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 protein (delta-endotoxin)
• • 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

Definitions

• the present invention relates to an isolated Bacillus thuringiensis strain, to its novel toxin encoding gene and to the insecticidal crystal protein toxin made by the gene, as well as to insecticidal compositions containing the protein that are toxic to coleopteran insects.
• Bacillus thuringiensis (hereinafter "B.t.") is a gram-positive soil bacterium that produces crystal
• compositions including B. t. strains which produce insecticidal proteins have been commercially available and used as environmentally acceptable
• the B. t. crystal protein is toxic in the insect only after ingestion. After ingestion, the alkaline pH and proteolytic enzymes in the insect mid-gut solubilize the crystal allowing the release of the toxic components.
• B. t. strains are active against insects of the order Lepidoptera, i.e., caterpillar insects.
• Other B. t. strains are insecticidally active against insects of the order Diptera, i.e., flies and mosquitoes, or against both lepidopteran and dipteran insects.
• a few B. t. strains have been reported as producing crystal protein that is toxic to insects of the order Coleoptera, i.e., beetles. The first isolation of a coleopteran-toxic B. t.
• B. t. var. tenebrionis is reported to be toxic to larvae of the coleopteran insects Agelastica alni (blue alder leaf beetle) and Leptinotarsa decemlineata (Colorado potato beetle).
• B. t. tenebrionis makes an insecticidal crystal protein reported to be about 65-70 kilodaltons (kDa) (U.S. Patent 4,766,203; see also K. Bernhard, FEMS
• a coleopteran-toxic strain designated B. t. var. san diego, is reported by C. Herrnstadt et al.,
• Another new B. t. strain, designated EG2158, is reported by W.P. Donovan et al., in Mo 1. Gen. Gene t. , 214 , pp.365-372 (1988) and in U.S. Patent No. 5,024,837 issued June 18, 1991, to produce a 73 kDa crystal protein that is insecticidal to coleopteran insects.
• the toxin-encoding gene from B. t. strain EG2158 was cloned and sequenced, and its sequence is identical to that reported by Sekar et al. (1987) for the cloned B. t. tenebrionis coleopteran toxin gene. This coleopteran toxin gene is referred to as the cry ⁇ IIA gene by H ⁇ fte et al., Microbiol . Rev. , 53 , pp.242-255 (1989).
• coleopteran toxin plasmid transferred from B. t. strain EG2158 by conjugal plasmid transfer.
• hybrid B. t. microorganism containing a plasmid from B. t. var. kurstaki with a lepidopteran toxin gene and a plasmid from B. t . tenebrionis with a coleopteran toxin gene.
• the hybrid B. t. produces crystal proteins
• European Patent Application Publication No. 0 318 143 published May 31, 1989, of Lubrizol Genetics, Inc. discloses the cloning, characterization and selective expression of the intact partially modified gene from B. t. tenebrionis, and the transfer of the cloned gene into a host microorganism rendering the microorganism able to produce a protein having toxicity to coleopteran insects.
• Insect bioassay data for B. t. san diego reproduced from Herrnstadt et al., Bio/Technology, 4 , pp.305-308 (1986) discussed above, is summarized. The summary also includes data for B. t. tenebrionis from another source; B. t.
• B. t. microorganism identified as B. t. PS86B1 which has insecticidal activity against the
• B. t. strain PS86B1 is identified via serotyping as being serovar tolworthi .
• European Patent Application No. 0 382 990 published August 22, 1990, of Plant Genetic Systems N.V., discloses two novel B. t. strains (btGSI208 and btGSI245) producing respective crystal proteins of 74 and 129 kDa that exhibit insecticidal activity against Colorado potato beetle larvae.
• Diabrotica corn rootworm
• Diabrotica virgifera western corn rootworm
• the B. t. strain of the present invention contains a novel toxin gene that expresses protein toxin having quantifiable insecticidal activity against the Diabrotica insects, among other coleopteran insects.
• One aspect of the present invention relates to a purified and isolated coleopteran toxin gene having a nucleotide base sequence coding for the amino acid
• cry ⁇ IIC (b) gene SEQ ID NO:1
• the cry ⁇ IIC(b) gene SEQ ID NO:1 has a coding region
• the Cry ⁇ IIC(b) protein (SEQ ID NO: 2) has the amino acid sequence, as deduced from the nucleotide sequence of the cry ⁇ IIC(b) gene (SEQ ID NO:1) from nucleotide bases 144 to 2099 that is shown in Figure 1.
• the protein exhibits insecticidal activity against insects of the order Coleoptera, in particular, Colorado potato beetle and insects of the genus Diabrotica .
• Still another aspect of the present invention relates to a biologically pure culture of a B. t. bacterium
• B. t. strain EG5144 is a wild-type B. t. strain that carries the cry ⁇ IIC(b) gene (SEQ ID NO:1) and produces the insecticidal Cry ⁇ IIC(b) protein (SEQ ID NO:1)
• B. t. strain EG5145 is also a wild-type B. t.
• Yet another aspect of this invention relates to insecticidal compositions containing, in combination with an agriculturally acceptable carrier, either the
• the invention also includes a method of controlling coleopteran insects by applying to a host plant for such insects an insecticidally effective amount of the
• Cry ⁇ IIC(b) protein SEQ ID NO: 2
• the method is applicable to a variety of coleopteran insects, such as the Colorado potato beetle, Japanese beetle larvae (white grubs), Mexican bean beetle and corn rootworm.
• Still another aspect of the present invention relates to a recombinant plasmid containing the cry ⁇ IIC(b) gene (SEQ ID NO:l), a biologically pure culture of a bacterium transformed with such recombinant plasmid, the bacterium preferably being B. t. , such as B. t. strain EG7237
• Example 6 described in Example 6, as well as a plant transformed with the cry ⁇ IIC(b) gene.
• Figure 1 comprises Figures 1-1 through 1-3 and shows the nucleotide base sequence of the cry ⁇ IIC (b) gene (SEQ ID NO:1) and the deduced amino acid sequence of the
• Cry ⁇ IIC(b) protein SEQ ID NO: 2.
• the putative ribosome binding site (RBS) is indicated. Restriction sites for Sspl and ffindIII are also indicated.
• Figure 2 is a photograph of an ethidium bromide stained agarose gel containing size fractionated native plasmids of B. t. strains EG5144 (lane 1), EG4961 (lane 2), EG2838 (lane 3) and EG2158 (lane 4).
• the numbers to the left of Figure 2 indicate the approximate sizes, in megadaltons (MDa), of the plasmids of B. t. strain EG5144.
• Figure 3 is a photograph of an autoradiogram made by transferring size fractionated DNA fragments from an agarose gel to a nitrocellulose filter, hybridizing the filter with a radioactively labeled 2.4 kilobases (kb) cryIIIB probe, and exposing the filter to X-ray film.
• the agarose gel contained size fractionated total DNA
• the lane labeled "stnd" is a size standard.
• Figure 4 is a photograph of a Coomassie stained sodium dodecyl sulfate (“SDS") polyacrylamide gel showing crystal proteins solubilized from B. t. strains EG5144 (lane 1), EG4961 (lane 2) , EG2158 (lane 3) and EG2838 (lane 4).
• the numbers to the left of Figure 4 indicate the approximate sizes in kDa of the crystal proteins produced by B. t. strain EG5144.
• Lane 5 contains protein molecular size standards.
• Figure 5 shows a restriction map of plasmid pEG271.
• the location and orientation of the cry ⁇ IIC(b) gene (SEQ ID NO:1) is indicated by the arrow.
• Plasmid pEG271 is functional in Escherichia coli (E. coli) , since it contains E. coli plasmid pUC18 (Ap r ), indicated by the segment marked pUC18.
• a one kilobase scale marker is also illustrated.
• Figure 6 aligned with and based on the same scale as Figure 5, shows a restriction map of plasmid pEG272.
• the location and orientation of the cry ⁇ IIC(b) gene (SEQ ID NO:1) is indicated by the arrow shown in Figure 5.
• Plasmid pEG272 is derived from plasmid pEG271 ( Figure 5) and contains the Bacillus plasmid pNN101 (Cm r Tc r ), indicated by the segment marked pNN101 and is incorporated into the SphI site of pEG271; this plasmid is functional in B. t. Abbreviations are the same as those for Figure 5.
• Figure 7 is a photograph of a Coomassie stained SDS-polyacrylamide gel. The gel shows protein bands
• Lane 2 contains a protein size standard and the numbers on either side of lanes 1 and 3 indicate approximate sizes, in kDa, of the crystal proteins produced by these strains.
• cry ⁇ IIC (b) gene SEQ ID NO:1
• coleopteran-toxic Cry ⁇ IIC(b) crystal protein SEQ ID NO: 2
• characterization of the new B. t. strain EG5144 which produces the Cry ⁇ IIC(b) protein are described at length in Examples 1-7.
• the utility of B. t. strain EG5144 and of the Cry ⁇ IIC(b) crystal protein (SEQ ID NO:2) in insecticidal compositions and methods is also illustrated in Examples 8-11.
• cry ⁇ IIC(b) gene (SEQ ID NO:1), has the nucleotide base sequence shown in Figure 1.
• the coding region of the cry ⁇ IIC(b) gene extends from nucleotide base position 144 to position 2099 shown in Figure 1.
• cry ⁇ IIC(b) gene coding region A comparison of the nucleotide base sequence of the cry ⁇ IIC(b) gene coding region with the corresponding coding region of the prior art cryIIIA gene indicates significant differences between the two genes.
• the cry ⁇ IIC(b) gene (SEQ ID NO:1) is only 76% homologous
• cryIIIC(b) gene SEQ ID NO:1 is 96% homologous (positionally identical) with the cryIIIB gene.
• Cry ⁇ IIC(b) protein that is encoded by the cry ⁇ IIC(b) gene (SEQ ID NO:1), has the amino acid sequence (SEQ ID NO:2) shown in Figure 1.
• references to the Cry ⁇ IIC(b) "protein” are synonymous with its description as a “crystal protein”, “protein toxin”, “insecticidal protein” or the like, unless the context indicates otherwise.
• the size of the Cry ⁇ IIC(b) protein (SEQ ID NO:2), as deduced from the DNA sequence of the cry ⁇ IIC(b) gene (SEQ ID NO:1), is 74,265 Daltons (Da).
• the size of the CryIIIB protein, as deduced from the sequence of the cryIIIB gene, is 74,237 Da.
• the prior art CryIIIA protein, encoded by the cryIIIA gene has a deduced size of 73,116 Da.
• Cry ⁇ IIC(b) protein (SEQ ID NO: 2) has been shown to be a different protein than the CryIIIB protein, based on its significantly improved insecticidal activity compared to the CryIIIB protein with respect to insects of the order Coleoptera and in particular, insects of the genus
• Diabrotica The Cry ⁇ IIC(b) protein (SEQ ID NO:2), unlike the CryIIIB protein, exhibits quantifiable insecticidal activity against corn rootworm larvae.
• the present invention is intended to cover mutants and recombinant or genetically engineered derivatives, e.g., truncated versions, of the cry ⁇ IIC(b) gene (SEQ ID NO:1) that yield a protein with insecticidal properties essentially the same as those of the Cry ⁇ IIC(b) protein (SEQ ID NO:2).
• cry ⁇ IIC(b) gene (SEQ ID NO:1) is also useful as a DNA hybridization probe, for discovering similar or closely related cryIII-type genes in other B. t. strains.
• the cry ⁇ IIC(b) gene (SEQ ID NO:1), or portions or
• hybridization probe e.g., with a radioactive label
• the labeled DNA hybridization probe may then be used in the manner described in the Examples.
• cry ⁇ IIC(b) gene (SEQ ID NO:1) and the
• B. t. strain EG5144 a novel B. t. isolate.
• the characteristics of B.t. strain EG5144 are more fully described in the Examples. Comparison of the plasmid arrays and other strain characteristics of B. t. strain EG5144 with those of the recently discovered B. t. strains EG2838 and EG4961 and those of the prior art B. t. strain EG2158 and B. t. var. tenebrionis (or the equivalent, B. t. var. san diego) demonstrates that each of these coleopteran-toxic B. t. strains is distinctly
• the plasmid array of B. t. strain EG5145 is similar to that of B. t. strain EG5144, and B. t. strain EG5145 exhibits the same insecticidal activity against coleopteran insects, e.g., Japanese beetle larvae, as that of B. t . strain EG5144 (see Example 11).
• cry I ⁇ IC(b) gene may be introduced into a variety of microorganism hosts, using procedures well known to those skilled in the art for transforming suitable hosts under conditions which allow for stable maintenance and expression of the cloned cry ⁇ IIC(b) gene.
• Suitable hosts that allow the cry ⁇ IIC (b) gene (SEQ ID NO:1) to be expressed and the Cry ⁇ IIC(b) protein (SEQ ID NO: 2) to be produced include Bacillus thuringiensis and other Bacillus species such as B. subtilis or B.
• cry ⁇ IIC (b) gene SEQ ID NO:1
• SEQ ID NO:1 can also contain other toxin genes present in the same microorganism and that these genes could concurrently produce insecticidal crystal proteins different from the CryIIIC(b) protein.
• the Bacillus strains described in this disclosure may be cultured using conventional growth media and standard fermentation techniques.
• the B. t. strains harboring the cry ⁇ IIC(b) gene (SEQ ID NO:1) may be fermented, as
• CryIIIC(b) crystal protein SEQ ID NO: 2
• fermentation is typically continued through the sporulation stage when the CryIIIC (b) crystal protein is formed along with spores.
• the B. t. fermentation culture is then typically harvested by centrifugation, filtration or the like to separate fermentation culture solids, containing the CryIIIC(b) crystal protein, from the aqueous broth portion of the culture.
• the B. t. strains exemplified in this disclosure are sporulating varieties (spore forming or sporogenous strains) but the cry ⁇ IIC(b) gene (SEQ ID NO:1) also has utility in asporogenous Bacillus strains, i.e., strains that produced the crystal protein without production of spores. It should be understood that references to
• “fermentation cultures” of B. t. strains in this disclosure are intended to cover sporulated B. t. cultures, i.e., B. t. cultures containing the CryIIIC(b) crystal protein and spores, and sporogenous Bacillus strains that have produced crystal protein during the vegetative stage, as well as asporogenous Bacillus strains containing the cryIIIC(b) gene (SEQ ID NO:1) in which the culture has reached the growth stage where crystal protein is actually produced.
• the separated fermentation solids are primarily
• CryIIIC(b) crystal protein (SEQ ID NO:2) and B. t. spores, along with some cell debris, some intact cells, and residual fermentation medium solids.
• the crystal protein may be separated from the other recovered solids via conventional methods, e.g., sucrose density gradient fractionation.
• Highly purified CryIIIC(b) protein (SEQ ID NO: 2) may be obtained by solubilizing the recovered crystal protein and then precipitating the protein from solution.
• the CryIIIC(b) protein (SEQ ID NO: 2), as noted earlier, is a potent insecticidal compound against
• coleopteran insects such as the Colorado potato beetle, Japanese beetle larvae (white grubs), Mexican bean beetle and the like.
• the CryIIIC(b) protein (SEQ ID NO: 2), in contrast to the CryIIIA and CryIIIB proteins, exhibits measurable insecticidal activity against Diabrotica insects, e.g., corn rootworms, which have been relatively unaffected by other coleopteran-toxic B. t. crystal
• the CryIIIC(b) protein (SEQ ID NO: 2) may be utilized as the active ingredient in insecticidal
• formulations or compositions typically contain
• the CryIIIC(b) protein may be employed in insecticidal formulations in isolated or purified form, e.g., as the crystal protein itself.
• the CryIIIC(b) protein may be present in the recovered fermentation solids, obtained from culturing of a Bacillus strain, e.g., Bacillus thuringiensis , or other microorganism host carrying the cry ⁇ IIC(b) gene (SEQ ID NO:1) and capable of producing the CryIIIC(b) protein.
• Bacillus hosts include B. t. strain EG5144 and genetically improved B. t. strains derived from B. t. strain EG5144. The latter B. t.
• strains may be obtained via plasmid curing and/or conjugation techniques and contain the native cryIIIC(b) gene-containing plasmid from B. t. strain EG5144. Genetically engineered or transformed B. t. strains or other host microorganisms containing a
• EG7237 which contains the cloned cry ⁇ IIC(b) gene (SEQ ID NO:1) on a recombinant plasmid.
• the recovered fermentation solids contain primarily the crystal protein and (if a sporulating B. t. host is employed) spores; cell debris and residual fermentation medium solids may also be present.
• fermentation solids containing the CryIIIC(b) protein may be dried, if desired, prior to incorporation in the insecticidal formulation.
• compositions or compositions of this invention containing the insecticidal CryIIIC (b) protein (SEQ ID NO: 2) as the active component are applied at an insecticidal CryIIIC (b) protein (SEQ ID NO: 2) as the active component.
• insecticidally effective amount which will vary depending on such factors as, for example, the specific coleopteran insects to be controlled, the specific plant or crop to be treated and the method of applying the insecticidally active compositions.
• An insecticidally effective amount of the insecticide formulation is employed in the insect control method of this invention.
• insecticide compositions are made by formulating the insecticidally active component with the desired agriculturally acceptable carrier.
• the formulated insecticide compositions are made by formulating the insecticidally active component with the desired agriculturally acceptable carrier. The formulated
• compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral) or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application.
• Suitable agricultural carriers can be solid or liquid and are well known in the art.
• the term "agriculturally acceptable carrier” covers all adjuvants, e.g., inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in insecticide formulation technology; these are well known to those skilled in insecticide formulation.
• the formulations containing the CryIIIC(b) protein (SEQ ID NO: 2) and one or more solid or liquid adjuvants are prepared in known manners, e.g., by homogeneously mixing, blending and/or grinding the insecticidally active CryIIIC (b) protein component With suitable adjuvants using conventional formulation techniques.
• insecticidal compositions of this invention are applied to the environment of the target coleopteran insect, typically onto the foliage of the plant or crop to be protected, by conventional methods, preferably by spraying.
• Other application techniques e.g., dusting, sprinkling, soaking, soil injection, seed coating,
• seedling coating or spraying, or the like are also feasible and may be required for insects that cause root or stalk infestation. These application procedures are well known in the art.
• cryIIIC(b) gene (SEQ ID NO:1) or its functional equivalent, hereinafter sometimes referred to as the
• cryIIIC (b) gene can be introduced into a wide variety of microorganism hosts.
• Expression of the cryIIIC (b) gene results in the production of insecticidal CryIIIC(b) crystal protein toxin (SEQ ID NO:2).
• Suitable hosts include B. t. and other species of Bacillus, such as B. ⁇ ubtilis or B. megaterium, for example.
• Plant- colonizing or root-colonizing microorganisms may also be employed as the host for the cryIIIC(b) gene (SEQ ID NO:
• cryIIIC(b) gene SEQ ID NO:1
• Various procedures well known to those skilled in the art are available for introducing the cryIIIC(b) gene (SEQ ID NO:1) into the microorganism host under conditions which allow for stable maintenance and expression of the gene in the resulting transformants.
• the transformants i.e., host microorganisms that harbor a cloned gene in a recombinant plasmid
• the transformants then can be tested for
• Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the gene into the host, availability of expression systems, efficiency of expression, stability of the CryIIIC(b) insecticidal protein in the host, and the presence of auxiliary genetic capabilities.
• the cellular host containing the insecticidal cry ⁇ IIC(b) gene may be grown in any convenient nutrient medium, where expression of the cry ⁇ IIC (b) gene is obtained and CryIIIC(b) protein (SEQ ID NO:2) produced, typically to sporulation.
• the sporulated cells containing the crystal protein may then be harvested in accordance with
• cry ⁇ IIC(b) gene (SEQ ID NO:1) may also be expressed as SEQ ID NO:1
• cry ⁇ IIC (b) gene SEQ ID NO:1
• Genetic engineering of plants with the cry ⁇ IIC (b) gene may be accomplished by introducing the desired DNA containing the gene into plant tissues or cells, using DNA molecules of a variety of forms and origins that are well known to those skilled in plant genetic engineering.
• An example of a technique for introducing DNA into plant tissue is disclosed in European Patent Application Publication No. 0 289 479, published November 2, 1988, of Monsanto Company.
• CryIIIC(b) protein may be delivered into the plant cells or tissues directly by infectious plasmids, such as Ti, the plasmid from Agrobacterium tumefaciens, viruses or microorganisms like A. tumefaciens, by the use of lysosomes or liposomes, by microinjection by mechanical methods and by other techniques familiar to those skilled in plant genetic engineering.
• infectious plasmids such as Ti
• the plasmid from Agrobacterium tumefaciens viruses or microorganisms like A. tumefaciens
• lysosomes or liposomes by microinjection by mechanical methods and by other techniques familiar to those skilled in plant genetic engineering.
• nucleotide base sequence SEQ ID NO:1
• the various amino acids forming the protein encoded by the gene usually may be determined by more than one codon, as is well known to those skilled in the art.
• there may be some variations or truncation in the coding regions of the cry ⁇ IIC(b) nucleotide base sequence which allow expression of the gene and production of functionally equivalent forms of the CryIIIC(b) insecticidal protein.
• the novel B. t. strain EG5144 was isolated following the procedure described in Example 1. The procedures described in Example 1 were also used to isolate the novel B. t. strain EG5145.
• Crop dust samples were obtained from various sources throughout the U.S. and abroad, typically grain storage facilities. The crop dust samples were treated by
• the filter was placed colony side up on an agar medium containing 0.5% (w/v) glucose.
• the transferred colonies were then allowed to grow on the agar-glucose medium for 5 hours at 30°C.
• Use of 0.5% glucose in the agar medium and the 5-hour, 30°C growth cycle were critical for assuring that the B. t. colonies were in a vegetative state and thus susceptible to lysis.
• a cloned coleopteran toxin gene was used as a
• the 2.9 kb HindIII cryIIIA DNA fragment containing the entire cryIIIA gene, was radioactively labeled with [alpha- P32 ]-dATP and Klenow enzyme, by standard methods.
• the nitrocellulose filters containing the DNA from each lysed colony were incubated at 65°C for 16 hours in a buffered solution that contained the radioactively labeled 2.9 kb HindIII cry ⁇ IIA DNA probe to hybridize the DNA from the colonies with the DNA from the radioactively labeled cry ⁇ IIA probe.
• the 65°C hybridization temperature was used to assure that the cryIII A DNA probe would hybridize only to DNA from colonies that contained a gene that was similar to the cry ⁇ IIA DNA probe.
• cryI-type genes encode lepidopteran-toxic, coleopteran-nontoxic crystal proteins with molecular masses of approximately 130 kDa.
• Computer- assisted comparisons of the sequence of the cryIIIA gene with the sequence of several cryI-type genes revealed that the 3'-end of the cryIIIA gene was partially homologous with portion of the cryI-type genes. This finding
• cry ⁇ IIA 3'-end of the cry ⁇ IIA gene was causing the 2.9 kb cry ⁇ IIA probe to hybridize to B. t. colonies containing cryJ-type genes.
• HindIII-XbaI fragment was purified that contained the cryIIIA gene minus its 3'-end.
• the 2.0 kb HindIII-XbaI fragment contains the 3'-truncated cryIIIA gene. When the 2.0 kb fragment was used in repeated colony hybridization experiments, it did not hybridize to cryI gene-containing B. t. colonies.
• strain EG2838 which has been deposited with the NRRL under Accession No. NRRL B-18603.
• B. t. strain EG2838 was found to be insecticidally active against coleopteran insects, notably, the Colorado potato beetle. B. t. strain EG2838 did not have
• cryIIIB gene A gene, designated the cryIIIB gene, was isolated from B. t. strain EG2838, and its nucleotide base sequence determined.
• the cryIIIB gene encoded a crystal protein, designated the CryIIIB protein, containing 651 amino acids having a deduced size of 74,237 Daltons.
• the size of the prior art CryIIIA protein had previously been deduced to be 73,116 Daltons (644 amino acids).
• the cryIIIB gene is 75% homologous with the cryIIIA gene, and the CryIIIB protein is 68% homologous with the CryIIIA protein.
• cryIIIB probe obtained from B. t. strain EG2838.
• the cryIIIB probe was radioactively labeled using the procedure set forth above with respect to the radioactively labeled cryIIIA probe.
• radioactively labeled cryIIIB probe consisted of a 2.4 kb Sspl restriction fragment of DNA from B. t. strain EG2838. The fragment contains the complete protein coding region for the coleopteran toxin cryIIIB gene of B. t. strain EG2838.
• the B. t. strains of the present invention designated B. t. strains EG5144 and EG5145, were isolated from a crop dust sample via B. t. colonies that specifically hybridized to the cryIIIB probe.
• B. t. strain EG5144 To characterize B. t. strain EG5144, several studies were conducted. One series of studies was performed to characterize its flagellar serotype. Additional studies were conducted to determine the sizes of the native plasmids in B. t. strain EG5144 and to ascertain which plasmids contained genes that encoded coleopteran-active insecticidal crystal proteins. DNA blot analysis was thereafter performed using size fractionated total DNA restriction fragments from B. t. strain EG5144, compared with similarly-processed total DNA from other B. t. strains containing cry III-type toxin genes, to demonstrate that B. t. strain EG5144 contains a unique coleopteran-active toxin gene. In addition, B. t. strain EG5144 was evaluated further by characterizing the crystal proteins it produces and by measuring the insecticidal activity associated with B. t. strain EG5144 and its crystal proteins. Examples 2 through 7 are directed to the procedures for
• Example 2 characterizing B. t. strain EG5144 and its unique cry ⁇ II- type gene, and Examples 8 through 11 are directed to the insecticidal activity of B. t. strain EG5144 and of B. t. strain EG7237, containing the cry ⁇ IIC(b) gene (SEQ ID NO:l) of this invention.
• Example 2
• Flagellar serotyping studies were carried out with B. t. strain EG5144, using an antibody mediated cell agglutinization assay (Craigie et al., J. Immunol . , 21 , pp.417-511 (1936)).
• Flagellar antibody reagents were prepared using purified flagella from B. t. var. kurstaki, morrisoni and tolworthi type-strains and from the novel coleopteran-active B. t. strain EG4961.
• the other coleopteran-active B.t. strains included B.t. var. tenebrionis, B.t. var. san diego, B.t. strain EG2158 (all containing the cryIIIA gene); B.t. strain EG2838 (containing the cryIIIB gene); and B.t. strain EG4961 (containing a novel coleopteran toxin-encoding gene designated as the cry ⁇ IIC(a) gene).
• the B.t. flagellar type-strains were B.t. var.
• israeltaki (HD-1, serotype 3ab), B.t. var. morrisoni (HD-12, serotype 8ab) and B.t. var. tolworthi (HD-13, serotype 9).
• B. t. var. kurstaki (HD-1) + - - - B. t. var. morrisoni (HD-12) + - B. t. var. tolworthi (HD-13) - - + -
• Table 1 show that cells of B. t. strain EG5144 gave a negative reaction with B. t. type-strain kurstaki , morrisoni and tolworthi flagella antibody reagents.
• B. t. strain EG5144 cells also gave a negative reaction with flagellar reagent from B. t. strain EG4961, a novel coleopteran-active strain that has been discovered to exhibit Diabrotica toxicity.
• B. t. strain EG5144 is not a kurstaki, morrisoni or tolworthi-type B. t. strain.
• B. t. strain EG5144 which is yet not known, is apparently different from that of B. t. strain EG4961, which has been serotyped as serovar kumamotoensis (serotype 18).
• Both B. t. strain EG5144 and B. t. strain EG4961 appear to have flagellar serotypes that are different from those of other coleopteran-toxic B. t. strains reported in the literature.
• B. t. strains may be characterized by fractionating their plasmids according to size by the well-known
• This procedure involves lysing B. t. cells with lysozyme and SDS,
• Plasmides electrophoresing plasmids from the lysate through an agarose gel and staining the gel with ethidium bromide to visualize the plasmids. Larger plasmids, which move more slowly through the gel, appear at the top of the gel and smaller plasmids appear toward the bottom of the gel.
• B.t. strain EG5144 contains native plasmids of approximately 145, 92, 12, 10 and 5.5 MDa, as indicated by the white horizontal bands. Plasmid sizes were estimated by comparison to plasmids of known sizes (not shown). Although not shown on Figure 2, B. t. strain EG5145 contains native plasmids of approximately 145, 92, 12 and 5.5 MDa. The cryptic 10 MDa plasmid found in B. t. strain EG5144 is not present in B. t. strain EG5145.
• Figure 2 further shows that the coleopteran-toxic B. t. strain EG4961 contains native plasmids of about 150, 95, 70, 50, 5 and 1.5 MDa and that the coleopteran-toxic B. t. strain EG2838 contains native plasmids of about 100, 90 and 37 MDa.
• Figure 2 also shows that the coleopteran- toxic B. t. strain EG2158 contains native plasmids of about 150, 105, 88, 72, and 35 MDa.
• strain EG2158 may not be visible in the photograph, although they are visible in the actual gel.
• Figure 2 demonstrates that the sizes of the native plasmids of B. t. strain EG5144 are different from the sizes of the native plasmids of B. t. strains EG2158, EG2838 and EG4961.
• B. t. strain EG5144 is
• B. t. strain EG5145 appears distinct from the coleopteran-toxic B. t. strains noted above based on plasmid array studies.
• the plasmids shown in Figure 2 were transferred by blotting from the agarose gel to a nitrocellulose filter using the blot techniques of Southern, J.Molec.Biol . , 98 , pp.503-517 (1975), and the filter was hybridized as described above with the radioactively labeled 2.4 kb cryIIIB DNA probe. After hybridization, the filter was exposed to X-ray film. Examination of the X-ray film confirmed that the cryIIIB probe specifically hybridized to the 92 MDa plasmid of B. t. strain EG5144. This result demonstrates that the 92 MDa plasmid of B. t.
• strain EG5144 contains a DNA sequence that is at least partly homologous to the cry ⁇ IIB gene and confirms that the 92 MDa plasmid contains a cryIII-type gene.
• the X-ray film also showed that the cry ⁇ IIB probe hybridized, as expected, to the 95 MDa plasmid of B. t. strain EG4961 and to. the 100 MDa plasmid of B. t. strain EG2838, and to the 88 MDa plasmid of B. t. strain EG2158.
• the 88 MDa plasmid of B. t. strain EG2158 has been previously shown to contain the
• coleopteran-toxin cry ⁇ IIA gene see Donovan et al., Mol . Gen. Genet. , 214 , pp.365-372 (1988)).
• the inventors have previously determined that the 100 MDa plasmid of B. t. strain EG2838 contains the coleopteran toxin cryIIIB gene and that the 95 MDa plasmid of B. t. strain EG4961 contains the novel coleopteran toxin cry ⁇ IIC(a) gene.
• Both chromosomal and plasmid DNA (total DNA) from B. t. strain EG5144 were extracted and digested with separate restriction enzymes, SspI , HindIII and EcoRI.
• the digested DNA was size fractionated by electrophoresis through an agarose gel, and the fragments were then visualized by staining with ethidium bromide.
• strains EG2158, EG2838 and EG4961 was processed in an identical manner. Examination of the resultant stained agarose gel showed that restriction digestions of total DNA from these B. t. strains with each of SspI , HindIII and EcoRI yield hundreds of DNA fragments of various sizes.
• the size fractionated DNA restriction fragments were transferred by blotting from the agarose gel to a
• FIG. 3 is a photograph of the autoradiogram where the numbers to the left indicate the size, in kb, of the DNA fragments of B.t. strain EG5144 that hybridized to the cryIIIB probe. These sizes were determined by comparison with the lane labeled "stnd" which contained phage lambda DNA digested with HindIII and radioactively labelled as size markers. Lanes in Figure 3 marked EG2158, EG5144, EG2838 and EG4961 contain size fractionated DNA fragments from these respective B. t.
• strains obtained by digestion with the restriction enzyme designated above the individual lanes.
• cryIIIB-hybridizing SspI restriction fragment for B. t. strain EG5144 is 3.4 kb, and this is unlike the corresponding Sspl restriction fragments for the other three B. t. strains: 2.8 kb for B. t. strain EG2158; 2.4 kb for B. t. strain EG2838; and 4.5 and 6.0 kb for B. t. strain EG4961. Similar differences are apparent for the DNA restriction fragments obtained using HindIII and EcoRI. These restriction pattern results suggest that B. t. strain EG5144 contains a cry III-type gene that is
• cryIIIA, cryIIIB and cry ⁇ IIC(a) genes of B. t. strains EG2158, EG2838 and EG4961 respectively.
• the cryIII-type gene of B. t. strain EG5144 has been designated cryIIIC(b; (SEQ ID NO:1) by the inventors.
• Total DNA from B. t. strain EG5144 and B. t. strain EG5145 was extracted and digested with six separate restriction enzymes (HindIII, EcoRI , AccI, DraI , SspI , Xbal) , and size fractionated by electrophoresis on an agarose gel. The size fractionated DNA restriction fragments were then transferred by blotting to a
• B. t. strain EG5144 was grown in DSMG sporulation medium at room temperature (about 21-25°C) until
• the DSMG medium is 0.4% (w/v) Difco nutrient broth, 25 mM K 2 HPO 4 , 25 mM KH 2 PO 4 , 0.5 mM Ca(NO 3 ) 2 , 0.5 mM MgSO 4 , 10 ⁇ M FeSO 4 , 10 ⁇ M. MnCl 2 and 0.5% (w/v) glucose.
• the sporulated culture of B. t. strain EG5144 was observed microscopically to contain free floating, irregularly shaped crystals in addition to B. t. spores.
• B. t. crystals are usually composed of proteins that may be toxic to specific insects. The appearance of the crystals of B.
• t. strain EG5144 differed from the flat, rectangular (or rhomboidal) crystals of B. t. strain EG2158, but partially resembled some of the irregularly shaped crystals of B. t. strains EG2838 and EG4961.
• TETX containing 10 mM Tris HC1 pH 7.5, ImM EDTA and 0.005% (w/v) Triton ® x-100
• the washed crystals were specifically solubilized from 250 ⁇ g centrifuged
• Figure 4 shows the results of this protein size fractionation analysis where the numbers to the left indicate the size, in kDa, of the crystal proteins
• B. t. strain EG5144 synthesized by B. t. strain EG5144. As shown in lane 1, a major protein of approximately 70 kDa and a minor protein of approximately 30 kDa were solubilized from centrifuged fermentation solids containing B. t. strain EG5144 spores and crystals. The approximately 70 kDa protein of B. t. strain EG5144 appears similar in size to the approximately 70 kDa coleopteran-toxic crystal proteins of B. t. strains EG4961 (lane 2), EG2158 (lane 3) and to the approximately 74 kDa coleopteran-toxic crystal protein of B. t. strain EG2838 (lane 4).
• the coleopteran-toxic crystal proteins of B. t. strains EG4961, EG2158 and EG2838 are each different.
• the CryIIIC(a) protein of B. t. strain EG4961 is coded by the cry ⁇ IIC(a) gene and has a deduced size of 74,393 Da.
• the CryIIIA protein of B. t. strain EG2158 is coded by the cryIIIA gene and has a deduced size of 73,116 Da.
• the CryIIIB protein of B. t. strain EG2838 is coded by the cryIIIB gene and has a deduced size of 74,237 Da.
• the coleopteran-toxic crystal protein of B. t. strain EG4961 is coded by the cry ⁇ IIC(a) gene and has a deduced size of 74,393 Da.
• the CryIIIA protein of B. t. strain EG2158 is coded by the cryIIIA gene and has
• EG5144 produced by the novel cryIIIC(b) gene is clearly different from the CryIIIA, CryIIIB and
• the minor crystal protein of approximately 30 kDa that is produced by B. t. strain EG5144 is roughly similar in size to small crystal proteins produced by B. t. strains EG4961, EG2158 and EG2838.
• the approximately 30 kDa minor proteins of B. t. strains EG2158, EG2838 and EG4961 appear to be related to each other and none has been found to exhibit measurable insecticidal activity towards
• coleopteran insects There is no reason to believe that the approximately 30 kDa protein of B. t. strain EG5144 possesses insecticidal activity against coleopteran insects.
• Example 6 The procedures set forth in Example 6 describe the determination of the nucleotide base sequence of the cry ⁇ IIC(b) gene (SEQ ID NO:1).
• a plasmid library of B. t. strain EG5144 was constructed by ligating size- selected DNA EcoRI-XbaI restriction fragments from B. t. strain EG5144 into the well-known E. coli vector pUC18. This procedure involved first obtaining total DNA from B. t. strain EG5144 by cell lysis followed by DNA spooling, then double digesting the total DNA with both EcoRI and XbaI restriction enzymes, electrophoresing the digested DNA through an agarose gel, excising a gel slice
• the plasmid library was then transformed into E. coli cells, a host organism lacking the gene of interest, as follows. After ligation, the DNA mixture was incubated with an ampicillin sensitive E. coli host strain, E. coli strain DH5 ⁇ , that had been treated with CaCl 2 to allow the cells to take up the DNA. E. coli, specifically strain DH5 ⁇ , was used as the host strain because these cells are easily transformed with recombinant plasmids and because E. coli strain DH5 ⁇ does not naturally contain genes for B. t. crystal proteins. Since pUC18 confers resistance to ampicillin, all host cells acquiring a recombinant plasmid would become ampicillin resistant. After exposure to the recombinant plasmids, the E. coli host cells were spread on agar medium that contained ampicillin. After
• the radioactively labeled 2.4 kb cry ⁇ IIB gene was then used as a DNA probe under conditions that permitted the probe to bind specifically to those transformed host colonies that contained the 7.0 kb EcoRI-XbaI fragment of DNA from B. t. strain EG5144.
• One cryIIIB-hybridizing colony, designated E. coli strain EG7236 was studied further.
• E. coli strain EG7236 contained a recombinant plasmid, designated pEG271, which consisted of pUC18 plus the inserted EcoRI-XbaI
• the cryIIIB probe specifically hybridized to the 7.0 kb DNA fragment insert in pEG271.
• a restriction map of pEG271 is shown in Figure 5.
• the 7.0 kb fragment of pEG271 contained HindIII fragments of 2.4 kb and 3.8 kb, and a BamHI-Xbal fragment of 4.0 kb that specifically hybridized with the cryIIIB probe.
• the 2.4 kb HindIII fragment was subcloned into the DNA sequencing vector M13mp18.
• the 4.0 kb BamHI -XbaI fragment was subcloned into the DNA sequencing vectors M13mp18 and M13mp19.
• nucleotide base sequence of a substantial part of each subcloned DNA fragment was determined using the standard Sanger dideoxy method. For each subcloned fragment, both DNA strands were sequenced by using
• cryIIIC(b) SEQ ID NO:1
• cry ⁇ IIC(b) gene is also clearly distinct from the cry ⁇ IIB gene.
• cry ⁇ IIC(b) gene SEQ ID NO:1
• the deduced amino acid sequence of the CryIIIC (b) protein SEQ ID NO : 2
• the protein coding portion of the cry I ⁇ IC (b) gene is defined by the nucleotides starting at position 144 and ending at position 2099.
• the probable ribosome binding site is indicated as "RBS" in Figure 1-1.
• CryIIIC(b) protein (SEQ ID NO: 2) encoded by the cryI ⁇ IC(b) gene, as deduced from the open reading frame of the cry ⁇ IIC(b) gene (SEQ ID NO:1), is 74,265 Da (652 amino acids). It should be noted that the apparent size of the CryIIIC(b) protein, as determined from SDS-PAGE, is approximately 70 kDa. Therefore, the CryIIIC(b) protein (SEQ ID NO:2) will be referred to in this specification as being approximately 70 kDa in size.
• the size of the CryIIIB protein has previously been determined to be 74,237 Da (651 amino acids).
• the nucleotide base sequence of the cry ⁇ IIC(b) gene (SEQ ID NO:1) was 96% positionally identical with the nucleotide base sequence of the cry ⁇ IIB gene and only 76% positionally identical with the nucleotide base sequence of the cryIIIA gene.
• the cry ⁇ IIC(b) gene (SEQ ID NO:1) is related to the cryIIIB and cryIIIA genes, it is clear that the cryI ⁇ IC (b) gene is distinct from the cryIIIB gene and substantially different from the cryIIIA gene.
• the deduced amino acid sequence of the CryIIIC(b) protein (SEQ ID NO: 2) was found to be 95% positionally identical to the deduced amino acid sequence of the
• CryIIIB protein but only 68% positionally identical to the deduced amino acid sequence of the CryIIIA protein. These differences, together with the differences in insecticidal activity as set forth below, clearly show that the CryIIIC(b) protein encoded by the cry ⁇ IIC(b) gene (SEQ ID NO:1) is a different protein from the CryIIIB protein or the CryIIIA protein.
• amino acids indicate the position of the amino acid in the sequence illustrated in Figure 1 and identified in SEQ ID NO: 2: His9, His231, Gln339, Ser352, Asn446, His449, Val450, Gly451, Ile600 and Thr624. These amino acid residues were selected as being of probable significance for the corn rootworm toxicity of the
• CryIIIC(b) protein (SEQ ID NO:2) because, after studying the amino acid sequences of several other CryIII proteins, the amino acids at the indicated positions fairly
• cry ⁇ IIC(b) gene SEQ ID NO:l
• site directed mutagenesis of the cry ⁇ IIC(b) gene may result in improved or enhanced corn rootworm toxicity for the resultant protein where one or more of the following amino acid modifications are effected:
• cry ⁇ IIC(b) gene SEQ ID NO:1
• SEQ ID NO:1 changes in the cry ⁇ IIC(b) gene (SEQ ID NO:1) may be made, via site directed mutagenesis or gene truncation or the like, that could yield a toxic protein which possesses essentially similar insecticidal activity (to corn rootworm and other coleopteran insects) as that exhibited by the CryIIIC(b) protein (SEQ ID NO: 2).
• Modifications to the cryI ⁇ IC (b) gene (SEQ ID NO:1) and CryIIIC(b) protein (SEQ ID NO:2) such as described above are intended to be within the scope of the claimed invention.
• Table 2 summarizes the relevant characteristics of the B. t. and E. coli strains and plasmids used during these procedures.
• a plus ( + ) indicates the presence of the designated element, activity or function and a minus (-) indicates the absence of the same.
• the designations s and r indicate sensitivity and resistance, respectively, to the antibiotic with which each is used.
• Amp ampicillin
• Cm chloramphenicol
• Cry crystalliferous
• Tc tetracycline
• E. coli cells harboring plasmid pEG271 described in Example 6 were analyzed and found not to produce
• the Bacillus vector pNN101 (Tc r Cm r Cry-) that is capable of replicating in B. t. was ligated into the SphI site of pEG271.
• the resultant plasmid was designated pEG272.
• the isolated plasmid pEG271 DNA was digested with SphI and was then mixed with the Bacillus vector pNN101 that had also been digested with Sphl . T4 DNA ligase and ATP were added to the mixture to allow pEG271 to ligate into the SphI site of the pNN101 vector.
• the DNA mixture was added to a suspension of E. coli strain DH5 ⁇ cells that had been treated with calcium chloride to permit the cells to take up plasmid DNA. After exposure to the recombinant
• the E. coli host cells were spread on an agar medium containing tetracycline. Only cells that had taken up a plasmid consisting of pEG271 ligated into the SphI site of pNN101 would grow on the tetracycline agar medium whereas cells that had not absorbed the plasmid would not grow.
• Plasmid was isolated from one tetracycline resistant colony, digested with SphI, and electrophoresed through an agarose gel.
• the plasmid consisted of two SphI DNA fragments of 5.8 kb and 9 kb corresponding to plasmids pNN101 and pEG271, respectively. This plasmid was
• E. coli strain GM2163 is a crystal negative (Cry-) and ampicillin sensitive (Amp s ) strain, constructed by the procedures of M.G. Marinus et al. in Mol. Gen. Genet., 192 , pp.28 ⁇ -2 ⁇ 9 (1983).
• Plasmid pEG272 was then isolated from the transformed E. coli strain GM2163, using the procedures described above. The isolated plasmid pEG272 was next transformed by electroporation into B. t. strain HD73-26. Cells of B. t. strain HD73-26 are crystal-negative (Cry-) and chloramphenicol sensitive (Cm s ). Using a BioRad Gene
• Pulser TM apparatus to carry out the electroporation, cells of B. t. strain HD73-26 in suspension were induced to take up pEG272 which was also added to the mixture.
• the transformed B. t. cells were spread onto an agar medium containing 5 ⁇ g
• sporulated culture of B. t. strain EG7237 contained spores and small free floating irregularly shaped crystals.
• the crystals in the centrifuge pellet suspension were solubilized by heating a portion of the centrifuge
• solubilization buffer (0.14 M Tris pH 6.8, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol and 0.1% (w/v) bromophenol blue) at 100°C for 5 minutes. After crystal solubilization had occurred, the mixture was applied to an SDS-polyacryamide gel and the solubilized proteins in the mixture were size fractionated by electrophoresis. After size fractionization, the proteins were visualized by staining with Coomassie dye.
• Lane 3 of the gel in Figure 7 shows that B. t. strain EG7237 produced a major protein of approximately 70 kDa and a minor protein of approximately 30 kDa. These proteins appeared to be identical in size with the major approximately 70 kDa protein and the minor approximately 30 kDa protein produced by B. t. strain EG5144, which are shown in the lane 1 of Figure 7 and which were prepared in a manner identical to B. t. strain EG7237. This result indicates that the 7.0 kb fragment of pEG272 contains two crystal protein genes: one for the approximately 70 kDa protein and one for the approximately 30 kDa protein.
• cryIIIC (b) gene The gene encoding the approximately 70 kDa protein is the cryIIIC (b) gene, and its encoded protein is the insecticidal CryIIIC(b) protein.
• the DNA sequence for the cry ⁇ IIC(b) gene SEQ ID NO:1
• the amino acid sequence for its corresponding deduced protein SEQ ID NO:2
• B.t. strain EG7237 produced approximately three times more 70 kDa protein, on a weight basis, than did B. t.
• strain EG5144 as is evident from the protein bands in Figure 7. Production of the minor 30 kDa protein in recombinant B. t. strain EG7237 was also increased, as compared with B. t. strain EG5144.
• the following Examples 8-11 describe the manner in which the insecticidal activities of B. t. strain EG5144, B. t. strain EG7237, and the CryIIIC(b) protein made by these strains were determined.
• the insecticidal activity of recombinant B. t. strain EG7237 which contains the cry ⁇ IIC (b) gene (SEQ ID NO:1) that produces the CryIIIC(b) toxin protein (SEQ ID NO: 2), was determined against southern corn rootworm (Diabrotica undecimpunctata howardi) and Colorado potato beetle
• B. t. strain EG7235 which contains the cryIIIA gene that produces the CryIIIA toxin protein
• recombinant B. t. strain EG7225 which contains the cryIIIB gene that produces the CryIIIB toxin protein.
• the fermentation broth was concentrated by microfiltration.
• the concentrated fermentation broth was then freeze dried to prepare a B. t. powder suitable for insect bioassay.
• the amount of CryIII-type toxin protein in each of the B. t. powders was quantified using standard SDS-PAGE techniques.
• Results are shown as the dose amount of CryIII-type protein (in ng CryIII protein per mm 2 of diet
• B. t. strains exhibit insecticidal activity against Colorado potato beetle larvae, with the CryIIIA toxin protein of B. t. strain EG7235 being
• Example 8 The insecticidal activity of recombinant B. t. strain EG7237, evaluated in Example 8, was also determined against Mexican bean beetle (Epilachna varivestis) . As in Example 8, recombinant B. t. strains EG7235 and EG7225 were included for comparison, and all B. t. powders were prepared as in Example 8.
• Soybean leaves were dipped into known treatment concentrations of the B. t. powder suspended in an aqueous 0.1% Triton ® X-100 solution. After excess material had dripped off, the leaves were allowed to dry. Leaves dipped in 0.1% Triton ® X-100 served as untreated controls. Twenty insect larvae were confined to a petri dish with treated leaves,
• insecticidal activity of specific CryIII-type toxin proteins varies widely for insect genera within the order Coleoptera.
• B. t. strain EG5144 The insecticidal activity of B. t. strain EG5144 was evaluated against Southern corn rootworm (Diabrotica undecimpunctata howardi ). For comparison, B. t. strain EG4961 which produces the CryIIIC(a) toxin protein was included in the bioassay study.
• the bioassay procedure for southern corn rootworm in this Example determined PLC 50 values, the concentration of CryIII-type protein required to kill 50% of the insects tested.
• the procedure was similar to the artificial diet bioassay carried out in the previous Example, using thirty-two first instar southern corn rootworm larvae per dose. Data from each of the replicated bioassays were pooled for probit analysis (R.J. Daum,
• Results are reported for two separate tests as the dose amount of CryIII-type protein (ng CryIII protein per mm 2 of diet surface) resulting in PLC50. Confidence intervals, at 95%, are given within parentheses following the PLC 50 values. In Test 1 four replications per dose were used, and in Test 2, carried out at a later date, two replications were used.
• the B. t. strains used in this Example were prepared as described for the B. t. strains in Example 8, except that the fermentation broth was concentrated by
• Test 2 6.4 1145 (773-2185)
• B. t. EG4961 Cry ⁇ IIC(a)
• Test 1 11.6 102 (66-119)
• B. t. strain EG5144 The insecticidal activity of B. t. strain EG5144 was evaluated against Japanese beetle larvae, also known as white grubs (Popillia japonica) .
• B. t. strain EG4961 which produces the CryIIIC(a) toxin protein was included in the bioassay study, as were B. t. strain EG215 ⁇ which produces the CryIIIA toxin protein and B. t. strain EG2 ⁇ 3 ⁇ which produces the CryIIIB toxin protein.
• the bioassay procedure in this Example was a
• Results were obtained at a single dose rate of CryIII-type protein: 1 mg CryIII-type protein per ml of diet;
• B. t. strain EG2158 and B. t. strain EG2838 B. t. strain EG5144 exhibited superior insecticidal performance against Japanese beetle grubs.
• B. t. strain EG5145 whose characteristics are similar to those of B.t. strain EG5144, has been found to exhibit insecticidal activity against Japanese beetle grubs equivalent to that of B. t. strain EG5144, although the bioassay data are not presented in this Example 11.
• MOLECULE TYPE DNA (genomic)

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