EA042498B1 - LIQUID BIOLOGICAL ANTIFUNGAL PREPARATION CONTAINING PYTHIUM OLIGANDRUM MICROORGANISM AND METHOD FOR ITS PRODUCTION - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to a liquid biological antifungal preparation containing the microorganism Pythium oligandrum.

The present invention also relates to a method for producing said drug.

Prerequisites for the invention

Over the past decades, chemical antifungals have been of ever-increasing concern in terms of their negative impact on the environment and certainly on users. However, it can be argued that the work to replace these drugs with effective biological drugs based on microbes, capable of purposefully and thoroughly destroying mold, was not carried out to the required extent. Currently, only 14 types of microbes are registered for this purpose in the EU countries, despite intensive research, as highlighted in a recently published article by Gerbore et al., 2014[1].

The practical application of a biological preparation that uses the antifungal action of the microorganism Pythium oligandrum has been known since the 1970s of the last century, and in this case we can refer to the classic works of Dreschler [2], 1943 and Deacon [3], 1976. devoted to the biology of the specified microorganism and containing a description of its mycoparasitic properties. It was at that time that we received U.S. Patent No. 4,259,317 [4], by Vesely and Hejdanek, priority date 5.7.1978, which discloses dry powder preparations containing Pythium oligandrum Dreschler oospores for protection against mold-related diseases affecting seedlings. sugar beet, while the content of oospores in the preparation is at least 1 million oospores per 1 g of the preparation, and the number of oospores in the range from 50 to 500 million per 1 g of the preparation is claimed to be optimal. As an appropriate alternative to dry powder preparations, the present inventors take a liquid suspension of oospores, however, they point out a number of disadvantages in the use of their preparation, in particular, the instability of such preparations, which limits their practical use to approximately 2 weeks. After a longer period of time, there is an unacceptable decrease in the number of oospores produced due to autolysis or contamination with superparasites, while the number of oospores is a significant factor in the application of the mycoparasitic defense mechanism. With regard to the source of individual strains of Pythium oligandrum, Baker and Lifshitz noted in their US patent 4574083 [5] the possibility of obtaining regionally suitable strains by isolating them from the soil in accordance with the standard published method. Over time, this method was recognized, and strains from various geographical regions are currently in public collections. For the determination of isolates at that time, the following biological criteria were mainly used: growth rate, optimal growth temperature - usually approximately 30 ° C, and the morphology of individual reproductive forms of the sexual and asexual cycle. From this point of view, the subsequent patent can be considered as innovative; in said patent, inventor Martin, US Pat. No. 5,961,971 [6], provides molecular characterization of individual isolates based on an accurate protocol for the sequences of the most important genes. The same patent document details individual formulations and methods of using the microorganism Pythium oligandrum in the field, liquid formulations after being grown on an inexpensive medium using industrial waste, and powder formulations made by encapsulating biomass dried after fermentation to convert them into alginates. , or in the form of dried and ground material, are mentioned in the patent document as the preferred formulations. Formulations in the form of wettable powders, standard powders, emulsified oils, and granules are another form of possible composition. In the patent CZ 303 908 and in the corresponding patent EP 2503892 [7], BIOPREPARATY, spol. s.r.o., 2009 focuses on post-harvest crop protection using the microorganism Pythium oligandrum. In the technological section of the patent, the emphasis is on the cultivation of the specified microorganism in standard fermenters on a liquid medium that contains cereals without the addition of other nutrients. The possibility of obtaining a pure concentrate of oospores without the presence of depleted nutrient medium or mycelial residues, which are removed during drying in a spray dryer at the last stages of the production process, is emphasized. Publication of information on the preparation of oospores from organisms other than Pythium oligandrum has been limited. In the patent application CN 102807957A [8], Liu X. et al., 2012, a preparation is described from the resulting oospores of the microorganism Phytophthora capsici in order to control resistance to various drugs or to conduct genetic studies. Liquid preparations intended for biological plant protection are indicated in US patent application No. 2014/0212387 [9], Luth, 2014, although they are exclusively liquid preparations that use polyethers of modified trisiloxanes as solvents.

In addition, the non-patent literature to a certain extent deals with the preparation and stabilization of oospores and other reproductive forms of microscopic oomycetes Pythium oligandrum. In 1990, McQuilken et al. [10] published a simple and reproducible method for obtaining oospores

- 1 042498 in liquid culture using molasses as an economically available substrate. However, the question of the ideal way to maintain the viability of cooked oospores has not been satisfactorily resolved in either the patent or non-patent literature. In 1992, McQuilken et al. [11] observed the effect of various osmotic agents, such as glycerol, NaCl, and KCl, on the rate of radial hyphal outgrowth and oomycete oomycete oospore formation in Pythium oligandrum by comparison with the osmotic pressure represented by a soil extract at -0.5 MPa. The addition of osmolytes contributed to a decrease in the growth rate in the radial direction, but had no effect on the formation of oospores up to a pressure value of approximately -2.0 MPa. The authors concluded that high performance of the applicable oospores can be achieved under the natural osmotic conditions at the application site if the conditions in terms of optimum temperature, typically 30°C, and acidity values, pH 6.0-7, are satisfied. ,5. From the point of view of the storage stability of the obtained oospores, the authors of Takenaka and Ishikawa [12], 2013, noted their high efficiency for a period of time up to 379 days when stored as a standard prepared suspension in distilled water in a refrigerator at 4°C.

According to experts, the main obstacle to the wider distribution of biological protection products is clearly the dependence of effectiveness on specific conditions of use and the solution of practical problems associated with the preservation and preparation of biological plant protection products in the field of crop production. For this reason, addressing the practical problems associated with the simple and effective use of Pythium oligandrum oospores is critical to the commercial success of biologics. In order to solve this problem, it would be desirable to develop liquid biological protection agents that are characterized by long-term stability even under standard storage conditions. However, the solutions published so far have a number of shortcomings that have not been successfully addressed even after several years of intensive research work. Most of the methods used in the laboratory preparation of biological plant protection products are complex and controlled exclusively at the laboratory level, which significantly limits the use of such methods in the field of industrial production of such products. So far, the preferred method of application (in the form of wettable powders), which has been used in preparations prepared by the Czech biotechnology company Biopreparaty, spol s.r.o. (Polyversum®, Polygandron, BIOREPEL®) and marketed worldwide, is not without its own disadvantages in terms of targeting protective measures and user protection. So far, no simple, effectively tested and consistently defined method has been published to create a composition with sufficient storage stability of prepared reproductive forms of the microorganism Pythium oligandrum, or oospores in a simple form, which can be freely used in industry.

Brief summary of the invention

The known disadvantages of the biological antifungal preparations used so far are eliminated or limited in accordance with the present invention, the essence of which is a liquid biological antifungal preparation containing the microorganism Pythium oligandrum in accordance with the independent claim 1 of the claims, including a stabilized suspension of Pythium oligandrum, and containing 0.05 -10.0 wt.% of the cultivated biomass of the microorganism Pythium oligandrum containing the culture medium, cell forms of the specified microorganism and substances produced by the specified microorganism; and 90.0-99.95% by weight of a stabilizer; while the number of dormant oospores in 1 ml of the specified liquid biological antifungal drug should be in the increased range from 1x103 to 2x10 ⁷ after standard normalization.

The essence of the liquid biological antifungal preparation containing the microorganism Pythium oligandrum in accordance with the 2nd independent claim of the present invention is that the preparation includes a stabilized suspension of Pythium oligandrum and contains: 0.05-10.0 wt.% of the cultivated biomass of the microorganism Pythium oligandrum containing culture medium, cell forms of said microorganism and substances produced by said microorganism; 79.77-99.95 wt% stabilizer; while the remainder up to 100 wt.% is at least one modifying/auxiliary substance selected from the group including filler, flavor, vitamin E; while the number of dormant oospores in 1 ml of the specified liquid biological antifungal drug should be in the increased range from 2.5x10 ⁴ to 1.0x10 6 after standard normalization.

Preferred is the strain Pythium oligandrum Dreschler ATTC 38472 of the microorganism Pythium oligandrum deposited in the Czech Collection of Microorganisms (CCM) at the Masaryk University in Brno under the name Pythium oligandrum M1.

The main advantage of the present invention lies in the creation of a new liquid biological

- 2 042498 a chemical antifungal preparation suitable for long-term use and achieving its long-term and significant stabilization effect, while the stabilizers used are available and provide an additional advantage in protecting the preserved biomass of the microorganism Pythium oligandrum from microbial contamination. The above stated content of dormant cells of the microorganism in the form of oospores provides for a practically tested and economically available subject of study, which simplifies the dosing of the drug for use in a wide range of areas. In this case, various types of biomass processed using a suitable preparation can be used. Lower concentrations of dormant oospores, for example, with tens or hundreds of dormant oospores, will also provide some results, although the effectiveness of this preparation will be lower. The predetermined number of dormant oospores can be adjusted if necessary by standard normalization, i.e. by dilution or concentration to the required value.

Preferably, the stabilizer contains at least one selected from the group consisting of water, saline, oil, or osmolyte solutions, which may be a polyol solution, such as a saccharide or saccharide alcohol, or saline solutions. The polyol solutions may be selected from the group consisting of metabolizable and non-metabolizable solutions of various oligosaccharides having a maximum of 10 saccharide units and a minimum of 2 saccharide units so as to ensure the use of maltodecaose, or maltononose, or maltooctose, or maltogelpaose, or maltohexaose, or maltopentose, or stachyose, or raffinose, or sucrose, or sucralose, or branched saccharides.

Ideally, the stabilizer is water in an amount, for example, from 30.0 to 99.9 wt.%, or saline in an amount, for example, 99.9 wt.%, or osmolite in the form of sucrose in an amount, for example, from 60.0 to 64.95 wt.%, or at least one of the oils selected from the group including paraffin oil, mineral oil, glycerin, sunflower oil, in an amount, for example, 79.77-99.9 wt.% .

The liquid biological antifungal preparation may contain other modifiers or excipients for specific applications, such as a biodegradable matrix of prolonged action, such as a carrier of oospores of the microorganism Pythium oligandrum, for example, polyvinyl alcohol in an amount of, for example, 16.0 wt.%; or filler based on silicon oxide in an amount of, for example, 16.0 wt.%; or other auxiliaries/modifiers such as sage or mint flavor in an amount of, for example, 0.96 wt.%, or vitamin E in an amount, for example, 0.4 wt.%.

The use of such excipients/modifiers depends on the application of the biological antifungal agent.

Therefore, a wide range of stabilizing agents of a hydrophilic or hydrophobic nature, such as commonly used water or oil, or solutions of various available osmolytes, can find use in a stabilized suspension of oospores of the microorganism Pythium oligandrum.

Osmolytes, due to their chemical properties, can be ideally adapted to the required specific application, as it has been demonstrated that osmolytes, such as solutions of various salts, can exhibit an ionic character, or, like solutions of polyols, a non-ionic character. It is advantageous that the polyols can be, for example, metabolizable and non-metabolizable solutions of various defined oligosaccharides suitable for a number of applications, since mixtures of appropriate natural or synthetic substances having the desired properties can be used for this purpose. The most widely distributed substance, such as sucrose, can also be advantageously used, however, if necessary, even widely distributed non-metabolizable analogues can be used. The liquid suspension composition is compatible with a wide range of other substances that can modify their beneficial properties or change their applications in the form of antioxidant substances, natural activators and vitamins, for example, silica as a filler, acetate (vitamin E) and other substances.

It has been surprisingly found that if a certain percentage of cells obtained by culturing in a liquid phase or on a solid substrate, at the end of the preparation process, is a suspension or a concentrated suspension, the reproductive forms of the microorganism Pythium oligandrum retain their original ability to germinate in such a suspension, at least , within six months when stored under standard laboratory conditions at normal temperature. This result becomes even more revealing when we consider the non-reproducible and inconsistent results presented in the published literature. In the literature, for example, it is stated that oospores in suspension prepared in distilled water and stored for 188 or 384 days retained the original ability to germinate when stored in a refrigerator at 4°C. However, the almost complete absence of a description of the method of preparation of such described preparations does not allow an assessment of their scientific reliability, not

- 3 042498 to mention their practical applicability, when under working conditions on farms it may not be possible to maintain a storage temperature of 4°C.

A liquid biological antifungal preparation containing the microorganism Pythium oligandrum is obtained using a preparation method according to the present invention, the essence of which is that a liquid medium including cereal extract, cane sugar molasses and other necessary nutrients is sterilized in a steam sterilizer by aerobic cultivation oomycete Pythium oligandrum in liquid phase. After cooling, the medium is inoculated with one of the selected strains of Pythium oligandrum. The biomass is collected and processed after completion of the cultivation process, which takes several days, preferably 13 days. After completion of the stage of cultivation of Pythium oligandrum in the liquid phase, the biomass is homogenized with a liquid medium so that the size of at least 95% of the particles in suspension is in the range from 0.050 to 0.300 mm, preferably 0.125 mm. A homogeneous suspension, characterized by a certain number of oospores, was concentrated or diluted depending on the number of oospores in solution to a predetermined concentration of oospores in a biological antifungal preparation. The suspension is preferably filtered after a homogenization and concentration or dilution step.

The aqueous suspension is stabilized during the homogenization process with the addition of osmolite and stored in a sterile large volume container at a temperature below 8°C.

As regards the anhydrous suspension of the microorganism Pythium oligandrum, the material obtained after completion of the solid phase or liquid phase culture step is homogenized until at least 95% of the particles in the suspension have a size of 0.050 to 0.300 mm, preferably 0.125 mm. The resulting homogeneous suspension, characterized by a certain number of oospores, was centrifuged. After removal of the supernatant, oil was added to the centrifuged material to achieve a predetermined concentration of oospores for the preparation of a biological antifungal preparation in such an amount that the final concentration of oospores in the biological antifungal preparation corresponded to the desired concentration, and the material thus obtained was further resuspended by homogenization.

The presented solution of the issue is, in all likelihood, acceptable even from the point of view of a significant simplification of the biotechnological process for obtaining the microorganism Pythium oligandrum, and, thus, also from the point of view of a significant reduction in the current costs of obtaining such products. Filtration or separation of cultivated biomass from the biomass preparation medium, its drying and subsequent grinding to the required particle size involves a number of relatively energy-intensive and technologically labor-intensive processes that are difficult to control and lead to possible contamination of the material, which, ultimately, can lead to significant damage to the end product. product, unless these processes are carried out under well-defined and strictly enforced strict industrial production conditions. In contrast, the technologically easy suspension of biomass, obtained in the form of dense and viscous solutions of osmolytes, provides an extremely stable product, directly suitable for practical applications in a number of areas. From a practical point of view, this is a significant advantage in that the prepared suspension of the microorganism Pythium oligandrum can be transferred from culture dishes or fermenters with virtually no waste, minimizing cleaning and maintenance costs, as well as minimizing the likelihood of product leakage. in the working environment of industrial premises. Moreover, one should take into account the fact that obtaining in this way a drug that remains stable for a long period of time even when stored at normal temperatures, also significantly reduces operating costs and simplifies the handling of the drug.

In addition, it was unexpectedly found that, in addition to the stabilizing effect of concentrated solutions of osmolytes, the preservative effect of such solutions, well known from industrial and common practice, is a significant factor in the particular case of suspensions of oospores of the microscopic oomycete Pythium oligandrum studied by us. Thus, a preparation stored under such conditions is protected from contamination by undesirable microorganisms, which, even in the extremely unlikely event of their entry into the environment of such stabilized suspensions, are unable to reproduce in the proposed chemical environment and, therefore, cannot contaminate the final product. This advantage is fundamental to existing manufacturing practices, in particular in light of increasingly stringent requirements for the level of accidental contamination by pesticides, biocides and cosmetics.

In addition to the function of a stabilizer and a preservative agent, osmolyte solutions also act as an adhesive, a property due to which the preparation can find practical application in many areas, either in the form of aqueous solutions after diluting the stabilized suspensions of oospores to an optimal concentration for their use as an impregnating material, or for application by spraying a finely dispersed medium of microscopic particles (the so-called fogging). Thus, in all the above examples of application, solutions of polyol promote effective adhesion of the drug and subsequent uniform application to the treated areas, plants or crops.

Last but not least, regarding the use of metabolizable osmolytes, these substances could act as a nutrient, which is essential in the process of colonization and colonization of target sites directly at the application sites, while these nutrients are exclusively important, in particular, during the first stage associated with the transformation of oospores into the form of mycelium. They can also further serve as nutrients that are essential for the long-term growth and maintenance of the microscopic oomycete Pythium oligandrum at the site of application of a nutrient of a more complex nature, represented by an insufficiently fully fermented substrate used in the process of biotechnological growth and reproduction of oospores in the process of biotechnological cultivation.

The specific content of oospores and other reproductive forms of microscopic oomycetes in suspensions in accordance with the presented solution is comparable to or exceeds the content of powdered preparations previously tested and sold on the market, which is important from the point of view of logistics and economic distribution of the preparation prepared in this way. However, there are also possibilities to use the new formulation at lower concentrations than the previous solution.

In fact, we have found a significant reduction in the complexity of the composition of a new liquid biological antifungal drug due to the technological process used, without being tied to any limitations in the functionality or effectiveness of the liquid drug, the dosage form of which was obtained using this method. As for the cellular and granulometric composition of the new proposed preparation, it almost completely includes stabilized oospores with an exceptionally low percentage of mycelial fragments, ground millet and other components. These components certainly do not settle in a dense medium created by concentrated osmoles, which, however, provide a good homogeneity of the preparation, incomparably higher than in the case of wettable powders used in the form of aqueous suspensions, while the preparation was extremely heterogeneous in terms of particle size, being in suspension. This applies in particular to the silica particles contained in the preparations made up to now; in contrast, the solution according to the present invention does not involve the use of silica in most cases (with the exception of toothpaste, in which it is used as an abrasive). Therefore, from the point of view of the requirements for practical use, clogging of filters and other technological equipment during the use of the drug is excluded.

If the presence of fewer metabolizable osmolytes is a negative factor in certain applications, these metabolizable osmolytes can be easily replaced by chemically identical non-metabolizable osmolytes that in no way affect the essential properties of the drug.

If from a technological point of view, the process of drying the resulting preparation is more acceptable, apparently, in this case, freeze-drying (lyophilization) will be a suitable process in a high-vacuum apparatus, because The use of concentrated solutions of osmolytes as cryopreservatives and protective agents in the lyophilization process is well known and widespread. As an example, we refer to osmolytes, widely used as protective cryopreservatives in a number of applications that require special attention, including the cryopreservation of human oocytes, described, for example, in an article by Wright [15], 2004.

A significant advantage of the preparations obtained in accordance with the present invention is that such preparations are suitable for all applications in which it would be preferable to use the anti-mould, mycoparasitic, elicitor and growth properties of the microorganism Pythium oligandrum. Thus, in particular, the use of this preparation as a plant biofungicide for use in clinical and veterinary medicine of the microorganism Pythium oligandrum to destroy biofilms and eliminate dysbiosis that occurs in the case of various diseases, as well as to protect public health from the effects of mold that forms in residential and /or a production environment is appropriate. Depending on the matrices used in the liquid suspension concentrate, the preparation can be used as a plant protection spray in the presence of a degradable biopolymer, which acts as a protective factor, and a gradually degradable matrix, which refers to the carrier of the biological preparation Polyversum with oospores of the active microorganism Pythium oligandrum .

Protection against molds, fungi, pathogenic bacteria and yeasts using a stabilized, concentrated suspension of oospores of the microorganism Pythium oligandrum, which involves applying such a stabilized, concentrated suspension in undiluted or diluted form to an area affected by mold, or fungi, or pathogenic bacteria, or pathogenic yeasts. fungi, or microbial dysbiosis, allows you to determine a wide

- 5 042498 range of applications for the stabilized liquid concentrate.

In the field of plant protection, the composition can be used to provide protection against mold, fungi or pathogenic bacteria and yeasts in such a way that the stabilized concentrated suspension, in diluted or undiluted form, is applied to plants or their surroundings, or seeds, crops , or fruit, either before harvest or after harvest, such as in warehouses; this protection includes the use of aerosols, self-dissolving capsules containing a concentrated suspension, additional feeding with fertilizers at the root of the plant, impregnation, spraying, dressing, spray irrigation of crops, the application of fertilizers and additional fertilizers as a component of complex fertilizers, as well as application in the form of hydroponics.

When protecting the oral cavity, a stabilized, concentrated suspension in diluted or undiluted form can be applied to an area affected by microbial plaque in the oral cavity or periodontal tissue disease, in case of infected non-healing wounds, in particular in diabetic patients, in case of infectious diseases caused by dermatophytes. and yeast fungi, as well as other symptoms associated with microbial dysbiosis of the skin and surface membranes in the human body.

When using the drug in order to protect public health, a stabilized concentrated suspension in a diluted or undiluted form is applied to areas of walls and brickwork affected by the fungus. Other emerging applications include antifungal protection of refrigeration equipment, air conditioning equipment, flood and water damage areas, and other areas where Pythium oligandrum is the target of frequent growth of fungi.

Brief description of the drawings

Below is a detailed description of the present invention on its illustrative examples of implementation, and given a detailed explanation with reference to the accompanying drawings.

In FIG. 1A-1C discuss in more detail the comparison of the growth rate of the phytopathogenic fungus Fusarium grammearum in the absence of the microorganism Pythium oligandrum and in the presence of said microorganism.

The drawings, in particular, illustrate the following:

fig. 1A - growth of a colony of phytopathogenic fungi Fusarium graminearum without additives (squares) and with simultaneous inoculation of Pythium oligandrum from dormant oospores and during cultivation in a liquid phase and on a solid substrate (asterisks and triangles);

fig. 1B - picture 4, examples of Petri dishes in which the microorganism Pythium oligandrum from dormant oospores was inoculated on the left side and the phytopathogenic fungi Fusarium graminearum (upper row) on the right side, and on the control dishes only Fusarium graminearum fungi were inoculated, while growth was registered after 4 days;

fig. 1C is a photograph of Petri dishes while inoculating the microorganism Pythium oligandrum from dormant oospores of the phytopathogenic fungus Fusarium graminearum, with growth recorded after 11 days.

Fig. 2A-2C are results of laboratory experiments on growing wheat in the presence of the phytopathogenic fungus Fusarium graminearum, the microorganism Pythium oligandrum, and a combination of the two.

The drawings, in particular, illustrate the following:

fig. 2A - Erlenmeyer conical flasks in which wheat was germinated on agar soil in the presence of the phytopathogenic fungus Fusarium graminearum only, small white dots recorded after 20 days of inoculation during wheat germination;

fig. 2B are Erlenmeyer conical flasks in which wheat was germinated on agar soil in the presence of both the phytopathogenic fungus Fusarium graminearum and the microorganism Pythium oligandrum; flasks were photographed after 20 days of simultaneous inoculation with wheat germinating;

fig. 2C - Erlenmeyer conical flasks in which wheat was germinated on agar soil in the presence of the microorganism Pythium oligandrum alone; the flasks were photographed after 20 days of inoculation of the indicated fungi during wheat germination.

Fig. 3A-3E show application of a plant protection antifungal agent according to Illustrative Embodiment 1 and subsequent analysis of population dynamics of the microorganism Pythium oligandrum observed during a field experiment on wheat and its surrounding soil, including an expression controlled response to the presence of the fungus. gene and genetic observations of the level of fungal contamination.

The drawings, in particular, illustrate the following:

fig. 3A - content of the microorganism Pythium oligandrum determined by genetic testing in plants (light gray column) and in the surrounding soil (dark gray column) at various times after application;

- 6 042498 fig. 3B is an increase in temperature levels measured using an infrared thermometer directly in the field at the time of sampling (light gray column) and temperature data recording performed by the local meteorological station (dark gray column);

fig. 3C - course of gene expression for cellulose (POCELL), endo-beta-glucanase (POENDO) and structural protein rich in serine and threonine (POSTRU) in individual parts of the plant and in the surrounding soil (So - soil, R - root, St - stem, L - leaf), observed in the time period of 6-120 h, while the level of gene expression was normalized to the expression of beta-tubulin (POTUBU) of the constitutive gene and expressed without the use of the drug in accordance with the specified method [12];

fig. 3D - the level of fungal contamination in identical samples as in panel C, measured by quantitative PCR with primers for general amplification for fungal amplification;

fig. 3E is a correlation plot that monitors correlations from gene expression for cellulose (POCELL) to fungal contamination.

Fig. 4A-4D are a test of the effectiveness of toothpastes according to illustrative embodiment 4 in terms of their ability to remove biofilms artificially created on hydroxyapatite-coated ceramic plates using the saliva of healthy individuals (at the top of the drawing) and using the saliva of individuals, diseased periodontal tissues (at the bottom of the drawing).

The drawings, in particular, illustrate the following:

fig. 4A is a photograph of a six-well plate using the saliva of healthy individuals after separating the washed biofilms in solution and removing the hydroxyapatite-coated ceramic plates, which were previously brushed without toothpaste, using Odol toothpaste, Enzycal toothpaste and toothpaste containing glycerol. , according to Illustrative Embodiment 4 without adding the microorganism Pythium oligandrum, the toothpaste containing glycerin according to Illustrative Embodiment 4 without adding silica, and the toothpaste containing glycerin according to Illustrative Embodiment 4;

fig. 4B - Quantification of biofilm content by measuring the intensity of staining in individual wells in FIG. 1A on a flatbed spectrophotometer at 562 nm;

fig. 4C is a photograph of a six-well plate using the saliva of persons affected by periodontal disease, after separation of the washed biofilms in solution and removal of hydroxyapatite-coated ceramic plates, which were previously cleaned with a toothbrush without using toothpaste, using Odol toothpaste, Enzycal toothpaste and the glycerin-containing toothpaste according to Illustrative Embodiment 4 without adding the microorganism Pythium oligandrum, the glycerol-containing toothpaste according to Illustrative Embodiment 4 without adding silica, and the glycerol-containing toothpaste according to Illustrative Embodiment 4 ;

fig. 4D - Quantification of biofilm content by measuring the intensity of staining in individual wells in FIG. 1C on a plate spectrophotometer at 562 nm.

Illustrative embodiments of the invention

The liquid biological antifungal preparation contains the microorganism Pythium oligandrum, and in all illustrative embodiments of the present invention, the microorganism Pythium oligandrum is Pythium oligandrum Dreschler strain ATTC 38472 deposited in the Czech Collection of Microorganisms (CCM) at the Masaryk University in Brno under the name Pythium oligandrum M1, namely under this name it is presented below in illustrative embodiments of the present invention.

- 7 042498 the sequence of the specified microorganism in accordance with

759

DNA

Pythium oligandrum strain M1

ITS rRNAgene

683

DNA

Pythium oligandrum strain M1

Mitochondrial COXII cytochrome oxidase

Below is the ST25_PCT protocol:

<160> <210> <211> <212> <213> <221> <400>

atcattacca cacctaaaaa ctttccacgt gaaccgttat aactatgttc tgtgcttcgt 60 cgcaagactt gaggctgaac gaaggtgagt ctgcgtctat tttggatgcg gatttgctga 120 tgttatttta aacacctatt acttaatact gaactatact ccgaatacga aagtttttgg 180 ttttaacaat taacaacttt cagcagtgga tgtctaggct cgcacatcga tgaagaacgc 240 tgcgaactgc gatacgtaat gcgaattgca gaattcagtg agtcatcgaa attttgaacg 300 catattgcac tttcgggtta tgcctggaag tatgcctgta tcagtgtccg tacatcaaac 360 ttgcctttct ttttttgtgt agtcaaaatt agagatggca gaatgtgagg tgtctcgcgc 420 tgtcttttta aagatggttc gagtcccttt aaatgtacgt tgattctttc ttgtgtctgc 480 gaattgcgat gctatgctct ttgtgatcgg tttagattgc tttgcgctgg tgggcgactt 540 cggttaggac atatggaagc aacctcaatt ggcggtatgt tcggctttgc ctgacgttaa 600 gctaagcgag tgtagttttc tgtcttttcc ttgaggtgta cctgtcgtgt gtgaggttga 660 tttaggctat atggttgctt ggttgtgtgg tttagcgttt tcagacgcct gcttcggtag 720 gtaaaggaga caacaccaat ttgggactga gagtttact 759 <210> <211> <212> <213 > <221 > <400>

atggaaggta ttattaactt tcatcatgat ttagtatttt ttttaattat tgtgactgtt 60 tttgtttgtt ggttattatt tagagtaatc gtattattcg atgaaaaaaa aaacccaata 120 cctgctacat ttgtacatgg agcaactatt gaaattattt ggacaacaat tccagcatta 180 attttattaa ccgtagcagt tccatctttt gctttattat attcaatgga tgaaattatt 240 gatccaatta taactttaaa agtaataggt agtcaatggt actggagtta tgaatattct 300 gataatttag aatttgcaga tgaaccttta atttttgata gttacatggt tcaagataat 360 gacttagaaa taggacaatt taggttatta gaagtagaca accgtgttgt tgtaccaact 420 aatagccata ttagagtttt aataacagct tctgacgttt tacattcatg ggctataccc 480 tctttaggtt taaaattaga tgcttgtcca ggtcgtttaa atcaaacttc aatgtttatt 540 aaaagagaag gtgtatttta cggtcaatgt agtgaaatat gtggtataaa tcatggtttt 600 atgccaatag ttgttgaagc agtttcatta gaagattatt tagtttggtt aaaaaacaaa 660 ttaattttga ttttaatgta taa 683

EXAMPLE 1 Liquid biological antifungal for spraying crops with a high percentage by weight of biomass.

Cooking process.

The biomass obtained by cultivating the microorganism Pythium oligandrum M1 on a solid-phase substrate was mixed in a homogenizer tank (industrial mixer) with demineralized water and homogenized. The homogenization process was carried out at a speed of 3000–5000 rpm for 3 min. Next, osmolite (sucrose) was added to the initial undiluted suspension in an amount corresponding to the final concentration by weight of 60 wt.%, and the required number of oospores. Next, the suspension with osmolite (sucrose) was homogenized in a mixer for 1 min at a speed of 2000 rpm. Further, the suspension was stored in sterile stainless steel containers at a temperature below 8°C.

It was more preferable to prepare a liquid biological antifungal preparation by combining the steps of homogenization in demineralized water and then adding osmolyte (sucrose) in one step so that the biomass obtained by culturing on a solid substrate is homogenized in a tank of an industrial homogenizer in an osmolyte solution (65% solution sucrose). The homogenization process was carried out at a speed of 3000–5000 rpm for 3 min. After determining the amount of oospores in the initial suspension, the specified suspension was diluted with osmolyte (65% sucrose solution) in the usual way so that the required concentration of oospores was achieved in the preparation. Further, the suspension was stored in sterile stainless steel containers at a temperature below 8°C.

The number of dormant oospores was determined microscopically using a Sedgewick-Rafter counting chamber, and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

The composition of the drug.

Sucrose as stabilizer 60% by weight.

Water as stabilizer 30% by weight.

Cultivated biomass 10 wt.%.

The number of dormant oospores of the microorganism Pythium oligandrum 5x105 per 1 ml.

Properties.

Stability: 6 months at temperatures below 25°C.

Example 2 Liquid biological antifungal preparation for spraying crops with a low percentage by weight of biomass.

Cooking process.

The biomass of Pythium oligandrum, obtained by cultivation in the liquid phase, was mixed in the tank of a homogenizer (industrial mixer) with osmolyte (sucrose) and homogenized in such a way that the required number of oospores and the concentration of osmolyte (sucrose) were achieved. The homogenization process was carried out at a speed of 3000–5000 rpm for 3 min. Further, the suspension was stored in sterile stainless steel containers at a temperature below 8°C. The number of dormant oospores was determined using a microscope using a SedgewickRafter counting chamber, and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

Composition.

Sucrose as stabilizer 64% by weight.

Water as stabilizer 35.9% by weight.

Cultivated biomass 0.1 wt%.

The number of dormant oospores of the microorganism Pythium oligandrum M1 5x105 per 1 ml.

Properties.

Stability: 6 months at temperatures below 25°C.

Example 3. Liquid biological antifungal preparation intended for dressing of grain by the method of immersion / seed coating.

Receiving process.

The biomass obtained by cultivating the microorganism Pythium oligandrum M1 on a solid-phase substrate was mixed in a homogenizer tank (industrial mixer) with demineralized water and homogenized. The homogenization process was carried out at a speed of 3000–5000 rpm for 3 min. Next, osmolyte (sucrose) was added to the initial undiluted suspension in an amount corresponding to the final concentration by weight of 60 wt.%, and the required number of oospores. The suspension with osmolite (sucrose) was further homogenized in a mixer for 1 min.

It was more preferable to prepare the preparation by combining the steps of homogenization in demineralized water and the subsequent addition of osmolyte (sucrose) in one step so that the biomass obtained by cultivation on a solid substrate is homogenized in a tank of an industrial homogenizer in an osmolyte solution (65% sucrose solution). The homogenization process was carried out at a speed of 3000–5000 rpm for 3 min. After determining the amount of oospores in the initial suspension, the specified suspension was diluted with osmolyte (65% sucrose solution) in the usual way so that the required concentration of oospores was achieved in the preparation. Further, the resulting suspension was stored in sterile stainless steel containers at a temperature below 8°C.

The number of dormant oospores was determined using a microscope using a Sedgewick-Rafter counting chamber, and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

Stability: 6 months at temperatures below 25°C.

Composition.

Sucrose as stabilizer 60% by weight.

Water as stabilizer 36% by weight.

Cultivated biomass 4 wt%.

The number of dormant oospores of the microorganism Pythium oligandrum M1 is 2.5x106 per 1 ml.

Example 4 Liquid biological antifungal preparation in the form of suspension concentrate with low water content.

Receiving process.

Biomass of Pythium oligandrum M1 obtained by liquid phase culture was centrifuged after harvest. The supernatant was removed after centrifugation, and paraffin oil was added to the centrifuged material so that the final number of oospores was 500,000 oospores per 1 ml of preparation. The mixture was further homogenized in a commercial mixer for 3 minutes. The suspension was stored in sterile containers at temperatures below 8°C. The number of dormant oospores was determined using a microscope using a Sedgewick-Rafter counting chamber, and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

Composition.

Cultivated biomass 0.1 wt%.

Paraffin oil as stabilizer 99.9% by weight.

The number of dormant oospores of the microorganism Pythium oligandrum M1 5x105 per 1 ml. Properties.

Stability: 6 months at temperatures below 25°C.

Example 5. Liquid biological antifungal drug used in the form of nanofibers forming a biodegradable matrix of prolonged action for the microorganism Pythium oligandrum M1.

Receiving process.

Biomass of Pythium oligandrum obtained by liquid phase cultivation was centrifuged after harvest. The supernatant was removed and osmolyte (65% sucrose) and biopolymer were added to the centrifuged material, then the material was processed using electrostatic rotation technology using non-toxic biodegradable polymeric polyvinyl alcohol (PVA). The number of dormant oospores was determined using a microscope using a Sedgewick-Rafter counting chamber, and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

Composition.

1. Main ingredient (liquid suspension of the microorganism Pythium oligandrum) 84% by weight;

containing:

sucrose as a stabilizer 63 wt.%, water as a stabilizer 36 wt.%, cultivated biomass 1 wt.%.

2. PVA carrier.

PVA as degradable carrier 16% by weight.

The number of dormant oospores of the microorganism Pythium oligandrum M1 5x106 per 1 ml. Properties.

Stability: 6 months at temperatures below 25°C.

Example 6 Liquid biological antifungal agent used in the form of an impregnating coating or spray.

Receiving process.

Biomass of Pythium oligandrum M1 obtained by liquid phase cultivation was homogenized after collection and filtered using filtration methods to separate particles up to 300 µm. After centrifugation, the obtained biomass was mixed with low-viscosity mineral oil in such a way that the required amount of oospores per 1 ml of the preparation was achieved. The number of dormant oospores was determined using a microscope using a Sedgewick-Rafter counting chamber, and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

Composition.

Cultivated biomass 0.1 wt%.

Mineral oil as stabilizer 99.9% by weight.

The number of dormant oospores of the microorganism Pythium oligandrum M1 2x105 per 1 ml.

Properties.

Stability: 6 months at 10-45°C (depending on the type of secondary substance).

Example 7 Liquid Biological Antifungal Used as an Air Conditioning Sanitary Product.

Receiving process.

The biomass of Pythium oligandrum M1 obtained by cultivation in the liquid phase was homogenized

- 10 042498 after collection and filtered using filtration methods to separate particles up to 100 µm in size. After centrifugation, the biomass was diluted with saline to the required amount of oospores per 1 ml of the preparation. The principle of application is rinsing or spraying filters or pipelines of air conditioning or cooling installations. The presence of the microorganism Pythium oligandrum M1 guarantees resistance to mold or fungal diseases. Cleaning anti-allergy filters:

The filter was immersed in a sanitary-hygienic solution and kept in it for 10-30 minutes and then rinsed. Alternatively, the same solution is sprayed as an aerosol onto the filter in its operating position. Dirt and agent residues are blown off with compressed air.

Sanitary and preventive treatment of aerosol devices and filters.

After cleaning the aerosol apparatus and / or filters, the drug solution is used without rinsing. An excellent sanitary and preventive effect is achieved even when the drug is used only as a flushing and disinfecting agent. The number of dormant oospores was determined using a microscope using a Sedgewick-Rafter counting chamber, and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

Composition.

Cultivated biomass 0.1 wt%.

Salt solution as a stabilizer 99.9 wt.%.

The number of dormant oospores of the microorganism Pythium oligandrum M1 5x105 per 1 ml.

Properties.

Stability: 6 months at 10-45°C (depending on the type of secondary substance).

Example 8 Liquid Biological Antifungal Used as Sanitary Product for Sludge Treatment - Silt Sites and Flooded Areas

Receiving process.

Biomass of Pythium oligandrum M1, obtained by cultivation in the liquid phase, was homogenized after collection and filtered using filtration methods to separate particles up to 400 μm. After centrifugation, the biomass was diluted with distilled or demineralized water to the required amount of oospores per 1 ml of preparation. The principle of operation is to create an aqueous suspension in accordance with the dilution rate indicated below, and to apply the suspension in areas affected by polluted silt and sediment deposits, as a consolidating and healing procedure in order to improve the properties of soils and soils. The number of dormant oospores was determined using a microscope using a Sedgewick-Rafter counting chamber, and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

Composition.

Cultivated biomass 0.1 wt%.

Distilled water as stabilizer 99.9% by weight.

The number of dormant oospores of the microorganism Pythium oligandrum M1 5x105 per 1 ml.

The amount of the agent used is from 250 g per 1 ha at a concentration of 500,000 oospores per 1 ml.

Properties.

Stability: 6 months at temperatures below 25°C.

Example 9. Liquid biological antifungal preparation in the form of a stabilized aqueous suspension of Pythium oligandrum M1 oospores.

Receiving process.

Biomass of Pythium oligandrum M1, obtained by cultivation in the liquid phase, was mixed with sterile distilled water and homogenized after centrifugation (4000 rpm, for 5 min) and removal of the supernatant. Homogenization was continued at high rotation speed (20,000 rpm) for 1 minute. Further, if necessary, the suspension can be filtered and concentrated in order to obtain a suspension concentrate containing the required amount of Pythium oligandrum M1 oospores per 1 ml, without the presence of residual original medium or residual mycelium. The resulting suspension material can be stored in sterile containers at a temperature ranging from 1 to 8°C. The number of dormant oospores was determined microscopically using a Sedgewick-Rafter counting chamber, and stability was measured by microscopically examining the number of viable oospores using the plasmolytic method, according to the published

- 11 042498 by Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

Composition.

Sterile distilled water as stabilizer 99.9% by weight.

Cultivated biomass 0.1 wt%.

The number of dormant oospores of the microorganism Pythium oligandrum M1 5x105 per 1 ml.

Properties.

Stability: at least 6 months at temperatures below 25°C.

Example 10 Liquid biological antifungal preparation in the form of a stabilized aqueous suspension with a high concentration of Pythium oligandrum M1 oospores.

Receiving process.

The biomass of Pythium oligandrum M1, obtained by cultivation in the liquid phase, was homogenized in the tank of a homogenizer (industrial mixer). The homogenization process was carried out at a speed of 20,000 rpm for 3 min. The suspension was then centrifuged and after removing the supernatant, the biomass was concentrated to the required number of oospores in 1 ml of the preparation by stirring with osmolyte (65% sucrose). The suspension concentrate was stored in sterile stainless steel containers below 8°C. The number of dormant oospores was determined using a microscope using a Sedgewick-Rafter counting chamber, and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

Composition.

Sucrose as stabilizer 60% by weight.

Water as stabilizer 35% by weight.

Cultivated biomass 5 wt%.

The number of dormant oospores of the microorganism Pythium oligandrum M1 2x10 ⁷ per 1 ml.

Properties.

Stability: 6 months at temperatures below 25°C.

Example 11. Biological antifungal preparation in the form of a paste based on glycerol.

Receiving process.

1197.1 g of pharmaceutical grade glycerol (glycerol 99% PharmEur) was poured into a laboratory homogenizer and 239.4 g of Sident® 22 S silica, 43.1 g of cultivated biomass, 14.4 g of vitamin E and 6 g of sage flavor were carefully sprinkled. The mixture was stirred at 60 rpm for 10 minutes at laboratory temperature. The mixture was then placed in plastic tubes with stoppers of 75 g each, so that a total of 20 such tubes were filled. The number of dormant oospores was determined using a microscope using a Sedgewick-Rafter counting chamber, and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

Composition.

Glycerin 99% Pharm Eur as stabilizer 79.77 wt%.

SiO ₂ Sident® 22 S as silica filler 16.00% by weight.

Cultivated biomass of Pythium oligandrum M1 2.87% by weight.

Acetate (vitamin E) as auxiliary substance 0.96% by weight.

FCO2 Bio 35% sage flavor 0.40 wt%.

The number of dormant oospores of the microorganism Pythium oligandrum M1 2.5x10 ⁴ per 1 ml.

Properties.

Stability: minimum 6 months at 25°C.

Example 12 Biological antifungal preparation in the form of an olive oil paste. Receiving process.

600 g of extra virgin olive oil were poured into a laboratory homogenizer and 120 g of Sident® 22 S silica, 21.6 g of cultivated biomass, 7.2 g of vitamin E and 3 g of mint flavor were carefully sprinkled. The mixture was stirred at 20 rpm for 10 minutes at laboratory temperature. The mixture was then placed in plastic tubes with stoppers, 75 g each, so that a total of 10 such tubes were filled. The number of dormant oospores was determined using a microscope using a Sedgewick-Rafter counting chamber, and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

- 12 042498

Composition.

Refined olive oil as a stabilizer 79.77% by weight.

SiO ₂ Sident® 22 S as silica filler 16.00% by weight.

Cultivated biomass of Pythium oligandrum M1 2.87% by weight.

Acetate (vitamin E) as auxiliary substance 0.96% by weight.

HNA peppermint essential oil as flavor 0.40 wt%.

The number of dormant oospores of the microorganism Pythium oligandrum M1 is 2.5x104 per 1 ml.

Properties.

Stability: 6 months at 25°C.

Example 13 Liquid biological antifungal for the protection of aquatic organisms.

The drug is intended for the preventive protection of fish and young fish (carp, roach, bream) from a number of fungal diseases (in particular, Aspergillus spp.). The drug is used prophylactically in spawning aquariums. The drug is also intended for preventive protection of juvenile fish from the effects of a number of fungal diseases (in particular, Fusarium spp., Aspergillus spp.).

Receiving process.

The biomass of Pythium oligandrum M1, obtained by cultivation in the liquid phase, was homogenized in the tank of a homogenizer (industrial mixer). The homogenization process was carried out at a speed of 20,000 rpm for 3 min. The suspension was then centrifuged and after removing the supernatant, the biomass was concentrated to the required number of oospores in 1 ml of the preparation by stirring with osmolyte (65% sucrose). The suspension was stored in sterile stainless steel containers below 8°C. The number of dormant oospores was determined using a microscope using a Sedgewick-Rafter counting chamber, and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

Composition.

Sucrose as stabilizer 63% by weight.

Water as stabilizer 36% by weight.

Cultivated biomass 1 wt%.

The number of dormant oospores of the microorganism Pythium oligandrum M1 5x106 per 1 ml.

Properties.

Stability: 6 months at temperatures below 25°C.

Example 14. Liquid biological antifungal preparation for the treatment of honeycombs.

The drug is intended for the preventive treatment of honeycombs in order to prevent exposure to a wide range of fungi, in particular the Aspergillus and Fusarium families. The product can be used to treat combs intended for storage outside bee hives and combs inside bee hives not occupied by bees.

Receiving process.

Biomass of Pythium oligandrum M1, obtained by cultivation in the liquid phase, was mixed in the capacity of a homogenizer (industrial mixer) with osmolyte (sucrose) and homogenized in such a way that the required number of oospores and the concentration of osmolyte (sucrose) were achieved. The homogenization process was carried out at a speed of 20,000 rpm for 3 min. Further, the suspension was stored in sterile stainless steel containers at a temperature below 8°C. The number of dormant oospores was determined microscopically using a SedgewickRafter counting chamber and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

Composition.

Sucrose as stabilizer 64% by weight.

Water as stabilizer 35.9% by weight.

Cultivated biomass 0.1 wt%.

The number of dormant oospores of the microorganism Pythium oligandrum M1 1x106 per 1 ml.

Properties.

Stability: 6 months at temperatures below 25°C.

Example 15. Liquid biological antifungal preparation in the form of an anhydrite suspension concentrate intended for spraying crops.

The biomass obtained by cultivating the microorganism Pythium oligandrum M1 on a solid-phase substrate was mixed in a homogenizer tank (industrial mixer) with demineralized water and homogenized. The homogenization process was carried out at a speed of 3000–5000 rpm for 3 min. After completion of the homogenization process, the resulting suspension was stirred with

- 13 042498 inorganic carrier (Sipernat), dried and crushed in a centrifugal mill to obtain a final particle size of not more than 350 microns. Next, the dry cultured biomass with the carrier was mixed with paraffin oil in order to achieve the required concentration of oospores in the preparation.

The suspension was stored in sterile containers at a temperature not exceeding 25°C.

The advantage of this preparation in comparison with example 1 is the longer stability of the preparation due to the presence of water. The silica-based inorganic carrier provides good suspension of the solid particles (biomass) in the preparation.

The number of dormant oospores was determined by microscope using a Sedgewick-Rafter counting chamber, and stability was determined by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011

Composition.

Cultivated biomass 1.5 wt.%.

Sipernat 22 as inorganic carrier 6.5% by weight.

Paraffin oil as stabilizer 92% by weight.

The number of dormant oospores of the microorganism Pythium oligandrum M1 1x106 per 1 ml.

Properties.

Stability: 24 months at temperatures not exceeding 25°C.

Example 16. Liquid biological antifungal preparation in the form of an anhydrite suspension concentrate, intended for spraying crops in the production of environmentally friendly agricultural products.

The biomass obtained by cultivating the microorganism Pythium oligandrum M1 on a solid-phase substrate was mixed in a homogenizer tank (industrial mixer) with demineralized water and homogenized. The homogenization process was carried out at a speed of 3000–5000 rpm for 3 min. After completion of the homogenization process, the resulting suspension was mixed with an inorganic carrier (Sipernat), dried and ground in a centrifugal mill to obtain a final particle size of not more than 350 μm. Next, the dry cultivated biomass with the carrier was mixed with sunflower oil in order to achieve the required concentration of oospores in the preparation. The suspension was stored in sterile containers at a temperature not exceeding 25°C.

The advantage of this preparation in comparison with example 1 is the longer stability of the preparation due to the presence of water. The silica-based inorganic carrier provides good suspension of the solid particles (biomass) in the preparation.

The number of dormant oospores was determined by microscope using a Sedgewick-Rafter counting chamber, and stability was determined by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011

Composition.

Cultivated biomass 1.5 wt.%.

Sipernat 22 as inorganic carrier 6.5% by weight.

Sunflower oil as stabilizer 92% by weight.

The number of dormant oospores of the microorganism Pythium oligandrum M1 1x106 per 1 ml.

Properties.

Stability: 24 months at temperatures not exceeding 25°C.

Example 17 Liquid biological antifungal preparation with reduced concentration of oospores.

Biomass of Pythium oligandrum M1, obtained by cultivation in the liquid phase, was mixed in the capacity of a homogenizer (industrial mixer) with osmolyte (sucrose) and homogenized in such a way that the required number of oospores and the concentration of osmolyte (sucrose) were achieved. The homogenization process was carried out at a speed of 20,000 rpm for 3 min. The suspension was further mixed with osmolyte (65% sucrose) so that the concentration of oospores in the preparation met the requirements for the preparation. Further, the suspension was stored in sterile stainless steel containers at a temperature below 8°C.

The number of dormant oospores was determined using a microscope using a Sedgewick-Rafter counting chamber, and stability was measured by microscopic examination of the number of viable oospores using the plasmolytic method, according to the published work of Etxeberria et al. [13], 2011. If necessary, the number of dormant oospores was stabilized in the usual way.

Composition.

Sucrose as stabilizer 64.95% by weight.

Water as stabilizer 35% by weight.

Cultivated biomass 0.05 wt%.

- 14 042498

The number of dormant oospores of the microorganism Pythium oligandrum Ml 1x10 ³ per 1 ml.

Properties.

Stability: 12 months at temperatures between 2 and 8°C; 6 months at temperatures below 25°C.

Example 18 Laboratory Test for the Efficacy of Liquid Biological Antifungals in Mycoparasitic Experiments.

Laboratory testing of Pythium oligandrum M1 proliferation from dormant oospores of the suspension concentrate according to illustrative embodiment 1 and subsequent mycoparasitism on the phytopathogenic fungus Fusarium graminearum were carried out on sterile malt extract live medium agar plates. The diluted concentrate was applied to an agar plate at the level of use, namely 0.2 ml of concentrate per 1 liter of water, and part of this plate was removed after germination and transferred to another agar, which was simultaneously inoculated with the fungus Fusarium graminearum.

The result of this test is shown in Fig. 1, which indicates an abrupt cessation of growth of the phytopathogenic fungi Fusarium graminearum after the addition of the microorganism Pythium oligandrum M1 when compared to normal growth rates (FIG. 1A). This situation is further illustrated using a row of Petri dishes photographed 4 days later, showing the combined growth of both microorganisms on the one hand and the growth of phytopathogenic fungi on the other hand (Fig. 1B). After 11 days of cultivation, there is a clear significant suppression of phytopathogenic fungi with simultaneous growth of the microorganism Pythium oligandrum M1. If the number of viable oospores did not decrease below 90% of the initial value, the drug was labeled as stable.

Example 19. The effectiveness of antifungal agents against phytopathogenic fungi, demonstrated in the course of laboratory experiments with wheat.

Cereal grains (wheat) were treated with a solution of Sava - the commercial name of a solution of sodium chloride (1-5% solution) and germinated on an extract of agar soil. After germination, the sprouts were again treated with 1% Sava solution and placed on a layer of phytoagar in a 250 ml conical Erlenmeyer flask. The flasks were inoculated with a piece of agar (5 mm) with the phytopathogen Fusarium graminearum or with the microorganism Pythium oligandrum M1 (dormant oospores in suspension concentrate) at an applicable concentration of 0.2 ml per 1 l and a volume of 0.2 ml in the following versions: 1) phytopathogen; 2) phytopathogen + Pythium oligandrum M1; 3) only one Pythium oligandrum M1; a control sample that was not inoculated. The flasks were covered with a thin layer of cellulose and gauze. Cultivation was carried out indoors at a temperature of 24°C. The growth of a colony of phytopathogens and the microorganism Pythium oligandrum M1 was monitored, as well as the state of the plant.

The results of this laboratory experiment are shown in FIG. 2. With regard to the addition of the actual phytopathogen, its significant growth in the environment of growing wheat is certainly evident, as well as plant damage caused by the pathogen (Fig. 2A). When the microorganism Pythium oligandrum M1 was added in addition to the phytopathogenic fungus, the presence of the phytopathogen was not observed, and the cultivated plants grew efficiently (Fig. 2B). A control experiment performed in the presence of only one microorganism, Pythium oligandrum M1, demonstrated normal growth of wheat, comparable to growth in experiments without the addition of any microorganism (Fig. 2C).

EXAMPLE 20 Efficacy test of a liquid biological antifungal agent for crop spraying according to Illustrative Embodiment 2 in a grow house test on Brassica napus rapeseed seeds.

Four sectors were used in the company's growing house in ύherce, in which rape Brassica napus, variety Lohana C1 was grown, and the drug was started in the growth and development phase of crops BBCh 15-21. In sector 1, a powdered biofungicide containing the mycoparasitic microorganism Pythium oligandrum M1 (Polyversum®) was applied, in sector 2, a new liquid fungicide was applied in accordance with illustrative embodiment 1 of the present invention, in sector 3, a new liquid antifungal preparation was applied in accordance with illustrative embodiment 2 of the present invention, and sector 4 was applied a new liquid antifungal drug in accordance with illustrative embodiment 4 of the present invention. When applied, the concentration of the drug in all cases was 0.5 g per 1 liter, or 0.5 ml per 1 liter, while such a concentrated drug was sprayed per 1 m ² of the treated area in an amount of 100 ml. In each sector, a sampling point was marked, covering the plant and soil between the rows. During each sampling, photographic documentation and recording of the temperature in the growing house was carried out. The selected samples were transferred to the laboratory within one hour and stored in a freezer at -20°C until analysis. Samples were taken before antifungal treatment and then after 1 h, 2 h, 7 h, 20 h, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 11 days, 14 days, 17 days and 20 days after application.

- 15 042498

Sample laboratory processing included photographic documentation of individual plants, leaf and root separation, sample wet weight determination, sample homogenization in DNA isolation buffer, centrifugation, and molecular analysis. Quantitative determination of the presence of the microorganism Pythium oligandrum M1 was carried out using a quantitative PCR method with a probe directed to the sequence of the internal transcribed spacer (ITS), while DNA quantification of rapeseed was performed using a similar method with a probe directed to the Fat(A)A sequence . Quantification involved performing 50 cycles of DNA amplification under standard conditions and calculating the concentration of target sequences using a calibration curve. The level of mycosis was determined in a similar way based on a standard protocol.

The results of the test for efficiency are given in table. 1 below. The efficacy achieved with the novel liquid antifungal formulations of the present invention is in all cases comparable to or better than the solid powder formulation. In all cases, foreign fungi were suppressed depending on the colonization and amplification of the microorganism Pythium oligandrum M1 at the site of application of the drug. The low level of fungal contamination was further maintained even after a decrease in the concentration of the microorganism Pythium oligandrum M1 at the end of the experiment. In this experiment, control over the dynamics of the population of the microorganism Pythium oligandrum M1 was carried out only for 15 days, which resulted in the absence of any comments about the long-term effect. However, from the point of view of the antifungal efficacy of the drug, there is no doubt that the ability to suppress fungi is significant and long-term: there was a decrease in the occurrence of such fungi by about one order of magnitude after 24 hours after the use of the drug and a further decrease by almost one order of magnitude after 24 more hours

Table 1

Determination of the content of the microorganism Pythium oligandrum M1 and determination of the content of the fungus on rapeseed leaves during the experiment in the growing house. The results are given as numerical values of the detected diploid genomes per 1 g of host rapeseed DNA. Collection time (h) Sector 1 Sector 2 Sector 3 Sector 4 Python Fungi Python Fungi Python Fungi Python Fungi 0 8x 10 ² 1 x ^10th 7x10 ² 1 x ^10th 8x 10 ² 2x ^10th 6x 10 ² 2x ^10th 2 2x10 ⁴ 8x ^10th 1 x 10 ⁴ 1 x ^10th 1 x 10 ⁴ 1 x ^10th 2x10 ⁴ 1 x ^10th 20 2x ^10th 1 x ^10th 2x ^10th 2x ^10th 2x ^10th Zx ^10th 2x ^10th 2x ^10th 26 5x ^10th 6x 10 ^{4 6x10th} 6x 10 ⁴ 7x10 ⁴ 5x10 ⁴ 6x ^10th 5x 10 ⁴ 44 1 x ^10th 2x 10 ⁴ 2x ^10th 2x 10 ⁴ 1 x ^10th 2x 10 ⁴ 2x ^10th 1 x 10 ⁴ 92 8x ^10th 1 x ^10th 8x ^10th 2x ^10th 7x ^10th 1 x ^10th 7x ^10th Zx ^10th 140 1 x 10 ⁴ 5x10 ² ZxYu ⁴ 6x 10 ² 4x10 ⁴ 6x 10 ² 4x10 ⁴ 5x 10 ² 212 1 x 10 ⁴ 5x 10 ² 2x10 ⁴ 5x 10 ² 2x 10 ⁴ 7x 10 ² 2x10 ⁴ 6x 10 ² 356 8x ^10th 6x 10 ² 1 x 10 ⁴ 4x 10 ² 1 x 10 ⁴ 5x 10 ² 1 x 10 ⁴ 4x 10 ²

Example 21 Efficacy test of a liquid biological crop spray antifungal according to Illustrative Embodiment 1 in a field trial on Triticum aestivum wheat.

Winter wheat Triticum aestivum was sown in October 2015 in a field owned by the company near the village of ύherce u Loun. The antifungal agent according to illustrative embodiment 1 of the present invention was applied to designated furrows in November 2015 using a standard spraying method at a rate of 100 ml per 1 ha in accordance with agricultural standards and with the accepted registration of the antifungal drug for plant protection Polyversum®. Samples were taken from 4 designated sampling points before the application of the drug and after a different number of days after the application of the drug, while samples of germinating wheat germ and soil were taken in the immediate vicinity of the sample. Samples were frozen immediately after collection and stored in sealed bags or test tubes at -20°C until analysis. Nucleic acids were extracted from the selected samples using the published method (Klimes R., Suchanek M., Mastalkova L. et al. /2016/ Comparison of the efficacy of treatment of dermatophytosis by chemical and biological antifungals: soil peronosporomycete Pythium oligandrum is as efficient as the antifungal enilconazole in the guinea pig model, Vet Dermatol, in print) and used for molecular analysis.

With regard to the dynamics of the emergence of the microorganism Pythium oligandrum M1 after spraying using a liquid preparation in accordance with illustrative embodiment 1 of the present invention, standardized concentrations on plants and the surrounding soil showed a similar relationship. Since only a few cells per gram of sample could be identified by the quantitative PCR genetic test before the drug was applied, this number increased to hundreds of cells per gram of the sample after the liquid drug was applied, which corresponds to the standard normalized number. Visible multiplication of the microorganism Pythium oligandrum M1 was observed under experimental conditions at the site later

- 16 042498 h, while this positive trend was interrupted in a special experiment with the onset of frost in early December 2015, after which the dynamic population growth was not restored (compare Figs. 3A and 3B).

Useful information was obtained from the analysis of gene expression profiles using the method of reverse transcription-quantitative PCR (RT-qPCR) during the peak phase of the experiment with the highest growth of the microorganism Pythium oligandrum M1. Transcript concentration values showed a rapid response to emerging pathogenic fungi in the first phase (essential gene expression for cellulose), followed by increased zoospore production (endoglucanase expression). Only after 48 and 120 h does a characteristic marker gene for increased sporulation appear, primarily in the surrounding soil and roots (Fig. 3C). At the same time, it was possible to observe a significant decrease in the relative content of the fungus 48 hours after the application of the drug (Fig. 3D), and this decrease was mutually inversely consistent with the gene expression for cellulose, which causes the breakdown of surface saccharides in fungi during the mycoparasitic action of the microorganism Pythium oligandrum M1 (Fig. 3E).

Example 22 Efficacy testing of toothpastes using the oral biofilm model with mechanical plaque removal.

Laboratory tests were carried out using the oral biofilm model with mechanical plaque removal in accordance with the published work of Verkaik M.J. et al. [14] 2010 to monitor the effectiveness of prepared toothpastes. Version (B) of the above published protocol was used, consisting of a 2 hour adhesion followed by overnight growth (16 hour biofilm) and mechanical cleaning. The effectiveness of glycerin toothpaste containing Pythium oligandrum M1 and olive oil and Pythium oligandrum M1 toothpastes in removing plaque microorganisms was compared with two types of the most common toothpastes (Odol and Enzycal), and mechanical cleaning with a toothbrush was used as a control. without any toothpaste (labeled Control). A similar toothpaste free of Pythium oligandrum and silica abrasive was used in the control experiment.

The results of this test are shown in the table in FIG. 2. Based on the results of the test, it was found that the toothpaste according to illustrative embodiment 4 had a significantly higher efficacy in removing dental biofilm and plaque compared to the alternative preparation used, even among individuals affected by periodontal tissue disease (Fig. 4A-4D ). While toothpastes commonly used to date have provided almost a halving of biofilm formation, the novel toothpastes of the present invention have achieved a reduction of up to one tenth. The effect of the Pythium oligandrum M1-free control dentifrice was comparable to that of conventional toothpastes, while the effect was significantly greater with the silica-free control dentifrice. The overall level of biofilm formation in a patient suffering from periodontal tissue disease was significantly higher, and there was also a significant difference in the effectiveness of individual toothpastes.

Example 23 Efficacy test of a liquid biological antifungal agent according to Illustrative Embodiment 1 in protecting residential areas from fungal spores.

The antifungal drug according to illustrative embodiment 1 was diluted with water to a concentration of 330,000 oospores per 1 liter of applied solution, i.e. 0.66 ml of liquid concentrate was added to 1 liter of solution. A control solution free of Pythium oligandrum M1 was also prepared under similar conditions; biomass was replaced with water. Thus, the prepared solutions were used in two small closed rooms at the farm ύherce u Loun No. 2 by spraying them in the form of a fine aerosol using a spray device. After spraying, both rooms were tightly closed and a dropout test was performed to determine the content of spores according to the Omeljansky method. The results were evaluated in the laboratory of Dr. Miroslav Kolarik at the Institute of Microbiology of the Academy of Sciences of the Czech Republic in Prague.

The results of the experiment are presented below in table. 2. The experiment was carried out in closed rooms without any air flow, for this reason the observed changes occurred at a slower pace, and their manifestation was observed only after a few months. However, it clearly follows from the results that in control room 1 with the use of auxiliary substances, the number of spores fluctuated, but at any time of observation did not fall below the limit set by WHO (World Health Organization) in the amount of 500 spores per 1 m ³ of air. On the other hand, there was a statistically significant reduction in the number of spores 3 months after application of the solution in room 2, and then there was a further decrease in the number of spores below the WHO limit 6 months after the date of application of the solution.

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table 2

Measurement of the number of fungal spores per 1 cubic meter of the room using the control preparation and in room 2 using the preparation containing the active ingredient Pythium oligandrum M1 Months after application (0 before application) 0 1 3 6 Room 1, control 5000 4500 3500 4500 Room 2 (drug according to illustrative embodiment 1) 5000 4000 1300 400

Example 24 Method for preparing a liquid biological antifungal preparation comprising Pythium oligandrum M1.

The nature of the liquid biological antifungal preparation, including Pythium oligandrum M1, in accordance with the present invention has been demonstrated in detail in the previous illustrative embodiments of the present invention.

Examples of the method for producing a liquid biological antifungal preparation, including Pythium oligandrum M1, in accordance with the present invention are presented below.

A medium including cereal extract, cane sugar molasses and other essential nutrients was used for aerobic cultivation of Pythium oligandrum M1 oomycetes. The liquid medium was sterilized in a steam sterilizer. After cooling, the medium was inoculated with one of the selected strains of Pythium oligandrum M1. After completion of cultivation (the cultivation process takes approximately 13 days), the biomass was collected and processed using the technology presented above in the exemplary embodiments of the present invention.

A medium was prepared for the cultivation of the microorganism Pythium oligandrum M1 on a solid phase substrate containing cereal grains, preferably hulled millet grains, for example, Panicum miliaceum L, which were mixed with a certain percentage of live liquid medium. The medium was sterilized in a culture apparatus in a steam sterilizer. After cooling, the medium was inoculated with one of the selected strains of Pythium oligandrum M1. After completion of the cultivation process (the cultivation process takes approximately 8 days), the collected biomass was further processed into a suspension concentrate, as described above in the illustrative embodiments of the present invention.

After completion of the stage of culturing Pythium oligandrum in liquid phase and in solid phase, the biomass is homogenized with a liquid medium, for example, in an industrial mixer, so that the size of at least 95% of the particles in suspension is in the range from 0.05 to 0.30 mm, preferably in the range from 0.050 to 0.125 mm. Thus, a homogeneous suspension was characterized by a certain amount of oospores. The suspension was normalized - concentrated or diluted - depending on the number of oospores to a predetermined concentration of oospores suitable for obtaining a liquid biological antifungal drug under consideration.

After completion of the phase of cultivation of the microorganism Pythium oligandrum M1 on a solid phase substrate, the biomass is homogenized, preferably in an industrial mixer in an appropriate volume of demineralized water, so that the final size of at least 95% of the particles in suspension is in the range from 0.050 to 0.300 mm, preferably in the range from 0.050 to 0.125 mm. Thus, a homogeneous suspension was characterized by a certain amount of oospores. The resulting suspension was concentrated or diluted, depending on the number of oospores, to a predetermined concentration of oospores suitable for obtaining a liquid biological antifungal drug under consideration.

The suspension is separated, for example, filtered after the homogenization phase and concentration or dilution in order to ensure the maximum allowable particle size within the appropriate range. Upon reaching a particle size of approximately 0.05-0.125 mm, there is a high probability of obtaining a concentrated suspension or a substantially pure concentrated suspension of Pythium oligandrum M1 oospores. In most of the above illustrative embodiments of the present invention, the particle size was predominantly in the range of 0.0500.125 mm.

The aqueous suspension can be stabilized during the homogenization process with the addition of osmolyte and can be stored in sterile large volume containers at temperatures below 8°C.

With regard to the anhydride suspension of the microorganism Pythium oligandrum M1, the material obtained after completion of the stage of cultivation on the solid phase substrate or in the liquid phase was homogenized, preferably in an industrial mixer, so that the final size of at least 95% of the particles in the suspension was in the range from 0.050 to 0.300 mm, preferably in the range from 0.050 to 0.125 mm. The obtained homogeneous suspension, characterized by a certain number of oospores, is then centrifuged and after removal of the supernatant, oil is added to the centrifuged material to achieve a predetermined concentration of oospores, if necessary to obtain a liquid biological antifungal drug under consideration. The resulting material is then resuspended, for example, using an industrial mixer.

Specific possible methods of obtaining are indicated in the above illustrative examples of implementation of the present invention, however, they are not limited to these examples.

Industrial Applicability

The novel liquid antifungal formulation comprising the stabilized oospore suspension can be produced in large volumes mainly because both the enzymatic manufacturing step and the dosage formulation step are technically easily controlled. The new liquid preparation can be used in all areas in which other forms of microscopic Pythium oligandrum omycetes have found application and are still used, in particular, in the field of biological plant protection, protection of public and residential buildings from fungi, mold and yeast suppression and achievement of physiological balance of microflora in the field of clinical and veterinary medicine. The use of the product as a plant protection spray using a biodegradable polymer acting as a protective factor and as a carrier of a biodegradable matrix of the long-acting microorganism Pythium oligandrum is extremely innovative. The preparation according to the present invention can also be effectively used for applying or spraying a coating on wooden structures, while the oil components of the preparation also provide an effective impregnation of the wood. The presence of the microorganism provides resistance to mold and fungal diseases and protects the wood from damage. One completely new area of application is the effective conservation of air treatment equipment, air conditioning units and cooling units. It is also preferable to use a specially developed preparation to reduce the level of fungal contamination in areas subjected to flooding, drought, and in areas adjacent to reclaimed lands and areas of reclamation construction.

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Claims (11)

CLAIM 1. Biological liquid antifungal preparation containing the microorganism Pythium. oligandrum, characterized in that the preparation contains a stabilized suspension of the microorganism Pythium. oligandrum, with long-term stability at temperatures below 25°C from 6 to 24 months, while the size of at least 95% of the particles of the suspension is in the range from 0.050 to 0.300 mm and includes 0.05-10.0 wt.% of the cultivated biomass of the microorganism Python. oligandrum containing a culture medium, cellular forms of the specified microorganism and substances produced by the specified microorganism, and 90.0-99.95 wt.% of the stabilizer, which contains oil or at least one component selected from the group consisting of water, saline solution or osmolyte solutions, while Pythium. oligandrum. is a strain of Pythium. oligandrum. Dreschler ATTC38472, and the predetermined number of dormant oospores in 1 ml of said liquid biological antifungal after normalization ranges from 1x103 to 2x10 ⁷ . 2. Biological liquid antifungal preparation containing the microorganism Pythium oligandrum, characterized in that the preparation contains a stabilized suspension of the microorganism Pythium oligandrum with long-term stability at temperatures below 25°C from 6 to 24 months, while the size of at least 95% of the particles of the suspension is in range from 0.050 to 0.300 mm, and includes 0.05-10.0 wt.% of the cultivated biomass of the microorganism Pythium oligandrum containing the cultivation medium, cell forms of the specified microorganism and substances produced by the specified microorganism, 79.77-99.95 wt.% a stabilizer that contains an oil or at least one component selected from the group consisting of water, saline or osmolyte solutions, while the remainder contains up to 100 wt.% at least one total modifying/auxiliary substance selected from the group including filler, flavor and vitamin E, while Pythium. oligandrum is a strain of Pythium oligandrum Dreschler ATTC38472 and the predetermined number of dormant oospores in 1 ml of said liquid biological antifungal after normalization is in the range of 2.5x104 to 1.0x106. 3. Biological liquid antifungal preparation according to claims 1, 2, characterized in that the size of at least 95% of the particles of the suspension is preferably in the range from 0.050 to 0.125 mm. 4. An antifungal preparation according to claim 1 or 2, characterized in that the microorganism Pythium oligandrum Dreschler ATTC 38472 is a strain deposited in the Czech Collection of Microorganisms (CCM) at the Masaryk University in Brno under the name Pythium oligandrum M1. 5. An antifungal preparation according to one of claims 1 to 4, characterized in that the stabilized suspension of the microorganism Pythium oligandrum includes an aqueous stabilizer, the content of which is in the range from 30.0 to 99.9 wt.%. 6. Antifungal preparation according to claim 5, characterized in that the stabilizer is only distilled water or sterile distilled water, the content of which is 99.9 wt.%. 7. Antifungal preparation according to claim 5, characterized in that the stabilizer of the aqueous suspension is a saline solution, the content of which is 99.9 wt.%. 8. An antifungal preparation according to claim 5, characterized in that the aqueous suspension stabilizer contains water and osmolite, and in particular water in an amount of from 30 to 36 wt.% and osmolite in the form of sucrose, the content of which is in the range from 60, 0 to 64.95 wt%. 9. Antifungal drug according to one of claims 1-4, characterized in that the stabilizer is at least one of the oils selected from the group including paraffin oil, mineral oil, glycerin or sunflower oil in the amount of 79.77-99, 9 wt%. 10. An antifungal preparation according to claim 2 or 4, characterized in that the preparation contains 16.0 wt.% of a biodegradable matrix of prolonged action as a carrier of oospores of the microorganism Pythium oligandrum, such as polyvinyl alcohol. 11. A method for producing a liquid biological antifungal preparation containing the microorganism Pythium oligandrum, including a stabilized suspension of the microorganism Pythium oligandrum according to one of claims 1-10, characterized in that: a) provide a liquid medium comprising cereal extract, cane sugar molasses and other essential nutrients for aerobic cultivation of the microorganism Pythium oligandrum oomycetes in liquid phase; b) the liquid medium is sterilized; c) after cooling, the medium is inoculated with a strain of the microorganism Pythium oligandrum; d) the microorganism Pythium oligandrum is cultivated in said medium; e) the biomass is collected and processed after the completion of the cultivation process; f) further, after completion of the phase of cultivation of the microorganism Pythium oligandrum in the liquid phase, the biomass is homogenized with a liquid medium so that the final size of at least 95% of the particles in suspension is in the range from 0.050 to 0.300 mm; g) the obtained homogeneous suspension, characterized by a certain number of oospores, is then normalized depending on the number of oospores to a predetermined concentration of oospores for a liquid biological antifungal drug. -