AU708560B2 - Mixtures of genetically modified insect viruses with chemical and biological insecticides for enhanced insect control - Google Patents

Description

WO 96/03048 PCT/US95/09525 1 MIXTURES OF GENETICALLY MODIFIED INSECT VIRUSES WITH CHEMICAL AND BIOLOGICAL

INSECTICIDES

FOR ENHANCED INSECT CONTROL Field of the Invention This invention relates to insecticidal compositions for use against insects comprising mixtures of genetically modified insect viruses with chemical and biological insecticides for enhanced insect control.

Background of the Invention Control of insect pests which infest commercially valuable crops has been the subject of a variety of approaches. Chemical insecticides have been widely used; however, several concerns have been raised about their use. Chemical insecticides may affect beneficial insect species in addition to target, non-beneficial insect species. Insects tend to acquire resistance to such chemicals, thereby requiring the development of new chemicals. Chemicals may persist in the environment for periods of time after their use.

In an effort to reduce the use of chemical insecticides, insect-specific viruses are being utilized to attack insects in their larval stages.

Insect-specific viruses include both DNA and RNA viruses. The DNA viruses include entomopox viruses and Baculoviridae viruses, such as nuclear polyhedrosis viruses granulosis viruses and Baculovirinae non-occluded baculoviruses and the like. The RNA viruses include togaviruses, flaviviruses, picornaviruses, cytoplasmic WO 96/03048 PCT/US95/09525 2 polyhedrosis viruses and the like. The Subfamily of double stranded DNA viruses Eubaculovirinae includes two genera, NPVs and GVs, which are particularly useful for biological control because they produce occlusion bodies in their life cycle.

Examples of NPVs include Lymantria dispar NPV (gypsy moth NPV), Autographa californica

MNPV,

Syngrapha falcifera NPV (celery looper NPV), Spodoptera litturalis NPV, Spodoptera frugiperda

NPV,

Spodoptera exigua NPV, Heliothis armigera NPV, Mamestra brassicae NPV, Choristoneura fumiferana

NPV,

Trichoplusia ni NPV, Helicoverpa zea NPV, etc.

Examples of GVs include Cydia pomonella GV (codling moth GV), Pieris brassicae GV, Trichoplusia ni GV, etc. Examples of NOBs are Orcytes rhinoceros NOB and Heliothis zea NOB. Examples of entomopox viruses include Melolontha melonotha EPV, Amsacta moorei EPV, Locusta migratoria EPV, Melanoplus sanguinipes

EPV,

Schistocerca gregaria EPV, Aedes aegypti EPV, Chironomus luridus EPV, etc.

Over 400 baculovirus isolates have been described as being present in invertebrates. The Autographa californica multiple nuclear polyhedrosis virus ("AcMNPV") is the prototype virus of the Family Baculoviridae and has a wide host range. The AcMNPV virus was originally isolated from Autographa californica cal.), a lepidopteran noctuid (which in its adult stage is a nocturnal moth), commonly known as the alfalfa looper. This virus infects 12 Families and more than 30 species within the order of Lepidopteran insects. It is not known to infect productively any species outside this order.

The life cycle of baculoviruses, as exemplified by AcMNPV, includes two stages. Each WO 96/03048 PCT/US95/09525 3 stage of the life cycle is represented by a specific form of the virus: Extracellular viral particles which are nonoccluded, and occluded virus particles The extracellular and occluded virus forms have the same genome, but exhibit different biological properties. The maturation of each of the two forms of the virus is directed by separate sets of viral genes, some of which are unique to each form.

In its naturally occurring insect infectious form, multiple virions are found embedded in a paracrystalline protein matrix known as an occlusion body which is also referred to as a polyhedron inclusion body The proteinaceous viral occlusions are referred to as polyhedra (polyhedron is the singular term). A polyhedrin protein, which has a molecular weight of 29 kD, is the major viral-encoded structural protein of the viral occlusions.

(Similarly, GVs produce OBs which are composed primarily of granulin, rather than polyhedrin).

The viral occlusions are an important part of the natural baculovirus life cycle, providing the means for horizontal (insect to insect) transmission among susceptible insect species. In the environment, a susceptible insect (usually in the larval stage) ingests the viral occlusions from a contaminated food source, such as a plant. The crystalline occlusions dissociate in the gut of the susceptible insects to release the infectious viral particles. These polyhedron derived viruses invade and replicate in the cells of the midgut tissue.

It is believed that virus particles enter the cell by endocytosis or fusion, and the viral DNA is uncoated at the nuclear pore or in the nucleus.

Viral DNA replication is detected within six hours.

WO 96/03048 PCT/US95/09525 4 By 10-12 hours post-infection secondary infection spreads to other insect tissues by the budding of the extracellular virus from the surface of the cell. The ECV form of the virus is responsible for cell to cell spread of the virus within an individual infected insect, as well as transmitting infection in cell culture.

Late in the infection cycle (12 hours polyhedrin protein can be detected in infected cells.

It is not until 18-24 hours p.i. that the polyhedrin protein assembles in the nucleus of the infected cell and virus particles become embedded in the proteinaceous occlusions. Viral occlusions accumulate to large numbers over 4-5 days as cells lyse. These polyhedra have no active role in the spread of infection in the larva. ECVs disseminate within the infected larva, leading to the death of the larva.

When infected larvae die, millions of polyhedra remain in the decomposing tissue, while the ECVs are degraded. When other larvae are exposed to the polyhedra, for example, by ingestion of contaminated plants or other food material, the cycle is repeated.

In summary, the occluded form of the virus is responsible for the initial infection of the insect through the gut, as well as the environmental stability of the virus. PDVs are essentially not infectious when administered by injection, but are highly infectious orally. The non-occluded form of the virus ECV) is responsible for viral viremia and cell to cell infection in tissue culture. ECVs are highly infectious for cells in culture or internal insect tissues by injection, but essentially not infectious by oral administration.

These insect viruses are not pathogenic to WO 96/03048 PCT/US95/09525 5 vertebrates or plants. In addition, the baculoviruses generally have a narrow host range. Many strains are limited to one or a few insect species.

The use of baculoviruses as bioinsecticides holds great promise. One of the major impediments to their widespread use in agriculture is the time lag between initial infection of the insect and its death.

This lag can range from a few days to several weeks.

During this lag, the insect larva continues to feed, causing further damage to the plant. A number of researchers have attempted to overcome this drawback by inserting a heterologous gene into the viral genome, so as to express an insect controlling or modifying substance, such as a toxin, neuropeptide and hormone or enzyme.

However, to date, 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 were not optimum in view of the limitations of wild-type viruses (Bibliography entries Researchers have also attempted to control insects with other biological control agents such as bacteria Bacillus thuringiensis), fungi, protozoans and nematodes, alone or in combination with insect viruses or chemical insecticides, but they have also not provided optimum results Therefore, there is a need to develop combinations of chemical insecticides and genetically modified insect viruses which will provide the benefits of both components while reducing the amount of chemicals used and reducing the time of kill from that obtained with wild-type viruses through the use of genetically engineered insect viruses.

WO 96/03048 PCTIUS95/09525 6 Summary of the Invention It is an object of this invention to provide insecticidal compositions for use against lepidopteran 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.

This invention provides insecticidal compositions comprising: an effective amount of a chemical insecticide selected from the class of chemicals consisting of pyrethroids, arylpyrroles, diacylhydrazines and formamidines; and an effective amount of a genetically modified Autographa californica nuclear polyhedrosis virus ("AcMNPV") which contains either: an inserted gene which expresses Androctonus australis insect toxin or (ii) a deletion in the gene encoding ecdysteroid UDP-glucosyl transferase of AcMNPV, wherein said compositions are used against lepidopteran insects, with the proviso that when the insects are Heliothis zea insects and the chemical insecticide is a formamidine, the genetically modified AcMNPV contains an inserted gene which expresses AaIT.

In one embodiment, this invention provides WO 96/03048 PCT/US95/09525 7 insecticidal compositions for use against Heliothis virescens insects comprising: an effective amount of a chemical insecticide selected from the class of chemicals consisting of pyrethroids and arylpyrroles; and an effective amount of a genetically modified AcMNPV which contains either: an inserted gene which expresses AaIT, or (ii) a deletion in the gene encoding EGT of AcMNPV.

In another embodiment, this invention provides insecticidal compositions for use against Heliothis zea insects comprising: an effective amount of a chemical insecticide selected from the class of chemicals consisting of arylpyrroles and diacylhydrazines; and an effective amount of a genetically modified AcMNPV which contains either: an inserted gene which expresses AaIT, or (ii) a deletion in the gene encoding EGT of AcMNPV.

In still another embodiment, this invention provides insecticidal compositions for use against Heliothis zea insects comprising: an effective amount of a chemical insecticide selected from the class of chemicals consisting of formamidines; and an effective amount of a genetically modified AcMNPV which contains an inserted gene which expresses AaIT.

This invention further provides a method for the control of lepidopteran insects which comprises WO 96/03048 PCT/US95/09525 8 administering to said insects or to a crop where said insects feed the insecticidal compositions described above.

Brief Description of the Fiqures Figure 1 is a graphical depiction of the data presented in Table 13 below, that is, percent mortality at 1, 4 and 10 days for the first three treatments set forth in Table 13, with the exception that the "Untreated check" data in Table 13 is not depicted in Figure 1.

Figure 2 is a graphical depiction of the data presented in Table 14 below, that is, percent mortality at 1, 4 and 10 days for the first three treatments set forth in Table 14, with the exception that the "Untreated check" data in Table 14 is not depicted in Figure 2. "AcMNPV AaIT-ins." in Table 14 is the same as "rNPV" in Figure 2.

Detailed Description of the Invention Insects such as Lepidoptera undergo a wellcharacterized sequence of events during their development from egg to adult insect. After hatching of the egg, the insect larva enters a period of extensive feeding. During this time, it molts several times to allow for continued growth. Stages between successive molts are referred to as instars. At the end of the larval growth period, the larva pupates and emerges as the adult insect. It is the goal of this invention to enhance the control of pestiferous insects during the larval stages. Lepidopteran families which are known to be important pests of crops include Noctuidae, Notodontidae, Arctiidae, WO 96/03048 PCT/US95/09525 9 Pyralidae, Plutellidae, Pieridae and Geometridae.

Two criteria are utilized to determine whether an insecticidal composition provides effective control of insect pests. One is the number of larvae killed over a period of time. This is referred to as mortality". Another is the speed of kill. Even if the mortality over the final time period is not improved, if more larvae are killed in the early stages of the time period, this is beneficial, in that there is less feeding time and thus less damage to the crop. Thus, if either the mortality or the speed of kill is improved, the composition tested can be said to be an improvement over existing compositions.

A combination of a genetically modified insect virus with a chemical or biological insecticide is said to be "synergistic" if the mortality of the combination is greater than the sum of the single components applied individually; "additive" if the mortality of the combination is equal to the sum of the single components applied individually; "subadditive" if the mortality of the combination is greater than either of the single components applied individually, but less than the sum of the single components applied individually; and "antagonistic" if the mortality of the combination is less than either of the single components applied individually.

Benefits are obtained when the combinations are either synergistic or additive. Even when the combination is additive, by reducing the dose of either or both of the components compared to the dose when applied individually, there is a savings in cost, as well environmental benefits such as reduction in the amount of chemical insecticide which reduces about persistence and development of resistance.

The insecticidal composition is beneficial WO 96/03048 PCTIUS95/09525 10 if it provides enhanced control of either or both permissive and semi-permissive insects. A permissive insect is generally 100-1,000 fold more susceptible to an insect virus or chemical insecticide than a semipermissive insect. For example, the tobacco budworm virescens) is permissive to AcMNPV, whereas the cotton bollworm zea) is semi-permissive to AcMNPV.

An ancillary benefit of this invention is that the combination of the chemical insecticide and insect virus is that more types of insects can be targeted than through the individual components alone.

Both chemical insecticides and insect viruses have specific host ranges. The combinations may expand the host range because of the presence of both components.

However, this effect is not due to any interaction between the insecticidal components.

A large number of classes of insecticidal chemicals are utilized to control insect pests. A summary of a number of these classes and a description of their mode of action will now be set forth.

Pyrethroids are compounds which bind to a sodium ion channel protein, which subsequently causes a change in the action potential across the axonal membrane. In turn, this disrupts proper functioning of the insect nervous system. Examples of pyrethroids include cypermethrin (a-cyano-3-phenoxybenzylcis/trans-3-( 2 ,2-dichlorovinyl)-2,2dimethylcyclopropanecarboxylate; FMC Corp.), PERMETHRIN' 3 -phenoxybenzyl-cis/trans-3-(2,2dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate; Coulston International Corp.), fenvalerate (a-cyano-3phenoxybenzyl-2-(4-chlorophenyl)-3-methylbutyrate) and cyhalothrin (a-cyano-3-phenoxybenzyl-3-(2-chloro- 3,3,3-trifluoro-prop-l-enyl)dimethylcyclopropanecarboxylate).

WO 96/03048 PCT/US95/09525 11 Formamidines are compounds which have several postulated modes of action, including binding to octopamine (a neurohormone/neurotransmitter) receptor and acting as an agonist, enhancment of cAMP production and induction of behavioral changes, or inhbition of mixed function or monoamine oxidases.

Examples of formamidines include Amitraz dimethylphenyl)-N-[[( 2 ,4-dimethylphenyl)imino]methyl]- N-methylmethanimidamide; NOR-AM, Schering AG) and chlordimeform 4 -chloro-O-tolyl)-N,Ndimethylformamidine).

Arylpyrroles are mitochondrial toxins which exert their lethal effects by uncoupling oxidative phosphorylation. Examples of arylpyrroles include 4- (trifluoromethyl)-pyrrole-3-carbonitrile Patent Number 5,310,938) and compounds described in U.S.

Patent Number 5,010,098.

Diacylhydrazines are non-steroidal insect growth regulants, whose primary mode of action is as an ecdysone agonist. Examples of diacylhydrazines include dibenzoyl-t-butylhydrazine (whose preparation is described in U.S. Patent Number 5,300,688) and

MIMIC

T (3,5-dimethylbenzoic acid 1-(1,1dimethylethyl)-2-( 4 -ethylbenzoyl) hydrazide; Rohm Haas Co.).

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

Carbamates act as inhibitors of cholinesterase. Examples of carbamates include thiodicarb (dimethyl-N,N- (thiobis(methylimino)carbonyloxy)- WO 96/03048 PCT/US95/09525 12 bis(ethanimidothioate); Rhone-Poulenc) and methomyl (S-methyl N-[(methylcarbamoyl)oxy] thioacetimidate).

Organophosphates act as inhibitors of cholinesterase. Examples of organophosphates include profenofos (O-4-bromo-2-chlorophenyl O-ethyl S-propyl phosphorothioate; Ciba-Geigy), malathion (O,0-dimethyl phosphorodithioate of diethyl mercaptosuccinate), sulprophos (O-ethyl O-[4-(methylthio)phenyl] S-propyl phosphorodithioate and dimethoate (O,0-dimethyl

(S-

methylcarbamoylmethyl)-phosphorodithioate.

Pyrazoles are inhibitors of mitochondrial respiration by acting specifically at Complex I of the electron transport system. Examples of pyrazoles include tebufenpyrad (N-(4-t-butylbenzyl)-4-chloro-3ethyl-l-methylpyrazole-5-carboxamide; Mitsubishi Kasei, American Cyanamid Company) and compounds described in published European Patent Application Number 289,879.

Nitroguanidines prevent binding of acetylcholine to certain acetylcholine receptors in the postsynaptic membrane; by binding to the receptors themselves, these compounds disrupt neurotransmission.

Examples of nitroguanidines include imidacloprid (1- [(6-chloro-3-pyridinyl)methyl]-N-nitro-2imidazolidinimine; Bayer) and its derivatives.

Milbemycins first bind to a site in the GABA receptor/chloride ion channel complex, and then induce paralysis and death in insects by inhibiting signal transmission at the neuromuscular junction. An example of a milbemycin is abamectin (mixture of avermectins containing >80% avermectin Bla and avermectin Blb; Merck, Sharp Dohme).

Benzoylphenylureas are insect growth regulators which interfere with chitin synthesis, thereby disrupting the process of cuticle formation WO 96/03048 PCT/US95/09525 13 during insect molting. An example of a benzoylphenylurea is diflubenzuron chlorophenyl)-3-(2,6-difluorobenzoyl) urea; Uniroyal Chemical Co., Inc.).

Amidinohydrazones are inhibitors of mitochondrial respiration by inhibiting electron transport at Complex II. An example of an amidinohydrazone is hydramethylnon dimethyl-2(1H)-pyrimidinone (trifluoromethyl)phenyll-1-[2-[4- (trifluoromethyl)phenyl]ethenyl]-2-propenylidene; American Cyanamid Company).

It will be understood by persons skilled in the art that additional examples of the foregoing classes of chemicals are known and available either from commercial suppliers or described in the patent and scientific literature.

In accordance with this invention, the insecticidal composition comprises an insecticidal chemical (or biological insecticide as described below) and a genetically modified insect virus.

In one embodiment of this invention, the genetic modification of the insect virus comprises the insertion of a gene which expresses an insect controlling or modifying substance at any suitable location within the viral genome. The substance, for example, is a toxin, a neuropeptide or a hormone, or an enzyme. A substance thus expressed enhances the bioinsecticidal effect of the virus.

Such toxins include the insect-specific toxin AaIT from the scorpion Androctonus australis a toxin from the straw itch mite species Pyemotes tritici Bacillus thuringiensis toxins and a toxin isolated from spider venom Examples of such neuropeptides or hormones include eclosion WO 96/03048 PCT/US95/09525 14 hormone prothoracicotropic hormone (PTTH), adipokinetic hormone, diuretic hormone and proctolin An example of such enzymes is juvenile hormone esterase (JHE) (14).

This invention is exemplified with a genetically modified AcMNPV which contains an inserted gene which expresses AaIT. The starting point for the genetic modification is the wild-type strain of AcMNPV designated E2 (ATCC VR-1344). The toxin inserted into this viral strain is AaIT, which is produced by the venom of the North African scorpion Androctonus australis Hector. The toxin is 70 amino acids in length and binds to sodium channels in insects and causes contractile paralysis at the nanogram to microgram range in insect larvae. Because AaIT does not bind to mammalian sodium channels, AaIT is a candidate for use as a bioinsecticide to protect crops because it can be safely ingested by humans.

The region upstream of the coding region of the AaIT gene includes a signal sequence which directs the secretion of AaIT from the cell. Specifically, the signal sequence directs the toxin through the secretory pathway to the cell surface where it is secreted from the cell. During transport, enzymes cleave the signal sequence, leaving the mature AaIT.

It has been found that heterologous signal sequences are useful in the expression and secretion of insect toxins, such as AaIT A preferred heterologous signal sequence is the cuticle signal sequence of Drosophila melanogaster (for an exoskeletal protein), which secretes a large quantity of associated mature proteins.

In turn, a codon optimized DNA sequence encoding the cuticle signal sequence and AaIT is used.

The degeneracy of the genetic code permits variations WO 96/03048 PCT/US95/09525 15 of the nucleotide sequence, while still producing a polypeptide having the identical amino acid sequence as the polypeptide encoded by the native DNA sequence.

The procedure known as codon optimization provides one with a means of designing such an altered DNA sequence to reflect the codon frequency utilized by the host insect. In this embodiment, codon use tables for Drosophila melanogaster are utilized to generate a codon optimized DNA sequence encoding the cuticle signal sequence and AaIT.

An additional means to improve AaIT expression is the use of the AcMNPV DA26 "early" promoter. This promoter is inserted upstream of the codon optimized DNA encoding the cuticle signal sequence and AaIT.

Samples of a genetically modified AcMNPV E2 strain containing the DA26 promoter and the codon optimized DNA encoding the cuticle signal sequence and AaIT are constructed in accordance with the procedures set forth in co-pending, commonly-assigned United States Patent Application Serial Number 08/070,164, which is hereby incorporated by reference. Samples of the resulting viral construct, designated AC1001, have been deposited with the American Type Culture Collection and have been assigned ATCC accession number VR-2404. Other constructs using wild-type AaIT DNA sequences, other heterologous signal sequences and other promoters can be generated by persons skilled in the art using conventional techniques.

Improvement of insect viral performance in controlling insects by genetic modification of the insect virus also takes the form of a deletion in a gene. An example is a deletion in the gene encoding ecdysteroid UDP-glucosyl transferase Miller et al. have reported the construction of such EGT- WO 96/03048 PCT/US95/09525 16 strains of insect viruses In particular, Miller described the construction of an AcMNPV EGT- strain.

Expression of the eat gene causes the production of EGT. EGT inactivates insect molting hormones (ecdysone), which prevents the insect larva from molting or pupating. When the egt gene is inactivated, such as by generating an EGT- strain, molting and pupation of the larva infected with the insect virus can proceed. In turn, this continued development of the insect results in such beneficial crop protection results as reduced feeding, reduced growth and more rapid death. This is because the EGTinsect virus fails to block larval molts and pupation, along with the cessation of feeding in preparation for these molting events. Consequently, the EGT- infected insects are much more prone to die earlier than wildtype (EGT infected insects when they attempt to molt while they are in an infected state. Thus, infecting insects with EGT- strains is more effective than infecting insects with wild-type virus in terms of LTS 0 values (the time it takes one-half of a group of insects to die after being infected with a virus).

The eat gene is inactivated by substituting in its place or inserting within it another gene such as the nonviral marker gene for P-galactosidase. Any DNA sequence can be used to disrupt the egt gene as long as it disrupts expression of the et coding sequence. Alternatively, all or part of the eqt gene can be removed from the genome by deleting or mutating an appropriate coding segment. In addition, the regulatory part of the genome that controls eat gene expression can be altered or removed. The result of these modifications is underexpression of the egt gene. Deletions inactivating the eat gene can also be produced by serial virus passage in insects or insect 17 cell culture. All of these insertions, deletions or mutations are achieved using conventional means. The resulting deleted insect viruses have the advantage that they contain no foreign DNA and differ from wild-type viruses only in that they lack a functional egt gene.

Miller exemplified an AcMNPV EGT- virus by the recombinant designated vEGTDEL, in which a portion of the egt gene was deleted. Miller obtained vEGTDEL by cotransfecting into SF cells a plasmid pEGTDEL (which is the product of cleavage of a plasmid containing the egt gene with EcoRI and XbaI so as to excise part of the gene) and DNA from the virus vEGTZ (which contains the lacZ gene inserted in frame with the preceding egt coding sequences). Homologous recombination results in the replacement of the egt-lacZ fusion gene in vEGTZ with the deleted egt gene from pEGTDEL, yielding the recombinant virus vEGTDEL which is EGT-.

Miller used a strain of AcMNPV designated L1, which is a clonal isolate of the originally isolated wild-type strain (ATCC VR-1345). More recently, a strain of AcMNPV designated V8 has been isolated and characterised. Samples of this V8 strain have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, on 22 July 1994, and have been assigned

ATCC

accession number VR 2465. The techniques described in Miller for constructing an L1 EGT- strain are readily applicable to constructing a V8 EGT- strain.

Conventional formulation technology known to persons skilled in the art is used to prepare the compositions of this invention. The compositions are in the form of wettable powders, granules, suspensions, emulsions, solutions, solutions for *o [n:\libc]01798:MEF WO 96/03048 PCT/US95/09525 18 aerosols, baits and other conventional insecticide preparations.

The compositions frequently include an inactive carrier, which can be a liquid such as water, alcohol, hydrocarbons or other organic solvents, or a mineral, animal or vegetable oil, or a powder such as talc, clay, silicate or kieselguhr.

The insecticidal compositions of this invention are applied using conventional techniques known to persons skilled in the art. These include exposing the insect pests to the compositions by inhalation (through spraying or dusting crops where said insects feed), ingestion or direct contact.

The insecticidal compositions are administered in several ways. The virus and chemical are administered at the same time, either in one dosage form or simultaneously with two dosage forms.

If two dosage forms are used, they are packaged separately and then admixed, if necessary in the presence of a diluent, to generate the final composition. Alternatively, one of the virus or chemical can be administered first to stress the insect, followed by the other component.

The insecticidal compositions of this invention are administered at dosages in the range of 2.4X10 8 -2.4X0 12 PIBs/hectare of genetically modified virus with 0.001-1.0 kg/hectare of chemical insecticide. These dosages represent dosage ranges established in the art for each component individually, as well as reductions made possible by the combination insecticidal compositions of this invention.

The concentrations of each of the active components of the compositions needed to produce optimum insecticidally effective compositions for WO 96/03048 PCT/US95/09525 19 plant protection depend on the type of organism, chemical and insect virus modification used and the formulation of the composition. These concentrations are readily determined by a person 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 as XENTARI and DIPEL T 2X.

Other biological control agents include protozoans such as Nosema polyvora, M. grandis and Bracon mellitor Still other biological control agents include entomopathogenic fungi and nematodes.

Nematodes are administered in a liquid formulation or dispersed in a gel where they are in dormant stage until ready for use.

In order that this invention may be better understood, the following examples are set forth. The examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention.

Examples Example 1 Bioassay Technique The bioassay technique used in these examples is the diet overlay method. The bioassays are carried out as follows. The insects used are H.

virescens (tobacco budworm) and H. zea (cotton bollworm). The larvae are reared on a soybean/wheat germ agar-based diet (Stoneville diet), adapted from the USDA Insectary Labs, Stoneville, MS. Each colony is kept at 28°C under constant fluorescent light. All bioassays are conducted on Stoneville diet with second WO 96/03048 PCT/US95/09525 20 instar larvae virescens four days old and H. zea three days old).

Bioassay trays (C-D International, Inc., Pitman, NJ) each contain 32 separate arenas. Each 4X4 cm arena contains 5 ml of Stoneville diet. Clear vented adhesive tops (C-D International, Inc.) enclose the insect in the arena following treatment and infestation. The clear tops allow for easy scoring.

For log/PROBIT TM (HRO Group, Inc.) analysis, serial dilutions are made from viral stock solutions in acetone:double distilled water prior to each experiment. The dilutions are made in log increments from 1X10 8 to 1X10 1 PIBs/ml, depending upon the species tested. Viral stocks are concentrated, when necessary, by centrifugation. Technical grade insecticides are prepared in a variety of concentrations, measured in parts per million ("ppm") based on weight of insecticide to volume of diluent.

To the surface of the artificial diet (which had hardened) is added by pipetting 0.4 ml of an acetone:water (60:40) solution of one of the following: viral solution, chemical solution, viral plus chemical solution or untreated solution. For viral solutions, the dilutions range from 1X10 8 to 1X10 1 PIBs/ml, in 10-fold dilutions, depending upon the insect species tested. The chemical applications range from 1000 ppm to 0.1 ppm, depending upon the chemical studied and the insect species tested. Each dilution is tested with 32 larvae and repeated with 3- 4 replicates. The applications are evenly distributed by rotation of the tray and solutions are allowed to dry in a fume hood. Once dried, one larvae is added to each test arena and allowed to feed for a period of 8 to 10 days. H. virescens are fed for 8 days; H. zea for 12 days. Bioassay trays are kept at 28C in WO 96/03048 PCT/US95/09525 21 continuous fluorescent light throughout the study period. Readings are taken once a day to observe early onset time of infection. At each reading, a larva is considered dead if it exhibits no movement even after shaking the diet tray or if the body becomes liquified. Chemical and viral LC 2 and LC, 0 values (concentration at which 20% or 50% mortality is observed) are calculated, based on 3-4 replicates.

Statistics are computed using the SAS log/PROBIT

T

program, mortality versus dose, at 8 or 10 days posttreatment. Once these PROBIT values are calculated, tests are conducted with the chemicals alone at the predicted

LC

20 and LC, doses, the viruses alone at the

LC,

2 and LC doses, and all possible chemical/virus permutations, using the same diet overlay method.

"LC

20 is the dose which is predicted to cause mortality of the larvae by application of the product, while is the dose which is predicted to cause mortality of the larvae by application of the product.

The concentration of PIBs/ml is indicated in the ensuing Tables, for example, as "5E4", which is 5X10 4 where means exponent. The term "DAT" in the Tables stands for day(s) after treatment. In these Tables, AcMNPV "AaIT inserted" is the genetically modified E2 strain containing the DA26 promoter with the codon optimized DNA encoding the cuticle signal sequence and AaIT.

Enhanced insect control is obtained from the compositions containing a combination of genetically modified insect virus and chemical insecticide when either (or both) increased mortality or improved speed of kill results.

Examples 2-5 present the results of experiments with Helicoverpa zea; Examples 6-8 present WO 96/03048 PCT/US95/09525 22 the results of experiments with Heliothis virescens.

Example 2 Combination of the Formamidine, Amitraz With Genetically Modified Insect Viruses In the first experiment, the formamidine, Amitraz is tested in combination with the insect virus AcMNPV, which is genetically modified to either contain AaIT or be EGT-. The results are presented in Tables 1 and 2.

pw~i~ o Table 1. Impact of the formamidine, amitraz, on virulence of AcMNPV-E2 "AaIT inserted" against second-instar Helicoverpa zea Treatment 1 Mean larval mortality DAT 8 DAT Amitraz at 100 ppm 1 2 AcMNPV "AaIT inserted" at 5E4 PIBs/ml 25 44 Amitraz at 100 ppm plus AcMNPV "AaIT inserted" at 5E4 PIBs/ml 542 702 SFour replications of each treatment in a diet-overlay assay; each replicate consists of 32 larvao.

2 indicates response is significantly different from additive (paired t-test, P 0.05).

I

0 Table 2. Impact of the formamidine, amitraz, on virulence of AcMNPV-V8 "EGT deleted" against second-instar Helicoverpa zea TreatmentMean larval mortality Treat t 5 DAT 8 DAT Amitraz at 100 ppm 1 2 AcMNPV "EGT deleted" at 5E4 PIBs/ml 29 56 Amitraz at 100 ppm plus AcMNPV "EGT deleted" at 5E4 PIBs/ml 222 482 1 Four replications of each treatment in a diet-overlay assay; each replicate consists of 32 larvae.

2 indicates response is not significantly different from additive (paired t-test, P 0.05).

r> i WO 96/03048 PCTIUS9509525 25 The conclusions are as follows: Amitraz at 100 ppm synergizes the bioactivity of AcMNPV "AaIT inserted" against H. zea larvae. The synergism of the aforementioned virus is somewhat dose-dependent, since combinations of this recombinant virus plus Amitraz at 1000 ppm produce additive, rather than synergistic, effects on H. zea.

In contrast, Amitraz has no significant effect on the bioactivity of AcMNPV "EGT deleted" against H. zea larvae. There is a numerical trend suggesting H. zea response to the formamidine/"EGT deleted" combination is slightly less than additive.

Example 3 Combination of an Arylpyrrole With Genetically Modified Insect Viruses In the next experiment, the arylpyrrole 4bromo-2-(p-chlorophenyl)-1-(ethoxymethyl)-5- (trifluoromethyl)-pyrrole-3-carbonitrile is tested in combination with the insect virus AcMNPV, which is genetically modified to either contain AaIT or be EGT-.

The results are presented in Tables 3 and 4.

Table 3. Impact of the arylpyrrole, 4-bromo-2- (p-chlorophenyl) -1-(ethoxymethy.) (trifluoromethyl)pyrr 3cabitrile on virulence of AcMNPV-E2 "'AaIT inserted" against second-instar Helicoverpa zea Mean larval mortality Treatment' 3 DAT 5 DAT 8 DAT Arylpyrrole at 1.7 ppm 29 41 43 AcMNPV "AaIT inserted" at 5E4 PIBs/ml 2 9 34 Arylpyrrole at 1.7 ppm plus AcMNPV "AaIT inserted" at 5E4 PIBs/ml 4 82 52' 63 3

N)

of 32 larvae.

2indicates response issignificantly different from additive (paired t-test, P 0.05).

3.indicates response is not significantly different from additive (paired t-test).

o Table 4. Impact of the arylpyrrole, 4-bromo-2-(p-chlorophenyl)-1-(ethoxymethyl) (trifluoromethyl)-pyrrole-3-carbonitrile, on virulence of AcMNPV-V8 "EGT deleted against second-instar Helicoverpa zea Treatment 1 Mean larval mortality Tre 3 DAT 5 DAT 8 DAT Arylpyrrole at 1.7 ppm 29 41 43 AcMNPV "EGT deleted" at 5E4 PIBs/ml 2 19 42 Arylpyrrole at 1.7 ppm plus AcMNPV "EGT deleted" at 5E4 PIBs/ml 40' 50 612 SThree replications of each treatment in a diet-overlay assay; each replicate consists of 32 larvae.

2 indicates response is not significantly different from additive (paired t-test, P 0.05).

~17 WO 96/03048 PCT/US95/09525 28 The conclusions are as follows: The arylpyrrole 4-bromo-2-(p-chlorophenyl)-1- (ethoxymethyl)-5-(trifluoromethyl)-pyrrole-3carbonitrile significantly hastens the speed-of-kill properties of AcMNPV "AaIT inserted" against H. zea larvae based on data taken at three days posttreatment]. However, at five and eight days posttreatment, H. zea response to this arylpyrrole/recombinant virus combination is additive (or slightly less than additive).

The arylpyrrole has no statistically significant effect on the mean mortality of AcMNPV-V8 "EGT deleted" against second-instar H. zea. However, there is a numerical trend (at 3 DAT) suggesting the arylpyrrole slightly hastens speed-of-kill properties of "EGT deleted" against H. zea larvae.

Example 4 Combination of a Diacylhydrazine With Genetically Modified Insect Viruses In the next experiment, the diacylhydrazine dibenzoyl-t-butylhydrazine is tested in combination with the insect virus AcMNPV, which is genetically modified to either contain AaIT or be EGT-. The results are presented in Tables 5 and 6.

t~f~S' Table 5. Impact of the diacylhydrazine, dibenzoyl-t-butylhydrazine, on virulence of AcMNPV-E2 "AaIT inserted" against a mixture of second- third-instar Helicoverpa zea 0 Treatment Mean larval mortality Treat t 3 DAT 5 DAT 8 DAT Diacylhydrazine at 200 ppm 11 45 82 AcMNPV "AaIT inserted" at 5E5 PIBs/ml 9 19 27 Diacylhydrazine at 200 ppm plus AcMNPV "AaIT inserted" at 5E5 PIBs/ml 452 633 843 SThree replications of each treatment in a diet-overlay assay; each replicate consists of 32 larvae.

2 indicates response is significantly different from additive (paired t-test, P 0.05) indicates response is not significantly different from additive (paired t-test, P 0.05).

0

U'

1

MIWT

o Table 6. Impact of the diacy1hydrazine, dibenzoyl-t-butylhydrazine, on virulence of AcNPV-V8 "EGT deleted against a mixture of second- third-instar Helicoverpa zea 0 Treatment i Mean larval mortality Treatmn3 DAT 5 DAT 8 DAT Diacylhydrazine at 200 ppm 11 45 82 AcMNPV "EGT deleted" at 5E5 PIBs/ml 6 20 39 Diacylhydrazine at 200 ppm plus AcMNPV "EGT deleted" at 5E5 PIBs/ml 292 543 89) 1 Three replications of each treatment in a diet-overlay assay; each replicate consists indicates response is significantly different from additive (paired t-test, 0.05) 3 indicates response is not significantly different from additive (paired t-test, P 0.05).

FA

IF

WO 96/03048 PCT/US95/09525 31 The conclusions are as follows: The diacylhydrazine, dibenzoyl-t-butylhydrazine significantly hastens the speed-of-kill properties of AcMNPV AaIT inserted" against H. zea larvae based on data collected at 3 DAT].

The diacylhydrazine also significantly hastens the speed-of-kill properties of AcMNPV "EGT deleted" against H. zea larvae based on data collected at 3 DAT].

Example Combination of a Benzoylphenylurea With Genetically Modified Insect Viruses In the next experiment, the benzoylphenylurea diflubenzuron is tested in combination with the insect virus AcMNPV, which is genetically modified to either contain AaIT or be EGT-.

The results are presented in Tables 7 and 8.

MITF

Table 7. Impact of the benzoylphenylurea, diflubenzuron, on virulence of AcMNPV-E2 "AaIT inserted" against second-instar Helicoverpa zeaI Treatment i Mean larval mortality Treat t 3 DAT 5 DAT 8 DAT Diflubenzuron at 25 ppm 9 12 16 AcMNPV "AaIT inserted" at 5E4 PIBs/ml 7 23 36 Diflubenzuron at 25 ppm plus AcMNPV "AaIT inserted" at 5E4 PIBs/ml 63 223 372 1 Three replications of each treatment in a diet-overlay assay; each replicate consists of 32 larvae.

indicates response is significantly different from additive (paired t-test, P 0.05) indicates response is not significantly different from additive (paired t-test, P 0.05).

hj

COI

d Table 8 Impact of the benzoylphenylurea, diflubenzuron, on virulence of AcMNPV-V8 "EGT deleted" against second-instar Helicoverpa zeaP 00 TreatmentMean larval mortality 3 DAT 5 DAT 8 DAT Diflubenzuron at 25 ppm 9 12 16 AcMNPV "EGT deleted" at 5E4 PIBs/ml 5 13 36 Diflubenzuron at 25 ppm plus AcMNPV "EGT deleted at 5E4 PIBs/ml 103 233 302 SThree replications of each treatment in a diet-overlay assay; each replicate consists of 32 larvae.

2 indicates response is significantly different from additive (paired t-test, p 0.05) Sindicates response is not significantly different from additive (paired t-test, P 0.05).

H f^L^ Ur

U'

WO 96/03048 PCT/US95/09525 34 The conclusions are as follows: The benzoylphenylurea, diflubenzuron does not improve activity of AcMNPV-E2 "AaIT inserted" against H. zea larvae; further, H. zea response to this combination is less than additive.

The benzoylphenylurea also does not improve activity of AcMNPV-E2 "EGT deleted" against H. zea larvae; further, H. zea response to this combination is less than additive.

Example 6 Combination of a Pyrethroid With Wild-Type or Genetically Modified Insect Viruses In the next experiment (in second instar H.

virescens), the pyrethroid, cypermethrin is tested in combination with the insect virus AcMNPV, which is either wild-type or genetically modified to either contain AaIT or be EGT-. The results are presented in Tables 9-14.

Table 9 depicts the combination of cypermethrin with the wild-type E2 strain of AcMNPV.

The combination utilizes a dosage equivalent to the predicted

LC

20 of each component used alone.

TABLE 9 MEAN MORTALITY

AT

1DAT 4DAT Cypermethrin (0.5 ppm) 11 19 19 AcMNPV "wild-type" (400 PIBs/ml) 0 13 27 Cypermethrin (0.5 ppm) AcMNPV "wild-type" (400 PIBs/ml) 5 31 41 Untreated Check 0 0 0 WO 96/03048 PCT/US95/09525 35 Synergism is not observed with the combination compared to the individual components, as was also reported by Aspirot Table 10 depicts the combination of cypermethrin with the V8 EGT- strain of AcMNPV. The combination utilizes a dosage equivalent to the predicted

LC

20 of each component used alone.

TABLE MEAN MORTALITY AT 1DAT 4DAT Cypermethrin (0.5 ppm) 11 19 19 AcMNPV "EGT del." (775 PIBs/ml) 0 2 3 Cypermethrin (0.5 ppm) AcMNPV "EGT del." (775 PIBs/ml) 23 27 34 Untreated Check 0 0 0 Synergism is observed with the combination compared to the individual components. This synergism contrasts with the lack of synergism observed with the combination of cypermethrin and the wild-type virus.

Table 11 depicts the combination of cypermethrin with the E2 "AaIT inserted" strain of AcMNPV. The combination utilizes a dosage equivalent to the predicted

LC

20 of each component used alone.

TABLE 11 MEAN MORTALITY AT 1DAT 4DAT Cypermethrin (0.5 ppm) 11 19 19 AcMNPV "AaIT ins." (1000 PIBs/ml) 0 6 22 WO 96/03048 PCT/US95/09525 36 Cypermethrin (0.5 ppm) AcMNPV "AaIT ins." (1000 PIBs/ml) 22 38 44 Untreated Check 0 0 0 Synergism is observed with the combination compared to the individual components at 1 and 4 DAT.

This earlier speed of kill is superior to that observed with the combination of cypermethrin and the wild-type virus.

Table 12 depicts the combination of cypermethrin with the E2 wild-type strain of AcMNPV.

The combination utilizes a dosage equivalent to the predicted LC 0 of each component used alone.

TABLE 12 MEAN MORTALITY AT 1DAT 4DAT Cypermethrin (1 ppm) 39 58 58 AcMNPV "wild-type" (1200 PIBs/ml) 0 25 53 Cypermethrin (1 ppm) AcMNPV "wild-type" (1200 PIBs/ml) 48 77 91 Untreated Check 0 0 0 Except for one DAT, synergism is not observed with the combination compared to the individual components.

Table 13 depicts the combination of cypermethrin with the V8 EGT- strain of AcMNPV. The combination utilizes a dosage equivalent to the predicted

LC

20 for cypermethrin and the predicted

LC,,

for the AcMNPV V8 EGT- strain.

WO 96/03048 PCT/US95/09525 37 TABLE 13 MEAN MORTALITY AT 1DAT 4DAT Cypermethrin (0.5 ppm) 11 19 19 AcMNPV "EGT del." (1100 PIBs/ml) 0 0 8 Cypermethrin (0.5 ppm) AcMNPV "EGT del." (1100 PIBs/ml) 34 47 Untreated Check 0 0 0 The results of Table 13 are also depicted graphically in Figure 1. Synergism is observed with the combination compared to the individual components.

This synergism contrasts with the lack of synergism observed with the combination of cypermethrin and the wild-type virus, even though a smaller dose of cypermethrin is used with the genetically modified virus.

Table 14 depicts the combination of cypermethrin with the E2 "AaIT inserted" strain of AcMNPV. The combination utilizes a dosage equivalent to the predicted LCso of each component used alone.

TABLE 14 MEAN MORTALITY AT 1DAT 4DAT Cypermethrin (1 ppm) 31 31 31 AcMNPV "AaIT ins." (5000 PIBs/ml) 0 6 16 Cypermethrin (1 ppm) AcMNPV "AaIT ins." (5000 PIBs/ml) 25 63 72 Untreated Check 0 0 0 WO 96/03048 PCT/US95/09525 38 The results of Table 14 are also depicted graphically in Figure 2. Synergism is observed with the combination compared to the individual components at 4 and 10 DAT. This synergism contrasts with the lack of synergism observed with the combination of cypermethrin and the wild-type virus.

Thus, the combination of cypermethrin with either the virus genetically modified to contain AaIT or be EGT- is superior to the combination of cypermethrin and the wild-type virus. These results are not predictable in view of the prior data using only combinations of the wild-type virus with pyrethroids Example 7 Combination of a Diacylhydrazine With Wild-Type or Genetically Modified Insect Viruses In the next experiment (in third instar H.

virescens), the diacylhydrazine, dibenzoyl-tbutylhydrazine is tested in combination with the insect virus AcMNPV, which is either wild-type or genetically modified to be EGT- (LI strain). The results are presented in Tables 15-16. The combinations utilize lower dosages than those used in Example 6.

Table 15 depicts the combination of the diacylhydrazine with the wild-type L1 strain of AcMNPV.

WO 96/03048 PCTIUS95/09525 39 TABLE Treatment/Dose Percent Mortality 1DAT 4DAT AcMNPV "wild-type" (1E2 PIBs/ml) 0 0 Diacylhydrazine (100ppm) 0 0 31 AcMNPV "wild-type" (1E2 PIBs/ml) Diacylhydrazine (100ppm) 0 19 69 Acetone:Water Check 0 0 0 Synergism is observed with the combination at 4 and 10 DAT.

Table 16 depicts the combination of the diacylhydrazine with the genetically modified EGT- (LI strain) of AcMNPV.

TABLE 16 Treatment/Dose Percent Mortality 1DAT 4DAT Recombinant (1E3 PIBs/ml) 0 6 88 Diacylhydrazine (100ppm) 0 0 31 Recombinant (1E3 PIBs/ml) Diacylhydrazine (100 ppm) 0 13 100 Acetone:Water Check 0 0 0 Observations indicate this response is slightly different than additive with the combination at 4 DAT.

WO 96/03048 PCT/US95/09525 40 Example 8 Combination of an Arylpyrrole With Wild-Type or Genetically Modified Insect Viruses In the next experiment (in second instar H.

virescens), the arylpyrrole, 4-bromo-2-(ppyrrole-3-carbonitrile is tested in combination with the insect virus AcMNPV, which is either wild-type or genetically modified to either contain AaIT or be V8 EGT-. The results are presented in Tables 17-19.

Table 17 depicts the combination of the arylpyrrole with the wild-type E2 strain of AcMNPV.

The combination utilizes a dosage equivalent to the predicted

LC

20 for each component used alone.

TABLE 17 MEAN MORTALITY

AT

1DAT 4DAT Arylpyrrole (1 ppm) 3 8 13 AcMNPV "wild-type" (400 PIBs/ml) 0 0 6 Arylpyrrole (1 ppm) AcMNPV "wild-type" (400 PIBs/ml) 2 19 41 Untreated Check 0 0 0 Synergism is observed with the combination at 4 and 10 DAT.

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

WO 96/03048 PCT/US95/09525 41 TABLE 18 MEAN MORTALITY AT 1DAT 4DAT Arylpyrrole (2 ppm) 20 52 84 AcMNPV "EGT del." (1100 PIBs/ml) 0 0 3 Arylpyrrole (2ppm) AcMNPV "EGT del." (1100 PIBs/ml) 33 50 72 Untreated Check 0 0 0 Results indicate that, with the combination at 1 DAT, an improved earlier speed of kill is observed compared to the combination of the arylpyrrole and the wild-type virus.

Table 19 depicts the combination of the arylpyrrole with the genetically modified AaIT inserted E2 strain of AcMNPV.

TABLE 19 MEAN MORTALITY AT 1DAT 4DAT Arylpyrrole (2 ppm) 20 52 84 AcMNPV "AaIT ins." (1000 PIBs/ml) 0 3 22 Arylpyrrole (2ppm) AcMNPV "AaIT ins." (1000 PIBs/ml) 39 69 78 Untreated Check 0 0 0 Synergism is observed with the combination at 1 and 4 DAT, indicating an improved earlier speed of kill compared to the combination of the arylpyrrole and the wild-type virus.

Thus, on an overall basis, the combination WO 96/03048 PCTIUS95/09525 42 of the arylpyrrole 4-bromo-2- (p-chlorophenyl) -1- (ethoxymethyl) (trifluoromethyl) -pyrrole-3carbonitrile with either the virus genetically modified to contain AaIT or be EGT- is superior to the combination of the arylpyrrole and the wild-type virus.

WO 96/03048 PCTIUS95/09525 43 Bibliog~raphy 1. Aspirot, et al., United States Patent Number 4,668,511.

2. Mohamed, et al.f E~nviron.

Entomolocv, 12, 478-481 (1983).

3. Mohamed, et al.f Environ.

Entomoloov, 1-2, 1403-1405 (1983).

4. Velichkova-Kozhukharova, et al., Rasteniev~ldni Nauki, 25, 80-86 (1988).

Jaques, et al., "Compatability of Pathogens with Other Methods of Pest Control and with Different Crops", Chapter 38, pages 695-715.

6. Geervliet, et al., Med. Fac.

Landbouww. Rijksuniv. Gent, 56, 305-311 (1991).

7. Zlotkin, et al., Toxicon, 9,.1-8 (1971).

8. Tomaiski, et al., United States Patent Number 5,266,317.

9. Martens, et al., App. Envir.

Microbioloa-v, 56, 2764-2770 (1990).

Federici, In Vitro, 28, (1992).

11. Jackson, et al., United States Patent Number 4,925,664.

12. Eldridge, et al., Insect Biochem., 21, 341-351 (1992).

13. Menn, et al., J. Acric. Food Chem., 37, 271-278 (1989).

14. Hammock, et al., Nature, 344, 458-461 (1990) United States Patent Application Serial No. 08/009,265, filed January 25, 1993.

16. Miller, et al., International Patent Application Number WO 91/00014.

Claims (15)

1. An insecticidal composition comprising: an effective amount of a chemical insecticide selected from the class of chemicals consisting of pyrethroids, arylpyrroles, diacylhydrazines and formamidines; and an effective amount of a genetically modified Autographa californica nuclear polyhedrosis virus ("AcMNPV") which contains either: an inserted gene which expresses Androctonus australis insect toxin or (ii) a deletion in the gene encoding ecdysteroid UDP-glucosyl transferase of AcMNPV, wherein said composition is used against lepidopteran insects, with the proviso that when the insects are Heliothis zea insects and the chemical insecticide is a formamidine, the genetically modified AcMNPV contains an inserted gene which expresses AaIT.

2. The insecticidal composition of Claim 1 wherein the composition comprises: an effective amount of a chemical insecticide selected from the class of chemicals consisting of pyrethroids and arylpyrroles; and an effective amount of a genetically modified AcMNPV which contains either: an inserted gene which expresses AaIT, or (ii) a deletion in the gene encoding EGT of AcMNPV, wherein said composition is used against Heliothis virescens insects. WO 96/03048 PCT/US95/09525

3. The insecticidal composition of Claim 1 wherein the composition comprises: an effective amount of a chemical insecticide selected from the class of chemicals consisting of arylpyrroles and diacylhydrazines; and an effective amount of a genetically modified AcMNPV which contains either: an inserted gene which expresses AaIT, or (ii) a deletion in the gene encoding EGT of AcMNPV, wherein said composition is used against Heliothis zea insects.

4. The insecticidal composition of Claim 1 wherein the composition comprises: an effective amount of a chemical insecticide selected from the class of chemicals consisting of formamidines; and an effective amount of a genetically modified AcMNPV which contains an inserted gene which expresses AaIT, wherein said composition is used against Heliothis zea insects.

5. The insecticidal composition of Claim 2 wherein the chemical insecticide is selected from the class of chemicals consisting of pyrethroids.

6. The insecticidal composition of Claim wherein the pyrethroid is a-cyano-3-phenoxybenzyl- cis/trans-3-(2,2-dichlorovinyl)-2,2- dimethylcyclopropanecarboxylate.

7. The insecticidal composition of Claim 2 or Claim 3 wherein the chemical insecticide is selected from the class of chemicals consisting of arylpyrroles. WO 96/03048 46 PCT/US95/09525

8. The insecticidal composition of Claim 7 wherein the arylpyrrole is 4 -bromo-2-(p-chlorophenyl)- 1-(ethoxymethyl)-5-(trifluoromethyl)-pyrrole-3- carbonitrile.

9. The insecticidal composition of Claim 3 wherein the chemical insecticide is selected from the class of chemicals consisting of diacylhydrazines. The insecticidal composition of Claim 9 wherein the diacylhydrazine is dibenzoyl-t- butylhydrazine.

11. The insecticidal composition of Claim 4 wherein the formamidine is 2 ,4-dimethylphenyl)-N- 4 -dimethylphenyl)imino]methyl]-N- methylmethanimidamide.

12. The insecticidal composition of any of Claim 2, Claim 3 or Claim 4 wherein the effective amount of the chemical insecticide is 0.001-1.0 kg/hectare.

13. The insecticidal composition of any of Claim 2, Claim 3 or Claim 4 wherein the genetically modified AcMNPV contains an inserted gene which expresses AaIT.

14. The insecticidal composition of any of Claim 2, Claim 3 or Claim 4 wherein the genetically modified AcMNPV contains a deletion in the gene encoding EGT of AcMNPV. The insecticidal composition of any of Claim 2, Claim 3 or Claim 4 wherein the effective amount of the genetically modified AcMNPV is 2.4X10 8 2.4X10 12 polyhedron inclusion bodies ("PIBs")/hectare.

16. A method for the control of lepidopteran insects which comprises administering to said insects or to a crop where said insects feed the insecticidal composition of Claim 1. JUN 11 '99 01:36PM SPRUSON FERGUSON 92615486 P.6 47

17. An insecticidal composition which includes a genetically modified Autographa californtca nuclear polyhedrosis virus, substantially as hereinbefore described with reference to any one of the examples. Dated 11 June, 1-999 American Cyanamid Company Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON S C 9*4 a 04 S a *0* 0 S at hO t 0 .me 0 000 OS (1L1[BFjW)15.dft:Mtff 11/06 '99 FRI 12:37 [TX/RX. NO 8226] 90j06