WO1994028724A1

WO1994028724A1 - Bacillus thuringiensis strains capable of producing large amonts of insecticidal crystal proteins

Info

Publication number: WO1994028724A1
Application number: PCT/US1994/006454
Authority: WO
Inventors: James A. Baum; Thomas M. Malvar; Thomas C. Currier; Susan M. Brussock
Original assignee: Ecogen Inc.
Priority date: 1993-06-10
Filing date: 1994-06-09
Publication date: 1994-12-22
Also published as: AU7102494A

Abstract

Bacillus thuringiensis strain designated as EG1351 and derivative strains thereof that exhibit high levels of insecticidal crystal protein production. Insecticide compositions containing such protein-overproducing Bacillus thuringiensis strains and the method of controlling insects with such insecticidal compositions are also within the scope of this invention.

Description

BACILLUS THURINGIENSIS STRAINS CAPABLE OF

PRODUCING LARGE AMOUNTS OF INSECTICIDAL

CRYSTAL PROTEINS

Field of the Invention

The present invention relates to insecfϊcidal Bacillus thuringiensis strains that are capable of producing relatively large amounts of insecticidal crystal proteins .

Background of the Inven ion Bacillus thuringiensis ( " B . t . " ) is a gram-positive soil bacterium that typically produces proteinaceous crystalline inclusions during sporulation. These B . t . crystal proteins are often highly toxic to specific insects and are the active ingredient in commercial B. t . - based biological insecticides . Crystal proteins from various B . t . strain isolates have been identified as having insecticidal activity against insect larvae from the insect orders Lepidoptera (caterpillars) , Coleoptera (beetles) and Diptera (mosquitos, flies) . In the last ten years, numerous B.t. isolates, i.e., wild-type B . t . strains, have been discovered and identified as having activity solely against coleopteran insects, e.g., Colorado potato beetles. The first of such reported strains was B . t . tenebrionis, described by Krieg et al . in U.S. Patent No. 4,766,203. B.t. tenebrionis is identical to the B. t . strain which is designated as B.t. san diego in U.S. Patent No. 4,771,131. Another novel coleopteran-toxic B.t. strain isolate is strain EG2158, described by Donovan et al. in U.S. Patent No. 5,024,837, which is a different strain from B.t. tenebrionis .

Several of the commercial B.t. bioinsecticide products currently being marketed for coleopteran insect control are based on these wild-type B . t . strain isolates, and their formulations typically contain dried B.t. fermentation cultures, with the insecticidal crystal protein serving as the active ingredient. The insecticidal activity of these B.t. bioinsecticides, like that of all B.t.-based commercial products, results from insect larvae feeding on the crystal protein, typically in sprayed-on deposits of the bioinsecticide on leaves or other plant surfaces. Ingestion of the crystal protein by the insect causes release of toxic components of the protein in the insect's midgut, leading to cessation of feeding and death.

The amount of crystal protein present in commercial formulations of B.t.-based bioinsecticides is desirably maximized, to provide for its economic and efficient utilization in the field. Increased concentrations of crystal protein in the formulated bioinsecticide promote use of reduced amounts of bioinsecticide per unit area of treated crop (without reducing the actual amount of crystal protein applied per unit area) , thereby allowing for more cost effective use of the bioinsecticide product.

Concentrations of the crystal protein produced during the fermentation production of B.t. strains are often dependent on the B.t. strain isolate selected for use. Previous efforts to create mutants of known B.t. strains that result in enhanced crystal protein production have had limited success in improving protein yields.

-2-

SUBSTΓΓUTE SHEET (RULE 26) European Patent Application Publication No. 0 099 301, published January 25, 1984 of Fitz-James, describes mutants of Bacillus thuringiensis var. israelensis, obtained via use of a chemical mutagen, that produces up to 1.5 times the amount of protein obtained from the progenitor strain.

European Patent Application Publication No. 0 228 228, published July 8, 1987 of Mycogen Corporation, describes asporogenous Bacillus thuringiensis mutants obtained by treatment of the progenitor strains with ethidium bromide. Although such B.t. mutants are described as being more efficient at producing the insecticidal crystal protein, no evidence of this is disclosed. PCT International Patent Application Publication No. WO 91/07481, published May 30, 1991 of Novo Nordisk A/S, describes a mutant of Bacillus thuringiensis tenebrionis, which was obtained by gamma irradiation and which produces 2 times the amount of protein obtained from the progenitor strain.

The present invention provides B.t. strain isolates that produce very high yields of crystal protein, and these strains were obtained without resort to conventional mutgenic agents. The use of these protein "overproducing" B.t. strains in insecticide compositions and for use in insect control is also taught.

Summary of the Invention

The Bacillus thuringiensis (B . t . ) strains of this invention include a biologically pure culture of a Bacillus thuringiensis bacterium deposited with the Agricultural Research Service Culture Collection, Northern Regional Research Laboratory (NRRL) , having accession number NRRL B-21003 and being designated as strain EG1351, or derivative strains thereof capable of overproducing insecticidal protein. B.t. strain EG1351 is an asporogenous isolate that produces insecticidal crystal protein in large amounts, and its CrylllA crystal protein is toxic to coleopteran insects.

The Bacillus thuringiensis strains of this invention also include a biologically pure culture of a Bacillus thuringiensis bacterium, deposited with the NRRL having accession number NRRL B-21004 and being designated as strain EG7651, or derivative strains thereof capable of overproducing insecticidal proteins encoded by one or more toxin genes on plasmids harbored by such derivative strains. B.t. strain EG7651 is an acrystalliferous derivative of B.t. strain EG1351, which can serve as a host strain for the introduction of toxin plasmids containing insecticidal B.t. toxin genes.

The B.t. strains of this invention are especially useful for their ability to produce high levels of insecticidal crystal protein, e.g., in the overproduction of Cryll, Crylll and CrylV insecticidal B.t. proteins. Recombinant B.t. strains of this invention include strains derived from B.t. strain EG7651, which can be transformed with a plasmid carrying a B . t . toxin gene selected from the group consisting of cr JI genes, crylll genes and cryTV genes. Expression of the selected B.t. toxin gene is desirably accomplished with a vegetative phase promoter, such as a cry III-type toxin gene promoter.

The invention also extends to insecticide compositions comprising the B.t. strains of this invention, insecticidal protein produced by such B.t. strains and an agriculturally acceptable carrier, and to the method of using such insecticidal compositions for insect control on plants.

Brief Description of the Drawings

Figure 1 is a photograph of an ethidium bromide stained agarose gel containing size fractionated resident plasmids for four B.t. strains: B.t. tenebrionis DSM2803 (lane 1), B.t. strain EG7651 (lane 2) , B.t. strain EG1351 (lane 3) , and B.t. strain EG2158 (lane 4) . The numbers on the right side of the agarose gel indicate the approximate sizes, in megadaltons (MDa) , of the plasmids of B.t. strain EG2158; the number on the left side of the gel indicates the 125 MDa plasmid present in B.t. strain EG1351 but absent from the other B.t. strains shown. Figure 2 shows a circular structural map of recombinant plasmid pEG600, a 15.0 kb plasmid containing the cryIIIB2 toxin gene, which was used to transform B.t. strain EG7651 and to produce CryIIIB2 crystal protein. Plasmid pEG600 contains the cryIIIB2 gene (shaded arrow) and is functional in B.t. since it contains the B.t. replication origin region oriβO (shaded segment) . Other components of pEG600 are an antibiotic resistance marker gene for chloramphenicol acetyltransferase (cat, solid arrow) , the E. coli replicon pTZ19u (open segment) , and a cryptic crystal protein gene cryX (open arrow) . Restriction endonuclease cleavage sites are also shown.

Figure 3 shows a circular structural map of recombinant plasmid pEG333, a 12.7 kb plasmid containing the cryllA toxin gene, which was used to transform B.t. strain EG7651 and to produce CryllA crystal protein. Plasmid pEG333 contains the cryllA gene (shaded arrow) , a cryIIIB2 promoter (PcryJJJB2, open arrow) , which directs

-5-

SUBSITTUTE SHEET (RULE 26) transcription of the cryllA gene, and the B.t. replication origin ori43 (shaded box) . Other components of pEG333 are a cat antibiotic resistance marker gene (solid arrow) and the E. coli replicon pTZ19u (open segment) . Restriction endonuclease cleavage sites are also shown.

Microorganism Deposits

To assure the availability of materials to those interested members of the public upon issuance of a patent on the present application, deposits of the following microorganisms were made prior to the filing of present application with the ARS Patent Collection, Agricultural Research Service Culture Collection, Northern Regional Research Laboratory (NRRL) , 1815 North University Street, Peoria, Illinois 61604:

Bacterial NRRL Accession Date of

Strain Number Deposit B. thuringiensis EG1351 NRRL B-21003 09/29/92

B . thuringiensis EG7651 NRRL B-21004 09/29/92

B. thuringiensis EG2158 NRRL B-18213 04/29/87

These microorganism deposits were made under the provisions of the "Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure". All restrictions on the availability to the public of these deposited microorganisms will be irrevocably removed upon issuance of a United States patent based on this application. Description of the Preferred Embodiments

B.t. strain EG1351 is a novel strain isolate that produces the coleopteran-toxic crystal protein conventionally designated as CrylllA. B.t. strain EG1351 is asporogenous, i.e., it does not produce spores during its growth cycle when crystal protein is being produced.

One characteristic of B.t. strain EG1351 that differentiates it from other known coleopteran-toxic B.t. strains is its ability to produce insecticidal crystal protein in high yields, a characteristic that is described as "overproduction" in this disclosure. When tested against other closely related coleopteran-toxic B.t. strains, B.t. strain EG1351 can produce ten times, or more, the amount of CrylllA crystal protein as produced by such other strains.

Most other B.t. strains reported as producing increased amounts of crystal protein, e.g., B.t. tenebrionis DSM5480 in PCT International Patent Application Publication No. WO 91/07481, are artificially-induced mutants created via chemical or electromagnetic radiation treatment of the precursor strain. B.t. strain EG1351 was discovered as a naturally-occurring isolate and is further characterized by producing significantly more crystal protein than the prior art mutant B.t. strains. The reason for this overproduction characteristic in B.t. strain EG1351 is presently unknown.

B.t. strain EG7651 is also a naturally-occurring isolate, being discovered in a cell culture of B.t. strain EG1351. B.t. strain EG7651 is similar in most respects to B.t. strain EG1351 except that it is acrystalliferous, i.e., it produces no crystal protein. The value of B.t. strain EG7651 lies in its ability to function as a host strain for one or more toxin genes harbored on a plasmid, or plasmids, that may be introduced into this host strain. These derivative strains of B.t. strain EG7651 are capable of overproducing insecticidal proteins encoded by the toxin gene(s) that reside on the introduced plasmid(s) .

The crystal protein overproducing derivatives of B.t. strain EG7651 may be produced by conventional conjugation techniques, i.e., through the introduction of the desired toxin plasmid from a donor B.t. strain. Alternatively, the overproducing derivatives of B.t. strain EG7651 may be constructed via conventional recombinant DNA techniques, via the use of recombinant plasmids introduced into B.t. strain EG7651.

The B.t. strain EG7651 derivatives may be used to overproduce many types of B.t. crystal proteins and are not limited to production of CrylllA (made by B.t. strain EG1351) or Crylll-type proteins, e.g., CrylllBl, CryIIIB2, CryIIIB3. Such derivatives of B.t. strain

EG7651 are especially useful for overproducing Cryll-type proteins, e.g., CryllA and CryllB (which are insecticidal to lepidopteran insects) , and CryIV-type proteins (which are insecticidal to dipteran insects) . The overproduction of crystal protein in B.t. strain EG7651 derivatives is best accomplished by linking the selected B.t. toxin genes with a vegetative phase promoter, to effect transcription of the gene from such promoters. Particularly preferred vegetative promoters are the cry III-type promoters, e.g., crylllA promoters, crylllBl promoters, cryIIIB2 promoters and cryIIIB3 promoters. The B.t. strains described in this disclosure may be cultured using conventional growth media and standard B.t. fermentation techniques. The B.t. strains of this invention may be fermented, as described in the Examples, until the B.t. cell culture reaches the stage of its growth cycle when crystal protein is formed. The B.t. strains of this invention, e.g., B.t. strain EG1351 and its crystalliferous derivatives of B.t. strain EG7651, are asporogeneous, so that crystal protein is typically formed during fermentation in the vegetative phase of their growth cycle. After crystal protein has been formed, the B.t. fermentation culture is then typically harvested by centrifugation, filtration or the like to separate the fermentation culture solids, including crystal protein, from the aqueous broth portion of the culture.

It should be under stood that references to "fermentation cultures" of the B.t. strains of this invention are intended to cover B.t. strains in which the cell culture has reached the growth stage where crystal protein is actually produced, whether such point is during the vegetative phase, e.g., for asporogenous B.t. strains and some sporogenous B.t. strains, or whether such point is during sporulation when both crystal protein and spores are formed.

The separated fermentation culture solids are primarily crystal protein and, if any, B.t. spores, along with some cell debris, some intact B.t. cells, and residual fermentation medium solids. If desired, the crystal protein could be separated from the other recovered solids via conventional solids-solids separation techniques, e.g., sucrose density gradient fractionation. Af er separation and recovery of the fermentation culture solids, these crystal protein-containing solids are normally dried by conventional methods, e.g., spray drying, to yield a dry, free-flowing powder that is suitable for use in insecticide formulations.

The formulations or compositions of this invention containing the insecticidal B.t. strains, typically as dried fermentation culture solids with the crystal protein serving as the active component, are applied at an insecticidally effective amount. The insecticidally effective amount of any particular B.t. bioinsecticide may be approximated by laboratory insect bioassay or greenhouse studies but will also depend on such factors as, for example, the specific 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. The insecticide compositions are made by formulating the insecticidally active B.t. 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 insecticidal B.t. strain fermentation culture solids and one or more solid or liquid adjuvants are prepared in known ways, e.g., by homogeneously mixing, blending and/or grinding the insecticidally active B.t. strain component with suitable adjuvants using conventional formulation techniques.

The insecticide compositions of this invention are applied to the environment of the target 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.

The present invention will now be described in more detail with reference to the following specific, non- limiting examples. The examples relate to work that was actually carried out, using techniques generally known in the art and using commercially available equipment.

Example 1

■B.t. Strain EG1351

B . t . strain EG1351 was isolated as a spontaneous variant of B.t. var. morrisoni strain EG2158, a wild-type coleopteran-toxic isolate which has previously been described by Donovan et al . in U.S. Patent No. 5,024,837. U.S. Patent No. 5,024,837 is hereby incorporated by reference into this disclosure, for its teachings relating to B.t. strain EG2158.

B.t. strain EG1351, like B.t. strain EG2158, is serovar morrisoni . Microscopic examination of cell cultures of this novel B . t . strain indicate that it is asporogenous (sporulation deficient) , unlike the parent B.t. strain EG2158 which is sporogenous . The sporulation frequency of B.t. strain EG1351 is approximately lxlO^-7 lower than that of its progenitor, B.t. strain EG2158. B.t. strains in general may be characterized visually by fractionating their resident plasmids according to size by agarose gel electrophoresis, using the well-known Eckhardt gel procedure described by Gonzalez et al. , Plasmid (1981) 5:351-365. This procedure involves lysing B.t. cells with lysozyme and sodium dodecyl sulfate, 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 .

The photograph of the ethidium bromide stained agarose gel in Figure 1 depicts the plasmid arrays for four B.t. strains: B.t. tenebrionis DSM2803 (the first reported coleopteran-toxic B.t. strain) identified as B.t.t. in lane 1; B.t. strain EG7651 (the acrystalliferous derivative of B.t. strain EG1351) in lane 2; B . t . strain EG1351 in lane 3; and B.t. strain EG2158 in lane 4. B.t. strain EG1351 exhibits a different plasmid array from B.t. strain EG2158, its progenitor strain. Figure 1 shows that B.t. strain EG2158 contains native plasmids of about 150, 105, 88, 72 and 35 megadaltons

-12-

SUBSTTTUTE SHEET (RULE 26) (MDa) in size. B.t. strain EG1351, on the other hand, lacks the 150 and 35 MDa plasmids of B.t. strain EG2158 but contains a new plasmid of about 125 MDa.

In both strains, the crylllA gene which makes the coleopteran-toxic CrylllA crystal protein appears to reside on the 88-MDa plasmid. The CrylllA crystal protein made by both of these strains is insecticidal against Colorado potato beetle larvae.

As shown in Figure 1, both B.t. strain EG2158 and B.t. strain EG1351, are readily distinguishable from B.t. tenebrionis on the basis of their plasmid arrays.

Example 2

B . t. Strain EG7651

B.t. strain EG7651 was isolated as a spontaneous acrystalliferous derivative of B.t. strain EG1351. B.t. strain EG1351, when grown up on nutrient media, e.g., NSM (lx nutrient broth, ImM MgCl₂, 0.7 mM CaCl2, 0.05 mM MnCl2) plates, has cell colonies that have an opaque appearance after 3-4 days of growth at a temperature of o

30 C. B.t. strain EG7651 was isolated as a translucent colony from NSM plates containing cell colonies of B.t. strain EG1351. B.t. strain EG7651 does not produce CrylllA crystal protein,- microscope examination of cell cultures of this

B.t. strain shows no evidence of crystals of any type being present.

In Figure 1, the plasmid array of B.t. strain EG7651 is essentially identical to that of B.t. strain EG1351, except that the 88 MDa plasmid band for B.t. strain

EG7651 is greatly reduced in intensity. Example 3 Crystal Protein Overproduction by B . t . Strain EG1351

B.t. strain EG1351 was evaluated for its crystal protein (CrylllA) production against two other coleopteran-toxic B.t. strains, B.t. tenebrionis DSM2803 and B.t. strain EG2158.

The three B.t. strains were grown under similar conditions, in NSM medium (see Example 2) . Ten milliliter (ml) NSM broth cultures of each of the B.t. o strains were grown for four days at a temperature of 30 C, with shaking. At this point, the cultures of B.t. tenebrionis DSM2803 and B.t. strain EG2158 were fully sporulated and lysed. The culture of the sporulation deficient B.t. strain EG1351 were passed through a French pressure cell press (SLM Instruments) to insure cellular lysis, thereby releasing the crystal protein.

To assess the levels of CrylllA crystal protein, aliquots of each of the lysed samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using conventional procedures. Quantitative estimates of CrylllA protein production were determined using purified CrylllA crystal protein as a standard and scanning the applicable bands in the resultant stained gel with a Molecular Dynamics Model 300A computing densitometer. Protein yields were expressed as micrograms (μg) of CrylllA protein per ml of NSM broth culture.

Results are summarized in the following table, and these data confirm that B.t. strain EG1351 produces significantly more CrylllA protein than does B.t. tenebrionis DSM2803 or B.t. strain EG2158: CrylllA Protein Yield

B.t. Strain Ml/ml EG1351 259 ± 1 DSM2803 12 ± 1 EG2158 26 + 1

Example 4

CryIIIB2 Crystal Protein Overproduc ion

Utilising B . t . Strain EG7651

B.t. strain EG7651, the acrystalliferous derivative of B.t. strain EG1351, is useful as a host strain for the overproduction of crystal proteins other than CrylllA (the crystal protein produced by B.t. strain EG1351) .

As a demonstration of the utility of B.t. strain EG7651 as a host strain for the production of other Crylll-type coleopteran-toxic proteins, a recombinant plasmid containing the cryIIIB2 gene was introduced into B.t. strain EG7651 via conventional electroporation techniques . The cryIIIB2 gene is described by Donovan et al. in U.S. Patent No. 5,187,091 and in PCT International Patent Application Publication No. WO 91/14778 published October 3, 1991 and was used in the construction of the recombinant plasmid.

The recombinant plasmid, designated pEG600 and shown as a circular structural map in Figure 2, was constructed using recombinant DNA techniques familiar to those skilled in the art. Construction of pEG600 was accomplished using plasmid shuttle vector pEG853, described by Baum in PCT International Patent Application Publication No. WO 91/18102 published November 28, 1991. The resultant recombinant strain obtained by incorporating recombinant plasmid pEG600, containing the

^■15-

SUBSTTTUTE SHEET (RULE 26) cryIIIB2 gene, into B.t. strain EG7651 was designated as B. t. strain EG1371.

The production of CryIIIB2 protein by B.t. strain EG1371 was evaluated using the same procedures described in Example 3. As a basis for comparison, B.t. strain EG4961 was included in this evaluation. B.t. strain EG4961 is the wild type strain from which the cryIIIB2 gene was isolated by Donovan et al . and is also described in PCT International Patent Application Publication No. WO 91/14778.

The results of this comparative analysis of B.t. strain EG1371 (derived from B.t. strain EG7651) and the wild type B.t. strain EG4961, are summarized below. These data demonstrate that CryIIIB2 protein production by B.t. strain EG1371 greatly exceeds the amount produced by B.t. strain EG4961:

CryIIIB2 Protein Yield

B.t. Strain μl/mj EG1371 119 ± 4 EG4961 13 ± 5

Example 5

CryllA Crystal Protein Overproduction

Utilizing B.t. Strain EG7651

B.t. strain EG7651 is also useful as a host strain for the overproduction of crystal proteins that are not coleopteran-toxic Crylll-type crystal proteins. In this example, B.t. strain EG7651 was employed to demonstrate the overproduction of CryllA crystal protein, a well known lepidopteran- and dipteran-toxic B.t. protein. A recombinant plasmid containing the cryllA gene was introduced into B.t. strain EG7651 via electroporation.

The recombinant plasmid, designated pEG333 and shown as a circular structural map in Figure 3, was constructed using recombinant DNA techniques familiar to those skilled in the art. Construction of pEG333 was accomplished using plasmid pEG862, described by Baum in PCT International Patent Application Publication No. WO 91/18102 published November 28, 1991. As is shown in Figure 3, a cryJJJB2 gene promoter region was inserted upstream of the cryllA operon so that the cryIIIB2 promoter directs transcription of the cryllA gene in pEG333.

The resultant recombinant B.t. strain obtained by incorporating recombinant plasmid pEG333, containing the cryJJA gene, into B.t. strain EG7651 was designated as B.t. strain EG7803.

The production of CryllA protein by B.t. strain EG7803 was evaluated using the same procedures described in Example 3. As a basis for comparison, B.t. strain HD- 263 was included in this evaluation. B.t. strain HD-263 is the wild type strain from which the cryllA gene was isolated by Donovan and is described in PCT International Patent Application Publication No. WO 88/08034 published October 20, 1988, which also describes the isolation of the cry II A gene.

The results of this comparative analysis of B.t. strain EG7803 (derived from B.t. strain EG7651) and the wild type B.t. strain HD-263 are summarized below. The data demonstrate that CryllA protein production by B.t. strain EG7803 greatly exceeds the amount produced by B.t. strain HD-263 :

•17-

SUBSTΓΓUTE SHEET (RULE 26) CryllA Protein Yield

B.t, Strain Ml/ml

EG7803 97 ± 15 HD-263 9 + 6

Example 6

Insect Bioassay of Overproduced

Crystal Proteins

The crystal proteins produced by the B.t. strains described in the previous examples were subjected to insect bioassay to evaluate their efficacy. The CrylllA and CryIIIB2 proteins were evaluated against a coleopteran insect, Colorado potato beetle larvae (Leptinotarsa decemlineata) , and the CryllA protein was evaluated against a lepidopteran insect, tobacco budworm larvae (Heliothis virescens) .

The B.t. strains were grown in a liquid growth medium for several days at a temperature of 30 C. After cell lysis had occurred, or was mechanically induced in the case of the asporogenous B.t. strains, broth samples were utilized for insect bioassay. The amounts of protein in each of the samples were quantified using standard SDS-PAGE techniques.

Activity against the insects larvae was determined by topically applying 50μl of serially diluted samples to 1.5 ml of an agar base artificial diet in a plastic feeding well (175 mm surface) . One larva was placed in each well and scored for mortality after seven days. For each sample, P C50 values (the protein concentrations in nanoliters per cup to achieve 50% mortality of the insects tested) were determined by probit analysis using an 8-dose testing procedure with 32 larvae per dose. Bioassay results are summarized below for each of the B.t. strains tested and are shown as PLC50 values, with 95% confidence intervals shown in parentheses. The protein yields obtained during production of these B . t . strains for this bioassay evaluation are also included with the tabulated data and are expressed as micrograms of protein per ml of liquid broth culture.

Protein

Yield

B.t. Strain Protein PLC502 (μg/ml)

EG1351 CrylllA 29 (22-46) 905

EG2158 CrylllA 31 (22-41) 203

DSM2803 CrylllA 44 (31-57) 217

EG1371 CryIIIB2 160 (123-198) 825

EG4961 CryIIIB2 66 (49-86) 123

EG7803 CryllA 31 (25-39) 748

HD-263 CryllA¹ 15 (13-18) 76³

Purified CryllA crystals were used since B.t. stain HD-263 also produces Cryl proteins.

Crylll proteins tested against Colorado potato beetle; CryllA protein tested against tobacco budworm.

³ Yield excludes Cryl protein.

These results confirm that the overproducing B.t. strains of this invention, B.t. strains EG1351, EG1371 and EG7803, each produce significantly large yields of

-19-

SUBSTΓΓUTE SHEET (RULE 26) their respective crystal proteins, and these proteins have demonstrated insecticidal activity.

•20-

SUBSTΓΓUTE SHEET (RULE 26)

Claims

We Claim:

1. A biologically pure culture of a Bacillus thuringiensis bacterium deposited with the NRRL having accession number NRRL B-21003 and being designated as strain EG1351, or derivative strains thereof that overproduce insecticidal proteins.

2. .An insecticide composition comprising the bacterium of claim 1, an insecticidal protein produced by such bacterium, and an agriculturally acceptable carrier.

3. The insecticide composition of claim 2 wherein the insecticidal protein is coleopteran-toxic.

4. A method of controlling insects which comprises applying to a host plant for such insects an insecticidally effective amount of the insecticide composition of claim 2 or 3.

5. A biologically pure culture of a Bacillus thuringiensis bacterium deposited with the NRRL having accession number NRRL B-21004 and being designated as strain EG7651, or derivative strains thereof that overproduce insecticidal proteins encoded by one or more toxin genes on plasmids harbored by such derivative strains.

6. The bacterium of claim 5 containing a plasmid harboring an expressible B.t. toxin gene which produces a protein toxic to insects.

7. The bacterium of claim 6 wherein the B.t. toxin gene is selected from the group consisting of cry II genes, cry III genes and cryJV genes.

8. The bacterium of claim 6 wherein the B.t. toxin gene has a vegetative phase promoter.

9. The bacterium of claim 8 wherein the vegetative phase promoter is a cry III-type toxin gene promoter.

10. The bacterium of claim 9 wherein the crylll-type gene promoter is selected from the group consisting of cryJTJA promoters, crylllBl promoters, cryIIIB2 promoters and cryl I IB 3 promoters .

11. An insecticide composition comprising the bacterium of claim 6, 7, 8, 9 or 10, an insecticidal protein produced by such bacterium, and an agriculturally acceptable carrier.

12. A method of controlling insects which comprises applying to a host plant for such insects an insecticidally effective amount of the insecticide composition of claim 11.