RU2534213C2 - Method of obtaining biological preparation for protection of plants against phytopathogens and nematodes based on genus trichoderma fungus strain and biological preparation obtained thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology. The method of obtaining a biological preparation for the protection of plants against phytopathogens and nematodes based on a genus Trichoderma fungus strain is realised by preparation of an inoculation material of the fungus strain, making the preparation in a liquid or friable form based on the inoculation material, and mixing the preparation with a mineral, organic or bacterial fertiliser.

EFFECT: biological preparation, resulting from the method, possesses the high antagonistic activity with respect to a wide spectrum of phytopathogens and nematodes.

6 cl, 5 ex

Description

The alleged invention relates to the field of microbiology, and in particular to producer strains of substances that optimize plant growth conditions and crop safety. Applicable in agriculture and forestry, food industry.

It is known that pathogens cause damage in crop production, for example, in the cultivation of crops, in the storage of harvested crops, in forestry when growing planting material in the form of seedlings. To prevent damage, plant and product protection is necessary.

It is known that in crop production damage is also caused by the stress experienced by plants. For example, stresses caused by sudden fluctuations in temperature, extreme temperatures, drought or excess humidity due to rains. To prevent such damage, plant growth regulators are needed. In this case, the means used must be safe for the environment. Most fully meet these requirements biological means and technologies for their use.

Known biological means and methods of plant protection, created on the basis of microorganisms existing in nature [1].

A drug is provided that provides protection of plants from diseases of various nature, as well as regulates growth [2]. Said preparation includes at least two strains of Trichoderma from Trichoderma asperellum, Trichoderma atroviride, and Trichoderma inhamatum.

Known strains of mycelial fungi Trichoderma lignorum, mixed with the products of their vital functions and collectively called the drug "Trichodermin." The drug is used on sheltered soil to combat root rot of plants [1, p. 42].

The TBD-93 strain of the Trichoderma lignorum fungus is also known. The drug Trichodermin, made from this strain, is used on sheltered ground to combat root rot of cucumber and fusarium of decorative culture - cloves [3].

A disadvantage of the known strain [3] is that it (the strain) has a relatively low antagonistic activity against plant pathogens (phytopathogens), acts only on a very limited range of phytopathogens, and does not inhibit the reproduction of pests, for example, nematodes. And therefore, it is only partially applicable for the protection of cereals, vegetables, and ornamental crops, with the exception of the above. In addition, the well-known strain noted phytotoxic properties in relation to some types of cereals and vegetables. Based on this, the disadvantage of the known strain is a very limited scope - only for two cultures and two types of diseases [3]. And the list of plant diseases and pests from which plants need to be protected contains hundreds of items.

The closest analogue is the strain Trichoderma Asperellum R1, known from patent application No. 2008108035 RU [4]. This publication describes methods for preparing seed from a strain culture and preparations based on cultured seed. The disadvantage of this solution is the limited ability of the drug on fertilized soils, since the drug does not include additional nutrients (organic and mineral) for plants and beneficial microorganisms Trichoderma Asperellum R1.

The aim of the invention is the creation of a biological agent with high antagonistic activity against the widest possible range of pests and phytopathogens, contributing to increased productivity and preservation of a wide range of agricultural, ornamental crops, preservation of the harvested crop during storage.

The goal is achieved due to the composition of the product, which includes a strain of the fungus of the genus Trichoderma, which during its life forms antibiotics and / or enzymes that destroy the cell wall of phytopathogenic fungi, and / or gibberelin-like substances, one-component and / or complex mineral fertilizers, as well as organic fertilizers and bacterial fertilizers.

Strains of the fungus Trichoderma are characterized by the following cultural-morphological and physiological-biochemical characteristics.

Biotechnological characteristic

In the process of life, the strain forms metabolites (all kinds of substances that are formed in the body in the process of metabolism - metabolism). A product from the structural elements of fungi (spores, chlamydospores, mycelium) of various degrees of maturity with their metabolic products is a useful preparation of the strain, which is used in crop production. The specified preparation contains a mixture of resting structures, mycelium, enzymes, antibiotics and plant growth regulators - metabolic products of Trichoderma fungi obtained after cultivation of the fungus on natural liquid, solid and granular nutrient media - substrates.

In a preparation, for example, in its liquid form, it contains:

- antibiotics, for example viridin, viridion having fungistatic action, secreted, for example, by strains of Trichoderma Viride, Trichoderma Virens IMI 304061. Trichoderma sp. YMF 1.02647, and others [5, 6, 7],

- enzymes, for example, cellulases, chitinases, proteases that destroy the cell wall of phytopathogenic fungi, isolated, for example, by strains of Trichoderma Asperellum T-203, Trichoderma Virens, Trichoderma harzianum CECT 2413, Trichoderma lignorum HKI 0257 [8, 9, 10, 11 , 13, 14],

- gibberellin-like substances that stimulate plant growth, accelerate flowering and increase the immunity of plants, secreted, for example, Trichoderma harzianum CECT 2413, Trichoderma Asperellum T-203 [15, 16, 9].

The introduction of a Trichoderma fungal preparation into the soil reduces the infectious background. The intensity of exposure depends on the type of soil. The greatest fungistatic effect in soil was shown, for example, in chernozem (50%) and sod-podzolic (40%). The smallest effect is shown in gray forest soils - 30%. An increase in the fungistatic effect in soils was noted with the introduction of a biological product for 5 consecutive years.

The regulation of plant growth by the metabolites of the fungus is expressed in stimulating the energy of seed germination and germination, increasing the mass of plants and roots, increasing yield, reducing plant damage and the content of mycotoxins in the crop obtained using the strain.

The absence of genotoxicity, toxicity to warm-blooded animals, bees and fish was shown.

Moreover, Trichoderma fungal strains are characterized by high survival rates, for example, in soils, resistance to bacterial contamination, manifestation of antagonistic activity to a wide variety of phytopathogens and antagonistic activity to pathogens and plant pests.

According to the invention, the preparation of a biological agent consists of the steps of preparing the seed of a strain of a fungus of the genus Trichoderma, preparing the drug in liquid or bulk form based on the seed, mixing the preparation with mineral and / or organic and / or bacterial fertilizer. The drug for the product is prepared as follows. The culture of the Trichoderma strain is maintained on an agarized wort medium (malt wort content of 5-6 ° Balling + 2% agar) and stored in a refrigerator at a temperature of from + 3 ° C to + 5 ° C.

Then seed is obtained in 2 stages.

First step. A spore suspension is obtained by washing micromycete colony with sterile water from the surface of an agarized wort medium.

Second phase. The seed is obtained: spore suspension is added to Erlenmeyer flasks with liquid potato-glucose medium at the rate of 5% -15% of the medium volume. The flasks are placed on a rocking chair with a circular type of mixing and the strain is cultured for 24 to 40 hours, at a temperature of from plus 19 ° C to plus 39 ° C.

As a result, the seed obtained contains mycelium, conidia, chlamydospores of micromycetes. Seeds are used to produce mushroom preparations of various types, for example, in liquid and granular form.

To obtain a liquid form of the drug with a concentration of 10 ⁶ to 10 ⁹ CFU / ml, the seed of the strain is transferred to a fermenter with a nutrient medium indicated below (g / l):

Glucose - 15-25

NaNO ₃ - 1.8-3.0

MgSO ₄ - 0.3-0.8

KH ₂ PO ₄ - 0.7-1.3

NACl - 0.2-1.0

Sunflower oil - 2.0-4.0

The rest is water (bringing g volume to 1 liter)

pH - 4.0

The seed of the strain in a fermenter is cultured in a nutrient medium and receive a biological product. Cultivation time: 18-30 hours at temperatures from plus 19 ° C to plus 39 ° C.

The loose form of the preparation is obtained using grains or husks of cereals, for example rye. For the manufacture of a substrate for the cultivation of the strain, grains are taken, moistened with a molasses-yeast suspension, and molasses are taken in an amount of 0.5% to 2% of the volume of the substrate, the yeast suspension is taken in an amount of 3% to 10% of the volume of the substrate. A container, for example a glass bottle, is filled with a substrate and sterilized in an autoclave for 0.5 to 1.5 hours at 0.5 kg / cm ² overpressure. After sterilization, the substrate is cooled. Then, the seed inoculum of the strain is introduced into the substrate in a volume of 5% to 20% of the substrate and the strain is incubated for 5 ... 7 days at a temperature of from + 19 ° C to plus 39 ° C and exposure to illumination from 10 to 20 hours per day, 2 -10 thousand lux. The resulting bulk preparation is dried using forced low-temperature drying, for example, at room temperature. If necessary, in one embodiment, the granular form of the drug is washed with water, which is collected to obtain a liquid form of the drug with a concentration of from 10 ⁶ to 10 ⁹ CFU / ml.

Further, to obtain a biological agent, the preparation in liquid or granular form is mixed with mineral, organic or bacterial fertilizer.

In one embodiment, the preparation is mixed in bulk form with mineral fertilizer in a weight ratio of 1/1 to 1/10 (mineral fertilizer / mushroom preparation) and packaged in sterile parchment bags.

In one embodiment, the preparation is poured in bulk form with organic fertilizer in a weight ratio of 1/1 to 1/50 (mushroom preparation / organic fertilizer) and packaged in sterile parchment bags.

In one embodiment, the preparation in bulk form is mixed with bacterial fertilizer in a weight ratio of 1/1 to 1/5 (mushroom preparation / bacterial fertilizer) and packaged in sterile parchment bags.

In one embodiment, the preparation in liquid form is obtained by washing off the strain and its metabolic products with water from the surface of the granular form of the drug, the concentration of the drug is from 10 ⁶ to 10 ⁹ CFU / ml.

In one embodiment, the preparations in liquid form are further mixed with a solution of bacterial fertilizers in a ratio of 1/1 to 1/10 (liquid form of the drug / fertilizer solution), at concentrations of solutions of the liquid form of the drug from 10 ⁶ to 10 ⁹ CFU / ml.

When using the invention, the following occurs.

As a result of the fungistatic action of a biological agent based on strains of the fungus of the genus Trichoderma (inhibiting the germination of spores of phytopathogens) of volatile and non-volatile products of the metabolism of the strain, growth (inhibition or retardation) of phytopathogens is inhibited (for example, Fusarium by 40-50% depending on the species, alternaria infection by 90%, helminthosporiosis by 70%, botrythiosis by 100% in in vitro experiments. The development of pests, for example, stem and soil nematodes, is suppressed. In the field, a steadily observed decrease in the infectious load occurs depending on climatic conditions, illumination, temperature and soil moisture.

The use of mineral, organic, and bacterial fertilizers (based on phosphate-soluble, nitrogen-fixing, and other bacteria) as a part of the product corrects the nutritional and energy functions of the soil, which is necessary for crop growth and the effective implementation of the technical result of the drug based on Trichoderma fungus strains.

The technical result is also expressed in increasing the resistance of crops on protected and open ground to phytopathogens of various species, reducing the infectious background in the soil and / or greenhouse substrate, increasing the stress resistance of plants, in stimulating the growth and development of soil nitrogen-fixing and phosphate-dissolving microorganisms. There is an increase in the content of humus in the soil, increasing the quality of the soil and improving the conditions for the growth of plants.

Reducing the incidence of plants, the abundance of soil nitrogen-fixing and phosphate-dissolving microorganisms and their metabolic products (in the form of natural nitrogen and phosphorus compounds necessary for plant nutrition) leads to increased plant yields, improved crop quality by reducing mycotoxins. In addition, the crop that is not infected with phytopathogens is preserved significantly better than the infected one and is less lost during storage. At the same time, food products made from raw materials - crops with a reduced nitrate content - have increased nutritional value and are environmentally friendly, harmless to consumers.

The use of strains with metabolic products can reduce the use of inorganic fertilizers, in many cases reduce the amount used or abandon the use of chemicals - to protect plants from pathogens, protect against stress during unforeseen changes in the environment, for example, temperature, humidity, to regulate plant growth , increase yield and crop safety.

The absence of genotoxicity, toxicity of the strain and its metabolic products to humans, warm-blooded animals, bees and fish was shown. Reducing the use of chemicals in agriculture or abandoning their use improves the quality of food produced while reducing environmental damage.

Example 1

The strain Trichoderma asperellum T-310, deposited in the All-Russian Collection of Microorganisms under the number VKM F4340D, is used to protect cereal crops from phytopathogens, for example wheat from fusariosis, as follows. Make the drug. A liquid biological agent (a preparation based on a strain of the Trichoderma fungus in liquid form, mixed in a 1/1 ratio with a solution of the RIZOAGRIN preparation) is filtered, diluted with water in a ratio of 1:10 and placed in a pickling machine used in agriculture for preplant seed treatment with pesticides .

Processing is carried out similarly to pesticides [21]. Seed grain is poured into the grain bin of the pickling machine. The liquid preparation is introduced into the mass of seed grain. Grain is thoroughly mixed. The grain mixed with the preparation is removed from the hopper of the dressing machine and sowing is carried out.

Example 2

Bulk form of biological product (bulk form of the drug based on the Trichoderma lignorum strain deposited at the Vector Research Institute of Cell Culture Collections of the State Scientific Center for Virology and Biotechnology under number F5, mixed with Mitoz-1 or Isabion preparations in a weight ratio of 1/25 (mushroom preparation / organic fertilizer)) is used to disinfect the soil by making the strain preparation before planting / planting, and after harvesting. The application rate is from 200 to 500 g / ha of the drug, depending on the infectious background of the soil.

Example 3

The loose form of the biological agent is used to protect plants, such as potatoes, on open and protected ground against late blight (Phythophtora infestans). For this, a bulk form of a biological product (a bulk form of the drug based on the Trichoderma asperellum T-310 strain deposited in the All-Russian Collection of Microorganisms under the number VKM F4340D, mixed with Mitoz-1 or Isabion preparations in a weight ratio of 1/30 (mushroom preparation / organic fertilizer) ) are applied to the soil during the pre-planting period in spring and in August-September (in winter) based on, for example, in the amount of or 300 to 1000 g / ha.

Example 4

The loose form of the biological agent is used to protect carrots from rot on the open ground. For this, a bulk form of a biological product (a bulk form of a preparation based on the Trichoderma lignorum strain deposited at the Vector Research Institute of Cell Culture Collections of the State Scientific Center for Virology and Biotechnology under number F5, mixed with ammonized superphosphate in a mass ratio of 1/8 (mineral fertilizer / mushroom preparation )) is applied to the soil during the pre-planting period in the spring and in August-September (in winter) at the rate of a double dose of the drug per hectare, for example, in an amount of 300 to 1000 g / ha. Subsequently, to protect plants during vegetation, a liquid form of a biological agent is used (the liquid form of the drug is based on the Trichoderma strain mixed with a solution of the drug bactophosphine in a volume ratio of 1/1).

Example 5

A loose form of a biological agent is used to protect plants, for example, cucumber, on an open and protected ground from a gall nematode by applying a loose form of a biological agent (a form of the drug based on the Trichoderma viride strain deposited in the All-Russian Collection of Industrial Microorganisms under number F262 mixed with cainite in mass ratio 1/10 (mineral fertilizer / mushroom preparation)) to the soil in the pre-planting period - based on, for example, from 400 to 1000 g / ha.

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Claims (6)

1. A method of obtaining a biological agent for protecting plants from phytopathogens and nematodes based on a strain of the fungus of the genus Trichoderma, including the preparation of seed of a strain of the fungus of the genus Trichoderma, the preparation of the drug in liquid or granular form based on the inoculum, as well as mixing the drug with mineral, organic or bacterial fertilizer, as a result of which a biological agent is obtained, and for the preparation of seed of a strain of a fungus of the genus Trichoderma, the museum culture of a strain of the fungus of the genus Trichoderma live on an agarized wort medium with a malt content of 5-6 ° according to Balling and 2% agar and store in a refrigerator at a temperature of + 3 ° C to + 5 ° C, then by washing the micromycete colony with sterile water from the surface of the agarized wort environment get a spore suspension, then a spore suspension is added to Erlenmeyer flasks with liquid potato-glucose medium at the rate of 5% -15% of the volume of the medium, the flasks are placed on a rocking chair with a circular type of mixing and the strain is cultivated from 24 to 40 hours, at a temperature of +19 ° C to + 39 ° C, then for the preparation of the drug in dkoy form at a concentration of 10 ⁶ to 10 ⁹ cfu / ml inoculum of the strain obtained is transferred to a fermenter containing a nutrient medium having the composition in g / l is specified below:
Glucose - 15-25
NaNO ₃ - 1.8-3.0
MgSO ₄ - 0.3-0.8
KH ₂ PO ₄ - 0.7-1.3
NaCl - 0.2-1.0
Sunflower oil - 2.0-4.0
The rest is water (bring the volume to 1 liter)
pH 4.0
and cultivated for 18-30 hours at a temperature of + 19 ° C to + 39 ° C, or, if it is necessary to obtain a drug in bulk form, make seed in a sterilized substrate in a volume of 5% to 20% of the substrate and incubate the strain for 5-7 days at a temperature of + 19 ° C to + 39 ° C and an exposure of illumination from 10 to 20 hours per day, 2-10 thousand lux, then the resulting bulk preparation is dried using forced low-temperature drying, as a result of which receive a preparation based on a strain of the fungus of the genus Trichoderma in bulk form, and for the manufacture of the substrate for the cultivation of the strain take grain or husk of cereal crops, characterized in that the grain or husk of grain is moistened with molasses-yeast suspension, and molasses is taken in an amount of from 0.5% to 2% of the volume of the substrate, yeast suspension is taken in an amount of 3% to 10% of the volume of the substrate or, if necessary, the preparation in bulk form is washed with water that is collected to obtain a liquid form of the drug with a concentration of from 10 ⁶ to 10 ⁹ CFU / ml. 2. The method according to claim 1, characterized in that the preparation in bulk form is mixed with mineral fertilizer in a mass ratio of mineral fertilizer to mushroom preparation from 1/1 to 1/10. 3. The method according to claim 1, characterized in that the preparation in bulk form is mixed with organic fertilizer in a mass ratio of mushroom preparation to organic fertilizer from 1/1 to 1/50. 4. The method according to claim 1, characterized in that the preparation in bulk form is mixed with bacterial fertilizer in a mass ratio of mushroom preparation to bacterial fertilizer from 1/1 to 1/5. 5. The method according to claim 1, characterized in that the drug in liquid form with a concentration of from 10 ⁶ to 10 ⁹ CFU / ml is mixed with a solution of bacterial fertilizer in the ratio of the volume of the liquid form of the drug to the fertilizer solution from 1/1 to 1/10. 6. Biological agent for protecting plants from phytopathogens and nematodes based on a strain of the fungus of the genus Trichoderma, obtained by the method according to claims 1-5.