WO1998040491A2

WO1998040491A2 - Pesticidal bacillus thuringiensis strains

Info

Publication number: WO1998040491A2
Application number: PCT/US1998/005185
Authority: WO
Inventors: Gregory A. Bradfisch; Brian Stockhoff; Judy Muller-Cohn
Original assignee: Mycogen Corp
Current assignee: Mycogen Corp
Priority date: 1997-03-13
Filing date: 1998-03-13
Publication date: 1998-09-17
Anticipated expiration: 1999-09-13
Also published as: AU6704098A; CA2281914A1; EP1003876A2; AR012067A1; JP2001515355A; US6048839A; AU740906B2; US5985831A; WO1998040491A3; BR9808848A

Abstract

This application pertains to Bacillus thuringiensis isolates and their toxins active against various lepidopteran, coleopteran or aphid pests. The isolates are PS17, PS28Q2, PS33F2, PS32B, PS54G2, PS50C, PS71M3, PS86BB1, PS86Q3, PS14OE2, PS169E, PS62B, PS158C2, PS167P, PS196S1, PS201T6, HD511, PS18, PS28K1, PS43A2, PS159E6, PS164H2, PS186EE, PS196Q3, PS198A2, PS225K1, KB6, KB19, HD977 and HD541.

Description

DESCRIPTION

PESΗCIDAL BACILLUS THURINGIENSIS STRAINS

Background of the Invention

The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering these B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner, F.H., L. Kim [1988] TIBTECH 6:S4-S7). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.

Until the last fifteen years, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces a crystalline δ- endotoxin which is toxic to the larvae of a number of lepidopteran insects.

In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely israelensis and morrisoni (a.k.a. tenebrionis, a.k.a. B.t. M-7, a.k.a. B.t. san diego), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F.H. [1989] "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms," in Controlled Delivery of Crop Protection Agents, R.M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255.). See also Couch, T.L. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis," Developments in Industrial Microbiology 22:61-76; and Beegle, C.C. (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Industrial Microbiology 20:97-104. Krieg, A., A.M. Huger, G.A. Langenbruch, W.

Schnetter (1983) Z. ang. Ent. 96:500-508 describe Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni. More recently, new subspecies of B.t. have been identified, and genes responsible for active δ-endotoxin proteins have been isolated (Hδfte, H., H.R. Whiteley [1989] Microbiological Reviews 52(2):242-255). Hofte and Whiteley classified B.t. crystal protein genes into four major classes. The classes were Cryl (Lepidoptera-specific), Cryll (Lepidoptera- and Diptera-specific), Crylll (Coleoptera-specific), and CrylV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J.S., J. Payne, L. Kim [1992] Bio/Technology 10:271-275). CryV has been proposed to designate a class of toxin genes that are nematode- specifϊc. Lambert et al. (Lambert, B., L. Buysse, C. Decock, S. Jansens, C. Piens, B. Saey, J. Seurinck, K. van Audenhove, J. Van Rie, A. Van Vliet, M. Peferoen [1996] Appl. Environ. Microbiol 62(l):80-86) describe the characterization of a Cry9 toxin active against lepidopterans. Published PCT applications WO 94/05771 and WO 94/24264 also describe B.t. isolates active against lepidopteran pests. Gleave et al. ([1991] JGM 138:55-62), Shevelev et al. ([1993] FEBS Lett. 336:79-82; and Smulevitch et al. ([1991] FEBS Lett. 293:25-26) also describe B.t. toxins. Many other classes of B.t. genes have now been identified. The cloning and expression of a B. t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf, H.E., H.R. Whiteley [1981] Proc. Natl. Acad. Sci. USA 78:2893-2897.). U.S. Patent 4,448,885 and U.S. Patent 4,467,036 both disclose the expression of B.t. crystal protein in E. coli. U.S. Patents 4,990,332; 5,039,523; 5,126,133; 5,164,180; and 5,169,629 are among those which disclose B.t. toxins having activity against lepidopterans. PCT application WO96/05314 discloses PS86W1, PS86V1, and other B.t. isolates active against lepidopteran pests. The PCT patent applications published as W094/24264 and WO94/05771 describe B.t. isolates and toxins active against lepidopteran pests. B.t. proteins with activity against members of the family Noctuidae are described by Lambert et al., supra. U.S. Patents 4,797,276 and 4,853,331 disclose B. thuringiensis strain tenebrionis which can be used to control coleopteran pests in various environments. U.S. Patent

No. 4,918,006 discloses B.t. toxins having activity against dipterans. U.S. Patent No. 5,151,363 and U.S. Patent No. 4,948,734 disclose certain isolates of B.t. which have activity against nematodes. Other U.S. patents which disclose activity against nematodes include 5,093,120; 5,236,843; 5,262,399; 5,270,448; 5,281,530; 5,322,932; 5,350,577; 5,426,049; and 5,439,881. U.S. Patent Nos. 5,262,159 and 5,468,636 disclose B.t. isolates PS157C1, PS86A1, and PS75J1 as having activity against aphids.

As a result of extensive research and investment of resources, other patents have issued for new B.t. isolates and new uses of B.t. isolates. See Feitelson et al., supra, for a review. However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art. U.S. Patent No. 5,506,099 describes methods for identifying unknown B.t. isolates. Also, U.S. Patent No. 5,204,237 describes specific and universal probes for the isolation of B.t. toxin genes.

Brief Summary of the Invention

The subject invention concerns materials and methods useful in the control of non- mammalian pests and, particularly, plant pests. In a specific embodiment, the subject invention provides new isolates and toxins useful for the control of lepidopterans, coleopterans, and/or aphids. In preferred embodiments, these pests are selected from the group consisting of diamondback moth, Bertha armyworm, tobacco budworm, band sunflower moth, sunflower beetle, red sunflower seed weevil, canola flea beetle, sunflower stem weevil, and greenbug.

Nucleotide sequences useful according to the subject invention encode pesticidal toxins. One embodiment of the subject invention concerns plants cells transformed with at least one polynucleotide sequence of the subject invention such that the transformed plant cells express pesticidal toxins in tissues consumed by target pests. Such transformation of plants can be accomplished using techniques well known to those skilled in the art and would typically involve modification of the gene to optimize expression of the toxin in plants.

As described herein, the toxins useful according to the subject invention may be chimeric toxins produced by combining portions of multiple toxins. Also, toxins of the subject invention may be used in combination to achieve enhanced pest control.

Alternatively, the B.t. isolates of the subject invention, or recombinant microbes expressing the toxins described herein, can be used to control pests. In this regard, the invention includes the treatment of substantially intact B.t. cells, and/or recombinant cells containing the expressed toxins of the invention, treated to prolong the pesticidal activity when the substantially intact cells are applied to the environment of a target pest. The treated cell acts as a protective coating for the pesticidal toxin. The toxin becomes active upon ingestion by a target insect.

Detailed Disclosure of the Invention The subject invention provides B.t. isolates and toxins active against diamondback moth, Bertha armyworm, tobacco budworm, band sunflower moth, sunflower beetle, red sunflower seed weevil, canola flea beetle, sunflower stem weevil, and/or greenbug.

A further aspect of the subject invention concerns novel B.t. isolates and the toxins and genes obtainable from these isolates. The novel B.t. isolates of the subject invention have been designated PS 18, PS28K1, PS43A2, PS159E6, PS164H2, PS186EE, PS196Q3, PS198A2, and PS225K1

Microorganisms useful according to the subject invention have been deposited in the permanent collection of the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoπa, Illinois 61604, USA The culture repository numbers of the deposited strains are shown in Table 1

Table 1.

Culture Repository No. Deposit Date

PS18 NRRL B-21954 March 12, 1998

PS28K1 NRRL B-21955 March 12, 1998

PS43A2 NRRL B-21956 March 12, 1998

PS159E6 NRRL B-21958 March 12, 1998

PS164H2 NRRL B-21959 March 12, 1998

PS186EE NRRL B-21960 March 12, 1998

PS196Q3 NRRL B-21961 March 12, 1998

PS198A2 NRRL B-21962 March 12, 1998

PS225K1 NRRL B-21963 March 12, 1998

KB6 NRRL B-18873 August 27, 1991

KB19 NRRL B-21964 March 12, 1998

The isolates of the subject invention have been deposited under conditions that assure that access to the cultures will be available dunng the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention m derogation of patent πghts granted by governmental action.

Further, the isolates of the subject invention will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i e., they will be stored with all the care necessary to keep them viable and uncontammated for a period of at least five years after the most recent request for the furnishing of a sample of a deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposit(s) should the depository be unable to furnish a sample when requested, due to the condition of the deposit(s). All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.

Certain isolates which are useful according to the subject invention are available to the public by virtue of the issuance of U.S. patents. These isolates, their deposit accession number, and their date of deposit are shown in Table 2(a). The corresponding U.S. patents that disclose these isolates are shown in Table 2(b).

Table 2(a). Pesticidal B.t. strains of the subject invention Culture NRRL Deposit Deposit Date

PS17 B- 18243 10 AUG 87

PS28Q2 B-18888 25 SEP 91

PS32B B-21531 1 APR 96

PS33F2 B- 18244 10 AUG 87

PS50C B- 18746 23 JAN 91

PS54G2 B-21543 1 APR 96

PS62B B-18398 7 SEP 88

PS71M3 B-18930 8 JAN 92

PS80JJ1 B-18679 1 AUG 90

PS86BB1 B-21557 2 MAY 96

PS86Q3 B-18765 13 FEB 91

PS140E2 B-18812 10 APR 91

PS158C2 B-18872 17 SEP 91

PS167P B-18681 1 AUG 90

PS169E B- 18682 1 AUG 90

PS196S1 B-18748 23 JAN 91

PS201T6 B- 18750 23 JAN 91

The isolates HD511, HD541, and HD977 are available from the USDA-ARS NRRL Culture Collection, Peoria, Illinois. Table 2(b)

U.S. Patent No. or Isolate Sequences (of Toxin(s) Pesticidal Activity Publication No. Disclosed and Gene(s)) Disclosed Disclosed

4,849,217 PS17 alfalfa weevils

5,281,530 PS17 17(a) and 17(b) nematodes

5,427,786 PS28Q2 certain coleopteran genera, including Hypera, Diabrotica, and Phyllotreta

4,849,217 PS33F2 alfalfa weevils

5,439,881 PS33F2 33F2 nematodes

5,707,619 PS33F2 various weevils

5,670,365 PS32B nematodes

5,277,905 PS50C coleopterans

5,366,892 PS50C 50C coleopterans

5,185,148 PS50C scarabs

5,457,179 PS50C coleopterans

5,554,534 PS50C 50C(a) and 50C(b) scarabs

5,667,993 PS54G2 nematodes

5,670,365 PS54G2 nematodes

4,849,217 PS62B alfalfa weevils

5,723,440 PS71M3 hemipterans

4,849,217 PS80JJ1 alfalfa weevils

WO94/23036 PS80JJ1 wireworms (click beetles)

5,632,987 PS80JJ1 80JJ1 (130kda) corn rootworm

5,589,382 PS80JJ1 80JJ1 (130kda) nematodes

5,670,365 PS80JJ1 80JJ1 (130kda) nematodes

WO 97/40162 PS80JJ1 80JJ1 (14 and 44kda) coleopterans

WO 98/00546 PS86BB1 86BBl(a), 86BBl(b), lepidopterans and 86BB 1(c)

5,596,071 PS86Q3 86Q3(a) and 86Q3(c) ants

5,427,786 PS140E2 certain coleopteran genera, including Hypera, Diabrotica, and Phyllotreta

5,596,071 PS140E2 ants

5,268,172 PS158C2 lepidopterans

5,723,758 PS158C2 158C2(a), 158c2(b), lepidopterans 158C2(c), and l58C2(d)

5,632,987 PS167P corn rootworms

5,707,619 PS167P various weevils

5,589,382 PS167P 167P nematodes

5,670,365 PS167P 167P nematodes

5,632,987 PS169E corn rootworm

5,707,619 PS169E various weevils

5,436,002 PS196S1 dipterans and corn rootworm

5,707,619 PS196S1 various weevils

5,635,480 PS196S1 dipterans and corn rootworm

5,436,002 PS201T6 201T6 (30 & 25 kda) dipterans and corn rootworm

5,707,619 PS201T6 various weevils

5,635,480 PS201T6 201T6 (30 &25 kda) dipterans and corn rootworm

5,723,440 PS201T6 hemipterans

5,262,324 HD511 coleopterans

5,286,486 HD511 HD511 coleopterans

5,306,494 HD511 coleopterans

These patents, with their disclosure of the indicated isolates as well as their toxins and genes, are incorporated herein by reference. Genes and toxins. The genes and toxins useful according to the subject invention include not only the full length sequences disclosed but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the toxins specifically exemplified herein. As used herein, the terms "purified toxin" and "isolated toxin" refer to toxins that have been affected by "the hand of man" and that are substantially free from naturally associated impurities, as the toxins would be found in their natural state. As used herein, the terms "variants" or "variations" of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having pesticidal activity. As used herein, the term "equivalent toxins" refers to toxins having the same or essentially the same biological activity against the target pests as the exemplified toxins.

It should be apparent to a person skilled in this art that genes encoding active toxins can be identified and obtained through several means. The specific genes exemplified herein may be obtained from the isolates deposited at a culture depository as described above. These genes, or portions or variants thereof, may also be constructed synthetically, for example, by use of a gene synthesizer. Variations of genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as BaBl or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which encode active fragments may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these toxins.

Equivalent toxins and/or genes encoding these equivalent toxins can be derived from B.t. isolates and/or DNA libraries using the teachings provided herein. There are a number of methods for obtaining the pesticidal toxins of the instant invention. For example, antibodies to the pesticidal toxins disclosed and claimed herein can be used to identify and isolate other toxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the toxins which are most constant and most distinct from other B.t. toxins.

Fragments and equivalents which retain the pesticidal activity of the exemplified toxins would be within the scope of the subject invention. Also, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention. As used herein, reference to "essentially the same" sequence refers to sequences which have amino acid substitutions, deletions, additions, or insertions which do not materially affect pesticidal activity. Fragments retaining pesticidal activity are also included in this definition.

A further method for identifying the toxins and genes of the subject invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences may be detectable by virtue of an appropriate label or may be made inherently fluorescent as described in International Application No. WO93/16094. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample have substantial homology. Preferably, hybridization is conducted under stringent conditions by techniques well- known in the art, as described, for example, in Keller, G.H., M.M. Manak (1987) DNA Probes,

Stockton Press, New York, NY., pp. 169-170. Detection of the probe provides a means for determining in a known manner whether hybridization has occurred. Such a probe analysis provides a rapid method for identifying toxin-encoding genes of the subject invention. The nucleotide segments which are used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures. These nucleotide sequences can also be used as PCR primers to amplify genes of the subject invention.

Certain toxins of the subject invention have been specifically exemplified herein. Since these toxins are merely exemplary of the toxins of the subject invention, it should be readily apparent that the subject invention comprises variant or equivalent toxins (and nucleotide sequences coding for equivalent toxins) having the same or similar pesticidal activity of the exemplified toxin. Equivalent toxins will have amino acid homology with an exemplified toxin. This amino acid identity will typically be greater than 60%, preferably be greater than 75%, more preferably greater than 80%, more preferably greater than 90%, and can be greater than 95%. The amino acid homology will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity. In this regard, conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table 3 provides a listing of examples of amino acids belonging to each class. TABLE 3

Class of Amino Acid Examples of Amino Acids

Nonpolar Ala, Val, Leu, He, Pro, Met, Phe, Trp

Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin Acidic Asp, Glu

Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the biological activity of the toxin. The toxins of the subject invention can also be characterized in terms of the shape and location of toxin inclusions, which are described above.

Recombinant hosts. The toxin-encoding genes harbored by the isolates of the subject invention can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. With suitable microbial hosts, e.g., Pseudomonas, the microbes can be applied to the situs of the pest, where they will proliferate and be ingested. The result is a control of the pest. Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, then can be applied to the environment of the target pest. A wide variety of ways are available for introducing a B.t. gene encoding a toxin into a microorganism host under conditions which allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in United States Patent No. 5,135,867, which is incorporated herein by reference.

Alternatively, a plant transformed to express a toxin of the subject invention can be used to contact the target pest with the toxin. Synthetic genes which are functionally equivalent to the toxins of the subject invention can also be used to transform hosts. Methods for the production of synthetic genes can be found in, for example, U.S. Patent No. 5,380,831.

Treatment of cells. As mentioned above, B.t. or recombinant cells expressing a B.t. toxin can be treated to prolong the toxin activity and stabilize the cell. The pesticide microcapsule that is formed comprises the B.t. toxin within a cellular structure that has been stabilized and will protect the toxin when the microcapsule is applied to the environment of the target pest. Methods for treatment of microbial cells are disclosed in United States Patent Nos. 4,695,455 and 4,695,462, which are incorporated herein by reference.

Growth of cells. The cellular host containing the B.t. insecticidal gene may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the B.t. gene.

These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.

The B.t. cells of the invention can be cultured using standard art media and fermentation techniques. Upon completion of the fermentation cycle the bacteria can be harvested by first separating the B.t. spores and crystals from the fermentation broth by means well known in the art. The recovered B.t. spores and crystals can be formulated into a 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 particular target pests. These formulations and application procedures are all well known in the art. Methods and formulations for pest control. Control of pests using the isolates, toxins, and genes of the subject invention can be accomplished by a variety of methods known to those skilled in the art. These methods include, for example, the application of B.t. isolates to the pests (or there location), the application of recombinant microbes to the pests (or their locations), and the transformation of plants with genes which encode the pesticidal toxins of the subject invention. Recombinant microbes may be, for example, a B.t. , E. coli, or Pseudomonas.

Transformations can be made by those skilled in the art using standard techniques. Materials necessary for these transformations are disclosed herein or are otherwise readily available to the skilled artisan.

Formulated bait granules containing an attractant and spores and crystals of the B.t. isolates, or recombinant microbes comprising the genes obtainable from the B.t. isolates disclosed herein, can be applied to the soil. Formulated product can also be applied as a seed- coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments of B.t. cells may be employed as liquids, wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like).

As would be appreciated by a person skilled in the art, the pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least about 1% by weight and may be 100%> by weight. The dry formulations will have from about 1-95% by weight of the pesticide while the liquid formulations will generally be from about 1-60% by weight of the solids in the liquid phase. The formulations will generally have from about 10² to about 10⁴ cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the pest, e.g., soil and foliage, by spraying, dusting, sprinkling, or the like.

Mutants. Mutants of the isolates of the invention can be made by procedures well known in the art. For example, an asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis of an isolate. The mutants can be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.

All of the U.S. patents cited herein are hereby incorporated by reference.

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 - Culturing of B.t. Isolates of the Invention

A subculture of the B.t. isolates, or mutants thereof, can be used to inoculate the following medium, a peptone, glucose, salts medium.

Bacto Peptone 7.5 g/1

Glucose 1.0 g/1

KH₂P0₄ 3.4 g/1 K₂HP0₄ 4.35 g/1

Salt Solution 5.0 ml/1

CaCl₂ Solution 5.0 ml/1 pH 7.2

Salts Solution (100 ml)

MgS0₄-7H₂0 2.46 g

MnS0₄-H₂0 0.04 g

ZnS0₄-7H₂0 0.28 g

FeS0₄-7H₂0 0.40 g CaCl₂ Solution (100 ml) CaC -2H,0 3.66 g

The salts solution and CaCl₂ solution are filter-sterilized and added to the autoclaved and cooked broth at the time of inoculation. Flasks are incubated at 30° C on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors by procedures well known in the art.

The B.t. spores and/or crystals, obtained in the above fermentation, can be isolated by procedures well known in the art. A frequently-used procedure is to subject the harvested fermentation broth to separation techniques, e.g., centrifugation.

The supernatant from these cultures can be used to obtain toxins according to the subject invention. Thus, the subject invention is not limited to crystal proteins; useful soluble proteins are also contemplated.

Example 2 — Activity Against Diamond Back Moth (Plutella xylostellά)

Certain B.t. isolates and toxins were found to be active against diamond back moth. These results are shown in Table 4.

Table 4.

Strain Toxin

PS86Q3 Cry5Ac

PS17 Cry5Aa

HD511 Cry7Ab2

Example 3 — Activity Against Bertha Armyworm (Mamestra conβguratά)

Certain B.t. isolates and toxins were found to be active against Bertha armyworm. These results are shown in Table 5. Table 5.

Strain Toxin

HD511 Cry7Ab I>S8603^^ Cry5AC

Example 4 — Activity Against Tobacco Budworm (Heliothis virescens)

Certain B.t. isolates and toxins were found to be active against tobacco budworm. These results are shown in Table 6.

Table 6.

Strain Toxin

PS86Q3 Cry5Ac

PS17 Cry5Aa

Example 5 - Activity Against Band Sunflower Moth (Cochylis hospes)

Certain B.t. isolates and toxins have been found to be active against banded sunflower moth. The results are shown in Table 7.

Table 7.

Strain Gene

PS17 Cry5Aa

Example 6 — Activity Against Sunflower Beetle (Zygogramma exclamationis,

Certain B.t. isolates and toxins have been found to be active against sunflower beetle. These results are shown in Table 8. Table 8.

Strain Toxin

PS80JJ1 80JJ1

PS50C Cry8B PS158C2 158C2(c)

The sequence of a 1227 amino acid 158C2(c) toxin is disclosed as SEQ ID NO. 8 in U.S. Patent No. 5,723,758. A 45 kda toxin from PS80JJ1 is an example of a preferred toxin.

Example 7 — Activity Against Red Sunflower Seed Weevil (Smicronvx fulvus)

The B.t. isolates designated PS28Q2, PS32B, PS54G2, and PS43A2 have been found to be active against red sunflower seed weevil.

Example 8 - Activity Against Canola Flea Beetle (Phyllotreta cruciferae) The B.t. isolates designated PS201T6, PS33F2, HD977, PS196Q3, PS225K1, PS164H2,

KB 19, PS86BB 1 , PS 186EE, KB6, and PS 198A2 have been found to be active against canola flea beetle. A cytlB toxin and a 130 kda toxin obtainable from PS201T6 are examples of preferred toxins for controlling Phyllotreta.

Example 9 — Activity Against Greenbug (Schizaphis graminum)

The B.t. isolates designated PS159E6, PS167P, PS71M3, PS201T6, PS196SI, HD541, PS28K1, PS 18, and PS50C have been found to be active against greenbug.

Example 10 - Activity Against Sunflower Spotted Stem Weevils (Cylindrocopturus adspersus) The5.t. isolates designated PS28Q2, PS43A2, PS62B, PS86B1, PS140E2, PS169E, and

PS201T6 have been found to be active against sunflower stem weevils.

Example 10 — Insertion of Toxin Genes Into Plants

One aspect of the subject invention is the transformation of plants with genes encoding insecticidal toxins. The transformed plants are resistant to attack by the target pest.

Genes encoding pesticidal toxins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well known in the art. For example, a large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, the sequence encoding the B.t. toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting 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, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted.

The use of T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in EP 120 516; Hoekema ( 1985) In: The Binary Plant Vector System,

Offset-durkkerij Kanters B.V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4:1- 46; and An et al. (1985) EMBOJ. A:211-2 1.

Once the inserted DNA has been integrated in the genome, it is relatively stable there and, as a rule, does not come out again. It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. The individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.

A large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or

Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [1978] Mo/. Gen. Genet. 163:181-187). The Agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension- cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection.

The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.

The transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.

In a preferred embodiment of the subject invention, plants will be transformed with genes wherein the codon usage has been optimized for plants. See, for example, U.S. Patent No.

5,380,831. Also, advantageously, plants encoding a truncated toxin will be used. The truncated toxin typically will encode about 55% to about 80% of the full length toxin. Methods for creating synthetic B.t. genes for use in plants are known in the art.

Example 11 — Cloning of B.t. Genes Into Insect Viruses

A number of viruses are known to infect insects. These viruses include, for example, baculoviruses and entomopoxviruses. In one embodiment of the subject invention, genes encoding the insecticidal toxins, as described herein, can be placed within the genome of the insect virus, thus enhancing the pathogenicity of the virus. Methods for constructing insect viruses which comprise B.t. toxin genes are well known and readily practiced by those skilled in the art. These procedures are described, for example, in Merryweather et al. (Merryweather, A.T., U. Weyer, M.P.G. Harris, M. Hirst, T. Booth, R.D. Possee (1990) J. Gen. Virol. 77:1535- 1544) and Martens et al. (Martens, J.W.M., G. Honee, D. Zuidema, J.W.M. van Lent, B. Visser, J.M. Vlak (1990) />/?/. Environmental Microbiol. 56^~(9):2764-2770). It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

Claims

A method for controlling lepidopteran pests, wherein said method comprises contacting said pest with a toxin obtainable from Bacillus thurinigiensis isolate PS 17.

2. The method, according to claim 1, wherein said toxin is a cry5Aa toxin.

3. The method, according to claim 1, wherein said toxin is expressed by a transformed plant.

4. The method, according to claim 1, wherein said lepidopteran pest is Plutella xylostella.

5. The method, according to claim 1, wherein said lepidopteran pest is Heliothis virescens.

6. The method, according to claim 1, wherein said lepidopteran pest is Cochylis hospes.

7. A method for controlling a pest of a genus selected from the group of genera consisting of Smicronyx and Cylindrocopturus, wherein said method comprises contacting said pest with a toxin obtainable from Bacillus thurinigiensis isolate PS28Q2.

8. A method for controlling a pest of the genus Phyllotreta, wherein said method comprises contacting said pest with a toxin obtainable from Bacillus thurinigiensis isolate PS33F2.

9. A method for controlling coleopteran pests, wherein said method comprises contacting said pest with a toxin obtainable from Bacillus thurinigiensis isolate selected from the group consisting of PS32B and PS54G2.

10. The method, according to claim 9, wherein said pest is Smicronyx fulvus .

11. A method for controlling a pest of the genus Zygoramma, wherein said method comprises contacting said pest with a Cry8B toxin obtainable from Bacillus thurinigiensis isolate PS50C.

12. A method for controlling an aphid, wherein said method comprises contacting said pest with a toxin obtainable from Bacillus thurinigiensis isolate PS71M3.

13. The method, according to claim 12, wherein said aphid is a Schizaphis graminum.

14. A method for controlling a pest of the genus Zygoramma, wherein said method comprises contacting said pest with a toxin obtainable from Bacillus thurinigiensis isolate PS80JJ1.

15. A method for controlling a coleopteran pest, wherein said method comprises contacting said pest with a toxin obtainable from Bacillus thurinigiensis isolate PS86BB 1.

16. The method, according to claim 15, wherein said toxin is expressed by a transformed plant.

17. A method for controlling a lepidopteran pest, wherein said method comprises contacting said pest with a toxin obtainable from Bacillus thurinigiensis isolate PS86Q3.

18. The method, according to claim 17, wherein said toxin is a Cry5Ac toxin.

19. The method, according to claim 17, wherein said toxin is expressed by a transformed plant.

20. The method, according to claim 17, wherein said pest is selected from the group consisting of Plutella xylostella, Mamestra configurata, and Heliothis virescens.

21. A method for controlling a pest of genus Cylindrocopterus, wherein said method comprises contacting said pest with a toxin obtainable from a Bacillus thurinigiensis isolate selected from the group consisting of PS 140E2, PS 169E, and PS62B .

22. A method for controlling a coleopteran pest, wherein said method comprises contacting said pest with a toxin obtainable from Bacillus thurinigiensis isolate PS158C2.

23. The method, according to claim 22, wherein said toxin is expressed by a transformed plant.

24. The method, according to claim 22, wherein said pest is Zygoramma exclamationis .

25. The method according to claim 22, wherein said toxin is a 158C2(c) toxin.

26. A method for controlling an aphid, wherein said method comprises contacting said aphid with a toxin obtainable from a Bacillus thurinigiensis isolate selected from the group consisting of PS50C, PS167P, PS196S1, and PS201T6.

27. The method, according to claim 26, wherein said toxin is expressed by a transformed plant.

28. The method, according to claim 27, wherein said aphid is a Schizaphis graminum.

29. A method for controlling a pest of a genus selected from the group of genera consisting oϊ Phyllotreta and Cylindrocopturus, wherein said method comprises contacting said pest with a toxin obtainable from Bacillus thurinigiensis isolate PS201T6.

30. The method, according to claim 30, wherein said toxin is expressed by a transformed plant.

31. A method for controlling lepidopteran pests, wherein said method comprises contacting said pest with a toxin obtainable from Bacillus thurinigiensis isolate HD511.

32. The method, according to claim 31, wherein said toxin is expressed by a transformed plant.

33. The method, according to claim 31, wherein said lepidopteran pest is Plutella xylostella.

34. The method, according to claim 31, wherein said lepidopteran pest is Mamestra configurata.

35. A biologically pure culture of a Bacillus thuringiensis isolate selected from the group consisting oϊ Bacillus thuringiensis isolates PS 18, PS28K1 , PS43 A2, PS 159E6, PS 164H2, PS186EE, PS196Q3, PS198A2, and PS225K1.

36. A biologically pure culture of a Bacillus thuringiensis isolate selected from the group consisting oϊ Bacillus thuringiensis isolates KB6 and KB19.

37. A method for controlling a coleopteran pest, wherein said method comprises contacting said pest with a toxin from a Bacillus thuringiensis isolate selected from the group consisting of PS43A2, PS164H2, PS186EE, PS196Q3, PS198A2, PS225K1, HD977, KB6, and KB19.

38. The method, according to claim 37, wherein said coleopteran pest is member of the genus Phyllotreta, and wherein said isolate is selected from the group consisting of PS164H2, PS186EE, PS196Q3, PS198A2, PS225K1, HD977, KB6, and KB19.

39. The method, according to claim 37, wherein said coleopteran pest is member of the genus Smicronyx, and wherein said isolate is PS43A2.

40. A method for controlling an aphid, wherein said method comprises contacting said pest with a toxin from a Bacillus thuringiensis isolate selected from the group consisting of PS159E6, PS28Kl, PS18, and HD541.

41. The method, according to claim 40, wherein said aphid is a Schizaphis graminum.

42. A polynucleotide sequence encoding a pesticidal toxin obtainable from a Bacillus thuringiensis isolate selected from the group consisting of PS 18, PS28K1 , PS43 A2, PS 159E6, PS164H2, PS186EE, PS196I3, PS198A2, PS225K1, KB6, and KB19.

43. A pesticidal toxin obtainable from a Bacillus thuringiensis isolate selected from the group consisting of PS18, PS28K1, PS43A2, PS159E6, PS164H2, PS186EE, PS19613, PS198A2, PS225K1, KB6, and KB 19.

44. The method, according to claim 29, wherein said toxin is selected from the group consisting of a cytlB toxin and a 130 kda toxin.

45. The method, according to claim 14, wherein said toxin is a 45 kda toxin.