HRP920198A2 - Mutants or variants of bacillus thuringiensis producing high yields of delta toxin - Google Patents

Description

The field of technology

This invention relates to mutants and variants of Bacillus thuringiensis, which produce a high yield of delta endotoxin for the biological control of insect pests, which is why it finds application in agronomy.

Technical problem defined

It is an object of the present invention to provide mutants and variants of Bacillus thuringiensis that produce high yields of delta endotoxin.

State of the art

Commercial preparations of Bacillus thuringiensis - a are used worldwide for biological control of insect pests. The advantages of these bacteriological insecticides are high selectivity for a limited number of insects and they decompose in nature.

Commercial preparations of Bacillus thuringiensis - a can be applied until harvest without harmful effects.

Bacillus thuringiensis is a rod-shaped, aerobic spore-forming bacterium characterized by the production during the sporulation process of one or more inclusions called parasporal crystals. These crystals consist of high molecular weight proteins, which are called delta endotoxins. Delta endotoxins are active ingredients that are available in commercial preparations of Bacillus thuringiensis - a.

.

Many Bacillus thuringiensis strains with different insect spectrum have been found. They are classified into different subspecies based on their flagellar antigens. Of particular importance is Bacillus thuringiensis subspecies kurstaki subspecies aizawi, which are used to control lepidopteran insect pests. Bacillus thuringiensis subspecies israelensis used to control dipteran pests and Bacillus thuringiensis subspecies tenebrionis used to control coleopteran insect pests.

The first isolation of the toxic coleopteran Bacillus thuringiensis was reported in 1983 (A. Krieg et al., Z. and Ent. 96, 500-508, European Patent Publication EP 0149162 A2).

This isolate, called Bacillus thuringiensis subspecies tenebrionis, was submitted to the German Collection of Microorganisms (German Collection of Microorganisms), where it can be found under the number DSM 2803. Bacillus thuringiensis subspecies tenebrionis was isolated in 1982 from the dead larva of the grain worm, Tenebrio molitor (Tenebrionidae Coleoptera). . This strain produces within each cell one spore and one or more insecticidal parasporal crystals that have a flat plate-like shape, and the length of one side is about 0.8 µm to 1.5 µm. It belongs to serotype H8 a, 8b and pathotype C of Bacillus thuringiensis (Krieg et al, System Appl. Microbiol. 9, 138-141, 1987, US patent 4, 766, 203, 1988). It is only toxic to the larval species of leaf-eating insects (Chrysomelidae), but has no effect on caterpillars (Lepidoptera), mosquitoes (Diptera) or other insects.

Bacillus thuringiensis subspecies tenebrionis proved to be an effective control agent for Colorado potato beetle larvae. After extraction of crystals and spores from Bacillus thuringiensis subspecies tenebrionis or isolated larval crystals, and to a certain extent mature insects of the Colorado potato beetle (Leptinotarsa decemlineata), feeding stops. Larval stages LI - L3 die within 1-3 days (Schnetter et al., in "Fundamental and applied aspects of invertebrate pathology" eds. R.A. Samson et al., Proceedings of the 4th Int. colloquium of Invertebratae Pathology, p. 555.1986).

Recently, Bacillus thuringiensis subspecies tenebrionis has been shown to also produce a second parasporal crystal that is fusiform, spheroidal, or plate-like with the addition of a coleopteran active crystal. (A.M. Huger and A, Krieg, J. Appl. Ent. 108, 490-497, 1989). The activity of the second crystal is not yet known.

Four commercial products of Bacillus thuringiensis subspecies tenebrionis were found to control coleopteran pests, N0V0D0R from Novo Nordisk A/S, TRIDENT from Sandoz and DiTerra from Abbott Laboratories Inc., Foil from Ecogen.

The isolation of another strain of the toxic coleopteran Bacillus thuringiensis was reported in 1986 (Hernstadt et al., Gio/Technology vol. 4, 305-308. 1986 US patent 4, 764, 372, 1988). This strain called Bacillus thuringiensis subspecies san diego M7 was submitted to the Northern Regional Research Laboratory (the Northern Regional Research Laboratory, USA under the number NRRL B-15939- A commercial product based on Bacillus thuringiensis subspecies san diego was discovered by Mycogen Corporation.

Comparative analyzes of Bacillus thuringiensis subspecies .tenebrionis DSM 2831 Bacillus thuringiensis subspecies san diego, NRRL-B including vegetative cell phenotypic characteristics and toxic parasporal crystal characteristics and plasmid DNA analysis nevertheless showed that Bacillus thuringiensis subspecies san diego is apparently identical to the previously isolated strain DSM 283 Bacillus thuringiensis subspecies tenebrionis (Krieg et al., : J.Appl.Ent. 104, 417-424.1987). Rather, both the nucleotide sequences and the deduced amino acid sequences of the active genes of the cleopteran deltaendotoxin are identical in Bacillus thuringiensis subspecies tenebrionis and Bacillus thuringiensis subspecies san diego.

Under the same culture conditions, the above type of crystal was also synthesized by Bacillus thuringiensis subspecies san diego (A.M. Huger and A. Krieg, J. Appl. Ent. 108, 490-497, 1989).

According to H. de Barjac and Frachon (Entomophaga 35(2), 233.240, 1990) the san diego isolate is similar to tenebrion and cannot be considered as a distinct subspecies.

The usefulness of Bacillus thuringiensis strains for the control of coleopteran pests depends on the efficient and economical production of active coleopteran toxins, as well as on the strength of such a product. This, in turn, depends on the amount of delta endotoxin that can be produced by fermentation of coleopteran-active Bacillus thuringiensis strains.

Bacillus thuringiensis has been used for many years in the production of insecticides, but although mutants of B. thuringiensis with an increased yield of delta endotoxin would be advantageous, similar mutants have not yet been described. Mutants that produce higher yields of delta endotoxin will provide more efficient and economical production of Bacillus thuringiensis toxin and the possibility of manufacturing Bacillus thuringiensis with increased potency at a single cost. This in turn would be an advantage to the user as reduced volumes of pesticide formulation must be stored and used for a given area. In addition to this, users will have less material to discard, thus reducing the negative impact on the environment.

Improvements of delta endotoxin products by Bacillus thuringiensis subspecies tenebrionis through mutation have not been previously reported.

One problem related to the use of especially B. thuringiensi subspecies tenebrionis in the control of insect larvae occurs due to the relatively lower strength or strength of the preparations that require the application of relatively large amounts of the preparation on the surfaces to be protected, such as 5.10 l/ha compared to 1 to 2 liters/ha of many other Bacillus thuringiensis products and many other insecticides.

Consequently, there was a need for products with improved strength.

One way to overcome this problem would be in the concentration of the preparation. However, this would add a lot to the cost of production compared to the savings obtained through storage and transportation.

A much more convenient variant would be to create mutants of the existing ones. Bacillus thuringiensis strains that are able to produce a significantly larger amount of delta endotoxin per cell.

Description of the solution to the technical problem

The present invention relates in one aspect to variants and mutants of Bacillus thuringiensis strains that can produce substantially greater amounts of toxins than their parent strains.

In another aspect, the invention relates to such highly productive variants or mutants of Bacillus thuringiensis strains belonging to the subspecies tenebrionis.

Further aspects of the invention relate to the use of such variants or mutants of Bacillus thuringiensis strains for the production of pesticide products and also to such pesticide ingredients (mixtures) containing the active ingredient delta endotoxin obtained by a variant or mutant of the Bacillus thuringiensis strains of this invention.

The invention also relates in one of its aspects to a method of controlling pests by applying the composition according to the invention in an area where there are pests sensitive to the activity of delta endotoxin in question and which should be controlled.

In a further aspect, this invention relates to methods of selection or mutation and selection of Bacillus thuringiensis strains to obtain such strains of Bacillus thuringiensis which are capable of producing substantially greater amounts of delta endotoxin than their parent strain.

DEPOSITION OF MICROORGANISMS

In order to describe the present invention in more detail, a mutant of Bacillus thuringiensis subspecies tenebrionis which produces high amounts of delta endotoxin has been filed with the "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH", Mascheroderweg Ib, D-3300 Braunschweig, Germany, for patent proceedings on the date indicated below.

Since DSM is an international depository bank according to the Budapest Agreement, a permanent deposit is allowed in accordance with law no. 9 of the aforementioned agreement.

Date of deposit: August 10, 1989

Deposit ref.: NB 176-I

DSM specification: DSM 5480

Mutant DSM 5480 was obtained by mutation of Bacillus thuringiensis subspecies tenebrionis strain DSM 5526, which is also known in the Budapest Agreement, as seen from the following:

Date of deposit: September 14, 1989

Deposit ref.: NB 125

DSM specification: DSM 5526

Detailed description of the invention

The present invention relates in its main part to variants or mutants of Bacillus thuringiensis which produce large amounts of active delta endotoxin compared to its parent strain.

In this context, the term "large amounts" means at least twice as much or more.

Both in this main aspect and in more specific aspects of the invention - indicated below - delta endotoxins will have activity directed against the same insect pests as against its parent strain, B. thuringiensis delta endotoxin, such as those used against lepidopterans (mutants from Bacillus thuringiensis subspecies kurstaki and subspecies aizawai), dipterans and (mutants from Bacillus thuringiensis subspecies israelensis) or coleopterans (mutants from B. thuringiensis subspecies tenebrionis).

In one specific embodiment of this aspect of the invention, B. thuringiensis belongs to the subspecies tenebrionis, and the delta endotoxin produced becomes active against cleopterans.

With this in mind, the claimed embodiment of the invention is a variant or mutant B. thuringiensis subspecies tenebrionis capable of producing three times more delta endotoxin.

Further embodiments of the present invention include variants and mutants of B. thuringiensis, subspecies tenebrionis - strains capable of producing a parasporal crystal having a mean edge length of about 2 µm or more.

A further embodiment of the present invention comprises variants or mutants of B. thuringiensis subspecies tenebrionis - strains that show a sporulation frequency of 10 - 100 or even 106 times lower than the sporulation of its parent strain, or strain DSM 2803.

Even more specific is the realization of this invention of the deposit mutant B. thuringiensis subspecies tenebrionis DSM 5430.

During the work on the invention, a mutant of B. thuringiensis subsp. tenebrionis (DSM 5480) with a more than two-fold increase in delta endotoxin production compared to its parent strain (DSM 5526). Phase contrast microscopy, electron microscopy, and transmission electron microscopy of this mutant show that the high productivity of this mutant can be attributed to changes in the regulation of the delta endotoxin product in relation to sporulation resulting in the production of protein crystals that are nearly five times larger than those produced by existing active coleopteran B thuringiensis strains. The close link between crystal formation and sporulation appears to be removed, and the mutant produces large amounts of delta endotoxin prior to sporulation.

In one of its aspects, the invention relates to the use of variant or mutant strains of the invention in a process for obtaining an insecticidal B. thuringiensis product, by which method the variant or mutant bacillus of the B. thuringiensis strain is produced in a suitable culture medium that has sources of carbon, nitrogen and other known components referred for the appropriate period of time after which delta endotoxins recover.

In a further aspect of the invention, the B. thuringiensis delta endotoxic products obtained in the above-mentioned manner are used in pesticide mixtures as an active component.

In such mixtures, the delta endotoxins of this invention can be used either alone or in combination with other biocidal active products.

The invention also relates to such pesticidal mixtures or preparations containing the B. thuringiensis delta endotoxic product of the invention in joint action with agriculturally acceptable diluents or carriers.

The invention also relates to such pesticidal mixtures or preparations containing B. thuringiensis which contain the product B. thuringiensis delta endotoxin, the pesticidal liquid mixture having a potency of at least 15,000 BTTU/G, corresponding to at least 3% w/w coleopteran insecticidal crystal protein or pesticidal a mixture in dry form having a strength of at least 50,000 BTTU/g, corresponding to at least 10% w/w coleopteran insecticidal crystalline protein.

In a specific embodiment, the invention relates to pesticide mixtures produced from DSM 5480 that have at least twice the potency of pesticide mixtures produced from DSM 2803 or other coleopteran active Btt strains.

The compositions of this invention may take any form known in the art of agricultural chemical formulation, such as suspension, spray, aqueous emulsion, spreadable powder, sprayable powder, emulsion concentrate or granules. In addition, it can be found in a suitable form for direct use either as a concentrate or as an initial mixture, which requires mixing with an appropriate amount of water or other solvents before use.

The concentration of insecticidally active B. thuringiensis delta endotoxin in the mixtures of the present invention, when used alone or in combination with another pesticide, as applied to plants, is from 0.5 to about 25% by weight, and especially from 1 to 15% by weight.

In a further aspect, the invention relates to a method of controlling pests, where pesticide compositions (mixtures) according to the previous aspect are applied to the area attacked by the aforementioned pests.

In a specific embodiment, the pest to be controlled belongs to a group containing lepidopterans, dipterans and coleopterans, especially coleopterans such as the Colorado potato beetle.

In a specific embodiment, the pest can be controlled by applying 1 quart of liquid pesticide mixture per acre, or by applying a dry pesticide mixture per acre.

The active B. thuringiensis preparation or mixtures of the invention can be applied directly to the plant, for example by dusting it or spraying it, and this at the time when the pest appears on the plant. The spraying method is mostly used. It is very important to get good pest control in the first larval stages, because this is the time when the plant will suffer the least damage.

In the process of changing B. thuringiensis strains by selecting such mutants that are capable of producing significantly larger amounts of delta endotoxin than their parent strains, the parent strain is:

1) treated with a mutagen

2) mutants treated in this way develop in an environment suitable for the selection of asporogenous and/or oligo-sporogenous strains

3) transparent colonies are selected and grown in a medium that does not turn into a liquid due to heating, and true asporogenous strains disappear when the colonies are heated.

According to the desired realization of this method, the colonies selected in this way are grown in a normal environment and the final selection for strains capable of increased production of delta endotoxin is performed.

In part (1) of the above procedure, the mutagen may be any conventional chemical mutagen, such as N-methyl-N-nitro-N-nitrosoguanidine or ethyl methaneusulfonate, or the parent strain may be treated with electromagnetic radiation, such as y or x rays or ultraviolet radiation.

In part (2), a suitable medium can be a modified nutrient sporulation medium, including phosphates (NSHP medium) as described by Johnson et al. in "Spores IV", eds. P. Gerhardt et al., pp. 248-254, 1975.

In part (3) of the above procedure a suitable medium would be NSMP supplemented with MgCl2 and Gelrite, Kelco.

Other methods for obtaining highly productive variants or mutants of this invention can be imagined as growing the parental strain in a liquid medium, thus selecting spontaneous mutants or variants after covering the culture medium on a gelatin substrate (medium), which is suitable for the selection of asporogenous and/or oligosporogenous mutants .

Other methods of finding highly productive mutants or variants of the invention can be envisioned by using the mass of these mutants directly through centrifugation or using other mass processing methods.

Example 1

Mutant B. thuringiensis subsp. tenebrionis with more than two-fold increased production of delta endotoxin was isolated. Phase contrast microscopy, radar electron microscopy of this mutant shows that the high productivity of this mutant is due to changes in the regulation of delta endotoxin production, which corresponds to sporulation, resulting in the production of protein crystals that are more than five times larger than the crystals produced by existing coleopteran active B. thuringiensis strains. The close correlation between crystal formation and sporulation appears to be removed and the mutant produces high amounts of delta endotoxin prior to sporulation.

Production of high productivity mutants

Spores of B. thuringiensis subspecies tenebrionis, strain DSM 5526 were irradiated with y rays to give a dose of 7 kGy. Irradiated spores were spread on HSMP agar (gelatin) plates (a modified nutrient sporulation medium containing phosphate as described by Johnson et al., in "Spores IV", eds. P. Gerhardt et al., pp. 248-254, 1975 )« Medium suitable for the selection of asporogenous and/or oligosporogenous mutants.

NSMP - agar plates incubated for 2 to 3 days at a temperature of 30°. Air colonies were removed and transferred to NSMP Gerlite plates (NSMP medium supplemented with MgC12" (0.57 g/l) and Gerlite, Kelco (20 g/l)). NSMP Gerlite plates were incubated for one hour at , and then for another day- two at a temperature of .

Mutants that grew well on NSMP Gerlite plates were isolated. In this way, all asporogenous mutants were eliminated since they did not grow after the temperature treatment

.

The selected mutants were grown in the laboratory. whisking bottles that contained commercial medium. The amounts of delta endotoxin thus produced were determined by the immunological methods listed below.

Only mutants capable of producing higher, i.e. significantly higher amounts of delta endotoxin than their parental strain.

The morphology of the selected mutants on solid medium and in liquid medium was examined by phase contrast microscopy (X 2500) and examination (screening), as well as by electron microscopy.

Among the mutants obtained, one DSM 5480 was selected for its ability to produce delta endotoxin.

The amount of delta endotoxin produced by mutant DSM 5480 was compared with DSM 2803, the original isolate of B. thuringiensis subsp. tenebrionis, strain NB176, isolated from Sandoz, B. thuringiens: tenebrionis product of TRIDENT in 1989, strain NB 198, isolated from Sandoz, B. thuringiensis subsp. tenebrionis product of TIRDENT from 1990, B. thuringiensis subvr. San Diego, M-One, 1990. As shown in Table L of Example 2, the production-enhanced mutant of the present invention produces 2 to 3.5 times as much delta endotoxin as the cleopteran active strains of B. thuringiensis available today. find.

Example 2.

In this example delta endotoxin production of B. thuringiensis subspecies tenebrionis, mutant DSM 5480 was compared with delta endotoxin production of B. thuringiensis strains DSM 2803 (original isolate of B. thuringiensis sub. tenebrionis), DSM 5526 (production strains from Novo-Nordisk) and NB 178 and NB 198 (production strains of Sandoz) and B. thuringiensis subvr. san diego, strain NRRL-B15939 and NB 197 (Mycogen production strain) in commercial media. Each of the strains was grown for 17 hours at temp. of 30° on agar slopes, the following mixtures expressed as grams per liter of distilled water.

Peptone, Difco

Beef extract, Difco

Agar, Difco

pH 7.0

5 ml of cell suspension from each strain was then transferred to a 500 ml Erlenmeyer flask. The production medium consisted of the following components in the amounts indicated (expressed in grams per liter of plain water).

Soya flour

Hydrolyzed starch

KH2PO4

K2HPO4

pH 7.0

The bottles with these substances were incubated with the cherry (250 times per minute). After 96 hours of incubation, the culture mixture was examined by immunological methods for delta endotoxic yields.

The amount of endotoxin produced by individual strains was determined by rocket immuno electrophoresis (RIE) and photometric immunoassays (PIA) using antibodies raised for use on purified protein crystals from the group B. thuringiensis subspecies tenebrionis.

400 mg of each culture mixture was weighed. 7 ml of trisodium phosphate buffer (, pH 12) was added to each sample. The suspensions were shaken for one hour to dissolve the delta endotoxin proteins.

The samples were then centrifuged - 3,500 times per minute for about 15 minutes and what was separated was tested for delta endotoxin by rocket immune electrophoresis on antiserum grown on purified protein crystals from B. thuringiensis subvr. tenebrionis. The amounts of delta endotoxin produced by individual strains were relatively determined to standards of known crystal protein content.

The concentration of crystal proteins was also determined by photometric immunoassay. Crystal proteins were dissolved in an alkaline solution. Soluble proteins were precipitated by their antibodies. The ratio of this reaction is determined turbo-dimetrically. The amount of delta endotoxin was relatively determined to a standard with known crystal protein content.

The obtained results are presented in the following tables la and lb. Delta endotoxin production fields are expressed as BTTU/g (units per g of culture mixture, determined by rocker immuno electrophoresis, RIE, or by photometric immuno assay, PIA). The value used for pure B. thuringiensis crystal protein is 500000 BTTU/g. The values indicated in table la are averages of 6-7 independent fermentations, and those in table lb are averages of three independent fermentations.

Table la

Production of delta endotoxin from strains of B. thuringiensis subvr. tenebrionis in shaker bottles

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Table 1b

Production of delta endotoxin from strains of B. thuringiensis subvr. teneterionis in whipping bottles

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From Tables 1a and 1b it appears that DSM 5480 produces more than three times delta endotoxin than the original strain B. thuringiensis sub. tenebrionis, DSM 2803 and B. thuringiensis subv. san diego, strain NRRL B15939 and more than twice the amount of delta endotoxin than the strains currently used to produce commercial products B. thuringiensis subv. tenebrionis.

Phase contrast microscopy, scanning electron microscopy and transmission electron microscopy of B. thuringiensis sub. tenebrionis, mutant DSM 5480 revealed that the protein crystals produced by this mutant were much larger than the corresponding protein crystals produced by B. thuringiensis subsp. tenebrionis, strains DSM 2803, DSM 5526, NB 178 and NB 198 and B. thuringiensis subv. san diego, strains NRRL-B 15939 and NB 197.

A cultural mixture of B. thuringiensis sub. tenebrionis mutant DSM 5430 was tested for activity against Colorado potato beetle larvae. The increased amount of delta endotoxin produced by the mutant DSM 5480, determined by immunological procedures, was reflected in the biological activity against the larvae of the Colorado potato beetle.

Example 3

In this example, sporulation and parasporal crystal formation in B. thuringiensis sub. tenebrionis, strains DSM 2803, DSM 5526, NB 178 and NB 198 and mutant DSM 5480, and B. thuringiensis subsp. san diego, strains NRRL-B 15939 and NB 197 were compared in liquid and solid media.

Each of the strains was grown for two days at a temperature of on agar plates with the following mixture, which was expressed as grams per liter of distilled water.

Peptone, Difco 5g

Beef extract, Difco 3g

Agar, Difco 20g

pH 7.0

Each of the strains was also grown in liquid medium. All strains were grown for 17 hours at a temperature of on agar slopes. 5 ml of cell suspension from each strain was then transferred to 500 ml Erlenmeyer flasks, each containing 100 ml of medium.

The medium consisted of the following components in the quantities indicated (expressed in grams per liter of plain water)

Current environment:

Yeast extract 5g

Tryptone 5g

Glucose lg

KH2PO4 0.6g

pH 7.0

Bottles with mixtures were incubated with shaking (250 times per minute) for 96 hours.

The morphology of the strains in solid and liquid media was examined every day with a phase contrast microscope (x 2500). The number of spores and crystals was counted, and the size of the parasporal crystals was determined. A few selected specimens were also studied by scanning and transmission electron microscopy.

B. thuidngiensis subspecies tenebrionis, strains DSM 2803, DSM 5526, NB 178 and B. thuringiensis sub. san diego, strains NRRL-B 15939 and NB 197 had good sporulation in both environments. Before lysis, each cell contained a sporal and a parasporal crystal. Crystal size was from 0.4 to 0.9-H/µm in length, until lysis. The average size of the protein crystals was 0.6-0.7 µm in length.

Mutant DSM 5430 produced only a few spores (<106 spores/ml) on solid medium and in some liquid medium. Before cell lysis, many cells contained a large protein crystal, but not a single spore. The size of the protein crystals varied from 0.4 to 0.7 µm to 5-0 µm, and the average protein crystal size was 2.2 to 2.3 µm in length.

Ultrastructural analysis of cells from these environments using a transmission electron microscope revealed that the sporulation process in the mutant had begun, but was not completed by the time of lysis. The sporulation process reached different stages in different cells. In cells where the sporulation process reached the second stage (forespore septum formation), protein crystals filled the entire cells.

In the production medium (example 2), the mutant produced a higher number of spores (10 7 -10 8 spores/ml). In this environment, the sporulation frequency of the mutant was 10-100 times lower than in the parental strain.

Thus, the mutant retained its ability to produce normal spores. However, the sporulation frequency of the mutant is highly dependent on the environment.

The size of protein crystals produced by individual strains is shown in Tables IIa and IIb.

Table IIa

Size of protein crystals produced by coleopteran active B. thuringiensis strains that can be found today.

[image]

Table Ilb

Size of protein crystals produced by coleopteran active B. thuringiensis strains that can be found today.

[image]

It is clear from Tables IIa and IIb that mutant DSM 5480 produces much larger protein crystals than any of the coleopteran active strains available today.

Based on the obtained data, it seems that the regulation of delta endotoxin production in relation to sporulation is changed in the mutant.

The mutant appears to produce protein crystals before spore development, thus giving the cells a longer period to produce delta endotoxin, resulting in the production of much larger protein crystals by the time of cell lysis than in the parental strain.

Depending on the available nutrients and the size of the protein crystals in the cells until sporulation, a normal spore will develop before the time of cell lysis.

Example 4

In this example, the highly productive Btt mutant DSM 5480 was used to produce a higher potency product to control Colorado potato beetle larvae.

DSM 5480 was fermented on the production fermentation medium described in Example 2, in an aerated (containing air) production fermentation tank. After 96 hours, the mixture was recovered after continuous centrifugation.

The concentrated cream containing active protein crystals was stabilized with the addition of microbial preservatives and the pH was adjusted to 5.0.

One portion of the concentrated cream was spray-dried and later used to formulate wet powder. The rest of the concentrated cream was used directly for the formulation of liquid floating concentrates (FC).

The wet powder was formulated as in Table III. The formulation of the two FCs is described in Table IV.

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Table IV NOVODOR PC formulations

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When using the value of 500,000 BTTU/g of pure crystalline protein, the content of active crystalline protein in the following formulations is:

Btt crystal protein

[image]

Detergents are chosen from a wide selection of suspending additives and wetting agents commonly used as pesticide products in agriculture.

The anticoagulation agent hydrophilic silica and the internal filler are selected from commonly used inert filters, such as bentonites, inorganic salts or types of loam.

The condoms used in PC were chosen from the group of condoms for food and cosmetics. The pH regulator is an inorganic acid.

Example 5

A single field trial was conducted to demonstrate the biological effect of the highly productive Btt mutant DSM 5480 on the target pest, Colorado potato beetle larvae, in comparison to two commercial products, Trident and M-one. The crop was tomatoes.

The crop was sprayed three times: on July 20, July 27 and August 3 (second generation of larvae).

Products and doses used:

[image]

Mean % control of CPB larvae compared to the untreated control is shown in Table V. Colorado potato beetle pressure was high in the untreated control group: 370 larvae on 20 plants on the first of August and 904 larvae on 20 plants on the eighth of August.

Table V

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These results clearly show that the products created using the high-producing mutant DSM 5480 are very effective in controlling Colorado potato beetle larvae in the field. Crystalline protein produced using a high yielding strain is fully active as 1.5 NOVODOR FC, gives just as good results as Trident at 4 qts and is just as good as M-one at 2 qts.

Claims (39)

1. A mutant or variant of B. thuringiensis characterized by the fact that it is capable of producing large amounts of insecticidal delta endotoxin compared to its parent strain. 2. A mutant or variant according to claim 1, characterized in that the mutant or variant of B. thuringiensis can produce as much or more insecticidal delta endotoxin compared to its parent strain. 3. Mutant or variant according to claim 1 or 2, characterized in that the mutant or variant of B. thuringiensis belongs to pathotype C of B. thuringiensis. 4. A mutant or variant according to claim 1, 2 or 3, characterized in that the B. thuringiensis mutant or variant will have activity directed against the same insect pest species as its parental B. thuringiensis delta endotoxins, such as against lepidopterans (mutants of B thuringiensis subspecies kurstaki and/or subspecies aizawai), dipterans (mutants of B. thuringiensis subspecies israelensis) or coleopterans (mutants of B. thuringiensis subspecies tenebrionis). 5. Mutant or variant according to claims 1, 2, 3 or 4, characterized in that B. thuringiensis belongs to the subspecies tenebrionis, and the delta endotoxin produced is active against coleopterans. 6. Mutant or variant according to claim 5, characterized in that the mutant or variant B. thuringiensis subspecies tenebrionis is capable of producing more than three times delta endotoxin than strain DSM 2803. 7. Mutant or variant according to claim 5 or 6, characterized in that the mutated or variant B. thuringiensis subspecies tenebrionis can produce a parasporal crystal that has a mean edge length twice that of its parent strain. 8. Mutant or variant according to claim 7, characterized in that the mutated or variant B. thuringiensis subspecies tenebrionis strain can produce a parasporal crystal having a mean edge length of 2 µm or more. 9. Mutant or variant according to any of claims 4 to 8, characterized in that the mutant or variant B. tharingiensis subspecies tenebrionis strain shows a sporulation frequency of 10 to 100 or even 106 times lower than the sporulation frequency of the parent strain. 10. Mutant or variant according to claim 9, characterized in that the mutated B. thuringiensis subspecies tenebrionis strain shows a sporulation frequency of 10 to 100 or even 10 times lower than the sporulation frequency of the DSM 2803 strain. 11. Mutant according to claim 1, characterized in that the mutant is a deposit mutant of B. thurigniensis subsp. tenebrionis DSM 5480. 12. The use of a mutated or variant strain according to claim 1 to 11, indicated by the fact that the insecticidal B. thuringiensis product is intended for production, where the mutant or variant B. thuringiensis strain is grown in a suitable culture medium containing sources of carbon, nitrogen and other components for an appropriate period of time, after which the insecticidal product containing delta endotoxins is recovered alone or in combination with cells and/or spores from the culture medium, and this product is acceptable in agriculture. 13. Pesticidal mixture according to claim 12, characterized in that it contains B. thuringiensis endotoxin product as an active component. 14. The mixture according to claim 13, characterized in that the delta endotoxin product is used either alone or in combination with other biocidal active products. 15. A pesticidal mixture or preparation according to claim 13 or 14, characterized in that it comprises the B. thuringiensis delta endotoxin product in a mixture with some available solution or carrier used in agriculture. 16. The pesticidal mixture according to claims 13, 14, or 15, characterized in that it has twice the potency of the pesticidal mixtures of the parent strain. 17. A liquid pesticidal mixture, characterized in that it has a potency of at least 15,000 BTTU/g relative to at least 3% w/w coleopteran insecticidal crystal protein. 18. A dry pesticide mixture, characterized in that it has a potency of at least 50,000 BTTU/g relative to at least 3% w/w coleopteran insecticidal crystal protein. 19. A pesticidal composition, characterized in that produced by DSM 5480 has at least twice the potency of pesticidal mixtures produced by DSM 2803 or other coleopteran active Btt strains. 20. A mixture according to any one of claims 13 to 19, characterized in that the diluent or carrier in the mixture is solid or liquid depending on the surfactant. 21. The pest control method according to claims 13 to 20, characterized in that the pesticidal mixture is applied to one area infected with said pest. 22. The method according to claim 21, characterized in that the pest belongs to the group that includes lepidopterans, dipterans and coleopterans. 23. The method according to claim 22, characterized in that the pest is a coleopteran, especially the Colorado potato beetle. 24. The method according to claim 22 or 23, characterized in that the pest can be controlled by using 1 quarter per morning of the liquid pesticide mixture. 25. The method according to claim 22 or 23, characterized in that the pest can be controlled by applying 0- per acre of dry pesticide composition. 26. The method of mutated B. thuringiensis strains, characterized in that this procedure selects such mutants that are capable of producing larger amounts of delta endotoxin than its parent strains, and the parent strain is: i) treated with a mutagen, ii) mutants treated in this way are grown in an environment suitable for the selection of asporogenous and/or oligosporogenous strains, iii) airy colonies are selected and grown in a medium that does not turn into a liquid when heated, and completely asporogenous strains are eliminated by subjecting the colonies to heat treatment. 27. The method according to claim 26, characterized in that the colonies selected in this way are grown in a normal production medium and the final selection for strains capable of increased production of delta endotoxin is performed. 28. The method according to claim 26 or 27, characterized in that in part i) the mutagen is any suitable chemical mutagen, such as N-methyl-N'-nitro-N-nitrosoguanidine or ethyl methanesulfonate. 29. The method according to claim 26 or 27, characterized in that the parental strain in part i) is treated with appropriate electromagnetic radiation (irradiation), such as y or x radiation or ultra-violet radiation. 30. The method according to claims 25 to 29, characterized in that in part ii) said medium is a modified nutrient medium (medium) that includes phosphate (NSMP medium). 31. The method according to claims 26 to 30, wherein in part (iv) it is seen that said medium is NSMP medium to which MgCl 2 and Gerlite, Kelco have been added. 32. The method according to claims 26-31, indicated by the fact that the mentioned parental strain belongs to the group that includes B. thuringiensis subspecies kurstaki, B. thuringiensis subvr. aizawai. B. thuringiensis subsp. israelensis and B. thuringiensis sub. tenebrionis. 33. The method according to claim 32, characterized in that the said parent strain is B. thuringiensis subsp. tenebrionis strain. 34. The method according to claim 33, characterized in that said parental strain of B. thuringiensis subspecies tenebrionis is strain DSM 5526. 35. The method, indicated by the fact that it selects variants or natural mutants of the B. thuringiens strain that is capable of producing significantly larger amounts of delta endotoxin than its parent strains, and the parent strain is: i) in the culture of the liquid medium, ii) the culture mixture from i) is placed in a medium suitable for the selection of asporogenous and/or oligosporogenous strains, iii) completely asporogenous strains are lost by exposing the colonies to heat treatment. 36. The method according to claim 35 characterized by the fact that the colonies selected in this way are grown in a normal production environment, and the final selection is achieved for strains capable of increased production of delta endotoxin. 37. The method according to claim 35 to 36, characterized in that the medium mentioned in part ii) is a modified sporulation medium containing phosphate (NSMP medium). 38. The method according to claim 35 - 37 characterized by the fact that in part iv) the medium mentioned is NSMP medium supplemented with MgCl 2 and Gerlite, Kelco. 39. The method according to claims 35 to 38, characterized in that said parental strain belongs to the group comprising B. thuringiensis subspecies. kurstaki, B. thuringiensis subspecies aizawai, B. thuringiensis subspecies. israelensis and B. thuringiensis subsp. tenebrionis.