MXPA99008362A - Pesticidal bacillus thuringiensis strains - Google Patents

Abstract

This application pertains to Bacillus thuringiensis isolates and their toxins active against various lepidopteran, coleopteran or aphid pests. The isolates are PS17, PS28Q2, PS33F2, PS32B, PS54G2, PS50C, PS71M3, PS86BB1, PS86Q3, PS14OE2, PS169E, PS62B, PS158C2, PS167P, PS196S1, PS201T6, HD511, PS18, PS28K1, PS43A2, PS19E6, PS164H2, PS186EE, PS196Q3, PS198A2, PS225K1, KB6, KB19, HD977 and HD541.

Description

DESCRIPTION PESTICIDE MATERIALS AND METHODS BACKGROUND OF THE INVENTION The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized by crystalline protein inclusions for spores. These inclusions often appear microscopically as clearly configured crystals. Proteins can be highly toxic to pests and specific in their toxic activity. Some B.t. toxin genes they have been isolated and sequenced, and B.t. products have been produced. based on recombinant DNA and have been approved for use. In addition, through the use of genetic engineering techniques, new embodiments are being developed to provide these B.t. endotoxins. to agricultural environments, including the use of plants genetically engineered with endotoxin genes to provide resistance to insects, and the use of intact microbial cells stabilized as delivery vehicles for B.t. endotoxin. (Gaertner, F.H.L. Kim [1988] TIBTECH 6: 84-87). Therefore, the endotoxin genes asylated B.t. they are increasingly commercially valuable. Until the last fifteen years, the commercial use of pesticides B.t. it has been very restricted to a narrow range of lepidopteran pests (caterpillars). Preparations of crystals and spores of B. Thuringiensis subsp. Kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. Thuringiensis var. Kurstaki! D-1 produces a crystalline d-endotoxin that is toxic to larvae of a variety of lepidopteran insects. In recent years, however, researchers have discovered pesticides B.t. with specificities for a much wider range of pests. For example, other species of B.t. that is, Israelensis and morrisani (a, ka tenebrionis, aka BtM-7, aka Bt san diego) have been used commercially to control insects of the order of Diptera and Coleoptera, respectively (Gaertner, FH [1989] "Cellular Delivery Systems for Insecticidal Proteins : Living and Non-Living Microorganism "in Controlled Delivery of Crop Protection Agents, RM Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255). See also Couch, T.l. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. Israelensis", Developments in Industrial Microbiology 22:61 -76; and Beegle, C.C. (1978) "Use of Entomogenous Bacteria in Agroecosyhstems", Developments in Industrial Microbiology 20: 97-104, Krieg, A., A-M.Huger, G.A. Langenbruch, W.Schnetter (1983) Z, ang. Ent. 96-500-508 describe Bacillus thuringiensis var. Tenebrionis, which is active against two beetles of the order of the Coleoptera. These are Colorado potato beetle, Leptinotarsa decemlineata, Agelastica alni. More recently, new subspecies of B.t. and the genes responsible for the active d-endotoxin proteins have also been isolated. (Hofte, H., H.R. Whiteley [1989] Microbiological Reviews 52 (2): 242-255). Hofte and Whiteley classified the B.t. crystal protein genes. in four main classes. The classes were Cryl (Specific to the Lepidoptera), Cryl I (Specimens of Lepidoptera and Diptera), Cryl 11 (Species of the Coleoptera), and CrylV (Specific to Diptera). The discovery of strains that are specifically toxic to other pests has also been mentioned. (Feitelson, J.S., J. Payne, L.Kim [1992] Bio / Technology 10: 271-275). CryV has been proposed to designate a class of toxin genes that are specific for nematodes. Lambert et al, Lambert, B., L., Buysse, C. Decock, S. Jansens, C. Piens, B. Sacy, J. Seurinck, K. Van Audenhove, J. Van Rie, A. Van Vliet, M. Peferoen [1996] Appl. Environ. Microbial 62 (1): 80-86) describe the characterization of a Cry9 toxin that is active against Lepidoptera. Published PCT applications WO 94/05771 and WO 94/24264 also describe isolates of B.t. active against lepidopteran pests. Gleave et al ([1991] JGM 138: 55-62), Sheveley et al, ([1993] FEBS Lett 336: 79-82; and Smulevith et al ([1991] FEBS Lett 293-25-26) also describes toxins of B.t. Many other classes of B.t. genes have now been identified. The cloning and expression of the crystal protein gene B.t. in Escherichia coli has been described in the published literature (Schnepf, H.E., II.R. Whiteley [1981] Proc. Nath. Acad. Sci. USA 78: 2893-2897.). U.S. Patent Nos. 4,448,885 and 4,467,036 describe the expression of the crystal protein B.t. in E. coli. U.S. Patents 4,990,332; 5,039,523; 5,126,133; 5,164,180; and 5,169,629 are among those which describe the toxins of B.t. that have activity against lepidoptera. The application WO96 / 05314 describes PS86W1, PS86W1 and other isolates of B.t. which are active against lepidopteran pests. The application WO96 / 05314 describes PS86W1, PS86V1, and other isolates of B.t. which are active against lepidopteran pests. PCT patent applications published as WO94 / 24264 and WO94 / 05771 describe isolates of B.t. and toxins that are active against lepidopteran pests. The proteins of B.t. with activity against members of the Noctuidae family have been described by Lambert et al, supra. The North American patents 4,797,276 and 4,853,331 describe the strain of β. Thuringiensis tenebrionis that can be used to control coleopteran pests in various environments. U.S. Patent No. 4,918,006 describes toxins B.t. that have activity against diptera. U.S. Patent Nos. 5,151,363 and 4,948,734 describe certain isolates of B.t. that have activity against nematodes. Other North American patents describing activity against nematodes include 5,093,120; 5,236,843; 5,262,399; 5,270,448; 5,281,530; 5,322,932; 5,350,577; 5,426,049 and 5,439,881. U.S. Patent Nos. 5,262,159 and 5,468,636 disclose Bt isolates. PS157C1, PS86A1, and PS75J1, as having activity against aphids. As a result of extensive research and investment of resources, other patents have been granted to provide new B.t. and new uses of the isolates B.t. See Feitelson et al, suipra, for a review of them. However, the discovery of new isolates B.t. and new uses of the isolates B.t. known remains an empirical and unpredictable technique. Patent No. 5,506,099 describes methods for identifying B.t. isolates. unknown. Also, U.S. Patent No. 5,204,237 describes specific and universal probes for the isolation of B.t. toxin genes.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to materials and methods that are useful in the control of pests that are not mammals and, particularly, to pests of plants. In a specific embodiment, the present invention provides novel isolates and toxins that are useful for the control of lepidoptera, coleoptera, and / or aphids. In preferred embodiments these pests are selected from the group consisting of diamond-backed loin moths, southern Bertha worm, tobacco budworm, sunflower stick, sunflower beetle, red sunflower seed weevil, flea beetle, gorse of the sunflower stem, and green bug. The nucleotide sequences that are useful according to the present invention encode pesticidal toxins. An embodiment of the present invention relates to cells of plants transformed with at least one polynucleotide sequence of the present invention so that the cells of the transformed plant express pesticide toxins in tissues consumed by the pests that constitute the target. Said plant transformation can be carried out using techniques that are well known to those skilled in the art and would typically involve modification of the gene to utilize the expression of the toxins in the plants. As described herein, the toxins that are useful in accordance with the present invention can be chimeric toxins produced by the combination of multiple toxin portions. Also, the toxins of the present invention can be used in combination to achieve better control of pests. Alternatively, the isolates of B.t. of the present invention, or the recombinant microbes expressing the toxins described herein, can be used to control pests. In that regard, the invention includes the treatment of B.t. substantially intact and / or recombinant cells coning the expressed toxins of the invention, treated in order to prolong the pesticidal activity when substantially intact cells are applied to the environment where the target pest is. The treated cell acts as a protective coating for the pesticide toxin. The toxin becomes active when it is ingested by the insect that is the target.

DEED DESCRIPTION OF THE INVENTION The present invention provides isolates of B.t. and active toxins against diamond-backed moths, southern Bertha worm, tobacco budworm, sunflower moth, sunflower beetle, red sunflower seed weevil, canola flea beetle, sunflower stem weevil, and chinch green. Other aspects of the present invention relate to new isolates of B.t. and the toxins they ob from these isolates. The new isolates B.t. of the present invention have been designated PS18, PS28K1, PS43A2, PS159B6, PS1641 12, PS186EE, PS196Q3, PS198A2, and PS2225K1. The microorganisms that are useful in accordance with the present invention have been deposited in the permanent collection of Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604.USA.

The deposit numbers of the cultures of the deposited strains are shown in table 1.

TABLE 1 Deposit Cultivation. No. Deposit Date PS18 NRRL B-21954 March 12, 1998 PS28K1 NRRL B-21955 PS43A2 NRRL B-21956 PS159E6 NRRL B-21958 PS164H2 NRRL B-21959 PS186EE NRRL B-21960 PS196Q3 NRRL B-21961 PS198A2 NRRL B-21962 PS225K1 NRRL B-21963 KB6 NRRL B-18873 August 27, 1991 KB19 NRRL B-21964 March 12, 1998 The isolates of the present invention have been deposited under conditions that ensure access to crops will be available as long as this patent application is pending. until the time determined by the patent and trademark commissioner authorized under 37 CFR 1.14 and 35 U: S: C 122. Deposits will be available as required for foreign patent laws in countries where counterparts of the present application are submitted, or his progeny. However, it must be taken into account that the availability of a deposit does not constitute a license to carry out the present invention by repealing patent rights guaranteed by governmental action. In addition, the isolates of the present invention will be stored and made available to the public in accordance with the provisions of the Budapest Treaty for the Deposit of Microorganisms, that is, they will be stored taking all the necessary precautions to keep them viable and without contamination for a period of time. at least five years after the most recent request for the provision of a sample of a deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the entire life in which it is in force any patent that could be granted and describing the crops. The depositor assumes the duty to replace the deposits if said depositor is unable to provide a sample when required, due to the condition of the deposits. All restrictions on the availability to the public of the present crop deposits were irrevocably eliminated upon grant of the patent that describes them. Some isolates that are useful in accordance with the present invention are available to the public by virtue of the granting of North American patents. These isolates, their accession number to the deposit and their deposit date are shown in table 2 (a). The corresponding North American patents describing these isolates are shown in table 2 (b).

TABLE 2 (a) Strains B.t. Pesticides of the present invention Culture Deposit. NRRL Date Deposit PS17 B-18243 10 AG. 87 PS28Q2 B-18888 25 SEP 91 PS32B B-21531 1 APR. 96 PS33F2 B-18244 10 AG. 87 PS50C B-18746 23 EN. 91 PS54G2 B-21543 1 AB. 96 PS62B B-18398 7 SEP. 88 PS71 M3 B-18930 8 EN.02 PS80JJ1 B-18679 1 AG. 90 PS86BB1 B-21557 2 MAY 96 PS86Q3 B-18765 13 FEB 91 PS140E2 B-18812 10 AB. 91 PS158C2 B-18872 17 SEP 91 PS167P B-18681 1 AG. 90 PS169E B-18682 1 AG. 90 PS196S1 B-18748 23 EN 91 PS201T6 B-18750 23 EN. 91 Isolates IID511, HD541 and HD977 are available from USDA-ARS NRRI., Culture Collection, Peorina, Illinois.

Table 2 (b) Patent E.U.A No. Sequences (of Isolated Activity O Toxin (s) and Gene (s) Pesticide Described Pubicac No. Descriptions Descripta 4,849,217 PS17 Alfalfa Weevil . 218,530 PS17 17 (a) and 17 (b) nematodes some genera of Coleoptera inc. ,427,786 PS28Q2 Hypera, Diabrotica, and Phyllotreta 4,849,217 PS33F2 alfalfa weevil ,439,881 PS33F2 33F2 nematodes ,707,619 PS33F2 several weevils ,670,365 PS32B nematodes ,277,905 PS50C Coleoptera ,366,892 PS50C 50C Coleoptera ,185,148 PS50C beetles ,457,179 PS50C Coleoptera ,554,534 PS50C 50C (a) and 50C (b) beetles ,667,993 PS54G2 nematodes ,670,365 PS54G2 nematodes ,723,440 PS71 M3 hemiptera 4,849,217 PS80JJ1 Alfalfa Weevil WO94 / 23036 PS80JJ1 centipede rootworm ,632,987 PS80JJ1 80JJ1 (130kda) corn ,589,382 PS80JJ1 80JJ1 (130kda) nematodes ,670,365 PS80JJ1 80JJ1 (130kda) menates 4,849,217 PS62B alfalfa weevil WO97 / 40162 80JJ1 (14 and 44 kDa) 86BB1 (a), 86BB1 (b), and WO98 / 00546 PS86BB1 86BB1 Lepidoptera (c) 5,596,071 PS85Q3 86Q3 (a) and 86Q3 (c) ants some genera of Coleoptera, nc. ,427,786 PS140F2 Hypera, Diabrotica, and Phyllotreta PS140E2 ants ,268,172 PS158C2 lepidoptera rootworm ,632,987 PS158C2 corn 158C2 (a) m 158C2 (b), 5,723,758 PS158C2 lepidopteran 158C2 (c), and 158C2 (d) ,707,619 PS167P several weevils ,589,382 nematodes PS167P 167P Table 2 (b) US Patent No. Sequences (O Activity of Isolated Toxin (s) and gene (s) Pesticide Described Pubicac. No. 5707.619 described Descripta PS167P Several weevils 5,589,382 PS167P 167P Nematodes Rootworm of the ,632,987 PS169E corn 5,707,619 PS169E Various weevils 5,670,365 PS167E 167P Diptera nematodes and worm of the ,436,002 PS196S1 corn root 5,707,619 PS196S1 Several dipterous weevils and worm of the ,635,480 PS196S1 corn root diptera and worm of the ,436,002 PS201T6 201T6 (30 &25 kda) corn root 5,707,619 PS201T6 Various dipterous weevils and worm of the ,635,480 PS201 T6 201T6 (30 & 25 kda) corn root PS201T6 Hemiptera 5,723,440 5,262,324 5,286,486 HD511 Coleoptera Coleoptera 5,306,494 HD51 1 HD511 HD511 coleopteran These patents, with their description of the indicated isolates as well as their toxins and genes, are incorporated herein by reference. Genes and toxins. Genes and toxins useful according to the present invention include not only the sequences described total length but also fragments of these sequence, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the toxins specifically exemplified herein. As used herein, the terms "purified toxin" and "isolated toxin" refers to toxins which have been affected by the hand of man and are substantially free of naturally associated, such impurities as toxins would be in its state natural. As used herein, the terms "variants" or "variations" of genes refer to nucleotide sequences that encode the same toxins or that encode equivalent toxins that have pesticidal activity. As used herein, the terms "equivalent toxins" refer to toxins that have the same or essentially the same biological activity against the pests as the toxins exemplified. It will be apparent to experts in the field that, the genes that code active toxins can be identified and obtained through various means. The specific genes exemplified herein can be obtained from the isolates deposited in a culture reservoir as described above. These genes, or portions of variants thereof, can also be constructed synthetically, for example, using a gene synthesizer. Variations of genes can be rapidly constructed using conventional techniques to prepare point mutations. Also, fragments of these genes can be prepared using exonucleases or endonucleases commercially available according to conventional procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut nucleotides from the ends of these genes. Also, the genes encoding the active fragments can be obtained using a variety of restriction enzymes. The proteases can be used to directly obtain the active fragments of these toxins. The toxins and / or equivalent genes that code for these equivalent toxins can be derived from the B.t. and / or DNA libraries using the teachings described here. There are a number of methods for obtaining the pesticidal toxins of the present invention. For example, antibodies to the pesticidal toxins described and claimed herein.

They can be used to identify and isolate other toxins from a mixture of proteins. Specifically, antibodies can be created for portions of the toxins that are more constant and more distinct from other B.t. toxins. Fragments and equivalents that retain the activity of the exemplified toxins would be within the scope of the present invention.

Also, due to the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences described here. It is within the skill of a person skilled in the art to create these alternative DNA sequences that encode the same, or essentially, the same toxins. These variant DNA sequences are within the scope of the present invention. As used herein, the reference to "essentially the same" refers to sequences that have substitutions, deletions, additions, or amino acid insertions that do not materially affect the pesticidal activity. Fragments that retain pesticidal activity are also included in this definition. Another method for identifying the toxins and genes of the present invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences may be detectable by virtue of appropriate label or may be inherently fluorescent as described in International Application No. WO93 / 16094.

As is well known in the art, if the probe molecule and nucleic acid sample are hybridized to form a strong bond between the two molecules, it can reasonably be assumed that the probe and sample have substantial homology. Preferably, the hybridization is carried out under stringent conditions by techniques that are well known in the art, such as has been described, for example, in Keller, G.H., M.M. Manak (1987) DNA Probes, Stockton Press. New York, NY, p. 169-170. Detection of the probe provides a means to determine in a known manner whether hybridization occurred. Said probe analysis provides a rapid method to identify genes encoding toxins of genes of the present invention. The nucelotides that are used as probes according to the invention can be synthesized using a DNA synthesizer and conventional methods. These nucleotide sequences can also be used as PCR primers to amplify genes of the present invention. Some toxins of the present invention have been specifically exemplified herein. Because these toxins are merely examples of toxins of the present invention it will be readily apparent that the present invention comprises toxins in equivalent variants (and nucleotide sequences encoding equivalent toxins) that have the same or similar pesticidal activity as the toxin exemplified. The equivalent toxins will have amino acid homology with an exemplified toxin. This amino acid identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, more preferably greater than 90%, and may be greater than 95%. The amino acid homology will be the highest in critical regions of the toxin that counts for the biological activity or that is involved in the determination of the three-dimensional configuration that is ultimately responsible for the biological activity. In this sense, conservative institutions by which an amino acid of one kind is replaced by another amino acid of the same type are within the scope of the present invention provided that said substitution does not materially alter the biological activity of the compound. Table 3 provides a list of examples of amino acids that belong to this class.

TABLE 3 In some cases, non-conservative substitutions may be made. The critical factor is that these substitutions should not deviate significantly from the biological activity of the toxin. The toxins of the present invention can be characterized in terms of configuration and location of the toxin inclusions, which have been described above.

Recombinant guests. The genes encoding toxins hosted by the isolates of the present invention can be introduced into a wide variety of plant or microbial hosts. The expression of the toxin gene results in direct or indirect, intracellular production and maintenance of the pesticide. With appropriate microbial hosts, for example pseudomonas, the microbes can be applied to the place where the pest is, where they will proliferate and will be ingested. The result is the control of the plague. Alternatively, the microbe harboring the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell that retains the toxic activity can then be applied to the environment where the target pest is. There are a variety of ways available to introduce a B.t. which encodes a toxin in a host microorganism under conditions that allow stable maintenance and expression of the gene. These methods are well known to those skilled in the art, and have been described, for example, in U.S. Patent No. 5,135,867 which is incorporated herein by reference. Alternatively, a plant transformed to express a toxin of the present invention can be used to contact the pest with the toxin. Synthetic genes that are functionally equivalent to the toxins of the present invention can also be used to transform hosts. Methods for the production of synthetic genes can be found in for example U.S. Patent No. 5,380,831.

Treatment of cells As mentioned above, the B.t. or recombinants that express a B.t. toxin. they can be treated to prolong the activity of the toxin and stabilize the cell. The pesticide microcapsule that is formed comprises the B.t. toxin. with a cellular structure that has been stabilized and that will protect the toxin when the microcapsule is applied to the environment of the target pest. Methods for the treatment of microbial cells have been described in US Pat. Nos. 695,455 and 4,695,462 which are incorporated herein by reference.

Development of cells The cell host containing the B.t. insecticidal gene. it can be cultured in any convenient nutrient medium where DNA construction provides a selective advantage, providing a selective medium for substantially all or all cells to retain the B.t. these cells can then be harvested according to conventional forms. Alternatively the cells can be treated before being harvested.

The B.t. cells of the research can be grown using conventional means and known fermentation techniques. Upon completion of the fermentation cycle of the bacteria, it can be harvested by first separating the spores and crystals of B.t. of the fermentation broth by means which are well known in the art. Spores B.t. recovered and the crystals can be formulated in a wettable powder, in a liquid concentrate in granules or in other formulations by the addition of surfactants, dispersants, inert carriers, and other components to facilitate handling and application to particular pests that constitute the target. These formulations and methods of application are all well known in the art.

Methods and formulations for the control of pests. Pest control employing isolates, toxins and genes of the present invention can be carried out by a variety of methods known to those skilled in the art. These methods include for example the application of the isolates B. t. to pests (or where they are), the application of recombinant microbes of pests (or where they are), and the transformation of plants with genes encoding the pesticide toxins of the present invention. The recombinant microbes can be for example Bt, E. coli, or Pseudomonas. The transformations can be carried out by those skilled in the art using conventional techniques.

The materials needed for these transformations have been described here or will be available in another way for the expert in the field. The formulated bait granules containing an attractant and spores and crystals of the B.t. or recombinant microbes comprising the genes obtainable from the isolates B.t. here described can be applied to the ground. The formulated product can also be applied to a seed coat or root treatment or to the treatment of the whole plant in later stages of the crop cycle. Treatments to plants and soil of B.t. cells can be used in the form of liquids, wettable powders, granules or powders by mixing with various inert materials such as inorganic minerals (phyllosilicates, carbonates, sulphates, phosphates and the like) or botanical materials (powdered corn husks, rice husks, walnut shells) and similar). Those skilled in the art will appreciate that the pesticidal concentration will vary depending on the nature of the particular formulation, particularly if it is a concentrate or if it should be used directly. The pesticide will be present in at least about 1% by weight and can be up to 100% by weight. The dry formulations will have from about 1-95% by weight of the pesticide while the liquid formulations will generally be from about 1-60% by weight of the solids in the liquid phase. The formulations will generally have from about 102 to about 10 4 cells / mg.

These formulations will be administered approximately 50 mg (in liquid or dry form) up to 1 kg or more per hectare. The formulations can be applied to the environment where the pest is, for example to soil and foliage, by spraying, sprinkling, watering or the like.

Mutants Mutants of the isolates of the invention can be prepared by methods well known in the art. For example, an asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis of an isolate. Mutants can be prepared using the ultraviolet and nitrosoguanidine by procedures well known in the art. All of the North American patents cited herein are incorporated by reference. Following are examples that illustrate the procedures for practicing the invention. These examples should not be considered as limiting. All percentages are given by weight and all proportions of the solvent mixtures are given in volume unless otherwise indicated.

EXAMPLE 1 Culture of isolates of B.t. of the invention A subculture of the B.t. isolates can be used. or mutants thereof to inoculate the following medium, a glucose peptone or a saline medium. Bacto peptone 7.5 g / l Glucose 1.0 g / l KH2P04 3.4 g / l Saline solution 5.0 ml / l CaCl2 solution 5.0 ml / l pH 7.2 Salt solutions (100 ml) MgSO4.7H20 2.46g MnSO4.H2O 0.04 g ZnSO4.7H2O 0.28 g FeSO4.7H2O 0.40 g CaCl2 solution (100 ml) CaCI2.2H2O 3.66 g Saline solutions and CaCl2 solution are sterilized by filtration and added to the broth subjected to autoclaving and cooked at the time of inoculation. The flasks are incubated at 30 ° C on a rotary shaker at 200 rpm for 64 hours. The above process can be rapidly carried out on a larger scale in large fermentors by processes well known to those skilled in the art. The spores and / or crystals of B.t. obtained in the appropriate fermentation, they can be isolated by methods well known in the art. A frequently used method consists in subjecting the harvested fermentation broth to separation techniques such as centrifugation. The supernatant of these cultures can be used to obtain toxins according to the present invention. Therefore, the present invention is not limited to useful crystalline proteins; soluble soluble proteins have also been contemplated.

EXAMPLE 2 Activity against the diamond-backed moth (Plutella xylostella) Certain isolates of B.t. and toxins were shown to be active against the diamond-backed moth. These results are shown in table 4.

TABLE 4 TOXIN HEAT PS86Q3 CrydAc PS17 CrydAa HD511 Cry7Ab2 EXAMPLE 3 Activity against the Southern Sciara (Mamestra configure) Certain isolates of B.t. and toxins proved to be active against Southern Bertha sciara. These results are shown in table 5.

TABLE 5 TOXIN HEAT HD51 1 Cry7Ab PS86Q3 CrydAC EXAMPLE 4 Activity against the tobacco budworm (Heliothis virescens) Some isolates B.t. and toxins proved to be active against the tobacco budworm. These results are shown in table 6.

TABLE 6 TOXIN HEAT PS86Q3 CrydAc PS17 CrydAa EXAMPLE 5 Activity against sunflower moth (Cochylis hospes) Some isolated B.t and toxins were shown to be active against the sunflower moth. These results are shown in table 7.

TABLE 7 TOXIN HEAT PS17 CrydAa EXAMPLE 6 Activity against sunflower beetle (Zyqoqramma exclamationis) Some isolated B-t- and toxins proved to be active against the sunflower beetle. These results are shown in table 8.

TABLE 8 CEPA TOXIN PS80JJ1 80JJ1 PS50C Cry8B PS1 d8C2 1 d8C2 (c) The sequence of a 1 d8C2 toxin (c) of an amino acid 122-7 is disclosed SEQ ID No. 8 in U.S. Pat. No. 5,723,758. A 4 kDa toxin of PS80JJ1 is an example of a preferred toxin.

EXAMPLE 7 Activity against the red sunflower seed weevil (Smicronyx fulvus) The isolates B.t. designated PS28Q2, PS32B, PS54G2, and PS43A2 proved to be active against the red sunflower seed weevil.

EXAMPLE 8 Activity against the Canola Beetle (Phyllotreta cruciferae) The isolate B.t. Designated PS201T6, PS33F2, HD977, PS196Q3, PS225K1, PS164H2, KB19, PS86BB1, PS186EE, KB6 and PS198A2 proved to be active against the beetle of the canola. A toxin c and TIB obtainable from PS201T6 is an example of a preferred toxin for controlling Phyllotreta and a toxin of 130 kDa.

EXAMPLE 9 Activity against green insects (Schizaphis gramimum) The isolates B.t. designated PS159E6, PS167P, PS71 M3, PS201T6, PS196S1, HD541, PS28K1, PS18 and PS50C demonstrated activity against green insects.

EXAMPLE 10 Activity against sunflower stem weevils (Cylindrocopturus adspersus) The isolates B.t. designated PS28Q2, PS43A2, PS62B, PS86B1, PS140E2, PS169E, and PS201T6 proved useful against sunflower stem weevils.

EXAMPLE 10 Insertion of toxin genes in plants One aspect of the present invention is the transformation of plants with genes that code for insecticide toxins. The transformed plants are resistant to the attack of the pests that constitute the target. The genes encoding pesticide toxins, as described herein, can be inserted into plant cells using a variety of techniques that are well known in the art. For example, a large number of cloning vectors comprising a replication system in E. coli and a marker allowing the selection of transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise for example pBR322, pUC series, d M13mp series, pACYC184, etc. Therefore the sequence encoding the B.t. toxin. it can be inserted into the vector at an appropriate restriction site. The resulting plasmid is used for transformation in E. coli. The E. coli cells are grown in an appropriate nutrient medium and then harvested and used. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis and other biological, biochemical and molecular methods are generally carried out as methods of analysis. After each DNA manipulation used it can be dissociated and linked to the next DNA sequence. Each plasmid sequence can be cloned in the same or in other plasmids. Depending on the method of insertion of the desired genes into the plant, other sequences may be necessary.

DNA If for example the Ti or Ri plasmid is used for the transformation of the plant cells then at least the right, but often the left border of the Ti or Ri plasmid of T-DNA must be bound to the flanking region of the cells. genes that should be inserted. The use of T-DNA for the transformation of plant cells has been intensively investigated and has been sufficiently described in European patent 120.d16; Hoekema (198d) ln: The Binary Plant Vector System, Offsetdurkkerij Kanters B.V. Alblasserdam, Chapter 5; Fralcy et al., Crit. Rev. Plant Sci. 4: 1-46; and An and others. (1985) EMBO J. 4: 277-287. Once the inserted DNA has been integrated into the genome, it is relatively stable and generally does not come out again. It usually contains a selective marker that gives the cells of transformed plants resistance to a biocide or an antibiotic, such as cenamycin. G 418, bleomycin, hygromycin, or chloromfenicol among others. The marker used individually should allow according to this selection of transformed cells instead of cells that do not contain the inserted DNA. A large number of techniques for inserting DNA into a host plant cell are available. Such techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (bombardment of microparticles), or electroporation, as well as other possible methods. If agrobacteria is used for the transformation, the DNA that must be inserted must be cloned in special plasmids, that is to say in an intermediate vector or in a binary vector. Intermediate vectors can be integrated into the Ti or Ri plasmid by d homologous recombination due to sequences that are homologous to the sequences in the T-DNA. The Ti or Ri plasmid also comprises the Ti region or Ri necessary for the transfer of T-DNA. The intermediate vectors can not replicate themselves in Agrobacteria. The intermediate vector can be transferred to Agrobacterium tumefaciens by an auxiliary plasmid 0 (conjugation). Binary vectors can replicate themselves in both E. coli and Agrobacteria. They comprise a selection marker gene and a linker or polylinker that are conformed by the left and right border regions of T-DNA. They can be directly transformed into Agrobacteria (Holsters et al. [1978] Mol. Gen. Genet 163: 181-187). The Agrobacterium used as the host cell must comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA to the cell of the plant. Additionally it may contain T-DNA. The bacterium transformed in this way is used for the transformation of the cells of 0 plants. Plant explants can be advantageously grown with Agrobacterium tumefaciens or Agrobacterium thizones for the transfer of DNA to plant cells. Then, whole plants can be regenerated from the infected plant material (eg, leaf pieces, stem segments, roots, but also protoplasts or cells grown in suspension) in an appropriate medium which may contain antibiotics or biocides for selection. The plants obtained in this way can then be tested for the presence of the inserted DNA. There is no special demand for plasmids in the case of injection and electroporation. it is possible to use ordinary plasmids such as for example pUC derivatives. The transformed cells develop inside the plants in the usual manner. They can form germ cells and transmit the transformed traits to the progeny plants. Said plants can be cultivated in the normal manner and can be crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties. In a preferred embodiment of the present invention, the plants will be transformed with genes in which the use of codons for plants has been optimized. See, for example, United States Patent No. d.380.831. Also, advantageously, plants that code for a truncated toxin may also be used. The truncated toxin encodes 0 typically about dd% to about 80% of the total length toxin. The methods for creating B.t. genes Synthetics to be used in plants are known in the art.

EXAMPLE 11 Cloning of B.t. in insect viruses A number of viruses that infect insects are known. These viruses include, for example, baculoviruses and entomopoxiviruses. In an embodiment of the present invention the genes encoding the insecticide toxins described herein, can be placed within the genome of the insect virus, thereby improving the pathogenicity of the viruses. Methods for the construction of insect viruses comprising B.t. toxin genes. they are well known and can be easily implemented by experts in the field. These methods have been described for example in Merryweather and others, (MMerryweather, AT, Weyer, MPG Harris, M. Hirst, T. Booth, RD Possee (1990) J. Gen. Virol. 71: 1d3d-1d44) and Martens and others (Martens, JWM, G.Honee, D. Zuidema, JWM van Lent, B Visser, JM (1990) Appl. Enviromental Microbiol. d6 (9): 2764-2770). It is to be understood that the examples and embodiments described herein are given for illustrative purposes only and that various modifications or changes in light thereof may be suggested to those skilled in the art and are included within the spirit and scope of this application and the scope thereof. of the appended claims.

Claims (4)

1. NOVELTY OF THE INVENTION CLAIMS 1 .- A method to control lepidopteran pests, wherein said method comprises contacting said pest with a toxin obtainable from PS17 isolated from Bacillus thurinigiensis.
2. 2. The method according to claim 1, wherein said toxin is a crydAa toxin.
3. 3. The method according to claim 1, wherein said toxin is expressed in a transformed plant.
4. 4. The method according to claim 1, wherein said plague of Lepidoptera is Plutella xylostella. d.- The method according to claim 1, wherein said plague of lepidoptera is Haliothis virescens. 6. The method according to claim 1, wherein said lepidopteran pest is Cochylis hospes. 7. A method for controlling a pest of a genus selected from the group of genera consisting of Smicronix and Cilindrocopturus, wherein said method comprises contacting said pest with a toxin obtainable from isolated Bacillus thurinigiensis PS28Q2. 8. - A method for controlling a pest of a Phillotreta genus, wherein said methods comprise contacting said pest with a toxin obtainable from isolated Bacillus thurinigiensis PS33F2. 9. A method for controlling coleopteran pests wherein said method comprises contacting said pest with a toxin obtainable from the isolate Bacillus thurinigiensis selected from the group consisting of PS32B and PS54G2. 10. The method according to claim 9, wherein said pest is Smicronix fulvus. 11. A method for controlling a pest of the genus Zygoramma, where said method comprises contacting said pest with a toxin. Cry8B obtainable from Bacillus thurinigiensis isolated PSdOC. 12. A method for controlling an aphid, wherein said method comprises contacting said pest with a toxin obtainable from Bacilius d thurinigiesnsis isolated PS71 M3. 13. The method according to claim 12, wherein said aphid is Schizaphis graminum. 14. A method for controlling a pest of the genus Zygoramma, wherein said method comprises contacting said pest with a toxin 0 obtainable from isolated Bacillus thurinigiensis PS80JJ1. 15. A method for controlling a coleopteran pest where said method comprises contacting said pest with a toxin obtainable from isolated Bacillus thurinigiensis PS86BB1. 16. - The method according to claim 15, wherein said toxin is expressed in a transformed plant. 17. A method for controlling lepidopteran pests, wherein said method comprises contacting said pest with a toxin obtainable from isolated Bacillus thurinigiensis PS86Q3. 18. The method according to claim 17, wherein said toxin is a CrydAc toxin. 19. The method according to claim 17, wherein said toxin is expressed from a transformed plant. 20. The method according to claim 17, wherein said pest is selected from the group consisting of Plutella xylostella, Mamestra configurata, and Heliothis virescens. 21.- A method to control a plague of the genre Cylindrocopterrus where said method comprises contacting said pest with toxin obtainable from a Bacillus thurinigiensis isolate selected from the group consisting of PS140E2, PS169E, and PS169R, and PS62B. 22. A method for controlling a coleopteran pest where said method comprises contacting said pest with a toxin obtainable from isolated Bacillus thurinigiensis PS158C2. 23. The method according to claim 22 wherein said toxin is expressed in a transformed plant. - 3d 24.- The method according to claim 22 wherein said pest is Zygoramma exclamationis. 2d.- The method according to claim 22 wherein said toxin is a 158C2 toxin (c). 26.- A method for controlling an aphid, wherein said method comprises contacting said aphid with a toxin obtainable from the isolate of Bacillus thurinigiensis selected from the group consisting of PSdOC, PS167P, PS196S1 and PS201T6. 27. The method according to claim 26 wherein said toxin is expressed in a transformed plant. 28. The method according to claim 27 wherein said aphid is Schizaphis graminum. 29. A method for controlling a pest of a genus selected from the group of genera consisting of Phyllotreta and Cylindrocopturus, where said method comprises contacting said pest with a toxin obtainable from Bacillus thurinigiensis isolated PS201T6. 30. The method according to claim 29 wherein said toxin is expressed in a transformed plant. 31.- A method for controlling lepidopteran pests, wherein said method comprises contacting said pest with a toxin obtainable from Bacillus thurinigiensis isolated HDd1 1. The method according to claim 31, wherein said toxin is expressed in a transformed plant. 33. - The method according to claim 31 wherein said plague of Lepidoptera is Plutella xylostella. 34.- The method according to claim 31, wherein said plague of Lepidoptera Mamestra configurata. 3d.- A biologically pure culture of a Bacillus thuringiensis isolate selected from the group consisting of Bacillus thuringiensis isolates PS18, PS28K1, PS43A2, PS169E6, PS164H2, PS186EE, PS196Q3, PS198A2 and PS225K1. 36.- A biologically pure culture of a Bacillus thuringiensis isolate selected from the group consisting of Bacillus thuringiensis isolates KB6 and KB19. 37.- A method for controlling a coleopteran pest where said method comprises contacting said pest with a toxin of a Bacillus thuringiensis isolate selected from the group consisting of PS43A2, PS164h2, PS186EE, PS196Q3, PS198A2, PS226K1, HD977, KB6 , Y KB19. 38. The method according to claim 37, wherein said plague of coleoptera in a member of the genus Phyllotreta and wherein said isolate is selected from the group consisting of PS164H2, PS186EE, PS196Q3, PS198A2, PS226K1, HD977, KB6 and KB19. 39.- The method according to claim 37, wherein said plague of coleoptera in a member of the Smicronyx genus, and wherein said isolate is PS43A2. 40. - A method for controlling an aphid, wherein said method comprises contacting said pest with a toxin from an isolate of Bacillus thuringiensis selected from the group consisting of PS169E6, PS28K1, PS18 and HD541. d 41. The method according to claim 40, wherein said aphid is Shizaphis graminum. 42.- A polynucleotide sequence encoding a pesticidal toxin obtained from a Bacillus thuringiensis isolate selected from the group of isolates designated PS18, PS28K1, PS43A2, PS169E6, PD164H2, PS186EE, PS196Q3, PS198A2, PS226K1, KB6, and KB19 . 43.- A pesticide toxin obtainable from an isolate of Bacillus thuringensis selected from the group of isolates designated PS18, PS28K1, PS143A2, PS19E6, PS186EE, PS196Q3, PS198A2, PS225K1, KB6, and KB19. d 44. The method according to claim 29, wherein said toxin is selected from the group consisting of cytlB toxin and a toxin. 130 kDa. 4d.- The method according to claim 14, wherein said toxin is a 4d kDa toxin.