sharp & Dohme).

Benzoylphenylureas are insect growth regulators that interfere with chitin synthesis, interrupting the cuticle formation process during insect stripping. An example of benzoylphenylurea is diflubenzuron (1- (4-chlorophenyl) -3- (2,6-difluorobenzoyl) urea, Uniroyal Chemical Co., Inc.).

Amidinohydrazones are inhibitors of mitochondrial respiration by inhibiting electron transport in Complex II. An example of amidinohydrazone is hydramethylnone (tetrahydro-5,5 dimethyl-2 (1H) -pyrimidinone 3- [4- (trifluoromethyl) phenyl] -1- [2- [4- (trifluoromethyl) -phenyl] ethenyl] -2-propenylidene , American Cyanamid Company).

It is clear to those skilled in the art that additional examples of the preceding groups of chemicals are known, which are either commercially available or described in the patent and scientific literature.

The insecticidal composition of the present invention consists of an insecticidal chemical (or biological insecticidal substance as described below) and a genetically modified insect virus.

According to one embodiment of the present invention, genetic modification of the insect virus consists of including a gene that expresses a substance controlling or modifying the insect at any suitable site in the viral genome. The substance, for example, is a toxin, neuropeptide or hormone or enzyme. The substance thus expressed increases the bioinsecticidal effect of the virus.

Such toxins include the insect-specific toxin Aa1T from the scorpion Androctonus australis (Zlotkin, E., et al., Toxicon, 9,1-8 (1971)), a toxin of the species Pyemotes tritici (Tomalski, MD, et al., US 5 266 317 ), Bacillus thuringiensis toxins (Martens, JWM, et al., App. & Envir, Microbiology, 56, 27642770 (1990), Federici, BA, In vitro, 28.50A (1992)) and toxin isolated from spider venom (Jackson, JR N., et al., US 4,925,664). Examples of such neuropeptides or hormones are ecclesiastical hormone (Eldridge, R., et al., Insect Biochem., 21,341-351 (1992)), prothoracicotropic hormone (PTTH), adipokinetic hormone, diuretic hormone, and proctolin (Menn, JJ, et al. al., J. Agric. Food Chem., 37,271-278 (1989). An example of such an enzyme is Juvenile Hormonesterase (JHE) (Hammock, B. D., et al., Nature, 344,458L61 (1990)).

Examples of genetically modified AcMNPV are included in the present invention, which contains an included gene expressing Aa1T. The starting point for genetic modification is a wild AcMNPV strain designated E2 (ATCC VR-1344). The toxin; included in this viral strain is Aa1T, which is derived from the poison of the North African scorpion Androctonus australis Hector. The toxin has a length of 70 amino acids and binds to the sodium channels in the insect and causes contractile paralysis ranging from ng to pg in the insect larva. Since AA1T does not bind to mammalian sodium channels, AA1T can be used as a bioinsecticide for crop protection since it is safe for human ingestion.

The upstream region of the Aa1T gene coding region includes a signal sequence that controls the secretion of Aa1T from the cell. In particular, the signal sequence directs the toxin through the secretory pathway to the cell surface, where it is secreted by the cell. During transport, enzymes cleave the signal sequence, releasing mature Aa1T.

These xenogeneic signal sequences have been found to be useful for the expression and secretion of insect toxins such as AalT (US 08/009 265, 25/01/1993). A preferred xenogeneic signal sequence is the signal sequence in the cuticle of Drosophila melanogaster (for exoskeletal protein), which secretes a large amount of associated mature proteins.

In turn, a codoptimized DNA sequence encoding the cuticular signal sequence and AalT cleavage of the genetic code allows variations of the nucleotide sequence, while the still producing polypeptide has the same amino acid sequence as the polypeptide encoded by the native DNA sequence. The procedure known as codon optimization provides, by means of design, such a modified DNA sequence that reflects the codon frequency used by the host insect. In this embodiment, Drosophila melanogaster tables are used to create a codon-optimized DNA sequence encoding the cuticular signal sequence and AalT.

An additional means of enhancing AalT expression is the use of the AcMNPV DA26 "early" promoter. This promoter is inserted upstream of codonoptimized DNA encoding the cuticular signal sequence and AalT.

Samples of a genetically modified AcMNPV E2 strain containing the DA26 promoter and codoptimized DNA encoding the cutaneous signal sequence and AalT were constructed according to the procedures set out in US 08/070164, which is incorporated herein by reference. Samples of the resulting viral construct, designated AC 1001, were deposited in American Type Culture Collection under number VR-2404. Using conventional techniques, other constructs can be created using a wild-type AalT DNA sequence, another xenogeneic signal sequence, and other promoters.

Improvement in the control of insect viruses by insects can be achieved by genetic modification of the insect virus by gene deletion. An example is a deletion in the gene encoding the ecdysteroid UDP-glucosyl transferase (EGT). Miller et al. reported the construction of such EGT strains of insect viruses (Miller, L. K., et al., W091 / 00014). In particular, Miller describes the construction of the AcMNPV EGT strain.

The expression of the egt strain causes the production of EGT. EGT inactivates the hormone of insect change (undressing) of the insect (ecdysone), which prevents the insect larvae from changing or becoming a cacophony. When the egt gene is inactivated, for example, by creating an EGT strain, changing the shell and converting the larvae into the tubercle when infected with an insect virus can take place. In turn, this continuous development of the insect leads to a favorable result in the protection of the crop, such as reduced nutrition, reduced growth, and rapid death. This is because the EGT 'insect virus fails to block the larvae shell change and turn it into a cacophony, along with stopping feeding in preparation for these undressing stages. As a result, EGT-infected insects are more likely to die earlier than insects infected with wild-type EGT * when trying to change their shell in an infection state. Thus, infecting insects with EGT strains is more effective than infecting insects with wild-type viruses given _LT10 values (the time it takes for half of the insects to die after being infected with the virus).

The egt gene is inactivated by replacing or inserting into it another gene, such as the non-viral marker gene for β-galactosidase. Any DNA sequence can be used to terminate the egt gene of such length that the expression of the egt coding sequence is interrupted. Alternatively, the entire egt gene or portion thereof may be removed from the genome by deletion or mutation of a suitable coding segment. In addition, the regulatory part of the genome that controls egt gene expression can be altered or removed. The result of these modifications is insufficient expression of the egt gene. Deletions inactivating the egt gene can also be accomplished through serial insect viral passages or insect cell culture. All these insertions, deletions or mutations are achieved using conventional means. Deletion viruses have the advantage that they do not contain foreign DNA and are different from the wild type viruses only in that they lack the functional egt gene.

Miller cites an example of an AcMNPV EGT bhrus obtained by recombinantly engineered vEGTDEL in which part of the egt gene is delegated. Miller obtained vEGTDEL by cotransfection in the SF of a pEGTDEL plasmid (which is a cleavage product of a plasmid containing the egt gene with EcoR1 and Xbal to cut a portion of the gene) and DNA from the vEGTZ virus (which contains a LacZ gene inserted into a frame with the previous egt coding sequence). Homologous recombination results in the replacement of the egt-LacZ fusion gene in vEGTZc obtained by deletion of the egt gene from pEGTDEL, resulting in recombinant vEGTDEL virus, which is EGT.

Miller uses a strain of AcMNPV called L1, which is a clonal isolate of the originally isolated wild-type strain (ATCC VR-1345). More recently, an AcMNPV strain called V8 was isolated and characterized. Samples of these V8 strains were deposited in American Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U. S. A. and are ATCC VR-2465. The technique described by Miller for constructing an Ll EGT strain is readily applicable to constructing a V8 EGT strain.

Generally, drug preparation techniques are used to prepare the compositions of the invention. The compositions are in the form of wettable powders, granules, suspensions, emulsions, solutions, aerosol solutions, baits and other insecticidal preparations.

The compositions often include an inactive carrier, which may be a liquid, such as water, alcohol, hydrocarbons or other organic solvents, or a mineral, animal or vegetable oil or powder such as talc, wax, clay, silicate or diatomaceous earth.

The insecticidal compositions of the present invention are administered according to known conventional techniques. These include exposure to the insect pests of the formulations by inhalation (by spraying or crushing the crop where the insects feed), ingestion or direct contact.

Insecticidal compositions are administered in several ways. The virus and the chemical are taken at the same time, either in the same dosage form, or in the same time as two dosage forms. If two dosage forms are used, they are packaged separately and then mixed, if necessary, in the presence of a diluent to obtain the final composition. Alternatively, one of the two components, the virus or chemical, can be applied to bring the insect into stress and then the other component is administered.

The insecticidal compositions of the present invention are administered at doses in the order of 2.4x10 ⁸ -2.4x10 ¹² PIB / ha genetically modified virus with 0.001 -1.0 kg / ha chemical insecticide. These doses are in the order of the doses set for each component individually, but reductions are possible through the combination of insecticidal compositions of the invention.

The concentration of each of the active components of the compositions necessary to obtain the optimum insecticidally effective plant protection composition depends on the type of organism, the chemical substance and the modification of the insect virus and the formulation of the formulation used. These concentrations are readily determined by those skilled in the art.

As an alternative to chemical insecticides, biological control agents are combined with insect viruses. Biological control agents include bacteria such as Bacillus thuringiensis, available for example from Abbott Laboratories such as XENTARI ™ and DIPEL ™ 2X. Other means of biological control are protozoa such as Nosema polyvora, M. grandis, and Bracon mellitor (Jagues, RP, et al., “Compatability of Pathogens with Other Methods of Pest Control and with Different Crops”, Chapter 38, pages 695-715) . Other biological control agents include entomopathogenic fungi (Jagues, R. R., et al., "Compatability of Pathogens with Other Methods of Pest Control and with Different Crops", Chapter 38, pages 695-715) and nematodes. The nematodes are applied in liquid form or dispersed in a gel where they are asleep until they are ready for use.

Examples of carrying out the invention

The following examples are provided for a better understanding of the present invention. These examples are for illustration only and do not limit the scope of the invention.

Example 1. Bioassay technique

The bioassay technique used in these examples is a method of covering food. The bioassay is performed as follows. Heliothis virescens and Helicoverpa zea insects are used. The larvae are reared on soybean / wheat germ (Food Stoneville) food adapted from USDA Insectary Labs, Stoneville, MS. Each colony was stored at 28 ° C under constant fluorescent light. All bioassays were performed on Stoneville food with a second larva larva (H. virescens four days old and H. zea three days old).

Bioassay trays (C-D International, Inc., Pitman, NJ), each containing 32 separate sites, were used. Each 4x4 cm pad contains 5 ml of Stoneville food. Transparent, ventilation openings, adhesive covers (C-D Intemationl, Inc.) enclose the insect on site after treatment and infection. Transparent covers make it easy to read results.

For log / PROBIT ™ (HRO Group, Inc.) analysis, serial dilutions of stored viral solutions in acetone: double-distilled water were prepared before each experiment. Dilutions are made in log increments from 1x10 ⁸ to 1x10 ¹ PIB / ml depending on the type tested. The stored viral solutions were concentrated, if necessary, by centrifugation. Insecticides of technical purity are prepared at various concentrations, measured in parts per million (ppm), based on the weight of the insecticide per volume of solvent.

To the surface of the artificial food (which is solidified) is added by pipette 0.4 ml of acetone solution: water (60:40) to one of the following: viral solution, chemical solution, virus plus chemical solution or untreated solution. Viral solutions are tested for dilution in the order of 1x10 * to 1x10 'RPZ / piv 10-fold dilutions depending on the species tested. The chemicals are administered in an amount ranging from 1000 ppm to 0.1 ppm, depending on the chemical being tested and the type of insect tested. Each dilution was tested for 32 larvae and repeated 34 times. Additions are distributed evenly by rotating the trays and allowing the solutions to dry in a laboratory fireplace. After drying, one larva is added to each test site and allowed to feed for 8 to 10 days. N. virescens are fed for 8 days and H. zea are fed for 12 days. The bioassay trays were stored at 28 ° C under continuous fluorescent light throughout the study period. Readings are made once a day to monitor the timing of the earliest onset of infection. At each reading, a larva is considered dead if it does not show any movement even after shaking the food tray or if the body begins to liquefy. Chemical and viral LC ₂₀ and LC ₅₀ values (the concentration at which 20% or 50% mortality was observed) were calculated on the basis of 3-4 retries. Statistical processing was performed on a computer using the SAS log / PROBIT ™ program for mortality at a dose of 8 or 10 days after treatment. After calculating these PROBIT ™ values, the experiments were performed with the chemical only, at pre-calculated LC ₂₀ and LC _J0 doses, only with the virus at LC _2q h LC _jo doses and all possible permutations of the chemical / virus using the same method of covering food. "LC _2o " is the dose that is intended to cause 20% mortality of the larvae upon application of the product, while "Ι-Χ ^" is the dose that is intended to cause 50% mortality of the larvae upon application of the product.

The concentration of PIB / ml is indicated in the tables given, for example, as "5E4", which is 5x10 ⁴ , where "E" stands for exponent. The designation “DAT” in the tables is days / days after treatment. In these tables, AcMNPV "AalT incorporation" is a genetically modified E2 strain containing a DA26 promoter with codon-optimized DNA encoding the cutaneous signal sequence and AalT.

Improved insect control is obtained from formulations containing a combination of a genetically modified insect virus and a chemical insecticide, when one (or both) increases mortality or improves killing results.

Examples 2-5 present the results of experiments with Helicoverpa zea, Examples 6-8 present the results of experiments with Heliothis virescens.

Example 2. Combination of formamidine, Amitraz, with genetically modified insect viruses

In the first experiment, formamidine, Amitraz, was tested in combination with an insect AcMNPV virus that was genetically modified to either contain AalT or be EGT '. The results are presented in Tables 1 and 2.

TABLE 1. Effect of formamidine, Amitraz, on the virulence of AcMNPV-E2 AalT incorporation ”on a second larva of Helicoverpa zea

Average% larval mortality 8 DAT CXI 44 O G- 5 DAT - 25 CM to Treatment ¹ Amitraz at 100 ppm AcMNPV AalT inclusion at 5E4 PIB / ml Amitraz at 100 ppm plus AcMNPV AalT inclusion at 5E4 PIB / ml

Four repetitions of each treatment when attempting to cover the food. Each repetition was performed with 32 larvae.

The answer given is significantly different from additive (double t-test, P - 0.05)

CM

TABLE 2. Effect of formamidine, Amitraz, on the virulence of AcMNPV-V8 EGT deletion ”on a second larva of Helicoverpa zea

Average% larval mortality 8 DAT see 56 _With 8V. I 5 DAT and .......... r · SG) CM Midrange CM CM Treatment ¹ Amitraz at 100 ppm Well m ο. m · ui with Q. C E K X 3 Φ R Φ Ct 1— 0 LU in > CL z s o o < Amitraz at 100 ppm plus AcMNPV EGT Deletion ”at 5E4 PIB / ml

Four repetitions of each treatment when attempting to cover the food. Each repetition was performed with 32 larvae.

The answer given was not significantly different from additive (double t-test, P = 0.05)

CM

The conclusions are as follows: Amitraz at 100 ppm synergizes the bioactivity of AcMNPV "AalT incorporation" on H. zea larvae. The synergism of said virus depends to some extent on dose, since combinations of this recombinant virus plus Amitraz at 1000 ppm have an additive rather than a synergistic effect on H. zea.

In contrast, Amitraz has a significant effect on the bioactivity of AcMNPV “EGT deletion” on N. zea larvae. There is a digital trend that says the response to the formamidine / EGT deletion combination is slightly weaker than additive.

Example 3. Combination of arylpyrrole with genetically modified insect viruses

In the following experiments, arylpyrrole, 4bromo-2- (p-chlorophenyl) -1- (ethoxymethyl) -5- (trifluoromethyl) pyrrole-3-carbonitrile was tested in combination with an insect virus AcMNPV that was genetically modified or contained AalT, or be 10 EGT. The results are shown in Tables 3 and 4.

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Average% larval mortality I <Q co ⁴³ i co n CO CO., LTD 5 DAT <l “CM Yu 3 DAT 29 CXJ CM 00 Treatment ¹ Arylpyrrole at 1.7 ppm AcMNPV AalT inclusion ”at 5E4 PIB / ml Arylpyrrole at 1.7 ppm plus AcMNPV AalT inclusion ”at 5E4 PIB / ml

TABLE 4. Effect of arylpyrrole, 4-bromo-2- (p-chlorophenyl) -1- (ethoxymethyl) -5 (trifluoromethyl) pyrrole-3-carbonitrile on the virulence of AcMNPV-V8 EGT deletion on a second larva of Helicoverpa zea

Average% larval mortality 8 DAT 43 42 _ SEE Yu 5 DAT 19 C | Oh Yu 3 DAT 29 sch 40 ² Treatment ¹ Arylpyrrole at 1.7 ppm AcMNPV · EGT deletion at 5E4 PIB / ml Arylpyrrole at 1.7 ppm plus AcMNPV EGT deletion at 5E4 PIB / ml

Three repetitions of each treatment when trying to cover the food. Each repetition was performed with 32 larvae.

The answer given was significantly different from additive (double t-test, P = 0.05)

The conclusions are as follows: Arylpyrrole, 4bromo-2- (p-chlorophenyl) -1- (ethoxymethyl) -5- (trifluoromethyl) pyrrole-3-carbonitrile significantly improves the AcMNPV "AalT incorporation" property of the rate of killing of H. zea larvae (ie on a database taken 3 days after treatment). However, at 5 and 8 days after treatment, the response of H. zea to the arylpyrrole / recombinant virus combination was additive (or slightly less than additive).

Arylpyrrole had no statistically significant effect on the average mortality of AcMNPV-V8 "EGT deletion" on second H. zea larvae. However, there is a digital tendency (at 3 days post-treatment) that indicates that arylpyrrole slightly improves the property of the "EGT deletion" rate of killing of H. zea larvae.

Example 4. Combination of diacylhydrazine with genetically modified insect viruses

In the following experiment, diacylhydrazine, dibenzoyl-tert-butylhydrazine was tested in combination with an insect AcMNPV virus, which is genetically 10-mod identified or contains AalT, or be EGT '. The results are shown in Tables 5 and 6.

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Average% larval mortality 1 8 DAT 82 27 σ> H CO., LTD 5 DAT 45 σ> CO CD 3 DAT D " σ> CM U> M ' Treatment ¹ Diacylhydrazine at 200 ppm AcMNPV 'AalT inclusion at 5E5 PIB / ml Diacylhydrazine at 200 ppm plus AcMNPV * AalT inclusion * at 5E5 PIB / ml

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TABLE 6. Effect of diacylhydrazine, dibenzoyl-tert-butylhydrazine on the virulence of AcMNPV-V8 * EGT deletion ”on a mixture of second and third larvae of Helicoverpa zea

Average% larval mortality 8 DAT CM co σ> CO CT σ> 00 5 DAT ο see ST • U · 45 3 DAT D " co see 0> SEE Treatment ¹ Diacylhydrazine at 200 ppm Well -. CD Q. 45 W 45 X Q. p S <To X ζί f f Ct ΙΟ w d > 0. Ζ S o < Diacylhydrazine at 200 ppm plus AcMNPV EGT deletion at 5E5 PIB / ml

Three repetitions of each treatment when trying to cover the food. Each repetition was performed with 32 larvae.

The answer given was significantly different from additive (double t-test, P = 0.05)

The answer given is not significantly different from the additive (double t-test, P = 0.05) cm η

The conclusions are as follows. Diacylhydrazine, dibenzoyl-tert-butylhydrazine, significantly improved the property of AcMNPV "AalT incorporation" rate of killing of H. zea larvae (based on data collected after 3 days of treatment).

Diacylhydrazine also significantly improves the AcMNPV "EGT deletion" property of the rate of killing of H. zea larvae (based on data collected after 3 days of treatment).

Example 5. Combination of Benzoylphenylurea with Genetically Modified Insect Viruses

In the following experiment, benzoylphenylcarbamide, diflubenzuron was tested in combination with an insect virus AcMNPV that was genetically modified or contained AalT or EGT. The results are shown in Tables 7 and 8.

Average% larval mortality 8 DAT <D 36 rco 5 DAT CM 23 <0 CM CM 3 DAT σ> h. e> (D Treatment ¹ Diflubenzuron at 25 ppm AcMNPV AalT inclusion at 5E4 PIB / ml Diflubenzuron at 25 ppm plus AcMNPV AalT inclusion ”at 5E4 PIB / ml

φ and φ s X φ Q н ю О с о

TABLE 8. Effect of benzoylphenylurea, diflubenzuron on the virulence of AcMNPV-V8 EGT deletion ”on a mixture of second and third

Average% larval mortality n <Q co co 36 CM Oh CO., LTD 5 DAT your co CO., LTD CM 3 DAT σ> CO., LTD CT O Treatment ¹ Well Q. Q. CO CM s a c X o Q. >> n X Ф co> © s AcMNPV EGT deletion at 5E4 PIB / ml Diflubenzuron at 25 ppm plus AcMNPV EGT deletion at 5E4 PIB / ml

Three repetitions of each treatment when trying to cover the food. Each repetition was performed with 32 larvae.

The answer given was significantly different from additive (double t-test, P = 0.05)

The answer given is not significantly different from the additive (double t-test, P = 0.05) cm cm

The conclusions are as follows. Benzoylphenylurea, diflubenzuron does not enhance the activity of AcMNPV-E2 "AalT incorporation" on H. zea. In addition, the H. zea response to this combination is less than additive.

Benzoylphenylurea also does not enhance the activity of AcMNPV-V8 EGT deletion on H. zea larvae. In addition, the H. zea response to this combination is less than additive.

Example 6. Combination of wild-type pyrethroid or genetically modified viruses

In the next experiment (in the second H. virescens larvae), the pyrethroid cypermethrin was tested in combination with an insect AcMNPV virus that is either wild-type or genetically modified, either containing AalT or RGT. " The results are shown in Tables 9 and 10.

Table 9 shows the combination of cypermethrin with the wild-type E2 strain of AcMNPV. In this combination, doses equivalent to the predicted LC ₂₀ were used for each component used alone.

Table 9.

Average% mortality at 1 DAT 4 DAT 10 DAT Cypermethrin (0.5 ppm) 11 19 19 AcMNPV wild type (400 PIB / ml) 0 13 27 Cypermethrin (0.5 ppm) + AcMNPV Wild Type ”(400 PIB / ml) 5 31 41 Untreated control 0 0 0

Synergism was not observed with the combination compared to the individual components, which has also been reported by Aspirot (US 4,668,521).

Table 10 shows the combination of cypermethrin with the V8 EGT 'AcMNPV strain. In this combination, doses equivalent to the predicted LC ₂₀ for each component used alone were used.

Table 10.

Average% mortality at 1 DAT 4 DAT 10 DAT Cypermethrin (0.5 ppm) 11 19 19 AcMNPV'EGT pp (775 PIB / ml) 0 2 3 Cypermethrin (0.5 ppm) + AcMNPV'EGT Del. ”(775 PIB / ml) 23 27 34 Untreated control 0 0 0

There is synergism with the combination as compared to the individual components. This synergism is in contrast to the lack of synergism observed with the combination of cypermethrin and wild-type virus.

Table 11 shows the combination of cypermethrin with the E2 “AalT on” strain of AcMNPV. In this combination, doses equivalent to the predicted LC ₂₀ for each component used alone were used.

Table 11.

Average% mortality at 1 DAT 4 DAT 10 DAT Cypermethrin (0.5 ppm) 11 19 19 AcMNPVAalT incl. (1000 PIB / ml) 0 6 22 Cypermethrin (0.5 ppm) + AcMNPVAalT incl. (1000 PIB / ml) 22 38 44 Untreated control 0 0 0

Synergism with the combination was observed as compared to the individual components at 1 and 4 days after treatment. This observed earlier killing rate is better than that observed with the combination of cypermethrin and wild-type virus.

Table 12 shows the combination of cypermethrin with the wild-type E2 strain of AcMNPV. For this combination, doses equivalent to the predicted LC _M doses for each component used alone were used.

Table 12.

Average% mortality at 1 DAT 4 DAT 10 DAT Cypermethrin (1 ppm) 39 58 58 AcMNPV'ahb type (1200 PIB / ml) 0 25 53 Cypermethrin (1 ppm) + AcMNPV-flHB type ”(1200 PIB / ml) 48 77 91 Untreated control 0 0 0

With the exception of one day after treatment, synergism with the combination was not observed compared to the individual components.

Table 13 shows the combination of cypermethrin with the V8 EGT 'AcMNPV strain. In this combination, doses equivalent to the predicted LC ₂₀ doses for cypermethrin and the predicted LC ^ doses for the AcMNPV-V8 EGT 'strain were used.

Table 13.

Average% mortality at 1 DAT 4 DAT 10 DAT Cypermethrin (0.5 ppm) 11 19 19 AcMNPV'EGT Del. ”(1100 PIB / ml) 0 0 8 Cypermethrin (0.5 ppm) + AcMNPV'EGT Del. ”(1100 PIB / ml) 34 47 50 Untreated control 0 0 0

The results of Table 13 are also shown graphically in Figure 1. The combination shows synergism compared to the individual components. This synergism contrasts with the lack of synergism observed with the combination of cypermethrin and wild-type virus, although smaller doses of cypermethrin with a genetically modified virus were used.

Table 14 shows the combination of cypermethrin with the E2 “AalT on” strain of AcMNPV. When this combination is used in doses equivalent to those provided _{LC} 0} doses of each component used alone.

Table 14.

Average% mortality at 1 DAT 4 DAT 10 DAT Cypermethrin (1 ppm) 31 31 31 AcMNPV ”AalT incl (5000 PIB / ml) 0 6 16 Cypermethrin (1 ppm) + AcMNPW'AalT incl. ”(5000 PIB / ml) 25 63 72 Untreated control 0 0 0

The results of Table 14 are also shown graphically in Figure 2. The combination shows synergism compared to the individual components at 4 and 10 days. This synergism contrasts with the lack of synergism observed with the combination of cypermethrin and wild-type virus.

Thus, the combination of cypermethrin with a genetically modified virus, which either contains AalT or is EGT ', is superior to the combination of cypermethrin and wild-type virus. These results are not predictable, given previous data using only combinations of wild-type virus with pyrethroids (Aspirot, J. et al., U.S. Pat. No. 4,668,521).

Example 7. Combination of wild-type diacylhydrazine or genetically modified insect viruses

In the following experiment (in a third larva of H. virescens), diacylhydrazine, dibenzoyl-tert-butylhydrazine was tested in combination with an insectogenic AcMNPV virus, which was either wild-type or genetically modified to be EGT (L1 strain). The results are presented in Tables 15-16. The combination uses smaller doses than those used in Example 6.

Table 15 shows the combination of diacylhydrazine and the wild-type L1 strain of AcMNPV.

Table 15.

T retention / Dose Average% mortality at 1 DAT 4 DAT 10 DAT AcMNPV wild type (1E2 PIB / ml) 0 0 25 Diacylhydrazine (100 ppm) 0 0 31 AcMNPV Wild Type (1E2 PIB / ml) + Diacylhydrazine (100 ppm) 0 19 69 Acetone: water control 0 0 0

The combination shows synergism at 4 and 10 days after treatment.

Table 16 presents the combination of diacylhydrazine with the genetically modified EGT '(L1 strain) of AcMNPY.

Table 16.

T retention / Dose Average% mortality at 1 DAT 4 DAT 10 DAT Recombinant (1 EC PIB / ml> 0 6 88 Diacylhydrazine (100 ppm) 0 0 31 Recombinant (1EZ P1B / t1) + Diacylhydrazine (100 ppm) 0 13 100 Acetone: water control 0 0 0

Observations show that the response is slightly different from additive at the 4-day combination.

Example 8. Combination of wild-type arylpyrrole or genetically modified insect viruses

In the following experiment (in the second H. virescens larva), the arylpyrrole, 4-bromo-2- (p-chlorophenyl) -1- (ethoxymethyl) -5- (trifluoromethyl) pyrrole-3-carbonitrile was tested in combination with the insect virus AcMNPV, which is either wild type or genetically modified to contain AA1T or to be V8 EGT. The results are shown in Tables 17-19.

Table 17 shows the combination of the wild-type E2 strain of AcMNPV. The combination is used at a dose equivalent to that provided for LC ₂₀ for each component used alone.

Table 17.

Average% mortality at 1 DAT 4 DAT 10 DAT Arylpyrrole (1 ppm) 3 8 13 AcMNPV wild type "(400 PIB / ml) 0 0 6 Arylpyrrole (1 ppm) + AcMNPV wild type (400 PIB / ml) 2 19 41 Untreated control 0 0 0

The combination shows synergism at 4 and 10 days after treatment.

Table 18 shows the combination of arylpyrrole with genetically modified EGT (V8 strain) of AcMNPV.

Table 18.

Average% mortality at 1 DAT 4 DAT 10 DAT Arylpyrrole (2 ppm) 20 52 84 AcMNPV 'EGT Del.' (1100 PIB / ml) 0 0 3 Arylpyrrole (2 ppm) + AcMNPV EGT Del '(1100 PIB / ml) 33 50 72 Untreated control 0 0 0

The results showed that the combination at 1 day after treatment showed an improvement in the earlier rate of killing compared to the combination of arylpyrrole and wild-type virus.

Table 19 shows the combination of arylpyrrole with the genetically modified Aa1T incorporation of the E2 strain of AcMNPV.

Table 19.

Average% mortality at 1 DAT 4 DAT 10 DAT Arylpyrrole (2 ppm) 20 52 84 AcMNPV 'AalT incl. (1000 PIB / ml) 0 3 22 Arylpyrrole (2 ppm) + AcMNPV 'AalT incl (1100 PIB / ml) 39 69 78 Untreated control 0 0 0

Synergism with the combination was observed at 1 and 4 days after treatment, with an improvement in the earlier rate of killing compared to the arylpyrrole and wild-type virus combination.

Thus, in all cases, the combination of arylpyrrole, 4-bromo-2- (p-chlorophenyl) -1- (ethoxymethyl) -5- (trifluoromethyl) pyrrole-3-carbonitrile with a genetically modified virus containing AA1T or EGT is superior to the combination of arylpyrrole with wild-type virus.

Claims (16)

Claims 1. An insecticidal composition comprising: (a) an effective amount of a chemical insect selected from the group of chemicals consisting of pyrethroids, arylpyrroles, diacylhydrazines and formamidines, and (b) an effective amount of a genetically modified Autographa califomica nuclear polynuclear virus (AcMNPV) that contains either (i) an inserted gene expressing the insect toxin Androctonus australis (AalT), or (ii) a deletion in the gene encoding ex4D transferase glycerol EGT) of AcMNPV, these compositions being used against lepidopteran insects, provided that when the insects are Heliothis zea and the chemical insecticide is formamidine, the genetically modified AcMNPV contains an inserted gene that expresses AalT. An insecticidal composition according to claim 1, characterized in that the composition comprises: (a) an effective amount of a chemical insecticide selected from the group consisting of pyrethroids and arylpyrroles, and (b) an effective amount of genetically modified AcMNPV that contains either (i) an inserted gene expressing AalT, or (j) a deletion in the gene encoding the EGT of AcMNPV, which composition is used against Heliothis virescens insects. An insecticidal composition according to claim 1, characterized in that the composition comprises: (a) an effective amount of a chemical insecticide selected from the group consisting of arylpyrroles and diacylhydrazines, and (b) an effective amount of genetically modified AcMNPV that contains either (i) an inserted gene expressing AalT or (j) a deletion in the gene encoding the EGT of AcMNPV, which composition is used against Heliothis zea. An insecticidal composition according to claim 1, characterized in that the composition comprises: (a) an effective amount of a chemical insecticide selected from the formamidine group, and (b) an effective amount of genetically modified AcMNPV that contains an inserted gene expressing AalT which composition is used against Heliothis zea insects. An insecticidal composition according to claim 2, characterized in that the chemical insecticide is selected from the group of chemicals consisting of pyrethroids. An insecticidal composition according to claim 5, characterized in that the pyrethroid is α-cyano-3-phenoxybenzyl-cis / trans-3- (2,2-dichlorovinyl) -2,2-dimethylcyclopropanecarboxylate. An insecticidal composition according to claim 2 or claim 3, characterized in that the chemical insecticide is selected from the group of chemicals consisting of arylpyrroles. 8. An insecticidal composition according to claim 7, wherein the arylpyrrole is 4-bromo-2- (p-chlorophenyl) -1- (ethoxymethyl) -5 (trifluoromethyl) pyrrole-3-carbonitrile. An insecticidal composition according to claim 3, characterized in that the chemical insecticide is selected from the group of chemicals consisting of diacylhydrazines. 10. An insecticidal composition according to claim 9, wherein the diacylhydrazine is dibenzoyl-tert-butylhydrazine. An insecticidal composition according to claim 4, wherein the formamidine is N- (2,4-dimethylphenyl) -N [[(2,4-dimethylphenyl) imino] methyl] -Nmethylmethanimidate. An insecticidal composition according to claim 2, 3 or 4, characterized in that the effective amount of the chemical insecticide is 0.001-1.0 kg / ha. An insecticidal composition according to claims 2, 3 or 4, characterized in that the genetically modified AcMNPV contains an inserted gene that expresses AalT. An insecticidal composition according to claims 2, 3 or 4, characterized in that the genetically modified AcMNPV contains a deletion in the gene encoding the EGT of AcMNPV. An insecticidal composition according to claim 2, 3 or 4, characterized in that the effective amount of the genetically modified AcMNPV is 2.4x10 ⁸ -2.4x10 ¹² polynuclear inclusion bodies (PIB) / ha. 16. An insect lepidopteran control method, characterized in that an insecticidal composition according to claim 1 is applied to the insects or to the crop that feeds the insects.