Isolates and Genes Encoding

Europäisches Patentamt *EP000612218B1* (19) European Patent Office

Office européen des brevets (11) EP 0 612 218 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.7: A01N 63/00, A01N 63/02, of the grant of the patent: C12N 15/32, C12N 1/20 04.10.2001 Bulletin 2001/40 (86) International application number: (21) Application number: 92925087.6 PCT/US92/09510

(22) Date of filing: 06.11.1992 (87) International publication number: WO 93/08693 (13.05.1993 Gazette 1993/12)

(54) NOVEL COLEOPTERAN-ACTIVE $i(BACILLUS THURINGIENSIS) ISOLATES AND GENES ENCODING COLEOPTERAN-ACTIVE TOXINS NEUES KOLEOPTERENAKTIVES ISOLAT VON BACILLUS THURINGIENSIS UND GENE DIE FÜR KOLEOPTERENAKTIVE TOXINE KODIEREN NOUVEAUX ISOLANTS DE $i(BACILLUS THURINGIENSIS) ACTIFS CONTRE LES COLEOPTERES, ET GENES CODANT LES TOXINES ACTIVES CONTRE LES COLEOPTERES

(84) Designated Contracting States: • FU, Jenny, M. AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL San Diego, CA 92130 (US) SE (74) Representative: Perry, Robert Edward et al (30) Priority: 06.11.1991 US 788638 GILL JENNINGS & EVERY Broadgate House (43) Date of publication of application: 7 Eldon Street 31.08.1994 Bulletin 1994/35 London EC2M 7LH (GB)

(73) Proprietor: MYCOGEN CORPORATION (56) References cited: San Diego, California 92121 (US) EP-A- 0 382 990 EP-A- 0 501 650 WO-A-90/13651 WO-A-91/16433 (72) Inventors: US-A- 4 996 155 • PAYNE, Jewel, M. San Diego, CA 92126 (US)

Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 612 218 B1

Printed by Jouve, 75001 PARIS (FR) EP 0 612 218 B1

Description

Background of the Invention

5 [0001] The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These often appear microscopically as distinctively shaped crystals. The proteins are highly toxic to pests and specific in their activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products produced and approved. In addition, with the use of genetic engineering techniques, new approaches for delivering B.t. endotoxins to agricultural environments are under development, includ- 10 ing 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. [0002] Bacillus thuringiensis produces a proteinaceous paraspore or crystal which is toxic upon ingestion by a sus- ceptible insect host. Over most of the past 30 years, commercial use of B.t. pesticides has been largely restricted to 15 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 crystal called a delta endotoxin which is toxic to the larvae of a number of lepidopteran insects. [0003] In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader 20 range of pests. For example, other species of B.t., namely israelensis and san diego (a.k.a. B.t. tenebrionis), 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 Micro- 25 biology 22:61-76; Beegle, C.C., (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Indus- trial Microbiology 20:97-104. Krieg, A., A.M. Huger, G.A. Langenbruch, W. Schnetter (1983) Z. ang. Ent. 96:500-508, describe a B.t. isolate named 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. [0004] Recently, many new subspecies of B.t. have been identified, and many genes responsible for active δ-endo- 30 toxin proteins have been isolated (Höfte, H., H.R. Whiteley [1989] Microbiological Reviews 52(2):242-255). Höfte and Whiteley classified 42 B.t. crystal protein genes into 14 distinct genes, grouped into 4 major classes based on amino- acid sequence and host range. The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to protozoan path- ogens, animal-parasitic liver flukes (Trematoda), or mites (Acari) has broadened the potential B.t. product spectrum 35 even further (see Feitelson, J.S., J. Payne, L. Kim [1992] Bio/Technology 10:271-275). With activities against unique targets, these novel strains retain their very high biological specificity; nontarget organisms remain unaffected. The availability of a large number of diverse B.t. toxins may also enable farmers to adopt product-use strategies that min- imize the risk that B.t.-resistant pests will arise. [0005] The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in the pub- 40 lished literature (see, for example, Schnepf, H.E., H.R. Whitely [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. Patent 4,853,331 discloses B. thuringiensis strain san diego (a.k.a. B.t. tenebrionis) which can be used to control coleopteran pests in various environments. [0006] EP-A-0 382 990 discloses a coleopteran-active crystal toxin. Further B.t. toxins are disclosed in WO-A- 45 90/13651, WO-A-91/16433 and US-A-4 996 155.

Brief Summary of the Invention

[0007] The subject invention concerns the discovery that certain known and publicly available strains of Bacillus 50 thuringiensis (B.t.) are active against coleopteran pests. This is a surprising discovery since these B.t. microbes were not known to have any insecticidal properties. [0008] The microbes of the subject invention were obtained from the Howard Dalmage collection held by the NRRL culture repository in Peoria, Illinois and are designated B.t. HD511 and B.t. HD867. These microbes, and variants of these microbes, as well as genes and toxins obtainable therefrom, can be used to control coleopteran pests. The 55 procedures for using these microbes are similar to known procedures for using B.t. microbes to control coleopteran pests. [0009] Further, the invention also includes the treatment of substantially intact B.t. cells, and recombinant cells containing a gene of the invention, to prolong the pesticidal activity when the substantially intact cells are applied to the

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environment of a target pest. Such treatment can be by chemical or physical means, or a combination of chemical or physical means, so long as the technique does not deleteriously affect the properties of the pesticide, nor diminish the cellular capability in protecting the pesticide. The treated cell acts as a protective coating for the pesticidal toxin. The toxin becomes available to act as such upon ingestion by a target insect. 5 [0010] Disclosed herein are specific toxins, and nucleotide sequences encoding these toxins, obtainable from the exemplified isolates. Advantageously, these nucleotide sequences can be used to transform other microbes or plants to create insecticidal compositions and plants.

Brief Description of the Sequences 10 [0011] SEQ ID NO. 1 is the nucleotide sequence encoding toxin HD511. [0012] SEQ ID NO. 2 is the amino acid sequence of toxin HD511. [0013] SEQ ID NO. 3 is the nucleotide sequence encoding toxin HD867. [0014] SEQ ID NO. 4 is the amino acid sequence of toxin HD867. 15 Detailed Disclosure of the Invention

[0015] A summary of the characteristics of the B. thuringiensis microbes of the subject invention is shown in Table 1.

20 Table 1. A comparison of the novel coleopteran-active strains with B.t.s.d. Strain Crystal Type Approx. Molecular Weight of Protein* Serotype HD511 Bipyramid 130, 143 15, dakota 25 HD867 Bipyramid 130 18, kumamotoensis

* as shown on a standard polyacrylamide gel.

[0016] The cultures disclosed in this application are on deposit in the Agricultural Research Service Patent Culture

30 Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA. [0017] In a preferred embodiment, the nucleotide sequence information provided herein can be used to make primers which, when using standard PCR procedures, can be used to obtain the desirable genes from the disclosed isolates. These procedures are well known and commonly used in this art. Alternatively, synthetic genes, or portions thereof, can be made using a "gene machine" and the sequence information provided herein.

35 [0018] The subject invention pertains not only to the specific genes and toxins exemplified herein, but also to genes and toxins obtainable from variants of the disclosed isolates. These variants would have essentially the same coleopteran activity as the exemplified isolates. Furthermore, using the DNA and amino acid sequence provided herein, a person skilled in the art could readily construct fragments or mutations of the genes and toxins disclosed herein. These fragments and mutations, which retain the coleopteran activity of the exemplified toxins, would be within the scope of

40 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 similar, toxins. These 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 coleopteran activity. Frag-

45 ments retaining coleopteran activity are also included in this definition. [0019] The coleopteran-active toxin genes of the subject invention can be isolated by known procedures and can be introduced into a wide variety of microbial hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. With suitable hosts, e.g., Pseudomonas, the microbes can be applied to the situs of coleopteran insects where they will proliferate and be ingested by the insects. The result is

50 a control of the unwanted insects. Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin produced in the cell. The treated cell then can be applied to the environment of target pest(s). The resulting product retains the toxicity of the B.t. toxin. [0020] Where the B.t. toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is important that certain host microbes be used. Microorganism hosts are selected

55 which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of

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the pesticide from environmental degradation and inactivation. [0021] A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/ or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Pseudomonas, 5 Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobac- terium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Ser- ratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas 10 campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R glutinis, R. marina, R aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharo- myces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureo- basidium pollulans. Of particular interest are the pigmented microorganisms. [0022] A wide variety of ways are available for introducing the B.t. gene expressing the toxin into the microorganism 15 host under conditions which allow for stable maintenance and expression of the gene. One can provide for DNA constructs which include the transcriptional and translational regulatory signals for expression of the toxin gene, the toxin gene under their regulatory control and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur. 20 [0023] The transcriptional initiation signals will include a promoter and a transcriptional initiation start site. In some instances, it may be desirable to provide for regulative expression of the toxin, where expression of the toxin will only occur after release into the environment. This can be achieved with operators or a region binding to an activator or enhancers, which are capable of induction upon a change in the physical or chemical environment of the microorganisms. For example, a temperature sensitive regulatory region may be employed, where the organisms may be grown 25 up in the laboratory without expression of a toxin, but upon release into the environment, expression begins. Other techniques may employ a specific nutrient medium in the laboratory, which inhibits the expression of the toxin, where the nutrient medium in the environment allows for expression of the toxin. For translational initiation, a ribosomal binding site and an initiation codon will be present. [0024] Various manipulations may be employed for enhancing the expression of the messenger, particularly by using 30 an active promoter, as well as by employing sequences, which enhance the stability of the messenger RNA. The initiation and translational termination region will involve stop codon(s), a terminator region, and optionally, a polyadenylation signal. [0025] In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, the construct can involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 35 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region. This sequence as a double strand may be used by itself for transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker, where the second DNA sequence may be joined to the toxin expression construct during introduction of the DNA into the host. 40 [0026] By a marker is intended a structural gene which provides for selection of those hosts which have been modified or transformed. The marker will normally provide for selective advantage, for example, providing for biocide resistance, e.g., resistance to antibiotics or heavy metals; complementation, so as to provide prototropy to an auxotrophic host, or the like. Preferably, complementation is employed, so that the modified host may not only be selected, but may also be competitive in the field. One or more markers may be employed in the development of the constructs, as well as 45 for modifying the host. The organisms may be further modified by providing for a competitive advantage against other wild-type microorganisms in the field. For example, genes expressing metal chelating agents, e.g., siderophores, may be introduced into the host along with the structural gene expressing the toxin. In this manner, the enhanced expression of a siderophore may provide for a competitive advantage for the toxin-producing host, so that it may effectively compete with the wild-type microorganisms and stably occupy a niche in the environment. 50 [0027] Where stable episomal maintenance or integration is desired, a plasmid will be employed which has a replication system which is functional in the host. The replication system may be derived from the chromosome, an episomal element normally present in the host or a different host, or a replication system from a virus which is stable in the host. A large number of plasmids are available, such as pBR322, pACYC184, RSF1010, pRO1614, and the like. See for example, Olson et al. (1982) J. Bacteriol. 150:6069, and Bagdasarian et al. (1981) Gene 16:237, and U.S. Patent Nos. 55 4,356,270, 4,362,817, and 4,371,625. [0028] Where no functional replication system is present, the construct will also include a sequence of at least 50 basepairs (bp), preferably at least about 100 bp, and usually not more than about 1000 bp of a sequence homologous with a sequence in the host. In this way, the probability of legitimate recombination is enhanced, so that the gene will

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be integrated into the host and stably maintained by the host. Desirably, the toxin gene will be in close proximity to the gene providing for complementation as well as the gene providing for the competitive advantage. Therefore, in the event that a toxin gene is lost, the resulting organism will be likely to also lose the complementing gene and/or the gene providing for the competitive advantage, so that it will be unable to compete in the environment with the gene 5 retaining the intact construct. [0029] A large number of transcriptional regulatory regions are available from a wide variety of microorganism hosts, such as bacteria, bacteriophage, cyanobacteria, algae, fungi, and the like. Various transcriptional regulatory regions include the regions associated with the trp gene, lac gene, gal gene, the lambda left and right promoters, the tac promoter, and the naturally-occurring promoters associated with the toxin gene, where functional in the host. See for 10 example, U.S. Patent Nos. 4,332,898, 4,342,832 and 4,356,270. The termination region may be the termination region normally associated with the transcriptional initiation region or a different transcriptional initiation region, so long as the two regions are compatible and functional in the host. [0030] The B.t. gene can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under the regulatory control of the initiation region. This 15 construct can be included in a plasmid, which could include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host. In addition, one or more markers may be present, which have been described previously. Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome. 20 [0031] The transformants can be isolated in accordance with conventional ways, usually employing a selection technique, which allows for selection of the desired organism as against unmodified organisms or transferring organisms, when present. The transformants then can be tested for pesticidal activity. [0032] Suitable host cells, where the pesticide-containing cells will be treated to prolong the activity of the toxin in the cell when the then treated cell is applied to the environment of target pest(s), may include either prokaryotes or 25 eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, 30 and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Ser- ratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter, Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and As- comycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like. 35 [0033] The cell will usually be intact and be substantially in the proliferative form when treated, rather than in a spore form, although in some instances spores may be employed. [0034] Treatment of the microbial cell, e.g., a microbe containing the B.t. toxin gene, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability in protecting the toxin. Examples of chemical reagents 40 are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment with al- dehydes, such as formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Bouin's fixative and Helly's fixative (See: Humason, Gretchen L. [1967] Animal Tissue Techniques, W.H. Freeman and Company); or a combination of 45 physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host animal. Examples of physical means are short wavelength radiation such as gamma- radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like. [0035] The cells generally will have enhanced structural stability which will enhance resistance to environmental conditions. Where the pesticide is in a proform, the method of inactivation should be selected so as not to inhibit 50 processing of the proform to the mature form of the pesticide by the target pest pathogen. For example, formaldehyde will crosslink proteins and could inhibit processing of the proform of a polypeptide pesticide. The method of inactivation or killing retains at least a substantial portion of the bio-availability or bioactivity of the toxin. [0036] Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the B.t. gene into the host, availability of expression systems, efficiency of expression, stability of the pesticide 55 in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide micro- capsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and han-

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dling, economics, storage stability, and the like. [0037] Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharo- myces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwinia sp. and Flavobacte- rium sp.; or such other organisms as Escherichia, Lactobacillus sp., Bacillus sp., and the like. Specific organisms 5 include Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Es- cherichia coli, Bacillus subtilis, and the like. [0038] The cellular host containing the B.t. 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 10 can be treated prior to harvesting. [0039] The B.t. cells may be formulated in a variety of ways. They may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phos- phates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations 15 may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers. [0040] 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 about 100% by weight. The dry formulations will have from about 1-95% by weight of the pesticide while 20 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. [0041] The formulations can be applied to the environment of the coleopteran pest(s), e.g., plants, soil or water, by spraying, dusting, sprinkling, or the like. 25 [0042] Following are examples which illustrate procedures, including the best mode, 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 B.t. microbes 30 [0043] A subculture of a B.t. microbe, as disclosed herein, can be used to inoculate the following medium, a peptone, glucose, salts medium.

Bacto Peptone 7.5 g/l 35 Glucose 1.0 g/l

KH2PO4 3.4 g/l K2HPO4 4.35 g/l Salt Solution 5.0 ml/l

40 CaCl2 Solution 5.0 ml/l

Salts Solution (100 ml)

MgSO4·7H2O 2.46 g 45 MnSO4·H2O 0.04 g ZnSO4·7H2O 0.28 g FeSO4·7H2O 0.40 g

CaCl2 Solution (100 ml)

CaCl2.2H2O 3.66 g pH 7.2

55 [0044] 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. [0045] The above procedure can be readily scaled up to large fermentors by procedures well known in the art.

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[0046] The B.t. spores and 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.

5 Example 2 - Testing of B.t. Microbes Spores and Crystals

[0047] The B.t. strains were tested against Leptinotarsa rubiginosa, a surrogate for Leptinotarsa decemlineata, the Colorado potato beetle. [0048] The bioassay was performed on two different Bacillus thuringiensis preparations. (1) The spore/crystal pellet 10 was resuspended in water. (2) The spore/crystal pellet was treated with 0.1M Na2CO3, pH 11.5, with 0.5% 2-mercap- toethanol for two hours at room temperature. Prep #2 was dialyzed against 0.1M Tris, pH 8, for three hours with three changes of 15 times the sample volume. Prep #1 and Prep #2 contained equal amounts of active ingredient. [0049] Leaves were dipped in the B.t. preparations, and first instar larvae were placed on the leaves. The larvae were incubated at 25°C for 4 days before mortality was determined. 15 Table 2. Percent Mortality Strain Prep #1 Prep #2 20 HD511 52% 92% HD867 92% 100% HD1011 36% 92% Control 0% 0%

25 Example 3 - Insertion of Toxin Gene Into Plants

[0050] One aspect of the subject invention is the transformation of plants with genes encoding a coleopteran toxin. The transformed plants are resistant to attack by coleopterans.

30 [0051] Genes encoding lepidopteran-active 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

35 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 ma- nipulation, 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

40 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. [0052] 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., Alblasser-

45 dam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4:1-46; and An et al. (1985) EMBO J. 4:277-287. [0053] 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 indi- vidually employed marker should accordingly permit the selection of transformed cells rather than cells that do not

50 contain the inserted DNA. [0054] 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, 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

55 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

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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] Mol. 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- 5 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. 10 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. [0055] 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 15 corresponding phenotypic properties.

Example 4 - Cloning of Novel B.t. Genes Into Insect Viruses

[0056] A number of viruses are known to infect insects. These viruses include, for example, baculoviruses and en- 20 tomopoxviruses. In one embodiment of the subject invention, lepidopteran-active genes, as described herein, can be placed with 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. 71:1535-1544) and Martens et al. (Martens, J.W.M., G. Honee, D. Zuidema, 25 J.W.M. van Lent, B. Visser, J.M. Vlak [1990] Appl. Environmental Microbiol. 56(9):2764-2770).

SEQUENCE LISTING

[0057] 30 (1) GENERAL INFORMATION:

(i) APPLICANT: Payne, Jewel M. Fu, Jenny M. 35 (ii) TITLE OF INVENTION: Novel Bacillus thuringiensis Gene Encoding a Coleopteran-Active Toxin

(iii) NUMBER OF SEQUENCES: 4

40 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: David R. Saliwanchik (B) STREET: 2421 N.W. 41st Street, Suite A-1 (C) CITY: Gainesville 45 (D) STATE: FL (E) COUNTRY: USA (F) ZIP: 32606

(v) COMPUTER READABLE FORM: 50 (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.25 55 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

8 EP 0 612 218 B1

(B) FILING DATE: (C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: 5 (A) APPLICATION NUMBER: US 07/788,638 (B) FILING DATE: 6-NOV-1991 (C) CLASSIFICATION:

10 (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Saliwanchik, David R. (B) REGISTRATION NUMBER: 31,794 (C) REFERENCE/DOCKET NUMBER: MA68.Cl 15 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 904-375-8100 (B) TELEFAX: 904-372-5800 20 (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

25 (A) LENGTH: 3414 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear

30 (ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO 35 (vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus thuringiensis (B) STRAIN: dakota 40 (C) INDIVIDUAL ISOLATE: HD511

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: Lamdagem (TM)-11 library of J.M. Fu 45 (B) CLONE: 511

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

9 EP 0 612 218 B1

10 EP 0 612 218 B1

15 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

20 (A) LENGTH: 1138 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

25 (ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO 30 (vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus thuringiensis (B) STRAIN: dakota 35 (C) INDIVIDUAL ISOLATE: HD511

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: Lamdagem (TM)-11 library of J.M. Fu 40 (B) CLONE: 511

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

11 EP 0 612 218 B1

12 EP 0 612 218 B1

13 EP 0 612 218 B1

14 EP 0 612 218 B1

(2) INFORMATION FOR SEQ ID NO:3: 10 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3414 base pairs B TYPE: nucleic acid pairs 15 (C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

20 (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: 25 (A) ORGANISM: Bacillus thuringiensis (B) STRAIN: kumamotoensis (C) INDIVIDUAL ISOLATE: HD867

30 (vii) IMMEDIATE SOURCE:

(A) LIBRARY: Lamdagem (TM)-11 library of J.M. Fu (B) CLONE: 867

35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

15 EP 0 612 218 B1

16 EP 0 612 218 B1

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS: 50 (A) LENGTH: 1138 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 55 (ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: YES

17 EP 0 612 218 B1

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

5 (A) ORGANISM: Bacillus thuringiensis (B) STRAIN: kumamotoensis (C) INDIVIDUAL ISOLATE: HD867

(vii) IMMEDIATE SOURCE: 10 (A) LIBRARY: Lamdagem (TM)-11 library of J.M. Fu (B) CLONE: 867

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 15

18 EP 0 612 218 B1

19 EP 0 612 218 B1

20 EP 0 612 218 B1

40 Claims

1. A method for controlling a coleopteran insect pest, which comprises contacting the pest with a Bacillus thuringiensis microbe selected from B.t. HD511 and B.t. HD867, or with a toxin obtainable from either of these microbes. 45 2. A method according to claim 1, wherein the microbe is B.t. HD511.

3. A method according to claim 1, wherein the microbe is B.t. HD867.

50 4. A method according to any of claims 1 to 3, wherein the pest is the Colorado potato beetle.

5. A composition comprising a Bacillus thuringiensis microbe or toxin as defined in claim 1, together with an inert carrier and in the form of a wettable powder, granules, a dust or a sprayable liquid.

55 6. A composition comprising a microbe or toxin as defined in claim 1, and a beetle phagostimulant or attractant.

7. A toxin, active against a coleopteran pest, which is obtainable from a microbe as defined in claim 1, or comprises the amino acid sequence of SEQ ID NO. 2 (from B.t. HD511) or SEQ ID NO. 4 (from B.t. HD867).

21 EP 0 612 218 B1

8. A toxin according to claim 7, which comprises the amino acid sequence shown in SEQ ID NO. 2 (from B.t. HD511).

9. A toxin according to claim 7, which comprises the amino acid sequence shown in SEQ ID NO. 4 (from B.t. HD867).

5 10. A nucleotide molecule comprising DNA encoding a toxin according to any of claims 7 to 9.

11. A nucleotide molecule according to claim 10, wherein the DNA has the sequence shown in SEQ ID NO. 1 (from B.t. HD511).

10 12. A nucleotide molecule according to claim 10, wherein the DNA has the sequence shown in SEQ ID NO. 3 (from B.t. HD867).

13. A bacterial or plant host transformed to express a toxin according to any of claims 7 to 9.

15 14. A bacterial host according to claim 13, which is a species of Pseudomonas, Azotobacter, Erwinia, Serratia, Kleb- siella, Rhizobium, Bacillus, Streptomyces, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,or Alcaligenes.

15. A host according to claim 14, which is pigmented and phylloplane-adherent. 20 16. A method for controlling a coleopteran insect pest, which comprises exposing the pest to a plant according to claim 13.

25 Patentansprüche

1. Verfahren zur Bekämpfung eines Coleoptera-Insektenschädlings, umfassend das Kontaktieren des Schädlings mit einem Bacillus thuringiensis-Mikroorganismus, der aus B.t. HD511 und B.t. HD867 ausgewählt ist, oder mit einem Toxin, das aus einem dieser Mikroorganismen erhältlich ist. 30 2. Verfahren nach Anspruch 1, worin der Mikroorganismus B.t. HD511 ist.

3. Verfahren nach Anspruch 1, worin der Mikroorganismus B.t. HD867 ist.

35 4. Verfahren nach irgendeinem der Ansprüche 1 bis 3, worin der Schädling der Colorado-Kartoffelkäfer ist.

5. Zusammensetzung, die einen Bacillus thuringiensis-Mikroorganismus oder ein Toxin wie in Anspruch 1 definiert zusammen mit einem inerten Träger umfaßt und in Form eines benetzbaren Pulvers, von Granula, eines Staubs oder einer sprühfähigen Flüssigkeit vorliegt. 40 6. Zusammensetzung, umfassend einen Mikroorganismus oder ein Toxin wie in Anspruch 1 definiert und ein(en) Käfer-Phagostimulans oder -Lockstoff.

7. Toxin mit Aktivität gegenüber einem Coleoptera-Schädling, welches aus einem Mikroorganismus wie in Anspruch 45 1 definiert erhältlich ist oder die Aminosäuresequenz von SEQ-ID-Nr. 2 (von B.t. HD511) oder SEQ-ID-Nr. 4 (von B.t. HD867) umfaßt.

8. Toxin nach Anspruch 7, welches die in SEQ-ID-Nr. 2 dargestellte Aminosäuresequenz (von B.t. HD511) umfaßt.

50 9. Toxin nach Anspruch 7, welches die in SEQ-ID-Nr. 4 dargestellte Aminosäuresequenz (von B.t. HD867) umfaßt.

10. Nukleotidmolekül, umfassend DNA, die für ein Toxin nach irgendeinem der Ansprüche 7 bis 9 kodiert.

11. Nukleotidmolekül nach Anspruch 10, worin die DNA die in SEQ-ID-Nr. 1 dargestellte Sequenz (von B.t.HD511) 55 aufweist.

12. Nukleotidmolekül nach Anspruch 10, worin die DNA die in SEQ-ID-Nr. 3 dargestellte Sequenz (von B.t. HD867) aufweist.

22 EP 0 612 218 B1

13. Bakterieller Wirt oder Pflanzenwirt, welcher transformiert wurde, um ein Toxin nach irgendeinem der Ansprüche 7 bis 9 zu exprimieren.

14. Bakterieller Wirt nach Anspruch 13, welcher eine Spezies von Pseudomonas, Azotobacter, Erwinia, Serratia, Kleb- 5 siella, Rhizobium, Bacillus, Streptomyces, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter oder Alcaligenes ist.

15. Wirt nach Anspruch 14, welcher pigmentiert ist und an der Phyllooberfläche haftet.

10 16. Verfahren zur Bekämpfung eines Coleoptera-lnsektenschädlings, welches umfaßt, daß der Schädling einer Pflan- ze nach Anspruch 13 ausgesetzt wird.

Revendications 15 1. Procédé de lutte contre un insecte coléoptère nuisible, qui comprend la mise en contact du nuisible avec un microbe Bacillus thuringiensis choisi parmi B. t. HD511 et B.t. HD867, ou avec une toxine pouvant être obtenue à partir de l'un ou l'autre de ces microbes.

20 2. Procédé selon la revendication 1, dans lequel le microbe est B. t. HD511.

3. Procédé selon la revendication 1, dans lequel le microbe est B. t. HD867.

4. Procédé selon l'une quelconque des revendications 1 à 3, dans lequel le nuisible est le doryphore. 25 5. Composition comprenant un microbe Bacillus thuringiensis ou une toxine selon la revendication 1 avec un support inerte, sous forme d'une poudre mouillable, de granulés, d'une poudre pour poudrage ou d'un liquide à pulvériser.

6. Composition comprenant un microbe ou une toxine selon la revendication 1, et une substance stimulant l'appétit 30 ou attractive pour les coléoptères.

7. Toxine active contre les coléoptères nuisibles, pouvant être obtenue à partir d'un microbe selon la revendication 1, ou comprenant la séquence d'aminoacides de SEQ ID N° 2 (de B.t. HD511) ou de SEQ ID N° 4 (de B.t. HD867).

35 8. Toxine selon la revendication 7, comprenant la séquence d'aminoacides de SEQ ID N° 2 (de B.t. HD511).

9. Toxine selon la revendication 7, comprenant la séquence d'aminoacides de SEQ ID N° 4 (de B.t. HD867).

10. Molécule nucléotidique comprenant de l'ADN codant pour une toxine selon l'une quelconque des revendications 40 7 à 9.

11. Molécule nucléotidique selon la revendication 10, dans laquelle l'ADN a la séquence indiquée par SEQ ID N° 1 (de B.t. HD511).

45 12. Molécule nucléotidique selon la revendication 10, dans laquelle l'ADN a la séquence indiquée par SEQ ID N° 3 (de B.t. HD867).

13. Hôte bactérien ou végétal transformé de façon à exprimer une toxine selon l'une quelconque des revendications 7 à 9. 50 14. Hôte bactérien selon la revendication 13, qui est une espèce de Pseudomonas, Azotobacter, Erwinia, Serratia, Klebsiella, Rhizobium, Bacillus, Streptomyces, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter ou Alcaligenes.

55 15. Hôte selon la revendication 14, qui est pigmenté et adhérent au phylloplan.

16. Procédé de lutte contre un insecte coléoptère nuisible, qui comprend l'exposition du nuisible à un végétal selon la revendication 13.