PL248032B1 - Method of producing a microbiological fertilizer product and a microbiological fertilizer product for conditioning seedlings, maintaining and/or improving the microbiological quality of soil, while simultaneously allowing for controlling phytopathogens in soft fruit cultivation - Google Patents

Abstract

The subject of the invention is a method for obtaining a microbiological fertilizer product and a microbiological fertilizer product for conditioning seedlings, maintaining and/or improving the microbiological quality of the soil, also exhibiting plant biostimulation properties, simultaneously allowing for the control of phytopathogens in the cultivation of soft fruits and showing an effect on the improvement of their quality parameters, containing bacterial strains of the genus Bacillus. The method is characterized in that three environmental bacterial isolates selected from the rhizosphere soil are used: Bacillus subtilis AF75AB2 - sequence no. 1, Bacillus sp. AF75BC - sequence no. 2, Bacillus sp. Spl 115AD - sequence no. 3 on the list of sequences that do not show mutual antagonistic effects, cultivated on a multiplication medium with whey and microelements, prepared in water, suspended in a liquid organic carrier or dried on a loose carrier or lyophilized on a loose carrier. In addition, a proper carrier is used in the form of molasses decoction, which is a by-product of the molasses fermentation process, enriched with dry humic acids for the liquid form of the biopreparation, or micronized dolomite, enriched with dry humic acids, ground mustard seeds and clove oil for the dry form of the biopreparation, insoluble in water, or maltodextrin, enriched with dry humic acids, ground mustard seeds and clove oil for the dry form of the biopreparation, soluble in water, or whey powder, preferably acid neutralized, enriched with dry humic acids, ground mustard seeds and xanthan gum for the gel form, i.e. dry, which after suspension in water forms a gel for conditioning the roots of soft fruit plant seedlings.

Description

The invention concerns a method for obtaining a microbiological fertilizer product and a microbiological fertilizer product for conditioning seedlings, maintaining and/or improving the microbiological quality of soil, while also allowing for the control of phytopathogens in soft fruit cultivation. The preparation contains Bacillus spp. bacterial strains, as well as carrier components, including humic acids and other additives. The preparation improves the quality parameters of soft fruit and also exhibits plant biostimulation properties.

Research on the development of organic fruit cultivation techniques focuses, among others, on the analysis of the biodiversity of microorganisms in the soil environment and changes in the intensity of processes occurring in the rhizosphere, as well as the use of bioproducts to protect plants against fungal pathogens and pests (Frąc, M., Hannula, E.S., Belka, M., Salles, J.F., Jedryczka, M., 2022. Soil mycobiome in sustainable agriculture. Front. Microbiol. 13:1033824. 10.3389/finicb. 2022.1033824). In organic farming, the use of natural fertilizers and substances that improve soil properties is permitted. These are inorganic and organic products that are friendly to the environment and people (EASAC, 2022. Regenerative agriculture in Europe: A critical analysis of contributions to the European Union Farm to Fork and Biodiversity Strategies. European Academies Science Advisory Council, policy report 44, April 2022, pp. 1-70, ISBN: 978-3-8047-4372-4, www.easac.eu). Organic fertilizers and soil conditioners based on plant extracts and compost are available on the European market. Bioproducts based on plant extracts increasingly contain the addition of beneficial microorganisms, which are also used to control diseases and pests (Pylak M., Oszust K., Frąc M. (2019). Review report on the role of bioproducts, biopreparations, biostimulants and microbial inoculants in organic production of fruit. Rev Environ Sci Biotechnol (2019) 18:597-616, https://doi.org/10.1007/s1157-019-09500-5).

The situation of organic farming is changing dynamically in Poland, Europe, and globally. However, despite the difficulties, many farmers continue to convert their farms to organic farming, and the upward trend in this type of farming is also observed and supported by the European Union (European Commission. Communication from the commission to the European Parliament, the council, the European economic and social committee, and the committee of the regions. A Farm to Fork Strategy for a Fair, Healthy, and Environmentally-friendly Food System, 2020, ISBN 9789896540821, European Commission, Brussels, Belgium). Between 2003 and 2013, the number of organic farms in Poland increased more than 11-fold, from 2,286 in 2003 to almost 26,598 in 2013. Furthermore, the size of organic farms is also systematically increasing (FAOSTAT, Central Statistical Office). However, in Poland, there are few natural fertilizers and protective substances based on microorganisms, especially those containing native bacterial strains suitable for organic farming and fertilization of fruit crops, especially those dedicated to soft fruit crops. Furthermore, Polish agriculture faces the challenge of developing sustainable development strategies that protect non-renewable natural resources, including soil. In recent decades, much attention has been paid to mitigating soil erosion through physical protection measures and providing additional nutrients and water to meet crop needs. Less attention has been paid to soil as a dynamic living organism, even though its condition is crucial for food production and for the global balance and functioning of the ecosystem. Soil quality and health determine the development of sustainable agriculture, maintaining environmental quality and, consequently, supporting the health of plants, animals and humans (Frąc, M., Hannula, S.E., Belka, M., Jędryczka, M., 2018. Fungal Biodiversity and Their Role in‘soil Health. Front. Microbiol. 9:707. doi: 10.3389/fmicb.2018.00707). Agricultural practices that use large amounts of external inputs such as inorganic fertilizers, pesticides and other artificial inputs can overcome specific soil limitations in crop production, but the adverse impact of these additives on the environment is increasingly observed, leading to continuous environmental degradation, including the loss of soil biodiversity. Therefore, sustainable agriculture, including organic agriculture, is currently one of the fastest growing branches of agriculture in the world, especially in the European Union (EASAC, 2022. Regenerative agriculture in Europe: A critical analysis of contributions to European Union Farm to Fork and Biodiversity Strategies. European Academies Science Advisory Council, policy report 44, April 2022, pp. 1-70, ISBN: 978-3-8047-4372-4, www.easac.eu).

Therefore, the development of innovative microbiological fertilizer products is necessary for the environment and expected by farmers and producers of fertilizers and bioproducts in order to change the production profile to a greener and more ecological one and to provide farmers with tools for sustainable production.

The genus Bacillus is an intensively studied group of bacteria with great potential for use in agriculture, mainly due to the fact that microorganisms belonging to this genus occur in almost all types of soil, are highly tolerant to high temperatures, multiply rapidly in liquid culture media and belong to spore-forming bacteria.

Current knowledge suggests that strains of Bacillus species exhibit high metabolic activity, useful in the transformation of organic matter, and antagonistic properties against various fungal phytopathogens. However, this is not a self-evident characteristic, and selecting suitable strains requires extensive research. Below are several examples of the use of Bacillus spp. strains in agricultural applications.

An example of the use of strains of the Bacillus genus is a preparation containing spores of the Bacillus amyloliquefaciens strain (NRRL B-50349), which effectively limited the growth of fungal phytopathogens such as: Aspergillus niger; Bremia lactucae, Erisphe necator, Rhizoctonia Solani, Sclerotinia sclerotiorum, Septoria apicola, Spatherotheca fuligniea, Spatherotheca macularis (Snyder A., Vance J., Gnanmanickam S., Bacillus amyloliquefaciens strain, 2016, US Patent: US 9234251B2).

Bacillus subtilis 4/7 (PCM B/00105) and/or Bacillus licheniformis 4/18 (PCM B/00106) isolates with high cellulolytic activity were used as components of a preparation for the mineralization of organic matter, in which these bacteria are embedded on a carrier in the form of an aluminosilicate mineral (Ciecierski W., Kardasz H., Szychowska K., Wilk R., Microbiological preparation for the mineralization of organic matter containing cellulose, especially post-harvest waste, and the use of the microbiological preparation in plant cultivation, Pat.230762).

The use of Bacillus methylotrophicus XT1 and XT2 strains with antagonistic activity against Verticillium dahliae, Botrytis cinerea and Phytophthora cactorum in plant protection is also known (Bejar Luque M.V., Llamas Company, Inmaculada, Ruiz Garcia C., Quesada Arroquia E., Use of Bacillus methylotrophicus as a stimulant of plant growth and biological control means, and isolates of said species, 2016, CA Patent: CA 2991678 A1). Bacillus subtilis and Bacillus licheniformis bacterial strains have also been used as components of a nematicidal preparation (Abilio A., Knap I., De Freitas Zambelli L.S., Nematicidal composition containing Bacillus subtilis and Bacillus licheniformis, EP2603086).

From the publication: Paweł Trzciński, Mateusz Frąc, Anna Lisek, Michał Przybył, Magdalena Frąc, Lidia Sas-Paszt, Growth promotion of raspberry and strawberry plants by bacterial inoculants, Acta Sci. Pol. Hortorum Cultus, 20(6) 2021, 71-82, https://doi.org/10.24326/asphc.2021,6,8: and the publication Drobek M, Cybulska J, Gałązka A, Feledyn-Szewczyk B, Marzec-Grządziel A, Sas-Paszt L, Gryta A, Trzciński P, Zdunek A and Frąc M (2021) The Use of Interactions Between Microorganisms in Strawberry Cultivation (Fragaria x ananasa Duch.). Front. Plant Sci. 12:780099. doi: 10.3389/fpls.2021.780099, as well as publications: Anna Gałązka, Anna Marzec-Grządziel, Jarosław Grządziel, Karolina Gawryjołek, Karolina Furtak, Jarosław Ciepiel, Magdalena Frąc, The use of microorganisms to protect plants against fungal pathogens in organic strawberry cultivation, 60th Scientific Session of the Institute of Plant Protection of the National Research Institute, Poznań, February 11-13, 2020, pp. 175-176, the use of, among others, bacterial isolates including Bacillus sp. AF75AB2, Sp115AD, AF75BC to control the growth of fungal pathogens of raspberry and strawberry plants such as: Verticillium dahliae, Botrytis cinerea, Phytophthora cactorum and Colletotrichum acutatum is known.

Publication: da Silva MSRA, Dos Santos BMS, da Silva CSRA, da Silva CSRA, Antunes LFS, Dos Santos RM, Santos CHB, Rigobelo EC. Humic Substances in Combination With Plant Growth-Promoting Bacteria as an Alternative for Sustainable Agriculture. Front Microbiol. 2021;12:719653. doi:10.3389/fmicb.2021.719653 and publication: Agnieszka Rutkowska, STUDIES AND REPORTS IUNG-PIB, Issue 48(2): 65-80, 2016, Biostimulants in modern plant cultivation, doi:10.26114/sir.iung.2016.48.05 describe the use of humic compounds in plant cultivation, confirming their high effectiveness.

Most often, patents concern biopreparations used to control pathogens of vegetable and cultivated plants, and there are no solutions covering microbiological fertilizer preparations for conditioning plant seedlings, simultaneously affecting the microbiological quality of the soil and showing antagonistic effects on key fungal pathogens and fungus-like oomycetes, also intended for use in organic cultivation of soft fruits and improving their quality, or these are solutions covering the control of single pathogens, e.g. from the Botrytis genus, not constituting a comprehensive solution for the four key phytopathogens (Botrytis spp., Colletotrichum spp., Phytophthora spp., Verticillium spp.), maintaining and/or improving soil microbiological biodiversity and supporting biostimulation of plant growth and fruit quality.

There are also no literature reports and patents regarding the combined use of microorganisms on carriers composed of humic acids and/or molasses decoction and/or ground mustard and/or xanthan gum and/or clove oil and/or micronized dolomite and/or maltodextrin and/or whey powder, depending on the form of application (liquid or dry or gel) of the fertilizing microbiological product.

The aim of the invention is therefore to develop a microbiological fertilizer product for conditioning seedlings, maintaining and/or improving the microbiological quality of the soil, which at the same time allows for the control of phytopathogens of the genera: Botrytis, Verticillium, Colletotrichum and oomycetes of the genus Phytophthora in the cultivation of soft fruits and has an impact on the improvement of fruit quality parameters and plant characteristics important in biostimulation.

While conducting research on microbiological fertilizer products containing strains of the Bacillus genus, it was unexpectedly found that three of them not only exhibit antagonistic properties towards the phytopathogens Botrytis spp., Verticillium spp., Colletotrichum spp. and Phytophthora spp., but when immobilized on appropriate carriers, they exhibit increased antagonistic properties towards these plant pathogens, influence the preservation and/or improvement of soil microbiological biodiversity, including its health, as well as exhibit features supporting biostimulatory effects on plant growth and development and fruit quality parameters.

Therefore, the above goal was achieved by developing a method for obtaining a microbiological fertilizer product for conditioning plant seedlings, ensuring and/or improving soil microbiological biodiversity while simultaneously controlling key pathogens for soft fruit cultivation, such as Botrytis, Verticillium, Colletotrichum, and Phytophthora. The research also led to the development of a microbiological fertilizer product composition. The research led to a unique, optimized composition of the liquid medium for bacterial cultivation, carriers, and other components on which the microorganisms are immobilized. This led to the creation of a unique composition of the individual components of the microbiological fertilizer product, which determine the properties and originality of the developed solution and constitute a patent claim.

The essence of the method for obtaining a fertilizing microbiological product for conditioning plant seedlings, maintaining and/or improving soil biodiversity and/or controlling pathogens: Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp. in the cultivation of soft fruits, showing biostimulating properties on plants and influencing the quality of fruit, using bacterial strains of the genus Bacillus, is that:

- three bacterial isolates selected from the rhizosphere of healthy plants, not showing mutual antagonistic activity: Bacillus subtilis AF75AB2 - sequence no. 1, Bacillus sp. AF75BC - sequence no. 2, Bacillus sp. Sp115AD - sequence no. 3 on the sequence list, cultivated on a multiplication medium with whey and microelements, prepared on water and containing in 1 liter: 20 g of whey powder, preferably acid neutralized, 6.6 g of Na2HPO4, 3 g of KH2PO4, 1 g of NH4Cl, 0.5 g of NaCl, 0.022 g of CaCl2, 0.18 g of MgSO4, 12.9 mg of FeCl2, within the limits of ± 10% of each of the medium components, suspended in a liquid organic carrier for liquid form or dried on a loose carrier or lyophilized on a loose carrier for the dry form;

- a proper carrier in the form of molasses vinasse, which is a by-product of the fermentation process of molasses for the liquid form, or micronized dolomite for the dry form, insoluble in water, or maltodextrin for the dry form, soluble in water, or whey powder, preferably acid-neutralized for the powder form for preparing a gel;

- addition of dry humic acids for the liquid form of the product or a mixture of dry humic acids, ground mustard seeds and clove oil for the dry form or a mixture of dry humic acids, ground mustard seeds and xanthan gum for the powder form for preparing a gel, as supplementary components of the proper carrier.

The method for obtaining a microbiological fertilizer product preferably comprises a method for culturing bacterial strains of the genus Bacillus, with sequences no. 1, 2 and 3, respectively, indicated in the sequence list, in which the bacterial strains are initially multiplied in a stationary culture on a Plate Count Agar medium at a temperature of 25-30°C for 24-48 hours, and then the liquid multiplication medium is inoculated with the inoculum thus prepared, in an amount of 5%-15% of the volume of the culture medium, and the multiplication culture is carried out under shaking culture conditions at a temperature of 30°C at 120 rpm.

Preferably, the method of culturing bacterial strains uses an inoculum with a transmittance of 90%.

Preferably, the expansion culturing is carried out for 48 hours.

It is preferred that the whey-based growth medium contains the following microelements: Mn, Zn, Cu, Co and Mo.

After obtaining a multiplication culture titer of not less than 109 cfu/ml, a sporulation induction step is preferably carried out, preferably by changing the salinity of the culture medium, by adding KCl, to obtain a salinity of 4% and after 48 hours of cultivation and production of spores by bacterial cells, the culture is pasteurized by incubation at 80°C for 20 minutes and the obtained spore suspension is used as the main component of the microbiological fertilizer product.

After culturing, to obtain a dry product, the prepared spore suspension is dried using a spray dryer on whey powder or freeze-dried with the addition of whey powder as a cryoprotectant, in the amount of 5% of the fresh mass of the graveled microorganisms.

Dried bacteria or bacterial lyophilisates with a concentration of not less than ¹⁰¹¹ cfu/g are preferably added in equal amounts to obtain a product with a concentration of each strain in the range of 108 - ¹⁰¹¹ cfu/g.

Alternatively, to obtain a liquid form, 1 liter of a liquid organic carrier in the form of molasses decoction is used as a carrier for the bacteria, with the addition of dry humic acids in the amount of 8-12 g/l of molasses decoction.

In the variant to obtain a dry form insoluble in water, a mixture of dry humic acids in the amount of 0.80-1.2 g/kg of product, ground mustard seeds in the amount of 4.0-6.0 g/kg of product and clove oil in the amount of 0.01 ml/kg of product is used as a carrier component, and micronized dolomite is used as a carrier for all components, constituting a supplement to 1 kg.

In the variant to obtain a dry, water-soluble form, maltodextrin is used as a cryoprotectant for bacteria in the spray-drying or freeze-drying process and as a carrier for all components of the microbiological fertilizer product.

In addition, for the dry water-soluble form, a mixture of dry humic acids in the amount of 0.80-1.2 g/kg of product, ground mustard seeds in the amount of 4.0-6.0 g/kg of product and clove oil in the amount of 0.01 ml/kg of product is used as a carrier component, and maltodextrin is added to the total amount of 1 kg.

In another variant, to obtain a product in the form of powder for preparing a gel, a mixture of dry humic acids in the amount of 8-12 g/kg of product, ground mustard seeds in the amount of 40-60 g/kg of product and xanthan gum in the amount of 30-50 g/kg of product is used as a carrier component, and whey powder, preferably acid neutralized, is used as a carrier for all components of the biopreparation, constituting a supplement to 1 kg.

The essence of the microbiological fertilizer product for conditioning plant seedlings, maintaining and/or improving the microbiological quality of the soil, simultaneously allowing the control of phytopathogens: Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp. in the cultivation of soft fruit, showing biostimulating properties on plants and improving fruit quality, containing strains of bacteria of the genus Bacillus, is that it contains three bacterial isolates selected from the rhizosphere of healthy plants, showing no antagonistic activity to each other: Bacillus subtilis AF75AB2 - sequence no. 1, Bacillus sp. AF75BC - sequence no. 2, Bacillus sp. Sp115AD - sequence no. 3 on the sequence list. It also contains a carrier in the form of molasses vinasse for the liquid form, or micronized dolomite for the dry, water-insoluble form, or maltodextrin for the dry, water-soluble form, or whey powder for the powder form for gel preparation, and contains the addition of dry humic acids as supplementary carrier components for the liquid form, or dry humic acids, ground mustard seeds, and clove oil for the dry, water-soluble and insoluble form, or dry humic acids, ground mustard seeds, and xanthan gum for the powder form for gel preparation. Bacterial isolates are cultured in a growth medium with whey and trace elements, prepared in water, and suspended on the carrier, dried on the carrier, or lyophilized on the carrier.

The microbiological fertilizer product contains the bacterial strains Bacillus subtilis AF75AB2 - sequence no. 1, Bacillus sp. AF75BC - sequence no. 2, Bacillus sp. Sp115AD - sequence no. 3 on the sequence list, preferably in an equal weight ratio.

Bacterial isolates are preferably cultured on a multiplication medium with whey powder, preferably acid-neutralized.

It is preferred that the concentration of each bacterial strain in a dry, water-soluble or insoluble form or in the form of a powder for preparing a gel is in the range of 108 - 1011 cfu/g, and in a liquid form in the range of 108 - 1011 cfu/ml.

In the liquid form, the microbiological fertilizer product contains 8-12 g of dry humic acids per 1 liter of molasses decoction.

In the dry, water-soluble or insoluble form, the microbiological fertilizer product contains an addition of 0.80-1.2 g of dry humic acids, 4.0-6.0 g of ground mustard seeds and 0.01 ml of clove oil per 1 kg of product.

In the powder form for preparing the gel, the microbiological fertilizer product contains the addition of 8-12 g of dry humic acids, 40-60 g of ground mustard seeds and 30-50 g of xanthan gum per 1 kg of product.

The dry, water-insoluble product form contains powdered whey as a cryoprotectant in the freeze-drying or spray-drying process and micronized dolomite as the actual carrier.

The dry, water-soluble form of the product contains maltodextrin as a cryoprotectant in the freeze-drying or spray-drying process and as the actual carrier.

The powder form for preparing the gel contains powdered whey as a cryoprotectant during freeze-drying or spray-drying and as the carrier. The Bacillus spp. strains (AF75AB2, AF75BC, and Sp115AD) used to develop the microbial fertilizer product are environmental isolates and were selected at the Institute of Horticulture - National Research Institute in Skierniewice and disclosed in the Institute's SYMBIO BANK collection.

The resulting microbial fertilizer product, available in liquid, dry, and powder forms for gel preparation, enables the conditioning of plant seedlings, maintaining and/or improving soil microbial biodiversity. It also influences plant growth characteristics and fruit quality, and controls four important soft fruit pathogens, including three fungal species from the genera Botrytis, Colletotrichum, and Verticillium, and one oomycete from the genus Phytophthora. It should be emphasized that there are no commercially available biopreparations for the conditioning of plant seedlings with proven effectiveness in maintaining and improving soil microbial biodiversity based on the analysis of rhizosphere microbiome data. Analysis of such data allows for determining the impact on soil microbial biodiversity, while analysis of the root microbiome allows for the assessment of the impact on root microbial biodiversity. Therefore, the approach presented in this patent application is unique and innovative and confirms the beneficial effect of the developed biopreparation on the soil environment and the plant, providing the basis for recognizing the developed bioproduct as a microbiological fertilizer. Depending on the form, the biopreparation is applied directly to the roots (gel form) or/and directly under the roots via fertigation (liquid form) or/and foliarly (by spraying or sprinkling) or/and under the plant by watering (dry form to be suspended/dissolved in water). In the case of a dry formulation, a suspension of the biopreparation in water should be prepared, preferably 2-5 kg of the biopreparation (with a bacterial concentration of 109 cfu/g) per 700-800 liters of non-chlorinated water per 1 ha of crop.

In a preferred method of maintaining and/or improving the microbiological biodiversity of soil and/or roots and/or features important for biostimulation of plant growth and development and fruit quality and/or control of plant pathogens, the biopreparation is used in an amount of preferably 2-5 kg/ha for suspension in water by introducing it directly onto and/or under the roots of plants when establishing new plantations, or/and by foliar and/or soil and/or soil spraying in the case of existing plantations, of the preparation containing at least ¹⁰⁹ cfu/g of bacteria isolated from the rhizosphere of plants according to the composition and/or the biopreparation, wherein in the form of watering and/or spraying/sprinkling/irrigation the biopreparation can be advantageously diluted with water depending on the needs. In a preferred method of applying the liquid form for fertigation, the microbiological fertilizer product is preferably applied in the form of three injections during the growing season in the amount of 15 l of biopreparation per injection per 15,000 l of water per 1 ha at 3-4 week intervals or in the form of 12 injections applied weekly for 12 weeks of plant vegetation in the amount of 2 l of biopreparation per injection per 2,000 l of water per 1 ha. In a preferred method of protecting plant roots with a gel formulation (a powder that transforms into a viscous liquid gel after the addition of an appropriate amount of water), a microbiological fertilizer product is applied to the roots of plant seedlings used during plantation establishment, preferably at a dose of approximately 2 ml for strawberry plants or 10-20 ml for raspberry plants. The formulation is preferably prepared by suspending 1 g of the powdered bioproduct in 10 ml of water. The microbiological fertilizer product is applied in the form of a gel (a suspension of the preparation in a gel carrier), which is applied to the roots of plant seedlings by conditioning them (soaking the seedlings in the prepared formulation). The formulation is prepared by suspending 100 g of the powdered formulation in 1 liter of tap water, preferably non-chlorinated, and mixing the suspension thoroughly. The biopreparation prepared this way is ready for application to plant seedlings. Conditioning seedlings in a developed microbiological fertilizer product has a positive effect on plant rooting and vegetative growth, as well as protecting developing plants against pathogen infections.

The invention is illustrated in the embodiment examples and in the drawing, where:

Fig. 1 shows the appearance of colonies of bacterial strains AF75AB2, AF75BC, Sp115AD on PCA (Plate Count Agar) medium, 48-hour culture;

Fig. 2 shows the metabolic profile of bacterial strains that are components of the microbiological fertilizer product, indicating the degree of consumption of substrates (A 590 nm) belonging to amino acids, sugar alcohols, carbohydrates, sugar acids, sugar derivatives and carboxylic acids;

Fig. 3 shows the growth intensity (A 750 nm) of bacterial strains that are components of the microbiological fertilizer product on substrates belonging to amino acids, sugar alcohols, carbohydrates, sugar acids, sugar derivatives and carboxylic acids;

Fig. 4 shows the chemical sensitivity of bacterial strains that are components of the microbiological fertilizer product, based on the inhibition of respiratory processes (A 590 nm), towards selected compounds, including antibiotics and dyes;

Fig. 5 shows the chemical sensitivity to selected compounds exhibited by bacterial strains that are components of the microbiological fertilizer product, based on the inhibition of growth intensity (A 750 nm);

Fig. 6 shows the enzymatic activity profiles of bacterial strains that are components of the microbial fertilizer product, determined on the basis of API ZYM tests (Biomerieux);

Fig. 7 is a table presenting the metabolic properties of bacterial strains included in the microbial fertilizer product;

Fig. 8 shows the antagonism of the bacterial gel biopreparation containing the bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD towards plant fungal pathogens of the genera Botrytis, Colletotrichum, Vertticillium and the fungus-like oomycete of the genus Phytophthora;

Fig. 9 presents a table concerning the antagonism of a microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD in liquid form for fertigation, dry water-insoluble, dry water-soluble and gel form, towards plant fungal pathogens of the genera Botrytis, Colletotrichum, Vertticillium and the fungus-like oomycete of the genus Phytophthora;

Fig. 10 shows the antagonism of the microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC,

Bacillus sp. Sp115AD in a dry, water-soluble form (carrier - maltodextrin) against plant fungal pathogens of the genera Botrytis, Colletotrichum, Vertticillium and the fungus-like oomycete of the genus Phytophthora after 1 month of storage of the biopreparation at room temperature of about 21°C;

Fig. 11 shows the antagonism of the microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD in a dry, water-insoluble form (carrier - micronized dolomite) towards plant fungal pathogens of the genera Botrytis, Colletotrichum, Vertticillium and the fungus-like oomycete of the genus Phytophthora after 3 months of storage of the biopreparation at room temperature (approximately 21°C), 4°C and 35°C;

Fig. 12 shows the antagonism of the microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD in liquid form for fertigation (carrier - molasses decoction) towards plant fungal pathogens of the genera Botrytis, Colletotrichum, Vertticillium and the fungus-like oomycete of the genus Phytophthora after 3 months of storing the biopreparation at room temperature (approximately 21°C) and after 6 months of storing the biopreparation at 4°C;

Fig. 13 shows the effective number of species (ENS) of bacteria in the raspberry rhizosphere after application of a microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD;

Fig. 14 shows the effective number of fungal species (ENS) in the root of raspberry plants after application of a microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD;

Fig. 15 shows the relative percentage composition of trophic groups of fungi occurring in the rhizosphere and roots of raspberry plants after application of a microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD;

Fig. 16 shows the occurrence of functional guilds in groups including fungal pathotrophs occurring in the rhizosphere and roots of raspberry plants after application of a microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD;

Fig. 17 shows the relative abundance of bacterial types occurring in the rhizosphere and roots of raspberry plants after application of a microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD;

Fig. 18 shows the relative abundance of bacterial classes occurring in the rhizosphere and roots of raspberry plants after application of a microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD;

Fig. 19 shows the relative abundance - biodiversity of orders, families, genera and species of bacteria occurring in the rhizosphere and roots of raspberry plants after application of a microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD, where the more colored bands, the greater the biodiversity of bacteria;

Fig. 20 shows the relative abundance of fungal types occurring in the rhizosphere and roots of raspberry plants after application of a microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. S115AD;

Fig. 21 shows the relative abundance of fungal classes occurring in the rhizosphere and roots of raspberry plants after application of a microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. S115AD;

Fig. 22 shows the relative abundance - biodiversity of orders, families, genera and species of fungi occurring in the rhizosphere and roots of raspberry plants after application of a microbiological fertilizer product containing selected bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. S115AD, where the more colored stripes, the greater the biodiversity of fungi;

Fig. 23 shows the form of the microbiological fertilizer preparation for preparing the gel and the method of applying the liquefied gel form to the roots of plants: a) powder before adding water, b) liquefied form of the gel for application to the roots, c) application to the root cuttings of a strawberry plant, d) the appearance of the root after application of the biopreparation.

EXAMPLES

Example 1. Identification and biochemical characterization of bacterial strains that are components of a microbiological fertilizer product

Three bacterial strains selected for use in a microbial fertilizer product, selected from rhizosphere soil and originating from the SYMBIO BANK collection of the Institute of Horticulture - National Research Institute in Skierniewice, were identified based on 16S rDNA gene sequencing. The nucleotide sequences obtained from Sanger sequencing were subjected to comparative analysis with nucleotide sequences found in the commercial MicroSe q ^ID database and the open NCBI database (https://www.ncbi.nlm.nih.gov/). Based on molecular and bioinformatic analysis, bacterial strain AF75AB2 (sequence no. 1) was identified as belonging to the species Bacillus subtilis, while strain AF75BC (sequence no. 2) and strain Sp115AD (sequence no. 3) were identified as belonging to the genus Bacillus.

Strains AF75AB2, AF75BC, and Sp115AD were characterized for their basic metabolic properties that influence the biochemical properties of the soil environment. The strains demonstrated excellent growth with a creamy color on PCA (Plate Count Agar) (Fig. 1). The biochemical activity of the strains included in the biopreparation was characterized based on their ability to utilize 71 carbon substrates, including amino acids, sugar alcohols, carbohydrates, sugar acids, sugar derivatives, and carboxylic acids (Fig. 2), as well as determining the rate of growth in the presence of these substrates (Fig. 3). The chemical sensitivity of strains AF75AB2, AF75BC, and Sp115AD to 23 chemical compounds, including antibiotics and dyes, was determined. The sensitivity of bacterial strains to these compounds was assessed based on the inhibition of their respiratory processes and presented as a respiratory intensity profile of individual chemical compounds (Fig. 4), as well as by assessing the inhibition of bacterial growth in the presence of the tested compounds (Fig. 5). Furthermore, using the API ZYM rapid biochemical test (Biomerieux), the activity of basic hydrolytic enzymes, including phosphatases, esterases, arylamidases, phosphohydrolases, β-glucosidase, β-galactosidase, mannosidase, and glucosaminidase, was determined in selected bacterial strains (Fig. 6). The biochemical capabilities of the strains were determined in accordance with the test methodological guidelines by preparing a bacterial suspension with a density of 5-6 on the McFerland scale. All three strains AF75AB2, AF75BC, and Sp115AD showed esterase, lipase esterase, naphthyl-AS-BI phosphohydrolase, and β-glucosidase activities. Strains AF75AB2 and AF75BC were characterized by high lipase, α-galactosidase, and N-acetyl-ε-glucosaminidase activities. However, strain Sp115AD was the only one of the three isolates tested to show alkaline phosphatase, leucine arylamidase, and α-glucosidase activities (Fig. 7).

Example 2. Determination of the antagonistic capacity of a microbiological fertilizer product containing the bacterial strains Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD against plant pathogens of the genera Colletotrichum, Botrytis, Phytophthora and Verticillium - a plate experiment.

The antagonistic capacity of the bacterial preparation in gel form was determined by measuring the zones of inhibition of pathogen growth. Petri dishes with a diameter of 90 mm and a PCA (Plate Count Agar) medium were inoculated with 300 μl of spore suspensions (with 70% transmittance) of the phytopathogenic fungi Colletotrichum sp. (G171/18), Botrytis sp. (G277/18), and Verticillium sp. (G297/18), as well as the fungus-like oomycete Phytophthora sp. (G408/18). The plates were incubated at 23°C for 24 hours. After initial incubation, the biopreparation in the form of a prepared gel (approximately 20-30 μl) was placed in the center of the dish. The plates were incubated at 23°C for 5-7 days and the zones of growth inhibition and/or sporulation inhibition were measured, confirming the antagonistic effect of the developed microbial fertilizer product on key phytopathogens of soft fruits (Fig. 8 - Fig. 9).

Storage studies were also conducted, during which the antagonistic properties of the biopreparation were determined periodically during 6 months of storage at various conditions, including room temperature (~21°C), refrigeration (4°C), and elevated storage temperature (35°C). The antagonistic properties of the biopreparation were determined on various carriers: liquid (molasses vinasse), water-insoluble (micronized dolomite), water-soluble (maltodextrin), and for gel preparation (whey with xanthan gum). The obtained results indicate that the antagonistic properties of the developed biopreparation formulations against key fungal phytopathogens and fungi-like oomycetes of soft fruit generally persisted during storage, but this effect depended on both the tested pathogen and the storage conditions used (Fig. 10-Fig. 12). The best antagonistic properties after 1 month of storage of the microbiological fertilizer product in which bacteria were immobilized on maltodextrin were obtained against the fungus-like oomycete Phytophthora sp., demonstrating biocidal properties and preventing the development of this pathogen, and for the tested fungal phytopathogens, a fungistatic effect of the bioproduct was observed, manifested by the inhibition of sporulation of fungi of the genera Botrytis, Colletotrichum, and Verticillium (Fig. 10). The microbiological fertilizer product immobilized on micronized dolomite after 3 months of storage, regardless of the applied temperature, demonstrated the most effective effect against phytopathogens of the genera Phytophthora, Botrytis, and Verticillium, and in the case of Colletotrichum sp. it caused the inhibition of sporulation of this fungus (Fig. 11). The best antagonistic properties after 6 months of storage of the liquid biopreparation for fertigation were obtained against phytopathogens of the genera Phytophthora, Botrytis and Verticillium, demonstrating biocidal properties and preventing the development of these pathogens, as well as fungistatic properties, manifested by the inhibition of sporulation of fungi of the genus Colletotrichum (Fig. 12).

Example 3. Determination of the number of bacteria in a microbiological fertilizer product

The bacterial count in the prepared microbiological fertilizer product was determined using the dilution method, by counting and determining the total bacterial count. A series of dilutions of the biopreparation were prepared, ranging from 10'1 to ^10'8 , and 0.1 ml of each dilution was plated onto a 90 mm Petri dish with PCA medium, spread evenly across the dish, and then incubated at 26°C for 72 hours. The grown colonies were counted, and the total bacterial count was determined in the individual formulations of the microbiological fertilizer product, including liquid CFU/ml and dry CFU/g.

After production, the preparation showed a total bacterial count of >109 cfu/ml of the bacterial biopreparation in liquid form, >109 cfu/g of the bacterial biopreparation in dry, water-soluble and insoluble forms, and >108 cfu/g of the bacterial biopreparation in gel form. Storage test results showed that the bacterial count in the preparation remained at a level of > ¹⁰⁸ cfu/g or >108 ^cfu /ml, or even > ¹⁰⁹ cfu/g of the biopreparation, depending on the storage conditions used, for a period of 6 months.

Example 4. Effect of a microbial fertilizer product on maintaining and/or improving soil microbial biodiversity and plant microbiome - pot tests

To determine the fertilizing effect of a microbial product on soil microbial biodiversity and plant microbiome, a greenhouse experiment was conducted on Polana raspberry plants. The plants were planted in 3-liter pots containing a growth medium consisting of 14 volumes of soil and 14 volumes of 7-20 mm peat. The biopreparation was applied to the root surface, and untreated plants, growing in a medium supplemented with 5 g of dried manure per pot, served as a control. In this example, the biopreparation was applied in gel form. To determine changes in biodiversity, studies were conducted to characterize bacterial and fungal communities using next-generation sequencing (NGS) with high-throughput sequencing using Illumina SBS (Sequencing-by-Synthesis) technology, using the MiSeq instrument, and bioinformatics tools such as QIIME2. The obtained results were analyzed based on sequencing of the 16S rDNA marker for bacteria and ITS1 for fungi. The diversity of microorganisms in the rhizosphere and plant roots was determined. Application of the bacterial biopreparation allowed for the assessment of changes in biodiversity indicators, including the effective number of species (ENS) of bacteria and fungi in the rhizosphere and roots of treated and untreated plants. The microbial fertilizer product increased bacterial biodiversity in the rhizosphere compared to the control (Fig. 13) and maintained fungal biodiversity at the control level. Furthermore, the biopreparation caused a reduction in the ENS of fungi colonizing the root compared to control root samples not treated with the bioproduct, which may indicate a protective effect of the gel biopreparation applied directly to the seedling roots (Fig. 14). Trophic groups and functional guilds of fungi were also analyzed using the FunGuild database. These results showed that in the roots of plants treated with the microbial fertilizer product, a reduction in mixed trophic modes, including pathotrophs: pathotrophs-saprotrophs and pathotrophs-saprotrophs-symbiotrophs, was observed, while an increase in saprotrophs and symbiotrophs, as well as the mixed saprotroph-symbiotroph mode in the root, was noted. A very large increase in the mixed mode of saprotrophic fungi was also demonstrated in the rhizosphere of raspberries treated with the microbial fertilizer product, as well as a decrease in pathotrophs and the mixed mode of pathotroph-saprotroph-symbiotroph (Fig. 15). Furthermore, it was demonstrated that after application of the bioproduct, the number of guilds assigned to plant pathogens decreased in both the plant rhizosphere and the root (Fig. 16). Analysis of the results obtained for bacterial communities generally showed an increase in the relative abundance of bacteria at various taxonomic levels after application of the biopreparation. This effect was noted in both the rhizosphere soil and the root (Fig. 17-Fig. 19). The analysis of the fungal community results confirmed the lack of an increase in the biodiversity of this group of microorganisms in the roots of raspberry plants treated with the biopreparation, and in the rhizosphere a similar level or a reduction in the abundance of fungi was maintained compared to the control not treated with the biopreparation, which may indicate a protective effect of the microbiological fertilizer product, limiting the penetration of fungi into the roots, especially those unfavorable for plants (Fig. 20-Fig. 22).

The advantage of this microbiological fertilizer product is its high versatility, confirmed by its intended use in conditioning plant seedlings, maintaining and/or improving soil microbial biodiversity and the microbiome of soft fruit plants, while simultaneously controlling phytopathogens found in these crops. The developed microbiological fertilizer product is effective against many pathogens found in soft fruit plantations, and its additional advantage is that it is based on natural ingredients, including native bacterial strains isolated from the rhizosphere of healthy plants, and is therefore environmentally friendly. An additional, very important advantage of the bacterial bioproduct is the multitude of ways of its application thanks to the liquid form intended for fertigation, the gel form dedicated to the conditioning of plant seedling roots, as well as the powder form to be suspended in water, enabling spraying, sprinkling or watering, which constitutes a comprehensive application technology depending on the needs and throughout the entire plant growing season, from plantation establishment to fruit harvest.

Example 5. The effect of a microbiological fertilizer product on the growth and development of plants and the quality of strawberry and raspberry fruit, the occurrence of phytopathogens in organic strawberry cultivation and microbiological biodiversity in raspberry cultivation - field tests.

To determine the effect of a microbial fertilizer product containing selected bacterial strains of Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, and Bacillus sp. Sp115AD on plant yield and the occurrence of phytopathogens, field experiments were conducted in which the microbial fertilizer product was incorporated into the cultivation technology as one component of the overall cultivation technology used in organic strawberry and raspberry cultivation. Tests were conducted on three strawberry varieties: Honeoye, Rumba, and Vibrant, using a dry, water-insoluble formulation in two cultivation methods: irrigated and non-irrigated in an organic production system, and on two strawberry varieties: Honeoye and Rumba, using a liquid formulation for fertigation, using three and twelve injections of the bioproduct during the growing season. Additionally, tests of the developed microbiological fertilizer product in gel and dry, water-insoluble form, introduced as part of the organic raspberry production technology, were conducted based on experiments with four raspberry varieties: Delniwa, Enrosadira, Rosalita, and Poemat. The obtained results indicate that the species and 12 plant varieties responded differently to the tested cultivation technologies, including the addition of the microbiological fertilizer product containing selected bacterial strains. In the irrigated and non-irrigated areas, the varieties in which the developed microbiological fertilizer product was added in the second year in dry, water-insoluble form, showed the greatest increase in strawberry yield. It was observed that in the non-irrigated area, the variant in which the bacterial biopreparation was added, showed a reduction in anthracnose infection.

The Vibrant cultivar, on the other hand, responded with a reduction in the incidence of strawberry leather rot. For the Delniwa, Poemat, and Enrosadira raspberry cultivars, the microbiological fertilizer significantly increased the content of anthocyanins and phenolic compounds in two growing seasons, indicating improved fruit quality parameters. Furthermore, the bioproduct was shown to improve strawberry fruit quality (Honeoye, Vibrant, Rumba), manifesting itself through increased vitamin C and anthocyanin content. The results indicate that for some raspberry cultivars, particularly Enrosadira Rosalita, an increase in the functional diversity of soil microorganisms, as well as those colonizing the plant and fruit, was observed. This effect primarily involved increased biodiversity indices, including AWCD (Average Well Color Development), a measure of overall microbiological activity, and R (Richness), which indicates the number of carbon substrates mobilized by microbial communities. The conducted research indicates that the metabolic activity of microorganisms present in the plant and fruit increased, in particular in relation to carbohydrates, in the variant in which the developed bacterial biopreparation in the form of a gel was used as one of the cultivation elements.

Example 6. Method for obtaining a microbiological fertilizer product - liquid formulation

To obtain a microbiological fertilizer product for conditioning plant seedlings, maintaining and/or improving the microbiological quality of the soil, influencing the growth and development of plants and fruit quality parameters, while also allowing for the control of phytopathogens (Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp.) in the cultivation of soft fruit, three non-exclusive bacterial strains selected from the rhizosphere of healthy plants were used: Bacillus subtilis AF75AB2 - sequence no. 1, Bacillus sp. AF75BC sequence no. 2, Bacillus sp. Sp115AD - sequence no. 3, on the list of sequences for which separate cultures were carried out, which were mixed in equal proportions in order to obtain a composition containing strains of saprophytic bacteria isolated from the rhizosphere, and the multiplication cultures were carried out at a temperature of 30°C.

After initial cultivation of the bacteria on Plate Count Agar for 24-48 hours at 25-26°C, the resulting inoculum was inoculated into the cultivation medium and the culture was carried out with shaking (120 rpm) for 48 hours at 30°C. The multiplication medium for the production of the biopreparation was an optimized medium containing: 6.6 g/L Na 2 HPO 4, 3 g/L KH 2 PO 4, 1 g/L NH 4 Cl, 0.5 g/L Na Cl, 20 g/L neutralized acid whey, 0.022 g/L CaCl 2, 0.18 g/L MgSO 4, 12.9 mg Fe Cl 2 and 10 ml of a microelement solution containing: 0.5 mM MnCl 2 * 4 H 2 O, 1.2 mM ZnCl 2, 0.27 mM CuCl 2 * 2 H 2 O, 0.23 mM CoCl 2 * 6 H 2 O, 0.23 mM Na 2 Mo O 4 * 2 H 2 O. After obtaining the multiplication culture titer, sporulation induction was performed by changing the culture conditions, which included changing the salinity of the culture medium by adding KCl to achieve a salinity of 4%. After 48 hours of cultivation, the cultures were pasteurized by incubation at 80°C for 20 minutes. The spore suspension thus prepared was freeze-dried with the addition of whey powder as a cryoprotectant, at a rate of 5% of the fresh weight of the centrifuged microorganisms, with the resulting lyophilizates having a count of 1011 cfu/g. To obtain a liquid form of the microbiological fertilizer product, 10 g of dry humic acids and 10 g of each bacterial lyophilisate (Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD) with a bacterial concentration of 1011 cfu/g were added to 1 liter of liquid organic carrier, which was vinassa - a molasses vinasse, a by-product of molasses fermentation. After combining the ingredients, a microbiological fertilizer product in a liquid formulation for fertigation was obtained, characterized by a bacterial count of ¹⁰⁹ cfu/ml of the bioproduct.

Example 7. Method for obtaining a microbiological fertilizer product - dry formulation insoluble in water

The method was carried out as in Example 6, but after the cultivation was completed and the lyophilisates were obtained, in order to obtain a water-insoluble dry formulation for spraying or watering, instead of a liquid organic carrier with dry humic acids, a carrier in the form of 991 g of micronized dolomite with the addition of 1 g of dry humic acids, 5 g of ground mustard seeds, 0.01 ml of clove oil and 1 g of each bacterial lyophilisate (Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD) with a bacteria concentration of ¹⁰¹¹ cfu/g was used.

Example 8. Method for obtaining a microbiological fertilizer product - dry formulation soluble in water

The method of obtaining the microbiological fertilizer product was carried out as in Examples 6 and 7, except that maltodextrin was added to the freeze-drying process instead of whey powder and maltodextrin was used instead of the micronized dolomite carrier.

Example 9. Method for obtaining a microbiological fertilizer product containing spray-dried bacterial strains

The microbiological fertilizer product obtained by the method according to Example 6 has a composition analogous to that in Example 6, except that spray drying is used instead of freeze-drying.

Example 10. Method for obtaining a microbiological fertilizer product - formulation for preparing a gel

The method of obtaining the bioproduct was carried out as in Example 6, except that after completion of the cultivation, it was subjected to spray drying instead of lyophilization and instead of a liquid organic carrier with dry humic acids, a carrier in the form of a mixture of 10 g of dry humic acids, 50 g of ground mustard seeds, 40 g of xanthan gum and 900 g of acidic neutralized whey powder containing three bacterial strains (Bacillus subtilis AF75AB2, Bacillus sp. AF75BC, Bacillus sp. Sp115AD) with a concentration of 109 cfu/g was used. The method of preparation and application of the biopreparation to the roots is shown in Figure 23 (Fig. 23).

Claims (23)

1. A method for obtaining a microbiological fertilizer product for conditioning plant seedlings, maintaining and/or improving the microbiological quality of the soil, simultaneously allowing for the control of phytopathogens Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp. in the cultivation of soft fruit, showing biostimulating properties on plants and improving the quality parameters of fruit, using strains of bacteria of the genus Bacillus, characterized in that: - three bacterial isolates selected from the rhizosphere of healthy plants, not showing mutual antagonistic activity: Bacillus subtilis AF75AB2 - sequence no. 1, Bacillus sp. AF75BC - sequence no. 2, Bacillus sp. Sp115AD - sequence no. 3 on the sequence list, cultivated on a multiplication medium with whey and microelements, prepared on water and containing in 1 liter: 20 g of whey powder, preferably acid neutralized, 20 g of whey, 6.6 g of Na2HPO4, 3 g of KH2PO4, 1 g of NH4Cl, 0.5 g of NaCl, 0.022 g of CaCl2, 0.18 g of MgSO4, 12.9 mg of FeCl2, within the limits of ± 10% of each of the medium components, suspended in a liquid organic carrier for the liquid form or dried on a loose carrier or lyophilized on a loose carrier for the dry form; - a proper carrier in the form of molasses vinasse, which is a by-product of the fermentation process of molasses for the liquid form, or micronized dolomite for the dry form, insoluble in water, or maltodextrin for the dry form, soluble in water, or whey powder, preferably acid-neutralized for the powder form for preparing a gel; - addition of dry humic acids for the liquid form of the product or a mixture of dry humic acids, ground mustard seeds and clove oil for the dry form or a mixture of dry humic acids, ground mustard seeds and xanthan gum for the powder form for preparing a gel, as supplementary components of the proper carrier. 2. A method for obtaining a microbiological fertilizer product according to claim 1, characterized in that it comprises a method for culturing bacterial strains of the genus Bacillus, with sequences No. 1, 2 and 3, respectively, indicated in the sequence list, in which the bacterial strains are initially multiplied in a stationary culture on a Plate Count Agar medium, at a temperature of 25-30°C for 24-48 hours, and then the liquid multiplication medium is inoculated with the inoculum thus prepared, in an amount of 5%-15% of the volume of the culture medium, and the multiplication culture is carried out under shaking culture conditions at a temperature of 30°C at 120 rpm. 3. A method for obtaining a microbiological fertilizer product according to claim 2, characterized in that an inoculum with a transmittance of 90% is used. 4. A method for obtaining a microbiological fertilizer product according to claim 2 or 3, characterized in that the multiplication culture is carried out for 48 hours. 5. A method for obtaining a microbiological fertilizing product according to any of the preceding claims, characterized in that the whey-based growth medium contains the following microelements: Mn, Zn, Cu, Co and Mo. 6. A method for obtaining a microbiological fertilizing product according to any of claims 2-5, characterized in that after obtaining a multiplication culture titer of not less than 109 cfu/ml, a sporulation induction step is carried out, preferably by changing the salinity of the culture medium by adding KCl to obtain a salinity of 4% and after 48 hours of culturing and production of spores by bacterial cells, the culture is pasteurized by incubation at 80°C for 20 minutes and the obtained spore suspension is used as the main component of the microbiological fertilizing product. 7. A method for obtaining a microbiological fertilizer product according to any of claims 2-6, characterized in that after the cultivation is completed, in order to obtain a dry product, the prepared spore suspension is dried using a spray dryer on whey powder or is subjected to freeze-drying with the addition of whey powder as a cryoprotectant, in the amount of 5% of the fresh mass of the graveled microorganisms. 8. A method for obtaining a microbiological fertilizer product according to any of claims 1-7, characterized in that dried bacteria or bacterial lyophilisates with a concentration of not less than 10 ¹¹ cfu/g are added in equal amounts to obtain a product with a concentration of each strain in the range of 10 ⁸ - 10 ¹¹ cfu/g. 9. A method for obtaining a microbiological fertilizer product according to any one of claims 1-7, characterized in that to obtain a liquid form, 1 liter of a liquid organic carrier in the form of molasses vinasse is used as a carrier for the bacteria, with the addition of dry humic acids in the amount of 8-12 g/l of molasses vinasse. 10. A method for obtaining a microbiological fertilizer product according to any of claims 1-8, characterized in that for the dry, water-insoluble form, a mixture of dry humic acids in the amount of 0.80-1.2 g/kg of product, ground mustard seeds in the amount of 4.0-6.0 g/kg of product and clove oil in the amount of 0.01 ml/kg of product is used as a carrier component, and micronized dolomite is used as a carrier for all components, constituting the complement to 1 kg. 11. A method for obtaining a microbiological fertilizing product according to any of claims 1-8, characterized in that to obtain a dry, water-soluble form, maltodextrin is used as a cryoprotectant for bacteria in the spray-drying or freeze-drying process and as a carrier for all components of the microbiological fertilizing product. 12. A method for obtaining a microbiological fertilizing product according to any of claims 1-8 or 11, characterized in that for the dry water-soluble form, a mixture of dry humic acids in the amount of 0.80-1.2 g/kg of product, ground mustard seeds in the amount of 4.0-6.0 g/kg of product and clove oil in the amount of 0.01 ml/kg of product is used as a carrier component, and maltodextrin constitutes the complement to 1 kg. 13. A method for obtaining a microbiological fertilizing product according to any of claims 1-8, characterized in that to obtain a product in the form of a powder for preparing a gel, a mixture of dry humic acids in the amount of 8-12 g/kg of product, ground mustard seeds in the amount of 40-60 g/kg of product and xanthan gum in the amount of 30-50 g/kg of product is used as a carrier component, and whey powder, preferably acid neutralized, is used as a carrier for all components of the biopreparation, constituting the complement to 1 kg. 14. A microbiological fertilizer product for conditioning plant seedlings, maintaining and/or improving the microbiological quality of the soil, simultaneously allowing for the control of phytopathogens: Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp. in the cultivation of soft fruits, showing biostimulating properties on plants and improving the quality of fruits, containing strains of bacteria of the genus Bacillus, characterized in that it contains three bacterial isolates selected from the rhizosphere of healthy plants, not showing mutually antagonistic effects: Bacillus subtilis AF75AB2 - sequence no. 1, Bacillus sp. AF75BC - sequence no. 2, Bacillus sp. Sp115AD - sequence no. 3 on the sequence list, a suitable carrier in the form of molasses decoction for the liquid form or micronized dolomite for the dry form insoluble in water or maltodextrin for the dry form water-soluble or whey powder for the powder form for preparing a gel and contains the addition of dry humic acids as supplementary components of the carrier appropriate for the liquid form or dry humic acids, ground mustard seeds and clove oil for the dry form soluble and insoluble in water or dry humic acids, ground mustard seeds and xanthan gum for the powder form for preparing a gel, wherein the bacterial isolates are cultivated on a multiplication medium with whey and microelements, prepared in water and suspended on a carrier or dried on a carrier or lyophilized on a carrier. 15. Microbiological fertilizer product according to claim 14, characterized in that it contains the bacterial strains Bacillus subtilis AF75AB2 - sequence no. 1, Bacillus sp. AF75BC - sequence no. 2, Bacillus sp. Sp115AD - sequence no. 3 on the sequence list, in an equal weight ratio. 16. A microbiological fertilizer product according to claim 14 or 15, characterized in that the bacterial isolates are grown on a multiplication medium with powdered whey, preferably acid-neutralized whey. 17. A microbiological fertilizing product according to any one of claims 14-16, characterized in that the concentration of each bacterial strain in a dry, water-soluble or insoluble form or in a powder form for preparing a gel is in the range of ¹⁰⁸ - ¹⁰¹¹ cfu/g, and in a liquid form in the range of ¹⁰⁸ - ¹⁰¹¹ cfu/ml. PL 248032 Β1 18. A microbiological fertilizing product according to any one of claims 14-17, characterized in that its liquid form contains 8-12 g of dry humic acids per 1 liter of molasses vinasse. 19. A microbiological fertilizing product according to any one of claims 14-17, characterized in that its dry form, soluble or insoluble in water, contains an addition of 0.80-1.2 g of dry humic acids, 4.0-6.0 g of ground mustard seeds and 0.01 ml of clove oil per 1 kg of the product. 20. A microbiological fertilizing product according to any one of claims 14-17, characterized in that its powder form for preparing a gel contains an addition of 8-12 g of dry humic acids, 40-60 g of ground mustard seeds and 30-0 g of xanthan gum per 1 kg of the product. 21. A microbiological fertilizing product according to any one of claims 14-17 or 19, characterized in that its dry, water-insoluble form contains whey powdered as a cryoprotectant in the freeze-drying or spray-drying process and micronized dolomite as a proper carrier. 22. A microbiological fertilizing product according to any one of claims 14-17 or 19, characterized in that its dry, water-soluble form contains maltodextrin as a cryoprotectant in the freeze-drying or spray-drying process and as a proper carrier. 23. A microbiological fertilizing product according to any one of claims 14-17 or 20, characterized in that its powder form for preparing a gel contains whey powdered as a cryoprotectant in the freeze-drying or spray-drying process and as a proper carrier.