JP2000511543A - Pest control - Google Patents

Abstract

  (57) [Summary] The present invention provides a method of controlling Ostrina flunacharis (Asia corn borer) species in cereals, but especially corn species, and other cereals, including but not limited to: corn, wheat , Barley, rye, oats, rice, sorghum, corn and related cereals, fodder, bamboo and sugarcane.

Description

DETAILED DESCRIPTION OF THE INVENTION                               Pest control   The present invention relates to Bacillus thuringiensis and / or other Bacillus spp. By using the obtained toxin protein, Lepidoptera mephida in cereals, Preferably for the control of Ostriana flunakaris (Asia corn borer) Related.   Bacillus thuringiensis is a large gram-positive, aerobic, endospore-forming bacterium. Belongs to the group. Bacillus cereus or Bacillus anthracis Unlike other Bacillus species that are very similar to Bacillus thuringiensis The majority, known as spores, are called crystals during their sporulation due to their crystalline structure. Forming parasporal inclusions This crystal is a pesticidally active crystal It consists of a protoxin protein, the so-called δ-endotoxin.   Protein crystals are responsible for Bacillus thuringiensis insect toxicity. δ- Endotoxin is taken orally in crystalline form, the latter being dissolved in the intestinal fluid of the target insect Until time, it does not show its insecticidal activity. In most cases, the actual toxic components are Release from protoxin as a result of proteolysis by the action of Will be issued.   Δ-Endotoxin from various Bacillus thuringiensis strains is High specificity for insects, especially various Lepidoptera, Coleoptera and Diptera larvae, and Larvae are characterized by a high activity against susceptible larvae.   Various insecticidal crystal proteins from Bacillus thuringiensis Microbiol. Rev. 53: 242-255 (1989), the classification reported at the time Crystal proteins were divided into four major classes. In general, the main class is its activity As defined by the spectrum, CryI protein is active in Lepidoptera and CryII protein Cry III protein against both Lepidoptera and Diptera, CryIII protein against Coleoptera Active, the CryIV protein is active against Diptera. Within each major class Where δ-endotoxins are classified according to sequence similarity.   CryI protein is typically produced as a 130-140 kDa protoxin protein. And it is proteolytically cleaved to produce insecticides of approximately 60-70 kDa in size. Produces sex toxin protein. The active part of δ-endotoxin is the full-length molecule N The proteins were classified into 6 groups, IA (a), IA (b), IA (c), IB, IC and ID. Was. Since then, it has been classified as CryIE, CryIF, CryIG, CryIH and CryIX The resulting protein has also been characterized.   Of the insecticidal activity of individual δ-endotoxins from Bacillus thuringiensis The spectra tend to be somewhat narrow, with some δ-endotoxins being present in some insects. Is only active. Specificity is the different steps involved in the production of active toxin proteins Is the result of the efficiency of and subsequently its ability to interact with insect gut epithelial cells .   Plants, preferably corn, wheat, rye, oats, rice, sorghum, Including but not limited to millet and related crops, fodder, bamboo and sugarcane For those who want to get rid of the cereal Ostriana flunacharis (Asia corn borer) It is one of the objects of the present invention to provide a method. The purpose of this is, surprisingly, Bacillus thuringiensis toxin protein such as CryI type toxin protein This is achieved within the scope of the present invention by administering the protein to the grain to be protected. In another embodiment of the present invention, a toxin protein obtained by vegetative culture of a Bacillus species Quality, ie VIP3 [EP-A-0690916; International Application No. EP / 03826, the Content is incorporated herein by reference in its entirety] ・ Insecticidal protein (VIP) is also available from Ostriana Flunakaris (A (Gia corn borer) Can be used for pest control.   The present invention therefore provides a method for protecting plants or plant seeds or plant growing areas with Chillus toxin protein, preferably the CryI or VIP type protein described above. Protein or pure or at least one such protein or toxin protein. A microorganism having at least one toxin gene to be administered, preferably a Bacillus -Insecticidal (including entomocid, including B. cereus strains) al) Ostriana flu, including administration directly or indirectly in the form of a composition Plants against damage caused by Nakaris (Asia corn borer) species (Including its descendants). The microbe used in the present method The product may be a naturally occurring strain or another containing a recombinant gene encoding a toxin. , A recombinant strain.   In a preferred embodiment, the transgenic plant is Plants to protect against damage caused by squirrel (Asia corn borer) species Used to administer toxins to objects. Such plants are Cry-type, preferably Bacillus such as CryI-type toxin protein or VIP-type protein Encoding a pesticidal toxin protein derived from Pseudomonas sp. When planted in an area where germination occurs, Ostriana flunakaris (Asia corn A toxin protein that expresses the toxin protein in an amount sufficient to provide Obtained from transformation with the gene.   Ostriana flunakaris (Asia corn borer) The insecticidal composition used in the method of the invention for protection may, for example, be combined with the active ingredient. And at least one Cry-type toxin protein, more preferably at least One CryI-type protein, even more preferably at least one CryI A-type toxin protein and most particularly preferably at least one SEQ ID NO: CryIA (b) type toxin protein according to 53 to 55, even more preferably Coding for Bacillus thuringiensis or at least one of said toxin proteins. A microorganism containing the gene to be loaded, preferably at least one Bacillus thuringiensis strain containing two genes, or a derivative or derivative thereof Of the carrier, diluent, surfactant or application-enhancing adjuvant Include with agricultural adjuvant. The active ingredient in the insecticidal active composition is EP-A VI-0609916 and VI as described in PCT International Application EP95 / 03826. P-type toxin protein or CryI-type and VIP-type protein It can be a combination. Preferred within the scope of protection is the VIP1A (a) protein Quality or VIP1-type protein such as VIP1 (b) protein, or VIP VIP2-type proteins such as 2 (a) protein or VIP2 (b) protein Or a combination of VIP1-type and VIP2-type proteins, Or VIP3-type such as VIP3 (a) protein or VIP3 (b) protein It is a protein. More preferred within the scope of protection are SEQ ID NOs: 1, 2, 4- 7, 17-24, 26-32, 35, 36, 39, 40, 42, 43, 45, 4 6, 49, 50, 51 or 52.   The composition may also include additional biologically active compounds. The compound may be a fertilizer or It can be a micronutrient donor or other formulation that affects plant growth. If desired Further agriculturally acceptable carriers and surfactants conventionally used in the field of formulation Herbicides, insecticides, fungicides, fungicides, nematicides, with application promoting adjuvants , A molluscicide or a combination of these preparations. Suitable carrier And adjuvants can be solid or liquid, and include materials commonly used in pharmaceutical processes. Corresponding, for example, natural or regenerated mineral substances, solvents, dispersants, wetting agents, stickiness Giving agents, binders or fertilizers.   The compositions may contain from 0.1 to 99% by weight of active ingredient, from 1 to 99.9% by weight of solids or May comprise a liquid adjuvant and 0 to 25% by weight of a surfactant component. Activation The composition comprising the active ingredient or the active ingredient can be used to protect the plant or crop to Agents or drugs (1993 Crop Proteciton Chemicals Reference, Chemical and Phar maceutical Press, Canada). Hoton Compatible with any other commonly used agricultural spray material, but with CryI-type poison If nitrogen is involved, it should not be used in very alkaline spray solutions. Powder , Suspensions, wettable powders or other substance forms suitable for agricultural applications.   Preferably, a Bacillus thuringiensis CryI-type toxin protein and And / or an active ingredient which is one of the above VIP-type proteins or the activity thereof (B) plants or parts of plants in an environment where pests can occur; The plant or part of the plant to protect it from damage caused by pests. Or (c) to protect plants that germinate from seeds from damage caused by pests To the seeds.   A preferred method of application in the area of plant protection is plant leaves (foliar surface treatment). And the rate of application depends on the planta being protected and the risk of infection with the pest.   The composition used in the method of the present invention may also be used for fruits, tubers or grains or cut plants. It is also suitable for protecting such plant propagation material, for example seeds, from pests. Breeding Treat with formulation before planting material: seeds can be soaked before sowing, for example . The active ingredient of the present invention may also be used to fill the grain (coating) with a liquid formulation, Or it can be applied by coating it with a solid formulation. The formulation breeds When planting the material, it can also be applied at the planting site, for example, on the ridges during sowing. Departure Ming also describes the method of treating plant propagation material and the plant propagation material thus treated. Also concerns.   Within the scope of the present invention, the composition is known for treating seed or soil with a bacterial strain. Can be applied. See, for example, U.S. Patent No. 4,863,866. Strains It is effective for ecological control even if the object is not alive. Preferably, but surviving Apply microorganisms.   Target grains protected within the scope of the present invention are Ostriana flunakaris (Asia (Corn-borer) is a host plant of corn, wheat, large Wheat, rye, oats, rice, sorghum, corn and related crops, fodder, bamboo and sato Including but not limited to cane.   The active ingredients of the present invention may be in unmodified form or with a suitable agriculturally acceptable carrier. Can be used. Such a carrier is an adjuvant commonly used in the field of agrochemical formulation. Bant and therefore a thick emulsion, coatable paste, directly sprayable or diluted Possible solutions, diluted emulsions, wettable powders, soluble powders, powders, granules and, for example, polymers It can be prepared in a known manner for encapsulation in a substance. Similar to the nature of the composition Spraying, spraying, dispersing, dispersing or injecting to the intended purpose and prevailing environment So choose. An advantageous rate of administration is in hectares ("ha", about 2.471 acres). -) Of about 50 g to about 5 kg of active ingredient (a.i.), preferably about 1 From 00 g to about 2 kg a.i./ha. The preferred rate of application is from 200 g to about 1 kga. i./ha or 200 g to 500 g a.i./ha. For seed dressing Advantageous application rates for the range from 0.5 g to 1000 g a.i./100 kg seed, Preferably, it is 3 g to 100 g ai / 100 kg seed. Most preferably, apply The proportion is between 10 g and 50 g a.i./100 kg seed.   Suitable carriers and adjuvants can be solid or liquid and are commonly used in pharmaceutical methods. Corresponding to the substance used, for example, natural or regenerated mineral substances, solvents, dispersants, Wetting agents, tackifiers, binders or fertilizers. A formulation, ie an insecticidal composition, Preparations or other active ingredients and, where appropriate, solid or liquid adjuvants The mixture is prepared in a known manner, for example, with an active ingredient and a bulking agent, such as a solvent, a solid carrier and In some cases, homogenous mixing and / or grinding of surfactant compounds (surfactants) Manufacturing.   Suitable solvents are: aromatic hydrocarbons, preferably fractions having 8 to 12 carbon atoms For example xylene mixtures or substituted naphthalenes, dibutyl phthalate or phthalates Phthalates such as dioctyl acid, fats such as cyclohexane or paraffin Aliphatic hydrocarbons, ethanol, ethylene glycol, monomethyl or monoethyl Alcohols and glycols such as ethers and their ethers and esthetics , Ketones such as cyclohexanone, N-methyl-2-pyrrolidone, dimethyl Strongly polar solvents such as sulfoxide or dimethylformamide and epoxy Vegetable oils such as sidified coconut oil or soybean oil or epoxidized vegetable oils; Or water.   For example, solid carriers used in powders and dispersible powders are typically calcite, talc , Natural mineral filling like kaolin, montmorillonite or attapulgite Agent. Highly dispersed silica or highly dispersed adsorptive polymer to improve physical properties Can also be added. Suitable granular adsorptive carriers are porous types, such as Is pumice, broken brick, sepiolite or bentonite; and the non-adsorbing carrier is It is a substance such as calcite or sand. In addition, many grains of inorganic or organic nature Conditioning substances, in particular dolomite or ground plant residues, can be used.   Depending on the nature of the active ingredient to be dispensed, suitable surfactant compounds will result in good emulsification, Nonionic, cationic and / or anionic surfactants with dispersing and wetting properties It is a sex component. The term “surfactant” is understood to include mixtures of surfactants. Understood. Suitable anionic surfactants include water-soluble soaps and water-soluble synthetic interfaces. It can be both active compounds. Suitable soaps are higher fatty acids (C10-C22 chains) Alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium Salts, such as the sodium or potassium salts of oleic or stearic acid, Is a natural fatty acid mixture obtainable, for example, from coconut oil or tallow . Further suitable surfactants are also fatty acid methyltaurine salts and modified or Non It is a modified phospholipid.   More often, however, so-called synthetic surfactants, especially fatty sulfo Acid esters, fatty sulfates, sulfonated benzimidazole derivatives or The use of rukariaryl sulfonic acid. Fatty sulfonic acid esters are usually In the form of metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts And a C8 chain-C22 chain alkyl group (this is also an acyl group alkyl group). For example, lignosulfonic acid, dodecylsulfonic acid, or Sodium or fatty alcohol sulfonic acid mixture obtained from natural fatty acids Is a calcium salt. These compounds are based on the fatty acids of sulfates and sulfonic acids. Also includes salts of fatty alcohol / ethylene oxide adduct. Sulfonated benzimi The dazole derivative preferably has two sulfonic acid groups and about 8 to 22 carbon atoms. Contains one fatty acid group. An example of an alkylaryl sulfonic acid is dodecylben Zensulfonic acid, dibutylnaphthalenesulfonic acid or naphthalenesulfonic acid / Sodium, calcium or triethanolamide of formaldehyde condensation product Salt. Also suitable are the corresponding phosphoric acids such as, for example, p-nonylphenol. The adduct is a salt of 4 to 14 moles of ethylene oxide phosphate. Non-a The on-surfactant is preferably an aliphatic or cycloaliphatic alcohol, or Induction of polyglycol ethers of saturated or unsaturated fatty acids and alkylphenols A conductor, the derivative comprising 3 to 30 glycol ether groups and 8 to 20 carbon atoms Atom to the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms to the alkylphenol Included in the alkyl portion of the   Further suitable non-ionic surfactants are polyethylene oxide and polypropylene. 1 for renglycol, ethylenediaminopropyleneglycol and alkyl chains A water-soluble adduct of alkyl propylene glycol containing from 10 to 10 carbon atoms, The adduct comprises 20 to 250 ethylene glycol ether groups and 10 to 100 Contains propylene glycol ether groups. These compounds are usually propylene Contains 1 to 5 ethylene glycol units per glycol unit. Nonionic surfactant Representative examples of the active ingredient are nonylphenol polyethoxyethanol and castor oil polyglycol. Coal ether, polypropylene / polyethylene oxide adduct, tributyl Phenoxypolyethoxyethanol, polyethylene glycol and octylph Enoxypolyethoxyethanol. Polyethylene sorbitan trioleate Fatty acid esters of polyethylene sorbitan, such as Component.   The cationic surfactant component is preferably at least one N-substituent. As a C8-C22 chain alkyl group and further substituents, lower unsubstituted or halo Quaternary ammogens containing alkyl benzyl, benzyl or hydroxyl lower alkyl groups Is a sodium salt. These salts are preferably halide, methyl sulfate, ethyl sulfate Salts, such as stearyltrimethylammonium chloride or benzyldi- ( 2-chloroethyl) ethylammonium bromide.   Surfactants commonly used in the field of formulation include, for example, "McCutchenon's Detergents and Emulsifiers Annual ”, MC Publishing Corp. Redgewood, NJ ., 1979; Dr. Helmut Stache, “Tensid Taschenbuch” (Handbook of Surfactants ), Carl Hanser Verlag, Munich / Vienna.   Other particularly preferred properties of the insecticidal compositions of the present invention are those applied to plants and soil. Is the continuity of the active ingredient when exposed. Possible causes of decreased activity include ultraviolet light, heat , Leaf exudates and inactivation by pH. For example, at high pH, especially reducing agents In the presence, the δ-endotoxin crystals dissolve and thus become inactive due to proteolysis. It is susceptible to sex. High leaf pH can also result in the leaf surface being particularly in the pH range 8-10. Is important in that The preparation of the insecticidal composition used in the method of the present invention comprises an active ingredient. Contains additives that help prevent loss of minutes or prevent active ingredients from inactivating These problems are addressed by encapsulation in a protected manner. capsule Encapsulation is chemical (McGuire and Shasha, J. Econ. Entomol. 85: 1425-1433, 1992). Or biologically (Barnes and Cummings, 1986; EP-A-0192319). Can be achieved. Chemical encapsulation involves the process of coating the active ingredient with a polymer. Biological encapsulation, while δ-endotoxin gene in microorganisms Including expression. As-is, containing toxin proteins for biological encapsulation Microorganisms are used as active ingredients in the formulation. UV protection reduces irradiation damage It is effective for Thermal inactivation is also controlled by the inclusion of suitable additives .   Preferred within the scope of the present invention are living microorganisms as active ingredients, growing cells or Are more preferably formulations containing spores when available. A suitable formulation is For example, it can consist of a polymer gel that crosslinks with multivalent cations and contains these microorganisms. You. This is the case, for example, when the alginate of the polymeric substance is D.C. R. Fravel et al. Phyt opathology, Vol. 75, No .. 7, 774-777, 1985. This publication It is also known that carrier materials can be used together. These preparations are Or a solution of a synthetic gel-forming polymer, such as alginate, present in the individual It is produced by mixing an aqueous salt solution of polyvalent metal ions in the form of droplets, and the microorganisms are It is possible to suspend in one of the two suspensions or in both reaction solutions. gel Formation begins with mixing into droplet form. Subsequent drying of these gel particles is possible. This method is called ionic gelation. Depending on the degree of drying, structurally polyvalent Crosslinks via thione and contains microorganisms and carriers present in excellent homogenous dispersion A dense hard particle of polymer is formed. The size of the particles is up to 5 mm.   In addition to the microorganism, it may comprise finely dispersed silicic acid as a carrier substance, with⁺⁺I Partially crosslinked polysaccharide-based compositions that occur via on It is described in EP-A1-0097571. The composition is not more than 0.3 Has water activity. W. J. Cornick et al. Reviewed the review literature [New Directions in Bio logical Control: Alternatives for Suppressing Agricultural Pests and Dise ases, p. 345-372, Alan R. Liss, Inc. (1990)] by ionic gelation method Describes the various formulation systems that have been produced, including granules and granules containing vermiculite as a carrier. And compact alginate beads. Such compositions are also described in D.S. R. Fravel , Pesticide Formulations and Application Systems: 11th Volume, ASTM STP 11 12 American Society for Testing and Materials, Philadelphia, 1992, p. 17 3-179, which can be used to formulate the recombinant microorganism of the present invention. Survival Further methods of formulating organisms are described in WO 96/02638.   The compositions of the present invention are prophylactic in the field of pest control, even at low proportions of application. And / or is effective for therapeutic treatment and is very resistant to warm-blooded species, fish and plants. It is tolerable, non-toxic and has a very favorable biocidal spectrum. The present invention The composition of Ostriana flunacharis (Asia corn borer) pest Active against all or individual stages of development. The insecticidal activity of the compounds of the invention is Directly, that is, by destroying the pest immediately or after a short time , Can be apparent.   The composition comprises a chemical mixture comprising the toxin protein in substantially pure form, Or as part of a microorganism or transgenic plant, at least one poison It can be provided in the form of a mixture containing the native protein. In a specific embodiment of the present invention, One of the sex components is directly applied to the plant, for example, by foliar treatment as described hereinabove. The second predisposition is previously formed by the gene encoding the second predisposition. It is provided by the plant itself by expressing the transformed gene.   The insecticidal composition used in the method of the present invention is usually from about 0.1 to about 99%, preferably from about 0.1% to about 99%. Or from about 0.1 to about 95% and most preferably from about 3 to about 90% of the active ingredient About 1 to about 99.9%, preferably about 1 to about 99%, and most preferably About 5 to about 95% solid or liquid adjuvant; and about 0 to about 25%, preferably Preferably from about 0.1 to about 25% and most preferably from about 0.1 to about 20% of the interface Contains active ingredients. The product is preferably prepared as a concentrate, but the end user Use a rather low concentration of the diluted formulation. Insecticidal compositions also include stabilizers, defoamers, Additional ingredients such as viscosity modifiers, binders, tackifiers and fertilizers, and especially It may also contain other active ingredients to achieve a certain effect.   The present invention also provides the active ingredient in the form of growing cells or, where available, spores. The present invention also relates to a preparation comprising a living microorganism present in the form of:   A further object of the present invention is to provide for the use of Ostriana flunakaris (Asia (Ra) C in the method of controlling grain against damage caused by seeds Bacillus thuringiensis toxin proteins such as ryI-type proteins For the use of recombinant microorganisms containing toxin genes encoding quality, Apply the toxin to the plant to be protected or recombinantly It is isolated from the product and is formulated as described before it is applied to the grain to be protected. Recombinant microorganism The products are as described in EP-A-690916 and in international application EP 95/03826. Toxin genes encoding different VIP-type toxin proteins or at least Gene encoding one Cry-type toxin and one VIP-type toxin May be included.   A host where the coding sequence was selected for recombinant production of the toxin protein in the host organism. Insert into an expression cassette designed primarily for you and into a recombinantly produced host . Promoter, signal sequence, 5 'and 3' untranslated sequences appropriate for the chosen host Specific regulatory sequences such as and enhancers are within the skill of the art. Suitable The resulting molecule, which contains the individual elements bound in the appropriate reading frame, is Insert into a transformable vector. Suitable sources for recombinant production of proteins Current vectors and methods are described in Escherichia coli (e.g., Studier and Moffatt). J. Mol. Biol. 189: 113 (1986); Brosius, DNA 8: 759 (1989)), yeast (eg, Schneider and Guarente, Meth. Enzymol. 194: 373 (1991)) and insect cells Hosts such as vesicles (see, eg, Luckow and Summers, Bio / Technol. 6:47 (1988)). Known in living organisms. Examples are pBluescript (Stratagene, La Jolla, CA), pFLAG (International Biotechnologies, Inc., New Haven, CT), pTrcHis (Invitrogen , La Jolla, CA) and baculovirus expression vectors such as autography Includes those derived from the genome of the californica nuclear polynucleosis virus (AcMNPV). Good The preferred baculovirus / insect system is pVI11392 / Sg21 cells (Invitrogen, La J. olla, CA).   Recombinant toxin proteins can be isolated and purified using a variety of standard methods . The actual method that can be used depends on the host organism used and the toxin protein used for secretion. Depends on what was designed and on such factors known to those skilled in the art (e.g., For example, Ausbel, F. et al., “Current Protocols in Molecular Biology”, John Wi ley & Sons, Inc. (See Chapter 16 of Publishing (1994).)   A preferred object of the present invention is to provide for the use of Ostriana flunakaris (Asia corn botanical). Bacillus thuringiensis toxin protein in an amount sufficient to control the species In particular, a transgenic plant containing and expressing a gene encoding a CryI-type toxin protein. Ostriana flunakaris (Asia corn borer), a sgenic plant For use in methods of protecting plants against damage caused by pests . The plant may be nuclear transformed or plastid transformed (see WO 95/24492). Can be provided by Particularly preferred is Cr from Bacillus thuringiensis. y Transgenic plants expressing IA (b) toxin protein. The present invention EP-A-690916 and the state of the art which are hereby incorporated by reference in their entirety. Toxin encoding VIP-type protein described in International Application EP 95/03826 It also relates to the use of transgenic plants containing the gene. The present invention also provides Sufficient to provide control of Ana Furnakalis (Asia corn borer) species Toxin gene encoding a Bacillus thuringiensis toxin protein in quantity But specifically contains, expresses, and expresses the VIP-type toxin gene. It also involves the use of transgenics that contain and express toxin genes that encode proteins. I do. The host plant expressing the toxin gene is Ostriana flunakaris (Acorn bowler) species have enhanced resistance to insect attack, Provide resistance to grain loss in response to such attacks.   In one preferred embodiment, one or more of the transgenic plants One or more Bt δ-endotoxins may contain one or more VIP-type proteins. Achieved by expression of protein. Pesticides in this same transgenic plant Co-expression of one or more pesticides may include all necessary genes and be expressed This is achieved by genetically engineering the plant. Alternatively, the parent 1 plant is replaced with VI Genetically engineered for expression of P-type proteins. The second plant that is parent 2 is B Engineering for tδ-endotoxin expression. Mating parent 1 and parent 2 Thereby obtaining a progeny plant, which expresses all genes inserted into parents 1 and 2 I do. Particularly preferably, Bt δ-endotoxin is included herein by reference. EP-A-0618976.   Also encompassed by the present invention are D-encoding Bacillus sp. Toxin proteins. An NA molecule, preferably the toxin protein of at least 10 nucleotides in length Oligonucleotides derived from said DNA molecule comprising a contiguous portion of a quality coding sequence A probe and a DNA molecule that hybridizes under stringent conditions Use of recombinant microorganisms or transgenic plants containing the gene to be transferred. The invention preferably comprises Bacillus thuringiensis or Bacillus cereus. A DNA molecule encoding a toxin protein, particularly a Cry-type protein. DNA molecule or VIP-type toxin protein, preferably CryIA (b) Encodes a DNA molecule that hybridizes to a toxin gene that encodes a protein The use of recombinant microorganisms or transgenic plants containing the gene.   Factors affecting the stability of hybridization include the hybridization strategy. Determine your agency. One such factor is DNA PROBES, Ge. orgeH. Keller and Mark M. Manak, Macmillan Publishers Ltd. 1993, Sectio none: Molecular Hybridizatoin Technology: Melting temperature T that can be easily calculated_mIt is.   The preferred hybridization temperature is the calculated melting temperature T_mRange of about 25 ° C And preferably the calculated melting temperature T_mIn the range of about 12-15 ° C below For oligonucleotides, the melting temperature T_mBelow about 5-10 ° C.   The invention further relates to Bacillus thuringiensis, preferably a Cry-type venom. Toxin encoding a native protein, more preferably a CryI-type toxin protein And at least one gene, wherein the toxin protein is Transgenic, which is expressed in sufficient quantities to control corn (Asia corn borer) Ostriana flunakaris (Asia corn borer), including cereals A product bag labeled with instructions for use for controlling pests. Scope of the invention Preferred among them are at least one Cry-type toxin protein as active ingredient. Transgene containing genes encoding proteins and VIP-type proteins This is a product bag containing the seeds of a plant. Particularly preferred are CryIA (b) toxin proteins It is a combination of protein and VIP-type protein.   It is a further object of the present invention to provide a cereal ostriana hull comprising an insecticidal composition of the invention. Label the instructions for use for the control of Lunakaris (Asia corn borer) Product bag.   The term plant refers to a plant species that can be genetically transformed by methods in the art. Ostriana flunacharis, in particular, including but not limited to the following plant species: (Asia corn borer) is a plant that is the host plant of the species: corn, small Wheat, barley, rye, oats, rice, sorghum, corn and related grains, fodder, bamboo Mogaya, Boletus genus etc.) and sugarcane.   Known methods for plant transformation are described below. The host plant is Including, but not limited to, those listed above.   The invention further encodes a Bacillus thuringiensis toxin protein. Gene and harmful toxin protein by Ostriana flunakaris (Asia Transgenic plants that are expressed in sufficient quantities to provide control of the species The present invention relates to a product seed and a product bag containing the seed. The term plant is corn, Wheat, barley, rye, oats, rice, sorghum, millet and related grains, fodder, bamboo Ostriana flunacharis (including but not limited to sugarcane and sugarcane) (Asian corn borer).   The codon usage of the native Bacillus thuringiensis toxin gene is It is clearly different from that of the gene. In particular, natural Bacillus thuringiensis remains The codon usage of the gene is very different from that of the maize gene. as a result However, mRNA derived from this gene is not fully utilized. Codon usage can be Affects gene expression at the level of transcription or mRNA processing. Toxin tampa Use codons to optimize protein in plants, e.g., corn Gender, a synthetic gene encoding the same protein found in the natural toxin gene sequence Most preferred codons in maize (maize preferred codons) Optimize by using Optimized maize priority codon handling is high It is effective for expressing Bell Bt insecticidal proteins. Corn optimized synthetic toxin Further details of the construction of genes can be found in WO 93, hereby incorporated by reference in its entirety. / 07278. Microbial toxin genes are also known as plant genes. Can be different. A plant gene is defined as one whose transcribed RNA is adjacent to the initiation methionine. The ribosome binding site, which has no sequence, differs from the gene found in microorganisms. Become. As a result, the microbial gene is the eukaryotic consensus translation initiator for ATG. It can be facilitated by the inclusion of eta. Clontech (catalog in 1993/1994, 2 10) is a consensus for the expression of the Escherichia coli uidA gene in plants. The sequence GTCGACC as a translation initiatorATGSuggests GTC. Further Joshi (Nucl. Acids Res. 15: 6643-6653 (1987)) describes a number of plants adjacent to the ATG. Product sequences, and consensus TAAACAATGGCT is shown. Inside the plant In the situation where expression of the microbial ORF is difficult in Insertion into the ATG can improve translation. In such cases, at least consensus Even three nucleotides are included in the modified sequence for modification of the secondary amino acid residues May not be appropriate. Sequences adjacent to the preferred initiating methionine may be different plants. Can vary by species. Maize genes present in the GenBank / EMBL database The nucleotide sequence adjacent to the ATG is converted to corn by measuring the sequence of Recognize that the inserted toxin gene should be modified to facilitate translation Can be.   In addition, removal of abnormal splice sites can increase expression and stability of the inserted gene. Is shown. Cloned from non-plant sources, ideal for expression in plants Unmodified genes can be recognized in plants as 5 'or 3' splice sites. Motifs. As a result, the transcription process stops prematurely and the truncated or deleted Produces RNA. The toxin gene identifies these abnormal splice sites in the art. It can be manipulated by removal using known methods.   Many δ-endotoxins from Bacillus thuringiensis are associated with protoxins Is expressed. These protoxins are soluble in the alkaline environment of the insect digestive tract. And then proteolytically cleaved by the protease to form the toxin core fragment. For the CryI class δ-endotoxin protein, the toxin core fragment Localizes to the N-terminal half of the protoxin. Full-length protoxin of a novel toxin protein Genes encoding the native or truncated toxin core fragment have insecticidal properties in host plants Within the scope of the present invention, it can be used for a plant transformation vector for imparting You. Recombinant DNA molecules can be inserted into plant cells in many art-recognized ways. Wear. One of skill in the art will appreciate that the choice of method depends on the type of plant targeted for transformation, i.e., monocotyledonous or Recognizes that it depends on dicotyledons. Suitable methods for transforming plant cells are described in Icroinjection (Crossway et al., BioTechniques 4: 320-334 (1986)), Electroporation (Riggs et al., Proc. Natl. Acad. Sci. USA 83/5602. -5606 (1986)), Agrobacterium-mediated transformation (Hinchee et al., Biotechnol ogy 6: 915-921 (1988)), direct gene transfer (Paszkowski et al., EMBO J. 3: 2717-2). 722 (1984)) and Agracetus, Inc. Madison, Wisconsin and Dupont, Inc., Wilmington, Delaware (see, for example, Sanford et al., U.S. Pat. No. 945,050; and cCabe et al., Biotechnology 6: 923-926 (1988)). Orbital particle acceleration using equipment available from Weissinger et al., A nnual Rev. Genet. 22: 421-477 (1988); Sanforde et al., Particulate Science and Technology 5: (27-3791987) (Onion); Chri stou et al., Plant Physiol . 87: 671-674 (1988) (soy); McCabe et al., Bio / Technology 6: 923-926 (1988) ( Soybean); Datta et al., Bio / Technology 8: 736-740 (1990) (US); Klein et al., Pro c. Natl. Acad. Sci. USA, 85: 4305-4309 (1988) (maize); Klein et al., Bio / Technology 6: 559-563 (1988) (maize); Klein et al., Plant Physiol . 91: 440-444 (1988) (maize); Fromm et al., Bio / Technology 8: 833-839. (1990); and Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) (Maize S); Svab et al., Proc. Natl. Acad. Sci. USA 87: 8526-8530 (1990) (tobacco leaves Chloroplast); Koziel et al. (Biotechnology 11: 194-200 (1993)) (maize); Shima moto et al., Nature 338: 274-277 (1989) (US); Christou et al., Biotechnology  9: 957-962 (1991) (U.S.A.); European Patent Application EP0332584 (Camogaya and other programs). Vaide et al. (Biotechnology 11: 1553-1558 (1993) (wheat); Weeks e t al. (Plant Physiol. 102: 1077-1084 (1993) (wheat); Wan et al. (Plant Phys. iol. 104: 37-48 (1994) (barley); Umbeck et al., (Bio / Technology 5: 263-266 (1987 See also) (cotton). Recombinant DNA molecule is inserted into corn by microprojectile bombardment One specific preferred embodiment is described in WO WO, incorporated herein by reference in its entirety. 93/07278. Further preferred embodiments are cited in their entirety. Corn, as described in application EP-A-292435, which is incorporated herein by reference. This is a method for protoplasmic transformation of sorghum.   The genetic characteristics engineered into the transgenic seeds and plants are based on sexual reproduction Or they are passaged in vegetative growth and are therefore maintained and propagated in progeny plants. In general, The maintenance and breeding is suitable for a specific purpose such as cultivation, sowing or harvesting Use of known agronomic methods developed in Special features such as hydroponics or greenhouse method Special purposes can also be applied. Attack and damage caused by growing crops by insects Or treatment for weeds, plants, because they are vulnerable to infection and to competition from weeds Do for the control of diseases, insects, nematodes and other adverse conditions and improve the yield . These are the mechanical treatment of soil arable or removal of weeds and infected plants, and Herbicides, fungicides, gameticides, nematicides, growth regulators, ripening agents and insecticides Including the application of pesticides.   The use of the advantageous genetic properties of the transgenic plants and seeds of the present invention is further If the pest, herbicide or load resistance, improved nutritional value, increased yield, Develop plants with improved structure resulting in reduced losses due to lodging or breakage Plant breeding for any purpose. The various breeding stages are dependent on the choice of line to be crossed and the parent line. Characterized by distinct human involvement such as pollination or selection of appropriate progeny plants Can be Different breeding treatments are performed, depending on the properties desired. Appropriate methods are known in the art Known, hybridization, inbreeding, backcrossing, multibred breeding, various Including but not limited to hybridization, interspecies hybridization, aneuploidy, etc. Absent. Hybridization methods also include plant sterilization and include mechanical, chemical, and Alternatively, a male or female sterile plant is produced by biochemical treatment. Of a different line from the male sterile plant Cross-pollination of pollen is male-sterile, but the genomes of female-fertile plants are homogenous in both parents. Ensure that the characteristics of the system are obtained. Accordingly, the transgenic species of the present invention Offspring and plants can increase the effectiveness of conventional methods such as, for example, herbicide or pesticide treatment. Or improved plants that, due to the properties of the modified gene, cause the practice of the method Can be used to breed lines. Alternatively, a new plant with an improved loading regime is Products that are not tolerant to equivalent adverse developmental conditions because of the modified gene "equipment" A good quality product can be harvested.   In seed production, germplasm and seed uniformity are essential product characteristics. Germ quality and uniformity of the seeds harvested and released by the farmer are not important. Other grains It is difficult to keep seeds away from cereals and weed seeds. To roll, and to produce good germinating seeds, rather large quantities, And clear seed production practices are the key to growing, conditioning and selling pure seed. Developed by experienced seed producers. Therefore, harvest from your own grain Buying guaranteed seeds that meet specific qualitative standards instead of using fresh seeds Is customary for farmers. Breeding materials used as seeds include herbicides, insecticides, Killing Protective agents including fungicides, fungicides, nematicides, molluscicides or mixtures thereof Processed conventionally with coatings. Conventionally used protective coatings are , Compounds provide protection against damage caused by bacteria, fungi or animal pests. To provide additional carriers, surfactants or other ingredients conventionally used in the field of pharmacy. Is formulated with an application-enhancing adjuvant. Protective coating, breeding liquid formulation Material or coat with a combination of wet or dry formulations Can be applied. Other applications, such as treating the shoots or fruits directly, are also possible. Noh.   Provides control of Ostriana flunakaris (Asia corn borer) The transgenic plant, transgenic plant material or plant of the present invention. New methods, such as those exemplified above, characterized by the use of transgenic seeds Providing an agricultural method is a further aspect of the present invention.   In order to grow progeny from a plant transformed by the method of the present invention, the following method is used. Can be used: corn plants produced as described in the Examples below are used in a greenhouse In a pot or soil, grow and flower as known in the art. Mature ear with pollen From the same plant, the same species, or any desired maize plant Use for powder. Similarly, the ears grown from the transformed plants are May be pollinated with pollen obtained from any desired corn plant. Got this way Transforming the progeny with the presence of the transgene and / or accompanying DNA (genotype), or The conferred phenotype can distinguish it from non-transformed progeny. The transformed progeny is also Self-pollinate or combine with other plants, as is usually done with plants that carry the desired properties. Can be mated. Similarly, tobacco or other transformed plants produced by this method can be Self-pollinate as is known in the art to produce progeny with the desired properties Or can be crossed. Similarly, combinations of the methods of the present invention with other methods known in the art. The transgenic organisms produced in the above are bred as is known in the art, and Can produce offspring of sex.Example   The following examples illustrate the materials and methods used to obtain specific embodiments of the present invention. It will be further described. They are set forth by way of explanation and should not be construed as limiting the description of the invention. Don't excuse me.Example 1 General law   DNA manipulations were performed using methods routinely practiced in the art. this These methods often involve modification and / or substitution without substantially altering the result. it can. Unless other cited references were identified, the method described in Sambrook et al., M. olecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Pres s, Second Edition, 1989.Example 2: Plant transformation vector   Plant transformation is described in WO 93/0727, both of which are incorporated herein by reference. 8 and Kozziel et al. (1993) [Biotechnology Vol. 11, 194-200] This is performed using the transformation vectors pCIB4431 and pCIB3064. pCIB4431 is corn It is a vector designed for transformation of Koshi. It originates in corn Containing two possible chimeric synthetic Bt CryIA (b) endotoxin genes, One of them is PEP carboxylase promoter / synthesis-CryIA (b) gene And the other contains the pollen promoter / synthetic CryIA (b) gene. pCIB4431 is It contains the synthetic CryIA (b) gene of SEQ ID NO: 1, 1815 North University Street, Peoria, Illinois Agricultural Research Services at the Southern Regional Research Center Service to Patent Culture Collection (NRRL) with accession number NRRL B-18998 and deposited on September 21, 1992. pCIB3064 is originally a strep Cloned from Tomyces hygroscopicus [Thompson et al. (1987) E MBO J. 6, 2519-2523] including the plant-expressable bar gene (615 bp). This is Phosph Encoding phosphinotricin acetyltransferase (PAT) Provides resistance to syn. The bar gene contains the CaMV35S promoter and Terminator [OW et al. (1987) Proc. Natl. Acad. Sci. USA 84, 4870-487 4] It is under control and provides resistance to phosphinothricin.Example 3 Transgenic tow containing synthetic corn CryIA (b) gene Production of sorghum plants   The following example demonstrates the use of microparticles to insert DNA-coated particles into corn cells. A gun plant is used to generate transformed plants therefrom. 3.1Organization   1.5-2.5 mm immature maize embryos are pollinated from the ears of genotype 6N615. Taken 14-14 days after mistake. The mother plant was grown in the greenhouse. Before removing, 2 ears Surface sterilized with 0% Clorox for 20 minutes and rinsed three times with sterile water. Individual embryos with scutellum side up In a 2 cm square area, callus initiation medium, 2DG4 + 5 chlorambene medium (N6 Plate 36 embryos with essential salt, B5 low salt, MS iron, 2% sucrose) 5 mg / l chlorambene, 20 mg / l glucose and 10 ml G4 additive (Table 1) Was added after autoclaving.Table 1 : G4 additive              component Per liter of medium              Casein hydrolyzate 0.5g              Proline 1.38g              Nicotinic acid 0.2mg              Pyridoxine-HCl 0.2mg              Thiamine-HCl 0.5mg              Choline-HCl 0.1mg              Riboflavin 0.05mg              Biotin 0.1mg              Folic acid 0.05mg              0.1 mg of Ca pantothenate              p-Aminobenzoic acid 0.05 mg              B12 0.136 μg 3.2Production of DNA for delivery   Microcarriers were manufactured essentially according to the instructions attached to the particle gun device . While vortexing 50 μl of a 10 μm microcarrier, pCIB4431 ( 1.23 μg / μl) [# 898] +2 μl pCIB3064 (0.895 μg / μl) [# 456], Then 2. 5M CaCl_Two  50 μl then 0.1 M spermidine (free base, TC gray) Was added. The resulting mixture is vortexed for 3 minutes and microcentrifuged for 10 seconds. did. The supernatant was removed and the microcarriers were washed twice with 100% EtOH (HPLC Vortex briefly with 250 μl, centrifuge and remove supernatant And washed. The microcarrier was resuspended in 65 μl of 100% EtOH . 3.3Irradiation   Tissue was irradiated using a PDS-100He Biolistics instrument. Hurt organization Placed on the shelf 8 cm below the shelf. Allow the tissue to dry on the macrocarrier One shot with 10 μl of the DNA / gold microcarrier solution. Used stop screen Lean by hand using a 10 × 10 stainless steel mesh screen Drilled. A 1550 psi rupture disc was used. After irradiation, the embryos are kept in the dark at 25 ° C. And cultured. 3.4Callus formation   Embryos were transferred to callus initiation medium containing 3 mg / PPT one day after irradiation. Irradiate the embryo 2 And counted after 3 weeks for callus formation. Callus maintenance culture after reaction The medium was transferred to 2DG4 + 0.5 2,4-D medium supplemented with 3 mg / L PPT. Callus The preservation medium was N6 major salt, B5 low salt, MS iron, 2% sucrose, 0.5m g / l 2,4-D, 20 mg / l glucose and 10 mg G4 additive Added after lave. Embryogenic callus every 3 weeks containing 3 mg / L PPT The culture was subcultured into a holding medium. All calli were incubated at 25 ° C. in the dark. Type I callus type The synthesis reaction was 15%. All callus-forming embryos develop individual lines Cultured as individual events. 3.5Playback   After 12 weeks of selection, the tissue is removed from the callus maintenance medium containing PPT and placed in regeneration medium. Was. The regeneration medium was 0.25 MS3S5BA (0.25 mg / l 2,4-D, 5 mg / l BA) for 2 weeks. P, MS salt, 3% sucrose) and subsequently transferred to MS3S medium for plant regeneration. Was replaced. After 4 to 10 weeks, the plants regenerated and were sown on GA7.Example 4: Analysis of transgenic corn plants 4.1ELISA assay   Detection of cryIA (b) gene expression in transgenic corn Acorn borer insect bioassay and the resulting CryIA (b) levels Follow up using ELISA analysis for quantitative measurement of. Transgenic Quantitative measurement of CryIA (b) insecticidal protein in plant leaves was performed using Clark MF, Lister RM. , Bar-Joseph M: ELISA Techniques. Weissbach A, Weissbach H (eds.) Methods in Enzymology 118: 742-766, Enzyme as described in Academic Press, Florida (1986) Performed using a binding immunosorbent assay (ELISA). Bacillus Thuringiensis An immunoaffinity purified polyclonal rabbit specific for the subspecies Krusutaki insecticidal protein And soluble protein from crude extracts of leaf samples using goat antibodies The amount of insecticidal protein is measured. The sensitivity of the double sandwich ELISA is Using 50 μg total protein per ELISA microtiter dish well , 1-5 ng of insecticidal protein per mg of soluble protein. Corn extraction The product is extracted with extraction buffer (50 mM Na)._TwoCO_ThreePH 9.5, 100 mM NaCl, 0.05% Triton, 0.05% Tween, 1 mM PMSF and 1 μM Leupe (Putin) in the presence of hand-held ball bearing homogenizer (AGDIA) Elkart IN.) By grinding the leaf tissue in a nylon bag lined with gauze. U. Perform protein measurements using the Bio-Rad (Richmond, CA) protein assay. U. 4.2Asia corn bowlersey   1-4 cm sections are cut from the extended leaves of the corn plant. 50 pieces of each leaf section Place on a damp filter disc in a × 9 mm Petri dish. Five newborn Asian animals Larvae on each leaf section (total 5-20 larvae per plant) . Incubate the Petri dish at 29.5 ° C. 24 damage and fatal days of eaten leaves, Score at 48 and 72 hours.Example 5: Ostriana flunakaris (Asia corn borer) field test Sussey   First, the transgenic seedlings tested for the presence and expression of the transgene Small peat pots were transplanted outdoors. Non-transgenic inbred in the same place And planted for 6 weeks and used as control and for pollination. Outdoors When plants reach 40 cm long leaves, lab-grown Ostriana flunacharis (Asia corn borer) Infects larvae with transgenic and non-transgenic Start with both sgenic control plants. Mixed with corn About 300 newborn larvae were used to transplant each plant using Davis Inoculator And insert. The infection lasted four weeks on a weekly basis and was the first in the Asian corn borer. Stimulated the development of Starting each week 2 weeks after the first infection, each plant is weekly for 4 weeks 1 to 9 points (1 = no visible leaf damage; 9 = most leaves have long lesions) Some leaves have broken central ribs, formed by an Asian corn borer (Possibly a plant whose length has been disturbed). Average Asian corn bow Color damage score for each transgenic and non-transgenic control Calculate with plants. As each plant has reached flowering, 300 larvae / plant are increased by 4 per week. Apply weekly to stimulate second generation infection. 100 new corn in the corn The live larvae were inserted into the axilla of the initial ear and on or below one of the nodes of the initial ear. Thus, a total of about 2400 larvae are applied to each plant. Of the first second generation infection After about 50 days, stems are harvested from all transplants and certain non-transgenic plants . The extent of internal second generation infection hole formation damage in all plants is determined.Example 6: The transformed protoplasmic extract, Ostriana flunakaris (Asia Insecticidal activity assay   Western blot analysis was performed using DN to express the maize-optimized gene. This is done using extracts obtained from corn cells transiently transformed with A. Set A qualitative insect toxicity test is performed using the recovered protoplasm. Suspension in bioassay Manufactured at each replicate to be tested. For example, replicate is Asian corn Using a bowler, Ostriana flunakaris, against the insects of the order Lepidoptera Test for activity. 100 μl of the plasma suspension in 0.1% Triton X-100 Artificial banana bait in a Petri dish with 50 mm x 10 mm snap and lid (Bioserv , Inc. Pipette onto the surface of Frenchtown, NJ; F9240). After air drying, 10 Add the newborn larva to the writing plate. Mortality is recorded approximately 4 days later.Example 7: Ostriana flunacharis (Asia corn borer) plant immersion test Trial 7.1Bacillus thuringiensis (Bt) crystal   Bacillus thuringiensis (Bt) crystals were prepared from the stock suspension with 22 ml of distilled water. Made. Store the suspension in the refrigerator. 7.2Parameter recording   Three-day-old Ostriana flunacharis (Asia corn borer) larva Feed sorghum leaves. Larvae have already eaten untreated leaves. 120 hours later, larva Record the number of dead. The type of damage injured by leaf plants is observed in each case. 7.3Test method   Two zea maize homozygous crosses susceptible to Asian corn borers (A and B). 9-10 day old seedlings were treated with various concentrations of Bt tan Soak in parkin suspension and release larvae to dried leaves of seedlings (5-10 larvae per plant) Used for feeding experiments. Cover the seedlings with a nylon mesh bag. Keep in the case. Test in quadruplicate at 4-5 concentrations to determine mortality. temperature At 21-30 ° C. 7.4result Table 2 : Result of A system Table 3: Result of B system  Two types of damage were clearly identified in maize leaves: Bt soak of seedlings The pickled leaves were slightly damaged, but the control seedling leaves were very damaged. The following LC₅₀Got the value: A system: LC₅₀= 23.412 ppm (range from 17.834 to 30.734) B type: LC₅₀= 12.234 ppm (range of 9.547 to 15.676)Example 8: Ostriana flunakaris (Asia corn borer) plant dipping ISSEY (VIP3A) 8.1VIP 3A protein   5 mg of VIP3A protein is used to prepare various concentrations of VIP3 protein. Adjusted with ml distilled water: 100 ppm, 50 ppm, 25 ppm, 12.5 ppm, 6.25 ppm And 0 ppm (for comparison). 8.2Ostriana Furnakalis (Asia corn borer)   Entology and Animal Science Division DOA from a farmer in Ratchaburi The resulting pupae are ordered for larval (L2) preparation for testing. 8.3Parameter recording   After keeping the data for 5 days, the number of dead larvae was counted, and the larval mortality was then estimated. Analyzed with a bit analysis program. 7.3Test method   Two zea maize homozygous crosses susceptible to Asian corn borers (B and C lines). Plant B was planted in pots (5 concentrations and And the C line was subjected to a leaf immersion test (100, 50, 25 pp). m for density and 4 for verification). Variety of 10-14 day old seedlings Immersed in VIP3A protein suspension at a concentration of 2. Per 5-10 larvae), used for feeding experiments. Seedlings in nylon mesh bags Cover and keep in nylon mesh case. Cut leaves into plastic blocks Four, four were used for this experiment, kept at a controlled room temperature. 7.4result Table 4 : Result of B system Table 5: Result of C system After 120 hours, the following LC₅₀Got the value: B type: LC₅₀= 29.558 ppm (range 21.298 to 41.022) C system: LC₅₀= 78.498 ppm (range 53.644 to 114.866) Deposit North University strike, Peoria, Illinois, United States 61604 REIT 1815, Agricultural Research Services, Patent Cal Char Collection (NRRL), Northern Regional Research Center Deposit in. Stock name Deposit number Deposit date Bacillus ceresius AB78 NRRL B-21058 March 18, 1993 Escherichia coli pCIB4431 NRRL B-18998 November 21, 1992

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG), EA (AM, AZ, BY, KG, KZ , MD, RU, TJ, TM), AL, AM, AT, AU , AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, G B, GE, GH, HU, IL, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, N O, NZ, PL, PT, RO, RU, SD, SE, SG , SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU (72) Inventors Whit de Willichen, Wilhelms             Petrus Maria             Brazil, Kodigo de Endere Samen             To Postal-38401-097 Minas Jella             Chair, Uberlandia, Rua Raphae             Le Rinaldi 281, apartment             1700, Edificio San Marco

Claims (1)

[Claims]   1. Active directly or indirectly on plants or plant seeds or plant habitats Ostrinia, including applying a Bacillus species toxin protein as an ingredient Protect plants (including progeny) against damage caused by flunacharis species Method.   2. Toxin protein is Bacillus thuringiensis toxin protein The method of claim 1.   3. The toxin protein is a Bacillus cereus toxin protein. The described method.   4. The toxin protein is a Cry-type protein, preferably a CryI-type protein; Protein, more preferably CryIA-type protein, and most preferably CryIA 2. The method according to claim 1, which is an IA (b) protein.   5. The toxin protein is a CryI-type protein according to SEQ ID NOS: 53 to 55. 5. The method of claim 4, wherein the method is quality.   6. The toxin protein is a VIP-type protein, preferably VIP1A (a) protein. VIP1-type proteins such as protein or VIPIA (b) protein, or VIP2-typeta such as VIP2A (a) protein or VIP2A (b) protein Combination of protein or VIP1-type protein and VIP2-type protein The method of claim 1, wherein   7. The toxin protein is a VIP3A (a) protein or a VIP3A (b) protein 2. The method of claim 1, wherein said VIP3-type protein.   8. The toxin protein is SEQ ID NO: 1, 2, 4-7, 17-24, 26-32, 3 5, 36, 39, 40, 42, 43, 45, 46, 49, 50, 51 or 52 The method according to claim 6 or 7, which is the VIP-type protein according to (1).   9. 2. The method of claim 1, wherein the toxin protein is applied to the plant in the form of an insecticidal composition. Method.   10. 10. The method of claim 9, wherein the insecticidal composition comprises a microorganism.   11. 10. The method of claim 1, wherein the toxin protein is a CryI-type protein. The method according to any of the above.   12. The insecticidal composition comprises at least one CryI-type toxin protein or A microorganism having at least one gene encoding the toxin protein, Preferably Bacillus thuringiensis and / or Bacillus cereus strains 10. A method according to claim 9, wherein the method comprises a suitable carrier.   13. The microorganism comprises at least one cryI-type encoding a toxin protein 13. The method of claim 12, which is a naturally occurring organism containing a putoxin gene.   14． The microorganism comprises at least one cry-type toxin gene, preferably cryI -Type toxin gene, more preferably cryIA-type toxin gene, and most 2. A naturally occurring organism, preferably comprising a cryIA (b) toxin gene. 0. The method of claim 0.   15. The microorganism has at least one cry recombinant form encoding a toxin protein 13. The method of claim 12, which is a recombinant organism containing an I-type toxin gene.   16. The microorganism is a cry-type toxin gene, preferably a cryI-type toxin gene. Offspring, more preferably the cryIA-type toxin gene, and most preferably cryIA (b) The method according to claim 10, which is a recombinant organism containing a toxin gene.   17． The insecticidal composition comprises at least one VIP-type toxin protein or A microorganism having at least one gene encoding a toxin protein, preferred Or Bacillus thuringiensis and / or Bacillus cereus strain The method according to claim 10, wherein the method is included together with a suitable carrier.   18. The microorganism has at least one gene encoding a VIP-type protein Preferably, the VIP1A (a) protein or VIP1A (b) protein gene, or Or VIP2 such as VIP2A (a) protein or VIP2A (b) protein gene 11. A naturally occurring organism having a -type protein gene. the method of.   19. The microorganism comprises at least one VIP3A (a) protein or VIP3A (b) protein; Has genes encoding VIP3-type proteins such as protein genes 11. The method of claim 10, wherein the organism is a naturally occurring organism.   20. The toxin protein is SEQ ID NO: 1, 2, 4-7, 17-24, 26-32, 35, 36, 39, 40, 42, 43, 45, 46, 49, 50, 51 or 5 20. The VIP-type protein according to claim 2, wherein the protein is a VIP-type protein. the method of.   21. The toxin protein is a Cry-type protein according to SEQ ID NOS: 53-55. 17. The method according to any of claims 11-16, wherein   22. The method according to any of claims 17-21, wherein the microorganism is a recombinant organism. .   23. Transform plants with a toxin gene encoding a Bacillus toxin protein When plants transformed in this way are planted in areas where pests can occur, Eliminate protein from Ostrinia flakalis (Asia corn borer) species Indirectly apply toxin proteins to plants by expressing them in sufficient amounts to A method according to any of the preceding claims.   24. Bacillus thuringiensis or Bacillus cereus 24. The method of claim 23, which encodes a toxin protein of the formula:   25. The toxin gene is a Cry-type toxin protein, preferably CryI-type Proteins, more preferably CryIA-type proteins, and most preferably A coding sequence encoding a CryIA (b) protein as set forth in SEQ ID NOs: 53-55. 24. The method of claim 23.   26. The toxin gene is VIP-type toxin protein, preferably VIPIA (a) protein; Protein or VIP1A (b) protein, or VIP2A (a) protein or VIP 3. Encoding a VIP2-type protein, such as the 2A (b) protein. 3. The method according to 3.   27. The toxin gene is different from the VIP3A (a) protein or VIP2A (b) protein. 24. The method of claim 23, which encodes such a VIP3-type protein.   28. The toxin gene is a synthetic gene and its codon usage is most preferred by plants. 28. The method according to any of claims 23 to 27, wherein the codons have been optimized by using different codons. The described method.   29. 24. From claim 23, wherein the toxin gene encodes a CryI-type protein. 29. The method according to any of 28.   30. The toxin gene is SEQ ID NO: 1, 2, 4-7, 17-24, 26-32, 3 5, 36, 39, 40, 42, 43, 45, 46, 49, 50, 51 or 52 The method according to claim 26 or 27, which encodes the VIP-type protein according to claim 26. Method.   31. 31. The method according to any of the preceding claims, wherein the plant to be protected is a grain. .   32. 32. The method according to claim 31, wherein the plant to be protected is a corn plant.   33. Cry-type toxin proteins can be directly or indirectly Combined with VIP-type toxin protein in growing areas of offspring or protected plants To control the Asian corn borer (Ostrina flunakaris) pests 33. A method according to any of the preceding claims, wherein the method applies an amount sufficient to:   34. 33. Any of claims 1 to 32 wherein the active ingredient is a Cry1A (b) protein. The method described in Crab.   35. 33. Any of claims 1-32, wherein the active ingredient is a VIP-type protein. The method described in Crab.   36. Ostrinia flunacharis (Asia corn borer) pest Use of an active ingredient according to any of claims 1 to 35 for controlling.   37. Ostrinia flunacharis (Asia corn borer) pest Use of the transgenic plant according to claim 23 for controlling.   38. Ostrinia flunacharis (Asia corn borer) pest Cry-type or VIP-type encoding each toxin protein for control Gene and DNA molecules that hybridize under moderately stringent conditions. 31. The recombinant microorganism or transgenic microorganism according to claim 23. Use of check plants.   39. It encodes a Bacillus thuringiensis toxin protein and is A sufficient amount of toxin to control Trinia flunakaris (Asia corn borer) Ostrine full of cereals, including seeds containing toxin genes that express proteins Label of instructions for its use for control of Nakaris (Asia corn borer) pests Product bag with   40. Seeds of a transgenic plant, or from claims 1 to 30, 32 and At least one toxin gene encoding a toxin protein as defined in any of 33. Ostrich flonacaris (Asia corn borer) Microorganisms that express small amounts of toxin proteins, especially Bacillus thuring Ostrich strains, including gensis and / or Bacillus cereus strains Lunakaris (Asia corn borer) instructions for its use for controlling pests Product bag with bell.   41. Bacillus toxin tan as defined in any of claims 23 to 32. It contains the toxin gene that encodes protein and contains (Dia corn borer) expresses sufficient amounts of toxin protein to control species Transgenic plant or progeny thereof, or transgenic seed thereof Use an agricultural method.   42. An amount sufficient to provide control of the sesame pest, at least one Cry- Protects type toxin proteins in combination with VIP-type toxin proteins The method according to any one of claims 18 to 26, wherein the method is expressed in a plant.   43. At least one Cry-type toxin protein and VIP as active ingredients -Comprising a pesticidally effective amount of a type protein together with an agriculturally acceptable carrier; Composition.