RU2793159C1 - Consortium of microorganisms designed to improve plant growth - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a consortium formed from strains of microorganisms Bacillus subtilis-8 VKPM B-5251, Bacillus amiloliquefacienc VKPM B-11265, Flavobacterium arborescens L-30 VKPM B-1847, Azospirillum brasilense S94-3 VKPM B-7501, Azotobacter chroococcum BKPM-3173.

EFFECT: invention improves plant growth.

1 cl, 1 tbl

Description

Technical field

The invention relates to the field of agriculture and is used as a microbiological fertilizer to be applied to the soil before sowing (planting), for pre-sowing seed treatment and as a top dressing for all crops and ornamental plantings on various types of soils.

State of the art

A composition is known for implementing a method for enhancing plant growth (RF patent No. 2659000, publication date 06/26/2018). The composition contains a carrier and one or more flavonoids. When using a liquid carrier, the liquid carrier may further comprise a growth medium for culturing one or more microbial strains and contain one or more agriculturally useful ingredients, including one or more beneficial microorganisms. While one or more beneficial microorganisms include one or more phosphate-solubilizing microorganisms. Phosphate solubilizing microorganisms include fungal and bacterial strains. In one embodiment, the phosphate solubilizing microorganism is a microorganism selected from the group consisting of Bacillus amyloliquefaciens and Bacillus subtilis. In one embodiment, the compositions described herein may additionally contain one or more fungicides. Fungicides suitable for the compositions may be biological fungicides, chemical fungicides, or combinations thereof. In specific embodiments, the biological fungicide may be a bacterium of the genus, including Flavobacterium.

The disadvantage of the known technical solution is insufficient acceleration of plant growth.

The closest technical solution (prototype) is a method of processing plants to improve the properties of a plant and a means for its implementation (RF patent No. 2615834, publication date 04/11/2017). The method of treating plants to improve the properties of the plant contributes to better growth, or an increase in yield, or an intensive development of the root system, or an increase in leaf area, or an increase in the intensity of landscaping, or obtaining stronger shoots. According to the method, the agent for its implementation is applied to plants and parts of plants or their environment, habitat or storage place. The agent for carrying out the method contains fluopyram, a spore-forming bacterium of the genus Bacillus, at least one biological control agent, provided that the spore-forming bacterium of the genus Bacillus and the biological control agent are not identical. The spore-forming bacterium of the genus Bacillus is selected from the group including, but not limited to, Bacillus amyloliquefaciens, Bacillus subtilis. At least one biological control agent is selected from the group consisting of bacteria, fungi or yeasts, protozoa, viruses, entomopathogenic nematodes, inoculants, plant materials, and harpin (produced by Erwinia amylovora). In a particular case, at least one biological control agent is selected from the group including, but not limited to, Bacillus amyloliquefaciens, Bacillus subtilis, Azospirillum brasilense, Azotobacter chroococcum.

The disadvantage of the prototype is the insufficient growth rate of plants.

The technical result of the invention is to increase the growth rate of plants.

The set technical result is achieved due to the fact that the consortium intended to improve plant growth is formed from strains of microorganisms Bacillus subtilis-8 VKPM B-5251, Bacillus amiloliquefacienc VKPM B-11265, Flavobacterium arborescens L-30 VKPM B-1847, Azospirillum brasilense S94-3 VKPM V-7501, Azotobacter chroococcum VKPM-3173.

Disclosure of invention

A consortium is a community of two or more microorganisms that exists as a whole, connected by food chains and microecology (https://nauchkor.ru/uploads/documents/5f3f2860cd3d3e000153d06c.pdf, p. various interdependent organisms acting as a whole when using a certain substrate, according to the definition given in the Dictionary of Microbiology at https://dic.academic.ru/dic.nsf/dic_microbiology/535, accessed 03/16/2021. Between microorganisms in the consortium, there are various forms of relationships that allow you to maintain a stable symbiosis and balance within this consortium for a certain time.

Microorganisms used in this consortium are deposited in the All-Russian Collection of Industrial Microorganisms (hereinafter referred to as VKPM).

The consortium of microorganisms intended to improve plant growth (hereinafter sometimes referred to as the consortium of microorganisms) includes the following strains: Bacillus subtilis-8 VKPM B-5251, Bacillus amiloliquefacienc VKPM B-11265, Flavobacterium arborescens L-30 VKPM B-1847, Azospirillum brasilense S94-3 VKPM V-7501, Azotobacter chroococcum VKPM-3173.

The strain, according to the Encyclopedic Dictionary on the website https://dic.academic.ru/dic.nsf/es/65494/strain, accessed 07.10.2021, is a pure culture of microorganisms of one species, isolated from any source and having special characteristics. properties.

The Bacillus subtilis-8 strain was deposited with the VKPM with the registration number in the VKPM collection B-5251. The strain Bacillus subtilis-8 VKPM V-5251 was isolated from the soil of the Moscow region, the city of Sergiev Posad.

Nutrient medium for cultivation of Bacillus subtilis-8 VKPM B-5251 - meat-peptone broth (hereinafter referred to as MPB).

Nutrient medium for storage of Bacillus subtilis-8 VKPM V-5251 (agar nutrient medium) - meat-peptone agar (hereinafter referred to as MPA).

Morphology of Bacillus subtilis-8 VKPM B-5251: cells are rod-shaped with rounded ends, single or connected in pairs. Cells are motile, size (0.49-0.76)×(2.07-3.30) µm, spore-forming. The colonies are rounded, the surface is folded, with a crater in the middle, beige-gray, the edge is wavy.

Bacillus subtilis-8 VKPM B-5251 is a biofungicide producing strain, has activity against phytopathogenic fungi from the genera Fusarium, Microdochium, Pyrenophora, Botrytis, Alternaria and Rhizoctonia, and also provides the synthesis of subtilin and B vitamins. Bacillus subtilis-8 VKPM B-5251 is not pathogenic, environmentally non-toxic.

The role of Bacillus subtilis-8 VKPM B-5251 in this consortium - inhibits the development of phytopathogenic fungi that cause plant diseases.

The Bacillus amyloliquefaciens strain was deposited with the VKPM, registration number of the VKPM collection B-11265. Bacillus amiloliquefacienc VKPM V-11265 from the rhizosphere of spring wheat growing in the Gafury region of the Republic of Bashkortostan.

As a nutrient medium for the cultivation of Bacillus amiloliquefacienc VKPM B-11265, MPA can be used, cultivated at a temperature of 30°C.

Nutrient medium for storage of Bacillus amiloliquefacienc VKPM V-11265 (agar nutrient medium) - MPA.

Morphology of Bacillus amiloliquefacienc VKPM B-11265 - strain cells are gram-positive aerobic spore-forming straight rods with rounded ends measuring (1.5-2.5) × (0.5-0.7) μm, usually arranged in pairs or singly , chains are less common, colonies are flesh-colored, rounded, with ribbed edges, matte. During sporulation, the cells do not swell. Spores are ellipsoid, located centrally.

Bacillus amiloliquefacienc VKPM B-11265 is a biofungicide producing strain, has activity against phytopathogenic fungi Claviceps purpurea (ergot pathogen), Rhizopus stolonifer, Cercospora beticola, Passalora fulva, and also provides the synthesis of brassinolide and catasterone. Bacillus amiloliquefacienc VKPM B-11265 is a protease producer. Bacillus amyloliquefaciens VKPM B-11265 is non-pathogenic and environmentally safe.

The role of Bacillus amiloliquefacienc VKPM B-11265 in this consortium: the strain affects phytopathogens by synthesizing substances that limit their growth, these substances are able to induce systemic plant resistance to a wide range of pathogens.

The Flavobacterium arborescens L-30 strain was deposited with the VKPM, registration number of the VKPM collection B-1847. Origin of Flavobacterium arborescens L-30 VKPM B-1847: Wastewater from aniline can also be isolated from the epiphytic microbiocenosis of the apical part of the rice root.

Conditions for cultivation of the strain Flavobacterium arborescens L-30 VKPM V-1847: L-medium (composition: yeast extract - 5.0 g, peptone - 15.0 g, NaCl - 5.0 g, agar - 15.0 g, distilled water - 1.0 l or other quantities, depending on the required total volume) temperature 28°C.

Nutrient medium for storage of Flavobacterium arborescens L-30 VKPM V-1847 (agar nutrient medium) - L-medium.

The strain Flavobacterium arborescens L-30 VKPM B-1847 is rod-shaped cells, do not form spores, are immobile. Colonies on cabbage agar are rhizoidal, up to 3 mm in diameter, flat, shiny, orange, smooth, uneven edges. The culture uses mineral and organic nitrogen, gelatin does not liquefy, weak growth on media with carbohydrates (lactose, arabinose) with acidification, is able to hydrolyze starch, there is no growth at a temperature of 37 ° C, they are catalase-positive, they are a facultative anaerobe. Temperature range of growth of Flavobacterium arborescens L-30 VKPM V-1847 (22-30)°С. The optimal temperature for the growth and reproduction of this strain is (25-28)°C.

Flavobacterium arborescens L-30 VKPM B-1847 is an active producer of physiologically active substances (indolyl-3-lactic acid (hereinafter referred to as IBA), indolyl-3-carboxylic acid (hereinafter referred to as ICA), indolyl-acetic acid (hereinafter according to the text - IUK)).

In this consortium, Flavobacterium arborescens L-30 VKPM B-1847 synthesizes a number of phenazine-type antibiotics that inhibit the growth and development of phytopathogenic fungi of the Fusarium genus and inhibit the development of pathogenic microflora. Flavobacterium arborescens L-30 VKPM B-1847 is not pathogenic and environmentally friendly.

The Azotobacter chroococcum strain was deposited with VKPM under the registration number in the collection VKPM-3173. Azotobacter chroococcum BKPM-3173 was isolated from urban soil at 52 Timiryazevskaya Street, Moscow.

Nutrient medium for cultivation of Azotobacter chroococcum VKPM-3173: Ashby medium or composition medium, wt %: molasses - 3.0; K ₂ HPO ₄ - 0.03; MgSO ₄ - 0.03; NaCl - 0.05; K ₂ SO ₄ - 0.02; Fe ₂ Cl ₃ - 0.001; CaCO ₃ - 0.5.

Nutrient medium for storage of Azotobacter chroococcum VKPM-3173 (agar nutrient medium): (wt.%) KN ₂ PO ₄ - 0.05; MgSO ₄ - 0.03; NaCl - 0.03; Fe ₂ (SO ₄ ) ₃ - 0.0005; MnSO ₄ - 0.0005; (NH ₄ ) ₂ MoO ₇ - 0.0005; CaCO ₃ - 0.35; molasses - 3.0; agar - 2.0.

Morphology of Azotobacter chroococcum BKPM-3173: when microscopic examination of the cell, small rods, weakly mobile; mature cells - small sticks, coccoid shape, immobile, form thick-walled cysts.

Azotobacter chroococcum BKPM-3173 inhibits the growth of Alternaria sp., Pythium de Baryanum, Fusarium oxysporum, Fusarium vasinfectum, Fusarium moniliforme, Fusarium lini, Cladosporium sp., Scolecotrychum sp., Helmintosporium sativum, Botrytis sp., Verticillium dahliae and Rhizoctonia sp. , Puccinia triticina, Septoria graminum. It has a high nitrogenase activity, the ability to synthesize indoline-acetic acid.

The role of Azotobacter chroococcum BKPM-3173 in this consortium: the strain ensures the formation of the optimal root system of the plant in relation to the conditions in which it is located (planting frequency, soil properties, moisture, etc.). Azotobacter chroococcum VKPM-3173 has a positive effect on the native microflora, increases enzymatic activity and soil fertility.

Azotobacter chroococcum BKPM-3173 is not pathogenic and environmentally safe.

The Azospirillum brasilense S94-3 strain was deposited with the VKPM under the registration number VKPM B-7501. Azospirillum brasilense S94-3 VKPM B-7501 was isolated from the maize rhizosphere.

Nutrient medium for the cultivation of Azospirillum brasilense S94-3 VKPM B-7501: nutrient medium used for the cultivation of microorganisms in the American Type Culture Collection, with the composition (g/l): KH ₂ PO ₄ - 0.4; K ₂ HPO ₄ - 0.1; MgSO ₄ ⋅7H ₂ O - 0.2; NaCl - 0.1; CaCl ₂ - 0.02; FeCl ₃ - 0.01; Na ₂ MoO ₄ ⋅2H ₂ O - 0.002; sodium malate - 5.0; yeast extract - 0.05; glucose - 5.0; distilled water - up to 1 liter; pH 7.0.

Nutrient medium for storage of Azospirillum brasilense S94-3 VKPM V-7501 (agar nutrient medium): potato agar (hereinafter referred to as KA), composition: 1 l decoction obtained from 200 g of peeled potatoes, obtained with 1 l of tap water, with the addition of 20 g of agar , 1 ml 1% alcohol solution of Congo red.

Morphology of Azospirillum brasilense S94-3 VKPM B-7501: cells are short, straight rods, negatively stained by Gram, have a characteristic helical movement. Granules of poly-β-hydroxybutyrate accumulate inside the cells.

Azospirillum brasilense S94-3 VKPM B-7501 is able to fix atmospheric nitrogen, as evidenced by the nitrogenase activity detected by the acetylene method.

The role of Azospirillum brasilense S94-3 VKPM B-7501 in this consortium: the strain stimulates the development of the root system and increases its absorption capacity. Azospirillum brasilense S94-3 VKPM B-7501 enhances plant uptake of NPK. Azospirillum brasilense S94-3 VKPM B-7501 mobilizes hard-to-reach nutrients from the soil.

Azospirillum brasilense S94-3 VKPM B-7501 is non-pathogenic and environmentally friendly.

Implementation of the invention

The invention is implemented as follows.

The following is an example of the implementation of the invention.

First, strains of Bacillus subtilis-8 VKPM B-5251, Bacillus amiloliquefacienc VKPM B-11265, Flavobacterium arborescens L-30 VKPM B-1847, Azospirillum brasilense S94-3 VKPM B-7501, Azotobacter chroococcum BKPM-3173 are grown on an agar nutrient medium (solid-phase cultivation).

Prepare for each of the strains (cultures) Bacillus subtilis-8 VKPM B-5251, Bacillus amiloliquefacienc VKPM B-11265, Flavobacterium arborescens L-30 VKPM B-1847, Azospirillum brasilense S94-3 VKPM B-7501, Azotobacter chroococcum BKPM-3173 agarized nutrient medium. Description of the agar nutrient medium for each strain is given above. The resulting agar nutrient media are sterilized for 30 minutes (at an elevated pressure of 1.0 atm or 0.5 atm).

After sterilization, the agar nutrient medium for each strain is placed in a separate sterile container (sterile Petri dishes and/or sterile test tubes). Agar culture media are dispensed hot. The obtained Petri dishes and/or test tubes with agar nutrient media are left for 6 hours to solidify.

Next, each individual culture is inoculated into a Petri dish and/or a test tube with frozen agar nutrient medium prepared for this culture. Sowing is carried out with a microbiological loop, by streaking into Petri dishes and/or test tubes with frozen agar medium. Then the obtained Petri dishes and/or tubes with cultures (strains) are incubated for 48 hours in a thermostat at 28°C.

Next, the quality (cultural and morphological properties) of the crops is assessed (the absence of contamination is checked).

To assess the quality, take one-day cultures grown on agar nutrient media. Washing with 0.9% NaCl solution transfer each culture into a separate sterile tube. The culture liquids obtained as a result of washing are taken in a volume of 0.1 ml each. Further, by means of microscopy, the presence of foreign microflora is assessed with each of the culture fluids. The concentration of microorganisms is determined in the Goryaev chamber. The concentration of bacterial cultures should be at least 1.0×10 ¹⁰ cells/ml.

Then grow the required number of cultures on nutrient media for cultivation (liquid medium). Take cultures grown on agar nutrient media. For each culture prepare a separate nutrient medium for cultivation. The description of the media for the cultivation of each strain is given above.

In a sterile culturing dish (hereinafter referred to as a sterile mattress) with a volume of 1 l, 250 ml of a nutrient medium is added for cultivating one culture. At the same time, separate sterile mattresses are prepared for each culture with a nutrient medium for cultivation corresponding to this culture. The obtained sterile mattresses with nutrient media for cultivation are autoclaved at an elevated pressure of 1.0 atm or 0.5 atm for 30 minutes.

Each culture is sown in a separate mattress with a nutrient medium for cultivation corresponding to this culture. At the same time, the ratio is observed: at least 1.0×10 ⁹ cells of microorganisms are introduced into 1 mattress. Cultivation of all cultures is carried out microaerophilically for 2-4 days at 28°C.

Next, evaluate the quality of each of the cultures (number of viable cells). A 0.1 ml sample of culture fluid grown in it is sterilely taken from each mattress. The obtained samples of each culture liquid are subjected to staining with a fluorescent dye propidium iodide. The fluorescent dye propidium iodide stains only microorganisms with impaired permeability due to penetration and interaction with nucleic acids. This method allows you to stain only dead and damaged microorganisms. Next, produce microscopy of the sample of each culture fluid. The number of viable cells should be more than 98% of the total number of cells in the bacterial culture.

A consortium of microorganisms is formed by combining cultures grown on nutrient media for cultivation. To form a consortium, the required volume of the culture liquid grown in it is sterilely taken from each mattress and transferred to a mixing vessel (bottle). In a particular case, for example, the volumes of culture liquids in the mixture are for Bacillus subtilis VKPM B-5251 - 0.25 l, Bacillus amiloliquefacienc VKPM B-11265 - 0.25 l, Flavobacterium arborescens VKPM B-1847 - 0.1 l, Azospirillum brasilense VKPM V-7501 - 0.2 l, Azotobacter chroococcum VKPM-3173 - 0.2 l, everything is mixed.

Purification of the obtained biological product is not required.

In the resulting biological product, the content of microorganisms is at least 1.0×10 ⁹ cells/ml.

Containers with a biological product are stored in closed warehouses, which exclude the ingress of precipitation, direct sunlight and groundwater, in places inaccessible to children, separately from pesticides. Containers with a biological product are stored at room temperature (20-25)°C for up to 12 months, and at a temperature from +4°C to +15°C - up to 18 months. It is not allowed to store containers with a biological product below +4°С and above plus +25°С. During storage, contact with alkalis, acids and petroleum products should be avoided.

For the introduction of a biological product, standard and special technical means are used in agricultural production, designed to perform this type of work.

Plants are treated with a working solution prepared on the basis of a biological product. To prepare the working solution, non-chlorinated water is used, with a temperature of (20-25) ° C. When preparing a working solution, water is poured into the container used (watering system or sprayer tank), approximately 2/3 of the volume. With the mixing device turned on, the required amount of the biological product is added. Add water to the calculated volume. The working solution is stirred and processed.

Presowing (preplanting) treatment of seeds (tubers) of cereals, legumes, industrial crops, potatoes should be carried out by spraying. With small volumes, it is possible to use concrete mixers.

Seeds, planting material of vegetable, flower and ornamental crops are soaked in glass, enameled, polyethylene dishes, as well as in containers made of stainless steel.

For foliar feeding of plants, any sprayers should be used. Root top dressing is recommended to be carried out through irrigation systems (drip irrigation, sprinkling and other installations).

It is not recommended to carry out foliar feeding in hot sunny weather.

Consumption rates of the working solution for foliar and root dressing of various crops in agricultural production are generally accepted. Some examples are shown in Table 1.

Table 1 Application dose culture, time,
application features As needed (concentration of 100 ml of the drug per 1 liter of water) Vegetable, flower-decorative, fruit and berry crops - soaking seeds before sowing for 8-12 hours; cuttings, root system of seedlings, seedlings - for 3-4 hours Consumption of the working solution - until the earthen coma is moistened (concentration of 50 ml of the drug per 1 liter of water) Vegetable, flower and ornamental crops (seedlings) - root feeding of plants 2-3 times with an interval of 7-10 days Consumption of working solution - 1l per 10-30m ² (concentration 20-60ml per 10l of water) Vegetable crops, potatoes, strawberries, flower and ornamental crops - foliar feeding of plants during the growing season 2-3 times with an interval of 10-15 days Consumption of working solution - (2-5) l per 1 m ² (concentration 20-40 ml per 10 l of water) Vegetable crops, potatoes, strawberries, fruit and berry, flower and ornamental crops - root feeding of plants during the growing season 1-3 times with an interval of 10-15 days Consumption of the working solution - 2-5l / m ² (concentration 60-80ml per 10l of water) Lawn grasses - root feeding of plants after mowing

To prevent the biological product from getting into the lower layers of the soil, root feeding of plants is carried out after the main watering.

When using fertilizer, it is necessary to comply with general safety requirements (including the use of personal protective equipment).

Thus, the consortium consisting of microorganism strains Bacillus subtilis-8 VKPM B-5251, Bacillus amiloliquefacienc VKPM B-11265, Flavobacterium arborescens L-30 VKPM B-1847, Azospirillum brasilense S94-3 VKPM B-7501, Azotobacter chroococcum BKPM-3173 improves plant growth.

Claims (1)

Consortium designed to improve plant growth, consisting of microorganism strains Bacillus subtilis-8 VKPM B-5251, Bacillus amiloliquefacienc VKPM B-11265, Flavobacterium arborescens L-30 VKPM B-1847, Azospirillum brasilense S94-3 VKPM B-7501, Azotobacter chroococcum BKPM-3173.