WO1997046105A1 - Method of controlling insect pests - Google Patents

Abstract

The present invention provides a method of controlling Ostrinia furnacalis (Asian Corn Borer) species in crop plants, but especially in maize and other cereal plants, including, but not limited to the following species of plants: maize, wheat, barley, rye, oats, rice, sorghum, millet and related crops, forage grasses, bamboo and sugar cane.

Description

Method of Controlling Insect Pests

The present invention relates to a method of controlling species of the Lepidoptera genus Ostrinia species preferably Ostrinia furnacalis (Asian Corn Borer), in crop plants by use of toxin proteins obtainable from Bacillus thuringiensis and/or other Bacillus species.

Bacillus thuringiensis belongs to the large group of gram-positive, aerobic, endospore-forming bacteria. Unlike other very closely related species of Bacillus such as B. cereus or B. anthracis, the majority of the hitherto known Bacillus thuringiensis species produce in the course of their sporulation a parasporal inclusion body which, due to its crystalline structure, is generally referred to as a crystalline body. This crystalline body is composed of insecticidally active crystalline protoxin proteins, the so-called δ-endotoxins.

The protein crystals are responsible for the toxicity to insects of Bacillus thuringiensis. The δ-endotoxin does not exhibit its insecticidal activity until after oral ingestion of the crystalline body, when the latter is dissolved in the intestinal juice of the target insects. In most cases the actual toxic component is released from the protoxin as a result of proteolytic cleavage caused by the action of proteases from the digestive tract of the insects.

The δ-endotoxins of the various Bacillus thuringiensis strains are characterized by high specificity toward certain target insects, especially with respect to various Lepidoptera, Coleoptera and Diptera larvae, and by a high degree of activity against such succeptible larvae. A further advantage of Bacillus thuringiensis δ-endotoxins resides in the fact that the toxins are harmless to humans, other mammals, birds and fish.

The various insecticidal crystal proteins from Bacillus thuringiensis have been classified based upon their spectrum of activity and sequence similarity. The classification put forth by Höfte and Whiteley, Microbiol. Rev.53:242-255 (1989) placed the then known insecticidal crystal proteins into four major classes. Generally, the major classes are defined by their spectrum of activity, with the Cryl proteins being active against Lepidoptera, Cryll proteins against both Lepidoptera and Diptera, Crylll proteins being active against Coleoptera, and CrylV proteins against Diptera.

Within each major class, the δ-endotoxins are grouped according to sequence similarity.

The Cryl proteins are typically produced as 130-140 kDa protoxin proteins which are proteolytically cleaved to produce insecticidally active toxin proteins about 60-70 kDa in size The active portion of a δ-endotoxin resides in the NH₂-termιnal portion of the full- length molecule. Hofte and Whiteley, supra, classified the then known Cryl proteins into six groups, IA(a), IA(b), IA(c), IB, IC, and ID. Since then, proteins classified as CrylE, CrylF, CrylG, CrylH and CrylX have also been characterized.

The spectrum of insecticidal activity of an individual δ-endotoxin from Bacillus thuringiensis tends to be quite narrow, with a given δ-endotoxin being active against only a few insects. Specificity is the result of the efficiency of the various steps involved in producing an active toxin protein and its subsequent ability to interact with the epithelial cells in the insect digestive tract.

It is one of the objects of this invention to provide a method of controlling Ostrinia furnacalis (Asian Corn Borer) species in plants, preferalbly cereal crops, including, but not limited to the species of maize, wheat, rye, oats, rice, sorghum, millet and related crops, forage grasses, bamboo and sugar cane. This object could surprisingly be achieved within the scope of the invention by administering a toxin protein of Bacillus thuringiensis such as a Cryl-type toxin protein, to the crop plant to be protected. In another embodiment of the invention toxin proteins obtainable from vegetative cultures of Bacillus species, so-called Vegetative Insecticidal Proteins (VIPs) such as VIP3 [EP-A0 690916; International Application no EP95/03826, the disclosure of which is incorporated herein by reference in its entirety], can also be used to control Ostrinia furnacalis (Asian Corn Borer) pests.

The present invention thus relates to a method for protecting plants including progeny thereof against damage caused by Ostrinia furnacalis (Asian Corn Borer) species comprising directly or indirectly administering to the plant or the plant seed or the growing area of the plant to be protected a toxin protein of Bacillus species, preferably a Cryl-type or a VIP-type protein mentioned above, either purely or in the form of an ento- mocidal composition comprising at least one of said proteins or a microorganism, preferably a Bacillus thuringiensis and/or a Bacillus cereus strain, containing at least one toxin gene encoding the toxin protein. Said microorganisms used in the method according to the invention may either be naturally occurring strains or, in the alternative, recombinant strains comprising a recombinant gene encoding the toxin.

In a preferred embodiment, transgenic plants are used to administer the toxin to the plants to be protected against damage caused by Ostrinia furnacalis (Asian Corn Borer) species. Such plants are obtained by transformation with a toxin gene encoding an insecticidal toxin protein from a Bacillus species such as a Cry-type, preferably a Cryl- type toxin protein or a VIP-type protein, and expressing said toxin protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species upon planting the so transformed plant in an area where said insect pest occurs.

Entomocidal compositions to be used in the method according to the invention for protecting crop plants against Ostrinia furnacalis (Asian Corn Borer) pests for example comprise as an active ingredient at least one Cry-type toxin protein, more preferred at least one Cryl-type toxin protein, even more preferred at least one CrylA-type toxin protein, particularly preferred at least one CrylA(b)-type toxin protein and most particularly preferred at least one crylA(b) type toxin protein according to SEQ ID NOS: 53 to 55, even more preferred of Bacillus thuringiensis or a microorganism containing at least one gene encoding said toxin protein, preferably a Bacillus thuringiensis strain containing at least one gene encoding said toxin protein, or a derivative or mutant thereof, together with an agricultural adjuvant such as a carrier, diluent, surfactant or application-promoting adjuvant. The active ingredient contained in the entomocidal composition may also be a VIP-type toxin protein as disclosed in EP-A-0690916 and the PCT International Application No EP95/03826 or a combination of Cryl-type and VIP-type proteins. Preferred within the scope of protection is aVIP1-type protein, such as a VIP1 A(a) protein or a VIP1A(b) protein, or a VIP2- type protein, such as a VIP2A(a) protein or a VIP2A(b) protein or a combination of a VIP1-type protein and a VIP2-type protein or aVIP3-type protein, such as a VIP3A(a) protein or a VIP3A(b) protein.

More preferred within the scope of protection are VIP-type toxin proteins as shown in SEQ ID NOS: 1 , 2, 4-7, 17-24, 26-32, 35, 36, 39, 40, 42, 43, 45, 46, 49, 50, 51 or 52.

The composition may also contain a further biologically active compound. Said compound can be both a fertilizer or micronutrient donor or other preparations that influence plant growth. It can also be a selective herbicide, insecticide, fungicide, bactericide, nematicide, molluscide or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.

The composition may comprise from 0.1 to 99% by weight of the active ingredient, from 1 to 99.9% by weight of a solid or liquid adjuvant, and from 0 to 25% by weight of a surfactant. The active ingredient or the composition containing said active ingredient, may be administered to the plants or crops to be protected together with certain other insecticides or chemicals (1993 Crop Protection Chemicals Reference, Chemical and Pharmaceutical Press, Canada) without loss of potency. It is compatible with most other commonly used agricultural spray materials but should not be used in extremely alkaline spray solutions if a Cryl-type toxin is involved. It may be administered as a dust, a suspension, a wettable powder or in any other material form suitable for agricultural application.

The active ingredient, that is preferably a Cryl-type toxin protein of Bacillus thuringiensis and/or one of the VIP-type proteins mentioned previously, or the composition comprising said active ingredient may be applied to (a) an environment in which the insect pest may occur, (b) a plant or plant part in order to protect said plant or plant part from damage caused by an insect pest, or (c) seed in order to protect a plant which develops from said seed from damage caused the pest.

A preferred method of application in the area of plant protection is application to the foliage of the plants (foliar application), with the number of applications and the rate of application depending on the plant to be protected and the risk of infestation by the pest in question.

The compositions to be used in a method according to the invention are also suitable for protecting plant propagating material, e.g. seed, such as fruit, tubers or grains, or plant cuttings, from insect pests. The propagation material can be treated with the formulation before planting: seed, for example, can be dressed before being sown. The active ingredient of the invention can also be applied to grains (coating), either by impregnating the grains with a liquid formulation or by coating them with a solid formulation. The formulation can also be applied to the planting site when the propagating material is being planted, for example to the seed furrow during sowing. The invention relates also to those methods of treating plant propagation material and to the plant propagation material thus treated. Within the scope of the invention the compositions may be applied in any method known for treatment of seed or soil with bacterial strains. For example, see US Patent No.4,863,866. The strains are effective for biocontrol even if the microorganism is not living. Preferred is, however, the application of the living microorganism.

Target crops to be protected within the scope of the present invention are those that are host plants for Ostrinia furnacalis (Asian Corn Borer) species and include but are not limited to the species of maize, wheat, barley, rye, oats, rice, sorghum, millet and related crops, forage grasses, bamboo and sugar cane.

The active ingredient according to the invention may be used in unmodified form or together with any suitable agriculturally acceptable carrier. Such carriers are adjuvants conventionally employed in the art of agricultural formulation, and are therefore formulated in known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations, for example, in polymer substances. Like the nature of the compositions, the methods of application, such as spraying, atomizing, dusting, scattering or pouring, are chosen in accordance with the intended objective and the prevailing circumstances. Advantageous rates of application range from about 50 g to about 5 kg of active ingredient (a.i.) per hectare ("ha", approximately 2.471 acres), and preferably from about 100 g to about 2 kg a.i./ha. Preferred rates of application are 200 g to about 1 kg a.i./ha or 200 g to 500 g a.i./ha.

For seed dressing advantageous application rates range from 0.5 g to 1000 g a.i. per 100 kg seed, preferably from 3g to 100 g a.i. per 100kg seed. Most preferred are application rate from 10 g to 50 g a.i. per 100 kg seed.

Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. The formulations, i.e. the entomocidal compositions, preparations or mixtures thereof with other active ingredients, and, where appropriate, a solid or liquid adjuvant, are prepared in known manner, e.g., by homogeneously mixing and/or grinding the active ingredients with extenders, e.g., solvents, solid carriers, and in some cases surface-active compounds (surfactants). Suitable solvents are. aromatic hydrocarbons, preferably the fractions containing 8 to 12 carbon atoms, e.g. xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolιdone, dimethylsulfoxide or dimethylformamide, as well as vegetable oils or epoxidised vegetable oils such as epoxidised coconut oil or soybean oil, or water.

The solid carriers used, e.g., for dusts and dispersible powders, are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand. In addition, a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverized plant residues.

Depending on the nature of the active ingredients to be formulated, suitable surface- active compounds are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" will also be understood as comprising mixtures of surfactants. Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds. Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (C sub 10-C sub 22), e.g. the sodium or potassium salts of oleic or steanc acid, or of natural fatty acid mixtures which can be obtained, e.g. from coconut oil or tallow oil. Further suitable surfactants are also the fatty acid methyltaurin salts as well as modified and unmodified phospholipids.

More frequently, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates. The fatty sulfonates or sulfates are usually in the forms of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and generally contain a C sub 8 -C sub 22 alkyl radical which also includes the alkyl moiety of acyl radicals, e.g. the sodium or calcium salt of Itgnosulfonic acid, of dodecylsulfate, or of a mixture of fatty alcohol sulfates obtained from natural fatty acids . These compounds also comprise the salts of sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing about 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product. Also suitable are corresponding phosphates, e.g. salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of ethylene oxide. Non-ionic surfactant are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.

Further suitable non-ionic surfactants are the water-soluble adducts of polyethylene oxid e with polypropylene glycol, ethylenediaminopolypropylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups. These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit. Representative examples of non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxid e adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxypolyethoxyethanol . Fatty acid esters of polyoxyethylene sorbitan, such as polyoxyethylene sorbitan trioleate, are also suitable non-ionic surfactants.

Cationic surfactants are preferably quaternary ammonium salts which contain, as N- substituent, at least one C sub 8 -C sub 22 alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl, benzyl or hydroxyl-lower alkyl radicals. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g., stearyltnmethylammonium chloride or benzyldι-(2-chloroemyl)ethylammonιum bromide.

The surfactants customarily employed in the art of formulation are described, e.g.,in "McCutcheon's Detergents and Emulsrfiers Annual", MC Publishing Corp Ridgewood, N.J., 1979, Dr. Helmut Stache, "Tensid Taschenbuch" (Handbook of Surfactants), Carl Hanser Verlag, Munich/Vienna.

Another particularly preferred characteristic of an entomocidal composition of the present invention is the persistence of the active ingredient when applied to plants and soil Possible causes for loss of activity include inactivation by ultra-violet light, heat, leaf exudates and pH. For example, at high pH, particularly in the presence of reductant, δ- endotoxin crystals are solubilized and thus become more accessible to proteolytic inactivation. High leaf pH might also be important, particularly where the leaf surface can be in the range of pH 8-10. Formulation of an entomocidal composition to be used in a method according to the present invention can address these problems by either including additives to help prevent loss of the active ingredient or encapsulating the material in such a way that the active ingredient is protected from inactivation. Encapsulation can be accomplished chemically (McGuire and Shasha, J Econ Entomol 85:1425-1433, 1992) or biologically (Barnes and Cummings, 1986; EP-A 0192319). Chemical encapsulation involves a process in which the active ingredient is coated with a polymer while biological encapsulation involves the expression of the δ-endotoxin genes in a microbe. For biological encapsulation, the intact microbe containing the toxin protein is used as the active ingredient in the formulation. The addition of UV protectants might effectively reduce irradiation damage. Inactivation due to heat could also be controlled by including an appropriate additive.

Preferred within the present application are formulations comprising living microorganisms as an active ingredient either in form of the vegetative cell or more preferable in form of spores, if available. Suitable formulations may consist, for example, of polymer gels which are crosslinked with polyvalent cations and comprise these microorganisms. This is described, for example, by D.R. Fravel et al. in Phytopathology, Vol.75, No.7, 774-777, 1985 for alginate as the polymer material. It is also known from this publication that carrier materials can be co-used. These formulations are as a rule prepared by mixing solutions of naturally occurring or synthetic gel-forming polymers, for example alginates, and aqueous salt solutions of polyvalent metal ions such that individual droplets form, it being possible for the microorganisms to be suspended in one of the two or in both reaction solutions. Gel formation starts with the mixing in drop form. Subsequent drying of these gel particles is possible. This process is called ionotropic gelling. Depending on the degree of drying, compact and hard particles of polymers which are structurally crosslinked via polyvalent cations and comprise the microorganisms and a carrier present predominantly uniformly distributed are formed. The size of the particles can be up to 5 mm.

Compositions based on partly crosslinked polysaccharides which, in addition to a microorganism, for example, can also comprise finely divided silicic acid as the carrier material, crosslinking taking place, for example, via Ca⁺⁺ions, are described in EP-A1-0 097571. The compositions have a water activity of not more than 0.3. W.J. Cornick et al. describe in a review article [New Directions in Biological Control: Alternatives for Suppressing Agricultural Pests and Diseases, pages 345-372, Alan R. Liss, Inc. (1990)] various formulation systems, granules with vermicuhte as the carrier and compact alginate beads prepared by the lonotropic gelling process being mentioned. Such compositions are also disclosed by D.R.Fravel in Pesticide Formulations and Application Systems:11th Volume, ASTM STP 1 112 American Society for Testing and Materials, Philadelphia, 1992, pages 173 to 179 and can be used to formulate the recombinant microorganisms according to the invention. Further methods for formulating living microorganism are described in WO96/02638.

The compositions according to the invention are valuable for preventive and/or curative treatment in the field of pest control even at low rates of application while being well tolerated by and non-toxic to warm-blooded species, fish and plants and have a very favourable biocidal spectrum. The compositions according to the invention are active against all or individual development stages of Ostrinia furnacalis (Asian Corn Borer) pests. The insecticidal action of the compounds according to the invention can become obvious either directly, i.e. by destroying the pests immediately or only after some time has elapsed.

The said composition can be provided in form of a chemical mixture comprising the toxin proteins in an essentially pure form or in form of a mixture comprising at least one of the toxin proteins as part of a microorganism or a transgenic plant.

In a specific embodiment of the invention, one of the active ingredients may be applied to the plant directly by, for example, leaf application as described herein previously, whereas the second active principle may be provided by the plant itself upon expression of a previously transformed gene encoding the said second principle.

The entomocidal compositions to be used in the method according to the invention usually contain from about 0.1 to about 99%, preferably from about 01 to about 95%, and most preferably from about 3 to about 90% of active ingredient; from about 1 to about 99.9%, preferably from about 1 to about 99%, and most preferably from about 5 to about 95% of a solid or liquid adjuvant, and from about 0 to about 25%, preferably about 0.1 to about 25%, and most preferably from about 0.1 to about 20% of a surfactant .

Whereas commercial products are preferably formulated as concentrates, the end user will normally employ dilute formulations of substantially lower concentration . The entomocidal compositions may also contain further ingredients, such as stabilizers, antifoams, viscosity regulators, binders, tackifiers as well as fertilizers or other active ingredients in order to obtain special effects.

The present invention also relates to formulations comprising living microorganisms as an active ingredient which are present in the form of vegetative cells or more in the form of spores, if available.

A further object of the invention relates to the use of recombinant microorganisms comprising a toxin gene encoding a toxin protein of Bacillus thuringiensis such as a Cryl-type protein, in a method of controlling crop plants against damages caused by Ostrinia furnacalis (Asian Corn Borer) species, which recombinant organisms are either applied directly to the plant to be protected or the recombinantly produced toxin protein is first isolated from the recombinant microorganism and formulated as described above before being applied to the crop plant to be protected. The recombinant microorganisms may also contain a toxin gene encoding a VIP-type toxin protein as disclosed in the EP-A-690 916 and the International Application No EP95/03826 or a combination of genes encoding at least a Cry-type toxin and a VIP-type toxin, respectively.

For recombinant production of the toxin protein in a host organism, the coding sequence may be inserted into an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced. The choice of specific regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated sequences, and enhancer appropriate for the chosen host is within the level of skill of the practioneer in the art. The resultant molecule, containing the individual elements linked in the proper reading frame, are inserted into a vector capable of being transformed into the host cell. Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms such as E. coli (see, e.g. Studier and Moffatt, J. Mol. Biol.189:113 (1986); Brosius, DNA 8:759 (1989)), yeast (see, e.g., Schneider and Guarente, Meth. Enzymol.194:373 (1991)) and insect cells (see, e.g., Luckow and Summers, Bio/Technol.6:47 (1988)). Specific examples include plasmids such as pBluescnpt (Stratagene, La Jolla, CA), pFLAG (International Biotechnologies, Inc., New Haven, CT), pTrcHis (Invitrogen, La Jolla, CA), and baculovirus expression vectors, e.g., those derived from the genome of Autographica californica nuclear polyhedrosis virus (AcMNPV). A preferred baculovirus/insect system is pVI11392/Sf21 cells (Invitrogen, La Jolla, CA).

The recombinantly produced toxin protein can be isolated and purified using a variety of standard techniques. The actual techniques which may be used will vary depending upon the host organism used, whether the toxin protein is designed for secretion, and other such factors a skilled artisan is aware of (see, e.g. chapter 16 of Ausubel, F. et al., "Current Protocols in Molecular Biology", pub. by John Wiley & Sons, Inc. (1994).

A preferred object of the invention relates to the use of transgenic plants comprising and expressing a toxin gene encoding a toxin protein of Bacillus thuringiensis, especially a Cryl-type toxin protein, in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species, in a method of protecting crop plants against damages caused by Ostrinia furnacalis (Asian Corn Borer) pests. The plants can be the result of nuclear transformation or plastid transformation (see WO 95/24492). Especially preferred are transgenic plants expressing a CrylA(b) toxin protein of Bacillus thuringiensis. The invention also relates to the use of transgenic plants comprising a toxin gene encoding a VIP-type protein as described in EP-A-690916 and International Application No EP95/03826, herein incorporated by reference in its entirety. The invention also relates to the use of transgenic plants comprising and expressing a toxin gene encoding a toxin protein of Bacillus thuringiensis, but especially a Cry-type toxin protein, and also comprising and expressing a toxin gene encoding a VIP-type protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species. A host plant expressing said toxin genes will have enhanced resistance to insect attack of Ostrinia furnacalis (Asian Corn Borer) species and will be better equipped to withstand crop losses associated with such attack.

In one preferred embodiment, expression of one or more Bt δ-endotoxins in a transgenic plant is accompanied by the expression of one or more VIP-type proteins. This co-expression of more than one insecticidal principle in the same transgenic plant can be achieved by genetically engineering a plant to contain and express all the genes necessary. Alternatively, a plant, Parent 1 , can be genetically engineered for the expression of VIP-type proteins. A second plant, Parent 2, can be genetically engineered for the expression of Bt δ-endotoxin. By crossing Parent 1 with Parent 2, progeny plants are obtained which express all the genes introduced into Parents 1 and 2. Particularly preferred Bt δ- endotoxins are those disclosed in EP-A 0618976, herein incorporated by reference.

Also comprised by the present invention is the use of recombinant microorganisms or transgenic plants comprising a gene encoding DNA molecules which hybridizes to a DNA molecule encoding a toxin protein of Bacillus species, but preferably to an oligonucleotide probe obtainable from said DNA molecule comprising a contiguous portion of the coding sequence for the said toxin protein at least 10 nucleotides in length, under moderately stringent conditions. The invention preferably comprises the use of recombinant microorganisms or transgenic plants comprising a gene encoding DNA molecules which hybridizes to a DNA molecule encoding a toxin protein of Bacillus thuringiensis or B cereus especially to a DNA molecule encoding a Cry-type protein or to a toxin gene encoding a VIP-type toxin protein, preferably to a CrylA(b) protein.

Factors that effect the stability of hybrids determine the stringency of the hybridization. One such factor is the melting temperature T_m which can be easily calculated according to the formula provided in DNA PROBES, George H. Keller and Mark M. Manak, Macmillan Publishers Ltd, 1993, Section one: Molecular Hybridization Technology; page 8 ff.

The preferred hybridization temperature is in the range of about 25°C below the calculated melting temperature T_m and preferably in the range of about 12-15°C below the calculated melting temperature T_m and in the case of oligonucleotides in the range of about 5-10°C below the melting temperature T_m.

The invention further relates to a commercial bag comprising seed of a transgenic plant comprising at least a toxin gene encoding a toxin protein of Bacillus thuringiensis, preferably a Cry-type toxin protein, more preferably a Cryl-type toxin protein, but most preferably a CrylA-type toxin protein and expressing the said toxin protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species, together with lable instructions for the use thereof for control of Ostrinia furnacalis (Asian Corn Borer) pests in crop plants. Preferred within this invention is a commercial bag comprising seed of a transgenic plant comprising as an active ingredient a gene encoding at least a Cry-type toxin protein and a VIP-type protein. Especially preferred is a combination of a CrylA(b) toxin protein with a VIP-type protein.

The further object of the invention is a commercial bag comprising an insecticidal composition according to the invention together with lable instructions for the use thereof for control of Ostrinia furnacalis (Asian Corn Borer) pests in crop plants.

By plant is meant any plant species which can be genetically transformed by methods known in the art, but especially those plants that are host plants for Ostrinia furnacalis (Asian Corn Borer) species including, but not limited to, the following species of plants: maize, wheat, barley, rye, oats, rice, sorghum, millet and related crops, forage grasses, bamboo (orchardgrass, fescue, and the like), and sugar cane.

Methods known in the art for plant transformation are discussed below. Host plants include, but are not limited to, those species previously listed as target crops.

The invention further relates to seed of a transgenic plant comprising a gene encoding a toxin protein of Bacillus thuringiensis and expressing said toxin protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species, and a commercial bag containing said seed.

By plant is meant any plant species that is a host for Ostrinia furnacalis (Asian Corn Borer) including, but not limited to, the species of maize, wheat, barley, rye, oats, rice, sorghum, millet and related crops, forage grasses, bamboo and sugar cane.

It has been discovered that the codon usage of a native Bacillus thuringiensis toxin gene is significantly different from that which is typical of a plant gene. In particular, the codon usage of a native Bacillus thuringiensis gene is very different from that of a maize gene. As a result, the mRNA from this gene may not be efficiently utilized. Codon usage might influence the expression of genes at the level of translation or transcription or mRNA processing. To optimize a toxin gene for expression in plants, for example in maize, the codon usage is optimized by using the codons which are most preferred in maize (maize preferred codons) in the synthesis of a synthetic gene which encodes the same protein as found for the native toxin gene sequence. The optimized maize preferred codon usage is effective for expression of high levels of the Bt insecticidal protein Further details for constructing maize-optimized synthetic toxin genes can be found in WO 93/07278, herein incorporated by reference in its entirety.

Toxin genes derived from microorganisms may also differ from plant genes. Plant genes differ from genes found in microorganisms in that their transcribed RNA does not possess defined nbosome binding site sequence adjacent to the initiating methionine Consequently, microbial genes can be enhanced by the inclusion of a eukaryotic consensus translation initiator at the ATG. Clontech (1993/1994 catalog, page 210) has suggested the sequence GTCGACCATGGTC as a consensus translation initiator for the expression of the E. coli uidA gene in plants. Further, Joshi (Nucl Acids Res 15: 6643- 6653 (1987)) has compared many plant sequences adjacent to the ATG and suggests the consensus TAAACAATGGCT. In situations where difficulties are encountered in the expression of microbial ORFs in plants, inclusion of one of these sequences at the initiating ATG may improve translation. In such cases the last three nucleotides of the consensus may not be appropriate for inclusion in the modified sequence due to their modification of the second amino acid residue. Preferred sequences adjacent to the initiating methionine may differ between different plant species. By surveying the sequence of maize genes present in the GenBank/EMBL database it can be discerned which nucleotides adjacent to the ATG should be modified to enhance translation of the toxin gene introduced into maize.

In addition, it has been shown that removal of illegitimate splice sites can enhance expression and stability of introduced genes. Genes cloned from non-plant sources and not optimized for expression in plants may contain motifs which can be recognized in plants as 5' or 3' splice sites. Consequently, the transcription process can be prematurely terminated, generating truncated or deleted mRNA. The toxin genes can be engineered to remove these illegitimate splice sites using techniques well known in the art.

Many δ-endotoxin proteins from Bacillus thuringiensis are expressed as protoxins. These protoxins are solubilized in the alkaline environment of the insect gut and are then proteolytically converted by proteases into a toxic core fragment (Höfte and Whiteley, Microbiol Rev.53:242-255 (1989)). For δ-endotoxin proteins of the Cryl class, the toxic core fragment is localized in the N-terminal half of the protoxin. It is within the scope of the present invention that genes encoding either the full-length protoxin form or the truncated toxic core fragment of the novel toxin protein can be used in plant transformation vectors to confer insecticidal properties upon the host plant.

The recombinant DNA molecules can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant, i.e. monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (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., Biotechnology 6:915-921 (1988)), direct gene transfer (Paszkowski et al., EMBO J. 3:2717-2722 (1984)), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wisconsin and Dupont, Inc., Wilmington, Delaware (see, for example, Sanford et al., U.S. Patent 4,945,050; and McCabe et al., Biotechnology 6:923-926 (1988)). See also, Weissinger et al., Annual Rev. Genet.22:421-477 (1988); Sanford et al., Paniculate Science and Technology 5:27-3791987)(onιon); Christou et al., Plant Physiol.87:671-674 (1988)(soybean); McCabe et al., Bio/Technology 6:923-926 (1988)(soybean); Datta et al., Bio/Technology 8:736-740 (1990)(rιce); Klein et al., Proc. 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)(maιze); Svab et al. Proc. Natl. Acad. Sci. USA 87:8526-8530 (1990) (tobacco chloroplast); Koziel et al. (Biotechnology 11:194-200 (1993)) (maize); Shimamoto et al. Nature 338:274-277 (1989) (rice); Christou etal. Biotechnology 9: 957-962 (1991) (rice); European Patent Application EP 0 332 581 (orchardgrass and other Pooideae); Vasil et al. (Biotechnology 11: 1553-1558 (1993) (wheat); Weeks etal. (Plant Physiol.102:1077- 1084 (1993) (wheat); Wan etal (Plant Physiol 104:37-48 (1994) (barley); Umbeck et al, (Bio/Technology 5:263-266 (1987) (cotton).

One particularly preferred set of embodiments for the introduction of recombinant DNA molecules into maize by microprojectile bombardment can be found in WO 93/07278, herein incorporated by reference in its entirety. An additional preferred embodiment is the protoplast transformation method for maize as disclosed in Application EP-A-292435, hereby incorporated by reference in its entirety.

The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants. Generally said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be applied. As the growing crop is vulnerable to attack and damages caused by insects or infections as well as to competition by weed plants, measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield. These include mechanical measures such a tillage of the soil or removal of weeds and infected plants, as well as the application of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulants, ripening agents and insecticides.

Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering. The various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants. Depending on the desired properties different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines which for example increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow to dispense with said methods due to their modified genetic properties. Alternatively new crops with improved stress tolerance can be obtained which, due to their optimized genetic "equipment", yield harvested product of better quality than products which were not able to tolerate comparable adverse developmental conditions.

In seeds oroduction germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harcested and sold by the farmer is not important. As it is difficult to keep a crop free from other crop and weed seeds, to control seedborne diseases, and to produce seed with good germination, fairly extensive and well-defined seed production practices have been developed by seed producers, who are experienced in the art of growing, conditioning and marketing of pure seed. Thus, it is common practice for the farmer to buy certified seed meeting specific quality standards instead of using seed harvested from his own crop. Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures thereof. Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD^®), methalaxyl (Apron^®), and pirimiphos-methyl (Actellic^®). If desired these compounds are formulated together with further carriers, surfactants or application- promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests. The protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit. It is a further aspect of the present invention to provide new agricultural methods such as the methods examplified above which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seed according to the present invention to provide control against Ostrinia furnacalis (Asian Corn Borer).

To breed progeny from plants transformed according to the method of the present invention, a method such as that which follows may be used: maize plants produced as described in the examples set forth below are grown in pots in a greenhouse or in soil, as is known in the art, and permitted to flower. Pollen is obtained from the mature tassel and used to pollinate the ears of the same plant, sibling plants, or any desirable maize plant. Similarly, the ear developing on the transformed plant may be pollinated by pollen obtained from the same plant, sibling plants, or any desirable maize plant. Transformed progeny obtained by this method may be distinguished from non-transformed progeny by the presence of the introduced gene(s) and/or accompanying DNA (genotype), or the phenotype conferred. The transformed progeny may similarly be selfed or crossed to other plants, as is normally done with any plant carrying a desirable trait. Similarly, tobacco or other transformed plants produced by this method may be selfed or crossed as is known in the art in order to produce progeny with desired characteristics. Similarly, other transgenic organisms produced by a combination of the methods known in the art and this invention may be bred as is known in the art in order to produce progeny with desired characteristics.

EXAMPLES

The following examples further describe materials and methods used to obtain specific embodiments of the present invention. They are offered by way of illustration, and should not be interpreted as limitating the disclosure of the specification.

EXAMPLE 1 : General Methods

DNA manipulations were done using procedures that are routinely practized in the art. These procedures can often be modified and/or substituted without substantively changing the result. Except where other references are identified, the procedures are described in general text books such as Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, second edition, 1989.

EXAMPLE 2: Plant Transformation Vectors

Plant transformation is accomplished using the transformation vectors pCIB 4431 and pCIB 3064 described in WO 93/07278 and Koziel et al (1993) [Biotechnology Vol 11 , 194-200], both disclosures being incorporated herein by reference.

pCIB4431 is a vector designed to transform maize. It contains two chimeric synthetic Bt crylA(b) endotoxin genes expressible in maize the one of them constituting a PEP carboxylase promoter/synthetic-crylA(b) gene, the other one a pollen promoter/synthetic- crylA(b) gene.

pCIB4431 contains the synthetic crylA(b) gene provided in SEQ ID NO: 1 and was deposited on September 21, 1992 with the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, U.S.A. under accession no NRRL B-18998.

pCIB3064 contains a plant expressible bar gene (615 bp), which was originally cloned from Streptomyces hygroscopicus [Thompson etal. (1987) EMBO J 6, 2519-2523]. It encodes a phosphinotricin acetyltransferase (PAT), conferring tolerance to phosphinotricin. The bar gene is under the control of the CaMV 35S promoter and terminator [OW et al (1987) Proc Natl Acad Sci USA 84, 4870-4874] to provide resistance to phosphinotricin. EXAMPLE 3: Production of transgenic maize plants containing the synthetic maize

CrylA(b) gene

The example below utilizes a biolistic device to introduce DNA coated particles into maize cells, from which transformed plants are generated.

3.1 Tissue

Immature maize embryos, approximately 1.5-2.5 mm in length, were excised from an ear of genotype 6N61514-15 days after pollination. The mother plant was grown in the greenhouse. Before excision, the ear was surface sterilized with 20% Clorox for 20 minutes and rinsed 3 times with sterile water. Individual embryos were plated scutellum side up in a 2 cm square area, 36 embryos to a plate, on the callus initiation medium, 2DG4+5 chloramben medium (N6 major salts, B5 minor salts, MS iron, 2% sucrose, with 5 mg/l chloramben, 20 mg/l glucose, and 10 ml G4 additions (Table 1) added after autoclaving.

3.2 Preparation of DNA for delivery

The microcarrier was prepared essentially according to the instructions supplied with the Biolistic device. While vortexing 50 μl 1.0 μm gold microcarrier, 5 μl of pCIB4431 (1.23 μg/μl) [#898] + 2 μl pCIB3064 (0.895 μg/μl) [#456] was added followed by 50 μl 2.5 M CaCl₂, then 20 μl 0.1 M spermidine (free base, TC grade). The resulting mixture was vortexed 3 minutes and microfuged for 10 sec. The supernatant was removed and the microcarriers washed 2 times with 250 μl of 100% EtOH (HPLC grade) by vortexing briefly, centrfiuging and removing the supernatant. The microcarriesr are resuspended in 65 μl 100% EtOH.

3.3 Bombardment

Tissue was bombarded using the PDS-1000He Biolistics device. The tissue was placed on the shelf 8 cm below the stopping screen shelf. The tissue was shot one time with the DNA/gold microcarriersolution, 10 μl dried onto the microcarrier. The stopping screen used was hand punched using 10x10 stainless steel mesh screen. Rupture discs of 1550 psi value were used. After bombardment, the embryos were cultured in the dark at 25°C.

3.4 Callus formation

Embryos were transferred to callus initiation medium with 3 mg/l PPT 1 day after bombardment. Embryos were scored for callus initiation at 2 and 3 weeks after bombardment. Any responses were transferred to callus maintenance medium, 2DG4+

0.52,4-D medium with 3 mg/L PPT. Callus maintenance medium is N6 major salts, B5 minor salts, MS iron, 2% sucrose, with 0.5 mg/l 2,4-D, 20 mg/l glucose, and 10 ml G4 additions added after autoclaving Embryogenic callus was subcultured every 2 weeks to fresh maintenance medium containing 3 mg/L PPT. All callus was incubated in the dark at 25°C.

The Type I callus formation response was 15%. Every embryo which produced callus was cultured as an individual event giving rise to an individual line. 3.5 Regeneration

After 12 weeks on selection, the tissue was removed from callus maintenance medium with PPT and was placed on regeneration medium. Regeneration medium is 0.25MS3S5BA (0.25 mg/l 2,4 D, 5 mg/l BAP, MS salts, 3% sucrose) for 2 weeks followed by subculture to MS3S medium for regeneration of plants. After 4 to 10 weeks, plants were removed and put into GA 7's.

EXAMPLE 4: Analysis of transgenic maize plants

4.1 ELISA Assay

Detection of crylA(b) gene expression in transgenic maize is monitored using Asian corn borer insect bioassays and ELISA analysis for a quantitative determination of the level of crylA(b) protein obtained.

Quantitative determination of crylA(b) insecticidal protein in the leaves of transgenic plants is performed using enzyme-linked immunosorbant assays (ELISA) as disclosed in Clark M

F, Lister R M, Bar-Joseph M: ELISA Techniques. In: Weissbach A, Weissbach H (eds)

Methods in Enzymology 118:742-766, Academic Press, Florida (1986). Immunoaffinity purified polyclonal rabbit and goat antibodies specific for the B. thuringiensis subsp. kurstaki insecticidal protein are used to determine the amount of insecticidal protein per mg soluble protein from crude extracts of leaf samples. The sensitivity of the double sandwich ELISA is

1-5 ng insecticidal protein per mg soluble protein using 50 μg of total protein per ELISA microtiter dish well.

Corn extracts are made by grinding leaf tissue in gauze lined plastic bags using a hand held ball-bearing homogenizer (AGDIA, Elkart IN.) in the presence of extraction buffer (50 mM

Na2Cθ3 pH 9.5, 100 mM NaCl, 0.05% Triton, 0.05% Tween, 1 mM PMSF and 1 μM leupeptin). Protein determination is performed using the Bio-Rad (Richmond, CA) protein assay.

4.2 Asian Corn Borer Assay

One to four 4 cm sections are cut from an extended leaf of a corn plant. Each leaf piece is placed on a moistened filter disc in a 50 x 9 mm petri dish. Five neonate Asian corn borer larvae are placed on each leaf piece (making a total of 5-20 larvae per plant). The petri dishes are incubated at 29.5°C. Leaf feeding damage and mortality data are scored after 24, 48, and 72 hours.

EXAMPLE 5: Ostrinia furnacalis (Asian Corn Borer) Field Testing Assay

Small peat pots containing transgenic seedlings which were first tested for the presence and the expression of the transgene, are transplanted into the field. Non-transgenic inbred lines are planted in the same field over a six week period, to serve as controls and for pollinations.

When plants in the field reach 40 cm of extended leaf height, infestation with laboratory- reared ostrinia furnacalis (Asian Corn Borer) larvae begins on both the transgenic and non- transgenic control plants. About 300 neonate larvae mixed with corn cob grits are

introduced into the whorl of each plant using a Davis inoculator. Infestations continues on a weekly basis for four weeks to stimulate first generation Asian Corn Borer. Starting two weeks after the initial infestation, each plant is rated weekly for four weeks using a 1 to 9 scale (1= no visible leaf injury; 9=most leaf with long lesions, several leaves with broken mid ribe, possibly stunted plants due to Asian Corn Borer feeding). A mean Asian Corn Borer damage rating score is calculated for each transgenic and non-transgenic control plant. As each plant reaches anthesis, 300 larvae/plant are applied weekly for four weeks to stimulate second generation infestation. One hundred of neonate larvae in corn cob grits are introduced into the leaf axil at the primary ear and at the leaf axil one node above and below the primary ear node. Therefore a total of approximatively 2400 larvae are applied to each plant. About 50 days after the initial second generation infestation, stalks from all transplanted and some non-transgenic plants are harvested. The extent of internal second generation infestation tunneling damage in the whole plants is determined.

EXAMPLE 6: Assay of extract from transformed protoplasts for insecticidal activity against Ostrinia furnacalis (Asian Corn Borer)

Western blot analysis is performed using extracts obtained from maize cells which had been transiently transformed with DNA to express the maize optimized gene.

Qualitative insect toxicity testing is carried out using harvested protoplasts. Suspensions are prepared for each replicate tested in the bioassays. A replicate is considered positive if it causes significantly higher mortality than the controls. For example, replicates are tested for their activity against insects in the order Lepidoptera by using the Asian corn borer, Ostrinia furnacalis. One-hundred μl of a protoplast suspension in 0.1% Triton X-100 is pipetted onto the surface of artificial Black cutworm diet, (Bioserv, Inc., Frenchtown, NJ; F9240) in 50 mm x 10 mm snap-cap petri dishes. After air drying 10 neonatal larvae are added to each plate. Mortality is recorded after about 4 days.

EXAMPLE 7: Ostrinia furnacalis (Asian Corn Borer) Plant Dipping Assay

7.1 Bacillus thuringiensis (Bt) crystals

Bacillus thuringiensis (Bt) crystals are prepared for stock suspension with 22 ml of distilled water. The suspension is kept in the refrigerator.

7.2 Parameters recorded

3 days old larvae of Ostrinia furnacalis (Asian Corn Borer) are allowed to feed on maize leaves. Larvae had previously been fed with untreated leaves.120 hours later the number of larvae dead is recorded. The kind of feeding injuries on leaf plants is observed in each case.

7.3 Method for testing

Plants of two homozygous inbred lines of Zea mays susceptible to Asian Corn Borer are used (Lines A and B). Seedling plants aged 9-10 days are dipped in various concentrations of Bt protein suspension and are used in feeding experiments, wherein larvae are released on dried leaves of seedling plants, 5-10 larvae per plant. The seedling plants are covered with nylon mesh bags and kept in a nylon mesh case.4-5 concentrations with 4 replications are tested and mortality is determined. The temperature is kept at 21-30°C. 7.4 Results

Two kinds of injuries were clearly distinguished in maize leaves: Bt dipped leaves of the seedling plants were lightly damaged, whereas the leaves of control seedlings were severely damaged.

The following LC₅₀-Values were obtained:

Line A: LC₅₀ = 23.412 ppm (range from 17.834 to 30.734)

Line B: LC₅₀ = 12.234 ppm (range from 9.547 to 15.676) EXAMPLE 8: Ostrinia furnacalis (Asian Corn Borer) Plant Dipping Assay (VIP3A)

8.1 VIP3A protein

5mg of VIP3A protein were prepared with 50ml of distilled water in order to prepare varying concentration of VIP3 protein: 100ppm, 50ppm, 25ppm, 12.5ppm, 6.25ppm and Oppm (check).

8.2 Ostrinia furnacalis (Asian Corn Borer)

The pupae collected from farmers field at Racha Buri by Entomology and Animal Science Division DOA is order to prepare Larvae (L2) for testing.

8.3 Parameters recorded

Data were collected after incubation 5 days by counting number of died larvae and then analyzed percentage of mortality of larvae by probit Analysis Program.

7.3 Method for testing

Plants of two homozygous inbred lines of Zea mays susceptible to Asian Corn Borer are used (Lines B and C). Line B was conducted for potted plant test (4 replications 5 rated concentation and check) and Line C was conducted for leaf dipping test (4 replications with 100, 50, 25ppm and check). Seedling plants aged 10-14 days are dipped in various concentrations of VIP3A protein suspension and are used in feeding experiments, wherein larvae are released on dried leaves of seedling plants, 5-10 larvae per plant. The seedling plants are covered with nylon mesh bags and kept in a nylon mesh case. Cut leaves were put in platic blocks and kept in control room four replications were applied for this experiment.

The following LC₅₀-Values were obtained after 120 hours Line B: LC₅₀ = 29.558 ppm (range from 21.298 to 41.022) Line C: LC₅₀ = 78.498 ppm (range from 53.644 to 114.866)

DEPOSITS

with the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, U.S.A.

Claims

What is claimed is:

1. A method for protecting plants including progeny thereof against damage caused by Ostrinia furnacalis species comprising directly or indirectly applying to the plant or the plant seed or the growing area of the plant as an active ingredient a toxin protein of Bacillus species.

2. A method according to claim 1 , wherein the toxin protein is a toxin protein of Bacillus thuringiensis.

3. A method according to claim 1 , wherein the toxin protein is a toxin protein of Bacillus cereus.

4. A method according to claim 1 , wherein the toxin protein is a Cry-type protein, preferably a Cryl-type protein, more preferably a CrylA-type protein, and most preferably a CrylA(b) protein.

5. A method according to claim 4, wherein the toxin protein is a Cryl-type protein according to SEQ ID Nos. 53 to 55.

6. A method according to claim 1 , wherein the toxin protein is a VIP-type protein, preferably a VIP1-type protein, such as a VIP1A(a) protein or a VIP1 A(b) protein, or a VIP2- type protein, such as a VIP2A(a) protein or a VIP2A(b) protein or a combination of a VlP1 - type protein and a VIP2-type protein.

7. A method according to claim 1 , wherein the toxin protein is a VIP3-type protein, such as a VIP3A(a) protein or a VIP3A(b) protein.

8. A method according to anyone of claims 6 to 7, wherein the toxin protein is a VIP-type protein according to SEQ ID Nos. 1 , 2, 4-7, 17-24, 26-32, 35, 36, 39, 40, 42, 43, 45, 46, 49, 50, 51 or 52.

9. The method according to claim 1 , wherein the toxin protein is applied to the plant in form of an entomocidal composition.

10. The method according to claim 9, wherein the entomocidal composition comprises a microorganism.

11. The method according to claim 1 or 9, wherein the toxin protein is a Cryl-type protein.

12. The method according to claim 9, wherein the entomocidal composition comprises at least one Cryl-type toxin protein or a microorganism, preferably a Bacillus thuringiensis and/or a Bacillus cereus strain, containing at least one gene encoding said toxin protein together with a suitable carrier.

13. The method according to claim 12, wherein the microorganism is a naturally-occurring organism containing at least one cryl-type toxin gene encoding said toxin protein.

14. A method according to claim 10, wherein the microorganism is a naturally-occurring organism containing at least one cry-type toxin gene, preferably a cryl-type toxin gene, more preferably a crylA-type toxin gene, and most preferably a crylA(b) toxin gene.

15. The method according to claim 12, wherein the microorganism is a recombinant organism containing at least one cryl-type toxin gene in recombinant form encoding said toxin protein.

16. A method according to claim 10, wherein the microorganism is a recombinant organism containing at least one cry-type toxin gene, preferably a cryl-type toxin gene, more preferably a crylA-type toxin gene, and most preferably a crylA(b) toxin gene.

17. The method according to claim 1 0, wherein the entomocidal composition comprises at least one VIP-type toxin protein or a microorganism, preferably a Bacillus thuringiensis and/or a Bacillus cereus strain, containing at least one gene encoding said toxin protein together with a suitable carrier.

18. A method according to claim 10, wherein the microorganism is a naturally-occurring organism containing at least one VIP-type protein gene encoding the said toxin protein, preferably a VIP1A(a) protein or a VIP1A(b) protein gene or a VIP2-type protein gene such as as a VIP2A(a) protein or a VIP2A(b) protein gene.

19. A method according to claim 10, wherein the microorganism is a naturally-occurring organism containing at least one VIP3-type protein gene such as as a VIP3A(a) protein or a VIP3A(b) protein gene encoding the said toxin protein.

20. A method according to anyone of claims 17-19, wherein the toxin protein is a VIP-type protein according to SEQ ID Nos.1 , 2, 4-7, 17-24, 26-32, 35, 36, 39, 40, 42, 43, 45, 46, 49, 50, 51 or 52.

21. A method according to anyone of claims 11-16, wherein the toxin protein is a Cry-type protein according to SEQ ID Nos. 53 to 55.

22. Anyone of claims 17 to 21 , wherein the microorganism is a recombinant organism.

23. The method according to claim 1 to 22, wherein the toxin protein is indirectly applied to the plant, by transforming said plant with a toxin gene encoding a toxin protein of Bacillus species and expressing said toxin protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species upon planting the so transformed plant in an area where said insect pest may occur.

24. A method according to claim 23, wherein the toxin gene encodes a toxin protein of Bacillus thuringiensis or Bacillus cereus.

25. A method according to claim 23, wherein the toxin gene encodes a Cry-type toxin protein, preferably a Cryl-type protein, more preferably a CrylA-type protein, and most preferably a CrylA(b) protein such as those provided in SEQ ID Nos. 53 to 55.

26. A method according to claim 23, wherein the toxin gene encodes a VIP-type toxin protein, preferably a VIP1 -type protein, such as a VIP1A(a) protein or a VIP1A(b) protein or a VIP2-type protein, such as a VIP2A(a) protein or a VIP2A(b) protein.

27. A method according to claim 23, wherein the toxin gene encodes a VIP3-type protein, such as as a VIP3A(a) protein or a VIP3A(b) protein.

28. The method according to claim 23-27, wherein the toxin gene is a synthetic gene the codon usage of which is optimized by using the codons which are most preferred in plants.

29. The method according to claims 23 and 28, wherein the toxin gene encodes a Cryl- type protein.

30. A method according to any one of claims 26 to 27, wherein the toxin gene encodes a VIP-type protein according to SEQ ID Nos.1 , 2, 4-7, 17-24, 26-32, 35, 36, 39, 40, 42, 43, 45, 46, 49, 50, 51 or 52.

31. The method according to any one of claims 1 to 30, wherein the plant to be protected is a cereal plant.

32. The method according to claim 31 , wherein the plant to be protected is a maize plant.

33. A method according to any one of claims 1 to 32, wherein at least a Cry-tpye toxin protein is directly or indirectly applied to the plant or the plant seed or the growing area of the plant to be protected in combination with a VIP-type toxin protein in an amount sufficient to provide control against Asian Corn Borer (Ostrinia furnacalis) pests.

34. The method according to any one of claims 1 to 32, wherein the active ingredient is a CrylA(b) protein.

35. The method according to any one of claims 1 to 32, wherein the active ingredient is a VIP-type protein.

36. Use of an active ingredient as defined in the previous claims for controlling Ostrinia furnacalis (Asian Corn Borer) pests in crop plants.

37. Use of a transgenic plant as defined in claim 23 for controlling Ostrinia furnacalis (Asian Corn Borer) pests in crop plants.

38. Use of recombinant microorganisms or transgenic plants according to anyone of claims 23 to 30 comprising a DNA molecule which hybridizes to a cry-type or a VIP-type gene encoding the respective toxin protein under moderate stringent conditions for controlling Ostrinia furnacalis (Asian Corn Borer) pests in crop plants.

39. A commercial bag comprising seed of a transgenic plant comprising a toxin gene encoding a toxin protein of Bacillus thuringiensis and expressing said toxin protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species, together with lable instructions for the use thereof for control of Ostrinia furnacalis (Asian Corn Borer) pests in crop plants.

40. A commercial bag comprising seed of a transgenic plant or a microorganism, but especially a Bacillus thuringiensis and/or a Bacillus cereus strain, comprising at least a toxin gene encoding a toxin protein as defined previously in claims 1 to 30, 32 and 33, and expressing the said toxin protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species, together with lable instructions for the use thereof for control of Ostrinia furnacalis (Asian Corn Borer) pests in crop plants.

41. An agricultural method, wherein a transgenic plant or the progeny thereof is used comprising a toxin gene encoding a toxin protein of a Bacillus species and expressing the said toxin protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species or transgenic seed thereof, as defined in anyone of claims 23 to 32.

42. A method accroding to any one of claims 18 to 26, wherein at least a Cry-tpye toxin protein is expressed in the plant to be protected in combination with a VIP-type toxin protein in an amount sufficient to provide control against Sesamia pests.

43. A composition comprising as an active ingredient at least a Cry-type toxin protein and a VIP-type protein in a insecticidally effective amount together with a agronomically acceptable carrier.