RU2366178C1 - Method of obtaining preparation based on microfungi chlamydospores for vegetation and animal parasitic nematode control - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention concerns biotechnology, particularly methods of obtaining preparation based on chlamydospores of nematophagous microfungus for vegetation and animal parasitic nematode control. Method involves submerged cultivation of microfungus in nutritive medium including carbon and organic nitrogen sources, salts and water, till maximum accumulation of vegetative mycelial mass, introduction of sporulation inducer, with further chlamydospore formation. Duddingtonia flagrans F-882 is used as microfungus, submerged cultivation is performed in gas vortex bioreactor until dry rate of biomass reaches at least 10.0 g/l. To obtain fluid form of preparation, water is added as sporulation inducer, diluting vegetative mycelial mass by 2-5 times, and/or glycerin is added in end concentration of 1-2 wt %, and additionally the biomass is incubated until chlamydospore accumulation in the culture reaches maximum. To obtain dry form of preparation, sterile foamed vermiculite is added to vegetative mycelial mass as sporulation inducer at the ratio of 1.0-4.0 ml of mycelial mass to 1 g of foamed vermiculite, obtained mix is stirred and additionally incubated to obtain chlamydospores.

EFFECT: enhanced efficiency of preparation.

6 cl, 10 tbl, 11 ex

Description

The invention relates to methods for producing a preparation based on chlamydospores of the microscopic nematophagous fungus Duddingtonia flagrans in liquid or dry form, which can be used in microbiology and agriculture for controlling parasitic nematodes of plants and animals.

A known method for producing fungal spores in liquid culture (Japanese Patent 2003079362, IPC A01N 63/04, published March 18, 2003), in which the process of spore formation in fungal culture is started when there is a lack of nutrients in the medium, i.e. To maintain the process of spore formation, it is enough not to add food components after they are exhausted in a nutrient medium, preventing the propagation of vegetative mycelial cells.

However, in Duddingtonia flagrans fungi, when nutrients are exhausted, very few chlamydospores are formed in the medium (see table 1), i.e. this method is not effective for use in the manufacture of preparations based on chlamydospores of the microscopic nematophage fungus Duddingtonia flagrans.

A known method of growing mycelial forms of microorganisms on a carrier (RF patent No. 1169359, SEC C12N 3/00, publ. 07/10/1999), involving periodic immersion of the carrier in a nutrient medium.

However, the implementation of this method requires complex hardware. The need for periodic immersion of the carrier in a nutrient medium is caused by the insufficient moisture capacity of the used carriers. The lack of water and nutrients adsorbed in the material of the carrier leads to a decrease in the yield of spores, which is offset by periodic immersion in a nutrient medium.

The closest analogue (prototype) is a method for producing trichodermin by deep cultivation of the microscopic fungus Trichoderma lignorum on a nutrient medium (RF patent No. 2035145, IPC A01M 63/04, publ. 05.20.1995) containing sources of carbon, organic nitrogen, phosphates, magnesium sulfate , ammonium nitrate and water, followed by concentration of the culture fluid. Glycerin and green molasses are used as a carbon source, and a protein-vitamin concentrate hydrolyzate is used as a source of organic nitrogen in the following ratio of components in the medium, wt.%:

Glycerin 0.3-2.0

Green molasses 0.5-3.0

The hydrolyzate of protein-vitamin concentrate 3.0-10.0

Phosphates 0.2-0.6

Magnesium sulfate 0.3-0.5

Ammonium nitrate 0.3-0.6

Water - The rest.

In the process of cultivation with a natural decrease in the pH of the culture fluid to 3.0-4.5, glycerol is additionally introduced in an amount of 0.3-1.0 wt.%. The cultivation of the fungus is carried out until the maximum accumulation of mycelial mass or spore formation, the concentration of the culture fluid is carried out to a solids content of 6-10%, 4-10 wt.% Glycerol and 0.1-1.0 wt.% Polyethylene oxide or 1-3 are introduced into the concentrate wt.% polyvinylpyrrolidone. The cultivation of the fungus is carried out to the maximum spore formation, the concentration of the culture fluid is carried out to a dry matter content of 20-30 wt.%, A mixture of NaX type zeolite and montmorillonite or polygorskite in a ratio of 1: 2 in an amount of 10-30 wt.% To dry substances is introduced into the concentrate. The resulting mixture was granulated and dried. The titer of chlamydospores in a liquid preparation is 10 ⁸ spores in 1 ml, and in dry form 10 ⁹ spores in 1 g.

However, when using the technology described above to obtain a preparation based on chlamydospores of the microscopic nematophagous fungus Duddingtonia flagrans, a high titer of the microscopic fungus is not ensured: in liquid form no more than 10 ³ spores / ml, and in dry form - no more than 10 ⁴ spores / g. This is due to biological differences between the fungi Trichoderma lignorum and Duddingtonia flagrans. In particular, the conditions of the process of spore formation differ, and the size of D. flagrans spores exceeds the size of T. lignorum spores by more than an order of magnitude. The use of zeolite, montmorillonite or polygorskite is also impractical due to the low sorption capacity of these carriers.

The technical result of the invention is the creation of a technology for producing a preparation based on chlamydospores of the microscopic nematophagous fungus Duddingtonia flagrans in liquid and dry form with a higher titer by using more effective additives (spore formation inducers) at the stage of formation of chlamydospores of the indicated nematophagous fungus.

The specified technical result is achieved by the fact that in the method for producing a preparation based on chlamydospores of a microscopic fungus for controlling parasitic nematodes of plants and animals, including its deep cultivation on a nutrient medium containing sources of carbon and organic nitrogen, salt and water, to the maximum accumulation of vegetative mycelial mass , the introduction of inducers of spore formation, followed by obtaining chlamydospores, according to the invention, the strain Duddingtoni is used as a microscopic fungus a flagrans F-882, deep cultivation is carried out in a bioreactor until a dry matter biomass content of at least 10 g / l is reached; upon receipt of a preparation based on chlamydospores in liquid form, water is introduced as a spore formation inducer, diluting the vegetative mycelial mass 2-5 times and / or glycerin is added to a final concentration of 1-2 wt.% and the biomass is incubated to the maximum content of chlamydospores in liquid form, and upon receipt of the drug, chlamydospores in dry form are introduced into the vegetative as an inducer of spore formation sterile expanded vermiculite in the ratio of 1.0-4.0 ml of mycelial mass per 1 g of expanded vermiculite, mix and the resulting mixture is incubated to obtain chlamydospores.

The deep cultivation of the indicated strain of Duddingtonia flagrans F-882 in the bioreactor is carried out 40-50 hours at a temperature of + 28 ± 2 ° C, vigorous stirring and aeration.

Upon receipt of chlamydospores in liquid form, additional incubation is carried out in the bioreactor for 4-7 days at a temperature of + 28 ° C.

Upon receipt of chlamydospores in dry form, additional incubation of a mixture of mycelial mass with expanded vermiculite is carried out at a temperature of + 28 ° C for 4-10 days.

Upon receipt of chlamydospores in dry form, the vegetative mycelial mass is concentrated, for example, by a factor of 5, before mixing with expanded vermiculite.

Upon receipt of chlamydospores in dry form, glycerin, or molasses, or starch, or flour in an amount of from 2.0 to 12.0 wt.% Are added to the obtained vegetative mycelial mass of the fungus before mixing with expanded vermiculite.

The strain of the fungus Duddingtonia flagrans T-89 was isolated from the soil of the Kirovets greenhouse plant in Novosibirsk in 1989, and was deposited in the microorganism collection institute of the Microorganism Culture Research Institute of the Federal Research Center of Scientific Research of the Scientific and Production Center “Vector” of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Koltsove, Novosibirsk Region) under the number F-882 and is protected by RF patent No. 2253671, IPC ⁸ C12N 1/14, A01N 63/00, publ. 11/27/2004.

The strain is characterized by:

- the formation in surface culture on various solid (agarized and granular) sporulation media mainly in the form of chlamydospores having a thick shell;

- chlamydospores are stored in a dry state without loss of the initial properties of the fungus and viability of at least two years, are easily restored even after 10 years of storage when placing the culture in a suspension of nematodes, which stimulate the germination of chlamydospores;

- has high efficiency in relation to different nematodes, exhibits a duration of action. The strain is highly effective in the destruction of gall nematodes of plants, pathogens of helminth infections of animals, the ability to maintain viability without loss of properties both when passing through the gastrointestinal tract of animals and in soil conditions, and also has the property of stimulating plant growth and development.

Expanded vermiculite is a loose porous material in the form of flake particles of silver and golden colors, obtained by accelerated firing (up to a temperature of 600 ° ÷ 1000 ° C) before expanding vermiculite - hydromica, containing bound water between the elementary layers. The steam generated from this water acts perpendicular to cleavage planes and pushes mica plates, increasing the initial grain volume by 18–25 times. The density of expanded vermiculite is 70 ÷ 150 g / l. Expanded vermiculite is able to absorb up to 400% of water, i.e. per 1 g of vermiculite can be applied up to 4 ml of culture fluid, and there is no free fluid. The advantages of expanded vermiculite include the absence of toxicity and practical sterility due to the high-temperature processing of natural material. In particular, it is used to improve the structure of the soil and as a substrate in hydroponic plant cultivation. Due to the high sorption capacity of expanded vermiculite, the amount of applied seed and nutrients can be varied over a wide range.

Description of a method for producing a preparation based on chlamydospores of a microscopic nematophage fungus in liquid or dry form

A sterile nutrient medium is prepared containing sources of carbon and organic nitrogen, salts and water, as well as a 2-day seed of strain F-882 of the fungus of the species Duddingtonia flagrans. The cultivation process is carried out in a bioreactor at a temperature of 28 ° C, under conditions of intensive mixing and aeration. Cultivation is carried out until the biomass content of the dry matter is not less than 10.0 g / l (40-50 hours).

Upon receipt of the drug based on chlamydospores in liquid form, a spore formation inducer is introduced into the vegetative mycelial mass, water is used as it, diluting the vegetative mycelial mass 2-5 times with it, and / or glycerin is added to a final concentration of 1-2 wt.%. Next, the biomass is additionally incubated in the bioreactor for 4-7 days at a temperature of + 28 ° C, vigorous stirring and aeration to the maximum accumulation of chlamydospores in the culture.

Upon receipt of the preparation of chlamydospores in dry form, a spore formation inducer is introduced into the vegetative mycelial mass, using sterile expanded vermiculite in a ratio of 1.0-4.0 ml of mycelial mass per 1 g of expanded vermiculite, mixed and the resulting mixture is further incubated at a temperature of +28 ° C for 10 days to obtain chlamydospores. The resulting wet preparation is dried. The moisture content of the finished product should be no more than 12%. The amount of chlamydospores reaches (2 ÷ 4) * 10 ⁶ spores / g of dry preparation.

Upon receipt of chlamydospores in dry form, the vegetative mycelial mass is concentrated 5 times before mixing with expanded vermiculite.

Upon receipt of chlamydospores in dry form to increase the titer of chlamydospores, the carbon source glycerin, or molasses, or starch, or flour in an amount of from 2.0 to 12.0 wt.% Is added to the obtained vegetative mycelial mass of the fungus before mixing with expanded vermiculite.

The following are specific examples of the method.

Example 1. The accumulation of the vegetative mycelium (first stage) of the D. flagrans F-882 strain was performed in a 5 L BIOK vortex bioreactor - 022 s. Cultivation was carried out in a nutrient medium of the following composition:

KH ₂ PO ₄ - 5 g MgSO ₄ - 2 g NaNO ₃ - 5 g corn extract - 7.5 ml molasses - 30 ml tap water - up to 1 liter

The pH was adjusted to 6.8.

4 l of culture medium and 400 ml of inoculum, a 2-day F-882 culture, were loaded into the bioreactor. Cultivation continued for 48 hours, under the conditions indicated below:

- aeration - 6 l / min,

- activator rotation speed - 2000 rpm,

- temperature - + 28 ° С.

At the end of the cultivation, a culture fluid containing 12 g / l of vegetative mycelium was obtained by dry weight.

Example 2. Investigated the dependence of the harvest of chlamydospores on the degree of dilution of the culture fluid (QOL). Vegetative mycelium was obtained as described in example 1. QOL was diluted with sterile tap water and poured into 100 ml flasks in a volume of 0.5 L. Samples were incubated on a shaker (at 180 rpm, + 28 ° C) for 4 days. Samples were taken, homogenized and the concentration of chlamydospores was counted. The results are shown in table 1.

Table 1
Chlamydospore content according to dilution with water Description of samples the amount of chlamydospores * 10 ⁴ in 1 ml of liquid the number of chlamydospores * 10 ⁴ per 1 ml of the original (not diluted) QOL not diluted QL 0.4 0.4 QOL diluted 2 times 4.6 9.2 QOL diluted 3 times 5.8 17.4 QoL diluted 4 times 6.2 24.8 QoL diluted 5 times 5,4 27.0

Table 1 shows the values averaged over three replicates, deviations from the average within 10%.

Chlamydospores are also formed in undiluted culture fluid (QOL), but there are very few of them. Dilution of the culture fluid with water stimulates the process of spore formation so that the yield of chlamydospores with 1 ml of QOL increases by 23-68 times in comparison with undiluted QL. The greater the degree of dilution of QOL, the higher the yield of chlamydospores formed from the vegetative mycelium. Probably the role of a trigger factor for the spore formation process is played by a sharp decrease in the concentration of nutrients in the medium. The higher the degree of dilution, the stronger the lack of nutrients in the mycelial cells, and the more mycelial cells are transformed into chlamydospores.

Example 3. Investigated the dependence of the harvest of chlamydospores on the additives of glycerol in the liquid culture of strain F-882. The experimental conditions are similar to those described in Example 2. The only difference is that glycerol was added to some of the samples, along with dilution with water. After 4, 7, and 14 days, samples were taken, homogenized, and the concentration of chlamydospores was calculated. The results are shown in table 2.

table 2
Data on the content of chlamydospores depending on the concentration in the QOL of glycerol No. Description of samples Sample incubation time / amount of chlamydospores * 10 ⁴ in 1 ml of QOL 4 days 7 days 14 days one Not diluted QL 0.3 0.3 0.3 2 Like No. 1 + glycerin up to 2% 0.1 2.2 33.0 3 QOL diluted 2 times 4,5 4,5 4,5 four Like No. 3 + glycerin up to 2% 4.6 13.7 14.0 5 QOL diluted 3 times 5.5 5.5 5.5 6 Like No. 5 + glycerin up to 2% 7.4 11.8 12.0

The table shows the values (concentration of chlamydospores in 1 ml of liquid * 10 ⁴ ), averaged over three replicates, deviations from the average within 10%.

Studies have shown that the addition of glycerol to a concentration of 1-2% significantly increases the yield of chlamydospores (see table 2). In undiluted QL, glycerol supplementation increases the chlamydospore yield by 2 orders of magnitude. Unfortunately, the duration of the process of formation of chlamydospores increases to 2 weeks. In a culture fluid diluted 2–3 times, the process of spore formation ends in 7 days, whereas without glycerol, the formation of chlamydospores takes 4 days. However, the addition of glycerol (+ dilution with water) leads to an increase in the yield of chlamydospores by a factor of 2–3, compared with samples without glycerol.

Thus, glycerin can be used either as an independent inducer of spore formation, or in parallel with dilution of the culture fluid.

Example 4. The vegetative mycelium of strain D. flagrans F-882 was grown, as described in example 1. At the same time, vermiculite was prepared - 10 g of vermiculite was poured into 0.5 L flasks, sterilized for 4 hours at 180 ° C. The culture fluid grown in the bioreactor was applied to vermiculite and thoroughly mixed.

The flasks closed with foil were placed in a thermostat at + 28 ° С for the duration of the spore formation process (4 days). Table 3 shows the average test results for 4 options, differing in the amount of QOL applied per 1 g of vermiculite. Each option was set in 4 replicates.

Table 3
Data on the content of chlamydospores depending on the ratio of QOL and vermiculite Option Description chlamydospore / g vermiculite * 10 ⁵ chlamydospore / ml of the original QOL * 10 ⁵ 1 ml of QOL per 1 g of vermiculite 1.7 1.7 2 ml of QOL per 1 g of vermiculite 3.2 1,6 3 ml of QOL per 1 g of vermiculite 4.9 1,6 4 ml of QOL per 1 g of vermiculite 6.2 1.5

Deviations from the indicated average values are ± 10%.

The crop of chlamydospores almost linearly depends on the amount of biomass of vegetative mycelium deposited on vermiculite. However, a further increase in the yield of chlamydospores due to an increase in the amount of QL applied to vermiculite is impossible, since the moisture capacity of vermiculite is limited to 400% by weight of the vermiculite itself. The amount of biomass in 1 ml of deep culture is also limited.

Example 5. Vegetative mycelium was grown and vermiculite was prepared as described in examples 1 and 4. The biomass was concentrated 5 times by filtration. The culture fluid concentrate was applied to vermiculite, 3 ml of concentrate per 1 g of vermiculite, and thoroughly mixed. The flasks closed with foil were placed in a thermostat at + 28 ° C for the duration of the spore formation process (10 days). Table 4 shows the averaged (over 4 replicates) test results.

Table 4
Data on the content of chlamydospores on vermiculite in the concentration of QOL Options chlamydospore / g vermiculite * 10 ⁵ chlamydospore / ml of the original QOL * 10 ⁵ Control - 3 ml of QOL per 1 g of vermiculite 4,5 1.5 3 ml of QOL concentrate per 1 g of vermiculite 42.0 2.8 * Deviations from the indicated average values are ± 10%.

The use of biomass concentrate allowed to increase the yield of chlamydospores by 9 times, while the culture fluid was concentrated only 5 times. An additional gain in yield is due to the fact that chlamydospores formed more intensively in concentrated biomass. Based on 1 ml of the original QOL in the concentrate, almost 2 times more chlamydospores were obtained. The results obtained indicate that the increased density of mycelium on the surface of vermiculite is an additional factor stimulating spore formation.

Example 6. A vegetative mycelium was grown and vermiculite was prepared as described in examples 1 and 4. One of the following carbon sources was added to the culture fluid obtained in the bioreactor: molasses, glycerin, starch or flour, in various concentrations. Enriched with nutrients, the culture fluid (QL) was introduced into containers with sterile vermiculite, at the rate of 2 ml of QOL per 1 g of expanded vermiculite, and thoroughly mixed.

The containers were placed in a thermal room at + 28 ° C and incubated for 13 days. Table 5 shows the averaged (over 4 replicates) test results.

The addition of carbon sources allows to increase the yield of chlamydospores by 2–11 times, while the amount of seed applied to vermiculite does not increase. The greatest effect is given by additives of 12% molasses, 10% potato starch, and especially 10% wheat flour.

Table 5
Data on the content of chlamydospores on vermiculite when a carbon source is added to the QOL Option Description chlamydospore / g vermiculite * 10 ⁶ The output of chlamydospores relative to control (taken as 1) Control - UK without additives 0.5 one QOL with 2% glycerol 0.9 1.8 QOL with 3% molasses 1,0 2.0 QOL with 6% molasses 1.9 3.8 QOL with 12% molasses 3,7 7.4 QOL with 10% starch 3.8 7.6 QOL with 10% flour 5,4 10.8 * Deviations from the indicated average values are ± 10%.

Supplements of components such as molasses and glycerin are mainly spent on the formation of chlamydospores, while additives of starch and flour, along with the formation of chlamydospores, stimulate the growth of the vegetative mycelium. The use of additives allows, firstly, several times to reduce the production costs for growing seed in a bioreactor, and, secondly, eliminates the operation of concentrating biomass before applying to vermiculite.

Example 7. Investigated the dynamics of the process of formation of chlamydospores on vermiculite in samples with various additives. The experiment was carried out according to the scheme described in example 6. But the sampling was done after 4, 7, 10 and 13 days of incubation of the samples. Table 6 shows the results - the dynamics of the growth of chlamydospores on vermiculite.

Table 6
Data on the content of chlamydospores on vermiculite when a carbon source is added to the QOL Description of samples Duration of incubation / number of chlamydospores * 10 ⁶ in 1 g of dry preparation 4 days 7 days 10 days 13 days Control - QOL without additives 0.5 0.5 0.5 0.5 QOL with 2% glycerol 0.8 0.9 0.9 0.9 QOL with 12% molasses 3,5 3,7 3,7 3,7 QOL with 10% starch 0.5 2.2 3,1 3.8 QOL with 10% flour 0.6 2.7 4.4 5,4

The table shows the content of chlamydospores per 1 g of dry preparation 10 ⁶ . In the control, where QL without additives is applied to vermiculite, the process of chlamydospore formation is completed within 4 days. Spore formation in samples with additions of glycerol and molasses also almost quickly ends. In samples with starch and flour additives, the process stretches for 13 days. The reason is that starch and flour primarily stimulate the growth of the vegetative mycelium, as a result of which the formation of chlamydospores begins much later than in the control. On the other hand, the addition of flour provides the highest content of chlamydospores in the preparation. The best option seems to be with the addition of 12% molasses, where a high content of chlamydospores is achieved in a minimum time of 4 days.

The resulting wet preparation is dried. The moisture content of the finished product should be no more than 12%. The amount of chlamydospores reaches (2-4) * 10 ⁶ spores / g of dry preparation.

Thus, examples 1-7 confirm that the technical result of the present invention provides a preparation based on chlamydospores of the microscopic nematophagous fungus Duddingtonia flagrans in liquid and dry form with a higher titer (up to 4 * 10 ⁶ spores / g of dry preparation) by using more effective additives (sporulation inducers) at the stage of formation of chlamydospores.

Example 8. In the soil of the Novosibirsk Metro greenhouse infected with gall nematodes (Meloidogyne incognita), dry and liquid forms of the preparation were applied based on the Duddingtonia flagrans strain F-882, simultaneously with the planting of seedlings of Korolek cucumber. At the end of the growing season, the root system damage score and the average yield per plant were taken into account. In each variant, 100 plants were counted. Table 7 presents the results of yield and root system damage in the experimental and control variants.

Table 7
The average yield per plant and the damage to the roots of the cucumber by meloidoginosis treated with preparative forms based on Duddingtonia flagrans F-882 Option The average yield of cucumber per plant, kg The score for the defeat of the roots of the cucumber by meloidoginosis Dry form (on expanded vermiculite) - 30 g / plant 1.92 ± 0.08 2 Liquid form - 30 ml / plant 1.78 ± 0.07 3 Control (no processing) 1.08 ± 0.09 5

As can be seen from the results obtained, the dry form of the drug showed the greatest effect, which is due to the high content of mushroom propagules in the introduced dose compared to the liquid form.

Example 9. For practicing doses of the dry form of the drug (on expanded vermiculite) based on the strain Duddingtonia flagrans F-882 against gall nematodes of plants, a vegetative experiment was laid. The soil for the experiment was taken from the site of severe damage to the root system of the cucumber by meloidoginosis (5 points), thoroughly mixed and packed into vessels containing 400 g of soil. The dose of the drug was 1%, 2% and 3% by weight of soil in the vessel. Vessels with untreated soil served as control. Each variant was represented by 20 vessels. Used sprouted seeds of cucumber varieties Korolek. After 1 month, the experiment was interrupted and measurements were taken of the plant height, leaf plate area, root length, and the root lesion score by meloidoginosis (Table 8).

Table 8
The results of the growing experiment to assess the optimal dose of the dry form of the drug based on Duddingtonia flagrans F-882 against gall nematodes Option Plant height, cm The area of the sheet plate, cm ² Root length, cm The root lesion score by meloidoginosis 1% of the drug 14.0 ± 0.3 64.0 ± 1.2 15.0 ± 0.3 one 2% of the drug 13.4 ± 0.2 62.6 ± 1.0 12.7 ± 0.3 one 3% of the drug 12.9 ± 0.2 51.6 ± 2.2 12.1 ± 0.3 one the control 12.5 ± 0.2 44.0 ± 2.5 10.5 ± 0.5 5

As can be seen from table 8, the root system in all experimental plants was mainly cleared of nematodes, however, the results on plant growth and development were best in the version with 1% preparation, which is probably due to the insignificant toxic effect of the biological product in increased doses at the initial stages of development of cucumber plants.

Example 10. In the area with a high level of soil contamination with the Globodera rostochiensis potato nematode (10-12 thousand larvae per 100 g of soil), a preparation based on the Duddingtonia flagrans F-882 strain was applied in dry and liquid form for the Svitanok Kievsky and Lyubava potato varieties "(Susceptible to the nematode)," Nikita "(resistant variety). The dose of the biological product was 150 g / m ² (dry and liquid forms). The drug was added to the well when planting potatoes in the month of May.

For all varieties at the germination stage, the stimulating effect of the drug was noted, especially in liquid form. Data on the effect of the drug on potato nematode infection are presented in Table 9.

As can be seen from Table 9, the decrease in infection with potato nematode in susceptible varieties was higher in variants with the drug on expanded vermiculite compared with the liquid form (68-70% versus 52-60%).

Example 11. The effectiveness of the liquid form of the drug based on the strain Duddingtonia flagrans F-882 was evaluated on the larvae of the pathogen Elaphostrongylus (Elaphostrongylus panticola) maral.

Table 9
The effect of the drug based on the strain Duddingtonia flagrans F-882 on the infection of plants with the potato nematode Globodera rostochiensis (average repetition) Option Accounting in the budding phase (July 13) Accounting for harvesting tubers (September 8) Potato variety Drug form Number of females per plant, ind. % of control Number of females per plant, ind. % of control Svitanok Kiev Liquid 16.3 ± 2.8 - 28.2 * 12.0 ± 1.8 -52.0 Dry 5.0 ± 0.8 -77.9 8.0 ± 1.2 -68.0 The control 22.7 ± 3.2 - 25.0 ± 4.1 - Lyubava Liquid 5.0 ± 0.8 -79.2 8.0 ± 1.1 -60.0 Dry 3.0 ± 0.5 -87.5 6.0 ± 0.9 -70.0 The control 24.0 ± 3.9 - 20.0 ± 3.2 - Nikita Liquid 0 0 0 0 Dry 0 0 0 0 The control 0 0 0 0 * (-) - reduction of infection relative to control

For this purpose maral feces were used, containing on average 18.5 larvae per 1 g. 30 ml of the preparation was added to the experimental variants placed in Petri dishes; the same amount of distilled water was added to the control plates. Samples were placed in a thermostat at a temperature of + 26.4 ° C. On days 3, 6 and 10, three samples from each group were examined, counting the number of E. panticola larvae in 1 g of feces according to the Berman-Orlov method (Table 10).

Table 10
The influence of the liquid form of the drug based on the strain Duddingtonia flagrans F-882 on the number of larvae of elaphostrongile maral Option The number of larvae per 1 g of feces, pcs. (by day of sampling) 0 3 6 10 Liquid preparation 18.5 0.2 0.1 0,07 The control 18.5 10.7 23.6 11.7

As can be seen from table 10, a significant decrease in the number of larvae was detected under the influence of the liquid form of the drug after 3 days. Compared with the control on the 3rd, 6th and 10th day, the number of larvae decreased 53, 236 and 167 times, respectively.

Claims (6)

1. A method of producing a preparation based on a chlamydospore of a microscopic fungus for controlling parasitic nematodes of plants and animals, including its deep cultivation on a nutrient medium containing sources of carbon and organic nitrogen, salt and water, to the maximum accumulation of vegetative mycelial mass, the introduction of spore formation inducers followed by obtaining chlamydospores, characterized in that the strain Duddingtonia flagrans F-882 is used as a microscopic fungus, deep cultivation is carried out in bi the reactor until the dry matter biomass content is not less than 10 g / l, upon receipt of the preparation based on chlamydospores in liquid form, water is introduced as a spore formation inducer, diluting the vegetative mycelial mass 2-5 times, and / or glycerin is added in a final concentration of 1 -2 wt.% And the biomass is incubated to the maximum content of chlamydospores in liquid form, and upon receipt of the preparation of chlamydospores in dry form as a spore formation inducer, sterile expanded vermiculi is introduced into the vegetative mycelial mass t in a ratio of 1.0-4.0 ml of mycelial mass per 1 g of expanded vermiculite, stirred and the resulting mixture was further incubated to obtain chlamydospores. 2. The method according to claim 1, characterized in that the deep cultivation of the specified strain of Duddingtonia flagrans F-882 in the bioreactor is carried out 40-50 hours at a temperature of 28 ° C, vigorous stirring and aeration. 3. The method according to claim 1, characterized in that to obtain the maximum content of chlamydospores in liquid form, incubation in the bioreactor is carried out for 4-7 days at a temperature of 28 ° C. 4. The method according to claim 1, characterized in that to obtain chlamydospores in dry form, incubation of a mixture of mycelial mass with expanded vermiculite is carried out at a temperature of 28 ° C for 4-10 days. 5. The method according to claim 1, characterized in that upon receipt of the chlamydospores in dry form, the vegetative mycelial mass is concentrated 5 times before mixing with expanded vermiculite. 6. The method according to claim 1, characterized in that when receiving chlamydospores in dry form, glycerol or molasses, or starch, or flour in an amount of from 2.0 to 12.0 wt. Is added to the obtained vegetative mycelial mass of the fungus before mixing with expanded vermiculite .%.