RU2409660C1 - Bacillus mucilaginosus bacteria strain exhibiting antagonist activity relatively to phytopathogenic microorganisms and ability to transform phosphorus difficult of plants access and application thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: Bacillus mucilaginosus strain BM-09 RNCIM No. B-10369 is recovered from garden soils of the Moscow region by multistage analytical selection. The strain can be applied to raise the level of crop yield and to improve stability to various diseases. The level of crop yield is up to 341.0 hundreds kilograms per hectare, and the degree of crop affection is 1.1-33.2 %.

EFFECT: strains shows antagonist activity relatively to phytopathogenic microorganisms and ability to transform phosphorus difficult of plants access.

2 cl, 4 tbl, 4 ex

Description

The invention relates to agricultural biotechnology and microbiology, in particular to the production of bacterial preparations used in crop production to increase crop yields and their resistance to various diseases.

Known strain of Bacillus mucilaginosus VKPM B-5987 (RF patent No. 2081867). The specified strain has the ability to transform insoluble and poorly soluble phosphates in a form accessible to plants. The disadvantage of the strain is its lack of antagonism with respect to phytopathogenic fungi and bacteria.

The known strain of Bacillus mucilaginosus Vas-10 VKPM B-8966, a prototype (RF patent No. 2289621), which increases the yield of many crops and increases their resistance to various diseases. The disadvantages of the strain include a low yield increase obtained for a number of crops.

The objective of the invention is to expand the range of strains with antagonistic activity against phytopathogenic microorganisms and increase crop yields.

The problem is solved by the fact that the obtained strain of Bacillus mucilaginosus BM-09 VKPM B-10369 with antagonistic activity against phytopathogenic microorganisms and the ability to transform insoluble and poorly soluble phosphates into a form accessible to plants, which allows its use in crop production to increase crop productivity and increase their resistance to various diseases.

The bacterial strain Bacillus mucilaginosus BM-09 VKPM B-10369 was isolated from garden soils of the Moscow region by the method of multi-stage analytical selection according to the following criteria: adaptability (strain ability to grow under production conditions), good productivity, including in regulatory environments, the ability to transform insoluble ( sparingly soluble) compounds of phosphorus and potassium, a wide range of inhibition of phytopathogenic microflora.

The resulting strain was deposited in the All-Russian collection of industrial microorganisms FSUE GosNIIgenetika and has a registration number VKPM V-10369.

The resulting strain is characterized by the following features.

Cultural and morphological characters.

During microscopy, the cells are fixed, large sticks of regular shape with rounded ends, single, surrounded by a capsule. In young culture, cells are found in pairs, sometimes in short chains.

Cell sizes vary with growth on various nutrient media in the range of 1.0-1.6 × 3-7µ. Gram-negative, but Gram stain may vary.

When grown on potato agar (wt.%: Potato broth - 98.0; agar - 2.0) and Alexandrov’s medium (wt.%: K ₂ HPO ₄ - 0.2; MgSO ₄ - 0.05; FeCl ₂ - traces; sucrose - 0.5; silicates - 0.1; CaCO ₃ - 0.01 (triturated with silicates); agar - 2.0) at 29 ± 1 ° C for 72-96 hours, large colonies with a diameter of 3- 8 mm, mucous, convex, transparent, strongly spread over the entire surface of the nutrient medium, the full development cycle ends with spore formation.

When grown on Ashby agar medium (wt.%: Sucrose - 2.0; K ₂ HPO ₄ - 0.02; NaCl - 0.02; MgSO ₄ - 0.02; CaCO ₃ - 0.5; K ₂ SO ₄ - 0.5; agar - 2.0) at a temperature of 29 ± 1 ° C for 96 hours, the colonies are convex, colorless, transparent, mucous, with a smooth edge, viscous consistency, 1-4 mm in diameter.

When grown on Surman medium with molasses (wt.%: Molasses - 0.5; K ₂ HPO ₄ - 0.03; MgSO ₄ - 0.01; NaCl - 0.05; KNO ₃ - 0.1; CaCO ₃ - 1.0) at a temperature of 29 ± 1 ° C for 48 hours, the culture is a viscous liquid; upon microscopy of the cell, large single sticks and spores up to 130-250 million / ml.

On media without a nitrogen source (Ashby medium) spores are not formed. When vegetative cells are transferred from Ashby's medium to potato agar or to Surman's medium with molasses, the ability to spore formation is restored. The spores are oval in shape 1.5-1.8µ in diameter.

On protein media (MPA, MPB, milk), there is no growth (sometimes very poor development is observed). Hydrolyzes starch. Gelatin does not thin.

Physiological and biochemical characteristics.

Mesophile, growth is possible in the range of 20-40 ° C, the optimum temperature for growth and spore formation is 29 ± 1 ° C.

Aerob, with deep fermentation on a liquid nutrient medium, the optimal aeration-mixing mode is 1 volume of air / 1 volume of nutrient medium, which corresponds to a sulfite number of 1.

Possible growth at pH 6.0-8.5, optimal for growth and spore formation, pH 6.8-7.2.

Attitude to carbohydrate sources.

It grows well on glucose, sucrose, fructose, maltose, galactose, xylose, arabinose. It absorbs sorbitol, mannitol, glycyrin, lactate, citrate from alcohols. Does not absorb cellulose, ethanol, butanol, acetate.

Attitude to nitrogen sources.

Ammonium nitrogen, urea and nitrates can be used as a source of nitrogen.

Relation to insoluble phosphorus compounds.

The strain has the ability to dissolve silicates, apatites, tricalcium phosphate, sodium nucleonate, biotite, zeolite (table 1).

Table 1
The effect of the strain of Bacillus mucilaginosus BM-09 VKPM B-10369 on insoluble phosphates No. p / p Name of indicator The numerical value of the indicator one. The number of viable cells (CFU) 200 mln / ml 2. The accumulation of phosphorus in liquid culture when introduced into the environment (in terms of P ₂ About ₅ ): - sodium nucleonate 83.0 mg / ml - tricalcium phosphate 25.0 mg / ml - fluorapatite 4.0-6.0 mg / ml - zeolite 3.0-5.0 mg / ml

Attitude to phytopathogenic fungi and bacteria.

Antagonistic activity was determined by the method of “holes” (Egorov NS Antibody microbes and biological methods for determining antibiotic activity. M: Higher school, 1965, p. 131) after 3-6 days of growth of test cultures on diagnostic media.

It was shown that the studied strain inhibits the growth of fungi Alternaria sp., Pythium sp. and bacteria Erwinia phytophtora, Rhizoctonia sp., Puccinia triticina, Erysiphe graminis, Septoria graminum, Actinomyces sp. (table 2).

table 2
The spectrum of antagonistic activity of the strain of Bacillus mucilaginosus BM-09 VKPM B-10369 Test culture Growth inhibition zone, mm Alternaria sp. 6 Pythium sp. 5 Erwinia phytophtora four Rhizoctonia sp. 10 Puccinia triticina 7 Erysiphe graminis 5 Septoria graminum 7 Actinomyces sp. 5

The strain of Bacillus mucilaginosus BM-09 VKPM B-10369 is stored in test tubes on a beveled agar medium of the following composition, wt.%: Starch - 1.0; K ₂ HPO ₄ - 0.02; NaCl - 0.02; NH ₄ NO ₃ - 0.1; agar - 2.0, at a temperature of 4 ± 2 ° C up to 3 years.

The invention is illustrated by the following examples.

Example 1. Cultivation of a strain of Bacillus mucilaginosus BM-09 VKPM B-10369 in laboratory conditions.

The strain Bacillus mucilaginosus BM-09 VKPM B-0000 is grown in flasks on a rocking chair on a liquid nutrient medium of the following composition (wt.%): Sucrose - 2.0; K ₂ HPO ₄ - 0.02; NaCl - 0.02; MgSO ₄ - 0.02; NH ₄ NO ₃ - 0.02; CaCO ₃ - 0.5; K ₂ SO ₄ - 0.5, water - the rest. The flasks are seeded with a seed culture at the rate of 10% of the volume of the nutrient medium and grown for 72 hours at a temperature of 29 ± 1 ° C with continuous stirring.

Get a viscous liquid culture containing 250 million spores / ml, which is used for tillage and seeds.

Example 2. Cultivation of a strain of Bacillus mucilaginosus BM-09 VKPM B-10369 in a fermenter.

The sterile culture medium of example 1 in an inoculator is seeded with a seed culture at the rate of 10% of the volume of the culture medium. Grown for 36 hours at a temperature of 29 ± 1 ° C, continuous stirring and aeration equal to 1 volume of air / 1 volume of nutrient medium per minute.

The resulting culture is seeded with a fermenter with a volume of 1 m ³ with a pre-sterilized liquid nutrient medium having the composition (wt.%): Molasses - 0.5; K ₂ HPO ₄ - 0.03; MgSO ₄ - 0.01; NaCl - 0.05; KNO ₃ - 0.1; CaCO ₃ - 1.0 at the rate of 10% of the volume of the nutrient medium. Cultivation is carried out at a temperature of 29 ± 1 ° C, continuous stirring - aeration equal to 1 volume of air / 1 volume of nutrient medium per minute. Duration of cultivation is 60 hours. Get a viscous liquid culture containing 200 million spores / ml, which is used for tillage and seeds.

Example 3. The effect of the drug on crop yields. The preparation obtained in example 2 is used for testing in the open field:

- on sugar beets and buckwheat at the Research Institute of Agro-Industrial Production (Kursk);

- on potatoes at the All-Russian Research Institute of Potato farming (Moscow region);

- on spring wheat in the Primorsky State Agricultural Academy (Vladivostok);

- on barley in the Kuban State Agrarian University (Krasnodar);

- on winter wheat at the Research Institute of Agro-Industrial Production (Kursk).

In each case, the soil treatment with the drug is carried out before sowing seeds on an area of 6 ha at the rate of 200 ml of the drug / ha. For ease of application, the liquid culture is diluted 1000 times with water before use.

As a control, the results obtained under similar conditions, but without preliminary treatment of the soil with the preparation are given.

The test results shown in table 3 show that the preparation obtained on the basis of the strain of Bacillus mucilaginosus BM-09 VKPM B-10369, has a positive effect on the yield of a number of crops.

Table 3 The effect of the drug on crop yields Cultivated crops Harvest, t / ha Yield increase the control a drug c / ha % Winter wheat 19.9 22.1 2.2 11.0 Spring wheat 13,4 15.3 2.1 14.1 Sugar beet 279.0 341.0 62.0 22.2 Buckwheat 9.2 10.5 1.3 14.1 Potatoes 236.0 319.0 83.0 35.0 Barley 17.8 21,4 3.6 12,2

Example 4. The effect of the drug on the degree of damage to crops by diseases.

Experiments to study the effect of the drug obtained in example 2 on the degree of damage to crops by diseases were carried out:

- for spring and winter wheat in Kursk NIIAPP and Ulyanovsk Scientific Research Institute of Agriculture;

- for potatoes in the Bryansk branch of VNIIKH (Bryansk).

In each case, the soil was treated with a preparation on an area of 5 ha at the rate of 200 ml / ha. Before use, the liquid culture is diluted with water 1000 times.

The results shown in Table 4 show that the degree of disease damage is significantly less in the case of soil pretreatment, i.e. the preparation obtained on the basis of the strain Bacillus mucilaginosus BM-09 VKPM B-10369, inhibits the development of various diseases of crops.

Thus, the obtained strain of Bacillus mucilaginosus BM-09 VKPM B-10369, characterized by:

- good growth in production environments in conditions of deep fermentation;

- the ability to transform phosphorus inaccessible to plants;

- a wide range of suppression of phytopathogenic microflora.

It is shown that the liquid culture obtained on the basis of the strain Bacillus mucilaginosus BM-09 VKPM B-10369 increases the yield of crops and increases their resistance to diseases.

Table 4 The effect of the drug on the degree of damage to wheat by various diseases Experience Options Spring wheat
Degree of defeat% Winter wheat
Degree of defeat% Potatoes Tops Tubers Brown rust,% Martyrs
flock of dew Root rot Brown rust,% Septoria,% Late blight,% Viral% Scab,% Ulyanovsk Scientific Research Institute of Agriculture The control 48,4 - 12.5 - - - - - A drug 33,2 - 7.5 - - - - - Kursk NIIAPP The control 25.5 2.5 - 26.5 46.9 - - - A drug 12.0 1,1 - 12.0 27.9 - - - Bryansk branch of VNIIKH The control - - - - - 8.3 14.9 6.3 A drug - - - - - 6.2 13.9 4.1

Claims (2)

1. The bacterial strain Bacillus mucilaginosus BM-09 VKPM B-10369, which has antagonistic activity against phytopathogenic microorganisms and the ability to transform phosphorus inaccessible to plants. 2. The use of the bacterial strain Bacillus mucilaginosus BM-09 VKPM B-10369 to increase crop yields and increase their resistance to disease.