RU2764695C1 - Strain of bacillus subtilis subsp. subtilis bacteria - antagonist of phytopathogenic micromycetes with growth-stimulating properties and microbial agent based thereon for increasing the productivity of agricultural plants and protecting from fungal diseases - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to agricultural microbiology and biotechnology. Proposed are a strain of Bacillus subtilis subsp. subtilis 12501 NK, an antagonist of phytopathogenic micromycetes with growth-stimulating properties, deposited in the Departmental Collection of Useful Agricultural Microorganisms of the Federal State Budgetary Scientific Institution National Scientific Research Institute of Agricultural Microbiology (FSBSI NSRIAM) under the registration number RCAM05209, and a microbial agent based on the claimed strain.

EFFECT: inventions provide an increase in the productivity of agricultural plants and protection thereof from fungal diseases.

2 cl, 9 tbl

Description

SUBSTANCE: group of inventions relates to agricultural microbiology and biotechnology, namely to microbiological means for controlling phytopathogens and optimizing the mineral nutrition of plants by treating seeds before sowing (or before storing) and (or) plants during vegetation. EFFECT: group of inventions makes it possible to provide bioprotection of plants against fungal diseases, stimulate plant growth and increase crop yields.

The protection of crops from fungal and bacterial diseases remains an urgent problem in crop production and agriculture. The widespread use of pesticides has led to problems of accumulation of their residues in soil, air, water and plants. As a result, crop production and other branches of agriculture are polluted, followed by the entry of harmful substances into the human body. In addition, agricultural practice is increasingly faced with factors reducing the effectiveness of drugs caused by the emergence of forms of phytopathogenic fungi and bacteria resistant to fungicides. The issue of protecting crops from fungal diseases remains relevant. In this regard, there is a need to search for alternative systems for protecting plants from phytopathogens.

Such effective systems include a biomethod, among the tasks of which is the search for new antagonist strains that inhibit the development and limit the spread of pathogens, and the creation on their basis of antifungal and antibacterial biological products that can bring the production of agricultural crops to an environmentally and economically more expedient level.

Experiences of recent years indicate the prospects for the use of spore-forming bacteria of the genus Bacillus, which exhibit high antagonistic activity against a wide range of phytopathogenic fungi and bacteria, do not have a negative effect on macroorganisms and do not pollute the environment. Bacillus genus bacteria are also effective for increasing crop yields while simultaneously increasing energy savings by refusing or reducing doses when using chemical plant protection products and mineral fertilizers, improving the phytosanitary condition of the soil and obtaining environmentally safe crop products.

Known bacterial strain Bacillus subtilis 26D, deposited in the collection of the All-Russian Research Institute of Agricultural Microbiology under number 128 (AS No. 1717156, publ. 03/07/1992), which is characterized by high antagonistic activity against a wide range of phytopathogenic bacteria and fungi. Based on the Bacillus subtilis 26D strain, dry and liquid forms of the phytosporin preparation have been developed (patent RU No. 2099947, publ. 12/27/1997, patent RU No. 2129375, publ. 27.04.1999), intended to protect various plant species from diseases.

The above sources describe the method of obtaining the drug, its use and effect on the pathogen (plant susceptibility), while there is no information about other beneficial properties of the producer strain, in particular, about its effect on plant productivity.

There are a number of strains of bacteria of the genus Bacillus used for the biological control of phytopathogens and increasing plant productivity (patent RU No. 2337955, publ. 10.11.2008; patent RU No. 2495119, publ. 10.10.2013; patent UA No. 121062, publ. 25.03 .2020), as well as to restore soil microbiocenosis (RF patent No. RU 2482174, publ. 20.05.2013).

The disadvantages of the known strains are insufficiently high fungicidal activity, manufacturability, a narrow range of properties useful for plants, which limits their use in agrocenoses of agricultural crops. It is important to note that for known strains, there are no or no features of the joint use of legumes with nodule bacteria, which, in turn, are sensitive to fungicides, but their introduction into the soil (rhizosphere) is a necessary element of the technology for growing chickpeas, soybeans, peas and others. .

The invention is based on the task of obtaining a more competitive and effective strain that has antagonistic activity against a wide range of phytopathogenic fungi and a wide range of properties useful for plants and soil, compatible with rhizobia, as well as the development of a microbial preparation based on it, which has a stimulating effect on growth and development. plants, increasing their productivity and protecting against fungal diseases.

The problem was solved by isolating a strain of Bacillus subtilis subsp. subtilis 12501 NK, an antagonist of phytopathogenic micromycetes with growth-stimulating properties, and the creation of a drug based on it to increase the productivity of agricultural plants and protect them from fungal diseases.

The inventive strain is isolated from the rhizosphere of virgin grasses, which is located near the village of Klepinino, Krasnogvardeisky district of the Republic of Crimea. The strain was isolated by a modified method of enrichment cultures (Berestetsky O.A., Sherstoboev N.K., Sherstoboeva E.V., Patyka V.F. Modified method of enrichment cultures for the isolation of symbiotrophic nitrogen-fixing microorganisms // Journal of Microbiology. K. 1985. P. 5-6). Primary identification was carried out according to the morphological, cultural and biochemical characteristics of the strain according to Krasilnikov N.A. (Krasilnikov N.A. Key to bacteria and actinomycetes. - Ed. of the Academy of Sciences of the USSR, 1949. - 830 p.) Under the editorship of J. Holt, N. Krieg, P. Sneath, J. Staley S. Williams. - M.: Mir, 1997. - 432 p.).

The genetic identification of the isolated strain was carried out by sequencing a part of the 16S rRNA gene at the All-Russian Research Institute of Agriculture in 2019. Based on the analysis, the strain was identified as Bacillus subtilis subsp. subtilis 12501 NK and deposited with the Departmental Collection of Agricultural Useful Microorganisms of the Federal State Budgetary Scientific Institution VNIISKhM under the registration number RCAM05209.

The claimed strain of Bacillus subtilis subsp. subtilis 12501 NK grows intensively on meat peptone agar (MΠΑ). Cells of a one-day culture of the strain Bacillus subtilis subsp. subtilis 12501 NKs are rod-shaped, 0.6-0.7×2-5 µm in size, mobile, Gram-positive. An endogenous spore of an elliptical shape of a central-terminal location, without a noticeable thickening of the cell. When growing on ΜΠΑ, the strain forms colonies, ranging in size from 2 to 4 mm (medium), rounded with a coarsely toothed or lobed edge, folded surface, matte, pink, bumpy.

The growth of bacterial biomass when streaked on pea agar is intensive, the streak with a pink tint is matte with the release of drops of a shiny liquid, does not coalesce with the medium. Spores are formed for 2-3 days.

The strain of Bacillus subtilis subsp. subtilis 12501 NC in relation to oxygen is defined as aerobic; positive reaction to milk with litmus; acidifies the medium with glucose, but does not form gas. It develops well on media with ammonium salts. On nitrogen-free media growth is weak, does not form spores. The strain of Bacillus subtilis subsp. subtilis 12501 NK grows within the temperature range from 10 to 35°C, the growth optimum is 22-28°C. The growth of bacterial culture is observed in media with a pH range of 6.0-8.5; optimum pH - 7.0-7.2.

The collection culture is stored on the ΜΠΑ medium or pea agar with reseeding once a year.

The strain produces exopolysaccharides (EPS) in the amount of 33-35 μg/cm ^{3 of the} medium, which was established by the phenol-sulfur method (Zakharova I.M, Kosenko, L.V. Methods for studying microbial polysaccharides. 1982. 67 p.) Exopolysaccharides increase the adaptive the potential of bacteria to negative environmental factors, as well as increase their adhesion to seeds, roots, leaves, etc.

Bacillus subtilis subsp. subtilis 12501 NK grows on Hutchinson's medium with cellulose (filter, straw) as the sole carbon source. This allows the strain to be used to decompose plant residues.

The strain has antagonistic activity against phytopathogenic micromycetes of the genera Fusarium, Bipolaris, Trichotecium. The antagonistic properties of the strain in relation to phytopathogenic micromycetes were studied by the method of perpendicular strokes and the method of agar blocks (Egorov N.S. Fundamentals of the doctrine of antibiotics. - M.: Publishing House of Moscow State University; Nauka, 2004. - 528 p.). The fungal suppression effect is 5-47 mm (Table 1).

The strain of Bacillus subtilis subsp. subtilis 12501 NK produces physiologically active substances - plant growth stimulants: auxin-like substances (table 2) and gibberellins (table 3). The ability of the strain to synthesize exogenous plant growth stimulants was determined by a phytotest using coleoptels of winter wheat cv. Albatros Odessa (auxins) and cucumber hypocotyl cv. Lyaluk (gibberellins). Distilled water and the corresponding authentic substances were used as controls.

In a series of vegetation experiments found that the proposed strain stimulates the growth and development of plants, which is reflected in the increase in the dry weight of winter wheat (table 4).

Under the conditions of a field experiment with spring wheat variety Kharkivskaya 46, inoculation with a strain of Bacillus subtilis subsp. subtilis 12501 NK provided an increase in grain yield of 10.5% (table 5).

The proposed strain is a bioagent of a microbial preparation, which is a liquid industrial culture with a cell content of at least 1 billion per 1 ml, metabolites. The biological product is made in two stages with the production of mother and working cultures.

The uterine culture is obtained by washing the biomass of the strain cells from the slant pea agar into a liquid nutrient medium of the following composition (g/l):

Pea decoction - 10.0; Food sugar - 20.0; (NH ₄ ) ₂ SO ₄ - 1.0; KH ₂ PO ₄ - 0.5; K ₂ HPO ₄ - 0.5; MgSO ₄ ⋅7H ₂ O - 0.3; CaCO ₃ - 1.0

The strain is grown for three days in flasks with a volume of 0.75 l or bottles with a volume of 3 l with filling 30% of the volume on a shaker at a speed of 200-220 per minute and a temperature of 27-30°C and reaching a titer of 1.0-4.0 billion cells in 1 ml.

To obtain a working culture (preparation), the components of the medium and cultivation conditions were optimized, which made it possible to increase the titer of the strain by 16%.

Medium composition (g/l):

Molasses - 25 Corn extract - 25 MgSO ₄ ⋅7H ₂ O - 0.3 CaCO ₃ - 1.0

Get a working culture in bottles or a fermenter by inoculation with a mother culture of a strain of Bacillus subtilis subsp. subtilis 12501 NK in the amount of 5-7% of the total volume of the nutrient medium and its cultivation for three days at a temperature of 27-30°C and 200-220 rpm to a total titer of 1.3-2.0 billion cells in 1 ml and titer of spores - 1-1.6-1.2-1.8 billion (80-90%), which ensures high quality and efficiency of the biological product.

Store the biological product at a temperature of 10-15°C - up to three months, at 4-8°C (without freezing) - up to six months or more.

Method of application - prepare a suspension and apply to the seeds. The consumption rate of the drug is 0.1 l, the working solution is 1.8-3.0% by weight of the seeds.

It has been established that seed treatment with the preparation before sowing increases grain productivity by 6.1-8.7% when growing winter wheat using intensive technology and by 7.3-13.0% - using biological technology (Table 6). Under intensive technology is meant the application of mineral fertilizers for the main plowing N ₆₀ P ₆₀ , early spring top dressing with mineral nitrogen N ₃₀ . The use of herbicides and Bi-58 to protect crops from weeds, phytopathogens and phytophages. The biological technology of cultivation provides for treatment with the preparation N ₁₅ P ₁₅ . The soil is southern chernozem, heavy loamy. The comparison was made with the chemical fungicide Vitavax 200 FF.

The strain of Bacillus subtilis subsp. subtilis 12501 NK and a preparation based on it are effective both on their own and in combination with nitrogen-fixing nodule bacteria, agrobacteria. The proposed strain contributes to an increase in the number of beneficial microbiota in the rhizosphere, taken into account in the flowering phase of wheat plants: microorganisms that assimilate nitrogen from mineral and organic compounds by 1.5 and 1.7 times, respectively. The potential nitrogen-fixing activity increases by 1.5 times and the content of mobile phosphorus by 1.3 times in comparison with the control. Therefore, the proposed strain helps to optimize the mineral nutrition of wheat plants.

Treatment with the claimed microbial preparation before storage contributes to an increase in the number of bacteria and a decrease in micromycetes in the epiphytic microbial cenosis of chickpea seeds (table 7). Seeds of the Alexandrite variety were treated with a suspension of the drug and compared with the chemical fungicide Vitavax 200 FF, as well as the well-known drug Fitosporin. Distilled water was absolute control. A day later, the number of bacteria on the surface of chickpea seeds increased by 4 and 6.3 times in comparison with the control and the chemical disinfectant, respectively, and also exceeded the known preparation. After six months of storage, the number of bacteria on chickpea seeds was 5.5 and 6.8 times higher than the absolute and chemical controls, respectively. The number of micromycetes decreased: in a day by 3.9 and 2.7 times in comparison with absolute and chemical controls, respectively; after six months by 2.2 and 1.7 times, respectively, and was also lower in comparison with the known drug.

Early treatment of chickpea seeds did not reduce their sowing qualities (table 8). Germination energy increased by 33.3%, germination by 9.7% in comparison with the control.

Bacillus subtilis subsp. subtilis 12501 NK does not show antagonism to nodule bacteria, which was established in the laboratory by the method of perpendicular cultures and the method of wells (Egorov N.S. Fundamentals of the study of antibiotics. - M .: Publishing House of Moscow State University; Nauka, 2004. - 528 p. - S.155-159). In vegetation experiments, it was found that the treatment of chickpea seeds with a preparation based on Bacillus subtilis subsp. subtilis 12501 NC, both early (six months) and presowing together with rhizobia, increases the symbiotic potential of plants (Table 9). The control is the treatment of chickpea seeds with a preparation based on nodule bacteria Mesohrizobium ciceri L. in the block.

The proposed drug based on a strain of Bacillus subtilis subsp. subtilis 12501 NK helps to increase plant productivity. For three years of field trials, the increase in grain yield was 9.1% compared to the control, both when used together with rhizobia, and with advance treatment with the drug. The drug is recommended for the treatment of seeds of grain crops before sowing, and the treatment of seeds of leguminous crops, in particular chickpeas, when storing.

Thus, the proposed strain of Bacillus subtilis subsp. subtilis 12501 NK has a complex of properties useful for plants and soil, is able to produce physiologically active substances - plant growth stimulants and is characterized by antagonistic activity against a wide range of phytopathogenic fungi. The microbial preparation developed on its basis can be used to increase the yield of agricultural crops and protect them from fungal diseases.

Claims (2)

1. The bacterial strain Bacillus subtilis subsp. subtilis 12501 NK, an antagonist of phytopathogenic micromycetes with growth-promoting properties, deposited in the Departmental Collection of Agricultural Useful Microorganisms of the Federal State Budget Scientific Institution All-Russian Research Institute of Agriculture under registration number RCAM05209. 2. Microbial preparation for increasing the productivity of agricultural plants and protecting them from fungal diseases based on the bacterial strain Bacillus subtilis subsp. subtilis RCAM05209 with a total titer of 1.3-2.0 billion cells per 1 ml and a spore titer of 1-1.6 - 1.2-1.8 billion.