RU2307158C2 - Bacillus subtilus M1 STRAIN WITH FUNGICIDAL AND FUNGISTATIC ACTIVITY TO CULTURED PLANT DISEASE EXCITANTS - Google Patents

Abstract

FIELD: biotechnology, agriculture.

SUBSTANCE: claimed strain represents microorganism culture, isolated from internal tissue of potato plant stem. Strain is used in form of aqueous cell and spore suspension in plant seed and tuber pretreatment.

EFFECT: effective plant protection from root rot; heavy crop.

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Description

The bacterial strain Bacillus subtilis M1, which has fungicidal and fungistatic activity against pathogens of cultivated plants.

The invention relates to a strain of endophytic bacteria and can be used to protect plants from damage by phytopathogenic fungi and bacteria.

Biological preparations are known that have fungicidal properties to protect plants from the black leg, root, and neck rot, for example, based on the fungus Trichoderma harzianum [1].

The disadvantage of this method is the low efficiency for protecting plants from diseases such as verticillus wilt, fusarium wilt, etc.

The prototype of the invention is a strain of Bacillus polymyxa 9A, isolated from the roots of potato plants, having fungicidal properties against pathogens, in particular verticillin wilt of potatoes and cotton [2].

The disadvantage of the strain is that it does not suppress black root rot and fusarium wilt.

The invention allows to obtain a new biological effect - pronounced fungicidal and fungistatic activity of a strain of a bacterium of the genus Bacillus against pathogens of plants.

This effect is achieved by the fact that the strain Bacillus subtilis M1, isolated from the internal tissues of potato plants, has fungicidal and fungistatic activity against pathogens of cultivated plants. Used for presowing treatment of seeds and tubers of plants with a suspension of a new living culture.

The strain of Bacillus subtilis M1 is isolated as follows. Slices of the stem of potato plants in a flowering phase 0.5 cm long are sterilized for 3 minutes with an 80% aqueous ethanol solution, then washed three times with sterile distilled water. After that, the integumentary tissues are removed from the stem pieces with a sterile scalpel and the slices are placed on potato-glucose agar (KGA) in Petri dishes, which are incubated at 28 ° C for 5-7 days. After that, colonies of light flesh color with smooth edges are selected and sown separately in Petri dishes as described above. The cultures of the bacteria that formed the colonies are evaluated for antagonistic activity as described by N. S. Egorov (Fundamentals of the doctrine of antibiotics, 2004).

According to the determinant of bacteria (ed. J. Houghton et al., 1997), the work of "Norris et al. (1981), the strain is assigned to the genus Bacillus, a species of Bacillus subtilis, deposited in the collection of the All-Russian Research Institute of Agricultural Microbiology (St. Petersburg - Pushkin) under the registration number "VNIISKHM 111 D 24".

The strain of Bacillus subtilis M1 is characterized by the following main cultural and morphological characteristics and biochemical properties. Gram-positive aerobic spore-forming bacilli producing catalase. Form endospores that do not inflate the cell. The cell size is 1.8 ÷ 1.9 × 0.5 μm. In smears of a 24-hour culture, straight rod-shaped cells with rounded ends are located, located singly, in pairs, less often in chains. The spores are ellipsoidal in the cell are centrally located, the size of 0.9 × 0.5 microns. When the strain is cultivated on meat and peptone agar (MPA) for 24 hours at 37 ° C, flat translucent, slightly shiny colonies with a dense center and uneven edges are formed. The culture produces catalase, utilizes glucose, arabinose, xylose, maltose, galactose, mannitol, sucrose to form acid; does not decompose dulcite, rhamnose, gives a positive Foges-Proskauer reaction, hydrolyzes starch and gelatin, does not hydrolyze urea; utilizes citrate, does not use propionate. The culture does not grow under anaerobic conditions, does not form the inclusion of poly-β-hydroxybutyric acid on MPA with glucose; does not form indole and hydrogen sulfide, does not have lycitinase, hyaluronidase and coagulating activity. The strain inhibits the growth and development of phytopathogenic microflora - pathogens of fungal and bacterial diseases of wheat, potatoes and other plants.

The strain culture that is periodically reseeded is stored on the jambs with Gromyko medium, wort agar, Gauze No. 2, pH 6.8-7.0 at room temperature for 2-3 months. Under mineral oil on Gromyko medium, wort agar, Gauze No. 2, pH 6.8-7.0 at room temperature for at least 1 year. In a freeze-dried state up to three years. For cultivation use Gromyko medium, wort agar, MPA, Gauze No. 2.

From the agar medium biomass is removed with physiological saline. As a protective medium, a sugar gelatin filler is used. Freeze drying from -25 ° C to + 32 ° C for 32 hours

After storage under a layer of liquid paraffin or after lyophilization, the culture forms colonies of the same type on the above media.

The titer of bacterial cells in the studied sample culture of the strain of 1 billion cells and spores in 1 ml of medium.

The microbial strain of Bacillus subtilis M1 belongs to the group of non-virulent, non-toxic, non-toxigenic microorganisms; do not have the ability to disseminate, as well as a dysbiotic effect. Allergenic activity of the strain was not detected. The strain does not irritate the mucous membranes of the eyes with local contact.

Bacillus subtilis M1 has no medical-hygienic contraindications for its intended use as an active principle of a microbial preparation for protecting agricultural plants from pests and diseases.

The strain Bacillus subtilis M1 was tested by presowing treatment of wheat seeds.

For processing seeds from a bacterial culture, a working suspension was prepared, bringing the titer to 1 billion cells and spores in 1 ml of medium. To process 1 ton of grain seeds, 10 L of working suspension was prepared. Processing began 12-18 hours before sowing. Seeds were poured into the container and watered with a suspension of the strain. Following the moistening of the seeds, they were carefully shoveled and covered with a tarp. Control seeds (1 t, control) were treated with 10 l of water.

Wheat plants of the variety Zhnitsa from seeds treated with strain M1 and water were grown under the same conditions in the experimental field of the Milovskoye training farm of the Bashkir State Agrarian University.

The effectiveness of pre-sowing treatment of wheat seeds with an experimental series of the preparation Bacillus subtilis M1 is given in Tables 1-3.

Strain M1 increased seed germination by 20%, stimulated the growth of seedlings, increased their mass, and reduced the seedling damage by pathogens of rot (Table 1). The biological effectiveness of strain M1 in protecting seedlings from rot was 34% (Table 2).

Table 1 The effect of seed treatment with biological products of strain B. subtilis M1 on the growth of seedlings and their susceptibility to rot pathogens Option Seed germination,% Sprout length, cm Mass of 100 seedlings, g Rot of seedlings,% The control 67 13.7 ± 0.3 3.4 28 Strain M1 88 17.2 ± 0.7 4,5 8

Evaluation of the yield and structure of the wheat crop revealed that the use of a preparation based on Bacillus subtilis M1 in terms of the number of productive stems by the time of harvesting is preferable (Table 3).

table 2 The effectiveness of strain M1 against root rot of wheat in the phase of complete physiological ripeness Option Prevalence,% The degree of development of the disease,% Biological efficiency,% The control 70 29th 0 Strain M1 62 19 34

The use of strain M1 was effective in protecting plants from root rot. The yield increase in comparison with the control was 0.4 t / ha.

Table 3 The effect of seed treatment with a strain of Bacillus subtilis M1 on the yield of wheat grain and its structure Option The number of productive stems, pcs / m ² Grain mass 1 spike, g Mass of 1000 grains, g Grain Harvest, t / ha The control 410 0.588 29.1 2.29 Strain M1 430 0.665 33.6 2.70

The experimental data indicate the high efficiency of strain M1 for presowing treatment of seeds of agricultural crops.

Information sources

1. Seiketov G.Sh. Mushrooms of the genus trichoderma and their use in practice. - Alma-Ata: Science, 1982.- 242 p.

2. Kado C. I., Schnathorst William C., Azad; Hamid R. Method of using Bacillus polymyxa 9A to protect plants against verticillium wilt // United States Patent No. 4663162. May 5, 1987 (prototype).

Claims (1)

The bacterial strain Bacillus subtilis VNIISHM 111 D 24, possessing fungicidal and fungistatic activity against pathogens of cultivated plants.