RU2237088C1 - Strain mycelia sterilia - producer of biologically active substances complex eliciting growth-regulatory properties - Google Patents

Abstract

FIELD: microbiological industry, agriculture.

SUBSTANCE: invention relates to the strain of fungus Mycelia sterilia storing in the All-Russian collection of industrial microorganisms, collection number is VKPM F-833. The strain is a producer of complex of biologically active substances and elicits growth-regulatory properties. Invention provides preparing the preparation used as an agent exhibiting biostimulating effect on plants growth.

EFFECT: valuable agricultural properties of strain.

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Description

The invention relates to the microbiological industry and relates to a new culture of microorganisms producing biologically active substances with growth-regulatory properties that can be used in agriculture in the processing of crops such as cereals, gooseberries, currants, potatoes, cucumbers, tomatoes, lawns, etc.

A community of microorganisms for producing plant growth regulators is known, consisting of micromycete Acremonium panaxeorum UHMU F-160, yeast Candida muscorum UHMU Y-411, Rhodotorula glutinis UHMU Y-42 and Azomonas macrocytogenes UHMU B-28 and Xantobacter sp UHMU B-28 and Xantobacter sp. . Of the Russian Federation No. 2100932). The disadvantage of this community is the complexity and complexity of its cultivation, the instability of the resulting drug.

Known drugs that stimulate the development and growth of plants obtained from pure cultures of endophytic fungi isolated from the roots of wheat, oats, potatoes, wheat grass, mother-and-machiha. Each drug consists of metabolic products of the corresponding endophyte dissolved in 70% ethanol. The treatment of wheat, cucumbers or potato seeds with one of the preparations indicated before the sowing contributed to an increase in yield (F. Geltser. New producers of stimulants for plants. Reports of VASKHNIL, No. 5, 1975).

Known drug "Symbiotic-2", stimulating plant productivity, obtained from the products of metabolism of endophytic fungi (a.s. of the USSR No. 921488).

As raw materials for its manufacture, endophytes isolated from the roots of sea buckthorn are used.

A disadvantage of known growth stimulants is the low activity of preparations obtained from endophytes of plants with the highest medicinal value.

Known drug Symbiont-1, made from endophytes isolated from ginseng roots. The drug is effective only under good conditions of plant growth (Av. St. USSR No. 370932).

A common disadvantage of known drugs is the inability to identify products obtained using known strains, the content of significant amounts of alcohol in the drug.

The closest technical solution to the claimed invention is a known strain of Cylinddrocarpon magntsianum, which is a producer of plant growth regulator (Ukrainian patent No. 17558). The strain isolated from the root system of sea buckthorn. As the nutrient medium used, g / l: glucose, KH ₂ PO ₄ , K ₂ NRA ₄ , MgSO ₄ , K ₂ SO ₄ , MnSO ₄ , FeSO ₄ , asparagine. Its main disadvantage is the difficulty of its production on an industrial scale, the strain is effective only in the root zone of a particular host plant, the presence of alcohol in the composition of the drug.

The aim of the invention is to expand the sources of raw materials from new strains and increase the activity of the growth regulator.

The goal is achieved by the fact that a new strain of Mycelia sterilia is isolated from the roots of ledum. The strain is stored in the All-Russian collection of industrial microorganisms, collection number VKPM F-833. The strain is designed to obtain a biostimulator of plant growth.

The strain is isolated from the roots of rosemary. The specific name of the culture is Mycelia sterilia var. Ledum., Name strain NZh-13. Pedigree of the strain: natural isolate. A similar type of isolated fungus is similar to “ericoid mycorrhiza” fungi.

Cultural and morphological features of the strain: Aerob. The fungus gives slowly growing pressed-velvety colonies of black color (growth coefficient according to Bukhalo, 1988 - 10). In cultures, not only superficial, but also substrate mycelium is developed. The color of the medium under the influence of the fungus does not change. Microscopic examination revealed that the fungal hyphae are dark-colored, with a high content of oil droplets (the diameter of the hyphae does not exceed 3 μm). On hyphae, a significant amount of intercalary chained, formed in chains of spherical chlamydospores, is formed.

The product synthesized by the strain is a complex of biologically active substances.

When the strain was cultured for 3 weeks in a Geltzer liquid medium with the subsequent isolation of a complex of secondary metabolites, the resulting preparation gives an increase in the germination rate of crops by 20-30%, as well as an increase in the hypocotyl of the experimental mustard line 30-60% to control (water).

Biological activity was determined using the standard method of biotests on plant seeds.

The strain was stored on agarized nutrient media at a temperature of 4-7 ° C with reseeding once every 3 months.

Gelzer agar medium (GA), g / l, pH 6.2-6.6:

Glucose 8.0

KN ₂ RO ₄ 0.6

K ₂ NRA ₄ 1.8

MgSO ₄ 0.2

K ₂ SO ₄ 0.1

MnSO ₄ Traces

FeSO ₄ Traces

L-Asparagine 0.02

Agar Agar 15.0

Water Up to 1000 ml

The propagation of the culture was carried out by reseeding on an agar medium Gelzer, the composition of which is presented above.

The conditions and composition of the medium for fermentation: on a liquid nutrient medium Geltzer, the cultivation time is 20-30 days at a temperature of 26 ± 1 ° C. The composition of the medium: liquid nutrient medium Gelzer, g / l, pH 6.0-6.4:

Glucose 8.0

KN ₂ RO ₄ 0.6

K ₂ NRA ₄ 1.8

MgSO ₄ 0.2

K ₂ SO ₄ 0.1

MnSO ₄ Traces

FeSO ₄ Traces

L-Asparagine 0.02

Water Up to 1000 ml

The strain is not zoopathogenic, is not phytopathogenic, does not pose a threat to the environment.

The drug based on strain NZh-13 was obtained by growing the culture in flasks. Cultivation was carried out in flasks, then coarse and fine filtration was carried out. Then the resulting product was freeze-dried. During biotests and chemical analyzes, lyophilized dried filtrate of the culture fluid was used.

The content of phytohormones in the preparation: cytokinins (CC), gibberelins (GA) - was determined by chromatographic method.

Cytokinins are the most active natural compounds involved in the hormonal regulation of plant ontogenesis and characterized by the ability to induce plant cell division.

Gibberellic acid GA _{3 is} most widely used in agriculture as a plant growth regulator. However, GA ₇ is of the greatest interest since has a wider spectrum of activity and higher physiological activity.

Indolylacetic acid (IAA) is a growth promoter, but due to its poor solubility in water and relatively high cost, it has a narrow range of recommended crops.

All of these compounds related to known plant growth regulators are described in detail in the literature / Plant Growth Regulators. Edited by G.S. Murovtseva. M., 1979 /.

The inventive strain allowed to obtain all of these compounds simultaneously, which significantly increased the biological activity of the drug and at the same time increased the economic efficiency of its use.

The biological activity of a dry preparation based on the inventive strain on bluegrass, cultivar Denim, (control-water) and the content of phytohormones in this preparation are shown in table 1.

The method of obtaining the strain allows you to scale the production process, which is a necessary condition for the commercialization of the project. The optimal strain concentration in the preparation is 10 ppm.

Table 2 shows the elemental composition of the lyophilized preparation.

Analysis of the elemental composition showed that the preparation contains almost no heavy metals. In the preparation, in comparison with the nutrient medium, the content of potassium and molybdenum significantly increases, the content of other elements decreases.

The content of free carbohydrates, mg / g dry. weight:

Sucrose 178

Glucose 376

Fructose 91

Mannitol 106

The following amino acids are contained in the preparation: lysine — 0.0066, histidine — 0.0547, asparagine — 0.0221, serine — 0.0050, glutamine — 0.0121, glycine — 0.0218, alanine — 0.0410, valine — 0.0513. Amino acids were determined by ion exchange chromatography. When carrying out hard hydrolysis (in order to determine the bound amino acids), a rather wide spectrum of amino acids present in small amounts is determined in the preparation. The qualitative composition of free amino acids is less diverse, they are also present in small quantities.

The preparation contains fatty acids, including arachidonic acid, which is an important component of the complex of metabolites that stimulates the growth and development of plants. The fatty acid content was determined by gas chromatography.

The preparation also contains ascorbic acid and citrine (vitamin P). They were determined by the color reaction method.

Data on the chemical composition of the drug, mg / g dry. weight:

Phytohormones 18,478

Amino acids 0.0660

Carbohydrate 751,000

Ash elements 27,737

The resulting biostimulant is precipitated with ethanol, acetone, heat-resistant up to 150 ° C, characterized by biological stability, resistant to microorganisms.

The invention is characterized by the following examples.

Example 1

The strain Muselia sterilia grows in a rich organic medium (liquid wort) under aeration conditions at 20-30 ° C, and within 48 hours it produces 25 g of second metabolites that exhibit biostimulating activity. The cultivation of the strain is carried out on a liquid medium Geltzer, the duration of the cultivation of 20-30 days at a temperature of 26 ± 1 ° C.

The composition of the medium: liquid nutrient medium Gelzer, g / l, pH 6.0-6.4:

Glucose 8.0

KN ₂ PO ₄ 0.6

K ₂ HPO ₄ 1.8

MgSO ₄ 0.2

K ₂ SO ₄ 0,1

MnSO ₄ Traces

FeSO ₄ Traces

L-Asparagine 0.02

Water Up to 1000 ml

As a source of carbon nutrition used glucose in a concentration of 1.0% or 2.0 wt.%.

When cultivated in a liquid nutrient medium, the inventive strain requires an additional growth factor - fermentation of BVK in an amount of 0.2%. Cultivation is carried out at 28-30 ° C, under aeration and stirring in flasks of 750 mm ³ volume with 100 ml of medium of the specified composition for 48 hours on a rocking chair (220 rpm). The optimal pH value was established for the growth of Mycelia sterilia culture 6.8-7.0 and for the synthesis of metabolites - 6.8-7.2.

The process is stable, the strain steadily retains the ability to synthesize metabolites in five consecutive fermentations.

The obtained liquid preparation based on the claimed strain was carefully mixed and used for processing (applying to the soil) plantings of seedlings of spruce, gooseberry, currant (foliar top dressing), potatoes (watering seedlings), late cabbage (top-dressing seedlings on the eve of planting in the ground), tomatoes (foliar top dressing), cucumbers (watering seedlings). We conducted morphophysiological and phenological observations, took into account the yield of products, determined some parameters characterizing the quality of products. The results for individual crops are shown in table 3. Control (100%) - indicators of production from the site, not processed by the claimed strain.

The inventive strain has a positive effect on plants through direct and immediate stimulation of plant growth due to the synthesis of various metabolites useful for plants. The drug, made on the basis of the inventive strain, will be widely used in modern agrobiotechnology.

Second metabolites, the composition of which is shown in table 1, synthesized by the claimed strain, was also used in the processing of grain crops by both seed treatment and seedling spraying. In addition, a positive result was obtained when processing crops of sunflower, sugar beets, buckwheat. Productivity in these crops increased by an average of 32-34%.

The inventive strain when conducting a patent and information search at the level of technology is not found, in connection with which we can conclude that the claimed invention meets the conditions of patentability stipulated by the Patent Law of the Russian Federation.

Claims (1)

The strain of the fungus Mycelia sterilia is a producer of a complex of biologically active substances with growth-regulatory properties, collection number VKPM F-833.