PL248033B1 - Method of producing a microbiological fertilizer product and a microbiological fertilizer product for maintaining and/or improving the microbiological quality of soil while simultaneously allowing for controlling phytopathogens: 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 for 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, using bacterial strains of the genus Bacillus, in which: two bacterial isolates selected from the rhizosphere of healthy plants, not showing mutual antagonistic effects: Bacillus sp. Sp115AD — sequence no. 1 and Bacillus sp. Sp116AC* — sequence no. 2 on the sequence list, cultivated on a multiplication medium with whey and microelements, prepared on water, suspended in a culture medium or dried on a carrier or lyophilized on a carrier, the carrier being a proper culture medium for the liquid form or micronized dolomite for the liquid form water-insoluble dry matter or water-soluble maltodextrin for the dry form, and the addition of liquid humic acids and yeast production effluent for the liquid form or dry humic acids for the dry form as supplementary components of the actual carrier. The invention also provides a microbiological fertilizer product for maintaining and/or improving the microbiological quality of soil, while also allowing for the control of phytopathogens Botrytis sp., Colletotrichum sp., Phytophthora sp., and Verticillium sp. in fruit cultivation, exhibiting biostimulative effects on plants.

Description

The subject of the invention is a method for obtaining a microbiological fertilizer product and a microbiological fertilizer product for maintaining and/or improving the microbiological quality of soil in soft fruit cultivation, containing Bacillus spp. bacterial strains with antagonistic properties towards phytopathogenic fungi of the genera: Botrytis, Verticillium, Colletotrichum and oomycetes of the genus Phytophthora, and also containing carrier components, including humic acids and/or yeast culture effluent and/or micronized dolomite and/or maltodextrin, depending on the form of application of the bioproduct, which also exhibits plant biostimulation properties.

Due to the fact that Poland is a key global producer of soft fruits and a leader in the European Union in the production of organic strawberries (FAOSTAT, GUS), there is still a need to provide appropriate tools, including bioproducts intended for the cultivation of these fruits. Soft fruit cultivation requires fertile soils characterized by high biodiversity, and at the same time healthy soils with a low percentage of pathogens (Daugovish, O., Knapp, S., Gordon, T., Fennimore, S., Muramoto, J. and Bolda, M. (2021). Soil pest management in current California strawberry production: a review. Acta Hortic. 1309, 701-710, https://doi.org/10.17660/ActaHortic.2021.1309.101). It should also be emphasized that sustainable plant production is gaining popularity, and what is more, this method of plant production imposes numerous restrictions on farmers, such as limiting and/or even abandoning the use of fertilizers and chemical plant protection products in favor of natural solutions, biological preparations and products approved for use in organic farming (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). Biopreparations and biofertilizers play an important role not only in improving soil condition or providing plants with nutrients, but also in the control of phytopathogens in both organic and conventional agriculture (Mącik M., Gryta A., Frąc M. (2020). Biofertilizers in agriculture: An overview on concepts, strategies and effects on soil microorganisms. Advanc. Agron. 162, 31-87, https://doi.org/10.1016/bs.agron.2020.02.001). Typical chemical solutions used in agriculture can cause soil depletion, a decrease in organic matter and a decrease in the biodiversity of soil microorganisms (Ju, X.-T., Xing, G.-X., Chen, X.-P., Zhang, S.-L., Zhang, L.-J., Liu, X.-J., Cui, Z.-L., Yin, B., Christie, P., and Zhu, Z.-L. (2009). Reducing environmental risk by improving N management in intensive Chinese agricultural systems. Proceedings of the National Academy of Sciences 106, 3041-3046; Zhang, Q.-C., Shamsi, I. H., Xu, D.-T., Wang, G.-H., Lin, X.-Y., Jilani, G., Hussain, N, and Chaudhry, A. N. (2012). Chemical fertilizer and organic Manure inputs in soil exhibit a pattern of microbial community structure. Applied Soil Ecology 57, 1-8). Limiting this process is one of the main goals of the 2030 Biodiversity Strategy, as well as a basic assumption of regenerative agriculture (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). Microbiological preparations increasingly used in agriculture can also stimulate systemic plant resistance, improve the microbiological quality of soils and increase the availability of micro- and macroelements for plants (PylakM., Oszust K., Frąc M. (2019). Reviewreport 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). Strawberry crops are exposed to diseases caused by phytopathogens, including: from the genera: Botrytis, Verticillium, Colletotrichum or Phytophthora, which can cause infection of entire plantations and plant withering, thus reducing the amount of harvested fruit and causing economic losses (Malarczyk D., Panek J., Frąc M. (2019). Alternative Molecular-Based Diagnostic Methods of Plant Pathogenic Fungi Affecting Berry Crops - A Review. Molecules 2019, 24, 1200; doi: 10.3390/molecules24071200). The European Union's Biodiversity Strategy until 2030 focuses on increasing the area of organic farming and reducing the use of mineral fertilizers and pesticides (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), which means that new solutions, including microbiological fertilizer products dedicated to specific crops, are expected not only by farmers, but also by fertilizer companies and producers of various types of bioproducts for agriculture and horticulture.

Current knowledge suggests that strains of Bacillus species exhibit antagonistic properties against various fungal phytopathogens and also contribute to the provision of nutrients to plants and the improvement of soil microbial biodiversity. However, this is not a self-evident feature, and selecting appropriate strains requires extensive research. Below are several examples of the use of Bacillus spp. strains in agricultural and horticultural applications.

The genus Bacillus is an intensively studied group of bacteria with significant potential for agricultural use, primarily because microorganisms belonging to this genus occur in almost all soil types, are highly tolerant to high temperatures, multiply rapidly in liquid culture media, and are spore-forming bacteria. In 2006, the United States Environmental Protection Agency registered over 10 different strains of Bacillus as biopesticides and biofungicides (The United States Environmental Protection Agency, EPA, 2006).

An example of the use of strains of the genus Bacillus is a dry formulation of a preparation containing spray-dried 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 9234251 B2).

Isolates of Bacillus subtilis 4/7 (pCM B/00105) and/or Bacillus licheniformis 4/18 (PCM B/00106) were characterized by cellulolytic activity. A preparation based on these bacteria immobilized on an aluminosilicate mineral carrier is used for the mineralization of organic matter (Ciecierski W., Kardasz H., Szychowska K., Wilk R., Microbiological preparation for the mineralization of organic matter containing cellulose, especially crop waste, and the use of the microbiological preparation in plant cultivation, Pat.230762).

To combat fungal plant diseases, Bacillus methylotrophicus strains XT1 and XT2 were also used, which showed antagonistic activity against Verticillium dahliae, Botrytis cinerea, and Phytophthora cactorum. (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). Abilio A. et al. (Abilio A., Knap I., De Freitas Zambelli L.S., Nematicidal composition containing Bacillus subtilis and Bacillus licheniformis, EP2603086) developed a nematicidal composition containing Bacillus subtilis and Bacillus licheniformis.

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. Sp115AD, Sp116AC* 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 the publication: Agnieszka Rutkowska, STUDIES AND REPORTS IUNG-PIB, Issue 48(2): 65-80, 2016, Biostimulators in modern plant cultivation, doi: 10.26114/sir.iung.2016.48.05 describe the use of microbiological inoculants - bacteria, fungi, humic compounds, seaweed extracts, protein hydrolysates and amino acids, in plant cultivation, their effectiveness has been confirmed.

Most often, patents concern biopreparations used to control pathogens of vegetable and crop plants, and there are no solutions covering microbiological fertilizer preparations affecting the microbiological quality of the soil while simultaneously having an antagonistic effect on key fungal pathogens and fungus-like oomycetes, also intended for use in organic cultivation of soft fruits, or these are solutions covering the control of single pathogens, e.g. of the Botrytis genus, not providing 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.

There are also no literature reports and patents regarding the combined use of microorganisms on carriers composed of humic acids and/or yeast culture effluent and/or micronized dolomite and/or maltodextrin, depending on the form of application of the fertilizing bioproduct in biopreparations and bioproducts.

The aim of the invention is therefore to develop a microbiological fertilizer product for maintaining and/or improving the microbiological quality of soil in soft fruit cultivation, exhibiting features important in the control of phytopathogenic fungi of the genera: Botrytis, Verticillium, Colletotrichum and oomycetes of the genus Phytophthora, and important in plant biostimulation.

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 that ensures and/or improves soil microbiological biodiversity while simultaneously controlling key pathogens for soft fruit cultivation, including the Botrytis, Verticillium, Colletotrichum, and Phytophthora genera. 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, the carriers themselves, 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 also constitute a patent claim.

The essence of the method for obtaining a fertilizing microbiological product for maintaining and/or improving soil biodiversity and/or controlling pathogens: Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp. in the cultivation of soft fruit, showing biostimulating properties on plants, 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 sp. Sp115AD - sequence no. 1 and Bacillus sp. Sp116AC* - sequence no. 2 on the sequence list, grown on a multiplication medium with whey and microelements, prepared in 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 culture medium or dried on a carrier or lyophilized on a carrier;

- a suitable carrier in the form of a culture medium for the liquid form or micronized dolomite for the dry, water-insoluble form or maltodextrin for the dry, water-soluble form;

- addition of liquid humic acids and yeast production effluent for the liquid form or dry humic acids for the dry form as supplementary components of the actual carrier.

Preferably, the method comprises culturing bacterial strains of the genus Bacillus, wherein the bacterial strains are initially multiplied in stationary culture on Plate Count Agar at a temperature of 25-30°C for 24-48 hours, and then the liquid multiplication medium is inoculated with the inoculum 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 and 120 rpm. Preferably, an inoculum with a transmittance of 90% is used and the multiplication culture is carried out for 48 hours.

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

Preferably, after obtaining a multiplication culture titer of no less than ¹⁰⁹ cfu/ml, a sporulation induction step is performed. This process is preferably carried out by lowering the pH of the culture medium with lactic acid to pH 5.0-5.5, and after 48 hours of cultivation and spore production by the bacterial cells, the culture is pasteurized by incubation at 80°C for 20 minutes. The obtained spore suspension is used as the main component of the microbiological fertilizer product.

After culturing, humic acids are added as a carrier component, which, depending on the product formulation, are in liquid or dry form.

The bacterial carrier for the liquid form is a multiplying culture medium obtained by combining 390-410 ml of a spore suspension of each bacterial strain: Bacillus sp. Sp115AD and Bacillus sp. Sp116AC*, no. 1 and 2 on the sequence list, with 45-55 ml of liquid humic acids and 145-155 ml of yeast culture effluent. The resulting liquid form is preferably stabilized to prevent spore growth by lowering the pH of the preparation to 4.0-4.5. Lactic acid is preferred for this purpose.

In a variant to obtain a dry form insoluble in water, the spore suspension is spray-dried 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 centrifuged microorganisms, obtaining bacterial lyophilisates with a concentration of not less than 1011 cfu/g, and dried or freeze-dried bacteria are added in equal amounts to obtain a product with the concentration of each strain in the range of 108 - 101 ¹ cfu/g.

Preferably, for the dry, water-insoluble form, dry humic acids are used as a carrier component in an amount of 0.55-0.65 g/kg of product, and micronized dolomite is used as a carrier for all components, making up to 1 kg of product. Alternatively, to obtain the dry, water-soluble form, maltodextrin is used as a cryoprotectant for bacteria during spray drying or freeze-drying and as a carrier for all components of the microbiological fertilizer product, and dried/freeze-dried bacteria with a concentration of no 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.

Preferably, for the dry, water-soluble form, dry humic acids are used as a carrier component in the amount of 0.55-0.65 g/kg of the microbiological fertilizing product, and maltodextrin as a bacterial cryoprotectant and the actual carrier constitutes the supplement to 1 kg.

The essence of the microbiological fertilizer product for maintaining and/or improving the microbiological quality of the soil, allowing at the same time to control phytopathogens (Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp.) in the cultivation of soft fruit, showing biostimulating properties on plants, containing strains of bacteria of the genus Bacillus, is that it contains two bacterial isolates selected from the rhizosphere of healthy plants, not showing mutual antagonistic activity: Bacillus sp. Sp115AD - sequence no. 1 and Bacillus sp. Sp116AC* - sequence no. 2 on the sequence list, a carrier in the form of a culture medium for the liquid form of the product or micronized dolomite for the dry form insoluble in water or maltodextrin for the dry form soluble in water and contains the addition of liquid humic acids and yeast culture effluent as supplementary components of the carrier appropriate for the liquid form or the addition of dry humic acids for the dry, water-soluble and water-insoluble forms. Bacterial isolates are cultured in a whey and microelement growth medium prepared in water and suspended on a carrier, or dried on a carrier, or lyophilized on a carrier.

The microbiological fertilizer product contains bacterial strains preferably in an equal weight ratio for the dry form or in an equal volume ratio for the liquid form.

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

Preferably, the concentration of each bacterial strain in liquid form is in the range of 108 - ¹⁰¹¹ cfu/ml.

Preferably, the liquid form contains 45-55 ml of added liquid humic acids and 145-155 ml of added yeast culture effluent per litre.

In the product variant in dry, water-soluble or insoluble form, the concentration of each bacterial strain is preferably in the range of 108 - ¹⁰¹¹ cfu/g.

Preferably, the dry form of the product, soluble or insoluble in water, contains 0.55-0.65 g of dry humic acids per 1 kg.

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

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

The Bacillus spp. strains (Sp115AD and Sp116AC*) used to develop the fertilizer product are environmental isolates and were selected at the Institute of Horticulture (10) in Skierniewice and disclosed in the SYMBIO BANK collection of this Institute.

The obtained microbiological fertilizer product in liquid and dry form enables the maintenance and/or improvement of soil microbiological biodiversity 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 products 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 impact of the developed product on the soil environment and the plant, providing the basis for recognizing the developed bioproduct as a microbial fertilizer. The product is applied foliarly (by spraying or sprinkling) and/or sub-plant by watering. In the case of a dry formulation, a suspension of the product in water should be prepared, preferably 2-5 kg of product (with a bacterial concentration of 109 cfu/g) per 700-800 liters of non-chlorinated water per hectare 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/or control of plant pathogens, the product is applied in an amount of preferably 5-10 l/ha (109 cfu/1) for a liquid preparation, wherein the product can preferably be diluted with water as needed, or 2-5 kg/ha for suspension in water by applying 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 a preparation containing at least 109 cfu/g of bacteria isolated from the rhizosphere of plants according to the composition and/or product, wherein in the form of watering and/or spraying/sprinkling/irrigation the product can preferably be diluted with water as needed.

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

Fig. 1 shows the appearance of colonies of bacterial strains Sp115AD and Sp116AC* 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 a bacterial liquid product containing selected strains of Bacillus sp. Sp115AD and Bacillus sp. Sp116AC* bacteria 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 strains of Bacillus sp. Sp115AD and Bacillus sp. Sp116AC* bacteria in liquid, dry water-insoluble and dry water-soluble form 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 containing selected bacterial strains Bacillus sp. Sp115AD and Bacillus sp. Sp116AC* in a dry, water-soluble form (carrier - maltodextrin) towards plant fungal pathogens of the genera Botrytis, Colletotrichum, Vertticillium and the fungus-like oomycete of the genus Phytophthora after 1 month of product storage at 35°C;

Fig. 11 shows the antagonism of a microbiological fertilizer product containing selected bacterial strains Bacillus sp. Sp115AD and Bacillus sp. Sp116AC* 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 product storage at room temperature (approximately 21°C), 35°C and 4°C;

Fig. 12 shows the antagonism of a microbiological fertilizer product containing selected bacterial strains Bacillus sp. Sp115AD and Bacillus sp. Sp116AC* 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 6 months of product storage at room temperature (approximately 21°C);

Fig. 13 shows the effective number of bacterial species (ENS) in the raspberry rhizosphere after application of a microbiological fertilizer product containing selected bacterial strains Bacillus sp. Sp115AD and Bacillus sp. Sp116AC*;

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 sp. Sp115AD and Bacillus sp. Sp116AC*;

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 sp. Sp115AD and Bacillus sp. Sp116AC*;

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 sp. Sp115AD and Bacillus sp. Sp116AC*;

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 sp. Sp115AD and Bacillus sp. Sp116AC*;

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 sp. Sp115AD and Bacillus sp. Sp116AC*;

Fig. 19 shows the relative abundance of bacterial orders, families, genera and species occurring in the rhizosphere and roots of raspberry plants after application of a microbiological fertilizer product containing selected bacterial strains Bacillus sp. Sp115AD and Bacillus sp. Sp116AC*;

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 sp. Sp115AD and Bacillus sp. Sp116AC*;

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 sp. Sp115AD and Bacillus sp. Sp116AC*;

Fig. 22 shows the relative abundance 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 sp. Sp115AD and Bacillus sp. Sp116AC*.

EXAMPLES

Example 1. Identification and biochemical characterization of bacterial strains that are components of a microbial fertilizer product for maintaining and/or improving soil microbial quality and/or biodiversity while controlling key pathogens (Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp.) in soft fruit cultivation

Two bacterial strains selected for use in the 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 to genus based on 16S rDNA gene amplification and Sanger sequencing. The resulting nucleotide sequences were analyzed using bioinformatics tools by comparing them with nucleotide sequences found in the commercial MicroSeq ^ID database and the open NCBI database (https://www.ncbi.nlm.nih.gov/). The bacterial strains selected for the product also possess metabolic properties that positively impact soil environmental properties, biodiversity, and participate in the processes of making mineral compounds available to plants.

The bacterial strains comprising the product, Sp116AD (sequence no. 1) and Sp116AC* (sequence no. 2), were identified as belonging to the genus Bacillus; the obtained nucleotide sequences are presented in the Sequence List. The identified Bacillus strains possess beneficial metabolic properties that influence the microbial biodiversity of the soil environment, limiting the growth and spread of fungal phytopathogens, and also participate in the processes of making nutrients available to plants. The bacterial strains are characterized by very good growth with a light cream color on PCA medium (Fig. 1). The biochemical activity of the strains comprising the product was characterized based on their ability to utilize 71 carbon substrates belonging to the following compounds: 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 Sp116AD and Sp116AC* 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 for 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, the bacterial strains were characterized in terms of their basic enzymatic activity using the API ZYM biochemical assay (Biomerieux). The activity of hydrolytic enzymes, i.e., phosphatases, esterases, arylamidases, phosphohydrolases, β-glucosidase, β-galactosidase, mannosidase, and glucosaminidase, was determined in the bacterial strains constituting components of the microbial fertilizer product (Fig. 6). The Bacillus sp. Sp116AD strain was characterized by high activity of esterase, lipase esterase, and naphthyl-AS-BI phosphohydrolase, while the Bacillus sp. Sp116AC* strain was characterized by high activity of naphthyl-AS-BI phosphohydrolase and medium or low activity of esterase, alkaline phosphatase, lipase esterase, α-chymotrypsin, acid phosphatase, α-galactosidase, and β-glucosidase (Fig. 7).

Example 2. Determination of the antagonism of Bacillus strains included in a microbiological fertilizer product towards plant fungal pathogens and fungus-like oomycetes - a plate experiment

The antagonistic capacity of a microbiological fertilizer product containing a consortium of Bacillus strains: Bacillus sp. Sp115AD and Bacillus sp. Sp116AC*, which are the main components of the product, was determined in relation to environmental pathogen isolates isolated from organic soft fruit plantations at the Laboratory of Molecular and Environmental Microbiology, Institute of Agrophysics, Polish Academy of Sciences. Bacterial antagonism studies were carried out against the following fungi: Botrytis sp. (G277/18 and/or G276/18), Verticillium sp. (G297/18 and/or G296/18), Colletotrichum sp. (G 166/18 and/or G171/18), and the fungus-like oomycete Phytophthora sp. (G408/18 and/or G184/21).

Antagonism was determined in 90 mm diameter Petri dishes on PDA (Plate Dextrose Agar) agar medium. The entire plate was inoculated with 300 μl or 100 μl of a phytopathogen spore suspension with a transmittance of 70%. Next, a sterile 5 mm diameter filter paper disc, soaked in a suspension of a bacterial product containing the selected bacterial strains: Bacillus sp. Sp115AD and Bacillus sp. Sp116AC*, was placed in the center of the plate. The plates were incubated at 27°C for 5-7 days, and the zones of mycelial growth inhibition and/or sporulation inhibition were measured (Fig. 8 - Fig. 9). Storage studies were also conducted, during which the antagonistic properties of the product were determined periodically 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 antagonistic properties of the product were determined on various media—liquid (culture medium), water-insoluble (micronized dolomite), and water-soluble (maltodextrin). The results indicate that the antagonistic properties of the developed product formulations against key fungal and fungi-like oomycete phytopathogens of soft fruit generally persisted during storage; however, 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, fungistatic activity of the bioproduct was observed, manifested by the inhibition of sporulation of fungi of the genera Botrytis, Colletotrichum, and Verticillium (Fig. 10). The best antagonistic properties after 6 months of product storage were noted for the preparation on a carrier - micronized dolomite, which was effective against phytopathogens of the genera Phytophthora, Botrytis, and Verticillium, demonstrating biocidal properties and preventing the development of these pathogens, as well as fungistatic activity, 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 by counting and determining the total bacterial count using the dilution plating method. From a series of dilutions of the bioproduct ranging from ^10-1 to ^10-8 , 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 incubated at 26°C for 72 hours. The grown colonies were counted, and the total bacterial count was determined in the liquid form of the bioproduct (CFU/ml) and in the dry formulations of the microbiological fertilizer product (CFU/g).

After production, the preparation showed a total bacterial count of > ¹⁰⁹ cfu/ml of the liquid bacterial product and > ¹⁰⁹ cfu/g of the dry, water-soluble and water-insoluble bacterial product. 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 product, 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 growing medium consisting of ½ volume of soil and ½ volume of 7-20 mm peat. The product 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 product was applied in liquid 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 product 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 product increased the ENS of root-colonizing fungi compared to untreated root control samples (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 the pathotrophic group and the mixed modes: pathotrophs-symbiotrophs and pathotrophs-saprotrophs-symbiotrophs was observed, as well as an increase in saprotrophs and symbiotrophs in the root. An increase in symbiotrophs and sapro-symbiotrophs was also observed in the rhizosphere of raspberries treated with the microbial fertilizer product, as well as a reduction in the mixed mode: pathotroph-saprotroph-symbiotroph (Fig. 15). Furthermore, the number of guilds assigned to plant pathogens decreased after product application (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 product application. This effect was noted in both the rhizosphere soil and the root (Fig. 17 Fig. 19). Analysis of the results for fungal communities confirmed that there was no increase in the biodiversity of this group of microorganisms in the roots of raspberry plants treated with the product, while in the rhizosphere, a similar level or a reduction in the abundance of fungi was maintained compared to the untreated control, which may indicate a protective effect of the microbial fertilizer product, limiting the penetration of fungi, especially those unfavorable to plants (Fig. 20 - Fig. 22).

The advantage of this microbial fertilizer product is its high versatility, confirmed by its dedicated use in maintaining and/or improving soil microbial biodiversity and the microbiome of soft fruit plants, while simultaneously controlling phytopathogens present 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.

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 the microbiological biodiversity of soil and fruit in raspberry cultivation - field tests

To determine the effect of a microbiological fertilizer product containing selected strains of Bacillus sp. Sp115AD and Bacillus sp. Sp116AC* 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, using two cultivation methods: irrigated and non-irrigated, in an organic production system. Additionally, tests of the developed microbiological fertilizer product, introduced as one component of the organic raspberry production technology, were based on experiments with four raspberry varieties: Delniwa, Enrosadira, Rosalita, and Poemat. Both liquid and dry, water-insoluble formulations of the bacterial product were tested in the experiments. 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 plot, the greatest strawberry yield increase was observed in the varieties in which the developed microbiological fertilizer product was added, applied in liquid form in the first year and in a dry, water-insoluble form in the second year. The Honeoye variety in the non-irrigated plot, in the variety in which the bacterial product was added, was observed to have a reduced anthracnose infection. The Vibrant variety, on the other hand, responded with a reduced infection with strawberry leather rot. The results indicate that for some raspberry varieties, particularly Enrosadira and Rosalita, an increased metabolic activity of microorganisms present in the plant and fruit, particularly in relation to carbohydrates, was observed in the variety in which the developed bacterial product was used as one of the cultivation elements. The overall soil microbiological diversity index increased in the variant in which the application of the developed bacterial product was included.

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

To obtain a microbiological fertilizer product for maintaining and/or improving the microbiological quality of the soil, which also allows for 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 sp. Sp116AD - sequence no. 1 and Bacillus sp. Sp116AC* - 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 NH4Cl, 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 lowering the pH of the culture medium using lactic acid to pH 5.0-5.5. After 48 hours of cultivation, the cultures were pasteurized by incubation at 80°C for 20 minutes. The spore suspension prepared in this way was the main component of the microbiological fertilizer product.

Carrier components in the form of liquid humic acids and yeast culture effluent were added to the bacterial culture (spore suspension). After combining the ingredients, the prepared product was protected against spore development into vegetative cells (stabilized) by changing the pH, lowering the pH of the microbial fertilizer product with lactic acid to 4.0-4.5.

Example 7. Method for obtaining a microbiological fertilizer product - a dry, water-insoluble, spray-dried formulation

The method was carried out as in Example 6, except that after the cultivation was completed, in order to obtain a water-insoluble dry formulation for spraying or watering, the cultures were spray-dried on whey powder, and instead of a liquid culture medium with liquid humic acids and a effluent from yeast production, a carrier in the form of micronized dolomite with the addition of dry humic acids was used as a carrier for the bacteria dried on whey.

Example 8. Method for obtaining a microbiological fertilizer product - a water-insoluble dry formulation containing lyophilized bacterial strains

The microbiological fertilizer product obtained by the method described in Example 7 for maintaining and/or improving the microbiological quality of soil, allowing at the same time to control phytopathogens (Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp.) in the cultivation of soft fruit, had a composition analogous to that indicated in Example 7, but instead of spray drying, the bacterial cultures were subjected to freeze-drying with the addition of whey powder as a cryoprotectant, in the amount of 5% of the fresh mass of centrifuged microorganisms.

Example 9. Method for obtaining a microbiological fertilizer product - dry formulation soluble in water

The method for obtaining the microbiological fertilizer product was carried out as in Example 7, except that the cultures were spray-dried on maltodextrin instead of whey powder to obtain a soluble dry formulation for spraying or sprinkling, and instead of micronized dolomite with added dry humic acids, a carrier in the form of maltodextrin with added dry humic acids was used as a carrier for the bacteria dried on maltodextrin.

Example 10. Method for obtaining a microbiological fertilizer product - a water-soluble dry formulation containing lyophilized bacterial strains

The microbiological fertilizer product obtained by the method according to Example 9 has a composition analogous to that in Example 9, but instead of spray drying on maltodextrin, freeze-drying with the addition of maltodextrin as a cryoprotectant is used.

Claims (23)

1. A method for obtaining a microbiological fertilizer product for maintaining and/or improving the microbiological quality of soil, which also allows for the control of phytopathogens Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp. in the cultivation of soft fruit, exhibiting biostimulating properties on plants, using bacterial strains of the genus Bacillus, characterized in that: - two bacterial isolates selected from the rhizosphere of healthy plants, not showing mutual antagonistic activity: Bacillus sp. Sp115AD - sequence no. 1 and Bacillus sp. Sp116AC* - sequence no. 2 on the sequence list, grown on a multiplication medium with whey and microelements, prepared in 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 culture medium or dried on a carrier or lyophilized on a carrier; - a suitable carrier in the form of a culture medium for the liquid form or micronized dolomite for the dry, water-insoluble form or maltodextrin for the dry, water-soluble form; - addition of liquid humic acids and yeast production effluent for the liquid form or dry humic acids for the dry form 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, 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 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 lowering the pH of the culture medium with lactic acid to pH 5.0-5.5 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 fertilizing product according to any of claims 2-6, characterized in that after the cultivation is completed, humic acids are added as a carrier component, which, depending on the product formulation, are in liquid or dry form. 8. A method for obtaining a microbiological fertilizer product according to any of the preceding claims, characterized in that a multiplying culture medium obtained by combining 390-410 ml of a spore suspension of each bacterial strain: Bacillus sp. Sp115AD and Bacillus sp. Sp116AC* with no. 1 and 2 on the sequence list and 45-55 ml of liquid humic acids and 145-155 ml of yeast culturing effluent is used as a bacterial carrier for the liquid form. 9. A method for obtaining a microbiological fertilizer product according to any of the preceding claims, characterized in that the obtained liquid form is subjected to stabilization to protect against the development of spores by lowering the pH of the preparation to pH = 4.0-4.5. 10. A method for obtaining a microbiological fertilizer product according to claim 9, characterized in that lactic acid is used. 11. A method for obtaining a microbiological fertilizer product according to any of claims 1-7, characterized in that in order to obtain a dry form insoluble in water, the spore suspension is subjected to spray drying on whey powder or freeze-drying with the addition of whey powder as a cryoprotectant in the amount of 5% of the fresh mass of centrifuged microorganisms, obtaining bacterial lyophilisates with a concentration of not less than ¹⁰¹¹ cfu/g, and dried or lyophilized bacteria are added in equal amounts to obtain a product with the concentration of each strain in the range of 108 - ¹⁰¹¹ cfu/g. 12. A method for obtaining a microbiological fertilizing product according to any of claims 1-7 or 11, characterized in that for the dry, water-insoluble form, dry humic acids are used as a carrier component in the amount of 0.55-0.65 g/kg of the product, and micronized dolomite is used as a carrier for all components, constituting the balance up to 1 kg of the product. 13. A method for obtaining a microbiological fertilizing product according to any of claims 1-7, 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, and dried/freeze-dried bacteria 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. 14. A method for obtaining a microbiological fertilizing product according to any of claims 1-7 or 13, characterized in that for the dry water-soluble form as a carrier component, dry humic acids are used in the amount of 0.55-0.65 g/kg of the microbiological fertilizing product, and maltodextrin as a cryoprotectant for bacteria and the actual carrier constitutes the complement to 1 kg. 15. A microbiological fertilizer product for maintaining and/or improving the microbiological quality of soil, allowing at the same time to control phytopathogens (Botrytis sp., Colletotrichum sp., Phytophthora sp., Verticillium sp.) in the cultivation of soft fruit, showing biostimulating properties on plants, containing strains of bacteria of the genus Bacillus, characterized in that it contains two bacterial isolates selected from the rhizosphere of healthy plants, not showing mutual antagonistic effects: Bacillus sp. Sp115AD - sequence no. 1 and Bacillus sp. Sp116AC* - sequence no. 2 on the sequence list, a carrier in the form of a culture medium for the liquid form of the product or micronized dolomite for the dry form insoluble in water or maltodextrin for the dry form soluble in water, and contains an addition of liquid humic acids and yeast culture effluent as supplementary components of the carrier for the liquid form or the addition of dry humic acids for the dry form soluble and insoluble in water, 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. 16. A microbiological fertilizer product according to claim 15, characterized in that it contains the bacterial strains Bacillus sp. Sp115AD - sequence no. 1 and Bacillus sp. Sp116AC* - sequence no. 2 on the sequence list, in an equal weight ratio for the dry form or in an equal volume ratio for the liquid form. 17. A microbiological fertilizer product according to claim 15 or 16, characterized in that the bacterial isolates are grown on a multiplication medium with powdered whey, preferably acid-neutralized whey. 18. A microbiological fertilizing product according to any one of claims 15-17, characterized in that the concentration of each bacterial strain in liquid form is in the range of 108 - ¹⁰¹¹ cfu/ml. 19. A microbiological fertilizer product according to any one of claims 15-18, characterized in that its liquid form contains in 1 liter 45-55 ml of added liquid humic acids and 145-155 ml of added yeast culturing effluent. PL 248033 Β1 20. A microbiological fertilizing product according to any one of claims 15-17, characterized in that the concentration of each bacterial strain in a dry, water-soluble or insoluble form is in the range of ¹⁰⁸ - ¹⁰¹¹ cfu/g. 21. A microbiological fertilizer product according to claim 15, 16, 17 or 20, characterized in that its dry form, soluble or insoluble in water, contains an addition of dry humic acids in the amount of 0.55-0.65 g per 1 kg. 22. A microbiological fertilizing product according to claim 15, 16, 17, 20 or 21, characterized in that it is in a dry form, insoluble in water, and contains whey powdered as a cryoprotectant in the process of freeze-drying or spray-drying, and micronized dolomite as the actual carrier. 23. A microbiological fertilizing product according to claim 15, 16, 17, 20 or 21, characterized in that it is in a dry form, soluble in water, and contains maltodextrin as a cryoprotectant in the freeze-drying or spray-drying process and as a proper carrier.