Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/50—Isolated enzymes; Isolated proteins
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/40—Viruses, e.g. bacteriophages
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/52—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing groups, e.g. carboxylic acid amidines
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
  • A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N53/00—Biocides, pest repellants or attractants, or plant growth regulators containing cyclopropane carboxylic acids or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts

Definitions

• 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.
• the DNA viruses include entomopox viruses ("EPV”), and Baculoviridae viruses, such as nuclear polyhedrosis viruses (“NPV”), granulosis viruses (“GV”), and Baculovirinae non-occluded baculoviruses (“NOB”), and the like.
• the RNA viruses include togaviruses, flaviviruses, picomaviruses, cytoplasmic 6/03048 PC17US95/09525
• CPV polyhedrosis viruses
• OBs occlusion bodies
• NPVs examples include Lyma ⁇ tria dispar NPV (gypsy moth NPV) , Autographa californica MNPV, ⁇ yngrapha falcifera NPV (celery looper NPV) , Spodoptera li tturalis NPV, Spodoptera frugiperda NPV, Spodoptera exigua NPV, Heliothis armigera NPV, Mame ⁇ tra 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 sanguinipe ⁇ EPV, Schistocerca gregaria EPV, Aedes aegypti EPV, Cbironomus luridus EPV, etc.
• 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 (A. cal . ) , a lepidopteran noctuid (which in its adult stage is a nocturnal moth) , commonly known as the alfalfa looper. This virus infects 12
• the life cycle of baculoviruses includes two stages. Each stage of the life cycle is represented by a specific form of the virus: Extracellular viral particles ("ECV") which are nonoccluded, and occluded virus particles (“OV”) .
• ECV Extracellular viral particles
• OV 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.
• OB occlusion body
• PIB 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.
• 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.
• a susceptible insect usually in the larval stage
• 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.
• 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. By 10-12 hours post-infection (“p.i.”), secondary infection spreads to other insect tissues by the budding of the extracellular virus (“ECV”) from the surface of the cell.
• ECV extracellular virus
• polyhedrin protein Late in the infection cycle (12 hours p.i.), 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.
• 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 i.e., ECV
• 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 vertebrates or plants.
• the baculoviruses generally have a narrow host range. Many strains are limited to one or a few insect species.
• 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.
• 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.
• compositions comprising:
• an effective amount of a genetically modified Auto rapha californica nuclear polyhedrosis virus which contains either: (i) an inserted gene which expresses Androctonus australis insect toxin (“AalT”) , or (ii) a deletion in the gene encoding ecdysteroid UDP-glucosyl transferase (“EGT”) of AcMNPV, wherein said compositions are used against lepidopteran insects, with the proviso that when the insects are fleliothis zea insects and the chemical insecticide is a formamidine, the genetically modified AcMNPV contains an inserted gene which expresses AalT.
• this invention provides insecticidal compositions for use against Heliothis virescens insects comprising:
• this invention provides insecticidal compositions for use against Heliothis zea insects comprising: (a) an effective amount of a chemical insecticide selected from the class of chemicals consisting of arylpyrroles and diacylhydrazines; and (b) an effective amount of a genetically modified AcMNPV which contains either:
• this invention provides insecticidal compositions for use against Heliothis zea insects comprising:
• This invention further provides a method for the control of lepidopteran insects which comprises 6/03048 PC17US95/09525
• 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 AalT-ins.” in Table 14 is the same as "rNPV” in Figure 2.
• Lepidopteran families which are known to be important pests of crops include Noctuidae, Notodontidae, Arctiidae, 96/03048 PC17US95/09525
• 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; “sub- additive” 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 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 semi- permissive insect.
• the tobacco budworm ⁇ H. virescens) is permissive to AcMNPV
• the cotton bollworm (H. 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.
• 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 ( ⁇ -cyano-3-phenoxybenzy1- cis/trans-3- (2,2-dichlorovinyl) -2,2- dimethylcyclopropanecarboxylate; FMC Corp.), PERMETHRINTM (3-phenoxybenzyl-cis/trans-3- (2,2- dichlorovinyl) -2,2-dimethylcyclopropanecarboxylate;
• 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.
• formamidines examples include Amitraz (N'-(2,4- dimethylphenyl) -N- [ [ (2,4-dimethylphenyl) imino]methyl] - N-methylmethanimidamide; NOR-AM, Schering AG) and chlordi eform (N' - (4-chloro-O-tolyl) -N,N- dimethylformamidine) .
• Arylpyrroles are mitochondrial toxins which exert their lethal effects by uncoupling oxidative phosphorylation.
• Examples of arylpyrroles include 4- bromo-2- (p-chlorophenyl) -1- (ethoxymethyl) -5-
• 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 MIMICTM (3,5-dimethylbenzoic acid 1-(1,1- dimethylethyl) -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-1,5,5,6,9,9- hexahydro-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) - bis (ethanimidothioate) ; Rhone-Poulenc) and methorny1 (S-methyl N- [ (methylcarbamoyl)oxy] thioacetimidate) .
• Organophosphates act as inhibitors of cholinesterase.
• organophosphates include profenofos (0-4-bromo-2-chlorophenyl O-ethyl S-propyl phosphorothioate; Ciba-Geigy), malathion (O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate) , sulprophos (O-ethyl O- [4- (methylthio)phenyl] S-propyl phosphorodithioate and dimethoate (0,O-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-3- ethyl-l-methylpyrazole-5-carboxamide; Mitsubishi asei, 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 neurotransmis ⁇ ion.
• Examples of nitroguanidines include imidacloprid (1- [ (6-chloro-3-pyridinyl) ethyl] -N-nitro-2- imidazolidinimine; 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 ⁇ 20% avermectin Bib; Merck, Sharp & Doh e) .
• Benzoylphenylureas are insect growth regulators which interfere with chitin synthesis, thereby disrupting the process of cuticle formation during insect molting.
• An example of a benzoylphenylurea is diflubenzuron (1- (4- chlorophenyl) -3- (2,6-difluorobenzoyl) urea; Uniroyal Chemical Co. , Inc.) .
• Amidinohydrazone ⁇ are inhibitors of mitochondrial respiration by inhibiting electron transport at Complex II.
• amidinohydrazone is hydramethylnon (tetrahydro-5,5- dimethyl-2 (1H) -pyrimidinone [3, - [4- (trifluoromethyl)phenyl] -1- [2- [4-
• the insecticidal composition comprises an insecticidal chemical (or biological insecticide as described below) and a genetically modified insect virus.
• 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 AalT from the scorpion Androctonus australis (7) , a toxin from the straw itch mite species Pyemotes tri tici (8), Bacillus thuringiensis toxins (9,10), and a toxin isolated from spider venom (11) .
• neuropeptides or hormones examples include eclosion hormone (12) , prothoracicotropic hormone (PTTH) , adipokinetic hormone, diuretic hormone and proctolin (13) .
• 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 AalT.
• 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 AalT, 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 AalT does not bind to mammalian sodium channels, AalT 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 AalT gene includes a signal sequence which directs the secretion of AalT 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 AalT.
• heterologous signal sequences are useful in the expression and secretion of insect toxins, ⁇ uch as AalT (15) .
• 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.
• a codon optimized DNA sequence encoding the cuticle signal sequence and AalT is used.
• the degeneracy of the genetic code permits variations 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.
• codon use tables for Drosophila melanogaster are utilized to generate a codon optimized DNA sequence encoding the cuticle signal sequence and AalT.
• AalT expression is improved by the use of the AcMNPV DA26 "early" promoter. This promoter is inserted upstream of the codon optimized DNA encoding the cuticle signal sequence and AalT.
• Samples of a genetically modified AcMNPV E2 strain containing the DA26 promoter and the codon optimized DNA encoding the cuticle signal sequence and AalT are constructed in accordance with the procedures set forth in co-pending, commonly-assigned United
• EGT inactivates insect molting hormones (ecdysone) , which prevents the insect larva from molting or pupating.
• insect molting hormones ecdysone
• molting and pupation of the larva infected with the insect virus can proceed.
• 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 EGT ' insect virus fails to block larval molts and pupation, along with the cessation of feeding in preparation for these molting events.
• EGT * wild- type infected insects
• infecting insects with EGT ' strains is more effective than infecting insects with wild-type virus in terms of LT 50 values (the time it takes one-half of a group of insects to die after being infected with a virus) .
• the egt gene is inactivated by substituting in its place or inserting within it another gene such as the nonviral marker gene for /S-galactosidase.
• Any DNA sequence can be used to disrupt the egt gene as long as it disrupts expression of the egt coding sequence.
• all or part of the egt gene can be removed from the genome by deleting or mutating an appropriate coding segment.
• the regulatory part of the genome that controls egt gene expression can be altered or removed. The result of these modifications is underexpression of the egt gene.
• Deletions inactivating the egt gene can also be produced by serial virus passage in insects or insect 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.
• V8 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 characterized. Samples of this V8 strain have been deposited with the American Type Culture
• compositions of this invention are in the form of wettable powders, granules, suspensions, emulsions, solutions, solutions for aerosols, baits and other conventional insecticide preparations.
• 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.
• an inactive carrier 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 ⁇ -2.4X10" 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.
• concentrations of each of the active components of the compositions needed to produce optimum insecticidally effective compositions for 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.
• biological control agents are combined with insect viruses.
• Biological control agents include bacteria such as Bacillus thuringiensis, available, for example from Abbott Laboratories as XENTARITM and DIPELTM 2X.
• Other biological control agents include protozoans such as Nosema polyvora , M. grandis and Bracon melli tor (5) .
• Still other biological control agents include entomopathogenic fungi (5) and nematodes. Nematodes are administered in a liquid formulation or dispersed in a gel where they are in dormant stage until ready for use.
• 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 instar larvae (H. 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.
• C-D International, Inc. Clear vented adhesive tops
• 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* 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.
• viral solutions the dilutions range from 1X10* 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.
• H. virescens are fed for 8 days; H. zea for 12 days.
• Bioassay trays are kept at 28°C in 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- 0 and LC 50 values (concentration at which 20% or 50% mortality is observed) are calculated, based on 3-4 replicates. Statistics are computed using the SAS log/PROBITTM program, mortality versus dose, at 8 or 10 days post- treatment.
• LC 30 is the dose which is predicted to cause 20% mortality of the larvae by application of the product
• LC 50 is the dose which is predicted to cause 50% 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 "E” means exponent.
• the term “DAT” in the Tables stands for day(s) after treatment.
• AcMNPV "AalT inserted" is the genetically modified E2 strain containing the DA26 promoter with the codon optimized DNA encoding the cuticle signal sequence and AalT.
• 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 6/03048 PC17US95/09525
• 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.
• the diacylhydrazine also significantly hastens the speed-of-kill properties of AcMNPV "EGT deleted" against H. zea larvae [i.e., based on data collected at 3 DAT] .
• 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.
• 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 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.
• 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 "AalT inserted" strain of AcMNPV.
• the combination utilizes a dosage equivalent to the predicted LC 20 of each component used alone.
• 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 50 of each component used alone.
• Table 13 depicts the combination of cypermethrin with the V8 EGT " strain of AcMNPV.
• the combination utilizes a dosage equivalent to the predicted LC-- for cypermethrin and the predicted LC 50 for the AcMNPV V8 EGT' strain.
• Table 14 depicts the combination of cypermethrin with the E2 "Aa T inserted" strain of AcMNPV.
• the combination utilizes a dosage equivalent to the predicted LC 50 of each component used alone.
• Table 15 depicts the combination of the diacylhydrazine with the wild-type LI strain of AcMNPV. TABLE 15
• Table 16 depict ⁇ the combination of the diacylhydrazine with the genetically modified EGT " (LI strain) of AcMNPV.
• 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 AalT inserted E2 strain of AcMNPV.
• 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.
• the combination of the arylpyrrole 4-bromo-2- (p-chlorophenyl) -1- (ethoxymethyl) -5- (trifluoromethyl) -pyrrole-3- carbonitrile with either the virus genetically modified to contain AalT or be EGT " is superior to the combination of the arylpyrrole and the wild-type virus.

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