CZ562990A3 - Mutant of Bacillus thuringiensis micro-organism deposited as subsp. tenebrionis DSM 5480, process of its preparation and pesticidal agent containing thereof - Google Patents

Abstract

Mutant or variant (I) of Bacillus thuringiensis capable of producing high amts. of insecticidal delta-endotoxins compared to its parent strain is new. Also claimed is a method of mutating B. thuringiensis strains, and selecting mutants which can produce larger amts. of delta endotoxins than their parent strains, where the parent strain is: (1) treated with a mutagen; (2) the resultant mutants are grown in a medium used for the selection of asporagenous and/or oligosporagenous strains; (3) transulcent colonies are selected and grown in a medium that does not fludise on heating; and (4) truly asporogenous strains are deselected by subjecting the colonies to a heat treatment.

Description

A mutant of Bacillus thuringiensis deposited as subsp. tenebrionis DSM 5480, a process for its preparation and a pesticidal composition containing it

Technical field

The invention relates to a mutant of Bacillus thuringiensis deposited as subsp. tenebrionis DSM 5480, a process for its preparation and a pesticidal composition containing it.

BACKGROUND OF THE INVENTION

Commercial products Bacillus thuringiensis are used worldwide for biological control of harmful insects. The advantages of these bacterial insecticides are their high selectivity for a very limited field of pest control and biological degradation.

Commercial preparations of Bacillus thuringiensis can be applied up to harvest time without any adverse effects.

Bacillus thuringiensis is a rod-shaped aerobic, spore-forming bacterium, uniquely characterized by the production of one or more inclusions in the sporulation process, referred to as parasporal crystals. These crystals consist of high molecular weight proteins, referred to as delta endotoxins. Delta endotoxins are the active ingredient of commercially available Bacillus thur ingi ensi s.

Many Bacillus thuringiensis strains with different host insect spectrum have been identified. They are classified into different subspecies based on their antigens. It is of particular importance

o ε subspecies of Bacillus thuringiensis kurstaki and Bacillus thuringiensis subspecies of Bacillus thuringiensis to control Lepidoptera butterflies, bac illus thurigiensis subspecies israelensis for winged insect control, and Bacillus thuringiensis, tenebrionis subspecies used to control beetles.

The first isolation for the beetles of toxic Bacillus thuringienisis was described in 1983 / A. Krieg et al., Z. and. Ent.

96, pp. 500 to: 508, European Patent Publication No. 0149162 A2).

Isolate, designated as Bacillus thuringiensis subs.p. tenebrionis, is stored in the German Collection of Microorganisrns. under the number 1SM 2803. Bacillus thuringiensis subsp. tenebrionis was isolated in 1982 from the killed pupae of the darkling beetle Tenebrio Mol.it, or / Tenebrionidae, Coleoptera / o The strain produces, in each cell, one spore and one or more inaecticidal parasporal crystals having a flat, platelet-shaped form approximately 0.8 / µm. up to 1.5 µm. It belongs to serotype H8a, 8b and pathotype C of Bacillus thuringiensis / Krieg et al., System. Appl. Microbiol., 9, pp. 138-141, U.S. Pat. No. 4,766,203 (1988).

It is toxic only to certain beetle larvae (Chrysomelidae) with leaf resin, but is ineffective against caterpillars (Bepidoptera), mosquitoes (Diptera) and other insects.

Bacillus thuringiensis subsp. tenebrionis is an effective agent against the larvae of the Colorado potato beetle. After ingestion of crystals and spores of Bacillus thurin-3 giensis, subsp. tenebrionis or isolated crystals by larvae, and to some extent by adults, the Colorado potato beetle (Leptinotarsa decemlineata) stops eating. Larval stage L1 - L3 dies within 3 to 3 days (Schnettsr et al., Fundamental and applied aspects of invertebrate pathology), RA Samson et al., Proceedings of the 4th Int. Colloquium of Invertebrate ^r athology, pp. 555, 1986 /.

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More recently, Bacillus thuringiensis subsp. tenebrionis produces, in addition to the active crystals against beetles, another parasporal crystal having a spindle, spherical or platelet-like shape (A). M. Huger and A. Krieg, J. Appl. Ent., 108, p. 490 to 497 (1989)]. The efficacy of this second crystal has not yet been known.

Four commercial formulations of Bacillus thuringiensis have been developed; subsp. tenebrionis for controlling the beetle; N0V0D0R spoft greed. NovoNordisk A / S, TRIDENT of Sandoz, DiTerra of Abbottt Laboratories Inc. and Ecogen's EOIL

The isolation of another beetle-toxic Bacillus thuringiensis strain was described in 1986 (Hernnstadt et al., Bio / Technology, Vol. 4, pp. 305-308, 1986, U.S. Patent No. 4,764,372, 1988). This strain, designated Bacillus thuringiensis subsp. San Diego, M7, is deposited at the Northern Regional Research Laboratory, USA under NRRL B-15939. Commercial product based on Bacillus thuringiensis subsp. san diego was developed by Mycogen Corp.

- 4 Comparative study of Bacillus thuringiensis subsp. ^ nebrio. ili ms, DSN 2803 and Bacillus thuringiensis subsp. san diego, NRRLB 15939, including phenotypic characterization of vegetative cells, parasporal crystal characterization, and DNA »w plasmid analyzes showed that Bacillus thuringiensis subsp. San Diego is apparently identical to the previously isolated strain of DSM 2803, Bacillus thuringiensis subsp. tenebrionis / Krieg et al., J Appl. Ent. 104, pp. 417 to. 424, 1987]. In addition, the nucleotide sequences and deduced amino acid sequences against the beetles of the active delta endotoxin genes of the acillus thugingiensis subsp.

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tenebrionis and ac acxlluns thuringiensis subsp. san diego are authentic.

and the same culture conditions, the above type of rat / talc is also synthesized by Bacillus thuringiensis subsp. san diego / A.M. Hunter and A. Krieg, J. Appl. Ent.,

1989, 10: 490-497.

According to H. de Barjac and E. Frachon (Entomophaga 35/2), pp. 233-240, 1990 (san diego isolate '' is similar to tenebrionis isolate and therefore cannot be considered as different subspecies ·

The usefulness of Bacillus: thuringiensis for controlling beetles depends on the efficacy and economic production of toxins effective against beetles and the potency of the product produced. This in turn is dependent on the amount of delta endotoxins that can be produced by the fermentation of Bacillus thuringiensis strains effective in controlling beetles.

The microorganism fiacillus thuringiensis can be used for many years to produce an insecticidally active substance, but in any way. mutants of B. thuringiensis with an increased yield of delta endotoxins would be advantageous, such mutants have not previously been described. Mutants producing delta endotoxins at higher yields would allow more efficient and economical production of B. thuringiensis toxins. and $ ožnost. production of products _of B thuringiensis with increased potency for the same price, it would on the contrary be advantageous for the user, since this would reduce the volume stored pesticidal formulations and would require Manip ^ Lacey reduced volume for a given acreage. In addition, users would have less packaging problems and would reduce the environmental burden on packaging.

Improving the production of delta endotoxins by fiscillus thuringiensin subsp. tenebrionis. mutations have not yet been described.

One problem associated with the use of, in particular, B. thuringiensis subspecies tenebrionis in controlling beetle larvae is the relatively low potency or aggressiveness of the formulations, requiring the use of a relatively large amount of formulation per treatment area, e.g. 10 liters / ha compared to 1-2 liters / ha for most other B. thuringiensis products and most other insecticides. Therefore, there appears to be a need for new products with increased efficiency.

One solution to this problem is to concentrate the preparations.

However, this greatly increases production costs compared to the savings achieved during storage and handling of the formulations.

A more elegant solution is. development of mutants of existing B. thuringiensis microorganisms capable of producing substantially larger amounts of delta endotoxin per cell.

The invention thus relates to a mutant of Bacillus thuringiensis strains capable of producing substantially more toxins than their parent strain.

SUMMARY OF THE INVENTION

The present invention provides a mutant of Bacillus thuringiensis deposited as subsp. tenebrionis DSM 5480, producing at least twice as many insecticidal beta-endotoxins than the parent strain.

A pesticidal composition for combating harmful insects, in particular from the group consisting of butterflies, diptera and beetles, and in particular combating Colorado potato beetle, according to the invention comprises a mutant of Bacillus thuringiensis subsp. tenebrionis DSM 5480, producing at least twice as many insect endotoxin insects as the parent strain. The pesticidal composition may additionally comprise agriculturally acceptable diluents and / or carriers and a surfactant.

The method for the preparation of a mutant strain of Bacil lus thurigiensis and the selection thereof is according to the invention in that the parent strain is

(i) processed by any chemically appropriate mutagen, such as N-methyl-N'-nitro-N-nitrosoguanidine or ethyl methanesulfonate, under the action of electromagnetic radiation, such as gamma rays, X-rays or ultraviolet radiation; iii) separating the translucent colonies and growing in an environment that does not liquefy when heated; and iv) selecting the true asporogenic strains by heat treating the colonies in the modified nutrient sporulation medium containing and phosphate supplemented with magnesium chloride and supplements of the fashioned nutrient sporulation medium.

The colonies thus separated are allowed to grow in a normal production environment and then the final isolation of strains capable of increasing delta endotoxin production is carried out.

The pesticidal composition is suitable for controlling harmful insects, in which the pesticide composition according to the invention is applied to the areas affected by the harmful insects.

Bacillus thuringiensis subsp. tenebrionis DSM 5480, which produces high amounts of delta endotoxins, was deposited by the Deutsche Sammlung von Microorganisms and Zellkulturen GmbH, Mascheroderweg 1b, D-3300 Braunschweig, Germany, for the purposes of the patent procedure based on the values below. DSM is an international depositary under the international Budapest Permanent Deposit Agreement under paragraph 9 of this International Agreement.

deposit day 10 August 1989 reference deposit NB 176—1 designation DSM DSM 5480

The DMS 5480 mutant is obtained by mutating Bacillus thuringiensis subsp. tenebrionis, strain DMS 5526, which is also deposited under the International Budapest Agreement, as follows: deposit date September 14, 1989 deposit reference NB 125 DSM DSM 5526

In general, the invention relates to mutants and variants of Bacillus thuringiensis producing high amounts of delta endotoxins as compared to its parent strain.

In this context, a high amount means at least a double amount but rather an even greater amount.

In general, however, the invention relates in particular to delta endotoxins produced by a mutant of Bacillus thuringiensis which acts on the same pest species as the delta endotoxins of the parental Bacillus thuringiensis as well as against butterflies (mutants of Bacillus thuringiensis susp. Kurstaki and subsp. Aizawai). (mutants of Bacillus thuringiensis susp. israelensis) or

- 74 beetles (mutants of Bacillus thuringiensis susp. Tenebrionis).

In particular, the invention relates to a microorganism Bacillus thuringiensis belonging to the subclass tenebrionis and an endotoxin delta produced by the microorganism and effective against beetles.

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In this regard, the invention particularly relates to a mutant or a mutant

and.

B. thuringiensis suhsp. terbionis, capable of producing more than three times the delta endotoxin in; comparison with strain DSM 2803.

strains

The invention ae. further relates to a variant of the mutant microorganism B, thuringiensis suhsp. tenebrionis capable of producing a parasporal crystal having an average edge length of at least 2 microns.

The invention also relates to a variant or mutant of B. thuringiensis subsp. tenebrionis, showing the frequency of sporulation

6.

• 40 to 100 times or even 4.0 times lower than the sporulation frequency. parent strain or DSM 2803 strain *

According to a particularly preferred embodiment, the invention relates to a deposited mutant of B. thuringiensis subsp. tenebrionis, DSM 5480 «,

In the present invention, a mutant of B. thuringiensis subsp. tenebrionis / DSM 5480 / s more than double. the amount of delta endotoxin produced compared to the parent strain (DSM 5526). Phase counter microscopy, scanning electron microscopy and transmission electron microscopy demonstrate that the high productivity of this mutant is conditioned by changes in the delta-endotoxin production control resulting in protein crystal production that is up to five times higher than crystal production by strains of an existing microorganism. Bacillus. This close relationship between crystal formation and sporulation appears to be removed and the mutant produces a high amount of delta endotoxin prior to sporulation.

The present invention relates to the use of a strain mutant of the strains according to the invention in the production of insecticidal B. thuringia microorganisms, wherein the variant or mutant of the B. thuringiensis strain is cultured in a suitable dsiiltivation. for-

The compounds containing carbon, nitrogen and other components known to those skilled in the art for an appropriate period of time then isolate delta endotoxins.

The invention also relates to the B. thuringiensis microorganism for the ducated delta endotoxin produced as described above and used as an active ingredient in pesticidal compositions.

In such compositions, the delta endotoxins may be used, either alone or in admixture with other biologically active agents.

The invention also relates to such pesticidal compositions or compositions comprising the delta endotoxin produced by the B. thuringiensis microorganism of the invention in admixture with agriculturally acceptable diluents or carriers.

The invention also relates to such pesticidal compositions or compositions comprising the delta endotoxin produced by B. thuringiensis, which in liquid form have an activity of at least 15,000 BTTU / g, corresponding to at least 3% by weight of the beetle insecticidal protein crystal or 000 BTTU / g corresponding to at least 10% by weight of the beetle insecticidal protein crystal.

In particular, the invention relates to pesticidal compositions produced by DSM 5480 having at least twice the effectiveness in comparison with pesticidal compositions produced by DSM 28Q3, or other Btt strains effective against beetles.

The compositions of the invention may take any known form. from the field of agrochemicals, for example suspensions, dispersions, aqueous emulsions, dusts, dispersible powders, emulsifiable concentrates or granules. In addition, they can be in. in a form suitable for direct use or in the form of concentrates or primary compositions which require dilution with an appropriate amount of water or other diluent prior to use.

The concentration of B. thuringiensis insecticidal endotoxins: in the compositions of the invention, used as such or in admixture, with other pesticides, for application to plants is preferably 0.5 to about 25% by weight in particular: 1 to 15% by weight.

The invention also relates to a method for controlling pests using the insecticidal compositions described above for insect infestation.

More particularly, the invention relates to the control of butterflies, double-winged insects and beetles, and more particularly to the control of Colorado potato beetle. Preferably 4.5 liters per acre of liquid pesticide or about 226 g per acre of dry pesticide are used.

The active preparation of B. thuringiensis or the composition according to the invention can be applied directly to the plant, for example by spraying or dusting while it is being treated; unwanted insects begin to appear on the plant. The preferred method of application is spraying. In general, it is important to achieve good control of harmful insects in the early stages of larval development, since in this case the plant is least damaged.

A method for mutating Bacillus thuringiensis strains and selecting substantially essential mutants that are capable of producing greater amounts of delta enotoxins than the parent strains comprises i) processing with the mutagen; they are grown in a suitable environment for the selection of asporogenic or oligosporogenic strains, iii) the translucent colonies are separated and grown in an environment which does not liquefy when heated, and iv / separated by truly asporogenic strains by heat treatment of the colonies.

In such a preferred method, the separated colonies are allowed to grow in a normal production environment and then the final separation of strains capable of increased production of delta endotoxin is performed.

In step (i) of the above process, the mutagen may be any suitable chemical mutagen, such as TT-methyl N'-nitro-N-nitrosoguanidine or ethyl methsulfonate, or the parent strain may be treated with electromagnetic radiation such as β-rays, X-rays or such as ultraviolet radiation *

In step (ii), a suitable medium may be the phosphate-containing modified sporulation medium (NSMP medium) described by Johnson et al. in Spores. VI, vyd.

P. Gerhard et al., Pp. 248-254, 1975.

In stage. (iv) of the above process may be a suitable medium of NSMP medium supplemented with magnesium chloride and Gelm rite (Keleo).

Another way of obtaining the highly productive variants or mutants of the invention may be to grow the parent strain in a liquid environment and select spontary mutants or pa variants. spreading the culture medium on an agar medium, suitable for selection, asporogenic. and / or oligosporogenic mutants.

Other methods of separating the highly productive variants or mutants of the invention, such as using the mass of these mutants directly by centrifugation, or other means of separating the mass, may also be used.

Example 1

A mutant of B. thuringiensis subsp. tpnebrionia with more than twice the delta endotoxin production. By phase contrast microscopy, scanning electron microscopy and transmission electron microscopy of this mutant, it was found that the high productivity of this mutant was conditioned by changes in control, delta endotoxin production with respect to sporulation, leading to protein crystals production up to five times higher than crystals produced by the current Bacillus thuringiensis strains active against beetles. The close relationship between crystal formation and sporulation appears to be removed and the mutant produces high amounts of delta endotoxin prior to sporulation.

High-yield mutant production

Spores of B. thuringiensis subsp. tenebrionis strain DSM 5526 is irradiated with gamma rays at a dose of 7 kGy. The irradiated spores are then spread on NSMP agar platelets / modified phosphorous-containing sporulation broth described by Johnson et al., In Spores Yl, edited by P. Gerhardt. et al., pp. 248-254, 1975]. Environment suitable for selection of asporogenic and / or oligosporogenic mutants.

NSMP-agar plates are incubated at 30 ° C for two to three days. The translucent colonies are collected and transferred to NSMP gelrite plates / NSMP medium supplemented with magnesium chloride - 0.57 g / l and Gelrit, Kelco - 20 g / l.

NSMP gelrite plates are incubated for one hour at 90 ° C and then for one to two days at 30 ° C.

Mutants that grow well on NSMP gelite plates are separated. In this way, all asporogenic mutants are separated as they do not grow after heat treatment.

Separate mutants were grown in shakers containing commercial media. The amount of delta s.e endotoxin produced is determined by immunological methods as described below.

Only mutants that produce significantly higher amounts of delta endotoxins than the parent strain are selected.

The morphology of the selected mutants in the solid medium, and in the liquid medium, is studied by phase contrast microscopy (magnification 2,500) and scanning electron microscopy and transmission electron microscopy. The number of spores and crystals is added and the protein crystal size is determined.

In addition to the obtained mutants, a mutant (DSM 5480) is selected for superior ability to produce delta endotoxin.

The amount of delta endotoxins produced by the DSM 5480 mutant is. compares with the amount produced by DSM 2803, the original isolate from Bacillus thuringiensis subsp, tenebrionis, Bacillus thuringiensis subsp. tenebrionis, strain DSM 5526, used to produce NOV0D0R, Bacillus thuringiensis. subsp. tenebrionis., strain KB 178, isolated from Sanzaozi

Bacillus thuringiensis tenebrionis product of TRIDENT 1989 strain NN '198, isolated from Sandozo B. thuringiensis. subs. tenebrionis. No. TRIDENT of 1990, Bacillus thuringiensis subsp. san diego strain NRRL-B-15939 and strain NB 197 isolated from Myeogen B. thuringiensis subs. san diego of the product M-ONE 'of 1990. As shown in Table I of Example 2, the improved mutant of the invention produces 2 to 3.5 times more delta endotoxins with the currently available beetle-resistant strains of Bcillus thuringiensis.

Example 2

The yield of delta endotoxins of the microorganism is compared

Bacillus. thuringiensis suhspecies tenebrionis, a DSM 5480 mutant

- 15 with delta endotoxin yields of scilius thuringiensis subspecies tenebrionis strain J DSM 2803 / original isolate from microorganism. ^ Acillus thuringiensis subsp. tenebrionis (DSM 5526) Novo-Nordisk production strain (NB 178 and NB). 198 (production strains of Sandoz (Bacillus thuringiensis subsp. San diego), strain NRRL-B 15939 and NB 197) (production strains of Mycogen) in the commercial environment. Each strain is grown for 17 hours at 30 ° C on inclined agar of the following composition, expressed as grams per liter of distilled water:

Pepton, Difco 5 g Beef extract, Difco 3 g Agar, Difco 20 g pH 7.0

Then. transfer 5 ml of cell suspension from each strain to 100 ml production medium in a 500 ml erlenmeyer flask. The production medium consists of the following components in the indicated amount, expressed as grams per liter of tap water soybean meal 50 g hydrolysed starch 40 g Potassium dihydrogen phosphate 1.77 g Potassium monohydrogen phosphate 4.53 g pH value 7.0

The inoculated flasks were incubated at 30 ° C with shaking (250 rpm). After 96 hours incubation, the culture broth is assayed for delta endotoxin content by immunoassays

The amount of delta endotoxins produced by each strain is determined by increasing immunoelectrophoresis. / rocket immuno electrophoresis RIE / and photometric. immunoassay (PIA) using antibodies against purified protein crystals of bacillus thuringiensis subsp. tenebrionis.

Weigh 400 mg of each culture medium

It will be added. 7 ml trisodium phosphate buffer. (0.125 M, pH 12) to each sample. The suspension is then shaken for one hour to dissolve the delta endotoxin proteins.

Samples made. then centrifuge at 3500 rpm for 15 minutes, and assay the supernatant. for the RIE endo toxin content by scophoresis against antiserum rising against purified phi microorganism protein crystals. thugingiensiá. subsp. tenebrionis. The amount of delta endotoxins is determined with reference to a standard with known crystalline protein content.

The crystalline protein concentration is also determined by a photometric immunoassay. The crystalline proteins are dissolved in an alkaline solution. Dissolved proteins are precipitated by their antibodies. The rate of this reaction is turbidimetrically. The amount of delta endotoxins is determined by reference to a standard having a known crystalline protein content.

Crystalline antigens for. production of antibodies used in the assays are obtained from crystals isolated from B. thuringiensis subsp. tenebrionis.

Polyplonal antibodies are raised by injecting rabbits subcutaneously every 14 days with 0.25 mg of crystalline antigen #

The results obtained are shown in Tables 1a and Ib below. Delta endotoxin yields are expressed as BTTU / g / unit per g of culture broth, as determined by electrophoresis. RIE or PIA Photometric Immunoassay. Value used for pure crystalline protein of B. thuringiensis subsp. tenebrionis, is 500 000 BTTU / g. This value means in Table 1a the mean value of six to seven independent fermentations and in Table Ib the mean value of three independent fermentations.

Table la

Delta endotoxin production by Bacillus thsiringiensis subsp. tenebrionis in a shaker.

The delta endotoxin yield determined by the method.

RIE PIA Strain. BTTU / g BTTU / g DSM 2803 676 1293 NRRL-B. 15939 747 1126 NB 178 986 1728 DSM 5526 1097 1860 DS.M 5480 2382 4169

Table Ib

AND

Delta endotoxin production by Bacillus thuringiensis subsp.

tenebrionis in a shaker Strain The delta endotoxin yield determined by the method RIE BTTU / g NE 197 1103 NB 198 1237 DSM 5480 2867

It is apparent from Tables Xa and 1b that DSM 5480 produces more than three times the delta endotoxin. comparison with the original strain. of Bacillus thaaringiensins subsp. tenebrionis, DSM 2803 and Bacillus thuringiensis subsp. san diego ', strain NRRLB 15939 and more than twice the amount of delta endotoxin compared to strains hitherto used to produce Bacillus thuringiensis subsp. nebrionis commercial products.

Phase-contrast microscopy, scanning electron microscopy and transmission electron microscopy of Bacillus thuringiensis subsp. tenebrionis, a DSM 5480 mutant, was found to have protein crystals produced by this mutant much larger than the corresponding protein crystals produced by Bacillus thuriéngiensis subsp. tenebrioVZ * nis, strains DSM 2803, DSM 5526-, NB 17.8 and NB 198 and Bacillus thuringiensis subsp. san diego, strains NRRL-B 15939 and NB 197 »

Culture medium of Bacillus thuringiensis. subsp. tenebrionis mutant DSM 5480 se. tested for efficacy against Colorado potato beetle larvae. It was determined. increased amounts of delta endotoxin produced by the DSM 5480 mutant assay were performed by immunological methods. This increased amount is manifested in biological activity against Colorado potato beetle larvae.

Example 3

Sporulation and parasporal crystal formation in Bacillus thuringiensis subsp. tenebrionis, strains DSM 2803, DSM 5526,

NB 178, NB 198 and the mutant DSM 5480 and B. thuringiensis subsp. san diego ”, strains NRRL-B 15939 and NB 197.

Each of these strains was grown for two days at 30 ° C on agar plates with the following composition, expressed as grams per liter of distilled water: Pepton, Difco 5 g

Beef extract, Difco 3 g

Agar, Difco 20 g

PH 7.0

Each strain is grown in a liquid medium. All strains are allowed to grow. for 17 hours at 30 ° C on agar slanted plates. Then transfer 5 ml of the cell suspension from each strain to a 500 ml bottomed erlenmeyer flask, each flask containing 100 ml of medium.

The medium shall consist of the following components, in terms of grams per liter of tap water:

liquid medium: yeast extract, 5.0 g tr.ypton 5.0 g glucose 1.0 g dihydrogen phosphate. potassium 0,8 g pH 7,0

The inoculated flasks were incubated at 30 ° C with shaking (250 rpm) for 96 hours.

The morphology of the strains is studied daily on solid medium and in liquid medium by phase contrast microscopy (at a magnification of 2500 times). The amount of spores and crystals is calculated and the size of the parasporal crystals is determined. Several selected samples are also studied by scanning electron microscopy and transmission electron microscopy.

B. thuringiensis subsp. tenebrionis, strains DSM 2803, DSM 5526, NB 17.8 and NB 198, and B. thuringiensis. subsp. san diego ”, strains NRRL-B 15939 and NB 197 highly sporulate: in both environments. Before the lysing cell, each cell contains a spore: a parasporal crystal. The crystal size is 0.4 to 0.9 to 1.1 microns in length at the time of cell lysis. The average length of the protein crystals is 0.6 to 0.7 microns.

* The mutant DSM 5480 produces only little spores (less than 10 spores / ml) on a solid medium and in a defined liquid medium. Before lysing cells most cells contain a huge protein crystal and no controversy. The length dimension of the protein crystals is 0.4 to 0.7 micrometers to 5.0 micrometers, with the mean length dimension of the protein crystal being. 2.2 to 2.3 microns.

Ultrastructural analysis of cells from this medium by transient electron microscopy shows that the sporulation process in the mutant is. it starts but does not stop at the time of cell lysis. The sporulation process reaches different degrees in different cells. In cells in which sporulation has reached only stage II (pre-spore septum formation), protein crystals fill the entire cell.

In a production environment / Example 2 / the mutant produces a larger number of spores / ^R 1O to 10 spores / ml /. In this environment it is; the mutant sporulation frequency is 10 to 100 times lower than in the parent strain.

Thus, the mutant retains its ability to produce normal spores. However, the sporulation frequency of the mutant appears to be strongly dependent on the environment. The length dimension of the protein crystals produced by each strain is given in Table Ha and IIb,

Table Ha _kffleny

R _c measure of protein crystals produced by B. thuringiensis currently available against the beetles

Protein crystal length in micrometers

minimal maximal medium strain value value value DSM 2803 0.4 0.9 0.7 NRRL-B 15939 0.4 0.9. 0.7 NB 17,8 0.5 0.9 0.7 DSM 5526 0.4 0.9 0.7 DSM 5480 0.7 5.0 2.3

Table lib

Size of protein crystals produced by sonaspasively available B thuringiensis strains against Beetles

Protein crystal length in micrometers.

minimal maximal medium strain value value value NB 19 # 0.4 0.7 0.6 NB. 198 0.4 1.1 0.7 DSM 548 ° o, A 4.2 2,0

It can be seen from Table IIa and IIb that the DSM 5480 mutant produces much larger protein crystals than any known B. thuringiensis, anti-beetle strain.

Based on the values obtained, it appears that production control, delta endotoxin with respect to sporulation has changed in the mutant ...

The mutant appears to produce protein crystals prior to developing sperm, thereby giving cells longer time to produce delta endotoxin, and this results in the production of much larger, protein crystals at the time of cell lysis compared to the parent strain.

Depending on the nutrients available and the size of the protein crystals at the time of sporulation, a normal spore is established before the lysis time.

Example 4

In this example, a high-yield Btt mutant DSM 5480 is used to produce highly effective products to combat the larvae of Colorado potato beetle.

In the production fermentation broth described in Example 2, DSM 5480 is fermented in an aerated, stirred, production, fermentation vessel. After 96 hours i < 1. , v

obtains the culture medium by continuous centrifugation.

The concentrated cream, which contains active protein crystals, is stabilized by adding microbial preservatives and adjusting the pH to 5.0.

One part of the concentrated cream is spray dried and the other is used to make a wettable powder. The remainder of the concentrated cream is used directly to formulate aqueous, melt concentrates (FC).

The wettable powder is formulated as characterized in Table III. The formulation of the aqueous melt concentrates is described in Table IV.

Table III

NOVODO1P ^ wettable powder formulation (weight percent)

% Ingredient concentrated spray-dried Btt cream 40 detergents 9 anti-caking agent 1 inert filler 50

Table IV

NO VOD 01c dispersible aqueous concentrate formulation FC . % By weight Component NOVODOLE FC 1% by weight concentrated cream Btt 80.00 55.00 preservatives 4.00 4.00 antifreeze agent 9.10 19.00 detergents 2.50 2.50 pH regulators 2.85 . 2.85 water 1, 55 16.65 100.00 100.00

If a value of 500 000 BTTU / g net is used; j crystalline protein, the active crystalline protein content of the preparations is as follows:

NOVODOI ^ WP

NOVODOk PC1 (.

NOVODOR ^ PC2

% Btt crystalline protein 70.8 KBTTU / g 14.16 24.7 KBTTU / g 4.94 14.2 KBTTU / g 2.84

Detergents are selected from a wide variety of suspending aids and wetting agents normally used in agricultural, pesticidal compositions. As anti-caking agents, they are. using hydrophilic silica and as an inert filler s.e. using conventional inert fillers such as bentonites, inorganic salts or clays. Preservatives for food and cosmetic products are used as preservatives in the dispersed aqueous concentrates. The means for adjusting the pH is inorganic acid.

Example 5

Field tests were performed to confirm the biological activity of the high yield Btt mutant DSM 5480 on male Colorado potato beetle larvae. The comparison is made with two commercial products Trident ^ and the M-one ^ culture plant are potatoes,

The culture plant is sprayed three times: Black 20th, 27th July and 3rd August (2nd generation larva). The products used and their dosage are shown in the following table:

Product volume per Efficiency Quantity of Btt white acre of land a tartaric crystal in the composition litx ’// acre KBTTU / g % M · R FC Novodor ² 1, 134 14.2 84 _f 1, 701 14.2 2.84 2,835 1.4.2 2.84 3,402 14.2 2.84 Trident ^ 4,536 5.5 1, 10 M-one ^ 2,268 8.9 1, 78

The mean limiting value of Colorado potato beetle larvae compared to untreated control plants is shown in Table V. Infestation of Colorado potato beetle was very strong for untreated plants: 370 larvae per 20 plants, August 8 and 904 larvae per 20 plants August 8 .

Table V

Treatment Product volume per Percentage < limitations Tl acre of land liter / acre 1st August 8th August NCVODO1 · PC 2 1,134 99 99 NOVODOLl 2 1,701 95 1 00 NOVODOI ^ PC 2 2,835 98 99 NOVODOR ^ PC 2 3,402 100 ALIGN! 100 ALIGN! TŘÍDĚNÍ ^ 4,536 94 98 M-They® 2,268 98 ’ 98 Results clearly show that that products with you sokový heavyweight mutant DSM 5480 are effective for destroying larvae Colorado beetles

potato on the fields. The crystalline protein produced by the high-yield strain is fully effective, as approximately 1.7 liters per acre of NOVODOR ^H FC provides as good results as approximately

4.5 liters per acre of Trident and as good as approximately 2.3 liters per acre of M-on.

Industrial applicability

A mutant of Bacillus thuringiensis deposited as subsp. tenebrionis DSM 5480 produces high levels of active delta endotoxins and is therefore highly suitable for insecticides to combat butterflies, double-winged insects and beetles such as potato beetles7? & C- (/ tfuKc)

Claims (6)

1. A mutant of Bacillus thurygensis deposited as subsp. tenebriosis DSM 5480, producing at least twice as many insecticidal beta-endotoxins than the parent strain. 2. A pesticidal composition for combating harmful insects, in particular from the group consisting of butterflies, diptera and beetles, and in particular for combating mandarin with a potato line, characterized in that it contains a mutant of Bacillus thuringiensis subsp. tenebrionis DSM 5480, according to claim 1, producing at least twice as many insecticidal delta-endotoxins than the parent strain. Pesticidal composition according to claim 2, characterized in that it additionally comprises agriculturally suitable diluents and / or carriers. 4. A pesticidal composition according to claim 2, further comprising a surfactant. 5. t hur ₍ ii) A method for the preparation of a mutant strain of Bacillus / giensis according to claim 1 and its selection, characterized in that the parent strain is treated with any chemically suitable mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine or ethyl methanesulfonate under the action of electromagnetic radiation such as gamma rays, X-rays or ultraviolet radiation, the mutants treated in this way are grown in a phosphate-containing nutrient sporulation medium and optionally as additions of 0.57 g / l magnesium chloride and 20 g / l gelrite, translucent colonies are separated and they are grown in an environment which does not liquefy when heated and the true asporogenic strains are selected by heat treatment of iv) colonies in a phosphate-containing modified nutrient sporulation medium supplemented with magnesium chloride and modified nutrient sporulation medium supplements. 6. A method according to claim 5, characterized in that the colonies thus separated are allowed to grow in a normal production environment and then further isolation of strains capable of increasing delta endotoxin production is carried out.