Insecticidal mixture for the control of insect populations and the method of regulation of insect populations

 

(57) Abstract:

The invention relates to the field of biotechnology. Insecticidal compositions include mixtures of genetically modified insect viruses with chemical and biological insecticides. Genetically modified virus contains insertional gene, which expresses an insect, or modifying substance, such as a toxin, a neuropeptide hormone or enzyme. Genetically modified virus has a deletion in the gene. Insecticidal mixture has a high biological activity. 2 S. and 3 C.p. f-crystals, 19 tab., 2 Il.

The technical field

The invention relates to mixtures of insecticides intended for the regulation of insect populations and including mixtures of genetically modified insect viruses with chemical and biological insecticides, to ensure more effective regulation of insect populations.

Art

Currently, there are various ways of dealing with insect pests and several approaches to control insect pests, affecting important for the national economy of culture. Widely used chemical insecticides, but note comah pests chemical insecticides can destroy beneficial insects. Gradually, the insects become resistant to these drugs that requires the development of new categories of insecticides. These drugs may not break down in the environment for a long period of time from the moment of their application, which leads to pollution.

With the aim of reducing the use of chemical insecticides to control insect populations in the larval stages using species-specific viruses. Species-specific viruses include both DNA and RNA viruses. DNA viruses include entomopoxvirus (Entomopoxvirus) and baculoviruses (Baculoviridae), including the nuclear polyhedrosis viruses (NPV), granules (VG) and baculovirus (Bacuiovirinae), the subgroup of baculoviruses, not forming inclusions (NEW). RNA viruses include togavirus, flavivirus, picornaviruses, viruses cytoplasmic polidroso (TDC), etc. Subfamily of viruses with double-stranded DNA Eubaculovirinae includes two kinds. viruses: nuclear polyhedrosis (NPV) and granulosa (VG), the most frequently used in the biological control of insect pests because they are in their life cycle form in the tissues of insects characteristic of Taurus-enable (TV).

Nuclear polyhedrosis virus amaze: Lymantria dispar (near the fera litturalis of, Spodoptera frugiperda NPV, Spodoptera exigua NPV, Heliothis armigera NPV, Mamestra brassicae (cabbage scoop) of, Choristoneura fumiferana (plum moth) NPV, Trichoplusia ni (scoop) NPV, Helicoverpa zea NPV, etc.

Examples VG can serve Cydia pomonella VG (Codling moth VG), Pieris brassicae VG, Trichoplusia ni VG etc. NEW Examples can serve Orcytes rhinoceros NEW and Heliothis zea NEW. Examples of viruses of entomopox (VEP) can serve Melolontha melonotha VEP, Amsacta moorei VEP, Locust migratoria (locust) VEP, Melanoplus sanguinipes VEP, Schistocerca gregaria VEP, Aedes aegypti VEP, Chironomus luridus VEP, etc.

More than 400 isolates of baculovirus has been described as present in invertebrates. The virus multiple nuclear polyhedrosis Autographa californica (Asmmap) is the prototype for the entire virus family Baculoviridae and has a wide range of hosts. Asmmap was originally isolated from Autographa californica (A cal.), the adult stage is night butterfly)), known as the alfalfa looper. This virus infects insects to 12 families and more than 30 species of Lepidoptera. There is no evidence that it can effectively engage any species not belonging to this group.

The life cycle of baculoviruses, for example Asmmap includes two stages. Each stage presents a specific form of the virus: vnekletocna cnie and enclosed in a shell of viral particles have the same genome, but different biological properties. The reproduction of each of the two forms of the virus is governed by a separate set of viral genes, some of which are unique to each form.

In naturally occurring affecting insect form of the virus numerous viral particles embedded in a matrix formed by the protein paracrystalline, and form the so-called bull-enable (TV), which also are called polyhedral cells-inclusions (PTV). Protein poliakin having a molecular weight of 29 KD, is the main structural protein of the viral membranes encoded by a viral genome. (Likewise viruses granulate form inclusions consisting mainly of granulin, not polyhedrin). Enclosed in a shell of a virus particle are an important part of the natural life cycle of baculoviruses, providing the possibility of horizontal (from one insect to another) transmission between susceptible insect species. In the environment affected insects (usually in the larval stage) absorb enclosed in a shell of the virus from infected food source, such as plants. Crystal shells are dissolved in the gut of susceptible insects with the formation of infective Viru is that viral particles penetrate into the cell by endocytosis or by diffusion, and that virus DNA is released from the membrane with nuclear pores or in the nucleus. Replication of viral DNA is found six hours later. After 10-12 hours after infection ("., and".) secondary infection spreads to other tissues of the insect by otokoyaku extracellular viruses (UTC) with the cell surface. UTC form of the virus is responsible for the transmission of the virus from cell to cell within a single infected insect, or for the transmission of infection in cell culture.

Later in the cycle of infection in 12 hours p. I. in infected cells can be detected protein poliakin. Not earlier than after 18-24 hours p. I., the build polyhedrin protein and viral particles are enclosed in a protein shell. Enclosed in a shell viruses collected in large quantities, and after 4-5 days is the lysis of the cells. These are enclosed in the shell of the polyhedrin viruses do not play an active role in the spread of infection in the larvae. UTC spread in infected larvae, which leads to her death. After the death of the infected larvae millions of prisoners in the membrane of the virus remain in the decaying tissue, while UTC destroyed. When the other cylinder absorbs enclosed in the shell of the polyhedrin the

So, encased in a shell form of the virus is responsible for the initial defeat of the insect through the intestines, as well as for the stability of the virus in the environment. PTV poorly infect cells when introduced by injection, but very active in oral introduction. Not enclosed in the shell form of the virus (i.e., UTC) is responsible for the spread of the virus inside the body and the transmission of the infection from cell to cell in culture. UTC efficiently infect cells in culture or within the insect when injected, but ineffective in oral introduction.

These insect viruses are not pathogenic for vertebrates and plants. In addition, the baculoviruses, as a rule, have a narrow host range. Many strains infect only one or a few species.

The use of baculoviruses as bioinsecticides promising. One of the obstacles to their widespread use in agriculture is the period of time between initial infection of the insect and his death. This period can vary from several days to several weeks. During this period the larva continues to feed harming the plant. Several researchers tried to overcome this drawback by introducing heterological toxin, neuropeptide hormone or an enzyme.

However, to date such genetically modified viruses are not used in combination with chemical insecticides as part of an integrated control mechanism of the pests. Described the use of a combination of wild-type viruses insects and chemical insecticides, but their results were not optimal because of the limitations of the wild-type viruses (bibliography, paragraphs 1-5). The researchers also tried to regulate the number of insects with other biological agents such as bacteria (e.g., Bacillus thuringiensis), fungi, protozoa and nematodes, taken separately or in combination with insect viruses or chemical insecticides, but they also have not achieved optimal results(2, 3, 5, 6). There is therefore a need to develop combinations of chemical insecticides and genetically modified insect viruses that will provide the benefits of both components and reduce the number of toxic chemicals and the time of destruction, than observed for wild-type viruses by using virus obtained by the methods of genetic engineering.

The invention

This invention is an insecticidal mixture, including:

(a) an effective amount of a chemical insecticide selected from a class of substances consisting of pyrethroids, arilpirolul, diacylhydrazines have and formamidines; and

(b) an effective amount of genetically modified virus multiple nuclear polyhedrosis Autographa californica ("ASMAP"), which contains: (i) introduced a gene expressing internal toxin (AaTH) Autographa californica, or (ii) a deletion in the gene encoding ecdysteroid UDP glucosyl a transferase (AGT") ASMAP,

these mixtures are used to control insect Lepidoptera when used for regulating the numbers of moths Heliothis zea, and chemical insecticide is formamidines,Oseni this invention is an insecticidal mixture for use against Heliothis virescens, including:

(a) an effective amount of a chemical insecticide selected from a class of substances consisting of pyrethroids and arilpirolul; and

(b) an effective amount of genetically modified Asmmap that contains: (i) introduced a gene expressing AaTH, or (ii) a deletion in the gene encoding AGT ASMAP.

In another embodiment, this invention is an insecticidal mixture for use against Heliothis zea, including:

(a) an effective amount of a chemical insecticide selected from a class of substances consisting of arilpirolul and diacylhydrazines have; and

(b) an effective amount of genetically modified Asmmap that contains: (i) introduced a gene expressing AAT, or (ii) a deletion in the gene encoding AGT ASMAP.

In another embodiment, this invention is an insecticidal mixture for use against Heliothis zea, including:

(a) an effective amount of a chemical insecticide selected from a class of substances composed of formamidines;

(b) an effective amount of genetically modified Asmmap that contains embedded gene expressing AAT.

This invention also provides a method kulturnye plants, which these insects feed described above insecticidal mixtures.

Brief description of Fig.1 and 2

Fig. 1 is a graphical depiction of the data presented in table 13, i.e., the percent mortality of the pest on 1, 4 and 10 days for the first three treatments are shown in table 13, except for the "Untreated control" from table 13 that are not reflected in Fig.1.

Fig. 2 is a graphical depiction of the data presented in table 14, i.e., the percent mortality of the pest on 1, 4 and day 10 for the first three treatments are shown in table 14, except for the "Untreated control" from table 14 that are not reflected in Fig. 2. "AsMAP with insertional AAT in table 14 is the same as "rNPV" in Fig. 2.

Detailed description of the invention

Insects in the process of development from egg to adult (imago) are several well-studied stages. After hatching from the egg, the larva of an insect, which in Lepidoptera referred to as a caterpillar entering a period of increased power. During this time, she repeatedly shedding, to ensure continuous growth. The intervals between successive linkami who called the P> The objective of the invention is improving the efficiency of insect pests during larval development. Order Lepidoptera, which includes the well-known pests of agricultural crops, includes family: Noctuidae, Notodontidae, Arctiidae, Pyralidae, technical Bulletin), Pieridae and Geometridae.

To determine whether insecticide mixture of effective control for pests, there are two criteria.

The first is the number of destroyed within a certain period of time caterpillars (larvae). It's called "% mortality".

The other is the rate of destruction. Even if the % mortality during the entire period of larval feeding is not increased, preference is given to the destruction of a large number of caterpillars in the early stages, because the time during which the larvae eat less, and therefore less harm the crop.

Thus, the test mixture has advantages over existing mixes, if its use increases mortality and the rate of destruction of insects.

A mixture of genetically modified insect viruses with chemical or biological insecticides called the accused separately; "additive" if the mortality rate for the mixture equal to the sum of deaths for components used separately; "subadditive" if the mortality rate for the mixture exceeds mortality for any of the components used separately, but less than the amount of deaths for individual components; "antagonistic", if the mortality rate for the mixture is less than the mortality for any of the components used separately.

Winnings can be obtained if the mixture is synergistic or additive. Even if the mixture is additive, by reducing the dose of one or both components compared with the dose for independent use, costs are reduced, and it turns out environmental gains, such as reduced chemical insecticide, which leads to lower persistence and sustainability education.

The use of insecticide mixtures has advantages if it provides improved control over one or both permissive and pauperising insects. Permissive insect, typically 100-1000 times more sensitive to the action of a virus or chemical insecticide than pauperising insect. For example, the tobacco budworm (H. virescens), permissive as far as the PTO of the present invention, what targets for mixtures chemical insecticide and virus insect may be a greater number of insects than in the case of independent use of each individual component. And chemical insecticide, and the virus insect have a limited host range. The mixture due to the presence of both components may have broader host range. At the same time, this effect is not due to interaction between the components of the insecticide mixture.

To control insect pests is a wide range of classes of substances. Below will be presented with a listing of these classes and the description of their mechanism of action.

Pyrethroids are compounds that bind to the protein of the sodium channel, resulting in a change of action potential at the axon membrane. This, in turn, disrupts the normal functioning of the nervous system of the insect. From the use of pyrethroids cypermethrin (-cyano-3-phenoxybenzyl-CIS/TRANS-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate; FMC, Inc.). PERMETHRINTM(3-phenoxybenzyl-CIS/TRANS-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate; Corporation Coulston International), fenvalerate (-cyano-3-phenoxybenzyl-2-(4-chlorophenyl)-3-methyl who CLASS="ptx2">

Formamidine are compounds with multiple mechanisms of action, including binding with the receptor of octopamine (neurohormone/neurotransmitter) and effect as those of his antagonist, increased education sump and induction of changes in behavior or inhibition of mixed function monoamin oxidase. Of formamidine use Amitraz (N'-(2,4-dimetilfenil)-N-[[(2,4-dimetilfenil)imino] methyl] -N-methylmethanamine; NOR-AM, Sobering AG) and Chlordimeform (N'-(4-chloro-O-tolyl)-N,N-dimethylformamide).

Arylpyrazole are mitochondrial toxins, lethal effect of which is to disrupt the process of oxidative phosphorylation. From arilpirolul using 4-bromo-2-(p-chlorophenyl)-1-(ethoxymethyl)-5-(trifluoromethyl)-pyrrole-3-carbonitrile (U.S. patent 5310938) and compounds described in U.S. patent 5010098.

Diacylhydrazine is a non-steroidal insect growth regulators, the main mechanism of action is that they are antagonists ecdysone. From diacylhydrazines have used Dibenzoyl-t-butylhydrazine (the production of which is described in U.S. patent 5300688) and MIMICTM(3,5-dimethylbenzoic acid 1-(1,1-dimethylethyl)-2-(4-ethylbenzoyl) hydrazide; Bldg. Rohm & Haas).

Cyclodiene contact with the hydro-6,9-methane-2,4,3-benzodioxathiepin 3-oxide; Hoechst).

Carbamates act as cholinesterase inhibitors. From carbamates use thiodicarb (dimethyl-N,N-THIOBIS methylimino)carbonyloxy)-bis(ethanimidothioic); Rhone-Poulenc) and methomyl (S-methyl N-[(methyl-carbarnoyl)oxy] thioacetimidate).

Organophosphates act as cholinesterase inhibitors. From organophosphates use profenofos(O-4-bromo-2-chlorophenyl O-ethyl S-propyl phosphorothioate; Ciba-Geigy), Malathion (O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinic), sulprofos (O-ethyl O-[4-(methylthio) phenyl] S-propyl of phosphorodithioate) and dimethoate (O,O-dimethyl S-methyl-carbamate)-phosphorodithioate).

Pyrazoles are inhibitors of mitochondrial respiration by specific actions on Complex I of the electron transport system. Of pyrazoles using tebufenpyrad (N-(4-t-butylbenzyl)-4-chloro-3-ethyl-1-metalpar-evil-5-carboxamide; Mitsubishi Kasei, American Cyanamid Company), and compounds described in published European patent 289879.

Nitroguanidine prevent the binding of acetylcholine to specific acetylcholine receptors on the postsynaptic membrane; by linking themselves receptors, these compounds disrupt the transmission of nerve impulses. From nitroguanidine use iminal the first contact with customers on complex GABA receptor/chloride ion channel, and then cause paralysis and death of the insect by inhibition of signal transmission in the neuromuscular junction. From milbemycin use abamectin (mixture of avermectins containing >80% embryo death WA and <20% of embryo death Blb; Merck, Sharp & Dohme).

Benzoyleneurea are insect growth regulators that violates the synthesis of chitin, thus disrupting the formation of the cuticle during molting insect. Of benzoyleneurea use diflorasone (1-(4-course)-3-(2,6-differentail) urea; Corporation Uniroyal Chemical Co.).

Amidinopropane are inhibitors of mitochondrial respiration by inhibition of electron transport at complex II. From amidinopropane use hydramethylnon (tetrahydro-5,5-dimethyl-2(1H)-pyrimidinone [3-[4-(trifluoromethyl)phenyl] -1-[2-[4-(trifluoromethyl) phenyl] ethynyl]-2-properties; American Cyanamid Company).

For the person skilled in the art will understand that other known examples of the above classes of chemicals, information about which can be obtained from commercial suppliers or from the patent or scientific literature.

In accordance with the present invention insecticidal composition includes a chemical insecti the>

In one embodiment of this invention, the genetic modification of the virus insect includes interturbine in any suitable location in the genome of the virus gene expressing controlling or modifying the substance of the insect. This substance may be, for example, a toxin, a neuropeptide, a hormone or an enzyme. Expressed this way the substance increases bioinsecticide action of the virus.

Such toxins include species-specific toxin ATN Scorpion Androctonus auatralls (7), the toxin scabby mite species of Pyemotes tritici (8), the toxin Bacillus thuringiensis (9, 10) and toxin isolated from spider venom (11). Examples of such neuropeptides or hormones can serve as hormone eclosion (12), prothoracicotropic hormone (ptth), adipokinetic hormone diuretic hormone and prochain (13). Examples of such enzymes may serve as the juvenile hormone esterase (AWG) (14).

For a description of the present invention is an example of using genetically modified Asmmap containing insertional gene expressing AAT. Source for genetic modification is the wild-type strain, denoted by E2 (ATSS VR-1344). Introducing the toxin is AAT derived from the venom of the North African the insects, causing paralysis in contact with the caterpillar from nanogramme to micrograms. As AAT is not associated with sodium channels in mammals, it can be used as bioinsecticide, without bringing harm to human health.

The region before the coding region of the gene AAT contains a signal sequence that provides secretion of AAT outside the cells. The signal sequence directs the toxin on secretory pathways on the cell surface, where the toxin is secreted outside the cells. During transport the enzymes remove the signal sequence, leaving active AAT.

It was found that heterologous signal sequences useful for expression and secretion of toxins insects, for example ATN (15). Preferred heterologous signal sequence is cuticular signal sequence of Drosophila melanogaster (for protein exoskeleton), which secretes a large number of related Mature proteins. In turn, uses a DNA sequence encoding a cuticle signal sequence with optimized codons. The degeneracy of the genetic code allows me the AK and the polypeptide, encoded by the native DNA sequence. The procedure, known as optimization of codons, ensures the availability of funds for the establishment of such a modified DNA sequence which reflects the frequency of codons, the body of the insect host. In this invention to generate DNA sequences with optimized codons encoding cuticular signal sequence and AutN used table of codons for Drosophila melanogaster.

Additional means of increasing the level of expression of AAT is the use of "early" promoter Asmmap DA26. This promoter insertyour before DNA sequence encoding a cuticle signal sequence and AutN, with optimized codons.

Samples of genetically modified Asmmap E2 strain containing the DA26 promoter and DNA-optimized codons encoding cuticular signal sequence and AutN, synthesized according to the procedure described in patent application U.S. 08/070164.

Samples obtained viral compounds indicated AC 1001, deposited in the American type culture Collection and is registered in the ATCC under the number VR-2404. Other compounds, idrugie promoters can be created by specialists in the field using conventional techniques.

Improving the efficiency of the virus insect by genetic modification of the virus may also consist in making deletions in the gene. An example is a deletion in the gene encoding ecdysteroid UDP glucosyl a transferase (AGT"). Miller and others have described the creation of such EGT-strains of viruses (16). In particular, Miller described the creation of ASMAP EGT-the strain.

The expression of the egt gene leads to the development of EGT. EGT inactivates the insect molting hormone (Edison), resulting in a caterpillar that does not shed and do not pupate. When egt gene inactivated, for example, by identifying EGT-strain, moulting and pupation of larvae infected may continue. This, in turn, the continued development of insect leads to such favorable for plant protection results as reducing power, slower growth and more rapid death. The reason for this is that EGT-the virus can't block molting and pupation of the insect, and with them, and stopping power. Therefore, insects, infected EGT-the virus is much more prone to earlier death in the transition to shedding in infected condition than insects, infected wild-type (ERG+m wild type in terms of value LT50(the time required for the deaths of half of the insects from the group after virus infection).

Gene egt inactivate by replacing or interturbine another gene, such as non-viral marker gene-galactosidase. For violations of the egt gene can be any DNA sequence that disrupts the expression of the coding sequence egt. On the other hand, all deletions or part of the sequence egt gene can be removed from the genome by deletion or introduction of mutations in certain regions of the coding sequence. In addition, you can edit or delete the regulatory portion of the genome that control the expression of the egt gene. The result of these changes is the lack of expression of the egt gene. Deletions inactivate egt gene may also be obtained by serial passages of the virus in insects or in the culture of insect cells. All these interturbine, deletions or mutations carried out using conventional means. Obtained by deletion viruses have the advantage that they do not contain foreign DNA and differ from wild-type viruses only because it does not have a functional egt gene.

Miller was cited as an example Asmmap EGT-virus recombinant, smidi pEGTDEL (which is the product of cleavage of the plasmid containing egt gene with EcoRI and XbaI, with deletion of the gene) and DNA from the virus vEGTZ (containing the lacZ gene, insertional without shifting the reading frame relative to the previous coding sequence egt). The result of homologous recombination is the replacement of concatenated genes egt-lacZ in vEGTZ on the egt gene from a remote site from pEGTDEL that gives the recombinant virus vEGTDEL, which is EGT-.

Miller used the strain Asmmap denoted by L1, which is a clonal isolate of the originally isolated strain wild type (ATSS VR-1345). Was later isolated and characterized strain Asmmap indicated V8. Samples of strain V8 were accepted for storage by the American type culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U. S. A., and were registered at ATCC under the number [VR 24651. Described by Miller's method of creation L1 EGT-apply to creating strain V8 EGT-.

To prepare the mixtures in accordance with this invention uses conventional technology development formulation, known to specialists in this field. The mixture is prepared in the form of wettable powders, granules, suspensions, emulsions, solutions, solutions, aerosols, baits and other common insecticidal p is p water, alcohol, hydrocarbons or other organic solvents or mineral, animal or vegetable oil or a powder such as talc, clay, silicate.

Insecticidal mixture, in accordance with this invention, are applied using conventional techniques known to experts in this field: the introduction of the virus into the body of the insect food by direct contact, inhalation (by spraying or dusting the plants that feed on insects).

Insecticidal mixtures used in several ways. The virus and chemical insecticide used simultaneously in a single dosage form or simultaneously in two dosage forms. If you use two dosage forms, they are packaged separately and then mixed, optionally in the presence of a solvent, to obtain the final mixture. Alternatively, virus or chemical insecticide may be applied first to suppress insect, and then applied the second component.

Insecticidal mixture, in accordance with this invention, used in a dose of from 2.4108-2.41012PTV/ha of genetically modified virus and 0.001-1.0 kg/ha of chemical insecticide. These standards represent the dose, the mouth of the ance with this invention, the cuts.

The concentration of each of the active components necessary to obtain optimally effective insecticidal mixtures of plant protection, depending on the type of organism, modifications of chemical insecticide and virus insects used in the formulation of the mixture. These concentrations are determined by the experts in this field.

As an alternative to chemical insecticides with insect viruses combine biological control agents. Biological control agents include bacteria, such as Bacillus thuringiensis, which can be obtained from Abbott Laboratories as XENTARITMand DIPELTM2X. Other biological control agents include protozoa, for example, Nosema polyvora, M. grandis and Bracon mellitor (5). Besides the biological control agents are entomopathogenic fungi (5) and nematodes. Nematodes used in liquid formulations or dispergirovannykh in the gel, where they are dormant until use.

For a deeper understanding of this invention, the following examples. (Examples are only illustrative and should not be construed as limiting the range of the invention.)

Examples

Example 1

Technique biologically is on the surface of the medium. Experiments are performed as follows. Are insects N. virescens (tobacco moth-Packed) and Heliothis. zea (boxed worm of cotton). Caterpillars are grown on a nutrient medium comprising soy seeds, wheat germ and agar (Wednesday Stoneville), obtained from the USDA Insectary Labs, Stoneville MS. Each batch of insects contained in the 28oC and under constant fluorescent lighting. All experiments are performed on the environment Stoneville, with caterpillars of the second age (four-day N. virescens and a three-N. zea].

Each cell of the experiments (Corporation C-D International, Pitman, New Jersey) contains 32 individual cells, with a size of 4 cm2containing 5 ml of medium Stoneville. After processing environment and infect insect cell served transparent lids, blurred at the edges with glue, with ventilation (Corporation C-D International). Clear lids allow you to easily make observations.

For the log-probit analysis (Corporation HRO Group) from the original Oceanology solutions virus preparing a dilution series: distil water twice. Breeding is carried out with step 11081101PTV/ml, depending on the studied species. If necessary, by centrifugation of the solutions of virus concentrate. Solutions chemical insecticides ready is rites.

On the surface of the artificial medium after it has cooled down, put a pipette 0.4 ml solution in acetone:water (60:40) one of the following components: solution a virus solution of a chemical insecticide, the solution of the virus plus the solution of chemical insecticide or untreated solution. For solutions of the virus dilution ranges from 11081101PTV/ml in 10-fold dilution, depending on the studied species of insect. The concentration used chemical insecticide varies from 1000 ppm to 0.1 ppm, depending on the type of chemical insecticide and species studied insect. Each of the studied solution check to 32 tracks 3-4-fold repetition. Apply the solution evenly distribute over the surface of the medium by rotation of the cell, the solution allowed to dry in a fume hood. After drying in each cell is placed a caterpillar and give her the opportunity to eat for 8-10 days. N. virescens eat for 8 days; H. zea 12 days. The cell for experiments contain at a temperature of 28oWith continuous fluorescent lighting during the entire period of study. Monitoring is performed once a day to see the start time of infection. In each observation the caterpillar is considered dead if it is not DWI is for virus and chemical substances (concentration, in which there is a 50% and 20% mortality) calculated on the basis of 3-4 replicates. Statistical results are analyzed using the SAS log-probit analysis, determining the value of the dose-mortality at 8 and 10 days after treatment. After determining probit values conduct experiments separately with chemical insecticides, using predefined dose LD50and LD20separately from viruses and LD20and LD50doses, and with all possible combinations of viruses with chemical insecticides, using the same method applied on the surface of the medium. "LD20"it's a dose, when there is a destruction of 20% of the caterpillars, "LC50"the dose, when there is a destruction of 50% of the caterpillars.

In the tables the following symbols and abbreviations. The concentration of PTV/ml is marked with the letter E, where "E" is the exponent. For example, A corresponds 5104. "DPO" corresponds to the day (days) after treatment. In these tables Asmmap "with insertional ATN" is a genetically modified strain E2 containing DA26 promoter and a DNA sequence with optimized codons encoding cuticular signal sequence and AutN.

About powervantage virus insects and chemical insecticide testified or an increase in mortality or the rate of destruction of insects, or both effects simultaneously. Examples 2-5 present the results of experiments with Helicoverpa zea; examples 6-8 present the results of experiments with Heliothis virescens.

Example 2

The combination of formamidine of amitraz genetically modified insect viruses

In the first experiment formamidine amitraz study in combination with the virus insect ASMAP, genetically modified and contains AAT or EGT-.

The results are presented in tables 1 and 2.

The results are shown in table 1 and 2 data indicate that amitraz at a concentration of 100 ppm synergize bioactivity Asmmap "with insertional AAT for larvae of H. zea. The synergy of the above virus to some extent depends on the dose, as the combination of this recombinant virus with nitraza at a concentration of 1000 ppm have additive rather than a synergistic effect on H. zea. On the other hand, amitraz no significant effect on the bioactivity Asmmap "with the remote AGT" caterpillars N. z. The death of caterpillars when using combination formamidine/virus with the remote AGT is slightly less than when skin is zirovanii viruses insects

In this experiment the arylpyrol 4-bromo-2-(p-chlorophenyl)-1-(ethoxymethyl)-5-(trifluoromethyl)-pyrrole-3-carbonitrile studied in combination with the virus insect ASMAP, which is genetically modified in such a manner that contains or AAT, or EGT-. The results are presented in tables 3 and 4.

The results are shown in tables 3 and 4 show that the arylpyrol 4-bromo-2-(p-chlorophenyl)-1-(ethoxymethyl)-5-(trifluoromethyl)-pyrrole-3-carbonitrile in combination with ASMAP "with insertional ATN" significantly increases the rate of destruction of caterpillars N. zea, which is evident from the analysis of data obtained on the third day after treatment. At the same time, on the fifth and eighth days after treatment, the mortality of the caterpillars when using a combination of the arylpyrol/recombinant virus additive (or slightly less than additive).

The arylpyrol no significant impact on mortality of larvae of H. zea second age when using it in combination with ASMAP-V8 "with deliciously EGT". However, the obtained results (mortality of larvae at 3 days after treatment) show that arylpyrol slightly increases the rate of destruction of caterpillars N. zea using virus with deliciously EGT".

Example 4

The combination of drain Dibenzoyl-t-butylhydrazine was studied in combination with the virus insect ASMAP, which is genetically modified in such a manner that contains or AAT, or EGT-. The results are presented in tables 5 and 6.

The results presented in tables 5 and 6 show that diacylhydrazine Dibenzoyl-t-butylhydrazine when using it in combination with ASMAP "with insertional ATN increases the rate of destruction of caterpillars N. zea, which is evident from the analysis of data obtained on day 3 after infection.

Diacylhydrazine also significantly increases the rate of destruction of caterpillars N. zea when used in a mixture with the virus Asmmap "with deliciously AGT" [based on the data obtained on the 3rd day after the treatment].

Example 5

The combination of benzoyleneurea genetically modified insect viruses.

In the following experiment benzoyleneurea diflorasone studied in combination with the virus insect ASMAP, which is genetically modified in such a manner that contains or AAT, or is EGT-. The results are presented in tables 7 and 8.

The results presented in tables 7 and 8 show that the addition of dimensionality of diflorasone not increase insecticidal action ASMAP-E2 "with the Insa the above additive.

Benzoperylene does not increase the effectiveness of ASMAP-E2 "deliciously AGT" caterpillars N. zea, moreover, the mortality of caterpillars N. zea when applying this combination is less than additive.

Example 6

The combination of PYRETHROID with wild type or genetically modified virus insects.

In the following experiment (using larvae of N. zea second age) PYRETHROID cypermethrin study in combination with the virus insect Asmmap that belongs to the wild type or genetically modified so that it contains or AAT, or is EGT-. The results are presented in tables 9-14.

Table 9 shows the data obtained when using the combination of cypermethrin with strain ASMAP-E2 wild-type. In this combination are used doses that cause when applying each component separately mortality 20% of caterpillars (LD20).

The results presented in table 9 show that when using a combination of cypermethrin with the virus Asmmap "wild-type" synergism was not observed (mortality of caterpillars when using the combination and the individual components were not significantly different). Similar results Inacio cypermethrin and strain Asmmap V8 EGT-. In this combination are used doses that cause when applying each component separately mortality 20% of caterpillars (LD20).

The results presented in table 10 show that when using a combination of cypermethrin with ASMAP "with deliciously AGT" the phenomenon of synergism unlike combination with cypermethrin Asmmap "wild-type".

Table 11 displays the data for the combination of cypermethrin and strain Asmmap E2 "insertional ATN". In this combination are used doses that cause when applying each component mortality 20% of caterpillars (LD20).

The results presented in table 11 show that when using a combination of cypermethrin with ASMAP "with insertional ATN" is observed, compared with the combination with cypermethrin Asmmap "wild type", the increase in the rate of destruction of caterpillars. Thus, mortality of larvae at 1 and 4 days after treatment when using cypermethrin with ASMAP "wild-type" was, respectively, 5 and 31%, and the use of cypermethrin with ASMAP "with insertional AAT respectively 22% and 38%.

Table 12 displays the data for the combination of cypermethrin and strain Asmmap E2 "wild type". In EUNIC (LD50).

The results presented in table 12, show that with the use of cypermethrin with ASMAP "wild-type" synergism is observed only on day 10 after treatment caterpillars.

Table 13 shows the data on mortality of caterpillars by using a combination of cypermethrin and strain Asmmap V8 EGT-. In this combination of the applied dose, cause when used separately cypermethrin mortality 20% of caterpillars (LD20), and for strain Asmmap V8 EGT-- mortality 50% of larvae (L50).

The results presented in table 13 show that when using a combination of cypermethrin with ASMAP "with deliciously AGT" mortality of larvae greater than when using each component of this combination separately, indicating synergism, in contrast to the observations when using a combination of cypermethrin with the virus "wild type". When the application of cypermethrin with a genetically modified virus used low dose concentrations of cypermethrin.

Table 14 shows the data on mortality of caterpillars by using a combination of cypermethrin and strain Asmmap E2 "insertional ATN". In this combination are used to the s, presented in table 14 show that when using a combination of cypermethrin with ASMAP "with insertional ATN" mortality of larvae at 4 and 10 days after treatment was higher than when using separately each component, indicating a synergism between the components applied, in contrast to the lack of synergy when using a combination of cypermethrin with ASMAP "wild-type".

So, the combination of cypermethrin with a virus that is genetically modified so that it contains or AAT, or is EGT-more effective against caterpillars N. zea than the combination of cypermethrin and virus "wild type". These results cannot be predicted on the basis of previously obtained data for combinations of viruses "wild-type" with pyrethroids (1).

Example 7

The combination of diacylhydrazine virus insects "wild-type or genetically modified

In the following experiment (with N. virescens third age) diacylhydrazine Dibenzoyl-t-butylhydrazine studied in combination with the virus insect ASMAP, which belongs to the "wild type or genetically modified in such a way that is EGT-(strain L1). The results are presented in TableTennis mortality of caterpillars by using a combination of diacylhydrazine and strain Asmmap L1 "wild-type".

The synergy of this combination is observed at 4 and 10 days after treatment.

Table 16 shows the data on mortality of caterpillars by using a combination of diacylhydrazine and genetically modified Asmmap EGT-(strain L1).

The results show that the use of this combination has led, in comparison with the use of the individual components, to a slight increase in mortality at 4 days after treatment.

Example 8

The combination arylpyrol virus insects wild-type or genetically modified

In the following experiment (with caterpillars N. virescens second age) arylpyrol 4-bromo-2-(p-chlorophenyl)-1-(ethoxymethyl)-5-trifluoromethyl)-pyrrole-3-carbonitrile studied in combination with the virus insect ASMAP, who belongs to the "wild type or genetically modified so that it contains AAT or V8 is EGT-. The results are presented in tables 17-19. Table 17 shows the data for the combination of diacylhydrazine and strain Asmmap E2 "wild type". In this combination are used doses that cause in the application of each agent separately death of 20% of the insects (LD20).

Analysis of data on mortality of caterpillars in table observed at 4 and 10 days after treatment.

Table 18 shows the data on mortality of caterpillars when using combination arylpyrol with genetically modified Asmmap EGT-(strain V8).

The analysis is shown in table 18 data shows that increasing the speed of the caterpillars when using arylpyrol with ASMAP "with deliciously AGT" compared with the combination arylpyrol with ASMAP "wild-type" (table 17), as evidenced by the mortality of larvae on day 1 after treatment, amounting to, respectively, 33% and 2%.

Table 19 shows the data on mortality of caterpillars when using combination arylpyrol with genetically modified strain Asmmap E2 with insertional ATN.

The analysis shown in tables 19 and 17 data shows that the use of combination arylpyrol with ASMAP "with insertional AAT was increased, compared with the combination arylpyrol with ASMAP "wild type", the rate of destruction of caterpillars, which was on the 1st and 4th days after treatment 39 and 69% (table 19) and (2) and 19% (table 17).

So, based on all the experiences we can say that the combination of arylpyrol 4-bromo-2-(p-chlorophenyl)-1-(ethoxymethyl)-5-(trifluoromethyl)-pyrrole-3-carbonitrile with the virus of modificirowan the arylpyrol virus "wild-type".

Sources of information

1. Aspirat J. and others, U.S. patent 4668511.

2. Mohamed A. I. and others, Environ. Entomology, 12, 478-481 (1983).

3. Mohamed A. I. and others, Environ. Entomology, 12, 1403-1405 (1983).

4. Velichkova-kojouharova M. and others, Rasteniev'dni Nauki, 25, 80-86 (1988).

5. JAX R. P. and others "Compatibility of pathogens with other methods of pest management and with different cultures, Chapter 38, pages 695-715.

6. Girlit J. B. F., and others, Med. Fac. Landbouww. Rijksuniv. Gent. 56, 305-311 (1991).

7. Lotkin E. and others, Toxicon, 9, 1-8 (1971).

8. Tomalski M. D. and others, U.S. patent 5266317.

9. Martens, J. U. M. and others, App.& Envir. Microbiology, 56, 2764-2770 (1990).

10. Federici B. A., In Vitro, 28, 50A (1992).

11. Jackson J. R. X. and others, U.S. patent 4925664.

12. Eldridge R. and others, Insect Biochem.. 21, 341-351 (1992).

13. Mann J. J. and other J. Agric. Food Chem., 37, 271-278 (1989).

14. Hammock B. D. and others, Nature, 344, 458-461 (1990).

15. The patent application U.S. serial number 08/009, 265, registered on January 25, 1993.

16. Miller, L. K. and other international patent application WO 91/00014.

1. Insecticidal mixture for the control of insect populations from a squadron of butterflies (LEPIDOPTERA) scoop (Noctuidae) of the genus Heliothis, including biological and chemical insecticides, different is she from genetically modified nuclear polyhedrosis virus of the geometrid moths of California alfalfa Autographa californica ("ASMAP"), containing insertional gene expressing the toxin ("Aetn") of the Australian Scorpion Androctonus australis or a deletion in the gene encoding ecdysteroid KDR-glycoside transferase (AGT") ASMAP, and as a chemical insecticide is an insecticide selected from the group consisting of arylpyrol, diacylhydrazine, PYRETHROID.

2. Insecticidal mixture under item 1, characterized in that as arylpyrol selected 4-bromo-2-(p-chlorophenyl)-1-(ethoxymethyl)-5-(trifluoromethyl)pyrrole-3-carbonitrile.

3. Insecticidal mixture under item 1, characterized in that as diacylhydrazine selected Dibenzoyl-t-butylhydrazine.

4. Insecticidal mixture under item 1, characterized in that as a PYRETHROID selected-cyano-3-phenoxybenzyl-CIS/TRANS-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate.

5. The method of regulation of insect populations from a squadron of butterflies (LEPIDOPTERA) scoop (Noctuidae) of the genus Heliothis, including the application of insecticidal mixture of biological and chemical insecticides on insects or culture that they eat, characterized in that as an insecticidal mixture use insecticidal mixture according to any one of paragraphs. 1-5.

 

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