MXPA99000653A - Bacillus thuringiensis scapes that show improved production of certain crystal proteins toxicas to lepidopt - Google Patents

Abstract

The present invention relates to Bacillus thuringiensis isolates active against lepidopteran pests produced and certain lepidopteran toxic crystal proteins produced through the Bacillus thuringiens isolates.

Description

BACILLUS THURINGI ENSIS STRAINS THAT SHOW IMPROVED PRODUCTION OF CERTAIN CRYSTAL PROTEINS TOXICOS A LEPIDÓPTERO FIELD OF THE INVENTION The present invention relates to strains of the variant Bacillus thuringiensis, (B. thuringiensis, or B. t.) Which produce increased amounts of certain insecticidal crystal proteins active at Lepidoptera and exhibit normal sporulation. The invention also relates to derivative strains that retain the ability to produce increased amounts of certain toxic crystal proteins to lepidoptera and normal sporulation.

BACKGROUND OF THE INVENTION The most widely used microbial pesticides are derived from the bacterium Bacillus thuringiensis. This bacterial agent is used to control a wide variety of caterpillars and leaf-eating beetles, as well as mosquitoes. Bacillus thuringiensis produces a proteinaceous parasporal body or crystal, which is toxic after being ingested by a susceptible insect host. For example, B. thuringiensis subsp. kurstaki HD-1 produces a crystal inclusion consisting of biotoxins called delta endotoxins or insecticidal crystal proteins (Cry) which are toxic to the larvae of a number of lepidopteran insects. The cloning, sequence and expression of a crystal protein gene HD1 B. t. in Escherichia coli have been described in the published literature (Schnepf, H. E. and Whitely, H.R., Proc. Nati, Acad.Sci. USA 1981, 78: 2893-2897; Schnepf et al.). The patents of E.U.A. Nos. 4,448,885 and 4,467,036 describe the expression of crystal protein B. t. in E. coli. Bacillus thuringiensis is a gram-positive bacterium that typically produces crystalline proteinaceous inclusions during sporulation. These crystal proteins B. t. or delta endotoxins are a large collection of insecticidal crystal proteins, which are highly toxic to specific insects and are an active ingredient in biological insecticides based on B. t. commercial. The crystal proteins of several isolates of strain B. t. have been identified as having insecticidal activity against insect larvae of the insects of the orders lepidoptera (caterpillar), coleoptera (beetle), diptera (mosquitoes, flies) and homoptera (aphids). These insecticidal crystal proteins (ICPs) from B. thuringiensis were originally classified as Cryl, Cryll, Crylll, and CrylV proteins based on their insecticidal activities (Hofte H. and HR Whiteley, Microbiol Rev. 1989, 53: 242-255 ). The most highly related proteins within each family were then assigned divisional letters such as CrylA, CrylB, CrylC, etc. The even more closely related proteins within each division were given names such as CrylCI, CrylC2, etc. (Hofte &Whitely, Microbiol, Rev. 1989, 53: 242-255). Cryl proteins, which encompass crystal proteins of approximately 130-140 kilodaltons (kDa) in molecular mass, exhibit toxicity to Lepidoptera. Cryll proteins are approximately 71 kDa in mass and can exhibit toxicity to both Lepidoptera and Diptera. Crylll proteins are approximately 73-74 kDa in mass and exhibit toxicity to Coleoptera. CrylV proteins represent a diverse group of proteins that exhibit dipterous toxicity. For the purpose of this description, the original Hofte and Whiteley nomenclature will be used. The bioinsecticide products of B. t. Commercials that are currently sold for the control of lepidopteran insects are based on strains of natural existence ("nature") or transconjugate. Transconjugate strains are created by transferring a plasmid encoding a crystal protein from a donor strain to a recipient strain through a conjugation-like process, resulting in a new strain of B. t. Plasmids can also be transferred from one strain to another through phage transduction. The natural and transconjugate strains are fermented in a broth medium, the spores and crystals are harvested, either through spray drying, centrifugation or otherwise, and are subsequently formulated for spray application or other application.

The insecticidal activity of these B. t. Bioinsecticides, as well as that of commercial products based on B. t., Is believed to result from the feeding of insect larvae on the crystal protein, typically in sprayed bioinsecticide deposits on leaves or other surfaces of the plant. The general details of the mode of action of insecticidal crystal proteins (ICPs) are evident. The ICPs contained within the proteinaceous crystals are released into the insect's midgut after ingestion and solubilization of the crystals at that site. In many cases, proteins are processed through the midgut proteases to a fully active degree. Activated toxins bind to the hairline boundary membranes (BBMs) of the midgut epithelium of the insect, a step that often requires the presence of fortuitous "receptor" proteins. This binding is followed by a case of apparent intercalation where the active toxin portion, or a portion thereof, contributes to the formation of ion channels as well as aggregates to form larger pores within the BBM, leading to osmotic imbalance , cell swelling and lysis. Intoxicated insect larvae stop feeding in minutes and eventually die. For many insect pests of lepidoptera, such as the devastating worm beet (Spodoptera exigua), the spores of B. t. present in the bioinsecticide formulation also contribute substantially to toxicity. The synergistic effect of the spores has been reported for a number of important lepidopteran insect pests, including S. exigua (Moar, WJ, et al., Appl. Environ Microbiol., 1995, 61: 2086-2092), Lymantria Dispar ( DuBois, N. and DH Dean, Biological Control 1995, 24: 1741-1747), and Plutella xylostella (Tang, JD, et al., Appl. Environ Microbiol., 1996, 62: 564-569). This spore effect on the insecticidal activity of B. t. it may be due to septicemia: the ability of the spore to germinate within the midgut of the insect, to penetrate the epithelium of the destroyed midgut, and to enter and proliferate within the "hoemcoel". For many insect pests lepidoptera, therefore, it is desirable that the bioinsecticide formulation B. t. contain a mixture of spores and crystals to achieve maximum efficiency. The amount of crystal protein produced in the fermentation must be increased as much as possible to the maximum in order to provide its economic and efficient use in the field. An increased concentration of the crystal protein in the formulated bioinsecticide promotes the use of reduced amounts of bioinsecticide per unit area of treated crop, without reducing the actual amount of crystal protein applied per unit area, thus allowing the most cost effective use of the product. bioinsecticide product. Alternatively, increased fermentation productions of crystal protein, which result in more concentrated formulations, can be used to increase the amount of crystal protein applied per unit area, thereby improving the performance of the bioinsecticide product. Previous efforts to create mutants or variants of B. strains. that show improved production of crystal proteins have been related mainly to the production of toxic glass proteins to coleoptera or diptera, not crystal proteins toxic to Lepidoptera Cryl. Also, most of these examples describe oligoesporogenic or asporogeneous variants (produce few, if any, spores) of B. t. which show an increased crystal protein production. As noted above, total spore production is a desirable aspect for strain B. t. activates lepidoptera used for the production of a commercial bioinsecticide. The patent of E.U.A. No. 5,006,336, issued to Payne, describes the isolate B. t. natural (PS122D3), active against coleopteran insects, which produces more toxic protein to Coleoptera (Cryl 11 A) than a B. t. toxic to coleoptera not related B. t. San Diego. Strain PS122D3 is not a variant of strain B. t. San Diego. The patent of E.U.A. No. 4,996,156, issued to Zaehner et al., describes a mutant strain of B. t. israelensis, active in dipterans, which produces crystal proteins but is asporogenic.

European Patent Application Publication No. 0 099 30 of Fitz-James, describes mutants of B. t. israelensis, obtained using a chemical mutagen, which produces up to 1.5 times the amount of toxic crystal protein to dipterans as does the progenitor strain. European Patent Application Publication No. 0 228 228 of Mycogen Corporation discloses asporogenic Bacillus thuringiensis mutants obtained through the treatment of progenitor strains with ethidium bromide. Said mutants B. t. they are described as being more efficient in the production of toxic glass-beetle protein (Cryll I A). PCT International Patent Application Publication No. WO 94/28724 of Ecogen, Inc., discloses a naturally occurring ascorbicnic Bacillus thuringiensis mutant that exhibits high levels of CrylllA crystal protein. PCT International Patent Application Publication No. WO 91/07481 by Novo Nordisk A / S, discloses a mutant of Bacillus thuringiensis tenebrionis, which was obtained through gamma irradiation and which produces twice the amount of the crystal protein toxic to Coleoptera (Cryl 11 A) obtained from the parental strain. The patent of E.U.A. No. 4,990,332, issued to Payne et al., describes a mutant strain B. t. toxic kapstaki to lepidoptera (PS85a1-168) that produces crystal proteins in amounts "equal to or greater than the wild type" but is asporogenic. The present invention relates to the variant of natural existence of strain B. t. kurstaki EG4923 which is more efficient in the production of toxic crystal protein to Cryl lepidopteran than the progenitor strain EG4923. The variant strain, designated EG4923-4, also exhibits efficient sporulation comparable to that of the progenitor strain, making it ideal for use as a toxic bioinsecticide to lepidoptera of cost-effective, particularly against insect pests such as Spodoptera exigua.

COMPENDIUM OF THE INVENTION The present invention relates to novel isolates of Bacillus thuringiensis, which have activity against all the plagues of lepidoptera tested. This invention also relates to certain toxin crystal proteins produced by the B.t. strains, said proteins being toxic to lepidopteran insects. The toxin crystal proteins are the active ingredient in insecticidal compositions, which are also the subject of the present invention. The genes encoding these toxin proteins can be transferred to the novel Bacillus thuringiensis strain of this invention via plasmid vectors. Specifically, the invention comprises the novel isolate of B. t. designated EG4923-4, derivatives thereof, and toxin crystal proteins derived from these B. t. isolates, which are active against lepidopteran pests. The Bacillus thuringiensis strains of this invention include a biologically pure culture of Bacillus thuringiensis bacteria deposited in the Agricultural Research Service Culture Collection, Northern Regional Research Laboratory (NRRL), having accession number NRRL B-21577 and assigned as EG4923- Four. Also included are the derived strains capable of overproducing toxic crystal proteins to Cryl lepidoptera. The strain of B. t. EG4923-4 sporulates efficiently and produces more toxic crystal protein than Cryl lepidoptera that makes it the progenitor strain EG4923. The Bacillus thuringiensis strains of this invention also include recombinant derivatives of strain EG4923-4: derivatives that have been transformed with a recombinant plasmid containing an insecticidal crystal protein gene and capable of overproducing the toxic crystal protein to Cryl lepidoptera. These include biologically pure cultures of the bacterium Bacillus thuringiensis, deposited with the NRRL, having accession numbers NRRL B-21506, NRRL B-21507, NRRL B-21578, and the strains designated EG11621, EG11622, and EG7841-1, respectively. The Bacillus thuringiensis strains of this invention also include derivatives of strain EG4923-4 which contain a natural plasmid containing an insecticidal crystal protein gene, transferred from a donor strain to a strain EG4923-4 through a method of type conjugation or by phage transduction, and are able to over produce the toxic crystal protein to lepidopteran Cryl. The invention also relates to insecticidal compositions comprising strains B. t. of this invention, insecticidal proteins produced by said strains B. t. and an agriculturally acceptable carrier, and methods for using said insecticidal compositions for the control of insects in plants.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 comprises a structural map of the recombinant plasmid pEG935, containing the crylC insecticidal crystal protein gene, and the pEG935 derivative, designated pEG935 ?, present in the EG11621 strain. Arrows in boxes indicate genes or functional DNA elements. The designations: pTZ19u = phagemid vector of E. coli pTZ19u, tet = tetracycline resistance gene, or¡60 = origin of replication of plasmid B. thuringiensis, crylC = insecticide crystal protein gene, ter = terminator region of crylF transcript, IRS = DNA fragment containing the internal resolution site region of transposon Tn5401. The abbreviations of restriction endonuclease: A = Asp718, B1 = B1nl, H = Hindlll, Nsi = Nsil, Nsp = Nspl, P = Pstl, Sp = Sphl, Xba = Xbal. Figure 2 comprises a structural map of the recombinant plasmid pEG361, which contains the insecticidal crystal protein gene CrylC-lAc, and the derivative of pEG361, designated PEG361 ?, present in strain EG11622. Arrows in boxes indicate genes or functional DNA elements. The designations: pTZ19u = phagemid vector of E. coli pTZ19u, tet = tetracycline resistance gene, ori60 = β plasmid replication origin. thuringiensis, crylC-lAc = insecticidal crystal protein gene, IRS = DNA fragment containing the internal resolution site region of transposon Tn5401. Abbreviations of restriction endonuclease: A = / Asp718, B1 = B1nl, H = Hindlll, Nsi = Nsil, Nsp = Nspl, P = Pstl, Sp = Sphl, Xba = Xbal. Figure 3 comprises a structural map of the recombinant plasmid pEG348, containing the insecticidal crystal protein gene CrylC, and the pEG348 derivative, designated pEG348 ?, present in the EG7841-1 strain. The arrows in the boxes indicate genes or functional DNA elements. Designations: pTZ19u = phagemid vector E. coli pTZ19u, tet = tetracycline resistance gene, or¡60 = origin of replication of plasmid B. thuringiensis, crylC = insecticide crystal protein gene, IRS = DNA fragment containing the internal resolution site region of the Tn5401 transposon. The abbreviations of restriction endonuclease: A = AspV \ 8, H = Hindlll, Nsi = Nsil, Nsp = Nspl, P = Pstl, Sp = Sphl. Figure 4 is a photograph of an agarose gel demonstrating that the isolated EG4923-4 does not have a plasmid of 130 megadaltons. Lanes 1 = HD1, 2 = EG11622, 3 = EG7841-1 (EG11730), 4 = EG7841-4 (EG11621), 5 = EG4923-4, and 6 = EG4923.

DETAILED DESCRIPTION OF THE INVENTION A novel preferred isolate B. thuring ens of the present invention is designated as EG4923-4. This isolate produces crystal proteins toxic to Lepidoptera and is active against Lepidoptera. The lepidoptera-toxic crystal proteins produced by EG4923-4 include CrylAc and CryllA. The derivatives of the B. thuringiensis isolate, EG4923-4, harboring recombinant plasmids containing at least one crystal protein nucleotide sequence, are designated EG11621, EG11622, and EG7841-1. These derivatives are capable of the increased production of other crystal proteins toxic to Lepidoptera in addition to the crystal protein CrylAc. The four strains are capable of the increased production of certain crystal proteins toxic to lepidoptera, preferably on a scale of about 125 to about 140 kilodaltons, including and not limited to CrylA and CrylC. B. thuringiensis EG4923-4, EG11621, EG11622, and EG7841-1 can be cultured using standard known means and fermentation techniques. After finishing the fermentation cycle, the bacteria can be harvested by separating spores B. t. and the crystals of the fermentation broth through means well known in the art, such as and not limited to centrifugation. The spores and crystals of B. t. recovered can be formulated in a wettable powder, a liquid concentrate, granule or other formulations through the addition of surfactants, dispersants, inert vehicles and other components to facilitate the handling and application for particular target pests. The formulation and application procedures are all well known in the art and are used with commercial strains of ß. thuringiensis (HD-1) active against lepidoptera, for example, caterpillars. The isolates of B. t. of the present invention can be used to control lepidopteran pests. A subculture of B. t. EG4923-4, EG11621, EG11622, and EG7841-1, was prepared according to the methods set forth in the examples and deposited in the permanent collection of the Agricultural Research Service Culture Collection, Peoria, Illinois on June 25, 1996. The numbers of access are as follows: Bacterial strain Accession number NRRL Deposit date EG4923-4 NRRL B-21577 May 21, 1996 EG11621 NRRL B-21506 November 15, 1995 EG11622 NRRLB-21507 November 15, 1995 EG7841-1 NRRLB-21578 May 21, 1996 The crops of the invention have been deposited under conditions that ensure that access to the crops will be available during the term of this patent application to someone determined by the director of patents and trademarks that is entitled under 37 C.F.R. §1.14 and 35 U.S.C. §122. Deposits are available as required by foreign patent laws in countries where counterparts of the present application or their progeny are presented. However, it should be understood that the availability of a deposit does not constitute a license to practice the present invention in derogation of the patent rights granted by governmental action. In addition, the culture deposits of the present will be stored and made available to the public in accordance with the provisions of the Budapest Treaty for the deposit of microorganisms, that is, they will be stored with all necessary care to keep them viable and uncontaminated during a period of time. period of at least 5 years after the most recent request for the development of a sample of the deposit, and in any case, for a period of at least 30 years after the date of deposit or for the required duration of any patent that can issue the description of crops. The depositor recognizes the obligation to replace the deposits if the deposit is unable to develop a sample when requested, due to the condition of the deposit. All restrictions on the availability to the public of the crop deposits herein will be irrevocably removed after granting a patent that describes these. The methods to classify strains B. t. which show improved production of certain toxic crystal proteins to lepidoptera comprise inoculating a culture medium with about 100 to about 500 colony-forming units of Bacillus thuringiensis, preferably B. t. strain EG4923. The culture medium can be solid as such and is not limited to starch agar plates or nutrient broth salt agar plates. The culture may be developed for about 1 to about 14 days, preferably to about 3 days, preferably about 5 days and most preferably about 7 days, at a temperature of about 25 ° C to about 37 ° C, preferably around 30 ° C. Certain white and crust-like and opaque colonies were observed as compared to the usual EG4923 colonies, which are creamy and bright white. One of these colonies was examined for crystal protein production and found to be increased as compared to the parental strain EG4923. Bacillus thuringiensis isolate derivatives, EG4923-4 can be prepared by recombinant methods including, but not limited to, recombinant plasmids containing an insecticidal crystal protein gene and through phage transduction or through a procedure similar to conjugation. The derivatives EG11621, EG11622, and EG7841-1 have been transformed with a recombinant plasmid containing an insecticidal crystal protein gene and are capable of overproducing the toxic crystal protein to Cryl lepidoptera. In addition, the derivatives of strain B. t. EG4923-4 contain a natural plasmid containing an insecticidal crystal protein gene transferred from a donor strain to the EG4923-4 strain through a conjugation-type procedure or through phage transduction and are capable of over producing the protein of toxic crystal to lepidoptera Cryl. A wide variety of forms are available to introduce a B. t gene. expressing a toxin in the host B. thuringiensis under conditions that allow the maintenance and stable expression of the gene. DNA constructs including transcriptional and translational regulation signals can be provided for expression of the toxin gene, the toxin gene under its regulatory control and a DNA sequence homologous with the sequence in the host organism, so that the integration will occur, and / or a replication system, which is functional in the host, so that it will occur Integration or stable maintenance. Transcriptional initiation signals will include a promoter and a transcriptional initiation initiation site. In some cases, it may be desirable to provide regulatory expression of the toxin, wherein the expression of the toxin will only occur after being released into the environment. This can be achieved with operators or a region joining an activator or enhancers, which are capable of induction after a change in the physical or chemical environment of the microorganisms. For example, a temperature-sensitive regulatory region may be employed, where organisms may develop in the laboratory without the expression of a toxin, but upon release into the environment, expression may begin. Other techniques may employ a specific nutrient medium in the laboratory, which inhibits the expression of the toxin where the nutrient medium in the environment could allow the expression of the toxin. For the initiation of translation, a ribosomal binding site and a start codon will be present. Various manipulations can be employed to improve the expression of messenger RNA, particularly by using an active promoter, as well as by employing sequences, which improve the stability of messenger RNA. The transcriptional and translational termination region will involve strain codons, a termination region and optionally, a polyadenylation signal. A hydrophobic "leader" sequence can be employed in the amino terminus of the translated polypeptide sequence in order to promote the secretion of the protein through the inner membrane. In the transcription direction, particularly in the 5 'to 3' direction of the coding or sense sequence, the construct will involve the transcriptional regulatory region, if any, and the promoter, wherein the regulatory region can be either 'or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codons, the polyadenylation signal sequence, if there is any , and the termination region. This sequence as a double structure can be used by itself for the transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker, where the second DNA sequence can be attached to the toxin expression construct during the introduction of DNA into the host. By marker is meant a structural gene that provides the selection of those hosts that have been modified or transformed. The label will normally provide selective advantages, for example, by providing biocidal resistance, for example, resistance to antibiotics or heavy metals; complementation, in order to provide prototropia to an auxotrophic host, or the like. If no functional replication system is present, the construct will also include a sequence of at least 50 peer bases (bp), preferably at least about 100 bp, most preferably at least about 1000 bp, and usually no more of approximately 2000 bp of a homologous sequence with a sequence in the host. In this way, the probability of legitimate recombination is improved, so that the gene will be integrated into the host and stably maintained by the host.

A number of transcriptional regulatory regions are available from Bacillus thuringiensis. Several transcriptional regulatory regions include the regions associated with the α-amylase gene, the phospholipase C gene, the exoproteinase gene, and the naturally occurring promoters associated with the toxin gene or other Cry toxin genes, where they are functional in the host. . See, for example, Chak et al., Appl. Environ. Microbiol., 1994, 60: 2304-2310, PCT WO94 / 25611 by Sandoz Ltd., and PCT WO92 / 14826 by Ciba Geigy. The termination region may be the termination region normally associated with the transcriptional initiation region or a different transcriptional initiation region, provided that the two regions are compatible and functional in the host. When maintenance or stable episomal integration is desired, a plasmid, which has a replication system that is functional in the host, will be employed. The replication system can be derived from the chromosome, an episomal element normally present in the host or a different host, or a replication system of a virus that is stable in the host. A large number of plasmids are available, such as PEG147, pHT3101, pEG597, pEG853, pEG854, pHV33, and the like, see for review, Gawron-Burke, C, and Baum, J.A., Genetic Engineering 1991, 13: 237-236. The gene B. t. it can be introduced between the transcriptional and translation initiation region and the translation transcriptional termination region, in order to be under the regulatory control of the initiation region. This construction will be included in a plasmid, which will include at least one replication system, but may include more than one, where a replication system is used for cloning during the development of the plasmid and the second replication system is necessary for operation on the final guest. In addition, one or more markers may be present, which have been previously described. When integration is desired, the plasmid will desirably include a sequence homologous to the host genome. Transformants can be isolated according to conventional forms, usually employing a selection technique, which allows the selection of the desired organism as against unmodified organisms or transfer organisms, when present. The transformants can then be tested for pesticidal activity. When desired, unwanted or transient DNA sequences can be selectively removed from the recombinant bacterium using a site-specific recombination system as described in the US patent. number 5,441, 884. The B cells. T. which contain a crystal protein gene B. t. they can be grown in any convenient nutrient medium that permits efficient sporulation, wherein the DNA construct provides a selective advantage, providing a selective medium such that substantially all or part of the cells retain the B. t gene. After fermentation, the spores and crystals can then be harvested according to conventional methods. The spores and crystals B. t. They can be formulated in a variety of ways. These can be used as moistening powders, granules or powders, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered ears, rice husk, peel) of walnut, and the like), the formulations may include extension-adhesion auxiliaries, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous or non-aqueous based and used as foams, gels, suspensions, emulsifiable concentrates, or the like. the ingredients may include biological agents, surfactants, emulsifiers, dispersants or polymers. The insecticide concentration will vary widely depending on the nature of the particular formulation, particularly if it is a concentrate or it is going to be used directly. The insecticide will be present in at least 1% by weight and can be 100% by weight. The dry formulations will have about 1% to 95% by weight of the pesticide, while the liquid formulations will generally be from about 1% to 75% by weight of the solids in the liquid phase. The formulations will be administered from about 1 gram (liquid or dry) to about 1 kg or more per hectare. The formulations can be applied to the environment of the lepidopteran pest, for example, plants, soil or water, by spraying, dusting, spraying, or the like, applications will be fixed with respect to the conditions specific to the pest and environment as such and it is not limited to cultivation, environmental conditions, pressure and insect population. The proportional ratio of active ingredient to vehicle will naturally depend on the chemical nature, solubility and stability of the insecticidal composition, as well as the contemplated dose. The insecticidal compositions of the invention can be employed in the methods of the invention individually or in combination with other compounds, including, but not limited to other pesticides, as such and not being limited to insect feremons. The method of the invention can also be used in conjunction with other treatments. The insecticidal composition of the present invention can be administered through any suitable route, including but not limited to topical sprays. To prepare resistant variants of B. t. Phage, an aliquot of phage lysate is spread on the nutrient agar and allowed to dry. An aliquot of the phage-sensitive bacterial strain is then placed on the plate directly on the dried lysate and allowed to dry. Plates were incubated at 30 ° C. The plates were incubated for two days and, in that time, the development of numerous colonies on the agar could be seen. Some of these colonies are collected and subcultured on nutrient agar plates. These apparent resistant crops were tested for resistance through scratching with phage lysate. A line of phage lysate is drawn on the plate and allowed to dry. The probable resistant cultures were then plotted through the phage line. Resistant bacterial cultures showed no lysis anywhere in the line crossing the phage line after incubation overnight at 30 ° C. The phage resistance was then reconfirmed by placing a turf of the resistant culture on a plate of nutrient agar on the plate. The sensitive strain was also placed on the plate in the same manner to serve as the positive control. After drying, a drop of the phage lysate was placed on the plate in the center of the plate and allowed to dry. The resistant cultures did not show lysis in the area where the phage lysate was placed after incubation at 30 ° C for 24 hours. The present invention is further described in the following examples. These examples are not constructed as limiting the scope of the appended claims.

EXAMPLE 1 Isolation of the EG4923 Variants Strain EG4923 of the subspecies kurstaki is a transconjugate derivative of strain EG3125, a natural strain of B. t. obtained from a sample of New York grain dust. The EG4923 strain contains three crylAc genes that encode toxic CrylAc crystal proteins to lepidoptera and a cryllA gene that encodes a CryllA crystal protein toxic to Lepidoptera and Diptera. Two copies of the crylAc gene and one copy of the cryllA gene are present on a plasmid of approximately 110 megadaltons in mass. The remaining crylAc gene is contained in an autotransmissible plasmid of approximately 56 megadaltons. Strain EG4923 was grown at 30 ° C in a DSM broth culture medium, described by Donovan et al., Appl. Environ. Microbiol. 1992, 58: 3921-3927, for three days, during which time sporulation and cell lysis occurred. The culture was diluted 1 / 10,000 with water and 40 microliters of diluent placed on the starch agar plates (DIFCO) supplemented with 5 g / liter of agar. Plates were incubated at 30 ° C overnight. The next day, the plates were examined for the colonies exhibiting an unusual morphology. The EG4923 colonies typically exhibited a creamy white glossy appearance. Four variants of colony morphology of the plates were isolated, traced on fresh starch agar plates, and designated EG4923-1, EG4923-2, EG4923-3, and EG4923-4. Isolates 1 and 2 appeared less opaque than the typical EG4923 colony, whereas isolates 3 and 4 appeared whiter and scab-like in texture when compared to the typical EG4923 colony. Further examination of the isolates through the phase contrast microscope suggested that isolates 1 and 2 were delayed in sporulation, while isolates 3 and 4 exhibited apparently normal sporulation. The four isolates produced bipiramide-like crystals typical of Cryl crystal proteins toxic to Lepidoptera. The arrangement of plasmids contained in the four EG4923 variants was determined through agarose gel electrophoresis using a modified Eckhardt agarose gel electrophoresis method, described by Gonzalez Jr. et al., Proc. Nati Acad. Sci USA 1982, 79: 6951-6955. This analysis showed that isolates 1, 2 and 4 contained the plasmids encoding the crystal protein of strain EG4923. In contrast, isolate 3 was determined to be a contaminating strain unrelated to strain EG4923. In addition, isolate 4, designated EG4923-4, was found not to have a plasmid of 130 megadaltons, said plasmid does not appear to contain a toxin gene, see figure 4. Although it is not intended to be bound by any particular theory of operation, it is believed that strains B. t. of the present invention can produce increased amounts of Cryl crystal proteins due to the lack of the plasmid of 130 megadaltons.

EXAMPLE 2 Production of Crystal Protein Through Variants EG4923 Strain EG4923 and variants EG4923-1, EG4923-2, and EG4923-4 were inoculated in 25 ml flasks containing 8 ml of broth culture consisting of 14.4 g / l soybean meal, 7.9 g / l peptone meat, 20.0 g / l cerelosa, 3.1 g / l anhydrous KH2PO4, 4.7 g / l K2HP04, 1X C2 salts (described by Donovan et al., J. Biol Chem. 1988, 263: 561-567), titrated to a pH of 7.5 with 1N NaOH. The cultures were grown in duplicate for three days at 25 ° C. after three days, during that time, sporulation and cell lysis occurred, the production of crystal protein was quantified through the SDS-polyacrylamide gel method (PAGE) described by Brussock, S.M. and Currier, T.C., 1990, "Use of Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis to Quantify Bacillus thuringiensis d-Endotoxins", in Analytical Chemistry of Bacillus thuringiensis. (L. A. Hickie and W.L.Fitch, eds.), The American Chemical Society, p. 78-87. The procedure was modified to eliminate the neutralization step with 3M HEPES. The crystal proteins resolved through SDS-PAGE were quantified through densitometry using a Molecular Dynamics Model 300A computation densitometer and the CrylAc purified crystal protein as a standard. The results shown in Table 1 demonstrate that the EG4923-4 variant produced 30% more CrylAc crystal protein than the progenitor strain EG4923. Variants EG4923-1, and EG4923-2 produced less crystal protein than progenitor strain EG4923.

TABLE 1 On Production of Crystal Protein Through Variant EG4923-4 Production of crystal protein in relation to the production of strain EG4923, defined as 1.0.

EXAMPLE 3 Construction of Recombinant Derivatives EG4923-4 Capable of Overproduction of Toxic Crystal Protein to Lepidoptera Cryl Strain EG4923-4 can be used as a host strain to produce, at high levels, crystal proteins toxic to Lepidoptera in addition to the crystal protein CrylAc. To demonstrate this, recombinant plasmids containing the crystal protein genes crylC and crylC-lAc were introduced into strain EG4923-4 using the electroporation procedure described by Mettus. A.M. and A. Macaluso, Appl. Environ. Microbiol. 1990, 56: 1128-1134. The recombinant plasmids containing CrylC and crylC-lAc were designated pEG935 (Figure 1) and pEG361 (Figure 2), respectively, and are structurally similar to the cryl plasmids described in the US patent. No. 5,441,884. The transformants of strain EG4923-4 containing the plasmids pEG935 and pEG361 were isolated on Luria plates containing 10 μg / ml of tetracycline. The recombinant plasmid DNAs of the transformants were isolated through the alkaline lysis procedure described by Baum, J.A., J. Bacteriol. 1995, 177: 4036-4042 and was confirmed through the above restriction analysis performed according to normal methods such as those established by Maniatis et al., 1982 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor. The plasmid arrangements of the transformants were further confirmed through the Eckhardt agarose gel analysis procedure described by Gonzalez Jr. et al., Proc. Nati Acad. Sci USA 1982, 79: 6951-6955. The derivatives of the recombinant EG4923-4 were designated as EG4923-4 / pEG935 and EG4923-4 / pEG361. For comparison, the plasmid pEG935 was introduced into the progenitor host strain EG4923 through electroporation to produce strain EG4923 / pEG935. The crystal protein productions obtained with the strains EG4923-4 / pEG935 and EG4923-4 / pEG361 were determined as previously described compared to those obtained with EG4923-4 / pEG935. As shown in Table 2, both recombinant strains retain the ability to overproduce lepidopteran toxic crystal protein when compared to the pEG935 plasmid that hosts the progenitor strain EG4923.

TABLE 2 Overproduction of Crystal Protein Through Recombinant Derivatives EG4923-4 Strain Production of relative crystal protein1 EG4923-4 / pEG935 1.32 EG4923-4 / pEG361 1.19 Production of crystal protein in relation to the production of strain EG4923 / pEG935, defined as 1.0.

EXAMPLE 4 Modification of Strains EG4923-4 / pEG935, EG4923-4 / pEG361 and EG4923-4 / pEG348 To Remove Strange DNA Elements Plasmids pEG935 (Figure 1), pEG361 (Figure 2), and pEG348 (Figure 3) contain duplicate copies of a site-specific recombination site or an internal resolution site (IRS) that serves as a substrate for a reaction of specific recombination in the in vivo site mediated by the Tnpl recombinan transposon Tn5401 (described in Baum, JA, J. Bacteriol, 1995, 177: 4036-4042). This reaction of site-specific recombination, described in the patent of E.U.A. No. 5,441,884, results in the elimination of non-B DNA elements. t. or foreign DNA of the recombinant plasmids encoding the crystal protein. The resulting recombinant Bacillus thuringiensis strains are free of foreign DNA elements, a desirable feature for genetically engineered strains intended for use as bioinsecticides for spray application. Strains EG4923-4 / pEG935, EG4923-4 / pEG361 and EG4923-4 / pEG348 were modified using this specific recombination system in vivo (SSR) to generate three new strains, designated EG11621, EG11622, and EG7841-1 , respectively (table 3).TABLE 3 Recombinant Strain Derivatives EG4923-4 These strains, designated EG11621, EG11622, and EG7841-1, retain the ability to over produce crystal proteins toxic to Lepidoptera Cryl. As an example, the production of crystal protein obtained with the strain EG7841-1 (aka EG4923-4 / pEG348?) Was compared with that obtained with the strain EG4923 / pEG348? in Table 4 using the same methods described in Example 2. These results show an increase of about 40% in the production of the toxic protein to Lepidoptera Cryl.

TABLE 4 Overproduction of Crystal Protein Across Strain EG7841-1 Production of crystal protein in relation to the production of strain EG4923 / pEG348 ?, defined as 1.0. The three strains listed in Table 3 produce a CrylC toxin protein, a toxin that is known to be effective against the devastating worm of Spodoptera Exigua beet, in addition to the CrylAc toxin protein. These strains can be used in bioinsecticide compositions to control many insect pests lepidoptera, particularly Plutella xylostella, Trichoplusia ni, and Spodoptera exigua. Efficient production of spores through these strains also contributes to their toxicity, particularly towards S. exigua.

The description of each patent, patent application and publication cited or described herein are hereby incorporated by reference in their entirety. Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Said modifications are also intended to fall within the scope of the appended claims.

Claims (15)

1. An isolated Bacillus thuringiensis strain, which produces increased amounts of Cryl crystal protein.

2. The Bacillus thuringiensis strain according to claim 1, characterized in that it has normal sporulation.

3. The Bacillus thuringiensis strain according to claim 1, characterized in that it produces increased amounts of CrylC, or CrylAc and CrylC.

4. The Bacillus thuringiensis strain according to claim 1, characterized in that it is a recombinant derivative.

5. Strain EG4923-4, characterized in that it has access number NRRL B-21577.

6. Strain EG11621, characterized in that it has access number NRRL B-2 506.

7. Strain EG11622, characterized in that it has accession number NRRL B-21507.

8. Strain EG7841-1, characterized because it has access number NRRL B-21578.

9. The Bacillus thuringiensis strain according to claim 1, transformed with a plasmid vector containing a Bacillus thuringiensis nucleotide sequence encoding a Bacillus thuringiensis crystal protein.

10. The Bacillus thuringiensis strain according to claim 1, characterized in that it has nucleotide sequences that encode crystal proteins Cryl and Cry

ll. 11. The Bacillus thuringiensis strain according to claim 10, wherein the Cry crystal proteins are Cryl and Cryll.

12. A lepidopteran toxic crystal protein produced through Bacillus thuringiensis of claim 1.

13. Lepidopteran toxic crystal proteins according to claim 12, which are a combination of Cryll and CrylAc, a combination of Cryll, CrylAc and CrylC, or a combination of Cryll crystal proteins, CrylAc, and CrylC-IAc.

14. An insecticidal composition comprising an insecticidally effective amount of a lepidopteran toxic crystal protein obtained from a strain of Bacillus thuringiensis capable of overproducing said protein, and a suitable vehicle.

15. An insecticidal composition comprising an insecticidally effective amount of a toxic crystal protein to lepidoptera and spores obtained from a Bacillus thuringiensis strain capable of overproducing said protein, and a suitable vehicle.