RU2161884C1 - Association of microorganisms for production of plant growth control agent, method of production thereof, and plant growth control agent - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: association of microorganisms consisting of micromycete Aeremonium sp., yeast Rhodotorula glutinis, bacteria Enterobacter agglomerans and Azotobacter beijerinckii is isolated from ginseng root and deposited in the Moscow State University collection under No.452. Plant growth control agent is prepared by culturing above association for 1 month on nutrient medium containing carbon and nitrogen sources and mineral salts. Culture broth is extracted with alcohol. Extract solution is allowed to stand for some time, after which precipitated mycelium is separated. Resulting colorless transparent or slightly opalescent solution with specific smell contains gibberellins, purine bases, and amino acids. Preparation increases germinating power of seeds, crop harvest, and reduces plant disease occurrence. EFFECT: optimized preparation procedure. 4 cl

Description

 The invention relates to biotechnology and for the receipt of funds that increase crop yields from a community of microorganisms isolated from ginseng roots.

 A community of microorganisms is known for obtaining a bioregulator of plant growth, including endophyte fungi isolated from the roots of ginseng, sea buckthorn (1-2). A disadvantage of known communities is the uncertain composition of microorganisms within the community.

 A community of microorganisms for producing a plant growth regulator is known, consisting of micromycete Acremonium panaxeorum UHMU F-160, yeast Candida muscorum UHMU U-411, Rhodotorula glutinis UHMU U-42 and bacteria Azomonas macrocytogenes UHMU B-28 and Xantobacter sp3 UHMU B-28 and Xantobacter sp3 )

 Known methods for producing plant growth regulators by cultivating one of the above communities on a nutrient medium containing a carbon source, mineral salts, amino acids, followed by the isolation of biologically active substances stimulating plant growth from biomass (1-4).

 Known methods were obtained preparations Symbiont - 1 (1), Symbiont-Universal (3). These preparations are alcohol extracts from biomass of microorganism communities. The aim of the invention is the expansion of sources of raw materials from new communities of microorganisms and increase the efficiency and stability of drugs.

 The goal is achieved by the fact that a new community of microorganisms is isolated from ginseng roots, consisting of micromycete Acremonium sp, yeast Rhodotorula glutinis, and bacteria of the species Enterobacter agglomerans and Azotobacter beijerinckii. Microorganisms in the community are in a ratio of 90: 2: 1: 1. The community is stored in the collection of Moscow State University under number 452.

 Microorganisms entering the community have the following characteristics.

Cultural and morphological characteristics of Enterobacter agglomerans bacteria. When grown on MPA and BSA, they form white colonies, mucous, smooth up to 3-4 mm in diameter. On a high glucose-rich Saburo medium, it forms exopolysaccharides. Culture on a liquid medium is a single movable sticks sometimes in pairs. Cells growing on a solid medium, in a day form short chains, pairs or are represented by single forms of different lengths. The size of the sticks is 1.2-3 microns, in diameter ~ 1 micron. Endospores do not form. The culture belongs to the group "Optional anaerobic gram-negative rods." The optimum growth temperature is 25-30 ^o C, growth is observed both at 37 ^o C and at 15 ^o C. It dilutes gelatin, casein hydrolyzes, starch does not hydrolyze, and does not form pigments. Uses ammonium salts, organic compounds and nitrate as a source of nitrogen. Restores nitrate to nitrite. It grows well on citrate, does not synthesize urease (there is no growth on the medium with urea). Hydrogen sulfide from cystine and indole from tryptophan does not form. It grows well on the Ashby medium and Fedorov’s liquid nitrogen-free medium. Culture has the ability to nitrogen fixation. It uses the following carbohydrates to form acid: glucose, glycerin, D-mannose, D-xylose, maltose, melibiosis, raffinose, arabinose, L-rhamnose, D-sorbitol and sucrose. Of many of these carbohydrates, it forms gas. Identification of the species of bacteria was carried out on the basis of the determinant Bergey's Manual of Systematic Bacteriology (in four volumes), 1984, Baltimore / London.

 Cultural and morphological characteristics of the yeast Rhodotorula glutinis.

When growing yeast on a solid agar medium containing 10 ^o B wort, and glucose-peptone medium with the addition of yeast extract, small pink colonies with a diameter of 2-3 mm are formed. The colonies are rounded, the edges are pink, the center of the colony is raised, the surface is dull, ring grooves are noticeable with age. The stroke is bright pink, smooth, shiny. Pseudomycelia does not form a culture. The cells are oval or oblong, 1.5 · 2.0-3.0 μm in size. Yeast propagates by budding. They do not form a dispute. It uses both organic and inorganic forms of nitrogen as a source of nitrogen for growth. It uses glucose, sucrose, maltose, cellobiose, raffinose, mannitol, ethanol and succinate as the sole source of carbon and energy. Does not assimilate lactose, melibiosis, inulin, soluble starch, rhamnose, citrate, methanol. Weak growth is observed on arabinose, xylose, glycerin, lactate. Does not need vitamins. The temperature range of growth of 10-40 ^o C. Optimum 15 - 28 ^o C.

 The identification of yeast culture was carried out on the basis of the determinant, edited by Babev I.P., Golubev V.I. "Methods for the isolation and identification of yeast", Moscow: Food Industry, 1979. The Yeasts / A taxonomic stady / Sec cd / by Lodder, Amsterdam-London, 1970.

Cultural and morphological characteristics of micromycete Acremonium sp. When growing Acremonium sp micromycete on Chapek’s agar medium, white colonies are formed, after a few days the colonies turn creamy, and in old cultures they are dark brown, right down to black. The mycelium is plentiful cotton-like. Poorly removed from agar. With age, the back of the colony becomes brown. And in old cultures black. On the aerial mycelium, single conidiophores form, sometimes in whorls. Conidia are formed at the apex of the conidiophore, solitary and / or in the false heads. Conidia are colorless, mostly cylindrical with rounded ends. The conidia length reaches 17 microns, a thickness of 5 microns. There are conidiospores with a septum. Along with large cylindrical conidia, small ones from oval to spherical are found. On a liquid medium, the mycelium of the fungus is represented by septated hyphae with many strands surrounded by a thick mucous membrane encrusted with bacterial cells. Growth in liquid culture is accompanied by the formation of chlamydospores. The optimum growth temperature is 20 ^o C. A pure micromycete culture is maintained and stored on Czapek agar medium and on wort agar. Micromycete is stored in the collection of microorganisms of Moscow State University under N 441.

 Micromycete identification was carried out on the basis of the following sources: M. Litvinov "Key to microscopic soil fungi", L.: Nauka, 1967 Pidoplichko N.M. "Mushroom parasites of cultivated plants", Kiev, "Naukova Dumka", 1977, volume 2, Gilmal J.C. "A manial of soil fungi" Second ed. The Iowa State Univ. Press Ames. Lowa, USA, 1984.

 Cultural-morphological and physiological-biochemical characters of Azotobacter beijerinckii.

During the cultivation of bacteria, Azotobacter beijerinckii on meat-peptone agar (MPA) forms dense white and smooth, slightly convex colonies, which acquire a beige tint upon aging. Colonies with a diameter of 1-2 mm, acquiring a wavy edge with age. The colonies are soft and easy to remove from agar. Cells of at least 1.5 μm in diameter, cocciform, are arranged in pairs, forming as if eights. The cells of the daily culture on both solid and liquid media are motionless. With negative ink staining, the mucous capsule surrounding the cells is clearly visible. With aging, the culture forms cysts. The culture is gram-negative, catalase- and oxide-positive, aerobic, able to grow with a low oxygen content in the medium. It grows well on Ashby. Culture forms a lot of mucus. It fixes molecular nitrogen in an acidic environment at pH 5.5 - 6.0. It grows well at 20-30 ^o C, at 37 ^o C no growth. Uses nitrate and restores it to nitrite; hydrogen sulfide does not form from cysteine. It does not dilute gelatin. Starch and casein do not hydrolyze. As a single carbon source, it assimilates fructose, glucose, sucrose, ethanol, lactic and propionic acids. Ramnose does not use.

 Identification of this type of bacteria was carried out according to Bergey's "Manual of systematic Bacteriology", 1984.

The invention is illustrated by the following examples:
Example 1: obtaining a plant growth regulator "Symbiont".

The microbial community, consisting of micromycete Acremonium sp, yeast Rhodorula glutinis, bacteria Azotobacter beijerinckii and Enterobacter agglomerans, KM MSU 452, seeded in medium containing (%):
Glucose - 0.8
K ₂ HPO ₄ - 0.03
MgSO ₄ - 0.02
K ₂ SO ₄ - 0.01
FeSO ₄ and MnSO ₄ - Traces
Asparagine - 0.001
Distilled water - Up to 1 L
Cultivated under stationary conditions at an initial pH of 6.5 and a temperature of 20 ^o C for a month. At the end of the cultivation process, the biomass is separated. Biologically active substances are extracted from biomass with alcohol, aged, the precipitate formed is removed, and the Symbionta preparation is obtained, which is a water-alcohol colorless, transparent or slightly opalescent solution of the products of the above-mentioned microorganism community with a specific odor. The preparation contains gibberillins not less than 0.05 mg / ml, purine bases not less than 0.02 mg / ml and amino acids not less than 1 mg / ml, the content of heavy metals does not exceed 0.001%, arsenic and mercury are absent, pH 5.4-5-5 ,8.

 The ethyl alcohol content is 48-54%.

 The dry residue is not less than 0.25%.

 Of the amino acids, the regulator contains glutamine, glycine, alanine, valine, histidine, cytisine, guanine.

 The resulting preparation stimulates the development of mycorrhiza, which leads to improved plant nutrition. The resulting mycorrhizal fungi are ahead of pathogenic ones in their development, occupying the same ecological niche, contributing to a decrease in the degree of damage by fungal phytopathogens.

 It protects the plant during the growing season. The speed of exposure is 8-10 days after the growing season.

 The regulator is compatible with all plant protection products: disinfectants, insecticides, fungicides, herbicides and plant growth regulators. The drug has been tested on various plants.

 Studies conducted during the cultivation of cabbage of the Slava variety showed that the soaking of seeds in the Symbiont solution with a consumption rate of 2 ml / 1 kg for 30 minutes and subsequent spraying of plants 3 weeks after transplanting into the soil at a consumption rate of 1 ml / ha provided a reliable increase in cabbage productivity by 11-30%. The use of the drug contributed to increasing the energy of seed germination, their germination and improving the quality of seedlings. The defeat of seedlings of cabbage "black leg" decreased by 15%, cabbage leaves by various bacterioses by 15-20%, persniporosis by 5-12%.

Studies of the biological effectiveness of "Symbiont" on a cucumber (grade Nezhensky). The most effective was the soaking of seeds in the plant growth regulator with a consumption rate of 1 ml / kg, a working solution consumption rate of 2 l / kg, a soaking time of 30 minutes, 2-fold spraying: 1st in the phase of formation of 1-2 leaves, 2 in the phase of formation of 4-5 leaves. The consumption rate of the drug is 1 ml / ha, the consumption rate of the working solution is 300 l / ha. The increase in the yield of cucumber was 16.7% or 1.53 kg / m ² . In field trials, the yield of cucumber increased by 33%.

 As a result of 2-year trials of the plant growth regulator on Bodrost pepper, the most effective regulations for the use of the growth regulator were determined: soaking the seeds for 30 minutes, the consumption of the drug 2 ml / kg, the flow rate of the working solution 2 l / kg and spraying in the flowering phase 1 ml / ha, at a rate of consumption of a working solution of 300 l / ha. Studies have shown that the use of "Symbiont" contributed to the increase in the yield of pepper - 31%, the number of pepper fruits on the plant increased, the weight of 1 fruit.

Tests carried out on Universal 3 eggplant showed that the most effective method of eggplant processing is to soak the seeds for 30 minutes in the Symbionta solution and spray the plants in the flowering phase at the above application rates. Eggplant productivity increased by 13.8-16.8% and amounted to 1.13 - 1.12 kg / m ² . More developed and healthy eggplant seedlings were obtained, the mass of 1 fruit and the amount on average per plant increased.

 The drug increases the yield of buckwheat by 9-11%, while the germination and energy of seed germination has increased, the weight of grain on average from 1 plant. Buckwheat productivity increased by 9-11%. The drug also increases the yield of sugar beets by 10-20%, the sugar content of root crops by 0.5-0.8%. The drug was tested in growing asters. The use of the regulator ensured an increase in the height of the aster plant, an increase in the number of inflorescences and their diameter,% of blossoming flowers, and a decrease in the affection of the black-legged aster seedlings.

 Treatment of watermelon seeds of the Ogonyok and Kherson early varieties with growth regulator at a dose of 4 ml / kg, with a working solution consumption of 2 l / kg, contributed to an increase in watermelon yield by 36-40%, yield increase was 42-80 c / ha, with control yield 105-220 kg / ha, respectively.

 From the above data it follows that the resulting preparation helps to increase crop yields and plant resistance to pathogens.

Sources of information:
1. Copyright certificate N 370932, 11/22/73, A 01 N 65/00.

 2. Copyright certificate N 921488, 04.23.82, A 01 N 65/00.

 3. Patent N 2100932, 01/10/98, A 01 N 63/00.

 4. Copyright certificate N 1824148, 06/30/93, A 01 N 65 / 00.3

Claims (4)

 1. A community of microorganisms consisting of Acremonium sp micromycete, Rhodotortula glutinis yeast, Enterobacter agglomerans bacteria and Azotobacter beijerinckii KM MSU 452 to obtain a plant growth regulator.  2. A method of obtaining a plant growth regulator, comprising cultivating a community of microorganisms with subsequent extraction of biologically active substances with alcohol from the resulting biomass, characterized in that the community according to claim 1 is used from the community of microorganisms.  3. Plant growth regulator containing an alcoholic extract of the biomass of the microorganism community, characterized in that it contains an alcoholic extract of the biomass of the microorganism community according to claim 1.  4. The plant growth regulator according to claim 3, characterized in that it contains gibberillins of at least 0.05 mg / ml, purine bases of at least 0.02 mg / ml, amino acids of at least 1 mg / ml, alcohol 48 - 54% , the dry balance of trace elements is not less than 0.25%, while arsenic and mercury are absent.