RU2177466C2 - Strain of bacterium azotobacter chroococcum zao "bioflora" n b-05 for preparing biopreparation for soil productivity increase, agriculture crop yield increase and soil sanitation, recovery of broken soil and biopreparation based on thereof for soil productivity increase and broken soil recovery - Google Patents

Abstract

FIELD: biotechnology, agriculture microbiology. SUBSTANCE: invention relates to preparing the biopreparation used for increase of soil productivity and recovery of broken soils. The novel strain Azotobacter chroococcum 2E-16 is obtained by method of analytical selection of natural isolates by principle of selection of high productive producers eliciting sufficiently high nitrogenase activity, antibiotic activity with respect to microorganism-phytopathogens and high adaptability in soil and grounds. Strain is deposited in collection of microorganisms AOZT "Bioflora" at N B-05. Strain is used as a producer for preparing the biopreparation used in agriculture for recovery of broken soils and for increase of soil productivity. The preparation has bacterial culture Azotobacter chroococcum 2E-16 grown in nitrogen free nutrient medium, substrate-carrier as turf or biohumus, sodium benzoate as a stabilizing agent, calcium propionate, ascorbate taken in the total amount 0.001-0.03% and trace elements as chelates of Zn, Mg, Mn, Fe, Co and Mo taken in the amount 0.0001-0.002%, potassium humate taken in the amount 0.1-3% and growth regulating agent as derivatives of arachidonic acid taken in the amount 10^-3-10^-5 %. Addition of calculated amount of bacterial culture Azotobacter chrococcum 2E-16 or the preparation based on thereof results to the soil sanitation, enhances its productivity. EFFECT: strain indicated above, valuable agriculture properties, decreased consumption of mineral nitrogen fertilizers, increased yield and enhanced quality of crops. 6 cl, 5 tbl, 6 ex

Description

 The invention relates to ecology, biotechnology, agricultural microbiology, in particular to the selection of microorganisms and the production of biological products based on them to increase soil fertility, improve the soil and restore (remedy) disturbed lands.

 Known strains of Azotobacter chroococcum, significantly differing in their properties. For example, the strain VKPM-6011 (1) increases the yield of amaranth, reduces the level of nitrate nitrogen, and survives well in the root zone of plants; strain 92 (2) has the ability to inhibit the growth of phytopathogenic fungi; strain 6010 (3) is effective only under green crops.

 The disadvantage of strain 92 is the relatively low productivity, and strains 6011 and 6010 are effective only in the root zone of a particular host plant.

 The aim of the present invention is to obtain a strain having high productivity and survival in the soil, as well as a wide spectrum of suppression of pathogenic microflora, i.e. healing effect along with sufficient nitrogenase activity. The productivity of the strain should be high enough so that the production of the drug on its basis, as well as the consumption per unit of cultivated area, are sufficiently economical.

 The strain Azotobacter chroococcum 2E-16 (CJSC Bioflora), collection N B-05), was isolated from cultivated podzolic soil of the Sosnovsky district of the Chelyabinsk region and was selected by analytical selection from the isolate according to the characteristics: high productivity (including production, regulatory environments ), high survival rate in the soil, a wide range of suppression of phytopathogenic microflora, high nitrogenase activity, a high level of synthesis of IAA. The strain is stored and maintained in the Museum of Culture of JSC Bioflora, has a collection number B-05.

 1. Cultural and morphological characteristics.

1.1. On an agarized nutrient medium having a composition, g / l: KH ₂ PO ₄ - 0.3; CaHPO ₄ 0.2; MgSO ₄ - 0.3; K ₂ SO ₄ 0.2; NaCl - 0.5; FeCl ₃ - 0.01; CaCO ₃ - 5.0; sucrose - 20.0; agar - 20.0; trace element mixture - 1 ml; distilled water - up to 1000 ml, pH 6.8-7.0. The culture forms round colonies of 2-3 mm, with a smooth shiny surface, convex, cream or light brown in color, with smooth edges, soft viscous consistency. At 24 hours of cell growth, small rods, cocciform; at 48 hours - 60 hours - encapsulated cocciform, 1 x 12 microns.

1.2. On solid nutrient medium Burke, having the composition, g / l: K ₂ HPO ₄ - 0.8; KH ₂ PO ₄ 0.2; MgSO ₄ 0.2; NaCl - 0.2; CaSO ₄ - 0.1; Fe ₂ (MoO ₄ ) - 0.01; glucose - 10.0; distilled water - up to 1000 ml, agar-agar - 20.0; pH 6.8-7.0. The culture forms round colonies of 2-3 mm, with a smooth shiny surface, convex, transparent, colorless, with smooth even edges, of a homogeneous structure. At 24 hours of growth - very small sticks, mobile, size up to 2 microns. After 24 hours of growth, the cells are motionless, cocciform, large 1 x 15 microns.

 1.3. There is no growth on a solid nutrient medium with Na benzoate as the only carbon source.

1.4. On a liquid nutrient medium Fedorov with molasses, having the composition, g / l: K ₂ HPO ₄ - 0.3; CaHPO ₄ 0.2; MgSO ₄ - 0.3; K ₂ SO ₄ 0.2; NaCl - 0.5; FeCl ₃ - 0.01; CaCO ₃ - 5.0; molasses - 40.0; trace element mixture - 1 ml; pH 6.8-7.0, cultivation at a temperature of plus 28 ± 1 ^o C and aeration mode - mixing 180-200 rpm ^-1 for 60 hours. The range of natural pH changes is 6.8-7.4. When microscopic examination at 24 hours of cultivation - small rod-shaped cells, weakly mobile, 2-3 microns, in the mucous environment, clearly observed during microscopy (negative contrast); Gram reaction for 18-24 hours - varies. Mature cells at the 60th hour of fermentation are cocciform, motionless, form thick-walled cysts; depending on the carbon source, it can form a brown (from light brown to dark) water-insoluble pigment; Gram reaction is negative.

 2. Physiological and biochemical characteristics.

Strain Az. chroococcum 26-16 - aerobic, optimal growth temperature plus (28 ± 1) ^o C.

 Attitude to carbon sources: very well absorbs glucose, fructose, often with the formation of pigment, sucrose, sorbitol; well assimilates mannitol, glycerin, trehalose; poorly assimilates (weak growth) maltose, sorbose, dextrin, ethanol; does not absorb (no growth) arabinose, xylose, mannose, galactose, lactose, cellulose, inositol, rhamnose.

Attitude to nitrogen sources: grows on nitrogen-free media, maintaining high productivity of at least 4.5–9 • 10 ⁹ cells • ml ^-1 and high nitrogenase activity from 210 to 430 nm • ml ^-1 , or up to 3000 • 10 ^-3 μg N ₂ . Good growth on media with (NH ₄ ) ₂ SO ₄ - up to 0.5%, with molasses, but nitrogenase (activity) decreases in these cases to 1000-600 • 10 ^-3 μg N ₂ , i.e. 3-5 times.

 Attitude to antibiotics: resistant to chloramphenicol, penicillin, griseofulvin, gramicidin. It is sensitive to ampicillin, oleandomycin, streptomycin.

 Attitude to phytopathogenic fungi: inhibits the growth of Verticillum dahliae, Helminthosporium sativum, Scolecothrihum sp., Botrytis cinerea, Fusarium sp. , Pythium de Barjanum, Fusarium lini, Fusarium oxysporum, Cladosporium.

 Attitude to phytopathogenic bacteria: inhibits the growth of Streptomyces globisporus, Pseudomonas aeruginosa, Micrococcus luteus, Bac. sphaericus, Ps. putida, Ps. lacrimans.

Storage of the strain: on a solid nutrient medium containing molasses and having a composition,%: CaCO ₃ - 0.5; CaHPO ₄ - 0.02; K ₂ SO ₄ - 0.02; NaCl - 0.05; K ₂ HPO ₄ - 0.03; molasses - 3; agar-agar - up to 2. Incubation for 72-84 hours at (28 ± 2) ^o C, storage temperature plus 4-10 ^o C.

 Characteristic features of strain 2E-16 are: high productivity on production (regulatory) media under conditions of deep fermentation, high safety when using a biological product based on it (gel, bulk form), antagonistic activity against many phytopathogenic microorganisms, high survival rate in basal zone of plants, a sufficiently high nitrogenase activity, the ability to synthesize IAA.

Example 1. To compare the productivity of the new strain 2E-16 with the known strain 92 was grown in flasks on a rocking chair at 180-200 rpm · ^-1 for 60 hours at a temperature of plus (28 ± 2) ^o C in a liquid nutrient medium of the following composition ,%: molasses - 3.0; K ₂ HPO ₄ - 0.03; CaHPO ₄ - 0.02; NaCl - 0.05; CaCO ₃ - 0.5; K ₂ SO ₄ - 0.02.

 The results are shown in table 1.

Example 2. To compare the antagonistic activity of pcs. 2E-16 and famous, pcs. 92, they were grown in flasks on a rocking chair on a liquid regulatory medium: fermentation time 60 hours, fermentation temperature (28 ± 2) ^o C, aeration-mixing mode 180-200 rpm • ^-1 ; composition of the medium,%: sucrose - 2.0; K ₂ HPO ₄ - 0.03; CaHPO ₄ - 0.02; NaCl - 0.05; CaCO ₃ - 0.5; K ₂ SO ₄ - 0.02; a mixture of trace elements for growing - 1 ml / l. Activity was determined by the "wells" method after 2-5 days of growth of test cultures on diagnostic media and expressed in mm of their absence growth zone (the size of the growth suppression zone was determined in mm).

 The results are shown in table 2.

 Based on this bacterial culture of Azotobacter chroococcum (strain number 2E-16, collection number in the Museum of Cultures of Bioflora CJSC B-05), a preparation was developed that is intended for use in agriculture - to increase the yield of agricultural crops, as well as rehabilitation of soils infected with phytopathogenic microflora, which developed as a result of long-term soil exploitation in the regime of the so-called "intensive farming", during restoration (reclamation, remediation) of disturbed lands. The commodity form of the drug is gel (liquid), bulk (dry, peat).

Example 3. 80 ml of liquid culture of Az. chroococcum pcs. 2E-16, grown as described in Example 2, was added to 420 g of sterile peat. At the same time, a mixture of trace elements Zn, Mg, Mn, Co, Fe, Mo, in the form of complexonates based on HEDP, was added in a total amount of 3 mg, as well as a growth regulator based on arachidonic acid in an amount of 10 ^-5 % and nutritional supplements - molasses 0.2 g., chalk 0.1 g., stabilizer (calcium propionate, sodium benzoate, ascorbate) in an amount of 150 mg in total, potassium humate in an amount of 14 ml. The resulting mixture was stirred on a shaker and left to grow at a temperature of 20 ± 2 ^o C for 7 days. After growing, the free-flowing preparation contained 12.2 x 10 ⁹ cells / g. The preparation was used to improve the greenhouse soil during the preparation of greenhouses at the rate of 80 • 10 ⁶ cells of azotobacter per 1 m ^{2 of the} area of the greenhouse. 3 days after applying the drug to the soil, a sample was taken and the content of phytopathogenic fungi was determined. The result was compared with the content of phytopathogenic fungi in the soil after "steaming" (heat treatment). The results of the experiments are shown in table 3; the initial amount of phytopathogenic fungi before making the drug is taken as 100%.

Example 4. In liquid culture Az. chroococcum pcs. 2E-16, containing 25 • 10 ⁹ cells • ml ^-1 , was added a stabilizer (calcium propionate, sodium benzoate, ascorbate) in an amount of 450 mg in total per 2 l of liquid culture, as well as trace elements in an amount of 12 g (total) in the form of complexonates Zn, Mg, Mn, Fe, Co, Mo, potassium humate in an amount of 40 ml; the resulting liquid preparation was carefully mixed and used for processing (applying to the soil) plantings of spruce seedlings (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 are shown in table 4. Control (100%) - product indicators from areas not processed by the claimed drug.

Example 5. The liquid culture of Az. chroococcum, pcs. 2E-16, containing 25 • 10 ⁹ cells • ml ^-1 , mixed with stabilizers and microelements, humate was added as described in Example 4. The resulting liquid preparation was dried on an Aeromatic unit (drying in a pseudo-boiling layer) by spraying liquid drug for vermicompost obtained by bio-conversion using vermiculture. The content of bacterial cells in the obtained preparation was 1000 ml of a liquid preparation and 2 kg of biohumus, when dried, 8.5 • 10 ⁹ cells • ml ^-1 , the moisture content of the preparation after drying was 9.2%. The resulting preparation was used to test the effectiveness in experiments on the restoration of areas disturbed by emissions of sludge from a mining and processing plant (Kachkanar, Northern Urals).

 Example 6. Samples of the drug obtained in example 5, was introduced into the sludge taken from areas disturbed by discharges of sludge from a mining and processing plant. 2 months after the application of the drug (the drug was applied at a rate of 5 kg / ha), the survival rate of the azotobacter in the basal zone of grass sown simultaneously with the application of the drug was determined. Observations in the treated area were carried out for three years. The results are shown in table 5.

 Thus, a new strain of Azotobacter chroococcum 2E-16 was obtained, which reveals several useful properties and can become the basis of drugs (various commodity forms), the composition of which is developed and the new drug can be used both to increase the yield of agricultural crops and in green construction (for landscaping settlements), while restoring disturbed lands, while discovering not only high survival rates even in lifeless soils (for example, in sludges after ore dressing), but also high efficiency in the suppression of phyto pathogenic microflora of the soil. The composition of the components introduced into the preparation is such that during the course of the action of the preparation they are completely consumed either by the microorganism itself or participate in the processes for which the preparation is intended for use.

Sources of information
1. RF patent N 2074158. Publ. Bulletin No. 6, 1997.

 2. Auth. USSR certificate and 922105. Published on 07/23/82. Bull. N 15.

 3. RF patent N 2074159. Publ. Bulletin No. 6, 1997.

 4. Abstracts of the meeting on July 28-29, 1995, Anapa.

Claims (6)

 1. The bacterial strain Azotobacter chroococcum CJSC "Bioflora" N В-05 to obtain a biological product to increase soil fertility, increase crop yield and quality, improve soil and restore disturbed lands.  2. Biological product for increasing soil fertility, increasing crop yield and quality, improving the soil, restoring disturbed lands based on Azotobacter chroococcum grown on a nitrogen-free nutrient medium, characterized in that it additionally contains trace elements, potassium humate and a stabilizer, and from bacteria Azotobacter chroococcum it contains the strain according to claim 1. 3. The biological product according to claim 2, characterized in that it further comprises a growth regulator in the form of derivatives of arachidonic acid in an amount of 10 ^-3 -10 ^-5 %.  4. The biological product according to claim 2, characterized in that it further comprises 70-85% peat, 0.02-0.05% chalk and nutritional supplements to preserve the properties of the strain according to claim 1 in an amount of 0.04-0.1% by weight of the drug.  5. The biological product according to claim 2, characterized in that it further comprises biohumus.  6. The biological product according to claim 2, characterized in that it contains a stabilizer in the form of calcium propionate, sodium benzoate and ascorbate in an amount of 0.001-0.03% in total, trace elements Zn, Mg, Mn, Co, Fe, Mo in the form of salts or complexonates in an amount of 0.0001-0.002% in total and 0.1-3% of potassium humate.

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