Strain cylindrocarpon radicicola - producer of biologically active substances with postregulatory properties

 

(57) Abstract:

The invention relates to the microbiological industry. The strain of the fungus Cylindrocarpon radicicola stored in the all-Russian collection of industrial microorganisms, collection number VKPM F-861, is a producer of biologically active substances with postregulatory properties. The invention allows to obtain a drug used as a bio-stimulator of plant growth. 3 table.

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

Known community of microorganisms for receiving plant growth regulators, consisting of micromycete Acremonium panaxeorum UHMU F-160, yeast Candida muscorum UHMU Y-411, Rhodotorula glutinis UHMU Y-42 and bacteria Azomonas macrocytogenes UHMU B-28 and Xantobacter sp UHMU B-29 /Pat. RF 2100932/. The disadvantage of this community is the difficulty and complexity of its cultivation, the instability of the drug received.

Known drugs that stimulate the development and growth rmati-and-machihe. Each product consists of dissolved in 70% ethyl alcohol products of metabolism associated endophyte. Processing one of the listed drug of wheat seeds, cucumbers or potatoes before sowing increased the harvest /geltzer F. Y. New producers stimulating substances to plants. Reports of agricultural Sciences, No. 5, 1975/.

Known drug “Symbiont-2”, stimulating the productivity of plants, obtained from the products of metabolism of fungi endophyte (ed.St. The USSR №921488). As raw materials for its manufacturing use endophyte isolated from the roots of sea-buckthorn.

A disadvantage of the known stimulators of growth is the low activity of preparations derived from endophyte plants with the highest medicinal value.

Known drug Symbiont-1 carrier made of endophyte isolated from ginseng root. The drug is effective only under good conditions and plant growth (ed.St. The USSR №370932).

A common disadvantage of known products is the inability to identify produced using known strains of products, the content of significant quantities of alcohol in the product.

The closest technical solution to tawlae the patent of Ukraine No. 17558/. The strain isolated from the root system of sea buckthorn. As the nutrient medium used, g/l: glucose, KN2RHO4TO2NRA4, MgS4TO2SO4, MnS4, FeS4, asparagine. Its main drawback is the complexity of its receipt on an industrial scale, the strain has efficacy only in the root zone of a particular host plant.

The aim of the invention is the expansion of sources of raw materials of new strains and increasing the activity of growth regulator.

The objective is achieved by the fact that from the roots of ginseng isolated a new strain of Cylindrocarpon radicicola. The strain is stored in the all-Russian collection of industrial microorganisms, collection number VKPM F-861 (certificate of Deposit attached). The strain is used for obtaining a biostimulant plant growth.

The strain isolated from ginseng root. Species the name of culture Cylindrocarpon radicicola Wollenweber, the name of the strain NTN-10. Pedigree strain: natural isolate.

Cultural and morphological characteristics of strain: aerobe, aerial mycelium ragged, yellowish-white, the colony on the reverse side of ochre. Macroconidia cylindrical, apex rounded (20-454-7 microns), typically with three poperen is crocodilia spherical (3-3,5 μm) in diameter. Chlamydospores intermediate (intercalary), spherical, numerous, chains, brown, 10-16 microns in diameter.

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

Under cultivation of strain for three weeks in liquid medium geltzer, followed by separation of complex secondary metabolites obtained the drug gives the growth of the hypocotyl experimental lines mustard 30-60% of control (water).

Biological activity was determined using the standard methods of biotests on the seeds of plants.

The strain was kept on agar nutrient medium at a temperature of 4-7C with re-seeding once in 3 months.

Agar medium geltzer (HA), g/l, pH 6,2-6,6:

Glucose 8,0

KN2RHO40,6

TO2NRA41,8

MgSO40,2

TO2SO40,1

MnSO4Traces

FeSO4Traces

L-asparagine 0,02

Agar-agar 15,0

Water Up to 1000 ml

The reproduction of culture carried out by subculture on agar medium geltzer, the composition of which is presented above.

Conditions and the composition of the medium for fermentation: liquid nutrient medium geltzer duration of cultivation is 0

KN2RHO40,6

TO2NRA41,8

MgSO40,2

K2SO4 0,1

MnSO4Traces

FeSO4Traces

L-asparagine 0,02

Water Up to 1000 ml

The strain is not zoopathogenic, is not pathogenic, there is no danger to the environment.

Drug-based strain NTN-10 received when grown in culture flasks. Cultivation in flasks was carried out in two stages, after the first cycle was selected culture fluid was added to fresh medium in the amount of 30% of the original volume and was carried out by the second cultivation cycle. When conducting biotests and chemical analyses used lyophilized culture filtrate liquid.

The content of phytohormones in the preparation: indelicacy acid (IAA), cytokinins (CC), gibberellin (HA) was determined by the chromatographic method.

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

Gibberellovaja acid HA3the most widely used in agriculture to the R action and physiological activity above.

Endoxana acid (IAA) is poststimulation, but due to poor solubility in water and relatively high cost has a narrow range recommended for cultivation of crops.

All these compounds are related to well-known plant growth regulators, are described in detail in the literature /plant growth Regulators. Edited by G. C. Muromtseva, M., 1979/.

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

The biological activity of the dry preparation on the basis of the proposed strain on the bluegrass, grade Denim (control water) and the content of phytohormones in this preparation are listed in table 1.

Table 2 shows the elemental composition of lyophilized.

Elemental composition analysis showed that the product contains almost no heavy metals. In the drug compared with the culture medium significantly increased the potassium content and molybdenum content of other elements is reduced.

The content of free carbohydrates, mg/g dry. weight: sucrose - 183; glucose - 366; fructose - 89; mannitol - 0,0050; glutamine - 0,011; glycine - 0,0340; alanine - 0,570; valine - 0,0500. Amino acids were determined by the method ionoobmennoi chromatography.

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

The drug also contains ascorbic acid and citrine (vitamin P). Was determined by the method of color reactions.

Data on the chemical composition of the drug (mg/g dry. weight: phytohormones - 19,034; amino acids - 0,543; carbohydrates - 743,000; ash elements - 24,737.

Received the biostimulator precipitated with ethanol, acetone, heat resistant to 150C, characterized biological stability, resistant to microorganisms.

The invention is characterized by the following example.

Example 1

Strain Cylindrocarpon radicicola grows on rich organic medium (liquid wort) in conditions of aeration at 20-30C, within 48 hours produces 25 g permeabilites showing biostimulating activity. Cultivation of strain operate on liquid nutrient medium geltzer, duration of cultivation 20-30 days the CLASS="ptx2">KN2RHO40,6

TO2NRA41,8

MgSO40,2

TO2SO4 0,1

MnSO4Traces

FeSO4Traces

L-asparagine 0,02

Water Up to 1000 ml

As a source of carbon supply used glucose in a concentration of 1.5 or 2.5 wt.%.

During cultivation in liquid nutrient medium declare strain requires additional growth factor - fermentolizat BVK in the amount of 0.2%. The cultivation is carried out at 28-30C, under conditions of aeration and mixing in the flasks with a volume of 750 mm3with 100 ml of medium of the composition within 48 h on a rocking chair (220 rpm). When this occurs, the acidification of the medium to a pH of 4.7 by releasing organic acids and in the culture fluid. It was found the optimum pH value for growth of culture Cylindrocarpon radicicola of 6.8-7.0 and for the synthesis of metabolites of 6.8 to 7.2.

Investigated the synthesis of metabolites declare strain in the temperature range 25-35C. To achieve the required pH values resulted in Podarok 10% NaOH solution. The glucose concentration of 2.5 wt.%.

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

Metab the grain sugar beets, potatoes, tomatoes, cucumbers, seedlings of red currants, gooseberries.

The liquid preparation on the basis of the proposed strain was carefully mixed and used for processing (soil) planting seedlings of spruce (top dressing), potato (watering of seedlings), late cabbage (fertilizing seedlings before planting in the ground), tomatoes (foliar application), cucumbers (watering of seedlings). Led morpho-physiological and phenological observations, take into account the yield of the products was determined by some parameters characterizing the quality of the products. The results of the individual tests are given in table 3. Control (100%) - performance products from the site, uncultivated declare strain.

The inventive strain carries a positive effect on plants through direct and stimulate plant growth through the synthesis of various metabolites that are useful for plants. The drug, made on the basis of the proposed strain, will find wide application in modern agricultural biotechnology.

Permeability, the composition of which is given in table 1, synthesized by the claimed strain was also used in the processing of crops by seed treatment, Solnechniy, sugar beet, buckwheat. The yields for these crops has increased by an average of 32-34%.

The inventive strain when conducting patent and information search on the technical level is not detected, and therefore it is possible to draw a conclusion on the compliance of the claimed invention for patentability under the Patent law of the Russian Federation.

The strain of the fungus Cylindrocarpon radicicola - producer of biologically active substances with postregulatory properties, collection number VKPM F-861.

 

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