RU2736424C1 - Bacterial strain bacillus amyloliquefaciens recb-95 for production of biopreparation for protection of agricultural plants from stresses, stimulation of their growth and increase of crop capacity - Google Patents

Abstract

FIELD: agriculture; biotechnology.SUBSTANCE: invention relates to agricultural biotechnology and microbiology. Bacillus amyloliquefaciens RECB-95 strain deposited in the National Bioresource Center the All-Russian Collection of Industrial Microorganisms (BRC VKPM) Kurchatov Institute National Research Center - State National Research Institute Genetics under registration number VKPM V-13417. Strain of bacteria Bacillus amyloliquefaciens VKPM V-13417 is capable to increase resistance of plants to abiotic stresses and suppress growth and development of phytopathogenic fungi.EFFECT: invention increases crop yield.1 cl, 6 dwg, 10 tbl, 5 ex

Description

The invention relates to the field of agricultural microbiology and biotechnology, in particular, to the production of biological products to protect plants from stress, and can be used to create biological products to increase resistance to stress, stimulate growth and increase crop yields.

Various stresses (abiotic, biotic and anthropogenic) cause significant damage to both yield and quality characteristics of agricultural crops [1]. In the control of various plant stresses, various biological products based on various microorganisms are essential [2]. It is known that biological products have a positive effect on the productivity and quality characteristics of agricultural crops [3]. To solve this problem, biological preparations based on various microorganisms are most often used. Bacteria of the genus Bacillus [4] act as promising microorganisms for stress control, many of which increase plant resistance to abiotic stresses and infectious diseases. Spore-forming bacteria of the genus Bacillus have an advantage, since their spores are stable at high temperatures, unfavorable environmental conditions, which makes it possible to create biological products with long shelf life without losing activity [5]. In this regard, strains of bacteria of the genus Bacillus are promising for the creation of new biological products to protect plants from stress.

A large number of bacterial strains of the genus Bacillus are known that have the ability to suppress the growth of phytopathogenic fungi and stimulate the formation of crop yields. In particular, these are Bacillus subtilis VNIISKhM 128 and the biological product Fitosporin, created on the basis of this strain [6]; B. subtilis BZR 517 [7]; B. subtilis B 93 VIZR [8], B. mojavensis Lhv-97 [9] and others.

The disadvantage of all of the above strains is insufficient activity in reducing the negative effect of various stresses on plants and an insufficiently studied effect on the yield and quality of agricultural crops.

Closest to the proposed invention is the Bacillus amyloliquefaciens OPS-32 strain, which has the ability to suppress the development of phytopathogens, stimulate plant growth, increase the availability of nutrients from the soil and increase productivity [10]. However, for this strain, the effect on plant resistance to abiotic stresses has not been studied.

The objective of the invention is to identify a strain suitable for obtaining a biological preparation with high activity in increasing the resistance of plants to various stresses, increasing the productivity of agricultural crops and the quality characteristics of the products obtained, as well as allowing to expand the range of biological plant protection against the action of various stress factors.

The problem is solved by isolating the Bacillus amyloliquefaciens RECB-95B strain, suitable for obtaining a biological product, increasing resistance to stress, increasing the yield and quality of agricultural crops. The new strain was isolated from tomato stems and deposited at the National Bioresource Center All-Russian, collection of industrial microorganisms (BRC VKPM) NRC "Kurchatov Institute" - GosNIIgenetics under registration number VKPM B-13417.

The main selection criteria were an increase in plant drought resistance, stimulation of growth processes, suppression of the growth of phytopathogenic fungi, a positive effect on plant productivity and product quality, lack of pathogenicity to warm-blooded animals and compatibility with other microorganisms. The species was determined using molecular genetic methods by the nucleotide sequence in 16S rRNA, as well as amplification of a species-specific fragment characteristic of bacteria of the species Bacillus amyloliquefaciens at the Kurchatov Institute - GosNIIgenetics.

The strain is characterized by the following morphological, cultural and physiological and biochemical characteristics.

The cells of the strain are gram-positive aerobic spore-forming straight rods with rounded ends measuring 1.5-2.5 × 0.5-0.7 μm; are arranged, as a rule, in pairs or singly, chains are less common. During sporulation, the cells do not swell, the spores are elliptical, located centrally.

On meat-peptone agar (MPA) after 2 days forms rounded colonies with a scalloped edge and a crater-shaped center, 6-7 mm in diameter; with a predominantly folded-grooved surface (the surface of the “crater” is smooth), matte, opaque, the color is mainly milky white, in the furrows it is grayish beige. The colonies have a convex base, relief folds, and a significantly elevated central crater; They have a viscous, viscous consistency, while they have an outer soft leathery layer, they do not grow into agar.

On potato-glucose agar (CHA) after 2 days forms round colonies with a scalloped edge and a center in the form of a narrow cone, 5-10 mm in diameter, folded, opaque, opaque, milky white. The colonies have a flat base, raised folds, and a rising center; They have a viscous, viscous consistency, while they have an outer soft leathery layer, they do not grow into agar.

Bacillus amyloliquefaciens RECB-95B is an aerobic, chemoorganoheterotrophic bacterium that does not require growth factors. It grows in the temperature range from 10 to 47 ° C with an optimal range of 28-32 ° C, at pH values from 4.5 to 8.5 with an optimum of 7.0-7.5, with sodium chloride concentration up to 7%.

Shows the activity of tryptophanedaminase and gelatinase. The activity of β-galactosidase (ortonitrophenyl-βD-galactopyranosidase), arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease was not detected. Does not produce indole and hydrogen sulfide and does not reduce nitrates. The Voges-Proskauer reaction (acetoin production) is positive.

Utilizes D-glucose, D-fructose, D-xylose, D-ribose, L-arabinose, D-mannose, D-mannitol, inositol, D-sorbitol, methyl-αD-mannopyranoside, methyl-αD as a source of carbon and energy -glucopyranoside, D-maltose, D-cellobiose, D-lactose, amygdalin, arbutin, esculin, salicin, D-melibiose, D-sucrose, D-trehalose, D-raffinose, starch, glycogen, gentibiose, glycerin, citrate.

Does not utilize erythritol, D-arabinose, D-adonitol, D-galactose, L-xylose, L-sorbose, L-rhamnose, methyl-βD-xylopyranoside, dulcitol, N-acetylglucosamine, inulin, D-melecytosis, xylitol, D- Turanose, D-lyxose, D-tagatose, D-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate.

The strain is stored at 4-6 ° C in test tubes with semi-liquid agar in a mineral medium (composition: disubstituted potassium phosphate 5.8 g / l, monosubstituted potassium phosphate 3 g / l, ammonium sulfate 1 g / l, glycerol 2 g / l with the addition of 0 , 7% agar, pH 7.0-7.2) under vaseline oil, which is poured into test tubes after 2 days of culture growth. Under such conditions, the shelf life of the strain without reseeding is at least 1 year.

The strain grows well on MPA, KingB, LB medium, wort agar (composition: unhopped beer wort concentrate diluted with distilled water to a total sugar concentration of 10% (10 ° Balling), pH 7.0-7.2 with the addition of 2.0 % agar-agar), Gromyko's medium, Gauze's medium No. 2 (composition: tryptone - 2.5 g; peptone - 5.0 g; NaCl - 5.0 g; glucose - 10.0 g; agar-agar - 20 g ; tap water - 1000 ml; pH - 7.0-7.4) and mineral medium (composition: disubstituted potassium phosphate 5.8 g / l, monosubstituted potassium phosphate 3 g / l, ammonium sulfate 1 g / l, glycerin 10 g / l with the addition of 0.7% agar, pH 7.0-7.2). Fermentation is carried out on a mixture of equal volumes of meat-peptone broth and 6 ° B unhopped beer wort (pH 6.9-7.2) at 30 ° C to 95% sporulation. The number of CFU is at least 5 × 10 ⁹ in 1 ml.

The study of the pathogenicity of the claimed strain for warm-blooded animals was carried out at the Kazan Federal University, as a result of which it was concluded that in terms of virulence, dissemination, toxicity and toxicity, the Bacillus mojavensis PS17 strain is not pathogenic for warm-blooded animals and meets the requirements for industrial microorganisms.

The essence of the described invention is disclosed in more detail in the examples below.

Example 1. Study of the antagonistic activity of the Bacillus amyloliquefaciens RECB-95B strain.

To identify the antagonism of the studied strain to phytopathogenic microorganisms, a confrontational method was used. In the center of Petri dishes with King B medium, a block with the mycelium of the tested fungus was laid out, the Bacillus amyloliquefaciens RECB-95 strain was injected at a distance of 2-3 cm, the Pseudomonas putida PCL1760 strain was used as a negative control, which was also inoculated with an injection, which does not emit any or antagonistic substances that suppress the development of micromycetes (Fig. 1-6). Petri dishes were placed for 48-72 hours in thermostats at 25 ° C so that micromycetes and bacteria could develop. Observations were carried out on days 2-5, depending on the growth rate of the phytopathogen.

The results of determining the antagonistic activity of the Bacillus amyloliquefaciens RECB-95B strain are shown in FIG. 1-6. On which the following are indicated as options: 1) - Bacillus amyloliquefaciens RECB-95B strain, 2) Pseudomonas putida PCL1760. As a test culture of micromycetes used: Fig. 1 - Aspergillus flavus, fig. 2 - Penicillum sp., Fig. 3 - Fusarium equisety, fig. 4 - Fusarium oxysporum ZUM2407, fig. 5 - Fusarium sporotrichoides, fig. 6 - Trichoderma asperellum RECB-74F.

According to the information provided, the Bacillus amyloliquefaciens RECB-95B strain is an active antagonist and has a broad spectrum of fungicidal activity, since all micromycetes used in the study showed sensitivity to the antagonistic strain.

Example 2. Laboratory tests of phytotoxicity and growth-stimulating activity of the Bacillus amyloliquefaciens RECB-95B strain.

To test the stimulating properties of the strain Bacillus amyloliquefaciens RECB-95B, wheat grains of the "Sim-bircit" variety were used. For this, the grains were soaked in a cell suspension grown in a rich LB medium, which consists of 10 g / L bactotrypton, 5 g / L yeast extract, and 10 g / L sodium chloride. The prepared medium was sterilized for 40 minutes at 110 ° C. after soaking, the seeds were placed in a cuvette on filter paper moistened with tap water. The seeds were germinated for 7 days at a temperature of 23 ° C with a day length of 16 hours. The plants were collected and the length of the roots was measured. According to the results of the experiments, it was shown that the strain Bacillus amyloliquefaciens RECB-95B increased the length of the stems and roots of wheat (Table 1).

Example 3. Evaluation of the effect of Bacillus amyloliquefaciens RECB-95B on plant drought resistance.

To assess the effect of Bacillus amyloliquefaciens on drought resistance, vegetation experiments were carried out. The object of research is spring barley of the Raushan variety. Spring barley seeds were treated with experimental preparations of the studied strains at a rate of 1.0 l / t. For germination of barley seeds, 5 l vessels were used. The vessels were pre-weighed, expanded clay was added to the bottom of the vessel at an angle of 45 °, bringing the weight of each vessel to 500 g with expanded clay, 3.05 kg of sand were placed in each vessel, moistened in a separate vessel with Knop's solution to 50%. Seeds were sown to a depth of 1.5-2 cm, in 27 variants, 35 seeds per vessel (seeds were pre-germinated in Petri dishes in distilled water for a day, and only normally germinated seeds were selected for planting). After the end of planting, the vessels were weighed and covered with cling film on top until shoots appeared. Untreated seeds were used as a control; the biofungicide Rizoplan was the standard.

To assess the effect of drugs on drought resistance, the content of proline in leaves was determined. Proline is one of the most versatile stress metabolites in plants. An increased level of proline in plants is often associated with drought resistance [11]. In addition, proline performs chaperone, antioxidant, signal-regulatory, and other functions [12]. Under the action of stressors, conditions are created for the most pronounced manifestation of these effects of proline [13]. The free proline content was determined by the method of Bates et al. [fourteen].

The results of determining the content of proline in leaves show that the use of Bacillus amyloliquefaciens RECB-95B leads to a significant increase in the indicator both in comparison with the control and in the level of the standard biofungicide.

Determination of the effect of the studied strain on drought resistance was carried out by the germination of treated seeds of spring barley in sucrose solutions with different osmotic pressure [15]. Control and treated seeds were laid out on filter paper in Petri dishes, moistened with sucrose solutions with different osmotic pressure - 0 (pure water) atm., 1.12 atm. (0.05 N sucrose solution), 2.24 atm. (0.1 N sucrose solution), 4.48 atm. (0.2 N sucrose solution), 8.96 atm. (0.4 N solution) and 11.2 atm. (0.5 N solution) After 7 days, germination was determined. The experiment was repeated three times. The results are shown in Table 3.

The determination results showed that the use of Bacillus amyloliquefaciens RECB-95B has a positive effect on seed germination even under conditions of strong osmotic stress (drought simulation).

Example 4. Evaluation of the effectiveness of treatment of spring wheat plants with the strain Bacillus amyloliquefaciens RECB-95B in the field.

Field experiments were carried out in 2018 on the experimental fields of the Kazan State Agrarian University. The object of the study was spring wheat of the Yoldyz variety. The effectiveness of treatment with the Bacillus amyloliquefaciens RECB-95B strain was determined by seed treatment. The drug based on the studied strain was used at rates of 0.5 l / t, 1.0 l / t, 1.5 l / t, and 2.0 l / t of seeds. The control was the variant without treatment, and the biofungicide Rizoplan (0.5 l / t) was used as a standard. The flow rate of the working fluid during seed treatment is 10 l / t.

The results of evaluating the effect of seed treatment on field germination showed that the use of Bacillus amyloliquefaciens RECB-95B with rates of 1.5 and 2.0 l / t stimulates the germination of spring wheat seeds in the field (Table 4).

The use of Bacillus amyloliquefaciens RECB-95B with rates of 0.5 l / t, 1.0 and 1.5 l / t significantly reduced root rot infestation of spring wheat plants, surpassing the standard biofungicide in efficiency (Table 5).

The use of a preparation based on Bacillus amyloliquefaciens RECB-95B for the treatment of seeds of spring wheat, depending on the rate of expenditure, increases the yield by 0.42-0.89 t / ha (Table 6). In terms of its influence on the yield, the obtained strain exceeds the indicators of the standard biofungicide Rizoplan.

Example 5. Evaluation of the effectiveness of treatment of seeds of spring barley strain Bacillus amyloliquefaciens RECB-95B in the field.

Field experiments were carried out in 2018 on the experimental fields of the Kazan State Agrarian University. The object of the study was the spring barley of the Raushan variety. The effectiveness of treatment with the Bacillus amyloliquefaciens RECB-95B strain was determined by seed treatment. The drug based on the studied strain was used at rates of 0.5 l / t, 1.0 l / t, 1.5 l / t, and 2.0 l / t of seeds. The control was the variant without treatment, and the biofungicide Rizoplan (0.5 l / t) was used as a standard. The flow rate of the working fluid during seed treatment is 10 l / t.

In all variants with the use of Bacillus amyloliquefaciens RECB-95B, the proline content increased, which indicates an increase in plant drought resistance (Table 7).

The minimum values of damage to plants by root rot were when using Bacillus amyloliquefaciens RECB-95B with rates of 1.0 and 2.0 l / t (Table 8).

The highest yield of spring barley was when using Bacillus amyloliquefaciens RECB-95B (1.5 l / t). The increase was - 0.99 t / ha (Table 9).

The use of Bacillus amyloliquefaciens RECB-95B (with the exception of the rate of 1.5 l / t) led to an increase in the protein content in barley grain (Table 10).

Thus, the technical result of the claimed invention is to stimulate the growth of agricultural plants and protect them from stress, resulting in an increase in their productivity and quality characteristics. It is achieved by the fact that the bacterium strain Bacillus amyloliquefaciens RECB-95B is used as a plant treatment agent, which has a set of useful properties, such as the ability to increase plant resistance to drought, suppress the growth of phytopathogenic fungi, stimulate seedling growth, and increase the yield and quality of agricultural crops.

The invention was created with the financial support of the Ministry of Education and Science of the Russian Federation within the framework of the Federal Target Program "Research and Development in Priority Areas of Development of the Scientific and Technological Complex of Russia for 2014-2020" under agreement No. 14.610.21.0017 of October 03, 2017, unique project identifier RFMEFI61017X0017.

Claims (1)

The bacterial strain Bacillus amyloliquefaciens RECB-95B, which has the ability to increase plant resistance to stress and antagonistic activity against phytopathogens, increasing the yield and quality of agricultural crops, was deposited at the National Bioresource Center All-Russian collection of industrial microorganisms (BRC VKPM) NRC "Kurchatov Institute" - registration number VKPM B-13417.

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