PL248034B1 - Method of producing a microbiological fertilizer product and a microbiological fertilizer product for conditioning the soil and improving its biological properties while controlling the pathogens Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp. 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 the soil and maintaining and/or improving the biological properties (health) of the soil ensuring the maintenance and/or improvement and/or protection of the microbiological biodiversity of the soil while controlling key pathogens (Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp.) in the cultivation of soft fruits exhibiting plant biostimulation properties, containing bacterial strains of the genus Bacillus. The method is characterized in that two environmental bacterial isolates, Bacillus subtilis AF75AB2 - sequence no. 1, Bacillus sp. AF75BC - sequence no. 2 on the list of sequences that do not show mutual antagonistic activity, are used, selected from the rhizosphere soil, 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 whey, preferably acid neutralized, enriched with wheat bran, preferably durum, dry humic acids, ground mustard seeds, rapeseed oil and clove oil for a dry product form for spreading, which can be pelleted to form a pellet for soil conditioning, or to obtain a form of a microbiological fertilizer product for suspension in water and spraying/irrigation, whey powder, wheat bran and rapeseed oil are replaced with micronized dolomite, and to obtain a form for dissolving in water and spraying/irrigation, whey powder, wheat bran and rapeseed oil are replaced with maltodextrin.

Description

The subject of the invention is a method for obtaining a microbiological fertilizer product and a microbiological fertilizer product for conditioning the soil and maintaining and/or improving the biological properties (health) of the soil, ensuring the maintenance and/or improvement and/or protection of the microbiological biodiversity of the soil while controlling key pathogens (Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp.) in the cultivation of soft fruits, exhibiting plant biostimulation properties, containing bacterial strains of Bacillus spp., as well as carrier components, including powdered whey, preferably acid neutralized, wheat bran, preferably durum, dry humic acids, ground mustard seeds, rapeseed oil and clove oil, which is used in a loose form or in the form of a pellet or after replacing the whey, bran and rapeseed oil with micronized dolomite or maltodextrin in the form of a spray. prepared from a dry, water-insoluble or water-soluble form.

Bacteria of the Bacillus genus are a group of microorganisms, including strains with properties that promote plant growth and development. Therefore, they are intensively studied microorganisms with significant potential for use in horticulture. It's worth noting that members of this genus inhabit diverse ecological niches, occur in almost all soil types, and are characterized by high tolerance to environmental stresses, including high temperatures. Bacteria of the Bacillus genus are spore-forming microorganisms and multiply rapidly in liquid culture media.

Current knowledge suggests that strains of Bacillus species stimulate plant growth and development through the production of various metabolites. They are characterized by high enzymatic and metabolic activity, which is useful in the transformation of organic matter, and possess antagonistic properties against potentially phytopathogenic microorganisms. However, these characteristics are not self-evident, and identifying suitable strains requires extensive research. Below are several examples of the use of Bacillus spp. strains in agriculture and horticulture.

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 Gnanmanickam S., Bacillus amyloliquefaciens strain, 2016, US Patent: US 9234251B2).

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 Garda 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).

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).

Research on the development of ecological techniques for growing fruit plants 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 or the use of bioproducts to protect plants against fungal pathogens and pests (Frąc, M., Hannula, E.S., Bełka, M., Salles, J.F., Jedryczka, M., 2022. Soil mycobiome in sustainable agriculture. Front. Microbiol. 13:1033824. 10.3389/fmicb.2022.1033824).

Organic farming allows the use of natural fertilizers and soil conditioners. These include inorganic and organic products that are environmentally and human-friendly (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/s11157-019-09500-5).

The situation of organic farming is changing dynamically in Poland, Europe, and globally. However, despite 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 European 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). However, numerous meetings and discussions with farmers, as well as diagnoses based on surveys, indicate that Poland lacks effective bioproducts based on microorganisms, particularly those containing native bacterial strains suitable for organic farming and fertilization of horticultural plants, especially those dedicated to soft fruit cultivation. Agriculture faces the challenge of developing a sustainable development strategy that protects non-renewable natural resources, including soil. In recent decades, much attention has been paid to mitigating soil erosion through physical protection measures and the provision of additional nutrients and water to meet crop needs. Less attention has been paid to soil as a dynamic living organism, although 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., Bełka, 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 utilize large amounts of external inputs such as inorganic fertilizers, pesticides, and other artificial inputs can overcome specific soil limitations in crop production. However, the adverse impact of these additives on the environment is increasingly observed, leading to ongoing environmental degradation, including the loss of soil biodiversity. Therefore, sustainable agriculture, including organic farming, is currently one of the fastest-growing sectors of agriculture in the world, particularly 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, new solutions, including the development of microbiological fertilizer products, are needed for the environment and expected by farmers and producers of fertilizers and bioproducts in order to change the production profile to a more green and ecological one and to provide farmers with tools for sustainable production.

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 ananassa 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, 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.

Patents most often concern biopreparations used to control pathogens of vegetable and crop plants. There are no solutions encompassing microbiological fertilizers that maintain and/or improve microbiological properties and soil biodiversity, simultaneously exhibiting antagonistic effects on key fungal pathogens and fungal-like oomycetes. These are also intended for use in organic soft fruit cultivation. These products are particularly suitable for preparing soil for newly established plantations by conditioning the soil with pelleted biopreparations containing microorganisms or in their powder form. Known solutions cover the control of individual pathogens, such as those of the Botrytis genus, but there are no comprehensive bioproducts effective against the four key phytopathogens (Botrytis spp., Colletotrichum spp., Phytophthora spp., Verticillium spp.), maintaining and/or improving soil microbial biodiversity, and supporting plant growth.

There are also no literature reports and patents regarding pelleted forms of biopreparations containing microorganisms and regarding the combined use of microorganisms on carriers composed of whey powder, wheat bran, dry humic acids, ground mustard seeds, rapeseed oil and clove oil, which create a composition of a microbiological fertilizing product to be applied in a loose or pelleted form directly under seedlings, but also to the soil in order to condition it and prepare it for the cultivation of soft fruits.

The aim of the invention is therefore to develop a microbiological fertilizer product for conditioning the soil and maintaining and/or improving its microbiological quality (health) of the soil, ensuring the maintenance and/or improvement and/or protection of soil microbiological biodiversity while controlling key pathogens (Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp.) in the cultivation of soft fruits, exhibiting plant biostimulation properties.

While conducting research on microbiological fertilizer products containing strains of the Bacillus genus, it was unexpectedly found that two 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.

Therefore, the above goal was achieved by developing a method for obtaining a microbiological fertilizer product for soil conditioning and maintaining and/or improving its microbiological quality (soil health), ensuring the maintenance and/or improvement and/or protection of soil microbiological biodiversity while simultaneously controlling key pathogens (Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp.) in soft fruit cultivation, demonstrating plant biostimulation characteristics. 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 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 soil conditioning and maintaining and/or improving its microbiological quality (health) of the soil, ensuring the maintenance and/or improvement and/or protection of soil microbiological biodiversity while controlling key pathogens (Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp.) in the cultivation of soft fruits showing plant biostimulation characteristics, using bacterial strains of the genus Bacillus, is that:

- two 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 on the sequence list, cultivated on a multiplication medium prepared in water with whey and microelements and containing in 1 liter: 20 g of whey powder, preferably acidic, neutralized, 6.6 g of Na2HPO4, 3 g of KH2PO4, 1 g of NH4O, 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, dried on a loose carrier or lyophilized on a loose carrier;

- a proper carrier in the form of a mixture of whey powder, wheat bran and rapeseed oil for the loose form for spreading or for the pellet form or micronized dolomite for the dry form insoluble in water or maltodextrin for the dry form soluble in water;

- addition of a mixture of dry humic acids, ground mustard seeds and clove oil as supplementary components of the actual carrier.

The method preferably comprises a method for culturing bacterial strains of the genus Bacillus, with sequence numbers 1 and 2, respectively, indicated in the sequence list, consisting of culturing them in a multiplication medium prepared with water and whey and microelements, suspending or drying them on a carrier, and supplementing them with additives in the form of dry humic acids, ground mustard seeds, and clove oil. The bacterial strains are initially cultured in a stationary culture on Plate Count Agar at a temperature of 25-30°C for 24-48 hours. The liquid multiplication medium is then inoculated with the inoculum prepared in an amount of 5%-15% of the culture medium volume, and the multiplication is carried out under shaking culture conditions at a temperature of 30°C and 120 rpm.

It is preferable to use an inoculum with a transmittance of 90% for culturing bacteria.

The expansion culture is preferably carried out for 48 hours.

Preferably, the whey-based growth medium contains the following microelements: Mn, Zn, Cu, Co and Mo.

After obtaining a multiplication culture titer of no less than 109 cfu/ml, a sporulation induction step is preferably performed, preferably by changing the salinity of the culture medium with KCl to achieve a salinity of 4%, and after 48 hours of cultivation and spore production by bacterial cells. The culture is then pasteurized by incubation at 80°C for 20 minutes, and the resulting spore suspension is used as the main component of the microbiological fertilizer product.

Preferably, after the cultivation is completed, the prepared spore suspension is dried in a spray dryer on whey powder or is subjected to lyophilization with the addition of whey powder as a cryoprotectant in the amount of 5% of the fresh mass of the graveled microorganisms.

It is advantageous when dried bacteria or bacterial lyophilisates with a concentration of not less than ¹⁰¹¹ cfu/g are added in equal amounts to obtain a product with a concentration of each strain in the range of 108-1011 ^cfu /g.

In the loose form variant for spreading use, a mixture of: wheat bran, preferably durum, in the amount of 80-120 g/kg of product, rapeseed oil in the amount of 40-60 g/kg of product, dry humic acids in the amount of 8-12 g/kg of product, clove oil in the amount of 0.1 ml/kg of product, ground mustard seeds in the amount of 8-12 g/kg of product, and powdered whey, preferably acid neutralized, is used as a carrier for all components, constituting a supplement to 1 kg.

To obtain pellets for soil conditioning, the prepared bulk form for spreading is pelletized, preferably at an input temperature of the pelleted material of 25°C-28°C and a moisture content of the pelleted material of 4-5%.

In the dry, water-insoluble form, micronized dolomite as a carrier component makes up 1 kg of the product.

In the dry, water-soluble form, maltodextrin as a carrier component makes up 1 kg of the biopreparation.

The essence of the microbiological fertilizer product for conditioning the soil and improving its biological properties, while controlling the pathogens Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp. in the cultivation of soft fruits, showing the features of plant biostimulation, using bacterial strains of the genus Bacillus, is that it contains two 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 on the sequence list, cultivated on a multiplication medium with whey and microelements, prepared on water, dried on a loose carrier or freeze-dried on a loose carrier, the carrier in the form of a mixture of whey powder, wheat bran and rapeseed oil for the loose form to for spreading application and for the pellet form of the biopreparation or micronized dolomite for the dry form, insoluble in water, or maltodextrin for the dry form, soluble in water, the addition of a mixture of dry humic acids, ground mustard seeds and clove oil as supplementary components of the proper carrier.

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

Preferably, bacterial isolates are grown on a multiplication medium with whey powder, preferably acid-neutralized whey.

It is preferred that the concentration of each dried or lyophilized bacterial strain is in the range of 108-1011 ^cfu /g.

In the loose form for spreading, the microbiological fertilizer product contains 80-120 g of wheat bran, preferably durum, 40-60 g of rapeseed oil, preferably organic, 8-12 g of dry humic acids, 8-12 g of ground mustard seeds and 0.1 ml of clove oil per kilogram, and as a carrier for all components of the biopreparation it contains powdered whey, preferably acid neutralized, constituting a supplement to 1 kg.

In another variant, the microbiological fertilizer product is in the form of pellets.

In yet another variant, its dry form, insoluble in water, contains whey powder as a cryoprotectant in the process of freeze-drying or spray-drying, and micronized dolomite as a carrier makes up the total weight of 1 kg.

In another variant, the dry form of the water-soluble microbiological fertilising product contains maltodextrin as a cryoprotectant in the freeze-drying or spray-drying process, and maltodextrin as a carrier is added to the 1 kg.

Strains of saprophytic soil bacteria of the genus Bacillus (Bacillus subtilis sp. AF75AB2 and Bacillus sp. AF75BC), which contain the fertilizing microbiological product, were isolated from the rhizosphere of plants, they are native strains from the SYMBIO BANK collection of the Institute of Horticulture - State Research Institute in Skierniewice.

The obtained microbiological fertilizer product in a loose form for spreading, in the form of pellets and in a dry, insoluble and water-soluble form enables soil conditioning and maintaining and/or improving its microbiological biodiversity, and also influences plant growth characteristics and influences the control of four important soft fruit pathogens, including three fungal species of the genera Botrytis, Colletotrichum, Verticillium and one oomycete of the genus Phytophthora.

It should be emphasized that there are no commercially available biopreparations for soil conditioning, especially in pellet form, 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 assessing the impact on root microbial biodiversity. Therefore, the approach presented in this patent application is unique and innovative and confirms the beneficial effects of the developed biopreparation on the soil environment and the plant, providing the basis for recognizing the developed bioproduct as a microbial fertilizer.

A biopreparation containing dry humic acids, clove oil, and ground mustard, prepared in a carrier containing whey powder (preferably acid-neutralized), wheat bran (preferably durum wheat), and rapeseed oil (preferably organic), is suitable for application directly to the soil or under plant seedlings, either as a spread or pelleted, or for dissolving and spraying the soil and/or plants. The whey powder, wheat bran, and rapeseed oil are replaced with micronized dolomite (water-insoluble) or maltodextrin (water-soluble). This microbiological fertilizer product in the form of dry pellets or dry granules enables the gradual release of active ingredients directly into the soil, significantly extending the biopreparation's duration of action. This form of biological preparation allows beneficial microorganisms and the components of the preparation to colonize the soil environment evenly over time. This type of application also provides greater benefit in terms of varying weather conditions that may occur during and after application to the crop field. It is recommended for use during plantation establishment, either as a spread under plant roots or as a soil-based application by mixing pellets with the soil, ensuring the maintenance and protection of soil biodiversity. Furthermore, the suspension or water-soluble and sprayable form facilitates the application of the biopreparation to existing plantations in the years following establishment, impacting both soil microbiological quality and plant growth parameters, as well as strengthening plant resistance to stress factors.

The developed biopreparation in dry, pellet or water-soluble suspension form is a comprehensive technology for soil conditioning, biostimulation and plant protection, intended for application to the soil surface (dry and pellet form) and plants (water-soluble and suspension form) in soft fruit crops, mainly strawberries and raspberries, in order to improve the biological properties (health) of the soil, ensuring the maintenance and protection of biodiversity while controlling dangerous plant pathogens such as: Botrytis spp., Verticillium spp., Colletotrichum spp., and Phytophthora spp., and is characterized by its composition as well as the method of its production.

The invention is illustrated in the embodiment examples in the figures, where:

Fig. 1 shows the appearance of colonies of bacterial strains AF75AB2 and AF75BC 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 biopreparation, 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 a bacterial biopreparation in the form of a pellet containing the bacterial strains Bacillus subtilis AF75AB2 and Bacillus sp. AF75BC towards plant fungal pathogens of the genera Botrytis, Colletotrichum, Vertticillium and the fungus-like oomycete of the genus Phytophthora;

Fig. 9 is a table presenting the antagonistic properties of a microbiological fertilizer product in the form of pellets containing selected strains of Bacillus subtilis AF75AB2 and Bacillus sp. AF75BC bacteria against plant fungal pathogens of the genera Botrytis, Colletotrichum, Vertticillium and the fungus-like oomycete of the genus Phytophthora;

Fig. 10 shows the antagonism of a microbiological fertilizer product in the form of pellets containing selected strains of Bacillus subtilis AF75AB2 and Bacillus sp. AF75BC bacteria towards plant fungal pathogens of the genera Botrytis, Colletotrichum, Vertticillium and the fungus-like oomycete of the genus Phytophthora after 6 months of storage of the biopreparation at room temperature (approximately 21°C), 4°C and 35°C;

Fig. 11 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 and Bacillus sp. AF75BC;

Fig. 12 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 and Bacillus sp. AF75BC;

Fig. 13 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 and Bacillus sp. AF75BC;

Fig. 14 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 and Bacillus sp. AF75BC;

Fig. 15 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 and Bacillus sp. AF75BC, where the more colored bands, the greater the biodiversity of bacteria;

Fig. 16 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 and Bacillus sp. AF75BC;

Fig. 17 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 and Bacillus sp. AF75BC;

Fig. 18 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 and Bacillus sp. AF75BC, where the more colored stripes, the greater the biodiversity of fungi;

Fig. 19 shows the appearance of the microbiological fertilizer preparation in dry form for spreading and in pellet form;

Fig. 20 shows the decomposition of the biopreparation in pellet form in the soil within 5 days of application.

EXAMPLES

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

Two bacterial strains selected for the pellet preparation were identified to species or genus using 16S rDNA gene amplification and Sanger sequencing. The nucleotide sequences obtained from sequencing were analyzed for comparison with nucleotide sequences in databases (the commercial MicroSeq ^ID database and the open NCBI database https://www.ncbi.nlm.nih.gov/). The bacterial strains AF75AB2 (sequence no. 1) and AF75BC (sequence no. 2) included in the product were identified based on molecular and bioinformatic analysis to the species Bacillus subtilis AF75AB2 (sequence no. 1) and to the genus Bacillus sp. AF75BC (sequence no. 2). The obtained nucleotide sequences are presented in the Sequence List.

The strains were shown to exhibit very good, cream-colored growth on PCA (Plate Count Agar) (Fig. 1). The AF75AB2 and AF75BC bacterial strains selected for the development of a microbiological fertilizer product were characterized in terms of their basic metabolic properties, which influence the properties of the soil environment. The biochemical activity of the strains included in the product 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 the AF75AB2 and AF75BC strains 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). The biochemical capabilities of selected bacterial strains were determined using API ZYM tests (Biomerieux), which enable the determination of the activity of hydrolytic enzymes involved in the metabolism of organic matter, including phosphatases, esterases, arylamidases, phosphohydrolase, β-glucosidase, β-galactosidase, mannosidase, and glucosaminidase (Fig. 6). Enzymatic capabilities were determined in cultures of bacterial strains with a density of 5-6 on the McFerland scale. Bacillus subtilis strain AF75AB2 sequence no. 1 in the sequence list showed high activity of alkaline phosphatase, esterase, lipase esterase, naphthyl-AS-BI phosphohydrolase, α- and β-glucosidase. Bacillus sp. strain AF75BC - sequence 2 in the sequence list, was characterized by high esterase activity and medium or low activity of lipase esterase, lipase, acid phosphatase, naphthyl-AS-BI phosphohydrolase, α-galactosidase, β-glucosidase, N-acetyl-ε-glucosaminidase (Fig. 7).

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

The antagonistic capacity of the product was determined by measuring the zone of inhibition of mycelial growth. 90 mm diameter Petri dishes with PC A (Plate Count Agar) were inoculated with 300 μl of a spore suspension 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) with a transmittance of 70%. The plates were incubated at 23°C for 24 hours. Then, after pre-incubation of the phytopathogens on the plate, a dry pellet of the product (moistened with sterile water) was placed in the center of the plate. The plates were incubated at 23°C for 5-7 days and the zones of mycelial growth inhibition and/or sporulation inhibition were measured, confirming the antagonistic effect of the developed microbial fertilizer product in the form of pellets against key phytopathogens of soft fruits (Fig. 8-Fig. 9).

Storage studies were also conducted, during which the antagonistic properties of the product were periodically determined over a 6-month period of product storage at various conditions, including room temperature (~21°C), refrigeration (4°C), and elevated storage temperature (35°C). The obtained results indicate that the antagonistic properties of the microbial fertilizer product against key fungal phytopathogens and fungal-like oomycetes of soft fruit generally persisted during storage; however, this effect depended on both the tested pathogen and the storage conditions used (Fig. 10). The best antagonistic properties after 6 months of storage of the microbiological fertilizer product in the form of pellets containing bacteria were obtained for the product stored at room temperature and under refrigeration conditions against the fungus-like oomycete Phytophthora sp., as well as the fungi Botrytis sp. and Verticillium sp., demonstrating biocidal properties and preventing the development or limiting the growth of these pathogens, and for the fungal phytopathogen Colletotrichum sp., a fungistatic effect of the product was observed, manifested by the inhibition of sporulation of this fungus (Fig. 10). The observed effects were weaker for the product stored at an elevated storage temperature of 35°C (Fig. 10).

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

The number of microorganisms in the prepared preparation was determined by counting and determining the total bacterial count using the dilution plating method. A series of dilutions of the microbiological fertilizer product 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 over the surface of the medium, and then incubated at 26°C for 72 hours. The resulting colonies were counted, and the total bacterial count (CFU/g) in the bioproduct was determined.

After production, the preparation showed a total bacterial count of >109 CFU/g of the bacterial product in both bulk and pellet form. Storage test results showed that the bacterial count in the preparation remained at > ¹⁰⁸ CFU/g, and even > ¹⁰⁹ CFU/g (pellets stored at an elevated storage temperature of 35°C), 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 effect of a microbial fertilizer 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 growing medium consisting of ½ volume of soil and ½ volume of 7-20 mm peat. The product was applied to the soil, and untreated plants, growing in a medium supplemented with 5 g of dried manure per pot, served as a control. In order to determine changes in biodiversity, studies were carried out, including the characterization of bacterial and fungal communities by means of next-generation sequencing (NGS) using high-throughput sequencing in the Illumina SBS (Sequencing-by-Synthesis) technology with the MiSeq apparatus and bioinformatics tools such as QIIME2. The obtained results were analyzed based on the sequencing of the 16S rDNA marker for bacteria and ITS1 for fungi, and the diversity of microorganisms in the rhizosphere and plant roots was determined.

Application of the bacterial product allowed us to assess changes in biodiversity, including the occurrence of amplicon sequence variants (ASVs) at different taxonomic levels of bacteria and fungi in the rhizosphere and roots of treated and untreated plants. Trophic groups and functional guilds of fungi were also analyzed using the FunGuild database (Fig. 11-Fig. 12). These results showed that a reduction in pathotrophs was observed in the roots of plants treated with the microbial fertilizer product, and an increase in saprotrophs was noted in the roots. A significant reduction in pathotrophs and mixed modes: pathotroph-saprotroph-symbiotroph and pathotroph-symbiotroph, as well as a significant increase in symbiotrophs and mixed mode saprosymbiotroph fungi in the rhizosphere of raspberries treated with the microbial fertilizer product, was also demonstrated (Fig. 11), confirming the validity of using the product for soil conditioning. Furthermore, it was demonstrated that after product application, the number of guilds assigned to all tested pathotroph groups significantly decreased, and in particular, a reduction in plant pathogens was observed in both the plant rhizosphere and the root (Fig. 12). Analysis of the results obtained for bacterial communities generally showed an increase in the relative abundance and biodiversity of bacteria at various taxonomic levels after product application. This effect was noted in both the rhizosphere soil and the root (Fig. 13-Fig. 15). 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 product, 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 product (Fig. 16-Fig. 18), but when compared with the results of the trophic groups of fungi (Fig. 11-Fig. 12) this is a favorable observation, which may indicate a protective effect of the microbiological fertilizer product, limiting the penetration of fungi into the roots, especially those unfavorable for plants.

The advantage of this microbial fertilizer product is its high versatility, confirmed by its dedicated use in soil conditioning and maintaining and/or improving the microbial biodiversity of the soil and the microbiome of soft fruit plants, while simultaneously controlling phytopathogens found in these crops. The developed microbial 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 product is the multitude of ways of its application thanks to the dry form for spreading, pellets for mixing with soil or applying under plant seedlings, as well as powder forms for suspending or dissolving in water, enabling spraying, sprinkling or watering, which constitutes a comprehensive application technology depending on the needs and throughout the entire plant vegetation season, from plantation establishment to fruit harvest.

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

To determine the effect of a microbiological fertilizer product containing selected strains of Bacillus subtilis AF75AB2 and Bacillus sp. AF75BC on plant yield and the occurrence of phytopathogens, field experiments were conducted in which the microbiological 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, with pellets applied directly under the plant seedlings. Additionally, tests of the developed microbiological fertilizer product in dry form for broadcast application and pellets, introduced as one component 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 plant species and varieties responded differently to the tested cultivation technologies, including the addition of a microbiological fertilizer product containing selected bacterial strains. In the non-irrigated area, in the varieties where the developed microbiological fertilizer product was added in the first year in pellet form, an increase in strawberry yield was observed for the Honeoye variety. In the non-irrigated area, in the variant where the bacterial product was added, the Rumba variety was observed to have a reduced incidence of leathery fruit rot. The Rumba and Vibrant varieties, on the other hand, responded with a reduced incidence of gray mold. Furthermore, an increase in the fresh root mass of strawberry plants was observed after the product application. The results indicate that for some raspberry varieties, particularly Enrosadira and 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 microbial activity, and R (Richness), which indicates the number of carbon substrates mobilized by microbial communities. The studies indicate that the metabolic activity of microorganisms present in the plant and fruit increased, particularly in relation to carbohydrates, in the variant in which the developed bacterial product in gel form was used as one of the cultivation elements.

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

To obtain a fertilizing microbiological product for soil conditioning and maintaining and/or improving the microbiological quality of the soil, influencing the growth and development of plants, while also allowing the control of phytopathogens (Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp.) in the cultivation of soft fruit, two 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, on the list of sequences for which separate cultures were carried out, which were mixed in equal proportions 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 growth medium for the production of the product was an optimized medium containing: 6.6 g/L Na2HPO4, 3 g/L KH2PO4, 1 g/L NH4O, 0.5 g/L NaCl, 20 g/L neutralized acid whey, 0.022 g/L CaCl2, 0.18 g/L MgSO4, 12.9 mg FeCl2 and 10 ml of a microelement solution containing: 0.5 mM MnCl2*4 H2O, 1.2 mM ZnCl2, 0.27 mM CuCl2*2 H2O, 0.23 mM CoCl2*6 H2O, 0.23 mM Na2MoO4*2 H2O. 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 prepared spore suspension was spray-dried on acidic neutralized whey powder, which constitutes the basic component of the product. To obtain 1 kilogram of a loose microbiological fertilizer product for spreading, 100 g of durum wheat bran, 50 g of organic rapeseed oil, 10 g of dry humic acids, 10 g of ground mustard seeds, and 0.1 ml of clove oil were added to 830 g of powdered whey containing >109 cfu/g of bacterial spores, including 415 g of whey with spores of the Bacillus subtilis AF75AB2 strain and 415 g of whey with spores of the Bacillus sp. AF75BC strain. After combining and thoroughly mixing the ingredients, a loose microbiological fertilizer product for spreading was obtained, characterized by a bacterial count of 109 ^cfu /g of product.

Example 7. Method of obtaining a microbiological fertilizer product - pellet form

The method was carried out as in Example 6, except that after combining and mixing all ingredients to obtain a pellet, they were pelletized using a Nawrocki Pelleting Technology GRV340 pelletizer. During the process, the pelleting rate, temperature, and moisture of the pelleted material were controlled to ensure a temperature of 26.6°C and moisture within the range of 4.35%-4.52%. Pelleting was carried out in two passages, with the initial temperature of the material in passage I being 65°C, and in passage II being 72.2°C. After completion of the pelleting process, a microbiological fertilizer product was obtained, with a pellet size of up to 1 cm, characterized by a bacterial count of 109 cfu/g of product. The product in the form of a mixture before pelleting, the finished pellet, and its decomposition in water and soil are shown in the figures (Fig. 19-Fig. 20).

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

The method was performed as in Example 6, except that after culturing, the prepared spore suspension was lyophilized with the addition of whey powder as a cryoprotectant, at a rate of 5% of the fresh weight of the graveled microorganisms. To obtain a water-insoluble dry formulation for spraying or irrigation, 10 g of lyophilized spores of each bacterial strain were used.

PL 248034 Β1 (Bacillus subtilis AF75AB2, Bacillus sp. AF75BC) with a bacterial content of ¹⁰¹¹ cfu/g of lyophilisate. Instead of whey powder, wheat bran, and rapeseed oil, 960 g of micronized dolomite carrier was used, to which the remaining ingredients present in the bulk product were added, including 10 g of dry humic acids, 10 g of ground mustard seeds, and 0.1 ml of clove oil.

Example 9. 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 Example 8, except that maltodextrin was added to the freeze-drying process instead of whey powder and maltodextrin was used instead of the micronized dolomite carrier.

Claims (20)

1. A method for obtaining a microbiological fertilizer product for conditioning the soil and improving its biological properties (while simultaneously controlling the pathogens Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp. in the cultivation of soft fruit, showing the characteristics of plant biostimulation, using strains of bacteria of the genus Bacillus, characterized in that: - two 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 on the sequence list, cultivated on a multiplication medium prepared in water with whey and microelements and containing in 1 liter: 20 g of whey powder, preferably acidic, 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, dried on a loose carrier or lyophilized on a loose carrier; - a proper carrier in the form of a mixture of whey powder, wheat bran and rapeseed oil for the loose form for spreading or for the pellet form or micronized dolomite for the dry form insoluble in water or maltodextrin for the dry form soluble in water; - addition of a mixture of dry humic acids, ground mustard seeds and clove oil as supplementary components of the actual 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 and 2, respectively, indicated in the sequence list, consisting in culturing them on a multiplication medium prepared on water with whey and with microelements, suspending or drying them on a carrier and supplementing them with additives in the form of dry humic acids, ground mustard seeds and clove oil, wherein the bacterial strains are initially cultivated 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 prepared in this way, 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 the preceding claims, 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 with the addition of 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, the prepared spore suspension is dried in 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 fertilizing product according to any of the preceding claims, characterized in that dried bacteria or bacterial lyophilisates with a concentration of not less than ¹⁰¹¹ cfu/g are added in equal amounts to obtain a product with a concentration of each strain in the range of 108-1011 ^cfu /g. 9. A method for obtaining a microbiological fertilizing product according to any of the preceding claims, characterized in that to obtain a loose form for spreading, a mixture of: wheat bran, preferably durum, in the amount of 80-120 g/kg of product, rapeseed oil in the amount of 40-60 g/kg of product, dry humic acids in the amount of 8-12 g/kg of product, clove oil in the amount of 0.1 ml/kg of product, ground mustard seeds in the amount of 8-12 g/kg of product is used as a carrier for all components, and whey powder, preferably acid neutralized, is used as a carrier for all components, constituting the complement to 1 kg. 10. A method for obtaining a microbiological fertilizer product according to claim 9, characterized in that the prepared loose form for spreading is pelletized to form a pellet for soil conditioning, preferably at an input temperature of the pelleted material of 25°C-28°C and a moisture content of the pelleted material of 4-5%. 11. A method for obtaining a microbiological fertilizing product according to any of claims 1-8, characterized in that for the dry, water-insoluble form, micronized dolomite as a carrier component constitutes the balance up to 1 kg of the product. 12. A method for obtaining a microbiological fertilizing product according to any of claims 1-8, characterized in that for the dry, water-soluble form, maltodextrin as a carrier component constitutes the balance up to 1 kg of the product. 13. A fertilizing microbiological product for conditioning the soil and improving its biological properties, while controlling the pathogens Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp. in the cultivation of soft fruit, showing plant biostimulation properties, using bacterial strains of the genus Bacillus, characterized in that it contains two 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 on the sequence list, cultivated on a multiplication medium with whey and microelements, prepared on water, dried on a loose carrier or lyophilized on a loose carrier, the carrier being a mixture of whey powder, wheat bran and rapeseed oil for the loose form for for spreading application and for the pellet form of the product, either micronized dolomite for the dry, water-insoluble form or maltodextrin for the dry, water-soluble form, the addition of a mixture of dry humic acids, ground mustard seeds and clove oil as supplementary components of the proper carrier. 14. A microbiological fertilizer product according to claim 13, characterized in that it contains the bacterial strains Bacillus subtilis AF75AB2 - sequence no. 1, Bacillus sp. AF75BC - sequence no. 2, in the sequence list, in an equal weight ratio. 15. A microbiological fertilizer product according to claim 13 or 14, characterized in that the bacterial isolates are grown on a multiplication medium with powdered whey, preferably acid-neutralized whey. 16. A microbiological fertilizing product according to any one of claims 13-15, characterized in that the concentration of each of the dried or freeze-dried bacterial strains is in the range of 108-1011 ^cfu /g. 17. A microbiological fertilizing product according to any one of claims 13-16, characterized in that its loose form for spreading application contains in 1 kilogram 80-120 g of wheat bran, preferably durum, 40-60 g of rapeseed oil, preferably organic, 8-12 g of dry humic acids, 8-12 g of ground mustard seeds and 0.1 ml of clove oil, and as a carrier for all product components it contains whey powder, preferably acid neutralized, constituting the complement to 1 kg. 18. A microbiological fertilizing product according to any one of claims 13-17, characterized in that it is in the form of a pellet. 19. A microbiological fertilizing product according to any one of claims 13-16, characterized in that its dry form, insoluble in water, contains whey powdered as a cryoprotectant in the process of freeze-drying or spray-drying, and micronized dolomite as a carrier constitutes the complement to 1 kg. PL 248034 Β1 20. A microbiological fertilizing product according to any one of claims 13-16, characterized in that its dry, water-soluble form contains maltodextrin as a cryoprotectant in the freeze-drying or spray-drying process, and the maltodextrin as a carrier constitutes the balance up to 1 kg.