CS235786B1 - Method of insecticide bactericide preparation production against evil-doers caterpillars from crystalliforming bacilli of species bacillus thuringiensis - Google Patents

Abstract

A method for producing an insecticidal bacterial preparation against caterpillars from crystalophores Bacillus bacilli thuringiensis submersible method under constant stirring and aerating at temperature 29-32 ° C by seeding into sterile liquid soil containing assimilable carbohydrates and an assimilable organic source nitrogen, characterized in that they are assimilable carbohydrate and assimilable source of nitrogen containing growth factors and vitamins is used in a ratio of 2 to 3: 1 at a starting pH of 7.5, after completion fermentation, the active ingredient is fermented the soil is isolated and after the addition of adhesive and protective the substances were dried.

Description

(54) A method for producing an insecticidal bacterial preparation for the caterpillar pest pests from the crystalline bacillus Bacillus thuringiensia species

A process for the production of an insecticidal bacterial preparation against caterpillars of crystalline phosphorous bacilli of the Bacillus thuringiensis group by the submersible method of stirring and aerating at 29-32 ° C by inoculation into a sterile liquid soil containing assimilable carbohydrates and assimilable organic nitrogen source. The nitrogen source containing growth factors and vitamins is used in a ratio of 2 to 3: 1 at an initial pH of up to 7.5, after the fermentation the active ingredient is isolated from the fermentation broth and dried after addition of wetting agent, adhesive and preservative.

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The present invention relates to the production of a novel bacterial preparation as a selective biological agent for controlling insect pests of caterpillars of the order Lepidoptera.

It is known that the chemical insecticidal agents used so far, such as DDT, HCH, induce insect resistance to them. In cases where these insecticides get db of human food, they cause unwanted reactions and irreversible disorders in humans ·

Unrestricted use of insecticides in addition to direct food contamination has been shown to cause contamination of the plants with which they enter milk, meat, animal fats, so food insecticide contamination is far more common than it would seem. Forests by destroying countless small insect species, along with the pest, are generally useful as parasites of pests and thus break the balance in nature.

These disadvantages have led to the search for more gentle ways to control pests, such as biological control, which exploits the natural enemies of insects specialized in a certain pest · They are diseases and parasites of insects · Artificial multiplication of infection on man-made soils forming crystals toxic to caterpillars · Bacillus thuringiensis is a very virulent repressant, and the invention is described below. Bacillus thuringiensis is a bacterium that causes selectively the Lepidopteran caterpillar to die, does not harm other beneficial insects and is toxic to humans and other warm-blooded organisms, birds , fish, even for plants.

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Already in 1962 a patent was developed

103416 for a method for producing an insecticidal bacterial preparation from Bac. thuringieneis, which has now been altered in the culture regime, is working on a progressive new form of finalization and a preparation based on later isolated new strains with an increased effect mainly on large caterpillars and moth caterpillars.

The method according to the invention allows the production of a standard bacterial preparation from Bacillus thuringienais species in an economical manner on a large scale using modern equipment in a short time.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a process for the production of an insecticidal bacterial preparation from crystalline Bacillus thuringiensis bacilli by the submersible method of constant agitation and aeration at 29 to 22 ° C by inoculation into sterile liquid media containing similable carbohydrates and an assimilable organic nitrogen source. the nitrogen source is used in a ratio of 2 to 3: 1 containing growth factors and vitamins at an initial pH of up to 7.5, the termination of the fermentation being conditioned by the release of the insecticidal component which is further isolated from the fermentation fluid and fabrics dry ·

The bacillus is cultured aerobically under submersive conditions in sterile liquid soil with constant aeration and stirring. The cultivation time is 48 h to 72 h ·, the optimum temperature is about 30 ° C, the initial pH ae can be between 6.5 and 7.5, preferably 7.0 to 7.2. During fermentation, the pH is kept within the same range by the addition of, for example, K ₂ HPO0 or CaCOCO or NaHCO ^. Various media containing assiable carbon and nitrogen may be used. The carbon source may be aimidable carbohydrates (mono-, di- or polysaccharides, eg glucose, eacharoaa, starch). An assimilable source of nitrogen is a protein material, preferably containing simultaneously vitamins and growth factors, eg yeast autolysate, fermentation extract, dried yeast, peptone, soya flour, corn flour, wheat flour, defatted cotton germ flour, casein hydrolyzate, corn extract, corn extract, corn extract after the production of alcoholic beverages, fish meal, etc.).

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It is desirable to add oligobiogenic elements such as Mg, Lin, Fe, Zn, Ca salts to the culture medium.

After the development and 90-95% of the bacillary culture has been sporulated while the insecticidal crystals are released simultaneously, the effective spore and crystal biomass is isolated and judged from the fermentation fluid. Before isolation, a preservative such as lactose or NaCl (5 to 20%), a wetting agent (5 to 10%) such as Triton X, Tween 20, Chevron, Slovaaol, and an adhesive (up to 5%) are mixed into the fermentation fluid with the biomass. eg molasses, casein, latex, syrup. The spore material is isolated by centrifugation or filtration. The fermentation fluid with biomass can be concentrated on a Lavall-type film evaporator or centrifuge. Drying can be carried out in several ways: by a stream of warm air, by freeze drying, in a vacuum oven or in a spray dryer, or by precipitation with acetone followed by drying. The spore and crystal material in the form of a dry powder retains its insecticidal properties for many years when stored in a dry and cool place.

Said production process of the insecticidal formulation also applies to other crystallo-bacilli of the Bacillus thuringiensie group, such as, for example, thuringiensis, alesti, kurstaki, aotto, dendrolimus, kenyae, galleriae, aubtoxicus, ntomocide8, morrisoni, nigeriae, tolworthi, darmstadtiensis , moritai and more.

By a biological test on Lepidopteran caterpillars (eg caterpillar, woodpecker, pepper moth, cabbage white, etc.) ₉ and by comparison with the standard and determine the biological activity of the preparation in biological units. The bioassay takes 7 to 10 days.

Example 1.

The broth, shake, fermentation and fermentation broth were composed of: 1.5% soy flour, 1.4% corn starch, 0.03% MgSO ₄ .7H ₂ O, 0.002% FeSO ₄ THgO, 0.002 ZnSO ₄ . 7H ₂ 0 _with 0.002% CaCl ₂ , 0.1% NaHCO 3 A 500 ml shaker flask was seeded with a Bac culture strain for 20-24 hours. thuringiensia grown on sloping agar with composition: starch 2%, peptone 0,75%, yeast autolysate 2,5%, K ₂ HP0 ₄ 0,68 trace elements 0,1% of basic solution (basic solution:

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K ₂ SO ₄ 17.4 g, MgSO ₄ . 7H ₂ 0 12.3 g, MnSO ₄ . 7H ₂ O 0.22 g;

FeS0 ₄ . 7H ₂ 0 2.0 g, ZnSO ₄ o 7H ₂ O 1.44 g, CaCl 3. HgO 18.3 g, H ₂ 0 1000 ml) Shake flasks are filled with 50 ml of soil and inoculated with 1 flask agar culture in 3 flasks each. shake culture · Fermentation tank is inoculated for 20-24 hours. pre-seed tank culture (2 to 10%) · The fermentation tank is cultivated at 28 to 32 ° C under aeration with 1/2 volume of air and stirring at 300 rpm. 70-72 hours until spores and crystals are released in 95% of the culture.

After fermentation, lactose (10-20% calculated on dry weight), Slovasol wetting agent (5-10%) and 1% molasses are mixed into the fermentation fluid with Bacillue thuringiensis blot, stirred for 30 min. and dried on a spray dryer (inlet 150 - 200 ° C, outlet maximum 80 ° C).

The bioassay on Ephestia cautella caterpillars determines the activity of the preparation in International Biological Units.

Example 2.

The broth consisting of 2.5% glucose, 3% corn extract and 0.68% K ₂ HPO ₄ at pH 7.0-7.2 was inoculated with 1 to 3 µl of a 24 h old vegetative bacillus inoculum in a pre-seed tank. grown on a shaker on nutrient broth containing 2% Starch, 0.75% PTFE, 2.5% QaA autolysate, 0.68% K ₂ HPO ₄ and 0.1% oligobiogenic (trace) elements (see Example 1), and cultured at 29-30 ° C under aeration 1/2 volume of air and stirred at 200 rpm. 20-24 h. The fermentation tank is inoculated with the inoculum prepared from the pre-treatment tank. Cultivation in the fermentation tank is carried out at 30 ° C under aeration 1/2 volume and stirring at 300 rpm for 54 to 72 hours until spores and inclusions are released in 95% of the culture. 10-20% lactose and Tween 20 5-10% are added to spore and inclusion biomass, concentrated on a film evaporator and spray dried as in Example 1. The number of units of activity is determined by bioassay on white cabbage bead in the preparation obtained.

Example 3.

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The medium in the pre-seed and fermentation tank of mine. composition of 1.5% soya flour, 0.5% glucose, 0.5% corn starch, 0.03% MgSO ₄ . 7 ^ 0, 0.002% FeSO ₄ . 7 ^ 0, with 0.002% ZnSO ₄ . 7Η ^ 0 and 0,1% CaCO ^, the flask broth for inoculation of the pre-seed tank has 2% glucose, 1.5% yeast autolysate, 0.75% peptone, 0.6% K ₂ HP0 ₄ and 0 , 1% oligobiogenic elements (see Example 1 ·) · The procedure for inoculation and fermentation is the same as in Example 1.

Biomass spores and inclusions are cooled to about 20 ° C and thickened on an Alfa Lavall type separator to increase the dry matter by about 6 times. · To the concentrate ae is added lactoea (5 to 25% au auine and Triton X 5 to 10%), stirred 30 min. · and dried in a spray dryer (see Example 1). · The preparation is tested for biological activity on the borer of the paprika and compared to an international standard.

Effect of bacterial preparation on L ₄ caterpillars of Bphestia cautella ended, prep. mortality

l.den 2 .den 3 «den <«Day 7 · day 8 «day 9« day 10, 0.5% - 60% 100% 0.3% - 60% 100% 0.1% - 30% 70% 100% 0.05% 10% 50% 60% 60% 60% (2 votes) control - MB - • B '' Β

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Claims (2)

Object of the invention ⁷⁸⁸ A method for the production of an insecticidal bacterial preparation against caterpillar pests from both crystalline Bac and Hus thuringienaia species, characterized in that ae variety thuringiensis, alesti, kurstaki, sotto, dendroliaua, kenyae, galleriae, subtoxieue, entomocidus, morriaadios, nigeriae, nigeriae, nigeriae toumanoffi, moritai cultured by submerged method with constant agitation and aeration at 29-32 ° C by inoculation into sterile liquid nutrient medium containing aaimilizable carbohydrates and an assimilable organic nitrogen source, wherein aaimilizable carbohydrates and an assimilable nitrogen source containing growth factors and vitamins ee used in ratio of 2 to 3: 1, at a starting pH of 6.5 to 7.5, wherein after fermentation and release of spores and crystals, these are isolated from the fermentation broth and dried at temperatures up to 80 "after addition of wetting agent, adhesive and preservative. C. 2. A process according to claim 1, wherein the preservative is lactose or sodium chloride.