RU1825300C - Strain of bacterium bacillus thuringiensis var, kurstaki for preparing of insecticide preparation against insects of "lepidoptera" order - Google Patents

Abstract

Usage: production of plant protection products from harmful insects. SUMMARY OF THE INVENTION The bacterial strain Bacillus thurlnglensls var. kurstakl was deposited in the strain collection of the U.S. Department of Agriculture under Nfe C1B12570. The strain is grown on a medium containing peptone broth - 8 ml; glucose - 6 g; yeast extract - 5 ml; xylose - 0.5 g; cotton grain flour extract - 30 ml; corn extract - 3.2 ml; solution of a salt mixture Mary Mendel - 1 ml. With stirring and a temperature of 30 ° C, the resulting biomass, spores and crystals are separated by centrifugation, suspended in 100 ml of water, then 4.9 ml of Morwet D-425 dispersant, suspending agent 4.9 ml of veegum HV, wetting agent are added sequentially to the suspension. , 9 ml, Tween-80 and 24.4 ml Sorbo antifreeze. This strain is 5.1 times more effective than the known ND-1. ё

Description

The invention relates to the production of plant protection products against harmful insects, in particular, the order Lepldoptera, and relates to a new bacterial strain Bacillus thuringlensis var. kurstakl for the preparation of an insecticidal preparation.

The purpose of the invention is to provide a new strain having properties similar to those of HD-1, but having higher insecticidal activity against a number of lepidopteran parasites.

Strain Bacillus thurlnglensls var. kurstakl A20 deposited in the National Assembly of Industrial and Marine Bacteria under No. MC1 B 12570

Strain A20 was isolated from the soil under cedars in Coburg, Ontario. According to its morphological and general biochemical properties, it is similar to HD-1. The morphologists of the strain are presented in Table 1. Difco nutrient agar and Bacillus cereus agar base (code CM617 from Oxoid. Canada) were used to describe the morphology of the strain.

The biochemical properties of the strains are compared in table. 2-4.

The strain grows on both an organic and inorganic source of nitrogen. When using an inorganic source of nitrogen, valine should be added to the medium.

Given this similarity, it was unexpected that strain A20 has a significantly higher insecticidal

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activity against a series of lepidopteran pests. than HD-;.

Strain storage.

Strain A20 can be stored in four different conditions: a culture suspension stored at -20 ° C in glycerol, an oblique nutrient agar at 4 ° C and a spore suspension stored at -20 ° C, and for long-term storage cultures - freeze-dried, stored at room temperature.

One way of implementing the method of this invention is that the insect-prone plant is given the ability to produce d-endotoxin in situ. This is achieved by cloning the 6-endotoxin gene from strain NC1MB 12570 or 12571 by known methods using a suitable promoter (e.g. CaMU3b3 promoter) that induces gene expression in plants and transforming plants using known methods (e.g. using T plasmids).

Insects that can be most effectively controlled by the method of this invention are Lepidoptera, for example, those shown in Table 1. 5.

The method of this invention can be used to protect a wide variety of plants susceptible to lepidoptera infection. Particular examples of commercially important plants protected by the method of the invention are maize (corn) and conifers, as well as rice, cereals (wheat and barley) and vegetables, including lettuce, tomatoes and sugar beets.

The invention is illustrated by examples.

Example 1. Isolation of a strain of Bacillus thurlngiensls var kurstaki NCIB-12570 (A20).

A soil sample taken under cedars in Coburg, Ontario was diluted, 0.5 g of the sample was placed in a bottle containing 45 ml of 0.05% peptone. Then, the sample was heat-treated, for which it was placed for 10 minutes in a water bath at a temperature of 60 ° C. Then serial dilutions were prepared, taking 0.5 ml of the most diluted sample, and placing it in 4.5 ml of 0.05% peptone (for example, putting 0.5 ml of dilution 1.10 in 4.5 ml of peptone gives a 1: 100 dilution). this operation was repeated until dilution 10 was achieved. B. cereus selective medium (agar base, Saclilus cereus code CM617 from Oxold,

Canada) and zskulin agar (containing, g / L water, esculin 1.0: iron citrate 0.5, peptone 10: NaCI 5; agar 20) are inoculated with suspensions diluted from 10 to 10. Inoculated samples were incubated at 30 ° C for 5 days until colonies were obtained. Colonies having dark-colored parasporal crystals having the shape of a bipyramid are selected. Crystal positive colonies were transferred onto nutrient agar to obtain pure colonies and incubated for another 5 days at 30 ° C. Colonies are checked for crystal presence and purity. The purified colonies were transferred to mowed nutrient agar and stored in a refrigerator at 40 ° C for future use.

Example 2. Growing a strain.

The culture of the strain from mowed agar is seeded in a 250 ml Erlenmeyer flask containing 100 ml of CRL No. 1 medium (containing, g or ml / l of water: peptone broth 8; glucose 6; yeast extract 5; xylose 0.5:

extract of flour of cotton grains 30 ml; corn extract 3.2 ml; saline mix Mary Mendel 1 ml). 300 rpm were incubated with stirring at 30 ° C for 24 hours. Then all 100 ml were inoculated into 1 L of the medium of the indicated composition in a 2 L flask, incubated for 5 days with 300 rpm and 30 ° C stirring. . The fermentation broth contains 10-109 spores / ml.

Example 3. The recipe according to this invention.

After the completion of the fermentation, the biomass spores and crystals were separated from the fermentation broth by centrifugation or microfiltration. The separated spores and crystals were suspended in 100 ml of water and a liquid concentrate was added, adding 4.9 Morwet D-425 (sodium naphthalene-formaldehyde condensate) (dispersing agent), 4.9 Veegum HV (magnesium aluminum silicate) (suspending agent), 4.9 ml Tween 80 (wetting agent) and 24.4 ml sorbitol (antifreeze). Each ingredient is added individually in that order. The product was stored at 40 ° C until use. Similarly received a preparation based on strain HD-1, which was used as a control.

EXAMPLE 4 Effectiveness of BT-strains A20 for protecting plants from insect damage.

Using the formulations described in example 3 based on mixtures of spores and crystals, strain A20. obtained as described in Example 2, an efficacy test was carried out against the spruce buds of a spider bud by spraying foliage. Branches of spruce, naturally infected with insects (larvae at the 4-5th stage of age), were sprayed using a model MRY-2 sprayer. For A20 and HD-1, the droplet size (diameter) is 20-120 microns with an average diameter of 32 microns. In all cases

put about 11 drops on a needle with a concentration of 30 x 109 1 spores / ha. Treated branches were incubated in a growth chamber until pupation. Results are expressed as percent mortality, number of dead insects (number of dead insects + number of live insects x 100).

 Mortality divided by the amount of drug (g) used in the formulations

In this example, from the point of view of the efficiency coefficient, A20 is 5.1 times more active than HD-1.

EXAMPLE 5 Additional Tests for A20 Toxicity to Pest Insects

  -

LCso in μg dry matter per ml feed, ratio / IKso c / IKso control.

 Method for determination of insecticidal activity.

In the tests, Trichoplusia nl larvae of 4 days old were used, which were fed for 4 days at 30 ° C, using the antibiotic-free Browsville diet for bioassays. A minimum of twenty-five 4-day-old T. ni larvae are exposed to the drug at various dosages. The larvae incubated for four days at 30 ° C and during this time mortality was determined. The value of the effective insecticidal (standard) activity is obtained based on at least three activity values obtained in trials in which the group

After 5 days of incubation in Example 3, the biomass, spores and crystals were separated by centrifugation and precipitated with acetone. The resulting powder was introduced into an artificial feed suitable for test insects. After feeding, insect mortality was recorded by a standard method. Comparative

tests were performed using HD-1.

the coefficient of variation is less than 20%. These tests are defined as tests with a minimum of five points on a curve. where at least two points

are higher, and two are lower than Lcc, significant regression, significant F values, and 95% reliable significance levels in less than the 3X interval. The activity of strain NICB-12570 is 2000-12000 lv / mg broth.

Claims (1)

SUMMARY OF THE INVENTION Bacillus thuringlensls Bacterial Strain var. kurstakl NC1B 12570 for insecticidal insecticide order Lepldoptera. Note. - negative reaction, + positive reaction, (+) partial reaction. Table 3 Biochemical markers on ID-IDENT boards Table 1 table 2 Note, -1- partial color change (difficult identify) ID-IDENT is the trade name of Analytas Products Table 4 Biochemical markers on API-ZYME boards Note: API-ZYME - Trade name of the company API ANA TAB PRODUCTS O - no reaction + - positive reaction Continuation of the table. 3 eleven Common name Caterpillar of spruce bud-leaf beetle Unpaired silkworm d Scoop no Scoop cottonova Camping worm Moth cabbage Moth corn 1825300 12 table 5 Latin name Chorlstoneura fumlferana Lymantria dlspar Trlchopliisia nl Heliothls Zea Spodoptera exlgua Plutella xylostella Ostrlnla nubllalls