RU2103872C1 - Yeast strain exophiala nigrum used for seed treatment and plant growing - Google Patents

Abstract

FIELD: biotechnology, agriculture. SUBSTANCE: yeast strain Exophiala nigrum A-26 is obtained by irradiation of the strain E. nigrum R-11 and deposited in All-Union Collection of industrial microorganisms at N Y-748. At culturing the strain on liquid nutrient media containing nitrogen, phosphorus and potassium at 18-20 C under aeration for 2-3 days titer is 1-5 x $$$ cells/ml. The obtained suspension is used in plant growing. Suspension has hormones (cytokinins) and enzymes (oxygenases), increases plants yield and their resistance to diseases. EFFECT: enhanced effectiveness of strain. 17 tbl

Description

 The invention relates to agricultural crop production, horticulture and forestry, and in particular to biotechnological approaches to regulating the reproduction, growth and development of plants; increase their stability and productivity through the use of special microorganisms.

 It is known to use strains of the genus Trichoderma for treating plant seeds when seeds are dusted with spore-mycelial preparation 1–3 days before sowing in order to suppress the development of pathogens of agricultural plants [1].

 A known strain of Bacillus thuringiensis IMP-215, having a fungicidal effect against phytopathogenic fungi [2].

 Known strain of Streptomyces hidroscopicus M A 500, producing antibiotics that are active against aspergillus, neurospore penicilli and a number of other fungi, including phytopathogens [9].

 Known strains of actinomycetes ATCC 39271, 39272, 39273 of the Streptomyces family, used in the form of an aqueous suspension of spores with a titer of 1 x 10 spores / ml, for application to the soil or seed treatment by immersion in suspension in order to prevent fungal diseases of plants [3].

 Known drug, which is prepared on the basis of non-pathogens from the Pseudomonaceae family with the addition of macro- and microelements and growth substances, which enhances plant growth, increases disease resistance. Intended for use in crop production, viticulture, horticulture and forestry [7].

 The disadvantage of this drug is that the use of even non-pathogenic strains of Pseudomonaceae poses a certain danger to human health, since through the plasmids it is possible to transfer the properties of virulence and pathogenicity from pathogenic and conditionally pathogenic strains of Pseudomonaceae to non-pathogenic. In addition, this drug is biotechnologically inconvenient: for the preparation of this drug requires a large number of complex and expensive additives, it is not effective enough.

 Growth regulators of microbial origin are more effective [8].

 A known strain of endomycorrhizal fungus Glomus sp. N 7, used to increase the yield of agricultural plants, used to inoculate seeds before sowing [4]. Inoculation increases the grain yield by improving the phosphorus nutrition of agricultural plants on sod-podzolic soils poor in mobile phosphorus, as well as in the case of a phosphorus deficiency in fertile soils when applying nitrogen-potassium fertilizers. This strain is taken as a prototype.

 The disadvantages of this strain is the difficulty in obtaining inoculation material, since the strain belongs to obligate symbionts and is not able to reproduce on artificial nutrient media, has a positive effect only for a limited set of families, genera and species, and is effective only in certain soil and climatic conditions. In addition, it has a relatively low ability to increase productivity, does not significantly affect the improvement of food and dietary properties of the resulting crop products.

 The purpose of the invention is to obtain a new strain that is convenient in biotechnological terms, providing inoculation with improved growth and development of plants, increasing their productivity, resistance to unfavorable abiotic and biotic factors, improving the quality of crop production, providing environmental benefits in crop production technology.

 The problem is solved due to the new strain Exophiala nigrum A-26, which increases plant productivity, is convenient in biotechnological terms, is able to reproduce on artificial nutrient media, while maintaining its properties during reseeding, it can stimulate plant growth and development with increased efficiency, increase immunity and overall resistance plants to the adverse effects of biotic and abiotic factors, significantly improves the quality of the products.

 The strain of yeast Exophiala nigrum A-26 obtained by UV irradiation. Strain A-26 is stored in the collection of the Clone Implementation Company. The strain is deposited in VKPM under the number U-748.

The strain has the following characteristics:
1. Nomenclature data of A-26;
Generic name: Exophiala, species - nigrum.

 Origin: obtained as a mutant induced by the action of UV radiation from strain R-11 R. nigrum Pavlenko V.V. in 1987. The original strain R-11 Exophiala nigrum was isolated from a sample of Baikal water taken from the layer surface 5 m from the coast in the region of the city of Baikalsk in September 1977. Preliminary identification of strain R-11, its study and description was carried out by V. Pavlenko. , Kharlamov V.M., Chemerilova V.I. (5). It is used to improve the wastewater treatment of the pulp and paper mill (6), and is stored in the laboratory of the Clone Implementation Company.

 2. A method of creating a strain of A-26 Exophiala nigrum.

UV mutagenesis with fractional exposure. On the surface of the agar plates of Petri dishes filled with nutrient medium, a lawn of cells of a 4-day old R-11 Exophiala nigrum culture was applied. Cells were distributed evenly over the surface with a spatula. 1-3 thousand cells of strain R-11 were applied to each plate. The surface of agar plates coated with cells of strain R-11 was irradiated with a BUF-60 lamp located at a distance of 60 cm from the surface of the plates. Irradiation was carried out dosed: 30 '- 30' with a break between exposures of 120 minutes After irradiation, the plates were incubated in a thermostat at 22 ^o C for 4 days. Under this regimen, on average 50-100 colonies grew on plates (survival rate about 10%).

Mutant A-26 was isolated as morphological, by visual analysis of the color and morphology of colonies grown after irradiation of R-11 cells. It differed from the initial strain with a lighter grayish-brown color. With repeated cloning, a new trait was preserved. In subsequent experiments, it was found that the frequency of reversal of strain A-26 is less than 1 • 10 ⁷ , which is another confirmation of the mutational nature of strain A-26.

 3. Biotechnological characteristics of strain A-26.

 Strain A-26 produces a complex of growth substances and enzymes that stimulate seed germination, root formation processes, productivity, and also increase plant immunity and resistance. Designed for use in the reproduction and cultivation of plants, for the treatment of seeds and planting material.

 In the table. 1 presents information about the important biotechnological properties of the proposed strain A-26. The lack of information about the biotechnological properties of the prototype due to the fact that the strain of Glomus sp. N 7 does not grow on artificial media, and therefore it is impossible to conduct a comparative study. In the table. 1 provides some information about the biotechnological properties of the initial strain R-11, which is used for wastewater treatment [6]. The ability of the initial strain to stimulate the growth and development of plants, increasing their productivity was revealed later. Comparative studies have shown that strain A-26 is superior in activity to the parent R-11 of E. nigrum.

 4. Cultivation conditions.

 Perhaps surface and deep cultivation in environments used for growing yeast.

 5. Morphological and cultural properties of strain A-26.

 Cells 8.4 x 4.2 - 7.0 x 2.8 μm in size, multiply by apical, less often by lateral budding, more often unilateral. The kidneys are oval or oval-pear-shaped.

 When cultivated on special media, it is able to switch to mycelial growth. Mycelium hyphae are formed from large cells. Mycelium septate, irregularly branched, mostly true, substrate or air forms oval-shaped chlamydospores, slightly elongated, the germination tube is located at the apex, the size of the chlamydospores is 10.0 x 6.3 microns - 11.5 x 7.3 microns.

 No sexual process detected.

 Growth on solid media.

On agarized media at a temperature of 22 ^o C yeast-type colonies appear on 2-3 days. The line is straight, the profile is convex, the edge is even, the color is grayish-brown, dark with shine. The consistency is pasty. After a month, the stroke is condensed, slightly folded, unevenly pubescent. The giant colony reaches a diameter of 18 - 26 mm, convex along the edges, pubescent, grayish-brown, the edge is even, the center is raised, the consistency is pasty, compacted. On poor (minimal) media, the edge is pubescent; on the YEP edge, the roller is dark in color.

 Growth in a liquid medium.

 When growing in a minimal environment under stirring conditions, turbidity is observed on the 2nd day, on the 3rd - 4th day, the suspension is dark brownish in color. In the absence of mixing at the bottom, a dark precipitate of yeast cells: no film or ring. A month later, in the absence of mixing on the surface of the turf of mecelium, an abundant precipitate at the bottom; odor, foaming is not observed, the pH shifts to the acidic side.

 6. Physiological and biochemical properties of strain A-26.

 a) The growth of the strain depending on the conditions in comparison with strain R-11 (initial) is characterized by the data given in table. 2.

 Prototype Glomus sp. 7 is supported on mycorrhized roots, which limits the possibility of studying physiological and biochemical properties.

 b) Relation to carbon sources: strain A-26 does not ferment sugar, is able to assimilate glucose, sucrose, arabinose, ramnose, dulcite, maltose, lactose (weak), galactose (weak), raffinose, beckons; able to utilize phenols and lignin.

 c) Relation to nitrogen sources: strain A-26 is able to use a wide range of nitrogen sources (Table 3).

d) Other features of strain A-26: strain A-26 is able to grow on a minimal medium without vitamins, does not need additional growth factors, exhibits weak proteolytic activity: layered gelatin liquefaction. Fat does not hydrolyze. Esters and starch-like compounds do not form. Aminolytic activity does not have
7. Marker signs of strain A-26.

Strain A-26 contains a mutation that blocks one of the stages of melanin synthesis. Instead of black, characteristic of the parent pigment strain, strain A-26 synthesizes a brownish pigment. It reverses spontaneously with a frequency below 1 • 10 ⁷ : - when analyzing a sample of 1 • 10 ⁷ colonies, spontaneous reversal to the original phenotype was not detected.

 When cultured on solid media, the pigment does not emit. It produces a complex of growth substances and enzymes that determine the property of the strain to stimulate the growth, resistance and productivity of plants. These properties are stable during storage for 3 months and are preserved during reseeding. The planting period for museum specimens of strain A-26 Exophiala nigrum is 3 months.

 8. The geno- and chemotaxonomic characteristics of strain A-26 have not been studied.

 9. When checking pathogenic, virulent, allergenic, toxicogenic and toxic properties, the complete safety of strain A-26 E. nigrum was established.

 Strain A-26 E. nigrum is recommended for practical use as safe for warm-blooded organisms and plants.

 10. Examples of the use of strain A-26 E. nigrum.

 Example 1. The preparation of the uterine culture of strain A-26 E. nigrum, necessary for the manufacture of a biological product, was carried out as follows. A museum specimen of E. nigrum strain A-26 was subcultured onto solid Petri dishes. On the 4th - 5th day after sowing, the lawn of the grown knees was examined and, in the absence of extraneous microflora, they were used to build up the A-26 uterine culture in a liquid medium. In 1-2 liter flasks, filled into 2/3 of the volume with sterile liquid medium, 1 agar plate with a lawn of cells of strain A-26 grown on it was lowered.

Flasks with medium inoculated with strain A-26 were placed on a rocking chair or magnetic stirrers and cultured with constant stirring for 24 hours at a temperature of 20 - 22 ^o C. On the second day, when the titer of the suspension reached 1-5 • 10 ⁸ cells / ml. The resulting uterine culture was transferred to larger fermenters filled with sterile liquid medium of the same composition. It is possible to use simplified versions of liquid media containing nitrogen, phosphorus and potassium in a ratio of 90: 60: 30.

The titer of the initial suspension after making uterine culture 1-10 • 10 ⁵ cells / ml. Cultivation in the fermenter at t 18 - 20 ^o C with constant or periodic (intervals 20'-20 ') sparging with air or mechanical stirring. After 2-3 days, the titer of the cell suspension reached 1 - 5 • 10 ⁸ cells / ml.

 The resulting suspension is used later as a drug used in crop production. In addition to living A-26 cells, it contains metabolites produced by cells: - hormones (IAA, cytokinin, etc.) and enzymes - oxygenases.

Example 2. Investigated the stimulating effect of strain A-26 on the germination of seeds of cucumber cultivar Nezhinsky. For processing, a suspension of strain A-26 was used with a titer of 1 • 10 ⁷ cells / ml (experiment No. 1) and with a titer of 5 • 10 ⁵ cells / ml (experiment No. 2). They were prepared from uterine culture. Depending on the titer, it was diluted 10 or 50 times for experiment No. 1 and 200 or 1000 times for experiment No. 2. A liquid minimal medium was used for dilution.

 Strain Glonus sp. N 7 cannot be used under similar conditions: its effect on seed germination is unknown.

 Suspension treatment and seed germination were carried out in Petri dishes. At the bottom of the cup were placed 10 calibrated seeds of cucumber and 25 ml of a suspension of the corresponding titer were added.

 The processing efficiency was evaluated by such indicators as the length of the root, stem and cotyledons; the number of subordinate roots, the weight of the seedling. In the table. 4 shows the test results.

The data presented in the table indicate that seed treatment with a suspension of A-26 (experimental version 1) and a suspension diluted 20 times with a titer of 5 • 10 ⁵ cells / ml (experimental version 2) have high stimulating properties. The seedlings have the largest mass, a shortened, highly developed root with the largest number of subordinate roots. Shortened, well-developed stem. Moreover, the best indicators of seedling development were observed in experiment variant 1, where the titer 1 • 10 ^{7 was used} . The decrease in the titer of A-26 cells below 5 • 10 ^{5 is} impractical, because the positive effects of strain A-26 on seed germination are significantly reduced. Increasing the titer of the suspension of strain A-26 above 1 • 10 ⁷ is also impractical, because in this case, inhibition of growth processes is observed due to excessive concentrations of growth substances produced by cells of strain A-26.

 Example 3. The effect of inoculation of endomicoricorrhizal fungi by the strain Glomus sp. N 7 (prototype) and strain Exophiala nigrum A-26 on the harvest of oat grain in vegetation experiments. Used cultivated arable land, the experiment was set as described for Glomus sp. N 7 in example N 1 [4].

Dry non-sterile soil with a humus content of 2.9%, total nitrogen of 0.19%, P ₂ O ₅ (according to Krisanov) 2.5 mg / 100 g of soil, pH 6.6 was placed in a 3-liter growing vessel. Then, a suspension of Exophiala nigrum A-26 cells with a titer of 1 • 10 ⁷ cells / ml at a rate of 200 ml per 1 kg of soil was introduced into the vessel as inoculation material. After thorough mixing with inoculation material, the soil was seeded with surface sterilized oat seeds. Watering the soil, as in the case of Glomus sp. N 7 was carried out up to 60% of the total moisture capacity. Experimental results compared with data for Glomus sp. N 7 [4] are presented in table. 5.

 The data obtained confirm that the strain A-26 E. nigrum is effective when used for inoculation of soil. The increase in oat yield in vegetation vessels filled with the previously inoculated strain A-26 soil is 26.1 - 27.2%, which is significantly higher than in the case of Glomus sp. N 7 - the ability to grow and multiply on artificial nutrient media, allows you to use it directly for inoculation of seeds and other planting material, and not just soil, as in the case of the prototype. Thanks to this, it is possible to use the proposed strain A-26 in production conditions over large areas without significantly complicating the usual technology for growing grain, fodder and vegetable crops: in horticulture and afforestation.

Example 4. Under production conditions, the effect of strain A-26 on the growth development, maturation rate and yield of wheat, barley and oats was investigated. In experimental variants used strain A-26. The mother culture was prepared as described in Example No. 1, then it was diluted 10-50 times to a titer of 1 • 10 ⁷ - 5 • 10 ⁵ cells / ml. The treatment was carried out on the day of sowing or 1 to 3 days before sowing using seed dressing machines: PS-10 or Mobitox. The consumption of cell-enzyme suspension A-26 10 - 15 l per ton of grain. For winter and spring crops, it was found that A-26 stimulates germination, reduces the percentage of rot-affected seeds. In the table. 6 presents data on the state of seedlings of winter wheat Bezostaya -1, as well as the percentage of healthy and non-germinated sprouts affected by rotting seeds. Accounting was carried out 1 month after sowing. The size of the accounting plots is 30 cm in a row, with five replicates in each variant.

 The treatment of cereal seeds before sowing the cell-enzyme suspension A-26 stimulates the formation of a powerful tillering site in plants. A powerful root system provides good contact with the soil, which, in turn, is the basis for a better supply of plants with moisture and mineral salts. Under the action of A-26, the laying and development of a larger number of stems is provided. In the table. 7 presents data on the increase in productive bushiness in different wheat varieties under the action of A-26.

Estimation of the value of productive bushiness was carried out 1-2 weeks before harvesting. Sheaves were taken from plots of equal size, most often the size of the plot was 1/16 m ² (0.0625 m ² ). In the experimental and control variants, 3 to 7 sheaves were selected and analyzed. Productive bushiness was estimated as the ratio of the number of stems with an ear to the number of plants in each of the sheaves. Table 7 presents the average values of productive bushiness for different varieties of wheat.

 For both winter and spring wheat, the productive bushiness in the experimental variants, where the seeds were treated with strain A-26 before sowing, is significantly higher than in the control. The excess of productive bushiness is 25.0-37.5%. At the same time, the difference in thickened crops was found to be less, and in thickened more. Given the facts of an increase in the percentage of seed germination under the action of A-26, on the one hand, and an increase in productive bushiness under the action of A-26, on the other hand, it is logical to lower the grain sowing rate in the case of the use of strain A-26. It seems most appropriate to reduce the seeding rate by 20-30 compared with the control, where inoculation was not used.

 In the table. Figure 8 presents the results obtained during field trials (plot tests), according to yield estimates in the experimental and control variants. In the experiment, the seeding rate was reduced by 20% compared with the control. The yields of barley (cultivar Dawn) and spring wheat (cultivar Skala) were evaluated. Seeds the day before sowing were inoculated with a suspension of A-26 E. nigrum.

 Thus, it was found that inoculation of seeds with strain A-26 before sowing provides a yield increase even when the seeding rate is reduced compared to untreated A-26 control. The yield increase for barley was 9.2 kg / ha (51.1%), and for wheat - 5.8 kg / ha (35.8%).

 Analysis of the structure of the crop revealed that the increase in yield is due to an increase in productive bushiness, an increase in the grains of ears and an increase in the mass of grain.

 In the table. Figure 9 shows the data obtained during production tests on the effect of strain A-26 on wheat yields.

 Compared with the control, where inoculation with the A-26 cell-enzyme suspension was not performed, the wheat yield in the experiment increased by 5.8 - 10.9 kg / ha (18.9 - 37.5%), for spring wheat and by 10, 3 - 11.2 kg / ha (26.6 - 45.1%) for winter wheat varieties.

 Strain A-26 has a positive effect on the quality of grain, flour and bread. Grain samples for a complete technological analysis of grain, flour and bread were taken during harvesting. The results of the analyzes are given in table. ten.

 The use of strain A-26 allows to obtain crop products that do not contain residual pesticides. This is primarily due to the fact that oxygenase enzymes produced by strain A-26 are capable of highly active destruction of many pesticides accumulated in the soil. Secondly, the use of A-26 eliminates seed poisoning with poisons, since it has been established that treating seeds with strain A-26 increases the overall resistance of plants, including disease resistance. In the experimental variants, root rot, smut and dust smut were absent or met much less frequently than in the control variants. Plants suffered less from brown rust.

 In the table. 11 presents data on the effect of A-26 on a decrease in the percentage of plants affected by smut smut. Plants developing from treated A-26 seeds "go away" from the smut.

 Thus, strain A-26 stimulates resistance to adverse conditions and diseases in cereals, increases bushiness, including productive bushiness, increases productivity, shortens the stages of developmental stages, accelerates maturation, improves the quality of grain and flour by increasing grain weight, increasing grain content of protein and gluten.

 Example 5. The effect of strain A-26 on resistance to viral and bacterial diseases, yield and potato ripening rate was investigated. Balls of potato were processed before planting with strain A-26 Exophiala nigrum (experiment) and strain R-11 (control-1), preparation as described in example 1. Additionally, variants were laid as control where the balls were treated with trichodermin (control-2) and the chemical drug cuprozan (control-3). The treatment of tubers was carried out at the time of planting. Through the nozzles of the SKM-4 planter planter, a preparation — a solution or suspension of cells and enzymes from the tank — was supplied to the coulter through a sprayer using a gear pump. Tubers falling through the exhaust window passed through the spray zones and were treated uniformly with the preparation.

 In the table. 12 presents data showing the positive effect of strain A-26 Exophiala nigrum on the resistance of potato plants to damage by viral, bacterial and fungal diseases. The percentage of diseased and removed plants in the A-26 variant is significantly lower than in the control variants. In addition, it was found that in the experimental version, where the balls were treated with strain A-26 before planting, the yield was 5–35% higher than in the control variants, and ripening occurred 10–15 days earlier.

 Example 6. The effect of strain A-26 on the yield and quality of the obtained products of vegetable crops was investigated. The use of strain A-26 in growing vegetables allows you to get diet products with a high content of vitamins and sugars; not containing excess nitrates.

 In the table. 13 presents data on the nitrate content of carrots and cabbage grown from seeds, treated before sowing A-26 (experiment A-26) and not processed (control b / o).

 The use of A-26 for growing tomatoes increases their resistance to diseases and pests, increases their productivity, improves their taste.

 In the table. 14 presents data on the content in fruits of tomatoes grown using A-26, sugars, vitamin C, as well as the dry matter content.

 The use of A-26 for growing sugar beets increases its productivity, increases sugar content, and increases resistance to diseases.

 The dietary properties of vegetables grown using strain A-26 E. nigrum are further enhanced by the fact that they do not contain residual pesticides in their metabolites. This is because A-26 allows you to grow vegetables without the use of pesticides or dramatically reduce their use. In addition, strain A-26 produces enzymes capable of degrading pesticides used to treat plants that have been preserved in soil from previous treatments.

 In the table. 16 presents data on increasing the resistance of cucumber plants to diseases. Cucumber seeds were soaked in a cell-enzyme suspension of strain A-26 (preparation of the drug is described in example 1). Seeds were placed for 3–10 h in a cool (15–18 C) place. Sown as usual. After emergence, a periodic treatment with strain A-26 was performed.

 Strain A-26 helps to grow organic vegetables, accelerating the destruction of pesticides. In the table. 17 presents the results of semi-production tests of the ability of strain A-26 to decompose synthetic sumi-alpha, which is used to combat insect pests, characterized by high persistence.

 Cabbage plants were treated with Sumi-alpha. After 24 hours, half of the site was treated with a cell-enzyme suspension of strain A-26 (experiment), the second with water (control).

 After 48, 96, and 144 h, plant samples of the experimental and control variants were taken, and the Sumi-alpha content was analyzed in them.

 Example 7. Strain A-26 was used to grow a meristem carnation. Before planting, clove seedlings roots were mulched in a specially prepared mash of clay soil, humus and a suspension of strain A-26 with a titer of 5 • 10 cells / ml. The suspension was prepared as described in example 1. The titer of the working suspension was adjusted to the desired one by dilution with water. During the first month after planting, the experimental beds were watered with a suspension of strain A-26 once a week. In the control, when planting clove seedlings, the roots of the plants were mulched with a mash mill prepared on water. Watering was carried out as usual.

When comparing the experimental and control options, it was noted:
a) in plants in the experiment, the first buds appeared much earlier than in the control;
b) the quality of the colors in the experimental version was significantly higher than in the control;
c) the number of colors in the experiment was 2.5 times greater than in the control;
d) the plants of the experimental version were distinguished by a healthy appearance, increased resistance to diseases and pests.

 e) color cultivation using strain A-26 made it possible to exclude treatment with poisons.

 Thus, the strain A-26 Exophiala nigrum compared with the prototype Glonus sp. N 7 is convenient for propagation on artificial media and long-term storage in museum collections, stably retains valuable properties for practical use in crop production. In addition, this strain is not demanding on the substrate. The complex of properties listed in examples 1 - 7 determines the advantages of using the proposed strain in the practice of crop production.

Sources of information
1. Methodological recommendations for the use of trichodermin in vegetable growing. Comp. Nikolaeva S.N., Buymistra P.D., Bondarenko A.N. Chisinau, 1987, p. 9-11.

 2. Auth. St. USSR N 1144377, class C 12 P 1/04 / (C 12 P 1/04; C 12 R 1: 07) 1983.

 3. Application (France) N 2524486, cl. C 12 N 1/20, A 01 N 63/04 // C 12 R 1/465, 1983.

 4. SU Auth. St. N 1145503, CL A 01 N 63/00, C 12 N 1/14 (C 12 N 1/14, C 12 R 1: 645) 1983 - prototype.

 5. Pavlenko V.V., Chemerilova V.I., Kharlamov V.Sh. The use of "black yeast" in toxic genetic experiments. In the book. Research on GNP genetics 9. Leningrad University Press, 1981, pp. 9-17.

 6. Auth. St. USSR N 1071637, class C 12 N 15/00 / C 02 F 3/34; C 12 R 1/00, 1982.

 7. Application (France) N 2594294, cl. A 01 N 63/03; 65/00. 1987.

 8. Muromtsev G.S. et al. Plant growth regulators of microbial origin - Advances in Microbiology M., 1984, no. 19, p. 106 - 135.

 9. Patent (USA) N 4575500, class. A 01 N 57/12, C 07 F 9/09, NKI 514/121, 1984.

Claims (1)

 The strain of yeast Exophiala nigrum VKPM U-748 used for seed treatment and plant growth.

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