MXPA01001547A - Pesticidal toxins and genes from bacillus laterosporus - Google Patents

Abstract

Disclosed and claimed are novel toxins and genes obtainable from Bacillus laterosporus isolates disclosed herein. In preferred embodiments, the subject genes and toxins are used to control Western corn rootworm.

Description

TOXINS AND GENES PESTICIDES OF BACILLUS LATEROSPORUS STRAINS BACKGROUND OF THE INVENTION Insects and other pests cost farmers billions of dollars per year in crop losses and the cost of keeping these pests under control. The losses caused by insect pests in agricultural production means include the decline in crop yields, the reduction of crop quality, as well as higher costs for harvesting. The corn rootworm (a plague of coleoptera insects) is a serious pest for plants. In the United States there is a lot of damage to the crops every year because the larvae of the corn rootworm (Diabrotica spp) feed on the roots. It has been estimated that approximately 9.3 million acres (3848 ha) of corn in the United States are infested each year with corn rootworm species complexes. The maize rootworm species complex includes the western corn rootworm (Diabrotica virgifera viríigera), the northern corn rootworm (Diabrotica barberi), and the southern corn rootworm (Diabrotica undecimpunctata howardi). aABw6 - l! - - -J- ~ The life cycle of all Diabrotica species is similar. The worm eggs from the roots of the corn are deposited in the soil. The newly incubated larvae (the first instar) remain in the soil and feed on the smaller branches of the maize roots. The later instars of the rootworms of the north and west corn invade the internal root tissues that transport water and mineral elements to the plants. In most cases, the larvae migrate to feed on the most recent roots. The digging of tunnels in the roots that the larvae carry out produces damages that can manifest as scars or brown and elongated on the surface of the root, tunnels open in the roots or different degrees of severing. Plants with severed roots are usually uprooted after storms that are accompanied by heavy rains and strong winds. Southern corn rootworm larvae feed on the roots in a manner similar to the 5 larvae of the western and northern corn rootworm. Southern corn rootworm larvae also feed on the growth point of the stem when it is still close to the soil line, causing the plant to wilt and die. After feeding for approximately 3 weeks, 0 larvae of the corn rootworm emerge from the roots and form pupae in the soil. Adult beetles emerge from the soil and can be fed corn pollen and many other types of pollen, as well as styles. Your feeding of green styles can reduce the level of pollination, which results in a diminished seed development and poor yield. The adult worm root of western corn also feeds on the leaves of corn, which can hinder the development of the plant and, in rare cases, kill the plants of some varieties of corn. The larvae that reside in the soil of these Diabrotica species feed on the roots of the corn plant, causing its lodging. Accommodation eventually reduces the yield of corn and often ends with the death of the plant. By feeding on corn styles, adult beetles reduce pollination and, therefore, deleteriously affect yield per maize plant. In addition, both adults and larvae of the genus Diabrotica attack the crops of cucurbits (cucumbers, melons, squash, etc.) and numerous horticultural and agricultural crops of commercial production, as well as those grown in domestic gardens. It is estimated that the annual cost of insecticides to fight corn rootworm and losses of annual crops caused by the damage caused by the rootworm of corn exceeds a total of 1,000 million dollars in the United States each year (Meycalf, R.L. [1986], in Methods for the Study of Pest Diabrotica, Drysan, J.L. and T.A. Miller [Edit.], Springer-Verlag, New York, NY, p. vii-xv). Annually, insecticides worth approximately US $ 250 million are applied to combat corn rootworms in the United States. In the Midwest, in 1990, insecticides were applied for U $ S 60 million and U $ S 40 million in Lowa and Nebraska, respectively. Even using insecticides, corn rootworms cause crop damage worth $ 750 million annually, which makes them a very serious insect pest in the Midwest. Maize root worm control has been partially addressed through cultivation methods, such as crop rotation and the application of high nitrogen levels to stimulate the development of an adventitious root system. However, chemical insecticides are very strongly relied upon to ensure the desired level of control. The insecticides are deposited on the ground or incorporated into it. Economic demands for the use of farmland restrict the use of crop rotation. In addition, the emergence characteristic after a two-year dormant period (or hibernation) of northern corn rootworms is preventing crop rotations in some regions. The use of insecticides to control the rootworm of corn also has several drawbacks. The continuous use of insecticides has led to the evolution of resistant insects. Situations such as extremely abundant populations of larvae, copious rains and improper calibration of the insecticide applicator equipment may result in inadequate control. The use of insecticides often raises environmental concerns such as contamination of .- & . éfi3 * ^ & t soil and the water supply as much superficial as the groundwater. The public has also begun to worry about the amount of residual chemicals that can be found in food. The work with insecticides imposes risks to the people who apply them. Therefore, synthetic chemical pesticides are being increasingly investigated, and rightly so, because of their potentially toxic environmental consequences. Examples of synthetic chemical pesticides in widespread use include organic chlorines, for example DDT, mirex, kepone, lindane, aldrin, chlordaran, aldicarb and dieldrin, organic phosphates, for example chlorpyrifos, parathion, malathion, diazinon, as well as the carbamates. The recent strict restrictions imposed on the use of pesticides and the feeding of some effective pesticides on the market could limit the economic and effective options for the control of burdensome pests. Because of the problems associated with the use of pesticides synthetic chemicals, there is a manifest need to limit the use of these agents and a need to identify alternative control agents. The replacement of synthetic chemical pesticides, or the combination of these agents, with biological pesticides could reduce the levels of toxic chemicals in the environment. 0 A biological pesticide agent that is enjoying increasing popularity is the soil microbe Bacillus thuringiensis (B.t). The soil microbe Bacillus thuringiensis (B.t.), is a Gram positive sporiferous bacterium. Most strains of B.t. does not exhibit pesticidal activity.

Some strains of B.t. they produce, and can be characterized by, parasitic crystalline protein inclusions. These "d-endotoxins" that typically have pesticidal activity are different from exotoxins, which have a range of non-specific hosts. These inclusions often appear, seen under a microscope, as distinctive shape crystals. Proteins can be extremely toxic to pests and specific in their toxic activity. Certain toxin genes of B.t. They have been isolated and sequenced. The cloning and expression of a crystal protein gene of B.t. in Escherichia coli were described more than 15 years ago in published works (Schmepf, H.E., H.R. Whiteley [1981] Proc. Nati Acad. Sci. USA 78: 2893-2897). In addition, with the use of genetic engineering techniques, new approaches are being developed to apply B.t. toxins. to agricultural environments, including the use of genetically modified plants with toxin genes from B.t. for resistance to insects and the use of stabilized intact microbial cells as vehicles for the administration of B.t. toxins. (Gaertner, F.H., L. Kim [1988] TIBTECH 6: S4-S7). Accordingly, the endotoxin genes of B.t. They are acquiring commercial value. In the last fifteen years, the commercial use of pesticides with B.t. It has been limited to a narrow range of lepidopteran pests (caterpillars). Spores and crystals preparations of the subsp. kurstaki of B. thuringiensis for many years as commercial insecticides to combat lepidopteran pests. For example, the variety of B. thuringiensis ^, ^ ^ r ^ S »^^^ ¡^^ j l ** < ** > * ^ ** > * - '< ^ a_ ~ ~ .. < A ^, ... kurstaki IID-1 produces a crystalline endotoxin d that is toxic to the larvae of a number of lepidopteran insects. However, in recent years researchers have discovered pesticides with B.t. which are specific to a much wider spectrum of pests. For example, other Bt species, such as israelensis and morrisoni (also known as tenebrionis, also known as Bt M-7) have been used commercially to combat insects of the order Diptera and Coleoptera, respectively (Gaertner, FH [1989] "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms "in Controlled Delivery of Crop Protection Agents, RM Wilkins, 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 Agroecosystems" Developments in Industrial Microbiology 20: 97-104. Krieg, A., A.M. Huger, G.A. Lagenbruch, W. Schnetter (1983) Z. ang. Ent. 96: 500-508 describe var. tenebrionis of Bacillus thuringiensis, which is said to be active against two beetles of the order Coleoptera. These are the Colorado potato bug, Leptinotarsa decemlineata and Agelastica alni. Lately, new subspecies of B.t. and the genes responsible for the active endotoxin d proteins have been isolated (Hófte, H., H, R. Whiteley [1989] Microbiological Reviews 52 (2): 242-255). Hófte and Whiteley classified the crystal protein of B.t. in four classes «& __. r ..- ^^. . ^^. ife ^^ te ^^^^^^^^ '^ = ^, fr «A ^ ag ^^ main. The classes were Cryl (specific against Lepidóptera), Cryll (specific against Lepidóptera and Diptera), Cryll (specific against the Coleoptera) and CrylV (specific against Diptera). The discovery of strains specifically toxic for other pests has been reported (Feitelson, J. S., J. Payne, L. Kim [1992] B / o / Technology 10: 271-275). It has been suggested that CryV designates a class of specific toxin genes against nematodes. Lambert et al. (Lambert, N., L. Buysse, C. Decock, S. Jansens, C. Piens, B. Saey, J. Seurinck, K. van Audenhove, J. Van Rie, A. Van Vliet, M. Peferoen [1996] Appl. Environ Microbiol. 62 (1): 80.86) and Shevelev et al. ([1993] FEBS Lett 336: 79-82) describe the characterization of active Cry9 toxins 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) and Smulevitch et al. ([1991] FEBS Lett 293: 25-26) also describe toxins of B.t. A number of other classes of B.t. genes have now been identified. The 1989 nomenclature and classification scheme of Hófte and Whiteley for crystal proteins was based on both the deduced amino acid sequence and the host spectrum of the toxin. That system was adapted to cover 14 different types of toxin genes that were divided into five basic classes. The number of sequenced Bacillus thuringiensis crystal protein genes now stands at more than 50. A revised nomenclature based exclusively on the identity of amino acids has been proposed (Crickmore et al. [1996] Society for Invertebrate Pathology, 29a. Annual Meeting, lller. International Colloquium on Bacillus thuringiensis, University of Córdoba, Córdoba, Spain, September 1-6, 1996, compendium). The term "cry" has been retained for all toxin genes except cytA and cytB, which continue to constitute a separate class. The Roman numerals have been changed by Arabic numbers in the primary range and the parentheses of the tertiary range have been eliminated. Many of the original names have been preserved, with the exceptions indicated, although a number of these genes have been reclassified. See also "Revisions of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins. "N. Crickmore, DR Ziegler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum and DH Dean, Microbiology and Molecular Biology Reviews (1998) Volume 62: 807 -813 and Crickmore, Zeigler, Feitelson, Schnepf, Van Rie, Lercelus, Baum and Dean, "Bacillus thuringiensis toxin nomenclature" (1999) 1 5 Htpp: //www.biols. susx.ac.uk/llome/Neil_Crickmore/Bt/index.html. This method uses the available software applications CLUSTAL W and PHYLIP. The NEIGHBOR application within the PHYLIP package uses a mathematical averaging algorithm (UPGMA). As a result of deep research and investment of resources, other patents have been granted on new insulations of B.t. and new uses of said isolated products of B.t. See Feitelson and others, already mentioned, for a review. However, the discovery of new 6feJÍ &? Aatefc «^».] ¿¿¿^^ isolated from B.t. and new uses for known isolates of B.t. it remains an empirical and unpredictable technique. Favret and Yousten [1985] J. Invert. Path. 45: 195-203) analyzed the insecticidal activity of Bacillus laterosporus strains, although they concluded that the low levels of toxicity demonstrated by these strains indicate that these strains were not potential candidates for biological control agents. Montaldi and Roth (172 J. Bac. 4, April 1990, pages 2168-2171) carried out electron microscopy examinations of parapolar bodies of sporangia of Bacillus laterosporus. Bone et al. (U.S. Patent No. 5,045,314) reports that the spores of several strains of S. laterosporus inhibit the incubation of the eggs and / or the larval development of a nematode that parasitizes the animals. Aronson et al. (U.S. Patent No. 5,055,293) describe a sporiferous Bacillus laterosporus designated P5 (ATCC 53694). In this study, Bacillus laterosporus NRS-590 is used as a negative control. Aronson and others propose that B.l. P5 can invade the worm larvae of very young maize roots for immediate or subsequent damage, or that blocks the root worm's reception or response to the root signal of the maize that directs them to the roots. Orlova et al. (64 Appl. Env. Micro 7, July 1998, pp. 2723-2725) report that crystalline inclusions of certain strains of Bacillus laterosporus could possibly be used as candidates for mosquito control. Among the obstacles to the agricultural use of toxins from B.t. it includes the development of resistance to toxins of B.t. for the insects. ^^^^ faith & i ^^^^^^^^^^^^^^^^^^^^^^ j ^^^^^^^^ _ ^ _ ^ _ ^ _ ^ = _ Sa _ = _ In addition, certain insects may be refractory to the effects of B.t. The latter include insects such as the cotton weevil and the black night caterpillar, as well as adult insects of most species that have not yet shown considerable apparent sensitivity to B.t. endotoxins. While there has been a great interest in developing strategies to control the resistance in the technology of transgenic plants, the need remains to develop genes that can be expressed satisfactorily at appropriate levels in plants in order to produce effective control of various insects. BRIEF DESCRIPTION OF THE INVENTION The present invention relates to materials and methods useful for the control of pests of non-mammalian animals and, in particular, of plant pests. In one embodiment, the present invention features novel pesticidal toxins and toxin-coding genes that can be obtained from Bacillus laterosporus isolates. In a preferred embodiment, the pests proposed as targets are corn worm pests. The toxins of the present invention include heat-soluble soluble toxins that can be obtained from the culture supernatant of the Bacillus laterosporus strains in question. The toxins of the present invention also include smaller thermolabile toxins that are obtained from those strains.

The present invention further presents nucleotide sequences that encode the toxins of the present invention. The nucleotide sequences of the present invention encode toxins that differ from the toxins described above. The nucleotide sequences of the present invention can also be used in the identification and characterization of genes that encode pesticidal toxins. In one embodiment of the present invention, the Bacillus isolates of the present can be cultured under conditions that give rise to a high multiplication of the microbe. After treating the microbes to produce single-stranded genomic nucleic acid, the DNA is characterized using nucleotide sequences according to the present invention. The characteristic fragments of the genes encoding the toxins are amplified by the method, thus identifying the presence of the gene or genes coding for toxins. In a preferred embodiment, the present invention relates to plants and plant cells transformed to produce at least one of the pesticidal toxins of the present invention in such a way that the transformed plant cells express the pesticidal toxins in the tissues consumed by the target pests. . In addition, mixtures and / or combinations of toxins according to the present invention can be used. The transformation of plants with the genetic constructions described in this document could be done using techniques i.s -i. JA known to those skilled in the art and would typically involve the modification of the gene to optimize the expression of the toxin in plants.

BRIEF DESCRIPTION OF THE SEQUENCES The SEC. ID. DO NOT. 1 is an MIS probe SEC. ID. DO NOT. 2 is a WAR probe SEC. ID. DO NOT. 3 is a forward MIS primer SEC. ID. DO NOT. 4 is a reverse primer MIS SEC. ID. DO NOT. 5 is a nucleotide sequence obtained from the toxin gene of strain MB438 of B.l. The SEC. ID. DO NOT. 6 is the nucleotide sequence of the MIS toxin gene of strain MB438 of B.l. The SEC. ID. DO NOT. 7 is the polypeptide sequence of the MIS toxin of strain MB438 of B.l. The SEC. ID. DO NOT. 8 is the nucleotide sequence of the WAR toxin gene of strain MB438 of B.l. The SEC. ID. DO NOT. 9 is the polypeptide sequence of the WAR toxin of strain MB438 of B.l. The SEC. ID. DO NOT. 10 is a nucleotide sequence of the toxin MIS of strain MB439 of B.l. ^^ gH & s ü i DETAILED DESCRIPTION OF THE INVENTION The present invention relates to materials and methods useful in the control of pests of non-mammalian animals and, in particular, pests of plants. In one embodiment, the present invention features novel pesticidal toxins and toxin-encoding genes that are obtained from Bacillus laterosporus (B.l.) isolates. In a preferred embodiment, the target pests are corn rootworm pests. Toxins of the present invention include thermolabile solubles that can be obtained from the culture supernatant of the Bacillus laterosporus strains in ration. The toxins of the MIS and WAR type that can be obtained from these strains are described in detail below. The present invention further presents nucleotide sequences that encode the toxins of the present invention. The nucleotide sequences of the present invention encode toxins that differ from the toxins described above. Other nucleotide sequences of the present invention can also be used in diagnostic and analytical procedures that are well known in the art. For example, probes, primers and partial sequences can be used to identify and characterize the genes that code for pesticidal toxins. In one embodiment of the present invention the relevant Bacillus isolates can be grown under conditions that result in a large multiplication of the microbe. After treating the microbes * gMgü ge ^ &: i ^^^^^? M ^? ^^ m ^ m to produce single-stranded genomic nucleic acid, the DNA is characterized using nucleotide sequences according to the present invention. The characteristic fragments of the genes encoding the toxins are amplified by the method, thus identifying the presence of the gene or genes coding for toxins. In a preferred embodiment, the present invention relates to transformed plant cells for producing at least one of the pesticidal toxins of the present invention in such a way that the transformed plant cells express the pesticidal toxins in the tissues consumed by the target pests. In addition, mixtures and / or combinations of toxins according to the present invention can be used. In some preferred embodiments, a MIS toxin is used in conjunction with a WAR toxin. The transformation of plants with the genetic constructions of the present can be carried out using techniques well known to those skilled in the art and would typically involve modification of the gene to optimize the expression of the toxin in the plants. Useful isolates according to the present invention are deposited in the permanent collection of the Agricultural Research Service Patent Culture Collection (NRRL), Regional Center of North Investigations, 1815 North University Street, Peoria, Illinois 61604, USA: The deposit numbers of the crops are as follows.

Efc - &;,. .... «^ ¿« - .. ^ JM ^ É ^ ,, ^ d ^. & ^ The crops that have been deposited for the purposes of the present patent application were deposited under conditions that guarantee access to the crops, during the period in which this patent application is pending, to the person designated by the Patent Commissioner. and Brands to do so under 37 CFR 1.14 and 35 USC. The deposits will be available as required by the foreign patent laws of the countries in which counterparts of the present invention or their successors are presented. However, it should be understood that the availability of a deposit does not constitute a license to practice the present invention in contravention of patent rights. 1 5 granted by government action. In addition, the crop deposits in question will be stored and made available to the public in accordance with the clauses of the Budapest Treaty for the Deposit of Microorganisms, that is, they will be stored with all the necessary care to keep them viable and without contamination for a period of at least five years from the most recent request to provide a sample of the deposit and, in any case, for a period of at least thirty (30) years from the date of deposit or during the validity of any patent that may be granted, which describes the crop (s). The depositor recognizes the duty to replace the deposit (s) in case of not being able to provide a sample when requested, due to the state of the deposit. All restrictions imposed on the availability to the public of the present crop deposits will be irrevocably lifted upon granting a patent describing them. The mutants of the isolates referred to herein can be prepared by methods well known in the art. For example, a sporogenic mutant can be obtained by means of mutagenesis with ethyl methane sulfonate (EMS) from an isolate. Mutants can be prepared using ultraviolet light and nitrosoguanidine by methods well known in the art. In one embodiment, the present invention relates to materials and methods that include nucleotide primers and probes for isolating, characterizing and identifying Bacillus genes that encode protein toxins that are active against pests of non-mammalian animals. The nucleotide sequences described herein may also be used to identify new Bacillus pesticide isolates. The invention also relates to genes, isolates and toxins identified by the methods and materials described in the present application. The new toxins and polynucleotide sequences presented here are defined according to several parameters. A characteristic of the toxins described herein is their pesticidal activity. In a specific modality, these toxins have activity against the worm of the West corn roots. The toxins and genes of the present invention can also be defined by their amino acid and nucleotide sequences. The sequences of the molecules can be defined in terms of homology with certain exemplified sequences as well as in terms of the ability to hybridize with, or be amplified by, certain probes and primers exemplified. In a preferred embodiment, the MIS toxins according to the present invention have a molecular weight of about 70 to about 100 kDa and, most preferably, these toxins have a size of about 80 kDa. Typically, these toxins are soluble and can be obtained from the supernatant of Bacillus cultures according to what is described herein. These toxicity toxins against pests of non-mammalian animals. In a preferred embodiment, these toxins have activity against the rootworm of western corn. MIS proteins are also useful because of their ability to form pores in cells. These proteins can be used with secondary entities such as, for example, other proteins. When used with a secondary entity, the MIS protein facilitates the entry of the second agent into a target cell. In a preferred embodiment, the MIS protein interacts with the MIS receptors in a target cell and causes the formation of pores in said target cell. The secondary entity may consist of a toxin or other molecule whose entry into the cell is convenient. iMteh > - «& JMfa, .-%. , **** - ^^^^^ A ^^^^^^^^ ^^ i ^ ^^ The present invention also relates to toxins of the WAR type which have a size of about 30-50 kDa and very typically have a size of about 40 kDa. Typically, these toxins are soluble and can be obtained from the supernatant of Bacillus cultures as described herein. The types of MIS and WAR toxins according to the present invention can be identified with the primers described in this application. Another singular type of toxin has been identified as being produced by the Bacillus strains according to the present invention. These toxins are much smaller than the toxins of the MIS and WAR type of the present invention. These toxins, like toxins of the MIS and WAR type, are thermolabile. However, these toxins are in the approximate size range of approximately 10 kDa to approximately 1 kDa. These toxins are also soluble and can be obtained from the supernatants of the Bacillus cultures described herein. With the description presented herein, one skilled in the art could easily produce and use the various toxins and polynucleotide sequences described herein.

Genes and Toxins The genes and toxins useful according to the present invention include not only the complete sequences but also fragments of these sequences, variants, mutants and fusion proteins that retain the pesticidal activity characteristic of the toxins specifically exemplified herein. For example, U.S. Patent No. 5,605,793 discloses methods for generating more molecular diversity by using DNA reassembly after random fragmentation. In addition, internal deletions may be made to the genes and toxins specifically exemplified herein, provided that the modified toxins retain the pesticidal activity. Chimeric genes and toxins produced by combining portions of more than one toxin or Bacillus gene. They can also be used according to what is described in the present invention. In the present, the terms "variants" or "variations" of genes are used to refer to nucleotide sequences that encode the same toxins or that encode equivalent toxins with pesticidal activity. The term "equivalent toxins" is used herein to refer to toxins that have the same or essentially the same biological activity against pests as the toxins exemplified. It should be apparent to those skilled in the art that genes encoding active toxins can be identified and obtained by various means. The specific genes exemplified herein can be obtained from isolates deposited in a culture reservoir according to the above described. These genes, or portions or variants thereof, can also be synthetically constructed, for example by the use of a gene synthesizer. Variations of genes can be easily constructed using standard techniques to effect mutations punctual In addition, fragments of these genes can be prepared using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Sa / 31 or site-directed mutagenesis can be used to systematically trim nucleotides from the ends of these genes. In addition, genes encoding active fragments can be obtained using a variety of restriction enzymes. Proteases can be used to directly obtain active fragments of these toxins. Toxins and / or equivalent genes encoding these equivalent toxins can be derived from Bacillus isolates and / or DNA libraries using the indications provided herein. There are a number of methods for obtaining pesticidal toxins according to the present invention. For example, antibodies against the pesticidal toxins described and claimed herein can be used to identify and isolate other toxins from a mixture of proteins. Specifically, antibodies can be cultured against portions of the toxins that are more constant and more differentiated from other Bacillus toxins. These antibodies can then be used to identify equivalent toxins with characteristic activity by immunoprecipitation, immunoenzymatic assays (ELISA) or western blotting (western blotting). The antibodies against the toxins described herein, or against equivalent toxins, or fragments of these toxins can be easily prepared using standard procedures in the ^^ ¡n kr Ú? technique. Then you can get the genes that code for these toxins from the microorganism. Equivalent fragments that retain the pesticidal activity of the exemplified toxins are within the scope of the present invention. In addition, under the redundancy of the genetic code, a variety of different DNA sequences can. encode the amino acid sequences described herein. It is within the competence of those skilled in the art to generate these alternative DNA sequences that encode them, or essentially the same toxins. These variant DNA sequences are within the scope of the present invention. In the present, the expression "essentially the same" refers to sequences that have substitutions, deletions, additions or insertions of amino acids that practically do not affect pesticidal activity. Fragments that have 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. The probes provide a rapid method for identifying toxin-encoding genes according to the present invention. The nucleotide segments that are used as probes according to the present invention can be synthesized using a DNA synthesizer and standard procedures. Certain toxins of the present invention have been specifically exemplified in this document. Since these toxins are merely examples of toxins of the present invention, it should be apparent that the present invention encompasses variant or equivalent toxins (as well as nucleotide sequences encoding equivalent toxins) having equal or similar pesticidal activity to that of the exemplified toxin. The equivalent toxins have amino acid homology with an exemplified toxin. This amino acid identity is typically greater than 60%, preferably it should be greater than 75%, more preferably greater than 80%, more preferably greater than 90% and may be higher than 95%. These identities are determined using standard alignment techniques, preferably those employed by Crickmore and others in accordance with that set forth in the background section of the present specification. The amino acid homology is higher in the critical regions of the toxin that are responsible for the biological activity or are involved in the determination of the three-dimensional configuration that is ultimately responsible for the biological activity. In this aspect, certain amino acid substitutions are acceptable and feasible if these substitutions take place in regions that are not essential for activity or are conservative amino acid substitutions that do not affect the three-dimensional configuration of the molecule. For example, amino acids can be located in the following classes: non-polar, polar without charge, basic and acid. Conservative substitutions by which an amino acid of one kind is replaced with another amino acid of the same type within the scope of the present invention so long as the substitution does not substantially alter the biological activity of the compound. Table 1 presents a list of the examples of amino acids that belong to each class TABLE 1 Amino acid class Examples of amino acids Non-polar Ala, Val, Leu, Lie, Pro, Met, Phe, Trp Polar without charge Gly, Ser, Thr, Cys, Tyr, Asn, Gin Asp Acids, Glu Basics Lys, Arg, His In some cases, non-conservative substitutions can also be made. The critical factor is that these substitutions should not cause a considerable reduction in the biological activity of the toxin. In the present, reference to "isolated" polynucleotides and / or "purified" toxins refers to these molecules when they are not associated with other molecules with which they would be found in nature. Consequently, the reference to "isolated and purified" represents the intervention of the "hand of man" according to what is described in the present. Toxins and chimeric genes also imply the "hand of man". m ^ i- and a¡j ^^^ Recombinant hosts The toxin-encoding genes of the present invention can be introduced into a wide variety of microbial or plant hosts. The expression of toxin genes results, directly or indirectly, in the production and conservation of the pesticide. The transformation of plant hosts is preferred. In this way, the pests that feed on the recombinant plant that expresses the toxin will come into contact with the toxin. With suitable microbial hosts, for example Pseudomonas, the microbes can be applied to the site of the pest, where they proliferate and are ingested. With any of the different techniques, the result is the control of the pest. On the other hand, the microbe that acts as a host of the toxin gene can be killed and treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, can then be applied to the environment of the target pest. The Bacillus toxin can also be applied by introducing a gene by means of a suitable vector into a microbial host and then applying said host to the site in vivo. A wide variety of ways of introducing a Bacillus gene encoding a toxin into a host are available under conditions that result in stable gene expression and maintenance. 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. i ^ eg ^^ j ^ í ^^ íi 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, for example, in U.S. Patent No. 5 5,380,831. In preferred embodiments, the genes of the present invention are optimized for expression in plants.

Treatment of the cells As mentioned above, Bacillus can be treated or the recombinant cells that express a Bacillus toxin to prolong the activity of the toxin and stabilize the cell. The pesticide microcapsule that is formed contains the Bacillus toxin within a cellular structure that has been stabilized and protects the toxin when the microcapsule is applied to the target pest field. The right host cells may include prokaryotes or eukaryotes. As hosts, prokaryotes and lower eukaryotes, such as fungi, are of special interest. Upon receiving treatment the cell is generally intact and substantially in its proliferative form rather than in its spore form. The treatment of the microbial cell, for example a microbe which contains the gene of the Bacillus toxin, can be made by chemical or physical means, or by a combination of chemical and / or physical means, provided that the technique does not adversely affect the properties of the toxin, nor reduce the cellular capacity of toxin protection. The methods for the treatment of microbial cells has been described in U.S. Patent Nos. 4,695,455 and 4,695,462 which are incorporated herein by reference.

Methods and formulations for pest control The control of pests with the use of toxins and genes of the present invention can be achieved by a variety of methods known to those skilled in the art. These methods include, for example, the application of Bacillus isolates to pests (or their habitat), application of recombinant microbes to pests (or their habitats) and the transformation of plants with genes encoding the pesticidal toxins of the present invention. The transformations can be carried out by those skilled in the art using standard techniques. The materials necessary for these transformations have been described in the present or of the The opposite can easily be obtained by trained technicians. The formulated bait granules containing an attracting agent and toxins from the Bacillus isolates, or recombinant microbes containing the genes that are obtained from the Bacillus isolates described herein may be applied to the soil. The formulated product can be also apply in the form of seed coat or root treatment or as a total treatment of the plant in later stages of the crop cycle. The treatments of the plant and the soil with Bacillus cells can be used in the form of wettable powders, granules or dusts, mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates and the like) or botanical materials (powdered corn cobs, rice husks, walnut shells, and so on). The formulations may include spreader-tackifiers, stabilizers, other additives for pesticides or surfactants. The liquid formulations can be water-based or non-aqueous and used as foams, gels, suspensions, emulsifiable concentrates or the like. The ingredients may include Theological agents, surfactants, emulsifiers, dispersants or polymers. As trained technicians will appreciate, the concentration of the pesticide can vary widely depending on the nature of the specific formulation, especially if it is a concentrate or should be used directly. The pesticide must be present at a rate of at least 1% by weight and can reach a proportion of 100% by weight. The dry formulations have from 1-95% by weight of the pesticide while the liquid formulations generally have from 1-60% by weight of solids in the liquid phase. Formulations containing cells generally have from 102 to about 10 4 cells / mg. These formulations are administered at a rate of approximately 50 mg (liquid or dry) to 1 kg. or more per hectare. The formulations can be applied to the field of the pest, for example to soil and foliage, by spraying, sprinkling, sprinkling, etc.

Polynucleotide probes It is a well-known fact that DNA has a fundamental property called base complementarity. In nature, DNA commonly exists in the form of pairs of antiparallel chains, 5 projecting the bases of each chain from that chain to the opposite. The adenine base (A) of one chain always opposes the thymine base (T) of the other chain, and the guanine base (G) opposes the cytosine base (C). The bases are maintained in apposition for their ability to bind hydrogen in this specific manner. Although each individual link is relatively weak, or the net effect of many adjacent bases linked with hydrogen, added to the effects of accumulation of bases, constitutes a stable union of the two complementary chains. These bonds can be broken by treatments such as high pH or high temperature, and these conditions produce the dissociation or "denaturation" of the two chains. If the DNA is then placed under conditions that make the hydrogen bond of the bases thermodynamically favorable, the strands of the DNA are conjugated or "hybridized" to reform the original double-stranded DNA. If carried out under appropriate conditions, this hybridization can be highly specific. That is, only chains with a high or complementary degree of base can form stable double-chain structures. The relationship between the specificity of hybridization and the reaction conditions is well known. Therefore, hybridization can be used to analyze whether two DNA segments are complementary »', -. ..-,., JASfeja ^ Mfe ^ a ^ a tei8ag ^? Ata «i -» - jy > fc..s > i ^ te £ -; . in its base sequences. It is this hybridization mechanism that facilitates the use of probes according to the present invention to detect and easily characterize the DNA sequences of interest. The probes can be RNA or DNA ANP (peptide nucleic acid). The probe normally has at least about 10 kisses, more usually at least about 17 bases, and can have up to about 100 bases or more. Longer probes can be easily employed, and these can have, for example, a length of several kilobases. The sequence of the probe is designed to be at least substantially complementary to a portion of a gene encoding a toxin of interest. The probe does not need to have perfect complementarity with the sequence to which it hybridizes. The probes can be labeled using techniques well known to those skilled in the art. An approach to use the present invention as probes consists first in identifying, by Southern blot analysis of a gene bank of the Bacillus isolate, all the DNA segments homologous with the nucleotide sequences described. It is thus possible, without the help of biological analysis, to know in advance the probable activity of numerous new isolates of Bacillus. This type of analysis with probes gives a rapid method to identify genes of insecticidal toxins of potentially commercial value within the various subspecies of Bacillus. An advantageous hybridization method according to the present invention typically includes the initial steps of isolating the sample of DNA in question and chemically purify it. Used bacteria or total fractionated nucleic acid isolated from the bacteria can be used. The cells can be treated using known techniques to release their DNA (and / or RNA). The DNA sample can be cut into segments with an appropriate restriction enzyme. The segments can be separated by size by means of gel electrophoresis, usually agarose or acrylamide. The pieces of interest can be transferred to an immobilizing membrane. The specific hybridization technique is not essential for present invention. As improvements are made in hybridization techniques, they can be easily applied. The probe and sample can then be combined in a hybridization buffer solution and kept at an appropriate temperature until annealing occurs. Next, the membrane is washed to release it from foreign materials, leaving the sample and probe molecules attached typically detected and quantified by autoradiography and / or liquid scintillation counting. As is known to the technicians, if the probe molecule and the nucleic acid sample hybridize forming a strong non-covalent binding between the two molecules, it can be presumed with reason that the probe and the sample are essentially identical. The detectable marker of the probe provides a means to determine in a known manner whether hybridization has occurred. '-fcSfc- -a * > . - - * > • ¿aá-fi ^ J ^ á ^ t ^^.? ^^ a ^^? ü_fenartriia? > taM, a In the use of the nucleotide segments as probes, the specific probe is labeled by any suitable marker known to those skilled in the art, including radioactive and non-radioactive labels. Typical radioactive labels include 32 P, 35 S or 5 similar. Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or peroxidases, or the various chemiluminescers such as luciferin or fluorescent compounds such as fluorescein and its derivatives. The probes can be prepared in an intrinsically fluorescent manner according to that described in the international application No. WO 93/16094. Various degrees of stringency can be employed in hybridization. The more severe the conditions, the greater the complementarity required for the formation of duplexes. The severity can be controlled by means of the temperature, the concentration of the probe, the probe length, ionic power, time and other factors. Preferably, the hybridization is carried out under stringent conditions by well known techniques in the medium according to what is described, for example, in probes DNA, by Keller, G.H., M.M. Manak (1987), Stockton Press, New York, NY, p. 169-170. This information is incorporated into this reference. In the present, the expression "moderate to extremely stringent" conditions of hybridization refers to conditions that produce the same, or approximately the same degree of hybridization specificity than the conditions employed by the applicants hereof. This document presents examples of moderate and high stringency conditions. Specifically, hybridization of immobilized DNA in Southern blots with 32 P-labeled gene probes was carried out by standard methods (Maniatis et al.). In general, hybridization and subsequent washings were carried out under moderate to extremely stringent conditions that resulted in the detection of target sequences with homology to the toxin genes in question. For the double-stranded DNA gene probes, hybridization was carried out overnight at 20-25 ° C below the melting temperature (Tm) of the DNA hybrid in 6X SSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg / ml of denatured DNA. The melting temperature is defined by the following formula (Beltz, GA, KA Jacobs, TH Eickbush, PT Cherbas and FC Kafatos [1983] Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [edit] Academic Press, New York 100: 266-285). Tm 81.5 ° C + 16.6 Log [Na +] + 0.41 (% G + C) - 0.61 (% formamide) - 600 / length of duplex in base pairs. The washes are typically carried out in the following manner: (1) Twice at room temperature for 15 minutes in 1X SSPE, 0.1% SDS (low stringency wash). (2) Once at a Tm - 20 ° C for 15 minutes in 0.2X SSPE, 0.1% SDS (wash of moderate stringency).

. Aa ^^ ^ A ^^ ...-,. ^^^^ p ^ &amp ^ £ ^ ¡^ ^^^? ^, ^^? ^^^^^ For oligonucleotide probes, hybridization was carried out overnight at 10-20 ° C below the melting temperature (Tm) of the hybrid in 6X SSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg / ml Denatured DNA The Tm for the oligonucleotide probes was determined according to the following formula: Tm (° C) = 2 (number T / A base pairs) + 4 (number G / C base pairs (Suggs, SV, T. Miyake , EH Kawashime, MJ Johnson, K. Itakura and RB Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, DD Brown [edit], Academic Press, New York, 23: 683-693). were typically carried out in the following manner: (1) twice at room temperature for 15 minutes 1X SSPE, 0.1% SDS (low stringency wash). (2) Once at the hybridization temperature for 15 minutes 1X SSPE, 0.1% SDS (moderately stringent wash) In general, the salt and / or temperature can be modified to change the stringency With a DNA fragment labeled of> 70 bases approximately in length, the following conditions can be used: Low: 1 or 2X SSPE, low ambient temperature: 1 or 2X SSPE, 42 ° C Moderate: 0.2X OR 1X SSPE, 65 ° C High: 0.1X SSPE, 65 ° C ¿¿¿^ ^ J ^ l ^^^^^ Duplex formation and stability depend on the substantial complementarity between the two strands of a hybrid and, as noted above, some degree of mismatch can be tolerated. Therefore, the sequences of the probes of the present invention include mutations (both single and multiple), deletions, insertions of the described sequences, as well as combinations thereof, wherein said mutations, insertions and deletions allow the formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions and deletions can occur in a given polynucleotide sequence in many ways, and these methods are known to those skilled in the art. In the future other methods may be disclosed. It is as well as the variants by mutation, insertion or deletion of the described nucleotide sequences can be easily prepared by methods well known to those skilled in the art. These variants can be used in the same way as exemplified primer sequences, provided that the variants have a substantial sequence homology with the original sequence. Herein, the expression "substantial sequence homology" refers to a homology that is sufficient to allow the variant probe to function with the same characteristics as the original probe. Preferably, this homology is greater than 50%; more preferably, the homology is greater than 75% and most preferably, this homology is greater than 90%. The degree of homology or identity necessary for the variant to work in its intended capacity depends on the use to which the sequence is intended. It is the competence of an expert in this technique to carry out mutations by mutation, insertion or deletion intended to improve the performance of the sequence or, otherwise, provide a methodological advantage.

PCR technology The polymerase chain reaction (PCR) is a repetitive, enzymatic synthesis and with primers of a nucleic acid sequence.

This method is well known and commonly used by those skilled in the art (see Mullis, U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159; Saiki, Randall K., Stephen Sharf, Fred Faloona, Kary B. Mulis, Glenn T. Horn, Henry A. Erlich, Norman Arnheim [1985] "Enzymatic Amplification of ß-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickie Cell Anemia "Science 230: 1350-1354.) PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to the opposite strands of The target sequence The primers are oriented with the 3 'ends facing each other. repeated cycles of thermal denaturation of the template, annealing of the primers with their complementary sequences and extension of the primers annealed with a DNA polymerase results in amplification of the segment defined by the 5 'ends of the primers of .

PCR Since the product of the extension of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of DNA fragment produced in the previous cycle. This produces the exponential accumulation of the specific target fragment, up to several 5 million times in a few hours. Using a thermostable DNA polymerase such as Taq polymerase, which is isolated from the thermophilic bacteria Thermus aquaticus, the amplification process can be completely automated. The other enzymes that can be used as are known to those skilled in the art. The DNA sequences obtained according to the present invention can be used as primers for PCR amplification. In the practice of PCR amplification some degree of discrepancy between the primer and the template can be tolerated. Therefore, mutations, deletions and insertions (especially the additions of nucleotides at the 5 'end) of the exemplified primers fall within the scope of the present invention. Mutations, insertions and deletions can be produced in a given primer by methods known to a person skilled in the art. All references cited in this document are incorporated herein by reference. Below are examples that illustrate the procedures for practicing the invention. These examples should not be considered as limiting. All percentages are expressed by weight and all proportions of solvent mixtures are by volume unless otherwise indicated.

EXAMPLE 1 Cultivation of Bacillus laterosporus isolates useful in accordance with the present invention Strains of Bacillus lateroporus and recombinants of B.t. expressing the MIS and WAR toxins of B.l. in liquid TB medium (+ glycerol) at 30 ° C and 300 rpm for 25 hours. The cells were tabletted by centrifugation and the supernatants ("SN") were decanted and stored. EDTA was added to 1 mM and the samples were stored at -20 ° C. Fresh samples were used for biological trials on the same day of harvest. The frozen samples were thawed at 4 ° C and centrifuged to granulate and remove solids and then used for biological testing or fermentation.

EXAMPLE 2 Preparation of Genomic DNA and Southern Blot Analysis 20 Entire cellular DNA was prepared from various cultivated Bacillus laterosporus strains to obtain an optical density of 0.5-0.8 at 600 nm, of visible light in Luria Bertani broth (LB). DNA was extracted using the Genomic-tip 500 / G or Genomic-Tip 20 / G kit from Qiagen and the Buffers Set of genomic DNA according to the protocol for Gram-positive bacteria (Qiagen, Inc., Valencia, CA). The total DNA prepared was digested with various restriction enzymes, subjected to electrophoresis on a 0.8% agarose gel and immobilized on a nylon-supported membrane using standard methods. (Maniatis, T., E. F. Fritsch, J. Sambrook [1982] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Novel toxin genes were detected using 32 P-labeled probes in standard Southern hybridizations or by non-radioactive methods using the DIG nucleic acid labeling and detection technique (Boehringer Mannheim, Indianapolis, IN). In the SEC. ID. No. 1 appears the MIS probe of approximately 2.6 kbp. The WAR probe of approximately 1.3 kbp appears in the SEC. ID. No. 2. These probes can be prepared in various ways, even using a "gene machine" or they can be cloned from the B.t. PS177C8 and amplify by PCR with homologous primers with the 5"and 3 'ends of each respective gene.In the latter case, DNA fragments were gel purified and approximately 25 ng of each DNA fragment was randomly labeled with 32P for the radioactive detection Approximately 300 ng of each DNA fragment was randomly labeled with the DIG High Prime kit for nonradioactive detection Hybridization of immobilized DNA with 32 P-labeled probes was performed randomly under standard conditions for formamide: 50% of formate, 5X SSPE, 5X Denhardt solution, 2% SDS, 0.1 mg / ml at 42 ° C overnight. The transfers were washed with a low level of stringency in 2X SSC, 0.1% SDS at 42 ° C and exposed to the film. The results of the restriction fragment length polymorphism (RFLP) of total cellular DNA of strains MB438 and MB439 of Bacillus laterosporus determined by Southern blot analysis with MIS or WAR probes, as shown in Table 2, are shown below. the indicated. The bands contain at least one fragment of the operon MIS or WAR of interest.

TABLE 2 fifteen EXAMPLE 3 Gene Cloning of Toxins -i 20 Lambda libraries of total genomic DNA from strains of Bacillus laterosporus MB349 or MB438 were prepared from partially digested DNA and fractionated by size in the size range of 9-20 kb. The specific digestion times were determined using the Ndell enzyme ff KS ^ SisS. HgÜJU diluted 1: 10 (approximately 0.5 units) in order to optimize the desired size range of the deferred DNA. The DNA was digested for the indicated time and then fractionated on a 0.7% agarose gel. The DNA was visualized using ethidium bromide staining and the DNA was excised from the gel in the size range of 9-20 kb. The gel fragment was placed in dialysis tubes (cut at 12-14,000 MW) together with 2 ml of 10 mM Tris-HCl, 1 mM EDTA buffer, pH 8.0 (TE). The gel fragment DNA was electroeluted in 0.1X TAE buffer at approximately 30 mA for one hour. The DNA was removed from the tube in TE buffer and purified using the Elutip column and protocol (Schleicher and Schuell; Keene, NH). The purified DNA was precipitated with ethanol and resuspended in 10 μl of TE. The purified and fractionated DNA was ligated into Lambda-GEM-11 arms digested with BamHI (Promega Corp. Madison, Wl) according to the protocol. The ligated DNA was then encapsidated in lambda phage using Gigapack lll Gold encapsidation extract (Stratagene Corp. La Jolla, CA) according to the protocol. The bacterial strain KW251 of E. coli was infected with the encapsidation extracts and applied on LB plates in agarose with LB aggregate. Plates were transferred to nitrocellulose filters and prepared for hybridization using standard methods (Maniatis et al. mentioned work). 32 P-labeled probe was prepared (see above) and the filters were hybridized and washed and washed in the manner described above. The plaques containing the desired clone were visualized by exposure of the filters to Kodak XAR-5 film. The bald spots were separated from the plates and the resuspended phage of the agar in SM buffer. DNA was prepared from the phage using LambdaSorb phage adsorbent (Promega, Madison, Wl). The PCR was run on the phage DNA to verify that it contained the target operon using SEC. ID. No. 3 and SEC. ID. No. 4 5 as primers. The PCR reactions gave a band of 1 kb in both DNA samples, confirming that those clones contain the mis-type gene. In order to identify a smaller fragment of DNA containing the operon in question that could then be subcloned into a bacterial vector for further analysis and expression, the phage DNAs were directed to various enzymes were fractionated on a 1% agarose gel and transferred for Southern analysis. Southern analysis was performed in the above described manner. A Hincll fragment with a size of approximately 10 kb was identified for MB438. This fragment was gel purified and cloned into the EcoRV site of pBluescript II (SK +): the resulting plasmid receives the designation PMYC2608 and the recombinant E. coli strain containing this plasmid receives the designation MR 975.

EXAMPLE 4 Sequencing of the MIS and WAR genes of MB438 20 A partial sequence for the mis gene of MB438 was determined on a DNA fragment amplified by PCR. PCR was carried out using the MIS primers (SEQ ID No. 3 and SEQ ID No. 4) in DNA Total cellular genomic of MB438 and MB439. MB438 gave a DNA fragment of approximately 1 kbp which was then cloned into the cloning plasmid TA of PCR DNA, pCR2.1, according to that described by the supplier (Invitrogen, San Diego, CA). Plasmids from recombinant clones of PCT MB 438 were isolated and analyzed for the presence of an approximately 1 kbp insert by PCR using plasmid primers, T3 and T7. Then, those containing the insertion were isolated to be used as sequencing templates using QIAGEN miniprep kits (Santa Garita, CA) as stipulated by the supplier.

Sequencing reactions were performed using the Dye Terminator Cycle Sequencing Ready Reaction Kit from PE Applied Biosystems. Sequencing reactions were performed on an ABI PRISM 377 Automatic Sequencer. Sequence data was collected, edited and assembled using the ABI PRISM 377 Collection, Invoice and AutoAssembler of PE ABI. In the SEC. ID. No. 5 a partial nucleotide sequence of the mis gene of MB438 is displayed. The complete sequences of the MIS and WAR genes of MB438 were determined by collecting sequence data from the random restriction fragments of pMYC2608 and by the primer spanning the insertion of DNA in pMYC2608. The DNA insert of the plasmid pMYC2608 was isolated by cleavage of the vector using the Notl and Apal polylinkant restriction enzymes, fractionation on a 0.7% agarose gel and purification from the agarose gel using the Qiaexll Kit (Qiagen Inc .; , CA).

The gel-purified DNA insert was then digested with the restriction enzymes A \, Mse \ and Rsa \ and fractionated on a 1% agarose gel. Fragments of 0.5 and 1.5 kb DNA were excised from the gel, fragments that were purified using the Qiaexll kit. The recovered fragments were ligated into pBluescript II digested with EcoRV and transformed on XLIOGold cells. Miniprep DNA was prepared from randomly chosen transformants, directed with Notl and Apal to verify the insertion and used for sequencing. Sequencing reactions were carried out using the dRhodamine Sequencing kit (ABI Prism / Perkin Elmer Applied Biosystems). The sequences were executed in a sequencing gel according to the protocol (ABI Prism) and analyzed using the Invoice and Autoassembler (ABI Prism) programs. In SEC. ID. DO NOT. 6 the complete nucleotide sequence of the m / s gene of MB438 is exposed; the MIS MB 438 peptide sequence deduced appears in the SEC. ID. DO NOT. 7. The The complete nucleotide sequence of the MB438 war gene appears in SEC. ID. DO NOT. 8, the WAR peptide sequence of MB 438 deduced appears in the SEC. ID. DO NOT. 9. A partial DNA sequence for the MB439 mis gene was determined from DNA fragments amplified by PCR. The PCR using primers SEC. ID. DO NOT. 3 and SEC. ID. DO NOT. 4 on total cellular genomic DNA of MB439. A DNA fragment of approximately 1 kbp was obtained which was then cloned into the cloning plasmid TA of PCR DNA, pCR-TOPO, in accordance with that described i «9 | ^? te «! g £ ^^ by the provider (Invitrogen, San Diego, CA). Plasmids from recombinant clones of MB 439 PCR were isolated and analyzed for the presence of an approximately 1 kbp insert by PCR using the plasmid primers, T3 and T7. Next, those containing the insertion were isolated to be used as sequencing templates using QIAGEN miniprep kits (Santa Garita, CA) according to the stipulations of the supplier. Sequencing reactions were performed using the Dye Terminator Cycle Sequencing Ready Reaction Kit from PE Applied Biosystems. The sequencing reactions were performed in an ABI PRISM 377 Automatic Sequencer. Sequence data were collected, edited and assembled using the ABl PRISM 377 Collection, Invoice and AutoAssembler software from PE ABl. In the SEC. ID. No. 10 a partial nucleotide sequence of the mis gene of MB439 is displayed.

EXAMPLE 5 Subcloning of the MIS and WAR toxins of MB438 for the expression of Bacillus thurinqiensis The expression of the MIS and WAR toxins of MB 438 was obtained in B.t. subcloning the cloned genomic DNA fragment from pMYC2608 into a reciprocal vector of high copy number suitable for replication in both E. coli and B.t. The reciprocal vector, pMYC2614 is a modified version of pHT370 (O. Arantes and D. Lereclus. 1991. Gene 108: 115-119), which contains the region with multiple cloning sites of pBluescript II (Stratagene). The insertion of genomic DNA containing the mis and war genes was excised from pMYC2608 using the Notl and Apal restriction enzymes, gel-purified and ligated at the Notl and Apal sites of pMYC2614. The reciprocal plasmid of B.t. The resultant received the designation pMYC2609. To analyze the expression of MB438 toxin genes in B.t, pMYC2609 was transformed into the acryliclic host (Cry-) of B.t., CryB (A. Aronson, Purdue University, West Lafayette, IN) by electroporation. This recombinant strain was designated MR557. The expression of the WAR toxin was demonstrated by immunoblotting with the antibodies generated against the WAR PS177C8 toxin. Preparations of the culture supernatant and the MR557 cell pellet against the western corn rootworm were analyzed according to what is described later in example 8.

EXAMPLE 6 Biosensives of MB438 and MB439 with the western corn rootworm Samples of the supernatant prepared according to that described in Example 1 were loaded from the top on an artificial diet at a rate of 215 μl / 1.36 cm2. These preparations were infested a .... ^^^, ^ ** ttfrr. then with worms of the roots of the western corn neonates and conserved for 4 days in the dark at 25 ° C. Unless otherwise indicated, samples were evaluated to determine mortality on day 4 post-infestation. Table 3 is related to the temporary courses for MB438 and NB439. MB438 and MB439 demonstrate evidence of activity around 22-30 hours (MB438) and 24-39 hours (MB439). All strains were cultured in TBG medium. None of these samples was heat treated. 10 TABLE 3 fifteen twenty The results published in Table 4 show that warming removes most or all of the activity present in the unheated fresh samples of 24-hour and 48-hour cultures of MB438 and MB439.

TABLE 4 The results presented in table 5 show that the activity of MB438 and MB439 responds to the dose. All strains were cultured in TBG medium. None of the samples was heat treated. All samples constituted 24-hour cultures.

TABLE 5 EXAMPLE 7 Bioassays of fractionated samples in the western corn rootworm For dialyzed samples, aliquots of the culture supernatant were transferred to cellulose dialysis tubes and dialysed with 25 mM MaP0, 1 mM EDTA, pH 7, with stirring overnight at 4 ° C. This eliminates the possible free-flowing components of the SN of a molecular weight lower than the nominal cut-off of the dialysis membrane. The pore sizes were 6-8 kD and 50 kD and these samples demonstrate the activity of the components retained in the dialysis membrane, which can be called "high molecular weight". Low molecular weight fractions were generated by ultrafiltration ("UF") through membranes with pore sizes of 1, 3 or 10 kD by nitrogen gas pressure at 4 ° C. This method produces solutions ~ 9gg! containing supernatant components of a size less than the nominal molecular weight cutoff of the UF membrane. These solutions are called "infiltrators". The results shown in Table 6 show that the less than 10 kD component of MB438 and MB439 exhibits activity. All samples were cultured in TBG medium. None of the samples was subjected to heat treatment. All samples constituted 24-hour cultures.

TABLE 6 The results shown in table 7 show that the MB438 and MB439 components exhibit activity that is moderated by high heat and that the removal of low molecular weight components with dialysis does not eliminate activity. All samples were cultured for 24 hours in TBG medium. £ 3aL- ~; aift .. «£. «S ^ j ^ a ^ ¿# ^ sg ^^ Ujgü ^^^^^^ TABLE 7 The results reported in Table 8 demonstrate that MB438 and MB439 have activity in a component of less than 10 kD that does not pass through a 1 kD UF membrane. All the samples were cultured for 24 hours in TBG medium. ggsg «| s¡¡ ^ ¿-« »- ttg = g ^ .- Mfr --- - TABLE 8 fifteen EXAMPLE 8 20 Biological activity of MR957 and MR557 Cultures of MR957 were carried out in 5.0 ml of media (Difco TB premix, 5 g / liter of glycerol) in plastic tubes of 16 x 150 mm with lid. & "--- * ~ ^" * «---- * ÉB ^ ..» ^ - t ggjf u &S8 ^ ti tíatás? &? The cultures were shaken in a rotating drum for 24 hours at 37 ° C. The cells were tabletted by centrifugation and the supernatants were decanted and stored. EDTA was added at 1 mM and the samples were stored at 20 ° C. To determine the cell density, the samples were vortexed and 100 μl of each culture broth was transferred to a falcon tube (14 ml, 17 x 100 mm). A 1: 50 dilution was prepared by adding 4.9 ml of distilled water to each tube and re-vortexed. DO readings were made using a spectrophotometer at 600 nm. Strains of B.t. recombinants according to that described in example 1. Bioassays were carried out for clone MR957 of E. co // and the clone MR 557 of B. thuringiensis (each of which contained the mis and war genes of MB438) using essentially the same experimental design described in Example 6. MR948 and MR539 are negative control strains that contain cloning vectors without gene insertions of toxins. To analyze the strains of E. coli, samples of the supernatant or whole culture were applied to the surface of the diet at a dose of 215 ul / 1.36 cm2, while the cell pellet samples were concentrated 5 times and loaded onto the diet at a rate of 50 ul / 1, 36 cm2 (table 9). In order to analyze the strains of Bt, samples of the supernatant were applied to the surface of the diet at a dose of 215 ul / 1.36 cm2, while the samples of the cell pellet were concentrated 5 times and loaded in different proportions (table 10). Approximately 6-8 larvae were transferred onto the diet immediately after the sample was evaporated. The bioassay plate was sealed with mylar sheets using a soldering iron and holes were drilled on each receptacle to give rise to gas exchange. Mortality was evaluated four days after the infestation. The results of both trials demonstrate a higher CRW mortality attributable to the cloned mis and war genes of MB438. Table 9 illustrates the qualitative activity of the cloned toxins of MB438 in preparations of crude cultures of E. coli against the rootworm of western corn. TABLE 9 10 Table 10 illustrates the dose-dependent activity of MB438 toxins cloned in crude culture preparations of B.t. against 15 the worm of the roots of the corn of the west. In tables 9 and 10, the bold numbers represent the percentage of mortality, the numbers in parentheses indicate the number of dead larvae divided by the total number of test larvae.

TABLE 10 EXAMPLE 9 Insertion of Toxin Genes in Plants One aspect of the present invention is the transformation of plants with genes encoding the insecticidal toxin of the present invention. The transformed plants are resistant to the attack of the target pest. The genes encoding pesticidal toxins, according to what is described herein, can be inserted into plant cells using a variety of techniques known in the art. These techniques include the 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. 15 If Agrobacteria are used for transformation, the DNA to be inserted has to be cloned into special plasmids, that is, into an intermediate vector or into a binary vector. Intermediate vectors can be integrated into the T or R plasmid by homologous recombination due to sequences that are homologous to the T-DNA sequences. Plasmid T or R also contains the vir region necessary for T-DNA transfer. Intermediate vectors can not replicate in Agrobacteria. The intermediate vector can be transferred to Agrobacterium tumefaciens by means of an auxiliary plasmid (conjugation). Binary vectors can be replicated in both E. coli and ^^^^^^^^^^^^^^^^^^^^ ^^^^^^ in Agrobacteria. These comprise a selection marker gene and a binder or polylinker framed by the right and left end regions of the T-DNA. They can be directly transformed into Agrobacteria (Holsters et al. [1978] Mol. Gen. Genet 163: 181-187). The Agrobacterium 5 used as the host cell must contain a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA to the plant cell. It may contain more T-DNA. The bacterium thus transformed is used for the transformation of plant cells. The plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of DNA to the plant cell. Then whole plants can be regenerated from the infected plant material (for example leaf pieces, stem segments, roots, but also protoplasts or cells grown in suspension) in a suitable medium that may contain antibiotics or biocides for selection. The plants thus obtained can be analyzed below to confirm the presence of the inserted DNA. There are no special requirements with respect to plasmids in case of injection and electroporation. It is possible to use common plasmids such as, for example, pUC derivatives. In the transformation by biolistics, plasmid DNA or linear DNA can be used. A large number of cloning vectors are available that consist of a replication system in E. coli and a marker that allows the selection of the transformed cells in order to prepare them for insertion of strange genes in higher plants. The vectors consist, for example, of the pBR322 series, the pUC series, the M13mp series, pACYC184, etc. Accordingly, the sequence encoding the Bacillus toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultured in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. The sequence analysis, restriction analysis, electrophoresis and other biological-biochemical-molecular methods are generally carried out as analysis methods. After every By manipulation, the DNA sequence used can be excised and linked to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of insertion of the desired genes into the plant, other DNA sequences may be necessary. Yes, for example, plasmid T or R is used for the transformation of the cell In the case of plants, at least the right border must be joined, although frequently both the right and left borders of the T-DNA of plasmid T or R as the flanking region of the genes to be inserted. The use of T-DNA for the transformation of plant cells has been intensively investigated and sufficiently described in EP 102 516; 20 Hoekema (1985) in: The Binary Plant Vector System, Offset-durkkerij Kanters B.V, Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4: 1-46; and An et al. (1985) EMBO J. 4: 277-287.

Once the inserted DNA has been integrated into the genome, it is relatively stable there and, in general, does not come back out. This normally contains a selection marker which confers on the transformed plant cells resistance to a bokid or an antibiotic such as kanamycin, G418, bleomycin, hygromycin or chloramphenicol, among others. The individually used marker must, therefore, allow the selection of transformed cells instead of cells that do not contain the inserted DNA. The transformed cells regenerate into morphologically normal plants in the usual way. If a transformation event involves a germline cell, in that case the inserted DNA and the corresponding phenotypic trait (s) are transmitted to the progeny plants. Such plants can be grown as they are usually done and crossed with plants that have the same hereditary factors transformed or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties. In a preferred embodiment of the present invention, the plants are transformed with genes in which the codon usage has been optimized for the plants. See, for example, United States Patent No. 5,380,831. It is to be understood that the examples and embodiments described herein are presented for illustrative purposes only and that technicians trained in the environment may suggest various modifications or changes to the complexion of the same and these must be included in the spirit and purpose of this application. ..-..-; 3 LIST OF SEQUENCES < 110 > Schnepf, Ernest 11 Narva, Kenneth E Stockhoff, Brian A Finstad I-ee, Stacey Walz, ikki Sturgis, Blake 10 < 120 > Toxins and pesticide genes from strains of Bacillus laterosporus < 130 > MA-719XC2 15 < 140 > < 141 > < 150 > 60 / 095,955 < 151 > 1998-08-10 20 < 150 > 60 / 138,251 < 151 > 1999-06-08 < 160 > 10 25 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 2645 30 < 212 > DNA < 213 > Bacillus laterosporus < 400 > 1 atgaagaaga agttagcaag tgttgtaacg tgtacgttat tagctcctat gtttttgaat 60 atgctgttta ggaaatgtga cgcagacagc aaaacaaatc aaatttctac aacacagaaa 120 aagagatgga aatcaacaga ccgaaaagga ttacttgggt attatttcaa aggaaaagat 180 tttagtaatc ttactatgtt tgcaccgaca ctcttattta cgtgatagta tgatcaacaa 240 aactattaga acagcaaata taaaaaacaa caagaatatc agtctattcg ttggattggt 300 ttgattcaga gtaaagaaac gggagatttc acatttaact tgaacaggca tatctgagga 360 40 tcaatgggaa attatagaaa aataaaggga aattatttct aagaaaagca agttgtccat 420 ttagaaaaag gaaaattagt tccaatcaaa atagagtatc aatcagatac aaaatttaat 480 attgacagta aaacatttaa ttatttaaaa agaacttaaa tagatagtca aaaccaaccc 540 agcaagatga cagcaagtcc actgagaaat cctgaattta atcacaggaa acaagaaaga 600 aaccatcgaa ttcttagcga aataaatctt ttcactcaaa aaatgaaaag ggaaattgat 660 45 gaagacacgg atacggatgg ggactctatt cctgaccttt gggaagaaaa tgggtatacg 720 attcaaaata gaatcgctgt aaagtgggac gattctytag caagtaaagg gtatacgaaa 780 atccgctaga tttgtttcaa aagtcacaca gttggtgatc cttatacaga ttatgaaaag 840 gcagcaagag acctagattt gtcaaatgca aaggaaacgt ttaacccatt ggtagctgct 900 tttccaagtg tgaatgttag tatggaaaag gtgatattat caccaaatga aaatttatcc 960 50 aatagtgtag agtctcattc atccacgaat tggtcttata caaatacaga aggtgcttct 1020 gttgaagcgg ggattggacc aaaaggtatt tcgttcggag ttagcgtaaa ctatcaacac 1080 tctgaaacag ttgcacaaga atggggaaca tctacaggaa atacttcgca attcaatacg 55 1140 ^^ ^ & ^^ ¿^ £ ^ gcttcagcgg gatatttaaa tgcaaatgtt cgatataaca atgtaggaac tggtgccatc 1200 tacgatgtaa aacctacaac aagttttgta ttaaataacg atactatcgc aactattacg 1260 5 gcgaaatcta attctacagc cttaaatata tctcctggag aaagttaccc gaaaaaagga 1320 caaaatggaa tcgcaataac atcaatggat gattttaatt cccatccgat tacattaaat 1380 aaaaaacaag tagataatct gctaaataat aaacctatga tgttggaaac aaaccaaaca October 1440 gatggtgttt ataagataaa agatacacat ggaaatatag taactggcgg agaatggaat 1500 aacaaatcaa ggtgtcatac ggctaaaaca gcgtctatta ttgtggatga tggggaacgt 1560 15 gtagcagaaa aacgtgtagc ggcaaaagat tatgaaaatc cagaagataa aacaccgtct 1620 aagatgccct ttaactttaa tatccagatg gaagctttca aaataaaaga aatagaggga 1680 ttattatatt ataaaaacaa accgatatac gaatcgagcg ttatgactta cttagatgaa 20 1740 aagaagtgac aatacagcaa caaacaatta aatgatacca taaagatgta ctgggaaatt 1800 atgatgtaaa agtcatttat actgactcca aaaatgaatg ttacaatcaa attgtctata 1860 25 ctttatgata atgctgagtc taatgataac tcaattggta aatggacaaa cacaaatatt 1920 gtttcaggtg gaaataacgg aaaaaaacaa tatt cttcta ataatccgga tgctaatttg 1980 cagatgctca acattaaata agaaaaatta aataaaaatc gtactattat ataagtttat 30 2040 atatgaagtc agaaaaaaac acacaatgtg agattactat agatggggag atttatccga 2100 tcactacaaa aacagtgaat gtgaataaag acaattacaa aagattagat attatagctc 2160 35 aagtaatcca ataatataaa atttcttcaa ttcatattaa aacgaatgat gaaataactt 2220 tattttggga tgatatttct ataacagatg tagcatcaat aaaaccggaa aatttaacag 2280 attcagaaat taaacagatt tatagtaggt atggtattaa gttagaagat ggaatcctta 40 2340 ttgataaaaa aggtgggatt cattatggtg aatttattaa tgaagctagt tttaatattg 2400 aaattatgtg aaccattgca acaaaatata aagttactta tagtagtgag ttaggacaaa 2460 45 acgtgagtga cacacttgaa tttacaagga agtgataaaa tgggacaatt aaatttgatt 2520 ttacaaaata tagtraaaat gaacaaggat tattttatga cagtggatta aattgggact 2580 tgctattact ttaaaattaa tatgatggta aagagatgaa tgtttttcat agatataata 50 2640 2645 aatag 55 < 210 > 2 < 211 > 1341 gffj¡ ^. * »« ^ ** ^ - ^^^^^^^ * - * '"- * H-" * 8 H ^^ < 212 > DNA < 213 > Bacillus laterosporus < 400 > 2 atgtttatgg tttctaaaaa attacaagta gttactaaaa ctgtattgct tagtacagtt 60 ttctctatat ctttattaaa taatgaagtg ataaaagctg aacaattaaa tataaattct 120 caaagtaaat atactaactt gcaaaatcta aaaatcactg acaaggtaga ggattttaaa 180 gaagataagg aaaaagcgaa agaatggggg aaagaaaaag aaaaagagtg gaaactaact 240 gctactgaaa aaggaaaaat gaataatttt ttagataata aaaatgatat aaagacaaat 300 tataaagaaa ttactttttc tatggcaggc tcatttgaag atgaaataaa agatttaaaa 360 agatgtttga gaaattgata taaaaccaat ctatcaaatt ctattatcac ctataaaaat 420 gtggaaccga caacaattgg atttaataaa tctttaacag aaggtaatac gattaattct 480 gatgcaatgg cacagtttaa agaacaattt ttagataggg atattaagtt tgatagttat 540 ctagatacgc atttaactgc tcaacaagtt tccagtaaag aaagagttat tttgaaggtt 600 acggttccga gtgggaaagg ttctactact ccaacaaaag caggtgtcat tttaaataat 660 agtgaataca aaatgctcat tgataatggg tatatggtcc atgtagataa ggtatcaaaa 720 aaggggtgga gtggtgaaaa gtgcttacaa attgaaggga ctttaaaaaa gagtcttgac 780 tttaaaaatg atataaatgc tgaagcgcat agctggggta tgaagaatta tgaagagtgg 840 gctaaagatt taaccgattc gcaaagggaa gctttagatg ggtatgctag gcaagattat 900 aaagaaatca ataattattt aagaaatcaa ggcggaagtg gaaatgaaaa actagatgct 960 atatttctga caaataaaaa tgctttaggg aagaaaccaa taccggaaaa tattactgtg 1020 tatagatggt gtggcatgcc ggaatttggt tatcaaatta gtgatccgtt accttcttta 1080 25 aaagattttg aagaacaatt tttaaataca atcaaagaag tatgagtaca acaaaggata 1140 agcttatcga gtgaacgtct tgcagctttt ggatctagaa aaattatatt acgattacaa 1200 gttccgaaag gaagtacggg tgcgtattta agtgccattg gtggatttgc aagtgaaaaa 30 1260 ttgataaaga gagatcctac tagtaaatat catattgata aagtaacaga ggtaattatt 1320 aaggtgttaa gcgatatgta g 1341 35 < 210 > 3 < 211 > 20 < 12 > DNA 40 < 213 > Bacillus laterosporus < 400 > 3 ggrttamttg grtaytattt 20 45 < 210 > 4 < 211 > 20 < 212 > DNA < 213 > Bacillus laterosporus 50 < 400 > 4 atatckwaya ttkgcattta 20 55 < 210 > 5 < 211 > 1062 < 212 > DNA < 213 > Bacillus laterosporus < 400 > 5 ttattttaaa taattggata ggaaaagagt ttaatcatgt tactttgttc gcaccaacac 60 gtgataatac ccttatttat gatcaacaaa cagtagattc cttattggat aaaaaacaac 120 atctattcga aagaatatca tggattggtt tgattcaaag taaagaaacg ggtgatttca 180 catttaactt atcagatgat aaaaatgcaa ttatggaaat agatacaaaa accatttcgc 240 gaacaaacaa ataaaggaca gttgttcact tagaaaaagg aaagttagtc ccgataaaaa 300 ttgagtatca accaagacca aatagtaaat agggatagta aaatctttaa agagtttaaa 360 ttattcaaag tagatagtaa gcaacaatct ccaccaagtt caactagatg aattaagaaa 420 ccccggagtt taataaaaaa gaaacacaac attccttaga aaaagsc cc aaaaacaaat 480 ccnttttnac mcmcvrgaac cattgaaaaa gagatgaggg atgcntamcg gnatacagat 540 atcycctgga kggagatyyt cctttgggga agaaaatggg tataccaatc caaaataaag 600 ttagctggtc aaagttggra kgattccatt ccccsccgyt aaaagggt t accaaaattt 660 yccattttga ggttycyyaa gttggagatc tagtcataca ttatgaaaaa cctatactga 720 gcagcaagag atttagactt ggcccaatgc aaaagaaaca tttaacccat tagtagctgc 780 gtgaatgtga ttttccaagt atttggaaaa agtaatatta tccccaaatg aggatttatc 840 taacagtgta gaatctca tt cgtctacaaa ttggtcttat accaatacag aaggagtttc 900 tatcgaagct gggagtggtc cattgggtat ttcttatgga gtgagtgcta attatcaaca 960 ctctgaaaca gttgcaaaag aatggggaac atctacagga aatacttcgc aatttaatac 1020 agcttcagca gggtatctaa atgccaatat tcgatataag ce 1062 < 210 > 6 < 211 > 2355 < 212 > DNA < 213 > Bacillus laterosporus < 400 > 6 tgaaaaaaaa atgacataca gttagttagt gttgtaacct gtacgttatt agccccaatg 60 tttttgaatg gaaatgtaaa tcctgtttat geagacaate aaacaaatca gctttctaca 120 accaagaaaa gcgcaggaaa agaggtagat cgaaaaggat tactcggcta ttattttaaa 180 ggaaaagagt ttaatcatct tactttgttc gcaccaacac gtgataatac ccttatttat 240 gatcaacaaa cagtagattc cttattggat aaaaaacaac atctattcga aagaatatca 300 tggattggtt tgattcaaag taaagaaacg ggtgatttca catttaactt atcagatgat 360 aaaaatgcaa ttatggaaat agatacaaaa accatttcgc ataaaggaca gaacaaacaa 420 gttgttcact tagaaaaagg aaagttagtc ttgagtatca ccgataaaaa accagatcaa 480 atagtaaata gggatagtaa aatctttaaa gagtttaaat tattcaaagt agatagtaag 540 accaagttea caacaatctc actagatgaa ttaagaaacc ctgagtttaa taaaaaagaa 600 tcttagaaaa acacaacaat agcatcaaaa ttacacagaa acaaatcttt catgaaaaga 660 gatgaggatg ctacggatac agatggagat tetattectg acctttggga agaaaatggg 720 tataccatcc aaaataaagt agctgtcaag tgggatgatt cattcgccgc taaagggtat 780 acaaaatttg tttetaatec atttgatagt catacagttg gagatcecta tactgattat 840 gaaaaagcag caagagat tt agacttggcc aatgcaaaag aaacatttaa eccattagta 900 gctgcttttc caagtgtgaa tgtgaatttg gaaaaagtaa tattatcccc aaatgaggat 960 ttatetaaca gtgtagaatc tcattcgtct acaaattggt cttataccaa tacagaagga 1020 gtttctatcg aagctgggag tggtccattg ggtatttctt atggagtgag tgetaattat 1080 caacactctg aaacagttgc aaaagaatgg ggaacateta caggaaatac ttcgcaattt 1140 aatacagctt cagcagggta tctcaatgcc aatgttcgat acaataatgt gggaacaggt 1200 gcgatttatg aggtgaaacc tacaacaggt tttgtgttag ataacgatac tgtagcaaca 1260 attaccgcaa aatcgaattc gacagcttta agtatatctc caggagaaag ttatccgaaa 1320 5 aaaggacaaa atgggattgc aattaataca atggatgatt ttaattccca tccgattaca 1380 ttaaataaac aacaattaga tcaaatattt aataataaac ctcttatgtt agaaacaaat 1440 caggcagatg gtgtttataa aataaaagat acaagcggta atattgtgac tggtggagaa 10 1500 tggaacggtg ttatccaaca aattcaagca aaaacagcct ctattatcgt tgatacggga 1560 gaaggtgttt cagaaaagcg tgtcgcagca aaagattatg ataatcctga ggataaaaca 1620 15 ccttctttgt ctttaaaaga ggcacttaaa cttggatatc cagaagaaat taaagaaaaa 1680 gatggattgt tgtactataa tgacaaacca atttacgaat ctagtgttat gacttatcta 1740 gatgagaata cagcaaaaga agtaaaagaa caattaaatg atatcactgg aaaatttaaa 20 1800 gatgtgaagc agttatttga tgtgaaactt acacctaaaa tgaattttac tatcaagtta 1860 gctacgctat atgatggagc tgaagatggg tcatctccta ctgatgtagg tatcagtagt 1920 25 cctttagggg aatgggcatt taaaccagat ataaataatg ttgaaggggg gaatactgga 1980 aaaagacaat accaattaag taaaaataaa gatggttatt actatggtat gttagctcta 2040 tcaccagagg tatcaaacaa gttgaaaaaa aattatcaat actatatcag tatgtctata 30 2100 aaagcagatg ctggtgtgga acctacagta acagttatgg ataatttaaa ttgtatagta 2160 gataaaaaat taaaattaag tagtaacggt tatcaaagat ttgatatttt agtagataat 2220 35 tctgaatccc atccaataaa tgtgatggta atcgatttag gtgtaagcag ccaagattat 2280 aacaattata gtaagaatat atacattgat gatataacaa ttacagaggt ttcagctatg 2340 aaagtgaaaa attag 40 2355 < 210 > 7 < 211 > 784 45 < 212 > PRT < 213 > Peptide sequence < 400 > 7 Met Thr Tyr Met Lys Lys Lys Leu Val Ser Val Val Thr Cys Thr Leu 50 1 5 10 15 Leu Ala Pro Met Phe Leu Asn Gly Asn Val Asn Pro Val Tyr Ala Asp 20 25 30 55 Asn Gln Thr Asn Gln Leu Ser Thr Wing Gln Glu Asn Gln Glu Lys Glu 35 40 45 Val Asp Arg Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Glu Phe 50 55 60 Asn His Leu Thr Leu Phe Wing Pro Thr Arg Asp Asn Thr Leu lie Tyr 65 70 75 80 Asp Gln Gln Thr Val Asp Ser Leu Leu Asp Lys Lys Gln Gln Glu Tyr 85 90 95 Gln Ser lie Arg Trp lie Gly Leu lie Gln Ser Lys Glu Thr Gly Asp 100 105 110 Phe Thr Phe Asn Leu Ser Asp Asp Lys Asn Ala Met Glu lie Asp 115 120 125 Thr Lys Thr lie Ser His Lys Gly Gln Asn Lys Gln Val Val His Leu 130 135 140 Glu Lys Gly Lys Leu Val Pro He Lys He Glu Tyr Gln Pro Asp Gln 145 150 155 160 He Val Asn Arg Asp Ser Lys He Phe Lys Glu Phe Lys Leu Phe Lye 165 170 175 Val Asp Ser Lys Gln Gln Ser His Gln Val Gln Leu Asp Glu Leu Arg 180 185 190 Asn Pro Glu Phe Asn Lys Lys Glu Thr Gln Gln Phe Leu Glu Lys Wing 195 200 205 Ser Lys Thr Asn Leu Phe Thr Gln Asn Met Lys Arg Asp Glu Asp Wing 210 215 220 Thr Asp Thr Asp Gly Asp Ser He Pro Asp Leu Trp Glu Glu Asn Gly 225 230 235 240 Tyr Thr He Gln Asn Lys Val Wing Val Lys Trp Asp Asp Ser Phe Wing 245 250 255 Wing Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Phe Asp Ser His Thr 260 265 270 Val Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Wing Ala Arg Asp Leu Asp 275 280 285 Leu Wing Asn Wing Lys Glu Thr Phe Asn Pro Leu Val Wing Wing Phe Pro 290 295 300 Ser Val Asn Val Asn Leu Glu Lys Val He Leu Ser Pro Asn Glu Asp 305 310 315 320 Leu Being Asn Being Val Glu Being His Ser Being Thr Asn Trp Being Tyr Thr 325 330 335 Asn Thr Glu Gly Val Ser He Glu Wing Gly Ser Gly Pro Leu Gly He 340 345 350 Ser Tyr Gly Val Ser Wing Asn Tyr Gln His Ser Glu Thr Val Wing Lys 355 360 365 5 Glu Trp Gly Thr Ser Thr Gly Asn Thr Ser Gln Phe Asn Thr Wing Ser 370 375 380 Wing Gly Tyr Leu Asn Wing Asn Val Arg Tyr Asn Asn Val Gly Thr Gly 10 385 390 395 400 Wing He Tyr Glu Val Lys Pro Thr Thr Gly Phe Val Leu Asp Asn Asp 405 410 415 15 Thr Val Wing Thr He Thr Wing Lys Ser Asn Ser Thr Wing Leu Ser He 420 425 430 Ser Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gln Asn Gly He Wing He 435 440 445 20 Asn Thr Met Asp Asp Phe Asn Ser His Pro He Thr Leu Asn Lys Gln 450 455 460 Gln Leu Asp Gln He Phe Asn Asn Lys Pro Leu Met Leu Glu Thr Asn 465 470 475 480 Gln Wing Asp Gly Val Tyr Lys He Lys Asp Thr Ser Gly Asn He Val 485 490 495 30 Thr Gly Gly Glu Trp Asn Gly Val He Gln Gln He Gln Wing Lys Thr 500 505 510 Wing Being He He Val Asp Thr Gly Glu Gly Val Ser Glu Lys Arg Val 515 520 525 35 Wing Wing Lys Asp Tyr Asp Asn Pro Glu Asp Lys Thr Pro Ser Leu Ser 530 535 540 Leu Lys Glu Wing Leu Lys Leu Gly Tyr Pro Glu Glu He Lys Glu Lys 40 545 550 555 560 Asp Gly Leu Leu Tyr Tyr Asn Asp Lys Pro He Tyr Glu Ser Ser Val 565 570 575 45 Met Thr Tyr Leu Asp Glu Asn Thr Wing Lys Glu Val Lys Glu Gln Leu 580 585 590 Asn Asp He Thr Gly Lys Phe Lys Asp Val Lys Gln Leu Phe Asp Val 595 600 605 50 Lys Leu Thr Pro Lys Met Asn Phe Thr He Lys Leu Wing Thr Leu Tyr 610 615 620 Asp Gly Wing Glu Asp Gly Ser Ser Pro Thr Asp Val Gly He Ser Ser 55 625 630 635 640 Pro Leu Gly Glu Trp Wing Phe Lys Pro Asp He Asn Asn Val Glu Gly 645 650 655 Gly Asn Thr Gly Lys Arg Gln Tyr Gln Leu Ser Lys Asn Lys Asp Gly 5 660 665 670 Tyr Tyr Tyr Gly Met Leu Ala Leu Ser Pro Glu Val Ser Asn Lys Leu 675 680 685 10 Lys Asn Tyr Gln Tyr Tyr He Ser Met Be He Lys Wing Asp Wing 690 695 700 Gly Val Glu Pro Thr Val Thr Val Met Asp Asn Leu Asn Cys He Val 705 710 715 720 15 Asp Lys Lys Leu Lys Leu Ser Being Asn Gly Tyr Gln Arg Phe Asp He 725 730 735 Leu Val Asp Asn Ser Glu Ser His Pro He Asn Val Met Val He Asp 20 740 745 750 Leu Gly Val Ser Ser Gln Asp Tyr Asn Asn Tyr Ser Lys Asn He Tyr 755 760 765 25 He Asp Asp He Thr He Thr Glu Val Ser Wing Met Lys Val Lys Asn 770 775 780 < 210 > 8 < 211 > 1356 35 < 212 > DNA < 213 > Bacillus laterosporus < 400 > 8 atggtatcta aaaagttaca attaattaca aaaactttag tgtttagtac agttttatct 60 40 ataccgttat tgaacaatag tgagataaaa gcggaacaat taaatatgaa ttctcaaatt 120 acttccaaaa aaatatccta tataaatatc gctgataagc cagtagattt taaagaggat 180 aaagaaaaag cacgagaatg gggaaaagaa aaggaaaaag agtggaaact aactgttact 240 aaataaatga gaaaaaggaa ttttttagat gataaagatg gattaaaaac aaaatataaa 300 gaaattaatt tttctaagaa ctttgaatat gaaacagagt taaaagagct tgaaaaaatt 360 45 aataccatgc tagataaagc aaatctaaca aattcaattg tcacgtataa aaatgttgag 420 taggattcaa cctacaacaa tcaatctttg attgaaggga atcaaattaa tgccgaagct 480 caacaaaagt tcaaggaaca caggatatta atttttagga aatttgatag ttatttggat 540 ctgaacaaaa atgcacttaa tgtttccagt aaagaaaggg ttattttaaa agttacagta 600 cctagtggga aaggttctac tcccacaaaa gcaggtgttg ttttaaataa taatgaatac 660 50 ttgataatgg aagatgttga atatgtacta catgtagaaa acataacgaa agttgtaaaa 720 aatgtttaca aaaggacagg agttgaagga acgttaaaaa agagcttgga ctttaaaaat 780 gtaagggaga gatagtgacg ttcctgggga aagaaaaatt acaaggaatg gtctgatact 840 ttaacaactg atcaaagaaa agacttaaat gattatggtg tgcgaggtta taccgaaata 900 aataaatatt tacgtgaagg tgataccgga aatacagagt tggaggaaaa aattaaaaat 960 55 atttctgacg cactagaaaa gaatcctatc cctgaaaaca ttactgttta tagatattgc 1020 .. ^ - ad t.at * JiAA. ^ - ^^ fiMMifc & LM? ^ HÉ. ggaatggcgg aatttggtta tccgattaaa cctgaggctc cttccgtaca agattttgaa 1080 gagagatttt tggatactat taaggaagaa aaaggatata tgagtacgag cttatccagt 1140 gatgcgactt cttttggtgc aagaaaaatt atattaagat tgcaagtacc aaaaggaagt 1200 tcaggagcat atgtagctgg tttagatgga tttaaacccg cagagaagga gattctcatt 1260 gataagggaa gcaagtatcg tattgataaa gtaacagaag tggttgtgaa aggtactaga 1320 aaacttgtag tcgatgctac attattaaca aaataa 1356 < 210 > 9 < 211 > 451 < 212 > PRT < 213 > Peptide sequence < 400 > 9 Met Val Ser Lys Lys Leu Gln Leu He Thr Lys Thr Leu Val Phe Ser 1 5 10 - 15 Thr Val Leu Ser He Pro Leu Leu Asn Asn Ser Glu He Lys Wing Glu 20 25 30 Gln Leu Asn Met Asn Ser Gln He Lys Tyr Pro Asn Phe Gln Asn He 35 40 45 Asn He Wing Asp Lys Pro Val Asp Phe Lys Glu Asp Lys Glu Lys Wing 50 55 60 Arg Glu Trp Gly Lys Glu Lys Glu Lys Glu Trp Lys Leu Thr Val Thr 65 70 75 80 Glu Lys Gly Lys He Asn Asp Phe Leu Asp Asp Lys Asp Gly Leu Lys 85 90 95 Thr Lys Tyr Lys Glu He Asn Phe Ser Lys Asn Phe Glu Tyr Glu Thr 100 105 110 Glu Leu Lys Glu Leu Glu Lys He Asn Thr Met Leu Asp Lys Wing Asn 115 120 125 Leu Thr Asn Ser He Val Thr Tyr Lys Asn Val Glu Pro Thr Thr He 130 135 140 Gly Phe Asn Gln Ser Leu He Glu Gly Asn Gln He Asn Wing Glu Wing 145 150 155 160 Gln Gln Lys Phe Lys Glu Gln Phe Leu Gly Gln Aep He Lys Phe Asp 165 170 175 Ser Tyr Leu Asp Met His Leu Thr Glu Gln Asn Val Ser Ser Lys Glu 180 185 190 ^ Arg Val He Leu Lys Val Thr Val Pro Ser Gly Lys Gly Ser Thr Pro 195 200 205 Thr Lys Wing Gly Val Val Leu Asn Asn Glu Tyr Lys Met Leu He 210 215 220 Asp Asn Gly Tyr Val Leu His Val Glu Asn He Thr Lys Val Val Lys 225 230 235 240 Lys Gly Gln Glu Cys Leu Gln Val Glu Gly Thr Leu Lys Lys Ser Leu 245 250 255 Asp Phe Lys As Asp Asp As Asp Gly Asp Gly Asp Ser Trp Gly Lys Lys 260 265 270 Asn Tyr Lys Glu Trp Ser Asp Thr Leu Thr Thr Asp Gln Arg Lys Asp 275 280 285 Leu Asn Asp Tyr Gly Val Arg Gly Tyr Thr Glu He Asn Lys Tyr Leu 290 295 300 Arg Glu Gly Asp Thr Gly Asn Thr Glu Leu Glu Glu Lys He Lys Asn 305 310 315 320 He Ser Asp Ala Leu Glu Lys Asn Pro He Pro Glu Asn He Thr Val 325 330 335 Tyr Arg Tyr Cys Gly Met Wing Glu Phe Gly Tyr Pro He Lys Pro Glu 340 345 350 Wing Pro Ser Val Gln Asp Phe Glu Glu Arg Phe Leu Asp Thr He Lys 355 360 365 Glu Glu Lys Gly Tyr Met Ser Thr Ser Leu Ser Asp Wing Thr Ser 370 375 380 Phe Gly Wing Arg Lys He He Leu Arg Leu Gln Val Pro Lys Gly Ser 385 390 395 400 Ser Gly Wing Tyr Val Wing Gly Leu Asp Gly Phe Lys Pro Wing Glu Lys 405 410 415 Glu He Leu He Asp Lys Gly Ser Lys Tyr Arg He Asp Lys Val Thr 420 425 430 Glu Val Val Val Lys Gly Thr Arg Lys Leu Val Val Asp Ala Thr Leu 435 440 445 Leu Thr Lys 450 < 210 > 10 < 211 > 1041 < 212 > DNA ? ^^^ g ^ | Síljg ^ jg ^^ g ^^^^^^ < 213 > Bacillus laterosporus < 400 > 10 attaattggg tattatttta aaggaaaaga ttttaatgat cttaccttgt ttgcaccgac 60 acgtgataat actcttattt atgaccaaca aacagcaaat acactagtag atcaaaagca 120 tcaagaatat cattctattc gctggattgg attgattcag agtagtgcaa caggagattt 180 cacatttaaa ttgtcagatg atgaaaatgc catcattgaa ttggatggga aagttatttc 240 tgaaaaaggt aacaataaac aaagtgttca tttagaaaaa ggacagttgg tgcaaataaa 300 aattgagtac caatcagacg atgcattaca tatagataat aaaactttta aagagcttaa 360 gttattcaag atagatagtc aaaatcactc tctacaagtt caacaagatg aactgagaaa 420 ccctgagttt aataagaaag aaacgcaaag aattcttaaa gaaagcatcg aaagcaaatc 480 tttttaccgc aaaaaaccga aaagagatat tgatgaagat acggatacag atggagattc 540 tatccctgat gcttgggaag aaaacgggta taccattcaa aacaaagtag cagtcaaatg 600 ggatgattcg ttagcaagta aagggtataa aaaatttact tctaatccac tagaagcaca 660 cacagttgga gatccctata gtgattatga aaaagctgca agagatatgc ccttatcgaa 720 tgcaaaagaa acttttaatc ctctggttgc cgcctttcca tcagtaaatg ttagtttaga 780 aaaggtgatt ttatccaaaa atgaagacct ttcccatagc gttgaaagca gtcaatctac 840 caattggtct tatacca ata ctgaaggcgt taacgtcaat gctggatggt caggcttagg 900 acctagtttt ggagtttctg ttaactatca acatagtgaa actgtagcca atgaatgggg 960 ttctgcgacg aatgatggca cacatataaa tggagcggaa tctgcttatt taaatgccaa 1020 tgtacgatat aagggcgaat t 1041

Claims (51)

NOVELTY OF THE INVENTION CLAIMS

1. An isolated polynucleotide that encodes a toxin that is an active against a corn rootworm pest where said toxin is selected from the group consisting of a MIS type protein obtained from the isolated MB438 from Bacillus laterosporus that has an accession number NRRLB-30085, a type WAR protein that is

10 obtains from MB438 isolate of Bacillus laterosporus having an accession number NRRLB-30085, a MIS type protein obtained from Bacillus Laterosporus isolate MB439 having an accession number NRRL B-30086, a WAR type protein which is obtained from the MB439 isolate of Bacillus laterosporus which has an accession number NRRL

15 B-30086, a protein of about 1-10 kDa which is obtained from the MB438 isolate of Bacillus laterosporus having an accession number NRRL B-30085, and a protein of approximately 1-10 kDa which is obtained from the isolated MB439 Bacillus laterosporus that have an accession number NRRL B-30086.

2. The toxin according to claim 1, further characterized in that said toxin is a MIS type protein that is obtained from the MB438 isolate of Bacillus laterosporus having an accession number NRRL B-30085.

3. - The toxin according to claim 1, characterized further perqué said toxin is a WAR type protein that is obtained from MB438 isolate Bacillus laterosporus having an accession number NRRL B-30085.

4. The toxin according to claim 1, further characterized in that said toxin is a MIS type protein that is obtained from the MB439 isolate of Bacillus laterosporus having an accession number NRRL B-30086.

5. The toxin according to claim 1, further characterized in that said toxin is a WAR type protein that is obtained from the MB439 isolate of Bacillus laterosporus having an accession number NRRL B-30086.

6. The toxin according to claim 1, further characterized in that said toxin is a protein of about 1-10 kDa that is obtained from the MB438 isolate of Bacillus laterosporus having an accession number NRRL B-30085.

7. The toxin according to claim 1, further characterized in that said toxin is a protein of approximately 1-10 kDa that is obtained from the MB439 isolate of 0 Bacillus laterosporus having an accession number NRRL B-30086.

8. The toxin according to claim 2, further characterized in that said MIS-type protein is obtained from the clone MR957 of E.coli having an accession number NRRL B-30048, said

^ ^^^ «^ ¿^ ^. ^ ^, ^^^^^^, ^, and. ^ t ^^ ^ ^, k ^

clone comprises a gene encoding said protein, wherein approximately a 1 kilobase portion of said gene is amplified by the polymerase chain reaction using the primers of

SEQ ID NO: 3 and SEQ ID NO: 4.

9. The toxin according to claim 8, further characterized in that said gene comprises the nucleotide sequence of SEQ ID NO: 5.

10. The toxin according to claim 8, further characterized in that said gene comprises the nucleotide sequence of SEQ ID NO: 6.

11. An isolated toxin that is an active against a worm larva of the roots of corn, further characterized because said toxin comprises an amino acid sequence that has at least 90% identity with SEQ ID NO: 7.

12. An isolated toxin that is an active against a rootworm pest of corn, further characterized in that said toxin comprises the amino acid sequence of SEQ ID NO: 7 or an active portion thereof.

13. The toxin according to claim 12, further characterized in that said toxin comprises the amino acid sequence of SEQ ID NO: 7.

, - 14.- The toxin according to claim 3, further characterized in that said WAR type protein is obtained from the clone MR957 of E.coli having an accession number NRRL B-30048.

15. The toxin according to claim 3,

5 further characterized because said protein is obtained from the clone

MR957 of E. coli having an accession number NRRL B-30048, said clone comprises a gene encoding said protein, where the SEQ ID probe

NO: 2 hybridizes with said gene.

16. The toxin according to claim 15, further characterized in that said gene comprises the nucleotide sequence of SEQ ID NO: 8.

17. An isolated toxin that is an active against a rootworm pest of corn, further characterized in that said toxin comprises an amino acid sequence that has at least 90% identity with SEQ ID NO: 9.

18. The isolated toxin that is an active against a worm larva of the roots of corn, further characterized because said toxin comprises the amino acid sequence of SEQ ID NO: 9 or an active portion thereof.

19. The toxin according to claim 18, further characterized in that said toxin comprises the amino acid sequence of SEQ ID NO: 9.

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20. - The toxin according to claim 4, further characterized in that the MIS type protein is obtained from the MB439 isolate of Bacillus laterospurus having an accession number NRRL B-30086, said isolate comprises a gene encoding said protein, wherein a portion of approximately 1 kilobase of said gene is amplified by the polymerase chain reaction using SEQ ID NO: 3 and SEQ ID NO: 4.

21. The toxin according to claim 20, further characterized in that said gene comprises the nucleotide sequence of SEQ ID NO: 10.

22. The toxin according to claim 5, further characterized in that said WAR-type protein is obtained from the MB439 isolate of Bacillus laterosporus which has an accession number NRRL

B-30086, said isolate comprises a gene encoding said protein, wherein the probe of SEQ ID NO: 2 hybridizes with said gene.

23. An isolated polynucleotide that encodes a toxin according to any of the preceding claims.

24. The polynucleotide according to claim 23, further characterized in that said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 5.

25. The polynucleotide according to claim 23, further characterized in that said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6.

26. - The polynucleotide according to claim 23, further characterized in that said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8.

27. The polynucleotide according to claim 23, further characterized in that said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 10.

28. A plant cell or a microbial cell comprising an isolated polynucleotide that encodes a toxin according to any of claims 1-22.

29. A plant cell or a microbial cell comprising a first sequence of isolated polynucleotides and a second sequence of isolated polynucleotides, wherein said first sequence of polynucleotides encodes a toxin according to any of claims 2, 4, 8-13 , 20 and 21; and wherein said second polynucleotide sequence encodes a toxin according to any of claims 3, 5, 14-19 and 22.

30.- A plant cell or a microbial cell comprising an isolated polynucleotide according to any of the claims 23-27.

31. The cell according to claim 28, further characterized in that said cell is a bacterial cell.

32. The cell according to claim 28, further characterized in that said cell is a plant cell.

33. - The cell according to claim 32, further characterized in that said plant cell is a corn cell.

34. The cell according to claim 32, characterized in that said plant cell is a root cell.

35.- The cell according to claim 32, further characterized in that said plant cell is a cell of a corn root.

36.- A method for controlling a pest of coleopteran insects, further characterized in that said method comprises i administering to said pest a toxin according to any of claims 1-22.

37. The method according to claim 36, further characterized in that said plague of coleopteran insects is a worm of the roots of corn.

38. The method according to claim 36, further characterized in that said toxin is present in or on the tissue I of a plant. 39.- The method of compliance with claim 38, further characterized in that said toxin is produced by said plant. ! I 40.- The method according to claim 38, further characterized in that said method comprises applying said toxin to said plant.

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41. - The method according to claim 36, further characterized in that said toxin is applied to the roots of said plant.

The method according to claim 36, further characterized in that said method comprises administering to said pest a first toxin and a second toxin, wherein said first toxin is defined by any of claims 2, 4, 8-13, 20 and 21; and wherein said second toxin is defined by any of claims 3, 5, 14-19 and 22.

43.- A biologically pure culture of a Bacillus laterosporus strain selected from the group consisting of MB438, which has a number of access NRRL B-30085; and MB439, which has an access number NRRL B-30086.

44. The culture according to claim 43, further characterized in that said strain is MB438.

45. The culture according to claim 43, further characterized in that said strain is MB439.

46.- A method for controlling a plague of coleoptera insects further characterized in that said method comprises administering to said pest an isolate Bacillus laterosporus selected from the group consisting of

MB438, which has an access number NRRL B-30085; and MB439, which has an access number NRRL B-30086.

47. - The method according to claim 46, further characterized in that said isolatecreß < -MB438

48. The method of compliance with claim 46, further characterized in that said isolate is MB439.

49.- A method for controlling a plague of coleoptera insects further characterized in that said method comprises administering to said pest a fraction of approximately 1-10 kDa of the supernatant of a culture of a Bacillus laterosporus strain selected from the group consisting of MB438, which has an access number NRRL B-30085, and MB439; which has an access number NRRL B-30086.

50. The method according to claim 49, further characterized in that said strain is MB438.

51. The method according to claim 49, further characterized in that said strain is MB439.