WO1993005657A1 - NOVEL BACILLUS THURINGIENSIS ISOLATE DENOTED B.t.PS158C2, ACTIVE AGAINST LEPIDOPTERAN PESTS, AND GENES ENCODING LEPIDOPTERAN-ACTIVE TOXINS - Google Patents
NOVEL BACILLUS THURINGIENSIS ISOLATE DENOTED B.t.PS158C2, ACTIVE AGAINST LEPIDOPTERAN PESTS, AND GENES ENCODING LEPIDOPTERAN-ACTIVE TOXINS Download PDFInfo
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- WO1993005657A1 WO1993005657A1 PCT/US1992/007713 US9207713W WO9305657A1 WO 1993005657 A1 WO1993005657 A1 WO 1993005657A1 US 9207713 W US9207713 W US 9207713W WO 9305657 A1 WO9305657 A1 WO 9305657A1
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- WIPO (PCT)
- Prior art keywords
- ps158c2
- toxin
- lepidopteran
- mutants
- gene
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/832—Bacillus
Definitions
- Bacillus thuringiensis This bacterial agent is used to control a wide range of leaf-eating caterpillars, and mosquitos.
- Bacillus thuringiensis produces a proteinaceous paraspore or crystal which is toxic upon ingestion by a susceptible insect host.
- B. thuringiensis var. kurstaki HD-1 produces a crystal called a delta toxin which is toxic to the larvae of a number of lepidopteran insects.
- the cloning and expression of a crystal protein gene from this Rt. strain in Escherichia coli has been described in the published literature (Schnepf, H.E. and Whitely, H.R.
- the subject invention concerns a novel Bacillus thuringiensis isolate designated B . PS158C2, and mutants thereof which are active against lepidopteran pests.
- delta endotoxin genes obtainable from the isolate wherein the genes encode proteins which are active against lepidopteran pests. These toxin genes can be transferred to suitable hosts via a plasmid vector.
- the invention comprises a novel R isolate denoted B.t. PS158C2, mutants thereof, and a novel delta endotoxin genes which encodes 47, 37, 34 and 32 kDa proteins active against lepidopteran pests.
- B.t. PS158C2 mutants thereof
- a novel delta endotoxin genes which encodes 47, 37, 34 and 32 kDa proteins active against lepidopteran pests.
- Brief Description of the Drawing Figure 1 is a photograph of a 9% SDS polyacrylamide gel showing alkali-soluble proteins of Bacillus thuringiensis PS158C2 compared to two typical lepidopteran-active strains.
- B.t. delta endotoxins with lepidopteran activity are of the cryl type (130-140 kDa), or cryll (approximately 70 kDa).
- B.t. PS158C2 has a totally different group of smaller proteins, the largest of which measure 47 kDa on SDS-PAGE.
- B. thuringiensis PS158C2, NRRL B-18872, and mutants thereof can be cultured using standard known media and fermentation techniques. Upon completion of the fermentation cycle, the bacteria can be harvested by first separating the B ⁇ . spores and crystals from the fermentation broth by means well known in the art. The recovered B ⁇ . spores and crystals can be formulated into a wettable powder, a 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. The formulation and application procedures are all well known in the art and are used with commercial strains of B. thuringiensis (HD-1) active against Lepidoptera. e.g., caterpillars. B ⁇ . PS158C2, and mutants thereof, can be used to control lepidopteran pests.
- HD-1 active against Lepidoptera. e.g., caterpillars.
- B ⁇ . PS158C2, and mutants thereof can be used to control
- the subject culture deposit 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., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the 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 culture.
- the depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition of the deposit.
- the toxin genes harbored by the novel isolates of the invention can be recovered by standard procedures and 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 lepidopteran insects where they will proliferate and be ingested by the insects. The result is 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 pests. The resulting product retains the toxicity of the B ⁇ . toxin.
- suitable hosts e.g., Pseudomonas
- the microbes can be applied to the situs of lepidopteran insects where they will proliferate and be ingested by the insects. The result is a control of the unwanted insects.
- microorganism hosts are selected 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 the pesticide from environmental degradation and inactivation.
- phytosphere phytosphere
- rhizosphere rhizosphere
- rhizoplane rhizoplane
- 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. Erwinia. Serratia.
- a wide variety of ways are available for introducing the B ⁇ . gene expressing the toxin into the microorganism host under conditions which allow for stable maintenance and expression of the gene.
- 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.
- 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.
- a temperature sensitive regulatory region may be employed, where the organisms may be grown up in the laboratory without expression of a toxin, but upon release into the environment, expression would begin.
- Other techniques may employ a specific nutrient medium in the laboratory, which inhibits the expression of the toxin, where the nutrient medium in the environment would allow for expression of the toxin.
- a ribosomal binding site and an initiation codon will be present.
- initiation and translational termination region will involve stop codon(s), a te ⁇ mnator region, and optionally, a polyadenylation signal.
- the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 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 te ⁇ ninator 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.
- 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.
- 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 for modifying the host.
- the organisms may be further modified by providing for a competitive advantage against other wild-type microorganisms in the field.
- genes expressing metal chelating agents may be introduced into the host along with the structural gene expressing the toxin.
- 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.
- 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.
- bp basepairs
- 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 retaining the intact construct.
- 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, the naturally-occurring promoters associated with the toxin gene, where functional in the host. See for 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.
- a plasmid 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, pR01614, 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. 4,356,270, 4,362,817, and 4,371,625.
- the B ⁇ . 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 construct will be included in a plasmid, which will 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.
- one or more markers may be present, which have been described previously.
- the plasmid will desirably include a sequence homologous with the host genome.
- 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.
- 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 pests, may include either prokaryotes or eukaiyotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals.
- 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.
- prokaryotes 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, and Proteus: Bacillaceae; Rhizobiceae, such as Rhizobium: Spirillaceae, such as photobacterium, Zymomonas, Serratia. Aeromonas. Vibrio. Desulfovibrio. Spirillum:
- Lactobacillaceae Pseudomonadaceae, such as Pseudomonas and Acetobacter: Azotobacteraceae and Nitrobacteraceae.
- fungi such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces: and Basidiomycetes yeast, such as Rhodotorula. Aureobasidium. Sporobolomyces. and the like.
- Characteristics of particular interest in selecting a. host cell for purposes of production include ease of introducing the B ⁇ . gene into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities.
- Characteristics of interest for use as a pesticide microcapsule 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 handling, economics, storage stability, and the like.
- Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwinia sp. and Flavobacterium sp.; or such other organisms as Escherichia. Lactobacillus sp., Bacillus sp., and the like. Specific organisms include Pseudomonas aeruginosa.
- Pseudomonas fluorescens Saccharomyces cerevisiae. Bacillus thuringiensis. Escherichia coli. Bacillus subtilis. and the like.
- 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.
- Treatment of the microbial cell 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.
- chemical reagents 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.
- aldehydes such as formaldehyde and glutaraldehyde
- anti-infectives such as zephiran chloride and cetylpyridinium chloride
- alcohols such as isopropyl and ethanol
- histologic fixatives such as Bouin's fixative and Kelly's fixative (See: Humason, Gretchen L., Animal Tissue Techniques, W.H. Freeman and Company, 1967); or a combination of 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.
- physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like.
- the cells generally will have enhanced structural stability which will enhance resistance to environmental conditions.
- the method of inactivation should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen.
- 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.
- the cellular host containing the B ⁇ . 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 ⁇ . gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.
- the B ⁇ . 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, phosphates, 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 may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like.
- the ingredients may include Theological agents, surfactants, emulsifiers, dispersants, or polymers.
- 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 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
- the formulations will generally have from about 10 2 to about 10 4 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 lepidopteran pests, e.g., plants, soil or water, by spraying, dusting, sprinkling, or the like.
- Mutants of PS158C2 can be made by procedures well known in the art.
- an asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis of PS158C2.
- EMS ethylmethane sulfonate
- the mutants can be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.
- a subculture of R PS158C2, NRRL B-18872, or mutants thereof, can be used to inoculate the following medium, a peptone, glucose, salts medium.
- the salts solution and CaCl 2 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 B i 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.
- novel genes coding for the novel insecticidal toxins can be inserted into plant cells using the Ti plasmid from Agrobacter tumefaciens. Plant cells can then be caused to regenerate into plants (Zambryski, P., Joos, H., Gentello, C, Leemans, J., Van Montague, M. and
- pEND4K Klee, H.J., Yanofsky, M.F. and Nester, E.W. [1985] Bio/Technology 3:637-642).
- This plasmid can replicate both in plant cells and in bacteria and has multiple cloning sites for passenger genes.
- the toxin genes for example, can be inserted into the BamHI site of pEND4K, propagated in E. coli, and transformed into appropriate plant cells.
- the novel genes of the invention can be cloned into baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV).
- Plasmids can be constructed that contain the AcNPV genome cloned into a commercial cloning vector such as pUC8.
- the AcNPV genome is modified so that the coding region of the polyhedrin gene is removed and a unique cloning site for a passenger gene is placed directly behind the polyhedrin promoter.
- Examples of such vectors are pGP-B6874, described by Pennock et al. (Pennock, G.D., Shoemaker,
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Abstract
A novel $i(B.t.) toxin gene toxic to lepidopteran insects has been cloned from a novel lepidopteran-active $i(B. thuringiensis) microbe. The DNA encoding the $i(B.t.) toxin can be used to transform various prokaryotic and eukaryotic microbes to express the $i(B.t.) toxin. These recombinant microbes can be used to control lepidopteran insects in various environments.
Description
DESCRIPTION
NOVEL BACILLUS THURINGIENSIS ISOLATE DENOTED B.t. PS158C2. ACTIVE AGAINST LEPIDOPTERAN PESTS. AND GENES ENCODING LEPIDOPTERAN-ACTIVE TOXINS
Background of the Invention The most widely used microbial pesticides are derived from the bacterium Bacillus thuringiensis. This bacterial agent is used to control a wide range of leaf-eating caterpillars, and mosquitos. Bacillus thuringiensis produces a proteinaceous paraspore or crystal which is toxic upon ingestion by a susceptible insect host. For example, B. thuringiensis var. kurstaki HD-1 produces a crystal called a delta toxin which is toxic to the larvae of a number of lepidopteran insects. The cloning and expression of a crystal protein gene from this Rt. strain in Escherichia coli has been described in the published literature (Schnepf, H.E. and Whitely, H.R. [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 . crystal protein in E. coli.
Brief Summary of the Invention
The subject invention concerns a novel Bacillus thuringiensis isolate designated B . PS158C2, and mutants thereof which are active against lepidopteran pests.
Also embodied within the subject invention are the delta endotoxin genes obtainable from the isolate wherein the genes encode proteins which are active against lepidopteran pests. These toxin genes can be transferred to suitable hosts via a plasmid vector.
Specifically, the invention comprises a novel R isolate denoted B.t. PS158C2, mutants thereof, and a novel delta endotoxin genes which encodes 47, 37, 34 and 32 kDa proteins active against lepidopteran pests.
Brief Description of the Drawing Figure 1 is a photograph of a 9% SDS polyacrylamide gel showing alkali-soluble proteins of Bacillus thuringiensis PS158C2 compared to two typical lepidopteran-active strains.
Detailed Disclosure of the Invention All previously described B.t. delta endotoxins with lepidopteran activity are of the cryl type (130-140 kDa), or cryll (approximately 70 kDa). B.t. PS158C2 has a totally different group of smaller proteins, the largest of which measure 47 kDa on SDS-PAGE.
Characteristics of B.t. PS158C2
Colony morphology-Large colony, dull surface, typical B.t. Vegetative cell morphology-typical Bjt Inclusion type-_Amorphic
Activity-B.t. PS158C2 kills all Lepidoptera tested.
Approximate molecular weight of proteins (kDa)-47, 37, 34, 32
Bioassay procedures and results: Spodoptera littoralis Bioassay-This assay was done with spray- dried powder of B.t. strains. First instar larvae were used with 1% agar diet containing 0.5% spray-dried powder. Mortality was read at 7 days. BΛ. PS158C2 gave greater than 80% mortality. Plutella xylostella Bioassay-Dilutions of a spray-dried powder of B.t. PS158C2 were incorporated in the diet, and third instar larvae were used. Mortality was read at 6 days. Rates greater than 300 μg powder per gram diet gave over 90% mortality.
Table 1: Comparison of B.t. PS158C2 with other lepidopteran-active strains
B. thuringiensis PS158C2, NRRL B-18872, and mutants thereof, can be cultured using standard known media and fermentation techniques. Upon completion of the fermentation cycle, the bacteria can be harvested by first separating the BΛ. spores and crystals from the fermentation broth by means well known in the art. The recovered BΛ. spores and crystals can be formulated into a wettable powder, a 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. The formulation and application procedures are all well known in the art and are used with commercial strains of B. thuringiensis (HD-1) active against Lepidoptera. e.g., caterpillars. BΛ. PS158C2, and mutants thereof, can be used to control lepidopteran pests.
A subculture of BΛ. PS158C2 was deposited in the permanent collection of the Northern Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois, USA on August 27, 1991. The accession number is as follows:
B.t. PS158C2 NRRL B-18872
The subject culture has been deposited under conditions that assure that access to the culture will be available during 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 USC 122. The deposit is 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 in derogation of patent rights granted by governmental action.
Further, the subject culture deposit 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., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the 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 culture. The depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition of the deposit.
All restrictions on the availability to the public of the subject culture deposit will be irrevocably removed upon the granting of a patent disclosing it.
The toxin genes harbored by the novel isolates of the invention can be recovered by standard procedures and 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 lepidopteran insects where they will proliferate and be ingested by the insects. The result is 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 pests. The resulting product retains the toxicity of the BΛ. toxin.
Where the BΛ. 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 essential that certain host microbes be used. Microorganism hosts are selected 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 the pesticide from environmental degradation and inactivation.
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. Erwinia. Serratia.
Klebsiella, Xanthomonas. Streptomyces. Rhizobium. Rhodopseudomonas. Methylophilius. Agrobacterium. Acetobacter, Lactobacillus. _ rthrobacter. 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. Serratia marcescens. Acetobacter xylinum. Agrobacterium tumefaciens. Rhodopseudomonas spheroides. Xanthomonas 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. laurentϋ. Saccharomyces rosei. S. pretoriensis. S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae. and Aureobasidium poUulans. Of particular interest are the pigmented microorganisms.
A wide variety of ways are available for introducing the BΛ. gene expressing the toxin into the microorganism 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.
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 up in the laboratory without expression of a toxin, but upon release into the environment, expression would begin. Other techniques may employ a specific nutrient medium in the laboratory, which inhibits the expression of the toxin, where the nutrient medium in the environment would allow for expression of the toxin. For translational initiation, a ribosomal binding site and an initiation codon will be present.
Various manipulations maybe employed for enhancing the expression of the messenger, particularly by using 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 teπmnator region, and optionally, a polyadenylation signal.
In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 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 teπninator 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.
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 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.
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 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 retaining the intact construct.
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, the naturally-occurring promoters associated with the toxin gene, where functional in the host. See for 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.
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, pR01614, 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. 4,356,270, 4,362,817, and 4,371,625.
The BΛ. 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 construct will be included in a plasmid, which will 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.
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. 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 pests, may include either prokaryotes or eukaiyotes, 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, and Proteus: Bacillaceae; Rhizobiceae, such as Rhizobium: Spirillaceae, such as photobacterium, Zymomonas, Serratia. Aeromonas. Vibrio. Desulfovibrio. Spirillum:
Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter: Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces: and Basidiomycetes yeast, such as Rhodotorula. Aureobasidium. Sporobolomyces. and the like.
Characteristics of particular interest in selecting a. host cell for purposes of production include ease of introducing the BΛ. gene into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule 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 handling, economics, storage stability, and the like.
Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwinia sp. and Flavobacterium sp.; or such other organisms as Escherichia. Lactobacillus sp., Bacillus sp., and the like. Specific organisms include Pseudomonas aeruginosa.
Pseudomonas fluorescens. Saccharomyces cerevisiae. Bacillus thuringiensis. Escherichia coli. Bacillus subtilis. and the like.
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.
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 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 aldehydes, 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 Kelly's fixative (See: Humason, Gretchen L., Animal Tissue Techniques, W.H. Freeman and Company, 1967); or a combination of 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.
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 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.
The cellular host containing the BΛ. 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 BΛ. gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.
The BΛ. 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, phosphates, 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 may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include Theological agents, surfactants, emulsifiers, dispersants, or polymers.
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 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 lepidopteran pests, e.g., plants, soil or water, by spraying, dusting, sprinkling, or the like.
Mutants of PS158C2 can be made by procedures well known in the art. For example, an asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis of PS158C2. The mutants can be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.
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 - Culturine B.t. PS158C2. NRRL B-18872
A subculture of R PS158C2, NRRL B-18872, 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/I
CaCl2 Solution 5.0 ml/1
Salts Solution (100 ml) MgS04.7H20 2.46 g MnS04.H20 0.04 g ZnS04.7H20 0.28 g FeS04.7H20 0.40 g
CaQ2 Solution (100 ml) CaCl2.2H20 3.66 g pH 7.2
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 i 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 various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. Also, methods for the use of lambda bacteriophage as a cloning vehicle, i.e., the preparation of lambda DNA, in vitro packaging, and transfection of recombinant DNA, are well known in the art. These procedures are all described in Maniatis, T., Fritsch, E_F., and Sambrook, J. (1982) Molecular Qoning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York.
Example 2 — Insertion of Toxin Genes Into Plants
The novel genes coding for the novel insecticidal toxins, as disclosed herein, can be inserted into plant cells using the Ti plasmid from Agrobacter tumefaciens. Plant cells can then be caused to regenerate into plants (Zambryski, P., Joos, H., Gentello, C, Leemans, J., Van Montague, M. and
Schell, J [1983] Cell 32:1033-1043). A particularly useful vector in this regard is pEND4K (Klee, H.J., Yanofsky, M.F. and Nester, E.W. [1985] Bio/Technology 3:637-642). This plasmid can replicate both in plant cells and in bacteria and has multiple cloning sites for passenger genes. The toxin genes, for example, can be inserted into the BamHI site of pEND4K, propagated in E. coli, and transformed into appropriate plant cells.
Example 3 - Cloning of Novel B. thuringiensis Genes Into Baculoviruses
The novel genes of the invention can be cloned into baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV). Plasmids can be constructed that contain the AcNPV genome cloned into a commercial cloning vector such as pUC8. The AcNPV genome is modified so that the coding region of the polyhedrin gene is removed and a unique cloning site for a passenger gene is placed directly behind the polyhedrin promoter. Examples of such vectors are pGP-B6874, described by Pennock et al. (Pennock, G.D., Shoemaker,
C. and Miller, L.K. [1984] Mol. Cell. Biol. 4:399-406), and pAC380, described by Smith et al. (Smith, G.E., Summers, M.D. and Fraser, M.J. [1983] Mol Cell. Biol. 3:2156-2165). The genes coding for the novel protein toxins of the invention can be modified with BamHI linkers at appropriate regions both upstream and downstream from the coding region and inserted into the passenger site of one of the AcNPV vectors.
Claims
1. A process for controlling lepidopteran insect pests which comprises contacting said insect pests with an insect-controlling effective amount of B. thuringiensis PS158C2 having the identifying lepidopteran active characteristics of NRRL B-18872, or mutants thereof.
2. The process, according to claim 1, wherein said insect pest is Spodoptera littoralis.
3. The process, according to claim 1, wherein said insect pest is Plutella xylostella.
4. The process, according to claim 1, wherein said insect pest is contacted with an insect-controlling effective amount of B. thuringiensis PS158C2, by incorporating said B. thuringiensis PS158C2 into a bait granule and placing said granule on or in the soil when planting seed of a plant upon which plant insect pest is known to feed.
5. A process for controlling soil-inhabiting insect pests of the order Lepidoptera which comprises (1) preparing a bait granule comprising B. thuringiensis PS158C2, or mutants thereof, spores or crystals; and (2) placing said bait granule on or in the soil.
6. The process, according to claim 5, wherein said bait granule is applied at the same time corn seed is planted in the soil.
7. The process, according to claims 1 or 5, wherein substantially intact R PS158C2 cells, or mutants thereof, are treated to prolong the pesticidal activity when the substantially intact cells are applied to the environment of a target pest.
8. A composition of matter comprising B. thuringiensis PS158C2, or mutants thereof, spores or crystals in association with an insecticide carrier.
9. The composition of matter, according to claim 8, wherein said carrier comprises phagostimulants or attractants.
10. A composition of matter comprising B. thuringiensis PS158C2, or mutants thereof, in association with formulation ingredients apphed as a seed coating.
11- Bacillus thuringiensis PS158C2, having the identifying lepidopteran active characteristics of NRRL B-18872, or mutants thereof, having activity against insect pests of the order Lepidoptera.
12. A gene encoding a toxin which is active against lepidopteran insects obtainable from Bacillus thuringiensis PS158C2, and mutants thereof.
13. A toxin encoded by a gene obtainable from Bacillus thuringiensis PS158C2, and mutants thereof, wherein said toxin is active against lepidopteran pests.
14. A plant transformed by a gene obtainable from Bacillus thuringiensis PS158C2, and mutants thereof, wherein said gene encodes a toxin active against lepidopteran pests.
15. A microbe transformed by a gene obtainable from Bacillus thuringiensis PS158C2, and mutants thereof, wherein said gene encodes a toxin active against lepidopteran pests.
16. A baculovirus transformed by a gene obtainable from Bacillus thuringiensis PS158C2, and mutants thereof, wherein said gene encodes a toxin active against lepidopteran pests.
17. The microorganism according to claim 15, which is a species of Pseudomonas, Azotobacter, Erwinia, Serratia. Klebsiella. Rhizobium, Rhodopseudomonas.Methylophilius, Agrobacterium, Acetobacter orAlcaligenes.
18. The microorganism according to claim 17, wherein said microorganism is pigmented and phylloplane adherent.
19. A method for controlling lepidopteran insects which comprises ac_t____mstering to said insects or to the environment of said insects a microorganism according to claim 17.
20. The method according to claim 19, wherein said administration is to the rhizosphere.
21. The method according to claim 20, wherein said administration is to the phylloplane.
22. The method according to claim 19, wherein said administration is to a body of water.
23. An insecticidal composition comprising insecticide containing substantially intact, treated cells having prolonged pesticidal activity when applied to the environment of a target pest, wherein said insecticide is a polypeptide toxic to lepidopteran insects, is intracellular, and is produced as a result of expression of a transformed microbe capable of expressing the BΛ. toxin according to claim 13.
24. The insecticidal composition, according to claim 23, wherein said treated cells are treated by chemical or physical means to prolong the insecticidal activity in the environment.
25. The insecticidal composition, according to claim 24, wherein said ceUs are prokaryotes or lower eukaryotes.
26. The insecticidal composition, according to claim 25, wherein said prokaryotic ceUs are selected from the group consisting of Enterobacteriaceae, BaciUaceae, Rhizobiaceae, SpiriUaceae, LactobaciUaceae, Pseudomonadaceae, Azotobacteraceae, and Nitrobacteraceae.
27. The insecticidal composition, according to claim 25, wherein said lower eukaryotic ceUs are selected from the group consisting of Phycomycetes, Ascomycetes, and Basidiomycetes.
28. The insecticidal composition, according to claim 24, wherein said ceU is a pigmented bacterium, yeast, or fungus.
29. Treated, substantiaUy intact uniceUular microorganism ceUs containing an intraceUular toxin, which toxin is a result of expression of a Bacillus thuringiensis toxin gene toxic to lepidopteran insects which codes for a polypeptide toxin according to claim 13, wherein said ceUs are treated under conditions which prolong the insecticidal activity when said ceU is appUed to the environment of a target insect.
30. The ceUs, according to claim 29, wherein the ceUs are treated by chemical or physical means to prolong the insecticidal activity in the environment.
31. The ceUs according to claim 29, wherein said microorganism is Pseudomonas and said toxin is a BΛ. toxin according to claim 13.
32. Pseudomonas ceUs according to claim 31, wherein said cells are treated with iodine.
33. The cells, according to claim 29, which are Pseudomonas fluorescens.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU26727/92A AU662563B2 (en) | 1991-09-13 | 1992-09-11 | Novel (bacillus thuringiensis) isolate denoted (B.t.)PS158C2, active against lepidopteran pests, and genes encoding lepidopteran-active toxins |
DE69223810T DE69223810T2 (en) | 1991-09-13 | 1992-09-11 | BACILLUS THURINGIENSIS'S NEW LEPIDOPTER ACTIVE ISOLATE, CALLED B.T. PS 158C2, AND GENES THAT ENCODE LEPIDOPTER-ACTIVE TOXINS |
EP92920804A EP0642305B1 (en) | 1991-09-13 | 1992-09-11 | NOVEL BACILLUS THURINGIENSIS ISOLATE DENOTED B.t.PS158C2, ACTIVE AGAINST LEPIDOPTERAN PESTS, AND GENES ENCODING LEPIDOPTERAN-ACTIVE TOXINS |
GR980400546T GR3026361T3 (en) | 1991-09-13 | 1998-03-13 | NOVEL -i(BACILLUS THURINGIENSIS) ISOLATE DENOTED -i(B.t.)PS158C2, ACTIVE AGAINST LEPIDOPTERAN PESTS, AND GENES ENCODING LEPIDOPTERAN-ACTIVE TOXINS. |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US07/759,247 US5268172A (en) | 1991-09-13 | 1991-09-13 | Bacillus thuringiensis isolate denoted B.t.PS158C2, active against lepidopteran pests, and genes encoding lepidopteran-active toxins |
US759,247 | 1991-09-13 |
Publications (1)
Publication Number | Publication Date |
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WO1993005657A1 true WO1993005657A1 (en) | 1993-04-01 |
Family
ID=25054948
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/US1992/007713 WO1993005657A1 (en) | 1991-09-13 | 1992-09-11 | NOVEL BACILLUS THURINGIENSIS ISOLATE DENOTED B.t.PS158C2, ACTIVE AGAINST LEPIDOPTERAN PESTS, AND GENES ENCODING LEPIDOPTERAN-ACTIVE TOXINS |
Country Status (10)
Country | Link |
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US (1) | US5268172A (en) |
EP (1) | EP0642305B1 (en) |
AT (1) | ATE161391T1 (en) |
AU (1) | AU662563B2 (en) |
DE (1) | DE69223810T2 (en) |
ES (1) | ES2112331T3 (en) |
GR (1) | GR3026361T3 (en) |
NZ (1) | NZ244285A (en) |
WO (1) | WO1993005657A1 (en) |
ZA (1) | ZA926944B (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
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US5723758A (en) * | 1991-09-13 | 1998-03-03 | Mycogen Corporation | Bacillus thuringiensis genes encoding lepidopteran-active toxins |
WO1994012642A1 (en) * | 1992-11-24 | 1994-06-09 | Novo Nordisk Entotech, Inc. | Bacillus thuringiensis isolates active against lepidopteran pests |
AU740906B2 (en) * | 1997-03-13 | 2001-11-15 | Mycogen Corporation | Pesticidal bacillus thuringiensis strains |
US6218188B1 (en) | 1997-11-12 | 2001-04-17 | Mycogen Corporation | Plant-optimized genes encoding pesticidal toxins |
KR100432140B1 (en) * | 2001-06-28 | 2004-05-17 | 제연호 | Bacillus thuringiensis k-1 strain isolated from soil and microorganism pesticide using same |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0366398A1 (en) * | 1988-10-25 | 1990-05-02 | Mycogen Corporation | Novel lepidopteran-active bacillus thuringiensis isolate |
EP0366397A1 (en) * | 1988-10-27 | 1990-05-02 | Mycogen Corporation | Novel bacillus thuringiensis isolate denoted B.t. PS81F, active against lepidopteran pests, and a gene encoding a lepidopteran-active toxin |
EP0367474A1 (en) * | 1988-11-01 | 1990-05-09 | Mycogen Corporation | Novel bacillus thuringiensis isolate denoted b.t. ps81gg, active against lepidopteran pests, and a gene encoding a lepidopteran-active toxin |
EP0401979A2 (en) * | 1989-05-18 | 1990-12-12 | Mycogen Corporation | Novel bacillus thuringiensis isolates active against lepidopteran pests, and genes encoding novel lepidopteran-active toxins |
EP0405810B1 (en) * | 1989-06-27 | 1996-03-13 | Mycogen Corporation | Novel bacillus thuringiensis isolate active against lepidopteran pests, and genes encoding novel lepidopteran-active toxins |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
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US4467036A (en) * | 1981-11-12 | 1984-08-21 | The Board Of Regents Of The University Of Washington | Bacillus thuringiensis crystal protein in Escherichia coli |
US4448885A (en) * | 1981-04-27 | 1984-05-15 | Board Of The Regents Of The University Of Washington | Bacillus thuringiensis crystal protein in Escherichia coli |
GB8425487D0 (en) * | 1984-10-09 | 1984-11-14 | Agricultural Genetics Co | Strain of bacillus thuringiensis |
US4910016A (en) * | 1987-08-03 | 1990-03-20 | Mycogen Corporation | Novel Bacillus thuringiensis isolate |
US5064648A (en) * | 1988-03-04 | 1991-11-12 | Mycogen Corporation | Use of Bacillus thuringiensis microbe for controlling lesser mealworm, Alphitobius diaperinus |
US5135867A (en) * | 1988-11-01 | 1992-08-04 | Mycogen Corporation | Gene encoding a lepidopteran-active toxin from Bacillus thuringiensis isolate denoted B.t. .PS81GG active against lepidopteran pests |
US5126133A (en) * | 1989-06-27 | 1992-06-30 | Mycogen Corporation | Bacillus thuringiensis isolate active against lepidopteran pests, and genes encoding novel lepidopteran-active toxins |
-
1991
- 1991-09-13 US US07/759,247 patent/US5268172A/en not_active Expired - Lifetime
-
1992
- 1992-09-10 NZ NZ244285A patent/NZ244285A/en unknown
- 1992-09-11 AU AU26727/92A patent/AU662563B2/en not_active Ceased
- 1992-09-11 WO PCT/US1992/007713 patent/WO1993005657A1/en active IP Right Grant
- 1992-09-11 AT AT92920804T patent/ATE161391T1/en not_active IP Right Cessation
- 1992-09-11 ZA ZA926944A patent/ZA926944B/en unknown
- 1992-09-11 ES ES92920804T patent/ES2112331T3/en not_active Expired - Lifetime
- 1992-09-11 DE DE69223810T patent/DE69223810T2/en not_active Expired - Lifetime
- 1992-09-11 EP EP92920804A patent/EP0642305B1/en not_active Expired - Lifetime
-
1998
- 1998-03-13 GR GR980400546T patent/GR3026361T3/en unknown
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0366398A1 (en) * | 1988-10-25 | 1990-05-02 | Mycogen Corporation | Novel lepidopteran-active bacillus thuringiensis isolate |
EP0366397A1 (en) * | 1988-10-27 | 1990-05-02 | Mycogen Corporation | Novel bacillus thuringiensis isolate denoted B.t. PS81F, active against lepidopteran pests, and a gene encoding a lepidopteran-active toxin |
EP0367474A1 (en) * | 1988-11-01 | 1990-05-09 | Mycogen Corporation | Novel bacillus thuringiensis isolate denoted b.t. ps81gg, active against lepidopteran pests, and a gene encoding a lepidopteran-active toxin |
EP0401979A2 (en) * | 1989-05-18 | 1990-12-12 | Mycogen Corporation | Novel bacillus thuringiensis isolates active against lepidopteran pests, and genes encoding novel lepidopteran-active toxins |
EP0405810B1 (en) * | 1989-06-27 | 1996-03-13 | Mycogen Corporation | Novel bacillus thuringiensis isolate active against lepidopteran pests, and genes encoding novel lepidopteran-active toxins |
Also Published As
Publication number | Publication date |
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GR3026361T3 (en) | 1998-06-30 |
ATE161391T1 (en) | 1998-01-15 |
AU2672792A (en) | 1993-04-27 |
AU662563B2 (en) | 1995-09-07 |
ZA926944B (en) | 1993-03-22 |
DE69223810D1 (en) | 1998-02-05 |
NZ244285A (en) | 1994-12-22 |
EP0642305B1 (en) | 1997-12-29 |
EP0642305A1 (en) | 1995-03-15 |
DE69223810T2 (en) | 1998-05-20 |
US5268172A (en) | 1993-12-07 |
ES2112331T3 (en) | 1998-04-01 |
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