Bacillus atrophaeus strain 119 and mineral fertilizers containing same

Abstract

The invention relates to agricultural biotechnology and microbiology, and more particularly to the biomodification of mineral fertilizers and soil enhancers using Bacillus atrophaeus strain 119, 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, increasing crop yield, inhibiting nitrification processes in the soil, and also reducing loss of mineral fertilizers.

Description

[0001] STRAIN BACILLUS ATROPHAEUS 119 AND MINERAL FERTILIZERS 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 known 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 provide increased efficiency in protecting plants from infectious diseases that penetrate along with mineral fertilizers.

[0007] The biological preparation "Fitosporin" is known for protecting plants from diseases (patent RU2099947, 15.1 1.1996). The basis of the biological preparation "Fitosporin" is the strain of bacteria Bacillus subtilis VNIISKhM 128 with a cell concentration of 10 9 -10 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 increase the efficiency of mineral fertilizers and ameliorants in enhancing the growth and productivity of plants, protecting them from phytopathogenic fungi and bacteria, increasing plant resistance to diseases, and inhibiting nitrifying bacteria. The objective of the present invention is also to develop and create biomodified mineral (biomineral) fertilizers and ameliorants.

[0011] The task is solved by using the Bacillus atrophaeus 119 strain for biomodification of fertilizers and ameliorants.

[0012] For this purpose, the Bacillus atrophaeus 119 strain is applied to ameliorants or mineral fertilizers used in both dry and liquid form. In some embodiments of the invention, the mineral fertilizer in dry form is a fertilizer in granulated, prilled, microgranulated, powdered form or in the form of a water-soluble mineral fertilizer.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] In some embodiments of the invention, the ameliorant is an ameliorant-sorbent, an ameliorant-structure-forming agent, a ameliorant fertilizer, including, for example, phosphogypsum, phosphate rock. In various embodiments of the invention, the bacteria are used in spore or vegetative form. In more preferred embodiments of the invention, the bacteria are used in a dormant (spore) form, since it ensures longer storage and survival of the bacteria.

[0017] The set task is also solved by developing a biomodifier of mineral fertilizers and ameliorants (a bacterial preparation for modifying mineral fertilizers and ameliorants), including a strain of bacteria Bacillus atrophaeus 119 m, optionally, at least one auxiliary substance.

[0018] In some particular embodiments of the invention, 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. In particular embodiments of the invention, the concentration of bacteria on the carrier is at least 10 4 CFU / g carrier.

[0019] In particular embodiments of the invention, the concentration of bacteria in the liquid biomodifier is not less than 10 5 CFU / ml of bacterial suspension.

[0020] The stated task is also solved by developing a biomodified mineral (biomineral) fertilizer and ameliorant for protecting plants from phytopathogenic (and toxigenic) fungi and bacteria, improved nutrition and stimulation of plant growth, as well as inhibition of nitrification processes, including mineral fertilizer and bacteria of the Bacillus atrophaeus 119 strain.

[0021] 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.

[0022] 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).

[0023] 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.

[0024] The present invention also includes the use of the Bacillus atrophaeus 119 strain for producing a biomodifier of mineral fertilizers and ameliorants.

[0025] The present invention also includes the use of a biomodifier based on the Bacillus atrophaeus 119 strain for obtaining a biomodified mineral (biomineral) fertilizer or ameliorant for protecting plants from phytopathogenic fungi and bacteria, improving nutrition, stimulating plant growth, increasing their resistance to diseases, increasing the efficiency of digestibility, respectively, of mineral fertilizers and / or ameliorants, and inhibiting nitrification processes in the soil.

[0026] The stated task is also solved by developing a method for biomodifying mineral fertilizers and ameliorants, including mixing bacteria of the Bacillus atrophaeus 119 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 atrophaeus 119 strain directly on the mineral fertilizer or ameliorant, as well as using a carrier.

[0027] In some particular embodiments of the invention, a bacterial suspension (or a bacterial suspension to which auxiliary substances have been previously added) is mixed with a liquid mineral fertilizer or directly applied to the surface of a solid mineral fertilizer or ameliorant.

[0028] 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 microorganisms in the bacterial suspension affects the number of CFU 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 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.

[0029] As a result of implementing the invention, the following technical results are achieved:

[0030] - a new effective approach to the biomodification of mineral fertilizers and ameliorants, as well as a biomodification method, has been developed;

[0031] - biomodified mineral fertilizers and ameliorants according to the invention provide protection against phytopathogenic (and toxigenic) fungi and bacteria, 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 atrophaeus 119 strain to produce antifungal substances, enzymes and other organic compounds capable of suppressing the development of phytopathogenic (and toxigenic) fungi and bacteria, 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;

[0032] - the key feature of the Bacillus atrophaeus 119 strain is the ability to suppress nitrifying bacteria in the rhizosphere zone of plants, as well as inhibit the development of phytopathogenic (and toxigenic) fungi and bacteria, stimulate the growth of the root and stem system of plants, therefore the use of the strain in the composition of biomineral fertilizers / ameliorants and biomodifiers ensures the inhibition of nitrification processes, protection of plants from phytopathogenic (and toxigenic) fungi and bacteria, an increase in the phytomass and productivity of plants;

[0033] - 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;

[0034] - a biomodifier of mineral fertilizers and ameliorants (biomodified mineral fertilizer and ameliorant) based on the Bacillus atrophaeus 119 strain allows to reduce the loss of mineral fertilizers (ameliorants) by their leaching and volatilization; the use of the Bacillus atrophaeus 119 bacterial strain in the composition of a biomodified mineral fertilizer and ameliorant allows to increase the efficiency of their assimilation by plants;

[0035] - 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;

[0036] - developed biomineral fertilizers / ameliorants and biomodifiers based on the Bacillus atrophaeus 119 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.

[0037] Terms and definitions

[0038] 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.

[0039] 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 atrophaeus 119 strain and to biomodifiers of mineral fertilizers or ameliorants, as well as to biomodified mineral fertilizers or biomodified ameliorants. According to the invention, the Bacillus atrophaeus 119 strain can be used not only in isolation, but also in consortia with other strains.

[0040] The term "and / or" means one, more, or all of the listed elements.

[0041] Also here, listing numeric ranges by endpoints includes all numbers within that range.

[0042] 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.

[0043] The strain Bacillus atrophaeus 119 means both the strain Bacillus atrophaeus 119, which was deposited in the Network Bioresource Collection in the Field of Genetic Technologies for Agriculture (RCAM) of the All-Russian Research Institute of Agricultural Microbiology under number RCAM00759 and in the collection of the All-Russian Research Institute of Plant Breeding under number B-14563, and the mutant strain obtained using the deposited strain as the source material and retaining all the properties of the original strain, i.e. possessing all the defining (identifying) characteristics of the strain Bacillus atrophaeus 119. Including, at least, being suitable for use in enhancing plant growth and productivity, protecting them from phytopathogenic bacteria, as well as from phytopathogenic and toxigenic fungi, increasing plant resistance to diseases, and inhibiting nitrifying bacteria.In particular, the Bacillus atrophaeus 119 strain has the following identifiable features, including 1) the ability to inhibit phytopathogenic bacteria, at least Pseudomonas syringae, Erwinia carotovora, Clavibacter michiganensis; 2) fungicidal activity against at least such phytopathogenic and toxigenic fungi as Fusarium sporotrichioid, Alternaria solani, Fusarium culmorum; 3) the ability to suppress the vital activity of nitrifying bacteria; 4) 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 atrophaeus 119, which can be used to confirm that the analyzed variant is Bacillus atrophaeus 119.

[0044] The term "mutant" or "variant" as used herein with respect to the strain Bacillus atrophaeus 1 19 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.

[0045] "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.

[0046] 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.

[0047] "Culture fluid" is a term used to describe the nutrient medium in which microorganisms are grown, which contains their metabolites and other target substances produced by the microorganisms, as well as residual nutrients, as opposed to "bacterial suspension", which in addition to the culture fluid contains cells of agronomically useful microorganisms.

[0048] In the present invention, a "biomodifier" ("bacterial preparation for modifying mineral fertilizers") is understood to mean a composition comprising bacteria of the Bacillus atrophaeus 119 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 atrophaeus 119 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.

[0049] 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.

[0050] 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.

[0051] 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 territories of the mining industry and Mining Information and Analytical Bulletin, 2021, No. 4, pp. 100-112). In some cases, the ameliorant according to the invention can be, for example, phosphogypsum, phosphate flour, etc.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] The concentrations of bacteria in the biomineral fertilizer, in the biomodified ameliorant, and also in the biomodifier refer to those at the beginning of storage (i.e. immediately after application or no more than one month after application), unless otherwise specified; or not less than those specified for a certain period.

[0056] Unless otherwise defined, technical and scientific terms in this application have the standard meanings generally accepted in the scientific and technical literature.

[0057] Detailed disclosure of the invention

[0058] The Bacillus atrophaeus 119 strain was deposited in the Network Bioresource Collection in the Field of Genetic Technologies for Agriculture (RCAM) of the All-Russian Research Institute of Agricultural Microbiology (VNIISKhM) on June 6, 2022 under number RCAM00759, and in the Bioresource Center of the All-Russian Collection of Industrial Microorganisms (BRC VKPM) of the National Research Center "Kurchatov Institute" on August 19, 2024 under number B-14563 (the document on international deposition is an appendix to this application).

[0059] The bacterial strain Bacillus atrophaeus 119 can also be identified as Bacillus subtilis based on modern methods.

[0060] Origin of the strain

[0061] Isolated from soil by a group of authors (Reznik S.R., Lapa S.V.).

[0062] Morphological, cultural and biochemical characteristics

[0063] 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.

[0064] The culture has pronounced fungicidal activity against a wide range of phytopathogenic fungi and bacteria, including, in particular, inhibiting phytopathogenic fungi Fusarium spp., Alternaria spp. (which also have toxigenic properties), inhibiting bacteria of the genus Pseudomonas syringae, Erwinia carotovora, Clavibacter michiganensis.

[0065] Method, conditions and composition of media for long-term storage of the strain

[0066] Long-term storage (3 years or more) is carried out in a cryopreserved state at -80°C. The culture is also stored on slanted meat-peptone agar (MPA), TSA medium or potato agar at a temperature of +4°C in the refrigerator, and requires periodic reseeding twice a year onto a fresh nutrient medium.

[0067] Strain characteristics

[0068] Bacillus atrophaeus 119 strain is designed to protect plants from phytopathogens, suppress nitrifying bacteria in the rhizosphere zone of plants, improve nutrition, accelerate growth and increase the productivity of vegetable, grain and industrial crops in agriculture, as well as in floriculture and forestry. Bacillus atrophaeus 119 bacteria belong to the PGPR (Plant Growth Promoting Rhizobacteria) group - rhizobacteria that promote plant growth. Bacillus atrophaeus 119 strain bacteria have a wide range of host plants, are able to form endospores, and can increase plant productivity. Bacillus atrophaeus 119 strain is not pathogenic, not virulent, not toxic, and does not disseminate into tissues.The key features of the Bacillus atrophaeus 119 strain, which ensure its high efficiency when used to modify mineral fertilizers, are the mechanisms of its positive effect on plants: the Bacillus atrophaeus 119 strain actively produces fungicidal substances that suppress the development of phytopathogens, suppresses the development of nitrifying bacteria in the rhizosphere of plants, and produces metabolites that stimulate plant growth. The growth of the root and sprout system allows for an increase in 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.

[0069] 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.

[0070] It should be understood that the examples provided in the application materials are not limiting and are provided only to illustrate the present invention.

[0071] Example 1. Study of fungicidal and bactericidal properties of Bacillus atrophaeus strain 119 on test cultures of phytopathogens

[0072] The fungicidal properties of the Bacillus atrophaeus 119 strain were evaluated in relation to strains of phytopathogenic and toxigenic fungi: Fusarium sporotrichioid, Alternaria solani, Fusarium culmorum (from the collection of Microbox LLC). For this purpose, the Bacillus atrophaeus 119 bacterial culture was grown on liquid R2A medium in a stationary manner at 20°C for 3 days, the 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 atrophaeus 119 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”.

[0073] Bactericidal activity was studied on 3 strains of phytopathogenic bacteria: Erwinia carotovora var. atroseptica, Pseudomonas syringae, Clavibacter michiganensis pv. sepedonicum (strains from the collection of Microbox LLC) using the "agar blocks" method on 2% potato agar. Cultures of antagonist bacteria Bacillus atrophaeus 119 were grown as a lawn for three days at a temperature of 28°C. Test cultures of phytopathogenic bacteria were seeded as a lawn on the surface of potato agar, then agar blocks cut from cultures of the antagonist strain were laid out on the surface. The crops were incubated for 48 hours at a temperature of 28°C, bactericidal activity was assessed by the diameter of the phytopathogen inhibition zones around the agar block.

[0074] The study of fungicidal activity allowed to establish the ability of Bacillus atrophaeus 119 to actively suppress all test objects of phytopathogenic fungi. The zones of phytopathogen suppression were established: Fusarium sporotrichioides - 29 mm, Alternaria solani - 28 mm, Fusarium culmorum - 10 mm. The edges of the zones were clearly defined, well expressed, and the sizes of the zones were characterized by the duration of the suppression effect.

[0075] It has been established that the Bacillus atrophaeus 119 strain is also effective against bacterial pathogens; zones of suppression of phytopathogenic bacteria have been established: Erwinia carotovora var. Atroseptica - 15 mm, Pseudomonas syringae - 4 mm, Clavibacter michiganensis pv. Sepedonicum - 10 mm.

[0076] Example 2. Determination of the ability of the Bacillus atrophaeus 119 strain to suppress the activity of nitrifying bacteria

[0077] To obtain enrichment cultures of nitrifying bacteria, a selective medium with nitrite was used, into which soil samples of various origins were added (1. Chernozem. Field with embedded straw, Samara Region 2. Bottom 3. Chernozem. Rhizosphere of corn, Samara Region). Samples weighing no more than 0.1 g were added to flasks. Enrichment cultures were incubated for 7 days at room temperature. The degree of residual nitrite content in the medium was estimated in selected 0.5 ml samples by adding an equal volume of Griess reagent. When the qualitative reaction to nitrite in the samples disappeared, the intensification of nitrification processes in the enrichment cultures was judged.

[0078] Consortia of nitrifying bacteria from the surface layer of the liquid medium were transferred to flasks with fresh medium. Bacterial consortia were examined microscopically. Then, the enrichment cultures were 3-fold seeded onto fresh media to obtain stable consortia. The obtained samples of consortia were taken for further work. After each passage, the degree of nitrite conversion was checked using a qualitative reaction. Nitrifying bacteria were characterized by the ability to grow on the surface of the liquid medium in the form of films. Test cultures of nitrifiers were grown on selective media containing nitrite. The activity of suppression of test cultures of nitrifiers by Bacillus atrophaeus 119 bacteria was assessed using the agar block method. The degree of suppression of nitrifying bacteria was assessed by the size of the zones of suppression of the "lawn" around the agar block 72 hours after sowing.A high degree of suppression of all test cultures of nitrifiers by the Bacillus atrophaeus 119 strain was established, the zone of growth inhibition of nitrifiers was 10-14 mm. Thus, based on the obtained results, it was established that the Bacillus atrophaeus 119 strain has the ability to suppress nitrifying bacteria.

[0079] Example 3. Application of bacterial culture to mineral fertilizer granules and determination of numbers

[0080] Since the strain of microorganisms Bacillus atrophaeus 119 is intended 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.

[0081] For processing mineral fertilizer granules (urea, ammophos and nitroammophoska), a bacterial suspension of the Bacillus atrophaeus 119 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. Repeated determination of the number of colony-forming units (CFU) of Bacillus atrophaeus 119 bacteria on the surface of mineral fertilizers was carried out 12 months after application.

[0082] Determination of the titer of Bacillus atrophaeus 119 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 atrophaeus 119 strain on the surface of granules with direct application at the beginning of storage (24 hours after application) was: on urea - 6*10 6CFU / g, on ammophos - 2.5*10 6 CFU / g, on nitroammophoska - 2.5*10 6 CFU / g. After 12 months of storage, the bacterial titer was: on urea - 2.0*10 3 CFU / g, on ammophos - 1.3*10 5 CFU / g, on nitroammophoska - 3.2*10 4 CFU / g. Thus, it was shown that the Bacillus atrophaeus 119 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.

[0083] Example 4. Application of bacterial culture to mineral fertilizer granules using a carrier

[0084] Preparation of components. The bacterial culture of the Bacillus atrophaeus 119 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. Granules of mineral fertilizer (urea, ammophos and nitroammophoska) were sterilized in a drying cabinet at 100°C for 10 hours. 5 g of zeolite were placed in a separate sterile 50 ml falcon. 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 that, drying was carried out at 60°C in a drying cabinet for 10 hours.

[0085] 15 g of mineral fertilizer were added to sterile 50 ml falcons. 0.06 g of dried zeolite with bacterial culture applied was added to the fertilizer. The mixture was thoroughly mixed on a vortex for 30 s.

[0086] 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 count showed, the bacterial cells of the Bacillus atrophaeus 119 strain on urea contained 4.3*10 6 CFU / g, on ammophos - 1.8*10 6 CFU / g, on nitroammophoska - 1.2*10 6 CFU / g.

[0087] Example 5. Determination of the activity of bacterial strains when applied together with mineral fertilizer granules

[0088] Model gnotobiotic systems were used to determine the activity of microorganism introduction into the rhizosphere of agricultural crops. Winter wheat test plants were used as test objects.

[0089] Granules of mineral fertilizers (urea, ammophos and nitroammophoska) were used immediately after treatment with a bacterial suspension of the Bacillus atrophaeus strain 119 - see example 3.

[0090] 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 / m 7 (VKPM B-12776), already used in the production of biopreparations.

[0091] 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.

[0092] The analysis showed that the tested strain Bacillus atrophaeus 119 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 with the strain Bacillus atrophaeus 119 was 2.7*10 for the root. 6, for the rhizosphere - 2.2*10 6 , while the values ​​for the control variants were - root 2.5*10 6 , rhizosphere - 2.0*10 6 CFU / 1 g of wheat root. Thus, it was experimentally established that the Bacillus atrophaeus 119 strain is capable of active colonization of higher plants when introduced as part of biomineral fertilizers.

[0093] Confirmation of the growth-stimulating properties of biomodified fertilizers based on the Bacillus atrophaeus 119 strain was carried out in a microvegetation experiment under artificial climate conditions.

[0094] A sand-peat mixture was used as a substrate. The seeds were sterilized by treating them twice with a bleach solution for 30 minutes and then repeatedly rinsing with water. 700 g of soil substrate were added to plastic containers. 10 sunflower seeds, hybrid ES AGORA, were placed on top with sterile tweezers. 200 g of soil substrate was sprinkled on top. Then 100 ml of water with granules of biomodified mineral fertilizer based on Bacillus atrophaeus 119 (BMF) dissolved in it were poured. Samples without the addition of BMF and with the addition of mineral fertilizer granules were placed as a control. The results of the experiment showed that the ammophos BMU together with the Bacillus atrophaeus 119 strain allows to increase the root mass of plants by 158.3%, and the mass of the green part of plants by 15.1% relative to the control (ammophos mineral fertilizer).The use of biologized urea allowed to increase the root weight of plants by 26%, and the length of the root of plants by 50% relative to the control (mineral fertilizer urea). In turn, the use of biologized mineral fertilizers ammonium nitrate based on the strain Bacillus atrophaeus 119 allowed to increase the root weight of plants by 25.5%, and the mass of the green part of plants - by 50.3% relative to the control (mineral fertilizer ammonium nitrate).

[0095] To study the effectiveness of the Bacillus atrophaeus 119 bacterial strain, a vegetation experiment with sunflower was also conducted. The experiment was conducted in a film greenhouse. For the study, 2 kg of soil were added to three-liter vessels. Then, 0.7 g of biomodified fertilizers based on the Bacillus atrophaeus 119 strain were spread in an even layer. As a result of the experiment, the effect of biomodified fertilizers on the growth and development of sunflower plants was established. Thus, the Bacillus atrophaeus 119 strain in the composition of BMU ammophos showed an increase in root weight by 158.3%, root length - by 17%, green part weight - by 156.1%, sunflower plant height - by 20.8% (compared to non-biomodified mineral fertilizers). Variants of Bacillus atrophaeus 119 BMU with urea showed an increase in root mass by 26%, root length by 50%, and sunflower plant height by 11% (compared to non-biomodified mineral fertilizers).Nitroammophoska together with the Bacillus atrophaeus 119 strain showed an increase in the height of sunflower plants by 13% (compared to non-biomodified mineral fertilizers).

[0096] A vegetation experiment was also conducted with sunflower (hybrid ES AGORA), soybean seeds (variety Alena, first reproduction), and winter wheat (variety Svetoch, elite). The experiment was conducted in a film greenhouse. For the study, 2 kg of soil were added to three-liter vessels. Then, 0.7 g of biomodified mineral fertilizers based on the Bacillus atrophaeus 119 strain - urea, ammophos, and nitroammophoska - were spread in an even layer (see example 3). 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. The experiment was conducted over 3 months, taking into account the effect of biomodified fertilizers based on the Bacillus atrophaeus 119 strain, as well as non-biomodified mineral fertilizers, on the height of plants, the length of the root part of plants and the weight of plants.As a result of the experiment, an improved effect of biomodified fertilizers on the growth and development of sunflower plants was established. Thus, the Bacillus atrophaeus 119 strain in the composition of BMU ammophos showed an increase in plant weight by 10.7% (compared to non-biomodified mineral fertilizers). Variants of BMU Bacillus atrophaeus 119 on winter wheat showed an increase in root length by 25%, the length of the above-ground part by 37.7%, an increase in plant weight by 15.7% (compared to non-biomodified mineral fertilizers). On soybeans, BMU ammophos together with the Bacillus atrophaeus 119 strain showed an increase in plant weight by 46.6% (compared to non-biomodified mineral fertilizers) and the length of the root system by 19.2%.

[0097] A field experiment was also conducted on spring wheat seeds (Daria variety) to determine the agrochemical efficiency of biomodified mineral fertilizers (BMF) based on the Bacillus atrophaeus 119 strain on yield and crop quality on sod-podzolic soil in the conditions of the North-West region. The results indicate that the introduced microbial strains in combination with mineral fertilizers have a positive effect on wheat yield compared to the use of fertilizers without strains. When individually accounting for the yield structure for 30 plants, statistically significant differences were obtained, indicating a positive effect of BMF urea and the Bacillus atrophaeus 1 19 strain - an increase of 11.2% in yield, an increased number of additional productive shoots (shoots), an increased protein content, an increased number and weight of grains from shoots per plant.

[0098] Thus, it was found that the Bacillus atrophaeus 119 strain has fungicidal and bactericidal properties, as well as a phytostimulating effect on plants, including various agricultural crops, such as sunflower, wheat and soybeans. The results of the experiments show that the Bacillus atrophaeus 119 bacterial strain is an effective biological agent for use in the biomodification of mineral fertilizers as an effective means of increasing plant productivity and the efficiency of mineral fertilizer assimilation, and also ensures a decrease in mineral fertilizer losses by suppressing nitrifying bacteria and the nitrification process.

[0099] Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific instances described in detail are 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 will be understood that various modifications can be made without departing from the spirit of the present invention.

Claims

CLAUSE OF INVENTION 1. Bacillus atrophaeus 119 bacterial strain for biomodification of mineral fertilizers and / or ameliorants.

2. Use of the bacterial strain Bacillus atrophaeus 119 for 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 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, potassium sulfate, potassium salt, sulfoammophos, diammophos, 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, in which the bacteria are used in spore and / or vegetative form.

14. A biomodifier for producing a biomodified mineral fertilizer or ameliorant, comprising the bacterial strain Bacillus atrophaeus 119 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 16, 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 atrophaeus 119 strain.

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 a 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.