Bacillus subtilis strain 2.2 and biomodified mineral fertilizer containing same

Abstract

The invention relates to agricultural biotechnology and microbiology, and more particularly to the production and use of Bacillus subtilis strain 2.2 in crop cultivation for the purpose of stimulating plant growth, protecting plants against phytopathogens, improving plant nutrition and increasing crop yield. The invention further relates to the biomodification of mineral fertilizers and soil enhancers using Bacillus subtilis strain 2.2, as well as to the production of biomodifiers and biomodified mineral fertilizers and soil enhancers for use in crop cultivation for the purpose of protecting plants against phytopathogens, stimulating plant growth, improving plant nutrition and increasing crop yield.

Description

[0001] STRAIN BACILLUS SUBTILIS 2.2 AND BIOMODIFIED MINERAL FERTILIZER CONTAINING IT

[0002] Field of technology

[0003] The invention relates to agricultural biotechnology and microbiology, in particular to the biomodification of mineral fertilizers and ameliorants and to the production of biomodifiers and biomodified mineral fertilizers and ameliorants for use in plant growing for the purpose of stimulating plant growth, improving nutrition and increasing the yield of agricultural crops.

[0004] State of the art

[0005] Currently, there are many microbiological preparations for agriculture for various purposes: growth-stimulating, as well as suppressing the development of phytopathogenic bacteria and fungi.

[0006] A method for producing biofertilizers is known (patent RU2241692, 11.10.2002), which consists of combining granulated mineral fertilizers with microbial biomass in the form of a bacterial preparation based on the Bacillus subtilis 4-13 strain, which has antagonistic properties to phytopathogens and a growth-stimulating effect. The combination is carried out by applying a bacterial preparation in the form of a dry powder or a liquid fraction to the surface of mineral fertilizer granules. These methods ensure increased efficiency of plant protection against infectious diseases that penetrate along with mineral fertilizers.

[0007] The biological preparation "Fitosporin" is known for protecting plants from diseases (patent RU2099947, 15.11.1996). The basis of the biological preparation "Fitosporin" is the strain of bacteria Bacillus subtilis VNIISKhM 128 with a cell concentration of 10 9 -1 O 10per 1 ml of saline solution in the amount of 92-98 vol.% and filler in the amount of 2-8 vol.%. At the same time, the Bacillus subtilis strain VNIISKhM 128 is characterized by high antagonistic activity against phytopathogenic bacteria and fungi, which allows it to be used to protect various types of agricultural, ornamental, and woody plants.

[0008] The strain of bacteria Bacillus pumilus A1.5 is known, used as a means of increasing plant productivity and protecting them from diseases caused by phytopathogenic microorganisms (patent RU2551968, 01.08.2013). The strain has fungicidal and bactericidal activity against phytopathogenic bacteria.

[0009] Despite the large number of developments and the use of various strains, the issues of increasing the yield of agricultural crops and their resistance to various diseases are still very relevant. Disclosure of the invention

[0010] The objective of the present invention is to obtain a strain of bacteria capable of enhancing the growth and productivity of plants, as well as protecting them from phytopathogenic fungi and increasing their resistance to diseases, and the objective of the present invention is to increase the effectiveness of mineral fertilizers and ameliorants in enhancing the growth and productivity of plants, protecting them from phytopathogenic fungi and increasing their resistance to diseases. The objective of the present invention is also to develop and create biomodified mineral (biomineral) fertilizers and ameliorants.

[0011] The task is solved by means of the bacterial strain Bacillus subtilis 2.2, deposited in the VKPM collection under number B-13292, characterized by plant growth stimulating activity, protecting plants from phytopathogens, improving nutrition, and increasing plant yields.

[0012] The subject of the present invention is also the use of the Bacillus subtilis 2.2 strain as a microbiological fertilizer in plant growing. The present invention also includes a microbiological fertilizer comprising the Bacillus subtilis 2.2 bacterial strain and, optionally, at least one auxiliary substance. In particular embodiments of the invention, the auxiliary substance is water. In various embodiments of the invention, the bacteria (microbiological fertilizer) are applied to the seeds and / or leaves and stems of the plant and / or introduced into the soil.

[0013] The subject of the present invention is also the use of the Bacillus subtilis 2.2 strain for the control of phytopathogens in plant growing, including for the control of phytopathogenic (and toxigenic) fungi.

[0014] The stated task is also solved by using the Bacillus subtilis 2.2 strain for biomodification of mineral fertilizers and / or ameliorants. For this purpose, the Bacillus subtilis 2.2 strain is applied to ameliorants or mineral fertilizers used both in dry (solid) and liquid form. In some embodiments of the invention, the mineral fertilizer in dry (solid) form is a fertilizer in granulated, prilled, microgranulated, powdered form or in the form of a water-soluble mineral fertilizer.

[0015] In various embodiments of the invention, the bacteria are applied directly to the mineral fertilizer or ameliorant or using a carrier. In some embodiments of the invention, the carrier is a mineral or organic natural or synthetic substance acceptable in agriculture. In some particular embodiments of the invention, the carrier is a natural or non-natural substance based on siliceous rocks. In some particular embodiments of the invention, the carrier is diatomite, zeolite, opoka, tripoli, kaolin, glauconite, silica, perlite, metakaolin, wollastonite, bentonite, inorganic microsilica, belite, white carbon black, vermiculite (expanded), talc or diabazite.

[0016] In some embodiments of the invention, when immobilizing bacteria, a means is used that increases (enhances) the adhesion of bacteria to a mineral fertilizer and / or carrier. In some particular embodiments of the invention, cationic starch, collagen glue, bone-gelatin glue, albumin glue, fish glue, cherry glue, dextrin, molasses, gum arabic, or liquid glass are used as a means that increases the adhesion of bacteria.

[0017] In some embodiments of the invention, the mineral fertilizer is a nitrogen, phosphorus, potassium, boron, molybdenum, complex, complex-mixed, water-soluble or micro fertilizer, or a mixture of one of the fertilizers with microelements. In some particular embodiments of the invention, the mineral fertilizer is urea, ammonium nitrate, ammonium sulfate, calcium nitrate, ammophos, sodium nitrate, diammonium phosphate, nitroammophoska, phosphate rock, potassium sulfate, potassium salt, sulfoammophos, diammophos, azophoska, nitrophos, nitrophoska, potassium nitrate, potassium chloride or potassium magnesium sulfate.

[0018] In some embodiments of the invention, the ameliorant is an ameliorant-sorbent, an ameliorant-structure former, an ameliorant fertilizer, including, for example, phosphogypsum, phosphate flour.

[0019] In various embodiments of the invention, the bacteria are used in spore form, since it ensures longer storage and survival of the bacteria.

[0020] The stated task is solved by developing a biomodifier of mineral fertilizers and ameliorants (a bacterial preparation for modifying mineral fertilizers and ameliorants), including the bacterial strain Bacillus subtilis 2.2 and, optionally, at least one auxiliary substance.

[0021] In some particular embodiments of the invention, the auxiliary substance is a carrier and / or an agent increasing the adhesion of bacteria to a mineral fertilizer and / or an ameliorant and / or a carrier. In particular embodiments of the invention, the auxiliary substance is water. In other particular embodiments of the invention, the carrier is diatomite, zeolite, opoka, tripoli, kaolin, glauconite, silica, perlite, metakaolin, wollastonite, bentonite, inorganic microsilica, belite, white carbon black, vermiculite (expanded), talc or diabazite.

[0022] In particular embodiments of the invention, the concentration of bacteria on the carrier is not less than 10 4 CFU / g carrier. In particular embodiments of the invention, the concentration of bacteria in the liquid microbiological preparation (in particular, microbiological fertilizer) and / or biomodifier is at least 10 5 CFU / ml of bacterial suspension.

[0023] In particular embodiments of the invention, the biomodifier additionally includes other strains of agronomically useful microorganisms.

[0024] The set task is also solved by developing a biomodified mineral (biomineral) fertilizer and ameliorant for protecting plants from phytopathogenic (and toxigenic) fungi, improved nutrition and stimulation of plant growth, including mineral fertilizer and bacteria of the Bacillus subtilis 2.2 strain.

[0025] In various embodiments of the invention, the mineral fertilizer is used in solid or liquid form. In some embodiments of the invention, the mineral fertilizer in solid form is a fertilizer in granulated, prilled, microgranulated or powdered form.

[0026] In particular embodiments, the bacteria are applied to the mineral fertilizer or ameliorant directly or using a carrier. In some embodiments of the invention, the concentration of bacteria in the biomodified mineral (biomineral) fertilizer (or, respectively, ameliorant) is at least 10 3 CFU per 1g of solid mineral fertilizer (ameliorant). In private cases - not less than 10 4 CFU per 1g of solid mineral fertilizer (ameliorant).

[0027] In some embodiments of the invention, the bacteria are mixed with liquid mineral fertilizer. In particular embodiments of the invention, the concentration of bacteria is at least 10 3CFU per 1 ml of liquid mineral fertilizer.

[0028] The present invention also includes the use of the Bacillus subtilis 2.2 strain for producing a biomodifier of mineral fertilizers and ameliorants.

[0029] The present invention also includes the use of a biomodifier based on the Bacillus subtilis 2.2 strain for obtaining a biomodified mineral (biomineral) fertilizer or ameliorant for protecting plants from phytopathogenic fungi, improving nutrition and stimulating plant growth, increasing their resistance to diseases, and increasing the efficiency of digestibility, respectively, of mineral fertilizers and / or ameliorants.

[0030] The stated task is also solved by developing a method for biomodifying mineral fertilizers and ameliorants, including mixing bacteria of the Bacillus subtilis 2.2 strain (the biomodifier according to the invention) with a mineral fertilizer or ameliorant, respectively. In some embodiments of the invention, said method includes immobilizing bacteria of the Bacillus subtilis 2.2 strain directly on the mineral fertilizer or ameliorant, as well as using a carrier. In some particular embodiments of the invention, a bacterial suspension (or a suspension of bacteria to which auxiliary substances have been preliminarily added) is mixed with a liquid mineral fertilizer or directly applied to the surface of a solid mineral fertilizer or ameliorant.

[0031] In other particular embodiments of the invention, the bacterial suspension is first applied to the carrier, after which the resulting biomodifier is applied to the mineral fertilizer (or ameliorant) by mixing. Since the content of CFU (colony forming units) of microorganisms in the bacterial suspension affects the number of CFU of microorganisms ultimately deposited on the carrier and the mineral fertilizer and ameliorant, their concentration is selected based on the amount that is the target for content in the biomodified mineral (biomineral) fertilizer / ameliorant. The more CFU in the bacterial suspension, the greater the number of agronomically useful microorganisms deposited on the carrier and the mineral fertilizer / ameliorant. In particular embodiments of the invention, bacteria are applied to the carrier until a concentration on the carrier of at least 10 is achieved. 4CFU per 1 g of carrier. In particular embodiments of the invention, the resulting biomodifier is applied to mineral fertilizer / ameliorant in a ratio of at least 1 kg per ton of mineral fertilizer / ameliorant.

[0032] The present invention also includes the use of the Bacillus subtilis 2.2 strain for producing a microbiological fertilizer.

[0033] As a result of implementing the invention, the following technical results are achieved:

[0034] - a strain of bacteria Bacillus subtilis 2.2 was obtained, which is capable of enhancing the growth and productivity of plants, as well as protecting them from phytopathogenic fungi and increasing their resistance to diseases;

[0035] - a new effective approach to the biomodification of mineral fertilizers and ameliorants, as well as a biomodification method, has been developed;

[0036] - biomodified mineral fertilizers and ameliorants according to the invention provide protection against phytopathogenic (and toxigenic) fungi, increased growth and productivity of plants, as well as increased resistance to diseases due to the effective combined effect caused by the ability of the Bacillus subtilis 2.2 strain to produce antifungal and auxin-like substances, enzymes and other organic compounds capable of suppressing the development of phytopathogens, stimulating the growth of the root and sprout system of plants - on the one hand, and providing plants with additional nutrients contained in mineral fertilizers (ameliorants) and soil - on the other;

[0037] - the key feature of the Bacillus subtilis 2.2 strain is the ability to suppress the development of phytopathogenic (and toxigenic) fungi and produce auxin-like substances that stimulate the growth of the root system of plants, therefore the use of the strain in individual microbiological preparations (in particular, in microbiological fertilizer) and / or in the composition of biomineral fertilizers / ameliorants and biomodifiers ensures the protection of plants from phytopathogens, an increase in the phytomass and productivity of plants;

[0038] - biomodified mineral (biomineral) fertilizers and ameliorants according to the invention are effective for use in growing various plants - vegetable, grain and industrial crops in agriculture, plants used in floriculture, forestry, and others, without restrictions;

[0039] - biomodifier of mineral fertilizers and ameliorants (biomodified mineral fertilizer and ameliorant) based on the Bacillus subtilis 2.2 strain allows to reduce the loss of mineral fertilizers (ameliorants) by their leaching and volatilization;

[0040] - the use of the bacterial strain Bacillus subtilis 2.2 in microbiological preparations (in particular, in microbiological fertilizer) and / or as part of a biomodified mineral fertilizer and ameliorant makes it possible to increase the efficiency of their absorption by plants;

[0041] - the biomodified mineral (biomineral) fertilizer and ameliorant according to the invention is characterized by the long-term viability of bacteria in its composition, at least for 3 months (and in most cases much longer), in concentrations that ensure the effectiveness of biomineral fertilizers and ameliorants;

[0042] - microbiological fertilizer based on the Bacillus subtilis 2.2 strain is effective for use in growing various plants - vegetable, grain and industrial crops in agriculture, plants used in floriculture, forestry, and others, without restrictions; the developed microbiological fertilizer and biomineral fertilizers / ameliorants and biomodifiers based on the Bacillus subtilis 2.2 strain expand the range of products for increasing yields and improving the growth of agricultural crops and other plants and their resistance to various diseases.

[0043] Terms and definitions

[0044] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as understood by those skilled in the art. References to techniques used in describing this invention refer to well-known techniques, including variations of these techniques and their replacement by equivalent techniques known to those skilled in the art.

[0045] In the documents of this invention, the terms "includes", "including", etc., as well as "contains", "containing", etc. are interpreted as meaning "includes, among other things" (or "contains, among other things"). These terms are not intended to be interpreted as "consists only of". This applies, inter alia, to the use of the Bacillus subtilis strain 2.2 and to biomodifiers of mineral fertilizers or ameliorants, as well as to biomodified mineral fertilizers or biomodified ameliorants. According to the invention, the Bacillus subtilis strain 2.2 can be used not only in isolation, but also in consortia with other strains.

[0046] The term "and / or" means one, more, or all of the listed elements.

[0047] Also here, listing numeric ranges by endpoints includes all numbers within that range.

[0048] The term "optional" or "optional" or "optionally" as used herein means that the subsequently described event or circumstance may, but need not, occur and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0049] The strain Bacillus subtilis 2.2 is understood to mean not only the strain Bacillus subtilis 2.2, which was deposited in the VKPM collection under number B-13292, but also a mutant strain obtained using the deposited strain as the source material and preserving all the properties of the original strain, i.e. possessing all the defining (identifying) characteristics of the strain Bacillus subtilis 2.2. Including, at least, the ability to produce auxin-like substances, suitable for use in enhancing plant growth and productivity, protecting them from phytopathogenic fungi, and increasing plant resistance to diseases. In particular, the strain Bacillus subtilis 2.2 has the following identifying features, including (but not limited to) 1) the ability to produce 3-indoleacetic acid and its derivatives on a medium with L-tryptophan; 2) the ability to suppress phytopathogenic and toxigenic fungi, such as Fusarium sporotrichioid, Alternaria solani, Fusarium culmorum; 3) the presence of essential genes with an identity of more than 95%, more than 96%, more than 97%, more than 98% or more than 99% with the essential genes of Bacillus subtilis 2.2, which can be used to confirm that the analyzed variant is Bacillus subtilis 2.2.

[0050] The term "mutant" or "variant" as used herein with respect to the strain Bacillus subtilis 2.2 means a modification of the parent strain in which the target biological activity is similar to the activity expressed by the parent strain. Mutants or variants may arise in nature without human intervention. They may also be produced by treatment with one or more methods and chemicals (compositions) known to those skilled in the art, or by gamma, X-ray or ultraviolet radiation, or any other means well known to those skilled in the art.

[0051] "Carrier" according to the invention is any substance that is not prohibited for use in agriculture. This may be, without limitation, both a mineral and an organic substance, both natural and synthetic. The main requirements for carriers according to the invention are a large specific surface area, acceptable for the application of the bacterial strain according to the invention, and safety for use in plant growing, in particular in agriculture. First of all, such carriers should not contain heavy metal cations and radioactive substances. In some non-limiting embodiments of the invention, the carrier is a natural or non-natural substance based on silicon-containing rocks. Non-limiting examples of carriers may include diatomite (kieselguhr), zeolite, opoka, tripoli, kaolin, glauconite, silica, perlite, metakaolin, wollastonite, bentonite, inorganic microsilica, belite, white carbon black, vermiculite (expanded), talc, and diabazite.

[0052] According to the invention, a "bacterial suspension" is understood to be a complex mixture obtained by culturing agronomically useful microorganisms in vitro and containing the cultured microorganisms, residual nutrients and metabolic products of these microorganisms. The bacterial suspension is obtained as a result of fermentation - a set of successive operations from the introduction of agronomically useful microorganisms into a previously prepared and heated to the required temperature nutrient medium and until the completion of the cell growth process. The composition of the nutrient medium for cultivation (the content of substances in an easily digestible form necessary to meet nutritional and energy needs), its temperature, cultivation time and other necessary conditions (including, for example, pH, the presence of growth factors, vitamins, buffering, etc.) are selected as optimal depending on the specific strain of the cultivated agronomically useful microorganisms.

[0053] "Culture liquid" is a term denoting a nutrient medium in which microorganisms were grown, which contains their metabolites and other target substances produced by microorganisms, as well as residual nutrients, in contrast to a "bacterial suspension", which in addition to the culture liquid contains cells of agronomically useful microorganisms. In the present invention, a "microbiological preparation" is understood to mean a composition including microorganism cells, live or in spore form, as well as their metabolic products, which, in particular, are either in the culture liquid or adsorbed on a neutral carrier. Bacteria of the Bacillus subtilis 2.2 strain, deposited in the VKPM collection under number B-13292, are used as microorganisms in the microbiological preparation according to the invention.The microbiological preparation according to the invention can be used, in particular, to combat phytopathogens, including to combat phytopathogenic (and toxigenic) fungi, and also as a microbiological fertilizer.

[0054] In the present invention, a "biomodifier" ("bacterial preparation for modifying mineral fertilizers") is understood to mean a composition comprising bacteria of the Bacillus subtilis 2.2 strain and, optionally, an auxiliary substance. In particular embodiments of the invention, the biomodifier according to the invention is a composition comprising a carrier complex with agronomically useful microorganisms of the Bacillus subtilis 2.2 strain immobilized thereon. The said biomodifier is intended for modifying mineral fertilizers and ameliorants, in particular, by mixing with a mineral fertilizer (liquid or solid) and an ameliorant. In particular embodiments of the invention, the biomodifier includes at least one auxiliary substance.

[0055] The term "adjuvant" as used in the present description refers to substances that do not interfere with the biological activity and properties of bacteria and are acceptable for use in agriculture (safe for use in plant growing, in particular in agriculture). Particular embodiments of adjuvants include dry carriers or liquid diluents, such as water. In some particular embodiments of the invention, the adjuvant is an agent that increases the adhesion of bacteria to a mineral fertilizer and / or an ameliorant and / or a carrier.

[0056] According to the invention, "mineral fertilizer" means compounds of inorganic nature that contain the necessary plant nutrition elements, i.e. fertilizer of industrial or fossil origin containing nutritional elements in mineral form. According to the invention, any mineral fertilizers in solid (dry) form (e.g. in the form of granules, powder, crystals, tablets, etc.) can be used without limitation, including water-soluble fertilizers (fertilizers in solid form that are completely dissolved in water before use; they are mainly intended for use through a plant irrigation system) and fertilizers in liquid form.Among other things, simple mineral fertilizers can be used - with a guaranteed content of only one main nutrient (for example, nitrogen, phosphorus, potassium, magnesium, boron, molybdenum), complex (including complex-mixed) mineral fertilizers - containing at least two main nutrient elements (for example, nitrogen-potassium, nitrogen-phosphorus-potassium (NPK), nitrogen-phosphorus-potassium fertilizers with the addition of sulfur (NPKS)), micronutrient fertilizers - in which the nutrient elements are micronutrients in a form accessible to plants. In this case, micronutrients can be additionally added to any mineral fertilizer.In some particular embodiments, urea, ammonium nitrate (ammonium nitrate), ammonium sulfate, calcium nitrate, ammophos, sodium nitrate, diammonium phosphate, nitroammophoska, phosphorite (phosphate) flour, potassium sulfate, potassium salt, sulfoammophos, diammonium phosphate azophoska, nitrophos, nitrophoska, potassium nitrate, potassium chloride, potassium magnesium sulfate, etc. can be used to obtain biomineral fertilizers according to the invention.

[0057] According to the invention, an "ameliorant" is a substance of industrial or fossil origin intended to improve the physicochemical properties and increase the fertility of acidic, saline and other soils. According to the invention, any ameliorants can be subjected to biomodification, including, for example, ameliorant-sorbents, ameliorant-structure formers, ameliorant fertilizers (see, for example, Petrova T.A. et al. Types of ameliorants for the reclamation of technogenically disturbed areas of the mining industry and Mining Information and Analysis Bulletin, 2021, No. 4, pp. 100-112). In some cases, the ameliorant according to the invention can be, for example, phosphogypsum, phosphate flour, etc.

[0058] The term "nutrient" ("active substance") means a chemical element of fertilizer necessary for plant growth and development. Nutrients are divided into three groups: main (basic) nutrients - N, P, K, macronutrients - N, P, K, Ca, Mg, S, micronutrients - B, Mn, Cu, Zn, Co, Mo, Fe, etc.

[0059] The term "biomodified mineral fertilizer" ("biomineral fertilizer") means a mineral fertilizer whose granules or particles are coated with bacteria that improve its properties. This term also includes liquid mineral fertilizers (for example, urea-ammonia mixture (UAM), but not only), into which bacteria are introduced to improve their properties.

[0060] The term "biomodified ameliorant" means an ameliorant whose granules or particles are coated with bacteria that improve its properties. This term also includes liquid ameliorants, into which bacteria are introduced to improve their properties.

[0061] The concentrations of bacteria in a microbiological preparation (in particular in a microbiological fertilizer) or in a biomineral fertilizer, in a biomodified ameliorant, as well as in a biomodifier, refer to those at the beginning of storage (i.e. immediately after application or no more than a month after application), unless otherwise specified; or not less than those specified for a certain period.

[0062] Unless otherwise defined, technical and scientific terms in this application have the standard meanings generally accepted in the scientific and technical literature.

[0063] Detailed disclosure of the invention

[0064] The Bacillus subtilis 2.2 strain was deposited in the Bioresource Center of the All-Russian Collection of Industrial Microorganisms (BRC VKPM) of the Kurchatov Institute National Research Center under number B-13292 as part of the national patent deposition on February 24, 2022, and as part of the international patent deposition on August 17, 2023 (the document on the international deposition is an appendix to this application).

[0065] Origin of the strain

[0066] Isolated by scientists from the Eurobiokhim company from the soils of the Krasnodar Territory in 2019.

[0067] Morphological, cultural and biochemical characteristics

[0068] Large gram-positive spore-forming rods 1.0-2.0 µm, straight with rounded ends. On potato agar they form colonies of creamy gray color with uneven scalloped edges, with a raised center, pasty consistency with concentric circles. The diameter of the colonies is 20-40 mm. The optimum temperature for growth is 37 ° C. At a temperature of more than 45 ° C and less than 15 ° C, growth is slow. The optimum pH of the medium is 6.8; growth occurs at a pH from 4.5 to 8.0. On potato agar they form colonies of creamy yellow color with an uneven scalloped edge with a raised folded center of leathery consistency.

[0069] It is intended to improve nutrition - accelerate growth and increase productivity of vegetable, grain and industrial crops in agriculture. When cultivated on a medium with tryptophan, it produces auxins: indoleacetic acid, indolecarboxylic acid and indole lactic acid.

[0070] Method, conditions and composition of media for long-term storage of the strain

[0071] Cultivation on liquid nutrient media (pea medium) using laboratory shakers and fermenters. Optimum conditions for fermentation: temperature + 36°C, stirrer speed 400-600 rpm, air supply 1 rpm, medium: pea flour, sucrose, MgSO4. Periodic reseeding twice a year on fresh nutrient medium GMF, MPA, TSA or potato agar.

[0072] Strain characteristics

[0073] Bacillus subtilis 2.2 strain is designed to protect plants from phytopathogens, improve nutrition, accelerate growth and increase the productivity of vegetable, grain and industrial crops in agriculture, as well as in floriculture and forestry. Bacillus subtilis 2.2 bacteria belong to the PGPR (Plant Growth Promoting Rhizobacteria) group - rhizobacteria that promote plant growth. Bacillus subtilis 2.2 strain bacteria have a wide range of host plants, are able to form endospores, and can increase plant productivity. Bacillus subtilis 2.2 strain is not pathogenic, not virulent, not toxic, and does not disseminate into tissues.

[0074] The key features of the Bacillus subtilis 2.2 strain, which ensure its high efficiency when used as a microbiological fertilizer or for modifying mineral fertilizers, are the mechanisms of its positive effect on plants: the Bacillus subtilis 2.2 strain actively produces fungicidal substances that suppress the development of phytopathogenic (and toxigenic) fungi, as well as auxin-like substances that stimulate the growth of the root and sprout system of plants, increasing the absorption capacity of the roots and the amount of absorbed mineral elements. Thus, the effect on the digestibility (availability) of mineral fertilizers by plants occurs through the production of various organic and inorganic acids by bacteria and an increase in the volume of the root system.

[0075] The possibility of objective manifestation of the technical result in the implementation of the invention is confirmed by reliable data given below in the examples containing experimental information obtained in the process of conducting research using methods accepted in this field.

[0076] It should be understood that the examples provided in the application materials are not limiting and are provided only to illustrate the present invention.

[0077] Example 1. Study of the ability of the strain according to the invention to produce 3-indoleacetic acid (IAA) and its derivatives on a medium with L-tryptophan

[0078] To isolate auxins, the culture liquid (1 ml) was acidified with 0.4 N hydrochloric acid to pH 3.0 and extracted twice with equivalent volumes of ethyl acetate. The resulting extracts were evaporated to dryness at 35°C on a vacuum rotary evaporator and dissolved in 0.5 ml of 18% acetonitrile. The resulting samples were filtered through nylon membrane filters (Corning Costar Spin-X centrifuge tube filters) with a diameter of nop 0.22 μm (Sigma, USA) and diluted 10 times with 18% acetonitrile for subsequent chromatographic analysis. Analysis of auxins in the obtained extracts was carried out using a Waters ACQUITY UPLC H-class high-performance liquid chromatography (HPLC) system with a fluorescence detector. Auxins were separated on a Waters ACQUITY UPLC BEH Shield RP18 reversed-phase column (Waters, USA) using isocratic separation in an acetonitrile: water: acetic acid (18:82:0) eluent.1 v / v / v) for 4 min, followed by washing the column with acetonitrile: water: acetic acid eluent (80:20:0.1 v / v / v) for 3 min. The eluent flow rate was 0.3 ml / min, the column temperature was 300°C, the fluorescence detector wavelengths were 280 nm excitation (Ex) and 350 nm emission (Em). Auxins were identified by comparing the retention times of the corresponding substances (Sigma-Aldrich, USA) in the samples and in the standard mixture. The amounts of auxins were determined by comparing the peak areas in the samples and a standard mixture with an acid concentration of 0.5 μg / ml (indolyl-3-lactic), 10 μg / ml (indolyl-3-carboxylic) and 4 μg / ml (indolyl-3-acetic). The degree of L-tryptophan utilization (%) was determined as the proportion of its molecules used for the biosynthesis of an equivalent amount of auxin molecules identified in the samples. Sterile media without additional sample preparation were used to determine the L-tryptophan content in the nutrient media.L-tryptophan was analyzed on a Waters ACQUITY UPLC BEH Shield RP18 reversed-phase column (Waters, USA) for 5 minutes in a linear gradient of acetonitrile : water : acetic acid eluent from 1% : 99% : 0.1% to 18% : 82% : 0.1%, followed by 3-minute isocratic elution and 3-minute column wash at a buffer component concentration of 80% : 20% : 0.1%. The eluent feed rate was 0.3 ml / min, the column temperature was 300°C, the fluorescence detector wavelengths were 280 nm excitation (Ex) and 350 nm emission (Em). L-tryptophan was identified by comparing its retention time in samples and in a solution of chemically pure substance (Sigma, USA). The amount of L-tryptophan was determined by comparing the peak areas in the samples and a standard mixture with a concentration of 0.01 μg / ml.

[0079] It was established that the Bacillus subtilis 2.2 strain according to the invention is characterized by high rates of production of auxins and auxin-like substances: indole-3-acetic acid - 333.7 ng / ml, indole-3-carboxylic acid - 19.6 ng / ml, indole-3-lactic acid - 127 ng / ml. Thus, the total production of auxins by the strain was 480.3 ng / ml.

[0080] Example 2. Study of growth-promoting activity on wheat seedlings

[0081] Winter wheat seeds (Svetoch variety) were soaked in a bacterial suspension containing cells of the Bacillus subtilis 2.2 strain of bacteria according to the invention at a concentration of 1 *10 5CFU / ml, for 30 min, then under in vitro experimental conditions they were placed in humid chambers on the surface of filter paper. For each variant, 25 seeds were placed in one humid chamber in three replicates. The seeds in the control variant were soaked in sterile physiological solution. The sprouts were grown for five days at 28°C. The control strain for comparing the growth-stimulating properties of bacteria was the B. subtilis strain At 7 (VKPM B-12776) from the collection of the laboratory of Microbox LLC, already used in the production of the biopreparation Nodix.

[0082] Growth stimulation of the Bacillus subtilis 2.2 strain according to the invention on wheat sprouts was: root - 60% more in relation to the control, stem - 39.6% more in relation to the control variant. Example 3. Study of fungicidal properties of the strain according to the invention on test cultures of phytopathogenic fungi

[0083] The evaluation of the fungicidal properties of the Bacillus subtilis 2.2 strain according to the invention was studied in relation to strains of phytopathogenic and toxigenic fungi: Fusarium sporotrichioid, Alternaria solani, Fusarium culmorum (from the collection of Microbox LLC).

[0084] The culture of Bacillus subtilis 2.2 bacteria was grown on liquid R2A medium in a stationary manner at 20°C for 3 days, fungicidal activity was determined using the “well” method (Magnusson and Schnurer, 2001) on potato dextrose agar (Difco, USA). For this purpose, a spore suspension with a titer of 10 was prepared by surface washing from the fungal colony. 5CFU / ml. 20 μl of the resulting suspension were added to 200 ml of melted potato dextrose agar cooled to 45°C. The nutrient medium was poured into dishes, after the medium had solidified, holes were made in it with a sterile cork drill. 100 μl of the Bacillus subtilis 2.2 bacterial culture were added to the resulting “wells”. The crops were incubated for 5 days at 25°C, after incubation, the fungicidal activity was assessed by the diameter of the phytopathogen inhibition zones around the “well”.

[0085] The study of fungicidal activity allowed to establish the ability of the Bacillus subtilis 2.2 strain according to the invention to actively suppress all test objects of phytopathogenic fungi. The zones of phytopathogen suppression were established: Fusarium sporotrichioides - >60 mm, Alternaria solani - >60 mm, Fusarium culmorum 50 mm. The edges of the zones were clearly defined, well defined, and the sizes of the zones were characterized by the duration of the suppression effect.

[0086] Example 4. Application of bacterial culture to mineral fertilizer granules and determination of numbers

[0087] Since the Bacillus subtilis 2.2 microorganism strain is designed to increase the efficiency of mineral fertilizer application, the ability of bacterial cells to maintain their viability on the surface of granules and influence plant growth and productivity indicators was determined.

[0088] For processing mineral fertilizer granules (urea, ammophos and nitroammophoska), a bacterial suspension of the Bacillus subtilis 2.2 strain was used, containing bacteria in a titer of at least 0.5*10 9CFU / ml. The treatment of mineral fertilizer granules with a bacterial suspension was carried out by applying it to the surface of the granules at a rate of 1 liter per ton (or 1 ml per kg). After 24 hours, the number of bacteria on the granules was determined by serial dilutions with sowing on solid nutrient media and culturing at 36°C for 48 hours, after which the grown colonies were counted. The established titer of the Bacillus subtilis 2.2 strain on the surface of the granules with direct application at the beginning of storage (24 hours after application) was: on urea - 29*10 6 CFU / g, on ammophos - 17*10 6 CFU / g, on nitroammophoska - 23*10 6 CFU / gr. Repeated determination of the number of colony-forming units (CFU) of bacteria of the Bacillus subtilis 2.2 strain on the surface of mineral fertilizers was carried out 12 months after application.

[0089] Determination of the titer of Bacillus subtilis 2.2 bacteria on the surface of mineral fertilizer granules allowed us to establish the ability of bacteria to maintain their vital activity after their application to mineral fertilizers. The established titer of the Bacillus subtilis 2.2 strain on the surface of granules after 12 months of storage was: on urea - 6*10 4 CFU / g, on ammophos - 4.1*10 4 CFU / g, on nitroammophoska - 3.2*10 6 CFU / g. Thus, it was shown that the Bacillus subtilis 2.2 strain is capable of maintaining its viability on the surface of mineral fertilizer granules in concentrations that ensure the effectiveness of biomineral fertilizers for at least 12 months.

[0090] Example 5. Application of bacterial culture to mineral fertilizer granules using a carrier

[0091] Preparation of components. The bacterial culture of the Bacillus subtilis 2.2 strain was grown in a liquid nutrient medium at 36°C for 3 days on a shaker at 400 rpm. The carrier (zeolite) was sterilized in a drying cabinet at 200°C for 8 hours. The following types of mineral fertilizers were used in the experiment: urea, ammophos, nitroammophoska. All types of mineral fertilizer granules were sterilized in a drying cabinet at 100°C for 10 hours.

[0092] 5 g of zeolite were introduced into a separate sterile falcon with a volume of 50 ml. 5 ml of bacterial suspension were added to it. The mixture was thoroughly mixed with a sterile glass rod and transferred to a glass Petri dish in a layer of no more than 5 mm. After this, drying was carried out at 60 ° C in a drying cabinet for 10 hours.

[0093] 15 g of mineral fertilizer were added to sterile 50 ml falcons. 0.06 g of dried zeolite with bacterial culture was added to the fertilizer. The mixture was thoroughly mixed on a vortex for 30 s.

[0094] The bacterial titer on the mineral fertilizer granules was determined 24 hours after application by serial dilutions with seeding on solid nutrient media and cultivation at 36°C for 48 hours, after which the grown colonies were counted. As the calculation showed, the number of bacterial cells on the mineral fertilizer granules was: on urea - 4.5*10 7 CFU / g, on ammophos - 3.1*10 7 CFU / g, on nitroammophoska - 4.0*10 7 CFU / g,

[0095] Example 6. Determination of the activity of bacterial strains when applied together with mineral fertilizer granules. Model gnotobiotic systems were used to determine the activity of introducing microorganisms into the rhizosphere of agricultural crops. Winter wheat test plants were used as test objects.

[0096] Granules of mineral fertilizers (urea, ammophos and nitroammophoska) were used immediately after treatment with a bacterial suspension of the Bacillus subtilis 2.2 strain - see example 4.

[0097] To identify the ability of the studied strain to actively colonize higher plants, the method of introducing treated granules, introducing them into the system without direct contact between the granule and the planted plant sprout, and subsequent cultivation of plants in sterile conditions of gnotobiotic systems was used. The control strain for comparing the colonization properties of bacteria was the strain B. subtilis Am 7 (VKPM B-12776), already used in the production of biopreparations.

[0098] The plant object for the study were Triticum aestivum wheat sprouts (Svetoch variety). Wheat seeds were sterilized first for 2 minutes in 70% ethanol, then washed in sterile water, and then kept twice in a 30% sodium hypochlorite solution for 30 minutes. After sterilization, the seeds were thoroughly washed in sterile water. Then the seeds were planted in systems and grown under gnotobiotic systems. To determine the number of introduced bacteria in the rhizoplane, the washed roots were suspended in a physiological solution and the number of microorganisms was taken into account by sowing on a solid R2A medium.

[0099] The analysis showed that the tested strain Bacillus subtilis 2.2 took root well both in the rhizosphere and on the roots of wheat, not inferior to, and sometimes exceeding the corresponding indicators of the control strain B. subtilis Am 7. Thus, the number of bacteria (CFU / 1 g of wheat root) for the variants of ammophos granules with the strain Bacillus subtilis 2.2 was 8.7 * 10 for the root.7 , for the rhizosphere - 12.4*10 6 , for control variants - root 6.0*10 6 , rhizosphere - 6.8*10 6 CFU / 1 g of wheat root. For urea granules containing the Bacillus subtilis 2.2 strain on the surface, the number of bacteria for the root was 5.2*10 6 , for the rhizosphere - 11, 6*10 6 , while the values ​​for the control variants were - root 1.4*10 6 , rhizosphere - 5.2*10 6 CFU / 1 g of wheat root. For nitroammophoska granules with Bacillus subtilis strain 2.2, the number of bacteria for the root was - 7.3*10 6 , for the rhizosphere - 4.4*10 6 , while the values ​​for the control variants were - root 4.0*10 6 , rhizosphere - 3.2*10 6 CFU / 1 g of wheat root.

[0100] Thus, the Bacillus subtilis 2.2 strain is capable of active colonization of higher plants when introduced as part of biomineral fertilizers.

[0101] In order to identify the phytostimulating activity of bacteria in the composition of biomineral fertilizers, a vegetation experiment was also conducted with winter wheat, Svetoch variety, elite, to study the effectiveness of the bacterial strain Bacillus subtilis 2.2. The experiment was conducted in a polycarbonate greenhouse. For the study, 2 kg of soil were added to three-liter vessels. Then 0.7 g of biomodified (based on the Bacillus subtilis 2.2 strain) mineral fertilizers urea, ammophos and nitroammophoska were spread in an even layer (see example 4). The same amount of non-biomodified mineral fertilizer was spread in control vessels. 1 kg of soil was poured on top. The seeds were planted in holes 4-5 cm deep. 15 seeds per vessel. The experiment was carried out over a period of 3 months, taking into account the effect of biomodified fertilizers based on the Bacillus subtilis 2.2 strain on plant height, length of the root part of plants and weight of plants.As a result of the experiment, a positive effect of biomodified fertilizers on the growth and development of wheat plants was established. Thus, the Bacillus subtilis 2.2 strain showed an increase in the weight of winter wheat plants on ammophos - by 18.9%, on urea - by 22.8% (compared to non-biomodified mineral fertilizers). Thus, it was experimentally established that the Bacillus subtilis 2.2 strain has a pronounced phytostimulating effect.

[0102] The results of the experiments show that the bacterial strain Bacillus subtilis 2.2 is an effective biological agent for use in the biomodification of mineral fertilizers as an effective means of plant protection, increasing their productivity and the efficiency of mineral fertilizer assimilation, and expanding the arsenal of such means.

[0103] Although the invention has been described with reference to the disclosed embodiments, it should be obvious to those skilled in the art that the specific instances described in detail are provided merely for the purpose of illustrating the present invention and should not be considered as limiting the scope of the invention in any way. It should be understood that various modifications can be made without departing from the spirit of the present invention.

Claims

CLAUSE OF INVENTION 1. The strain of bacteria Bacillus subtilis 2.2, deposited in the collection of the All-Russian Collection of Plant Materials under number B-13292, is characterized by plant growth-stimulating activity, protecting plants from phytopathogens, improving nutrition, and increasing plant yields.

2. The use of the bacterial strain Bacillus subtilis 2.2 according to paragraph 1 for the biomodification of mineral fertilizers and / or ameliorants.

3. The use according to item 2, in which the biomodification provides improved nutrition and / or stimulation of plant growth, increased resistance to diseases, increased efficiency of digestibility, respectively, of mineral fertilizers and / or ameliorants.

4. The use according to any of paragraphs 2 or 3, in which the mineral fertilizer or ameliorant is used in solid or liquid form.

5. The use according to claim 4, wherein the mineral fertilizer in solid form is a fertilizer in granulated, prilled, microgranulated, powdered form or in the form of a water-soluble mineral fertilizer.

6. Use according to any of paragraphs 2-5, in which the bacteria are applied directly to the mineral fertilizer or ameliorant by direct application of a bacterial suspension or using a carrier and / or an agent that increases the adhesion of the bacteria to the mineral fertilizer and / or ameliorant and / or carrier.

7. The use according to claim 6, wherein the carrier is a mineral or organic natural or synthetic substance acceptable in agriculture.

8. The use according to claim 7, wherein the carrier is diatomite, zeolite, opoka, tripoli, kaolin, glauconite, silica, perlite, metakaolin, wollastonite, bentonite, inorganic microsilica, belite, white carbon black, vermiculite (expanded), talc or diabazite.

9. The use according to item 6, in which cationic starch, dextrin, collagen glue, bone gelatin glue, albumin glue, fish glue, cherry glue, molasses, gum arabic or liquid glass are used as the agent that increases bacterial adhesion.

10. Use according to any of paragraphs 2-9, wherein the mineral fertilizer is a nitrogen, phosphorus, potassium, boron, molybdenum, complex, complex-mixed or micro fertilizer, or a mixture of one of the fertilizers with microelements.

11. The use according to claim 10, wherein the mineral fertilizer is urea, ammonium nitrate, ammonium sulfate, calcium nitrate, ammophos, sodium nitrate, diammonium phosphate, nitroammophoska, phosphate rock, sulfate potassium, potassium salt, sulfoammophos, diammonium phosphate, azophoska, nitrophos, nitrophoska, potassium nitrate, potassium chloride or potassium magnesium sulfate.

12. Use according to any of paragraphs 2-4, 6-9, wherein the ameliorant is an ameliorant-sorbent, an ameliorant-structure former, an ameliorant fertilizer, including phosphogypsum or phosphate flour.

13. Use according to any of paragraphs 2-12, wherein the bacteria are used in spore form.

14. A biomodifier for producing a biomodified mineral fertilizer or ameliorant, comprising a strain of bacteria Bacillus subtilis 2.2 according to item 1 and, optionally, at least one auxiliary substance.

15. A biomodifier according to claim 14, wherein the auxiliary substance is water.

16. A biomodifier according to item 14, wherein the auxiliary substance is a carrier and / or an agent that increases the adhesion of bacteria to a mineral fertilizer and / or an ameliorant and / or a carrier.

17. A biomodifier according to claim 15, wherein the carrier is diatomite, zeolite, opoka, tripoli, kaolin, glauconite, silica, perlite, metakaolin, wollastonite, bentonite, inorganic microsilica, belite, white carbon black, vermiculite (expanded), talc or diabazite.

18. A biomodifier according to any of paragraphs 14-17, additionally including other strains of agronomically useful microorganisms.

19. A biomodified mineral fertilizer or biomodified ameliorant, comprising, respectively, a mineral fertilizer or ameliorant, and bacteria of the Bacillus subtilis strain 2.

2.

20. A biomodified mineral fertilizer or biomodified ameliorant according to claim 19, obtained by biomodifying, respectively, a mineral fertilizer or ameliorant using for this purpose a biomodifier according to any of claims 14-18.

21. A biomodified mineral fertilizer or biomodified ameliorant according to item 19, additionally including other strains of agronomically useful microorganisms.

22. A biomodified mineral fertilizer or biomodified ameliorant according to any of paragraphs 19-21, in which, respectively, the mineral fertilizer or ameliorant is used in solid or liquid form.

23. A biomodified mineral fertilizer or a biomodified ameliorant according to claim 22, in which the mineral fertilizer in solid form is a fertilizer in granulated, prilled, microgranulated, powdered form or a water-soluble mineral fertilizer.

24. A biomodified mineral fertilizer or biomodified ameliorant according to any of paragraphs. 19-21, in which the mineral fertilizer is a nitrogen, phosphorus, potassium, boron, molybdenum, complex, complex-mixed or micro fertilizer or a mixture of one of the fertilizers with microelements, and the ameliorant is an ameliorant-sorbent, an ameliorant-structure-forming, or a fertilizer-ameliorant.

25. A biomodified mineral fertilizer or a biomodified ameliorant according to claim 24, wherein the ameliorant is phosphogypsum or phosphate flour.