WO1990006999A1

WO1990006999A1 - New strain of bacillus thuringiensis

Info

Publication number: WO1990006999A1
Application number: PCT/EP1989/001539
Authority: WO
Inventors: Marnix Peferoen; Bart Lambert; Henk Joos; Jacques Mahillon
Original assignee: E.I. Du Pont De Nemours And Company
Priority date: 1988-12-12
Filing date: 1989-12-11
Publication date: 1990-06-28
Also published as: AU635728B2; AU4949090A; KR910700343A; EP0447493A1; JPH05500151A

Abstract

A novel Bacillus thuringiensis strain which produces crystal proteins during sporulation that are toxic to Lepidoptera. The crystal proteins comprise 130 kDa and 135 kDa protoxins which can yield 60 and 63 kDa toxins, respectively, as trypsin-digestion products. The strain, itself, or its crystals or its mixtures of protoxins or toxins can be used as the active ingredient in an insecticidal composition for combatting Lepidoptera.

Description

NEW STRAIN OF BACILLUS THURINGIENSIS

Background of the Invention

This invention relates to a new strain of Bacillus thurinqiensis (the "BtPGSI387 strain") which produces crystallized proteins (the "BtPGSI387 crystal proteins") which are packaged in crystals (the "BtPGSI387 crystals") during sporulation. The BtPGSI387 strain was deposited under the provisions of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen ("DSM") , Mascheroder Weg IB, Braunschweig, Federal Republic of Germany, under accession number 4783 on August 29, 1988.

This invention also relates to an insecticide composition that is active against Lepidoptera, particularly Noctuidae such as Spodoptera littoralis and that comprises the BtPGSI387 strain, as such, or preferably the BtPGSI387 crystals or the BtPGSI387 crystal proteins or the active component(s) thereof as an active ingredient. This invention further relates to a transformed B. thurinqiensis ("Bt") strain obtained by electroporation of the BtPGSI387 strain with a vector carrying all or part of a gene which comes from another Bt strain and which encodes a foreign Bt toxin or protoxin having insecticidal activity against Lepidoptera, Coleoptera and/or Diptera.

B. thurinqiensis ("Bt") is a gram-positive bacterium which produces endogenous crystals upon sporulation. The crystals are composed of proteins which are specifically toxic against insect larvae. Three different Bt pathotypes have been described: pathotype A that is active against Lepidoptera, e.g., caterpillars; pathotype B that is active against certain Diptera, e.g., mosquitos and black flies; and pathotype C that is active against Coleoptera, e.g., beetles (Ellar et al, 1986). That conventional submerged fermentation techniques can be used to produce Bt spores on a large scale has made this bacterium commercially attractive as a source of insecticidal compositions.

suirnnary of the invention

In accordance with this invention, a new B_. thurinqiensis strain of pathotype A, i.e., the BtPGSI387 strain, is provided. The BtPGSI387 crystal proteins, as well as BtPGSI387 protoxins and toxins, produced by this strain during sporulation, possess insecticidal activity against Lepidoptera in general, especially against Noctuidae such as Spodoptera sp. , Aqrotis sp. and Heliothis sp., Pyralidae such as Ostrinia nubilalis and Diatraea randiosella. and Gelechiidae such as Manduca sexta. Phtorimaea operculella and Pectinophora ossypiella. The BtPGSI387 crystals and/or BtPGSI387 crystal proteins are particularly useful against the cotton leafworm, Spodoptera littoralis_f and the tobacco budworm, Heliothis virescens. which are major pests of economically important crops.

Also in accordance with this invention, the BtPGSI387 strain is transformed by electroporation with a foreign DNA sequence encoding a Bt protoxin or toxin.

Detailed Description of the Invention

In accordance with this invention, the BtPGSI387 strain has been deposited at the DSM under accession number 4783. This strain produces crystals which are a mixture of two crystal proteins: a 130 kDa protein (the "first BtPGSI387 protoxin") that is particularly toxic to Manduca sp. and

Heliothis sp. ; and a 135 kDa protein (the "second BtPGSI387 protoxin") that is particularly toxic to Spodoptera sp. Trypsin digestion of the first and second BtPGSI387 protoxins produces, respectively: a 60 kDa protein (the "first BtPGSI387 toxin") and a 63 kDa protein (the "second BtPGSI387 toxin") .

The BtPGSI387 strain, its crystals, its protoxins and/or its toxins can be used as the active ingredient in an insecticide composition used to control insect pests belonging to the order of Lepidoptera. For example, the BtPGSI387 crystals can be isolated from sporulated cultures of the BtPGSI387 strain (Mahillon and Delcour, 1984) , and then, the respective protoxins and mixtures thereof, can be isolated from these crystals according to the method of Hofte et al (1986).

The BtPGSI387 strain can be transformed with one or more foreign Bt genes, such as the bt2 gene (U.S. patent application 821,582, filed January 22, 1986, an European patent application 86/300291.1 which are incorporated herein by reference) or another bt gene coding for a protoxin or toxin active against other Lepidoptera, and/or the bt!3 gene (European patent application 88/402115.5 which is also incorporated herein by reference) or another bt gene such as the btPGSI208 gene or btPGSI245 gene (European patent application 89/400428.2 which is also incorporated herein by reference) coding for a toxin active against Coleoptera. Thereby, a transformed Bt strain can be produced which is useful for combatting an even greater variety of insect pests, e.g., additional Lepidoptera and/or Coleoptera. Transformation of the BtPGSI387 strain with a foreign Bt gene, incorporated in a conventional cloning vector, can be carried out in a well known manner, preferably using conventional electroporation techniques (Chassy et al, 1988).

The BtPGSI387 strain can be fermented by conventional methods (Dulmage, 1981) to provide high yields of cells. Under appropriate conditions which are well understood (Dulmage, 1981) , the BtPGSI387 strain sporulates within 24 hours to provide the BtPGSI387 crystal proteins in high yields.

An insecticide composition of this invention can be formulated in a conventional manner using the BtPGSI387 strain or preferably its crystals, crystal proteins, protoxins and/or toxins, together with suitable carriers, diluents, emulsifiers and/or dispersants. This insecticide composition can be formulated as a wettable powder, pellets, granules or a dust or as a liquid formulation with aqueous or non-aqueous solvents as a foam, gel, suspension, concentrate, etc. The concentration of the BtPGSI387 strain, crystals, crystal proteins, protoxins or toxins in such a composition will depend upon the nature of the formulation and its intended mode of use. Generally, an insecticide composition of this invention can be used to protect a cotton field for 2 to 4 weeks against Lepidoptera, with each application of the composition. For more extended protection (e.g., for the whole growing season) , additional amounts of the composition would have to be applied periodically.

It is preferred that a composition of this invention for combatting insects comprise an insecticidal amount of the BtPGSI387 crystals or preferably a mixture of the first and second BtPGSI387 protoxins, particularly a mixture of the first and second BtPGSI387 protoxins of the BtPGSI387 crystals. A method for controlling Lepidoptera in accordance with this invention preferably comprises applying, to the locus (area) to be protected, an insecticidal amount of the BtPGSI387 crystals or preferably their mixture of the first and second BtPGSI387 protoxins.

In use, the BtPGSI387 crystals or a mixture of the first and second BtPGSI387 protoxins or toxins, or the isolated first or second BtPGSI387 protoxin or toxin or intact cells of the BtPGSI387 strain containing the first and second BtPGSI387 protoxins can be applied to the locus to be protected. The locus to be protected can include, for example, the habitat of the insect pests or growing vegetation or an area where vegetation is to be grown. To obtain the mixture of the first and second BtPGSI387 protoxins or toxins of the BtPGSI387 crystals or the individual first or second BtPGSI387 protoxin or toxin, cells of the BtPGSI387 strain can be grown in a conventional manner on a suitable culture medium and then lysed using conventional means such as enzymatic degradation or detergents or the like. The mixture of protoxins or the individual protoxins can then be separated and purified by standard techniques such as chromatography, extraction, electrophoresis, or the like. The mixture of toxins or the individual toxins can then be obtained by trypsin digestion of their respective protoxin mixture or individual protoxins. Alternatively, the BtPGSI387 cells can be harvested and then applied intact, either alive or dead, preferably dried, to the locus to be protected. In this regard, it is preferred that a purified BtPGSI387 strain (either alive or dead) be used, particularly a cell mass that is 90.0 to 99.9% BtPGSI387 strain.

BtPGSI387 crystals, a mixture of the first and second BtPGSI387 protoxins or toxins, individual BtPGSI387 protoxins or toxins, and/or the harvested cells of the protoxin-containing BtPGSI387 strain can be employed in an insecticidal composition. Such an insecticidal composition can be formulated in a variety of ways, using any number of conventional additives, wet or dry, depending upon the particular use. Additives can include wetting agents, detergents, stabilizers, adhering agents, spreading agents and extenders. Examples of compositions include pastes, dusting powders, wettable powders, granules, baits and aerosol compositions. Other Bt protoxins and toxins, insecticides, as well as fungicides, biocides, herbicides, and fertilizers, can also be employed along with the cells of the BtPGSI387 strain and/or the BtPGSI387 protoxins or toxins to provide additional advantages or benefits.

Such insecticidal compositions can be prepared in a conventional manner. The amount of protoxins, toxins and/or cells of the protoxin-containing BtPGSI387 strain employed depends upon a variety of factors, such as the insect pest targeted, the composition used, the type of area to which the composition is to be applied and the prevailing weather conditions. Generally, the concentration of the insecticidal protoxins and/or toxins will be at least about 0.1% of the weight of the formulation to about 100% by weight of the formulation, more often from about 0.15% to about 0.8% weight percent of the formulation.

In practice, some insects can be fed the BtPGSI387 protoxins or toxins or mixtures thereof in the protected area, that is, the area where the protoxins and/or toxins have been applied. Alternatively, some insects can be fed intact and alive cells of the BtPGSI387 strain or transformants thereof, so that the insects ingest some of the BtPGSI387 protoxins and suffer death or damage.

The following Examples illustrate the invention. The figures referred to in the examples are as follows:

Figure 1: SDS-PAGE and immunoblot of trypsinized crystal proteins from the strains: 1) Bt HD-127, 2) BtPGSI387 and 3) BtSl.

Figure 2: a schematic drawing of the pWP37 plasmid of Example 3, showing its restriction sites.

Unless otherwise stated in the Examples, all procedures for making and manipulating recombinant DNA are carried out by the standardized procedures described in Maniatis et al. Molecular Cloning - A laboratory Manual. Cold Spring Harbor Laboratory (1982) .

Example 1: Characterization of the BtPGSI387 strain

The BtPGSI387 strain was isolated by the method of Travers et al (1987) from forest soil sampled in Kumba, Ca eroun, Africa and has been deposited at the Deutsche Sammlung von Mikroorganismen, accession No. 4783 on August 29, 1988.

The strain can be cultivated on conventional standard media, preferably LB medium (tryptone 10 g/1, yeast extract 5 g/1, NaCl 10 g/1 and agar 15 g/1), preferably at 28'C. For long term storage, it is preferred to use LB liquid medium containing 50% glycerol at -70*C or lyophilization. For sporulation, the use of T3 medium (tryptone 3 g/1, tryptose 2 g/1, yeast extract 1.5 g/1, 5 mg MnCl₂, 0.05 M Na₂P0₄ pH 6.8 and 1.5% agar) is preferred for 24 hours at 28*C, followed by storage at 4*C. During its vegetative phase, the BtPGSI387 strain can also grow under facultative anaerobic conditions, but sporulation only occurs under aerobic conditions.

Sterilization of the BtPGSI387 strain occurs by autoclave treatment at 120'C (1 bar pressure) for 20 minutes. Such treatment totally inactivates the spores and the crystalline BtPGSI387 toxins. UV radiation (254 nm) inactivates the spores but not the crystal proteins.

The morphological and biochemical characteristics of the BtPGSI387 strain are as follows. The strain has a flat irregular colony with undulate to lobate margin, opaque and white appearance, the cells of which sporulate within 24 hours in Nutrient agar ("NA" from Difco Laboratories, Detroit, MI, USA) at 28*C while other Bt strains tested sporulate after 3 days under identical conditions. The specific growth rate of BtPGSI387 strain in T3 medium equals that of BtHDl (Bt Kurstaki Dipel, from Abbott Laboratories, Abbott Park, North Chicago, 111., USA) with a typical value of u max=1.79h^_1. However, the exponential growth phase of BtPGSI387 lasts longer in T3 medium, resulting in more biomass than Dipel Bt HD-1 strain. The BtPGSI387 crystal proteins produced during sporulation are packaged in bipyra idal crystals. These properties together with the early initiation of sporulation of the BtPGSI387 strain, make this strain well suited for fermentation.

For the following tests, well known methods were used as described, for example, by Sneath et al (1986). Growth was observed in Nutrient Broth ("NB", Difco) supplemented with 2 and 5% NaCl. No growth was observed in presence of 7% NaCl. The BtPGSI387 strain grew well on NA agar at 20, 28 and 37*C, but not at 4, 10, 50 and 60'C. The strain grew on NB at pH = 5, pH = 6 and pH = 7 and on NB containing 100 units of lysozyme (Sigma Chemical Company, St. Louis, MO, USA) per ml of NB. Growth on NA under anaerobiosis was very weak.

The BtPGSI387 strain forms gram-positive rods (1.7-2.4 x 4.4-7.0 urn) which form ellipsoidal spores centrally. One-day old cells are weakly catalase-positive, but they shown no gelatinase and tryptophan deaminase activity (i.e., they are GEL- and TDA-) with API-20E test strips (API Systems S.A., Montalieu-Vercieu, France). The cells also show no beta-galactosidase, lysine decarboxylase or ornithine decarboxylase activity (i.e., they are ONPG-, LDC- and 0DC-) with API-20E test strips, and they do not use citrate as their sole C-source (i.e. they are CIT-) according to API-20E test strips. Nor do the cells form

H₂S or mdole with the API-20E test strips (i.e., they are H S- and IND-) . The BtPGSI387 strain showed arginine dehydrolase and urease activity (i.e., it is ADH+ and

URE+) with API-20E test strips, it rapidly decomposed casein in skim-milk agar, and it weakly deaminated phenylalanine using the procedures of Sneath et al (1986) . Acid production from different sugars after 24 hours, as shown by API-50CHB test strips (API System SA) , is set forth in Table 1 below.

Table 1: Acid production of the BtPGSI387 strain compared to other known bacilli (+ » acid production; - = no acid produced; w=weak reaction)

Table 1: (continued)

Bt tene= Bacillus thurinqiensis subsp. tenebrionis from

DSM under accession number no. 2803 Bt darm= Bacillus thurinqiensis subsp. darmstadiensis from

Institut fur Landwirtschaftliche Bacteriologie und Garungsbiologie der Eidgenόssische Technische

Hochschύle, Zurich, Switzerland ("LBG") , under accession no. 4447. B.cer= Bacillus cereus from Laboratorium voor

Microbiologie, Ghent, Belgium ("LMG") , under accession no. 2098. B.subt= Bacillus subtilis from Agricultural Research

Culture Collection, Peoria, Illinois, USA, under accession no. NRRL B-237. The sensitivity towards different antibiotics was tested using Oxoid Susceptibility Test Discs on Oxoid Isosensitest agar ("CM 471" of Oxoid Ltd., Basingtoke, Hampshire, England) . The results are given in Table 2 below.

Table 2: Diameter (in mm) of inhibition zones observed after 24 hours on agar seeded with different bacilli.

The enzyme spectra of the BtPGSI387 strain and other bacilli are shown in Table 3 below. The results in Table 3 were obtained by using the extended API-ZYM test strips (API Systems S.A.). Esterase-, peptidase- (API, AP2, AP3, AP4, AP5 and AP6 test strips) and osidase-test strips were inoculated with 50 ul cell suspension (10⁷ cfu/ml) . The results of the osidase reaction were obtained after 4 hours incubation (28^βC) with 25 ul 0.1N NaOH, and the other results were obtained with 25 ul ZYM A and ZYM B reagent (API no. 7048) . Table 3 only shows some of the results. Table 3: The enzyme spectra of the BtPGSI387 strain and other bacilli.

BtSl= Bacillus thurinqiensis from DSM under accession no. 4288

Meaning of Enzyme Spectra in Table 3

0= no reaction

1= very weak reaction (5 nM substrate hydrolysis)

2= weak reaction (10 nM substrate hydrolysis)

3= reaction (20 nM substrate hydrolysis)

4= strong reaction (30 nM substrate hydrolysis)

5= very strong reaction (> 40 nM substrate hydolysis)

The BtPGSI387 crystals were isolated from the BtPGSI387 strain by density gradient centrifugation (Hόfte et al.,1986). Alternatively, the crystals could have been solubilized selectively from the spore crystal mixture in 50mM Na₂C0₃ and 5mM dithiotreitol ("DTT") at pH 10, and this suspension could then have been filtered through a filter with 0.45 urn diameter pore size.

The antigenic properties of the BtPGSl387 crystal proteins are similar to those of the crystal proteins from the well-known Bt HD-127 strain (Hόfte et al, 1988), pathotype A toxin (Fig. 1) , when tested against monoclonal and polyclonal antibodies, and distinct from those of the crystal proteins from the well-known Bt HD-1 (Yamamoto et al, 1983), Bt berliner (Hόfte et al, 1986), Bt tenebrionis and Bt israelensis (Chungjatupornchai et al, 1988) strains. The polyclonal and monoclonal antisera, utilized against the crystal proteins from the Bt strains were raised in rabbits and mice (Hόfte et al, 1988), and the reactions of these antisera were tested in ELISA (Engvall and Pesce, 1978) . The results are summarized in Table 4 below. Table 4: ELISA of solubilized crystal proteins of different B.t.strains, assayed with monoclonal and polyclonal antibodies.

Antibodies

Table 4 (continued)

Antibodies 82.1, 2H7, 54.1, 1B12, 5D11, 4F11 and 9H11 are monoclonal antibodies raised against Bt berliner crystals.

Antibodies 2D11, 4B10 and 4A2 are monoclonal antibodies raised against the purified 63 kDa toxin of Bt HD-127.

Antibody 2A10 is a monoclonal antibody raised against Bt tenebrionis.

Antibody ABt2 is a polyclonal antibody raised against purified Bt2 toxin.

Antibody ABtten is a polyclonal antibody raised against purified Bt tenebrionis toxin.

Antibody ABtl5 is a polyclonal antibody raised against purified Btl5 toxin.

The pattern of the trypsinized crystal proteins from the BtPGSI387 strain was also compared to the patterns of the crystals from Bt strains HD-127 and BtSl as shown in

Fig. 1. The crystal proteins from each Bt strain were trypsin-digested by: dissolving the crystals overnight at

37^*C in 50 mM Na₂C0₃, pH 10, 5 π DTT. Before trypsin was added (1 ug trypsin/25 ug protein) , the pH was adjusted to pH 9 by addition of 0.5 M HCl. The trypsin-digestion revealed the presence of two tryptic fragments of 63 kDa and 60 kDa respectively (Fig. 1) in the BtPGSI387 crystals. Immunoblotting (Peferoen, 1988) with the monoclonal antibody 2H7 (which shows the presence of a type A protein toxic to Manduca sexta and to

Heliothis sp.) and the monoclonal antibody 4B10 (which shows the presence of a type C protein toxic to Spodoptera sp.) demonstrated that the BtPGSI387 crystal proteins are immunologically related to the crystal proteins of Bt

HD-127 and confirmed the presence of both a type A (60 kDa) and a type C (63 kDa) toxin in the BtPGSI387 crystal proteins. The crystal proteins were also analyzed on a

12.5% SDS-PAGE gel (Laemmli, 1970) stained with Coomassie brilliant blue R-250 according to the procedure of Lambert et al (1987) .

Southern blotting analysis of total DNA isolated from HD-127 and BtPGSI387, digested with EcoRI, revealed a different hybridization pattern using a 620bp Clal-Bglll fragment from the btl5 gene (European patent application 89/401499.2) as a probe. This clearly demonstrates that the two strains are different. Example 2: Insecticidal activity of the BtPGSI387 crystals

A preculture of the BtPGSI387 strain was grown in 10 ml LB medium in a 100 ml baffled flask shaken gently (200 rpm) overnight at 28*C. 125 ml of T3 medium was then inoculated with enough of the preculture to provide a culture with an optical density of 12.5. The culture was then grown for 24 h at 28^*C with gently agitation (120 rpm) in a 1 liter baffled flask. After sporulation the spore-crystal mixture was recovered by centrifugation and dried with acetone according to the method of Dulmage (1981) . The spore-crystal mixture was suspended in water and the suspension was coated onto the surface of the artificial diet and allowed to dry for two hours.

The insecticidal activity of the coating containing the BtPGSI387 spore-crystal mixture was evaluated against Lepidoptera larvae. The larvae were placed on the artificial diet coated with the BtPGSI387 spore-crystal mixture and on the artificial diet coated with aqueous suspension containing other Bt spore-crystal mixtures. The LC50 of the BtPGSI387 crystals was significantly lower than the LC50s of the other Bt spore- crystal mixtures, tested. The results are summarized in Table 5 below.

Table 5: Toxicity of spore-crystal mixture from BtPGSI387, Bt HD-127 and Bt HD-1 strains against larvae of Spodoptera littoralis and Heliothis virescens .

Activity on S. littoralis

LC50: ug/cm of spore-crystal mixtures causing 50% mortality

The BtPGSI387 crystal proteins of the BtPGSI387 spore-crystal mixture caused larvae to stop feeding after a few hours and die within a few days, and the death rate was a function of the concentration of crystal proteins on the artificial diet. Upon ingestion by larvae, the crystals dissolved in the alkaline conditions of the insect's midgut and released the respective BtPGSI387 protoxins. These protoxins were proteolytically processed by the insect's midgut proteases to produce the BtPGSI387 toxins. Example 3: Electroporation of the BtPGSI387 strain

In order to enhance the insecticidal spectrum of the strain, it is electroporated, as described below, with plasmid pWP3713. pWP3713 is derived from plas id pWP37 shown in Fig. 2. pWP3713 contains the bt!3 gene (European patent application 88/402115.5) which encodes a crystal protoxin having insecticidal activity against Coleoptera, particularly the Colorado potato beetle. pWP37 is constructed by inserting the EcoRI/Pstl polylinker fragment from pLK37 (Botter an and Zabeau, 1987) into the EcoRI/Pstl site of the EcoRI/bovine alkaline phosphatase ("BAP)-treated pGKV2 plasmid (Van der Vossen et al, 1985). The bt!3 gene is cloned into the Hindlll restriction site of the BAP-treated pWP37 to produce pWP3713.

The Electroporation Procedure:

1. From a fresh culture (on LB plate)of the BtPGI387 strain, inoculate 20 ml TLB (tryptone 2 %; yeast 1 %; NaCl 2%, pH 7.3) in a 100 ml Erlenmeyer flask overnight at 37* C with shaking at 180 rpm.

2. Inoculate 400 ml prewarmed TLB (in a 2 L flask) with 4 ml of the preculture from step 1 and incubate at 37* C with shaking at 180 rpm for 3 h 30 - 4 h.

3. Centrifuge the cells at 10,000 rpm for 10 minutes at 4* C, using a GS3 rotor.

4. Suspend the pellet into 200 ml sterile distilled water and centrifuge as in step 3.

5. Suspend the pellet into 30 ml of sterile distilled water and transfer the cells into a pre-weighted tube and centrifuge in a S34 rotor at 10,000 rpm for 10 minutes at 4* C. 6. Discard the supernatant and recover the cell pellet in a 30 % solution of polyethylene glycol 1000 (Merck Schuchardt, Munich, Federal Republic of Germany) , with a volume (ml) corresponding to 5 times the cell pellet weight (grams) .

7. Mix in an Eppendorf tube 100 ul of bacterial cells from step 6 with 10 ul of DNA solution in water or in Tris lOmM pH 7.5 and EDTA 1 mM; keep the tube on ice until electroporation is carried out.

8. Transfer the cell suspension to an electroporation cuvette (2 mm width) of a BioRad Gene Pulser coupled to a Biorad Pulse Controller (BioRad Chemical Division, 1414 Harbor Way, South Richmond, CA 94804 USA) ; apply a single electric pulse ranging from 1000 to 1400 volts using a capacitance of 25 uF and with the controller set at 400 Ohms.

9. Recover the electroporated cells with 1.9 ml LB medium (dilution 1/20) and transfer to a 15 ml sterile plastic tube.

10. Incubate for 90 min.at 37* C on a rotary shaker.

11. Plate out the transformed (erythromycin-resistant) cells on LB-agar dishes containing erythromycin.

12. Incubate overnight at 37" C.

Using this electroporation procedure, a transformation efficiency between 10² and 10⁴ transformants/ug DNA can be obtained. However, other plasmids, such as pC194 (Mahillon et al, 1989), can also be suitably used in such an electroporation procedure. The BtPGSI387 strain transformed with pWP3713 has insecticidal activity against Lepidoptera, as well as against Coleoptera. The transformed BtPGSI387 strain or mixtures of its protoxins and/or toxins can therefore be used as the active ingredient in an insecticidal composition against Lepidopteran and Coleopteran insect pests. Moreover, the presence of flanking sequences in pWP37, which show homology with chromosomal DNA of the BtPGSI387 strain or with plasmid DNA of the BtPGSI387 strain, allows homologous recombination resulting in a transformed Bt strain having the new gene as a chromosomal insert or as an insert in the host plasmid.

Needless to say, this invention is not limited to the BtPGSI387 strain, deposited at the DSM under accession number 4783. Rather the invention also includes any mutant or variant of the BtPGSI387 strain which produces crystal proteins, protoxins or toxins having substantially the same properties as the BtPGSI387 crystal proteins, protoxins or toxins. In this regard, variants of the BtPGSI387 strain include variants which: 1) produce acid on a glycerol substrate and/or 2) do produce acid on mannose and saccharose substrates. Also in this regard, variants of the BtPGSI387 strain also include variants whose trypsinized crystal protein pattern differs somewhat from that of the BtPGSI387 strain as shown in Fig. 1.

References

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Claims

1. The BtPGSI387 strain, particularly in purifed form, ■quite particularly 90.0 to 99.9% pure cells of the BtPGSI387 strain.

2. A BtPGSI387 crystal or a mixture of the first and second BtPGSI387 protoxins or toxins, particularly a mixture of the first and second BtPGSI387 protoxins of the BtPGSI387 crystal.

3. An insecticidal composition, particularly against Lepidoptera, which comprises an active ingredient selected from the group consisting of the BtPGSI387 strain, the BtPGSI387 crystals or a mixture of the first and second BtPGSI387 protoxins or toxins.

4. A process for controlling an insect pest, especially Lepidoptera, particularly Noctuidae, Gelechiidae and Pyralidae, and quite particularly Spodoptera sp., Heliothis sp., A rotis sp., Ostrinia nubilalis. Diatraea qrandiosella. Phthori aea operculella and Pectinophora qossvpiella. characterized by: contacting the pest with the insecticidal composition of claim 3.

5. A process for transforming the BtPGSI387 strain with a foreign DNA sequence encoding a Bt protoxin or toxin,

I characterized by: electroporating the BtPGSI387 strain with the foreign DNA incorporated in a cloning vector.

6. A transformed BtPGSI387 strain made by the process of claim 5.

7. An insecticidal composition which comprises the transformed BtPGSI387 strain of claim 6 or the crystals or crystal proteins produced by the transformed BtPGSI387 strain.

8. A method of producing the BtPGSI387 crystal or the mixture of the first and second BtPGSI387 protoxins or toxins of claim 2 or the first and/or second BtPGSI387 protoxins or toxins, characterized by the steps of culturing the BtPGSI387 strain and, optionally, then isolating and purifying the BtPGSI387 crystal and, optionally, then isolating and purifying the mixture of the first and second BtPGSI387 protoxins or the first and/or second BtPGSI387 protoxins from the BtPGSI387 crystal and, optionally, then trypsin digesting the first and/or second BtPGSI387 protoxins to form the first and/or second BtPGIS387 toxins.