MXPA00001353A - Materials and methods for controlling homopteran pests - Google Patents

Abstract

The subject invention concerns materials and methods for the control of non-mammalian pests. In specific embodiments, the subject invention concerns materials and methods useful in the control of insects from the Order Homoptera. More specifically, the subject invention provides novel Bacillus thuringinesis (B.t.) isolates, or strains, toxins, and toxin-encoding genes that are useful for the control of homopterans. The strains HD969, PS66D3, and PS50C are specifically exemplified herein as yielding mortality against homopterans. In a preferred embodiment, the target pests are selected from the group consisting of leafhoppers and planthoppers.

Description

MATERIALS AND METHODS FOR THE CONTROL OF PESTS OF HOMOPTEROS BACKGROUND OF THE INVENTION The soil microbe Bacillus thuringiensis. { B.t.) is a Gram-positive spore-forming bacterium that is characterized by its crystalline protein inclusions. These inclusions often appear microscopically as crystals of distinctive shapes. Proteins can be highly toxic to pests and specific in their toxic activity. Certain toxin genes of B.t. have been isolated and sequenced, and B.t. products have been produced and approved for use. based on recombinant DNA. In addition, with the use of genetic engineering techniques, new approaches to administer B.t. toxins are being developed. to agricultural environments, including the use of genetically engineered plants with endotoxin genes for insect resistance and the use of intact microbial cells stabilized as vehicles to transport the B.t. toxin. (Gaerther, F.H., L. Kim [1988] TIBTECH 6: S4-S7, Beegle, C.C., T. Yamamoto, "History of Bacillus thuringiensis Berliner research and development" Can.In.124: 587-616). Accordingly, the toxin genes of B.t. isolated have increasing commercial value. Until recently, the commercial use of B.t. it has been largely restricted to a narrow range of lepidopteran pests (caterpillars). Preparations of the spores and crystals of B. Thuringiensis subsp. Kurstaki as commercial insecticides for lepidopteran pests. For example, the variety kurstaki HD-1 of B. Thuringiensis produces a crystalline d-endotoxin that is toxic to the larvae of a number of lepidopteran insects. Researchers have recently discovered B.t. pesticides. with specificity for a wider range of pests. For example, other species of ß.í. for example israelensis and morrísoni (also known as tenebrionis, also known as Bt M-7, also known as Bt san diego) to fight insects of the orders 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 Pathogenecity 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. Langenbruch, W. Shnetter (1983). Z. Ang. Ent. 96: 500-508 describe Bacillus thuringiensis var. tenebrionis, which supposedly is active against two beetles of the order of coleoptera. These are Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni.

More recently, new subspecies of B.t. and the genes responsible for the active d-endotoxin proteins have been isolated (Hophé, H, H.R. Whiteley [1989] Microbiological Reviews 52 (2): 242-255). Hófte and Whiteley classified crystal protein genes of B.t. in four main classes. The classes were cryl (specific for Lepidoptera), cryll (specific for Lepidoptera and Diptera), crylll (specific for coleoptera) and cryLV (specific for Diptera). The discovery of strains specifically toxic for other pests has been reported (Feitelson, JS, J. Payne, L. Kim [1992] Bio / Technology 10: 271-275), and has been proposed to designate a class of toxin genes that They are specific for nematodes Lambert et al. (Lambert, B., L. Buyssc, 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) describe the characterization of an active Cry9 toxin against lepidoptera, published PVT applications WO 94/95771 and WO 24264 also describe isolates of Bt active against lepidopteran pests, U.S. Patent No. 5,273,746 describes several Bt isolates, including PS192M4, as active against lice, Gleave et al. ([1991] JGM 138: 55-62), Shevelev et al. ([1993] FEBS Lett 336: 79-89; and Smulevitch et al. ([1991] FEBS Lett 293: 25-26) also describe Bt toxins. many more kinds of B.t. genes The cloning and expression of a crystal protein gene of B.t. in Escherichia coli in the published literature (Schnepf, H.E., H.R. Whiteley [1981] Pro. Nati, Acad. Sci. USA 78: 2893-2897). U.S. Patent No. 4,448,885 and U.S. Patent No. 4,467,036 describe the expression of crystal protein of ß.i. in coli. U.S. Patent Nos. 4,990,332, 5,039,523, 5,126,133, 5,164,180 and 5,69,629 are among those which describe ß.i. toxins. that has activity against lepidoptera. U.S. Patent Nos. 5,262,159 and 5,468,636 describe isolations of ß.í. PS157CI, PS86AI, and PS75JI for use against aphids. U.S. Patent Nos. 5,277,905 and 5,457,179 describe the use of ß.i. PS50C for use against coleopteran pests. The United States patent no. 5,366,892 describes the sequence of the 50C (a) toxin of ß.i. U.S. Patent No. 5,286,485 describes the use of PS50C against lepidopteran pests. U.S. Patent No. 5,185,148 describes the use of PS50C against beetle pests. U.S. Patent No. 5,554,534 describes the sequence of the 50C (b) toxin of β.í. U.S. Patent Nos. 5,262,158 and 5,424,410 describe the use of PS50C against acarids. As a result of extensive investigations and investment of funds, other patents have been granted for new isolates of ß.í. and new uses of them. See Feitelson et al., Mentioned above, for a review. However, the discovery of new isolates of ß.í. and new uses of the isolates of ß.í. known remains empirical and unpredictable. Insects that belong to the order Homoptera include drilling and sucking insects such as the cicada of leaves or plants. The cicadas of the leaves and the cicadas of the plants share a close evolutionary relationship. The cicadas of the leaves and the cicadas of the plants are found all over the world and cause serious economic losses in crops or ornamental plants by means of the damage by feeding and being vectors of disease. A specific example of plant cicada is the brown rice cicada. { Nilaparvata lugens). Due to their drilling and suction feeding habits, leaf cicadas and plant cicadas are not easily susceptible to the foliar applications of Bacillus thuringiensis proteins. { B.t.) in its original states, in crystals.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to materials and methods for the control of pests of non-mammalian animals. In specific embodiments, the present invention relates to materials and methods useful for combating insects of the Homoptera order. More specifically, the present invention presents novel isolates, or strains, toxins and genes coding for Bacillus thuringiensis toxins. { B.t.) that are useful for combating homoptera. Strains of ß.í. HD969, PS66D3 and PS50C are specifically exemplified in this document for their toxicity to Homoptera. In a preferred embodiment, the white pests are selected from the group consisting of leaf cicadas and plant cicadas. The nucleotide sequences useful according to the present invention encode pesticidal toxins. One embodiment of the present invention relates to plant cells transformed with at least one polynucleotide sequence of the present invention in such a way that the transformed plant cells express pesticidal toxins in tissues consumed by the white pests. Such transformation of plants 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 plants. On the other hand, ß.í isolates can be used. according to the present invention, or the recombinant microbes that express the toxins described that were described therein, to combat pests. In this regard, the invention includes the treatment of ß.í. or substantially intact recombinant cells containing the toxins expressed according to the present invention, treated to prolong the pesticidal activity when substantially intact cells are applied to the environment of the white pest. The treated cell acts as a protective coating for the pesticidal toxin. The toxin is activated by being ingested by a white insect.

The protein toxins of ß.í. Crystallized can be used in agricultural applications for pest control, with application methods and formulations well known in the art. In one embodiment, the present invention further presents protein toxins that are solubilized. The toxins of the present invention are distinguished from the β-γ exotoxins. that have broad spectrum non-specific activity. In accordance with what is described herein, toxins useful in accordance with the present invention can be chimeric toxins produced by combining multiple toxin portions. In addition, the toxins of the present invention can be used in combination to obtain a better pesticidal control.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides ß.í. and toxins active against homoptera, including cicadas of the leaves and cicadas of plants. Specific isolates useful in accordance with the present invention have been designated PS50C, PS66D3 and HD969. Table 1 shows some of the characteristics of these strains.

TABLE 1 Strain Type of inclusion Serotype H Protein profile by SDS-PAGE PS50C Spherical 1.8-kumamotoen-sis 133,128 PS66D3 Flattened, almost 8 75.66, (58) square HD969 BP to lemon at 6 130 (s) amorphous PS66D3 is a novel strain. This microorganism has been deposited in the permanent collection of the Agricultural Research Service Patent Culture Collection (NRRL), Research Center of the Northern Region, 1815 North University Street, Peoria, Illinois 61604. United States. The crop replacement numbers of the deposited strains are: Culture Replacement number Deposit date Bacillus thuringiensis N RRL B-21657 February 19, 1997 PS66D3 The isolates for use in accordance with the present invention have been deposited under conditions that guarantee the possibility that a person appointed by the Patent and Trademark Commissioner to access the crops during the period in which this patent application is pending, under 37 CFR 1.14 and 35 U.S.C. 122. Deposits are available in accordance with the requirements imposed by foreign patent laws in the countries where the counterparts of this application or their successors are presented. However, it should be understood that the availability of a deposit does not constitute a license to carry out the present invention by repealing the patent rights granted by the government action. The next asylee, as well as the clones that contain genes that encode it, are available to the public by virtue of the granting of United States patents. These isolates and their corresponding patents of the United States are: These patents, together with their description of the indicated isolates, as well as their toxins and genes, are incorporated herein by reference. The HF969 isolate is available from the USDA-ARS NRRL Culture Collection, Peoria, Illinois. HD969 has numerous Cry1 genes, including 1Ac, B and 1C. PCR tags have also been found for one or more genes of the cry7, 8, 9 class. PS66D3 constitutes a series of 72 and 64 kDa proteins (a typical 3A, 3B pattern).

Strains of HD969, PS66D3 and PS50C produced substantially higher mortality of nymphs of N. lugens after 72 hours than that obtained with negative controls.

Genes and Toxins Another aspect of the present invention relates to novel toxins and genes that are obtained from the isolates for use in accordance with the present invention. The toxins and polynucleotide sequences of the present invention are defined according to various parameters. An essential characteristic of the toxins described here is their pesticidal activity. In a specific embodiment, these toxins have activity against homopteran pests. The toxins and genes of the present invention can be further defined by their amino acid and nucleotide sequences. The sequences of the molecules can be defined in terms of homology or identity with certain sequences exemplified, as well as in terms of their ability to hybridize with, or be amplified by, certain probes and primers exemplified. The toxins provided herein can also be identified on the basis of their immunoreactivity with certain antibodies. With the teachings presented herein, a person skilled in the art could easily produce and use the various toxins and polynucleotide sequences described herein.

The genes and toxins useful according to the present invention include not only the sequences but also the fragments of these sequences, variants, mutants and fusion proteins that retain the pesticidal activity characteristic of the novel toxins specifically exemplified herein. In the present, the terms "variants" or "variations" of genes refer to nucleotide sequences that encode the same toxins or that encode equivalent toxins with pestidic activity. Herein, the term "equivalent toxins" refers to toxins that have equal or essentially the same biological activity against the white 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 the isolates deposited in a culture reservoir as described above. These genes, or portions or variants thereof, can also be constructed synthetically, for example by using a gene synthesizer. Variations of genes can be easily constructed using standard techniques to prepare point mutations. In addition fragments of these genes can be prepared using commercially available exonucleases or endonucleases according to normal procedures. For example, enzymes such as ßa / 31 or site-directed mutagenesis can be used to systematically trim the nucleotides at 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. Equivalent toxins and / or genes encoding these toxins equivalent to ß isolates can be obtained. í. and / or DNA libraries using the instructions provided in the present application. There are a number of methods for obtaining the pesticidal toxins of the present invention. For example, antibodies to the pesticidal toxins described and cited herein can be used to identify and isolate other toxins from a protein mixture. Specifically, antibodies can be cultured for the portions of toxins that are more constant and more different from the other toxins of ß.í .. These antibodies can then be used to specifically identify equivalent toxins with characteristic activity by immunoprecipitation, linked immunosorbent assay with enzymes (ELISA) or western blotting. Antibodies can be readily prepared for the toxins described herein, or for equivalent toxins or fragments thereof using standard procedures in this art. Subsequently, genes that encode these toxins can be obtained from the microorganisms. Fragments and equivalents that retain the pesticidal activity of the exemplified toxins would be within the scope of the present invention. In addition, because of the redundancy of the genetic code, there is a variety of different DNA sequences that can encode the amino acid sequences described herein. It is up to the person 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 reference to "essentially the same" sequence refers to sequences that have substitutions, deletions, additions or insertions of amino acids that do not noticeably affect pesticidal activity. Fragments that conserve pesticidal activity are also included within this definition. Certain toxins of the present invention have been specifically exemplified herein. Since these toxins are merely exemplifying of the toxins of the present invention, it should be evident that the present invention also relates to variants or equivalents of novel genes and toxins that have the same or similar pesticidal activity as the novel toxins exemplified. The equivalent toxins have amino acid homology with a novel exemplified toxin. These equivalent genes and toxins will typically have greater than 60% identity with the sequences specifically exemplified herein; preferably they will have more than 75% identity, more preferably more than 80%, most preferably more than 90%, and the identity may be greater than 95%. The amino acid homology will be 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 regard, certain substitutions of amino acids are admissible and can be expected if these substitutions are found in regions that are not critical 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 by another amino acid of the same type fall within the scope of the present invention so long as the substitution does not significantly alter the biological activity of the compound. Table 2 presents a list of examples of the amino acids that belong to each class.

TABLE 2 In some cases, non-conservative substitutions can also be made. The critical factor is that these substitutions should not cause a decrease in the biological activity of the toxin.

The toxins of the present invention can also be characterized in terms of the shape and location of the toxin inclusions, described above. While crystal proteins are normally used in the art, isolates can also be cultured according to the present invention under conditions that facilitate the secretion of toxins. Accordingly, the supernatant of these cultures can be used to obtain toxins according to the present invention. Accordingly, the present invention is not limited to crystal proteins; soluble soluble proteins are also contemplated. 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. Therefore, the designation "isolated and purified" represents the implication of the "hand of man" as described. Chimeric toxins and genes also involve the "hand of man" The use of oligonucleotide probes provides a method for identifying the toxins and genes of the present invention, and others, novel genes and toxins. The probes provide a rapid method to identify genes encoding toxins. The nucleotide segments that are used as probes according to the present invention can be synthesized using a DNA synthesizer and standard procedures, for example.

Chimeric toxins Chimeric toxins and genes, produced by combining portions of more than one toxin or B.t. gene, can also be used in accordance with the teachings of the present invention. Methods have been developed for preparing useful chimeric toxins by combining portions of crystal proteins of ß.i. The portions that are combined do not need to be pesticides per se, as long as the combination of portions generates a chimeric protein that is pesticidal. This can be done using restriction enzymes, as described, for example, in European Patent 0 228 838; Ge, A. Z., N.I., Shivarova, D.H. Dean (1989), Proc. Nati Acad. Sci. USA 86: 4037-4041; Ge, A.Z., D. Rivers, R. Milne, D.H: Dean (1991) J. Biol. Chem. 266: 17954-17958; Schnepf, H.E., K. Tomczak, J.P., Ortega, H.R. Whiteley (1990) J. Biol. Chem. 265: 209233-20930; Honee, G., D., Convents, J. Van Rie, S., Jansens, M. Peferoen, B Visser (1991) Mol. Microbiol. 5: 2799-2806. On the other hand, recombination can be used using cell recombination mechanisms to obtain similar results. See, for example, Caramori, T., A.M. Albertini, A. Galizzi (1991) Gene 98: 37-44; Widner, W.R., H.R. Whiteley (1990) J. Bacteriol 172: 2826-2832; Bosch, D., B. Schipper, H. van der Kliej, R.A. from Maagd, W.J. Stickema (1994) Biotechnology 12: 915-918. A number of other methods by which such chimeric DNAs can be prepared are known in the art. The present invention includes chimeric proteins that utilize genes and toxins of the present application.

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 maintenance of the pesticide. With suitable microbial hosts, for example Pseudomonas, the microbes can be applied to the situs of the pest, where they proliferate and are ingested. The result is the control of the plague. On the other hand, the microbe that acts as host of the toxin gene can be exterminated 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 white plague. Where the gene for the ß.í. it is introduced by means of a suitable vector into a microbial host and said host is applied to the environment in the living state, the use of certain host microbes is essential. Selected microorganism hosts are selected for occupying the "phytosphere" (phylloplane, phyllosphere, rhizosphere and / or rhizoplane) of one or more crops of interest. These microorganisms are selected to compete satisfactorily in the determined medium (culture and other insect habitats) with the wild type microorganisms, to produce a stable maintenance and expression of the gene that expresses the polypeptide pesticide and, conveniently, to give a better protection of pesticide degradation and environmental deactivation.

A large number of microorganisms are known to inhabit the phylloplane (the surface of the leaves of plants) and / or the rhizosphere (the soil surrounding the roots of plants) of a wide variety of important crops. These microorganisms include bacteria, algae and fungi. Of particular interest are microorganisms such as bacteria, for example the genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc and Alcaligenes, fungi, especially yeast, for example the genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula and Aureobasidium. Of particular interest are the bacterial species of the phytosphere, such as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus and Azotobacter vinlandii; as well as yeast species of the phytosphere such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S.pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are pigmented microorganisms. A wide variety of methods are available to introduce a ß.í. which encodes a toxin in a host microorganism under conditions that result in stable maintenance and expression of the gene.

These methods are well known to those skilled in the art and are described, for example, in U.S. Patent No. 5,135,867, which is incorporated herein by reference. Homopteran control can be performed using the isolates, toxins and genes of the present invention by a variety of methods known to those skilled in the art. These methods include, for example, the application of ß.í isolates. to pests (or their habitat), the application of recombinant microbes to pests (or their habitats) and the transformation of plants with genes that code for the pesticidal toxins of the present invention. Recombinant microbes can be, for example, βß, E. co // or Pseudomonas. The transformations can be carried out by those skilled in the art using standard techniques. The materials needed for these transformations are described herein or else they can be easily obtained by trained technicians. Synthetic genes that are functionally equivalent to the novel 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. 5,380,831 Treatment of the cells As mentioned above, ß.í. or recombinant cells expressing a β. tox toxin. to prolong the activity of the toxin and stabilize the cell. The pesticide microcapsule that is formed contains the ß.i. within a cellular structure that has been stabilized and protects the toxin when the microcapsule is applied to the environment of the white pest. Suitable host cells can include prokaryotes or eukaryotes, which are typically limited to those cells that do not produce substances toxic to higher organisms such as mammals. However, organisms that produce substances toxic to higher organisms could be used, where the toxic substances are unstable or the level of application is sufficiently low to avoid any possibility of toxicity to a mammalian host. As hosts, prokaryotes and lower eukaryotes, such as fungi, are of special interest. Upon receiving treatment the cell will generally be intact and substantially in its proliferative form, although in some cases spores can be used. The treatment of the microbial cell, for example a microbe containing the gene for the ß.i. toxin, can be carried out by chemical or physical means, or by a combination of chemical and / or physical means, provided that the technique does not adversely affects the properties of the toxin, nor reduce the cellular capacity of protection of the toxin. Examples of chemical reagents are halogenating agents, especially halogens of atomic No. 17-80. More specifically, iodine can be used under moderate conditions and for sufficient time to obtain the desired results. Other suitable techniques include treatment with aldehydes such as glutaraldehyde; anti-infectives such as zephirano chloride and cetylpyridinium chloride, alcohols such as isopropanol and ethanol; various histological fixatives such as iodine Lugol, Bouin's fixative, various acids and Helly's fixative (see: Humason, Gretchen L. Animal Tissue Techniques, W.H. Freeman and Company, 1967); or a combination of physical agents (heat) and chemicals that preserve and prolong the activity of the toxin produced in the cell when it is administered to the host medium. Examples of physical means are short wave radiation, such as gamma radiation and X radiation, freezing, UV irradiation, lyophilization and the like. Methods of treating microbial cells are described in U.S. Patent Nos. 4,695,455 and 4,695,462 which are incorporated herein by reference. Cells generally have greater structural stability that increases resistance to environmental conditions. In cases where the pesticide is presented in a proforma, a method of cell treatment should be chosen that does not inhibit the processing of the proform to the mature form of the pesticide by means of the target pest pathogen. For example, formaldehyde will cross-link proteins and could inhibit the processing of the proforma of a polypeptide pesticide. The treatment method must retain at least a substantial portion of the bioavailability or bioactivity of the toxin.

Characteristics of special interest in the selection of a host cell for the purpose of production include the ease of introducing the ß.í. in the host, the availability of expression systems, the efficiency of expression, the stability of the pesticide in the host and the presence of auxiliary genetic abilities. Characteristics of interest for use as pesticide microcapsules include the protective qualities of the pesticide, such as thick cell walls, pigmentation, and intracellular envelope or the formation of inclusion corpuscles: survival in aqueous media; the lack of toxicity for mammals, the uptake of pests for their ingestion, the ease of killing and fixing without harm to the toxin and the like. Other factors to consider include ease of formulation and carry, economy, storage stability and the like.

Development of the cells The cellular host that contains the insecticidal gene of ß.í. it can be developed in any suitable nutrient medium, in which the DNA construct provides a selective advantage, giving rise to a selective medium so that all or substantially all cells retain the ß.i. These cells can be harvested subsequently according to conventional methods. On the other hand, cells can be treated before harvesting. The cells of ß.í. in accordance with the present invention can be cultured using standard fermentation techniques and means in the art. When the fermentation cycle is completed, the bacteria can be harvested by first separating the spores of ß.í. and the crystals of the fermentation broth by means known in the art. Spores and crystals of ß.í. they can be recovered using well-known techniques and used as a conventional preparation of β-endotoxin. For example, spores and crystals can be integrated into a formation of wettable powder, liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers and other components to facilitate handling and application for specific white pests. These formulations and methods of application are well known in the art. On the other hand, the supernatant of the fermentation process can be used to obtain toxins according to the present invention. The secreted soluble toxins are then isolated and purified using generalized knowledge techniques.

Methods and formulations for pest control The control of homopterans using the isolates, 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 ß.í isolates. to pests (or their habitat), the application of recombinant microbes to pests (or their habitats) and the transformation of plants with genes encoding the pesticidal toxins of the present invention. Recombinant microbes can be, for example, ß.í., E. Coli or Pseudomonas. The transformations can be carried out by those skilled in the art using standard techniques. The materials needed for these transformations have been described herein or else they can be easily obtained by trained technicians. The formulated bait granules containing an attracting agent and toxins from the ß.í. isolates, or recombinant microbes containing the genes obtained from the ß.í. described herein may be applied to the soil. The formulated product can also be applied in the form of seed coat or root treatment or total treatment of the plant in later stages of the crop cycle. Treatments of the plant and soil of ß.í. can be used in the form of wettable powders, granules or dusting agents, mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulphates, phosphates and the like) or botanical materials (powdered corn cobs, rice husks, walnut shells, and the like). The formulations may include tackifying builders, stabilizing agents, 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 rheological 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 will be present in at least 1% by weight and can be 100% by weight. The dry formulations will have from 1-95% by weight of the pesticide, while the liquid formulations will generally have from about 1-60% by weight of solids in the liquid phase. The formulations will generally have from 102 to 10 4 cells / mg. These formulations containing cells will be 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 environment of the pest, for example to the soil and foliage, by spraying, sprinkling, sprinkling, etc.

Mutants Mutants of novel isolates that are obtained according to the present invention can be prepared using methods well known in the art. For example, a sporogenic mutant can be obtained by means of the mutagenesis with ethylmetansulfonate (EMS) of an isolate. Mutants can be prepared using ultraviolet light and nitrosoguanidine by methods well known in the art. A smaller percentage of sporogenous mutants remain intact and do not undergo lysis during prolonged periods of fermentation; these strains are designated as lysis minus (-). The less lysis strains can be identified by classifying sporogenous mutants in agitator bottle medium and selecting the mutants that are still intact and contain toxin crystals from the fermentation. The less lysis strains are suitable for a cell treatment process that produces an encapsulated and protected toxic protein. To prepare a fagorresistant variant of said sporogenic mutant, an aliquot of the phage used is spread on nutrient agar and allowed to dry. An aliquot of the phage-sensitive bacterial strain is then applied directly to the dry lysate and allowed to dry. The plates are incubated at 30 ° C. The plates are incubated for two days and, after that period, numerous colonies can be seen developing on the agar. Some of these colonies are harvested and subcultured on agar plates with nutrients. These seemingly resistant cultures are analyzed for resistance to cross-application with the phage lysate. A line of phage lysate is applied to the plate and allowed to dry. The crops are then applied presumably through the phage line. Resistant bacterial cultures show no lysis of the line across the phage line after incubation overnight at 30 ° C. Phage resistance is subsequently reconfigured by applying a layer of the resistant culture on the nutrient agar plate. The sensitive strain is also applied in the same way to serve as a positive control. After drying, a drop of the phage lysate is placed in the center of the plate and allowed to dry. The resistant cultures do not show any lysis in the area where the phage lysate has been placed after incubation at 30 ° C for 24 hours.

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 propellers, projecting the bases of each helix from that helix to the opposite. The adenine base (A) in one helix will always oppose the thymine base (T) in the other helix, and the guanine base (G) will oppose the cytosine base (C). The bases are held in opposition by their ability to bind hydrogen in this specific manner. Although each individual link is relatively weak, the net effect of many bases with adjacent hydrogen bonds, together with the effects of base accumulation, constitutes a stable union of the two complementary helices. 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 helices. If the DNA is then placed under thermodynamically favorable conditions in hydrogen bonding of the bases, the DNA helices will conjugate or "hybridize" and reform the original double helix DNA. If carried out under appropriate conditions, this hybridization can be highly specific. That is, only propellers with a high degree of base complementarity can form stable double helix structures. The ratio of the hybridization specificity to the reaction conditions is well known. Accordingly, hybridization can be used to analyze whether two DNA segments are complementary in their base sequences. It is this hybridization mechanism that facilitates the use of probes to detect and easily characterize the DNA sequences of interest. The probes can be RNA or DNA. The probe will normally have at least about 10 bases, more usually at least about 18 bases, and can have up to about 50 bases or more, generally no more than about 200 bases if the probe is prepared synthetically. However, 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. A useful hybridization procedure typically includes the initial steps of isolating the DNA sample in question and chemically purifying 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 ADB 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 transed to an immobilizing membrane in order to maintain the geometry of the pieces. The membrane can then be dried and prehybridized to equilibrate it for subsequent immersion in a hybridization solution. The manner in which the nucleic acid is fixed to a solid support may vary. This fixation of the DNA for the subsequent processing has great value for the use of this technique in field studies, far from the laboratory facilities. The specific hybridization technique is not essential for the present invention. As improvements are made in hybridization techniques, they can be easily applied. As is known to the technicians, if the probe molecule and the nucleic acid sample hybridize to form a strong non-covalent bond between the two molecules, it can be logically presumed 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. The polynucleotides of the present invention, as well as the probes derived from segments thereof, can be synthesized using DNA synthesizers by means of standard procedures. 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 32P, 35S or the like. A probe labeled with a radioactive isotope can be constructed from a nucleotide sequence complementary to the DNA sample by a conventional translation reaction using DNases and DNA polymerase. The probe and sample can then be combined in a hybridization bufsolution and maintained at an appropriate temperature until the anelation occurs. Then, the membrane is washed to release it from foreign materials, leaving the molecules of the sample and the ligated probe typically detected and quantified by autoradiography and / or liquid scintillation counting. For synthetic probes it may be more advisable to use enzymes such as polynucleotide kinase or terminal transferase to label the DNA at the end for use as probes. Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or peroxidases, or the various chemiluminescent ones 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. The probe can also be marked at both ends with different types of markers for ease of separation, such as, for example, using an isotopic label at the end mentioned above and a biotin label at the other end. The amount of labeled probe present in the hybridization solution varies widely, depending on the nature of the label, the amount of labeled probe that can reasonably be attached to the filter and the stringency of the hybridization. In general, considerable excesses of probe will be used to increase the binding coefficient of the probe to the fixed DNA. Various degrees of stringency hybridization can be employed. The more severe the conditions, the greater the complementarity required for double training. The severity can be controlled by means of the temperature, the concentration of the probe, the length of the probe, the ionic power, the time and other factors. Preferably, the hybridization is carried out under stringent conditions by means of well known techniques in the medium according to what is described, for example, in DNA Probes, by Keller, G.H., M.M. Manak (1987), Stockton Press, New York, NY, p. 169-170. Herein, the expression "stringent" conditions of hybridization refers to conditions that produce the same, or approximately the same degree of hybridization specificity as the conditions employed by applicants herein. Specifically, the hybridization of DNA immobilized in Southern blots with probes of specific 32 P-labeled genes was carried out by standard methods (Maniatis et al.). In general, hybridization and subsequent washings were carried out under stringent conditions that resulted in the detection of target sequences with homology to the toxin genes in question. For genetic double-stranded DNA 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 has been described 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 [eds.] Academic Press , New York 100: 206-285). Tm 81.5 ° C + 16.6 Log [Na +] + 0.41 (% G + C) - 0.61 (% formamide) - 600 / double length 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 Tm - 20 ° C for 15 minutes in 0.2X SSPE. 0.1 SDS (washing of moderate rigor). 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 (numbers G / C base pairs) (Suggs, S: V: , T. Miyake, EH Kawashime, MJ Johnson, K. Itakura and RB Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, DD Brown [ed.], Academic Press, New York, 23: 683- 693). Washes were 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 the hybridization temperature for 15 minutes in 1X SSPE, 0.1% SDS (wash of moderate stringency). Duplex formation and stability depend on the substantial complementarity between the two helices of a hybrid and, as noted above, some degree of mismatch can be tolerated. Therefore, the nucleotide sequences of the present invention include mutations (both single and multiple), deletions, insertions and 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. Known methods include, but are not limited to: (1) synthesizing chemically or otherwise an artificial sequence that is a mutation, insertion or deletion of the known sequence; (2) using a nucleotide sequence according to the present invention as a probe to obtain, by means of hybridization, a new sequence or mutation, insertion or deletion of the sequence of the probe; and (3) effecting the mutation, insertion or deletion of an in vitro or in vivo assay sequence. It is important to note that variants by mutation, insertion or deletion generated from a given probe can be more or less efficient than the original probe. Despite such differences in efficiency, these variants are within the scope of the present invention. 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 also be used as a substantial sequence with the original sequence. In the present, the term "sequence homology" refers to a homology that is sufficient to allow the variant to function with the same characteristics as the original probe. Preferably, the variants have amino acid or nucleotide identity with the exemplified sequences greater than 50%; more preferably, there is more than 75% identity and most preferably, there is more than 90% identity. The degree of homology necessary for the variant to function in its intended capacity depends on the intended use of the sequence. It is the competence of a person trained in this technique to carry out mutations by mutation, insertion or deletion designed 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 with primers of a nucleic acid sequence. This process 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 Scharf, Fred Faloona, Kary B. Mulis, Glenn T Horn, Henry A. Eriich, Norman Arnheim [1985] "Enzymatic Amplification of ß-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle 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 helices of the target sequence. The primers are oriented with the 3 'ends facing each other. The repeated cycles of heat denaturing of the pattern, the annealing of the primers to their complementary sequences and the extension of the primers annelated with a DNA polymerase results in the amplification of the segment defined by the 5 'ends of the PCR primers. Since the product of the extension of each primer can serve as a standard 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 white fragment, up to several million times in a few hours. By using a thermostable DNA polymerase such as Taq polymer, which is isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated. The DNA sequences obtained according to the present invention can be used as primers for the PCR amplification. In carrying out the PCR amplification some degree of mismatch between the primer and the standard can be tolerated. Therefore, mutations, deletions and insertions (especially additions of nucleotides to the 5 'end) of primers were obtained in light of the description herein which is included within the scope of the invention herein. Mutations, insertions and deletions can be produced in a given primer by methods known to one skilled in the art. It is important to note that variants by mutation, insertion and deletion generated from a certain sequence of primers can be more or less efficient than the original sequences. Despite such differences in efficiency, these variants are within the scope of the present invention. All of the US patents cited herein are incorporated herein by reference. Below are examples that illustrate the procedures for carrying out the invention. These examples should not be considered as limiting. All percentages are expressed by weight and all proportions of the solvent mixtures are by volume unless otherwise indicated.

EXAMPLE 1 Preparation of the isolates Strains of ß.í. in a peptone medium, glucose salts until they were fully sporulated. They were harvested by centrifugation and sprayed by lyophilization. The toxin proteins were extracted from the samples by extraction in a buffer of sodium carbonate, 2-mercaptoethanol with a pH of 10.7 to 11.0 during the extraction at 37 ° C. The protein extracts were recovered by centrifugation, dialysed with 0.01 M sodium carbonate, pH 9.5 and, if necessary, concentrated to at least 1 mg / ml protein using a "spin column" microconcentrator. Protein concentrations were estimated by laser densitometry using bovine serum albumin as a parameter. Under these conditions, the extract of strain HD969 contained proteins in a size range of 130 kDa to approximately 62 kDa (as well as a number of smaller bands), PS66D3 had an important band at approximately 64 kDa and bands less than approximately 52 kDa and 30 kDa, PS50C had an important band at approximately 62 kDa and numerous minor bands.

EXAMPLE 2 Bioassay of preparations against Nilaparvata lugens, the cicada of the brown rice plant The biological tests consisted in exposing insects to an artificial diet containing the preparations of ß.í. from example 1 diluted to 1 mg ml of the toxin of ß.i .. Insects were exposed, as nymphs, to the test solutions for 72 hours, and after 24 hours the insects that were not fed satisfactorily, judging by the lack of production of sweet secretion were discarded.The insects were examined at 0, 24, 48 and 72 hours and survival was classified.Each bioassay was replicated three times with 10 insects used in each replication. buffer, using once again 10 insects, as part of each replicated biological test.The results of these tests are represented in table 3.

TABLE 3 Percentage of living Sample n Oh 24h 48h 72h HD969 31 100 71 45 16 66D3 30 100 93 57 33 50C 30 100 93 70 40 Control in 30 100 93 83 62 Buffer to Control in 31 100 87 63 60 Buffer b Control in 31 100 87 84 74 Buffer c EXAMPLE 3 Transformation of plants One aspect of the present invention is the transformation of plants with genes encoding the insecticidal toxins of the present invention. The transformed plants are resistant to attack by white pests. The genes encoding the pesticidal toxins, as described herein, can be inserted into plant cells using a variety of techniques known in the art. For example, a large number of cloning vectors is available consisting of a replication system in E. coli and a marker that allows the selection of transformed cells for the preparation for the insertion of foreign genes into higher plants. The vectors consist, for example, in the pBR322 series, the pUC series, the M13mp series, pACYC184, etc. Consequently, the sequence encoding the toxin of B. t. it 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 grown in a suitable nutrient medium, then harvested and subjected to analysis. The plasmid is recovered. The sequence analysis, restriction analysis, electrophoresis and other biological biochemical and molecular methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be excised and linked to the next DNA sequence. Each plasmid sequence can be cloned into the same or other plasmids. Depending on the method of insertion of the desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right border must be joined, although frequently both the right and left borders of the T-DNA of the Ti or Ri plasmid as the region of flanking the genes to insert. The use of T-DNA for the transformation of plant cells has been intensively investigated and sufficiently described in EP 120 516; Hoekema (1985) in: The Binary Plant Vector System, Offset-durkkerij Kanters B.V., Alblasserdam, Chapter 4; Fraley eí al., Crit. Rev. Plant Sci. 4: 1-46; and An eí al. (1985) EMBO J. 4: 277-287. Once the inserted DNA has been integrated into the genome, it is relatively stable there and, as a general rule, does not come back out. They usually contain a selection marker that gives transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hrythromycin 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. A large number of techniques are available to insert DNA into a plant host cell. 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. If Agrobacteria are used for transformation, the DNA to be inserted has to be cloned into special plasmids, that is, it is an intermediate vector or a binary vector. Intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination due to sequences that are homologous to the T-DNA sequences. The Ti or Ri plasmid 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 Agrobacteria. These comprise a selection marker gene and a binder or polylinker that are formed 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 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. Advantageously, plant explants can be grown 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, pieces of leaves, segments of stem, meristematic tissue, roots, but also protoplasts or cells grown in suspension) in a suitable medium that can contain antibiotics or biocides for the selection. The plants thus obtained can be analyzed below to confirm the presence of the inserted DNA. No special demands were made regarding the plasmids in the case of injection and electroporation. It is possible to use common plasmids such as, for example, pUC derivatives. In transformation by biolistics, plasmid DNA or linear DNA can be used.

The transformed cells are regenerated to form morphologically normal plants in the usual way. If a transformation event involves a germline cell, the inserted DNA and the corresponding phenotypic traits are transmitted to the plants of the progeny. Such plants can be cultivated as usual and crossed with plants that have the same hereditary factors transformed and 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; U.S. Patent No. 5,380,831. In addition, advantageously, plants that code for a toxin are used truncate The truncated toxin typically encodes about 55% to about 80% of the complete toxin. The methods to create genes of ß. í. Synthetics for use in plants are known in the art. 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 in light thereof and these should be included in the spirit and purpose of this application. and the scope of the appended claims.

Claims (18)

NOVELTY OF THE INVENTION CLAIMS

1. A method for controlling a pest of homopterous insects, which consists in contacting said pest with a toxin obtained from an isolated strain of Bacillus thuringiensis selected from the group consisting of HD969, PS66D3 and PS50C.

2. The method according to claim 1, further characterized in that said isolated strain is HD969.

3. The method according to claim 1, further characterized in that the isolated strain is PS66D3.

4. The method according to claim 1, further characterized in that said isolated strain is PS50C.

5. The method according to claim 1, further characterized in that said toxin is expressed in plant.

6. A toxin obtained from an isolated strain of Bacillus thuringiensis selected from the group consisting of HD969, PS66D3 and PS50C, or an active portion against homoptera of said toxin, a toxin that is toxic to a pest of homopteran insects.

7. The toxin according to claim 6, further characterized in that said isolated strain is HD969.

8. - The toxin according to claim 6, further characterized in that the isolated strain is PS66D3.

9. The toxin according to claim 6, further characterized in that said isolated strain is PS50C.

10. A gene that encodes a toxin obtained from an isolated strain of Bacillus thuringiensis selected from the group consisting of HD969, PS66D3 and PS50C, or an active portion against homoptera of said toxin, a toxin that is toxic to an insect pest Homoptera.

11. The gene according to claim 10, further characterized in that said isolated strain is HD969.

12. The gene according to claim 10, further characterized in that the isolated strain is PS66D3.

13. The gene according to claim 10, further characterized in that said isolated strain is PS50C.

14. A host cell transformed with a toxin obtained from an isolated strain of Bacillus thuringiensis selected from the group consisting of HD969, PS66D3 and PS50C, or an active portion against homopterans of said toxin, toxin that is toxic to a pest of homopterous insects.

15. The transformed host cell according to claim 14, further characterized in that said isolated strain is HD969.

16. The transformed host cell according to claim 14, further characterized in that the isolated strain is PS66D3.

17. - The transformed host cell according to claim 14, further characterized in that said isolated strain is PS50C.

18. The isolated strain PS66D3 of Bacillus thuringiensis, available under deposit number NRRL B-21657.