RU2514217C1 - Biological preparation for leguminous crop of liquid form based on nodule bacteria - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: biological preparation comprises the culture fluid containing the strain of nitrogen-fixing nodule bacteria with a titre of not less than 10⁷ CFU/ml, and an aqueous solution containing (wt %): sodium carboxymethyl cellulose (2.0-5.0), potassium sorbate (0.5-3.0) and glycerol (1.0-5.0), mixed in a ratio of from 1:1 to 1:2.

EFFECT: invention enables to increase the productivity of leguminous crops and to increase the shelf life of the resulting preparation.

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Description

The invention relates to agricultural biotechnology and can be used to obtain a liquid form of microbiological preparations based on nodule bacteria that increase the yield of legumes.

The invention is known “Bradyrhizobium sp. (Lotus) for the production of bacterial fertilizer for Lyophonidae horned "according to the certificate of authorship No. 1458359 with priority dated 02.27.1987, MKI C05F 11/08, published 02.15.1989.

The materials for this copyright certificate describe the technology for producing a biological product based on peat - Rizotorfin, which includes the preparation of culture fluid based on a strain of nodule bacteria Bradyrhizobium sp. (Lotus) 476, preparation of peat (grinding, packaging, sterilization), followed by infection with culture fluid with rhizo-bacteria and growing bacteria in peat.

In a similar way, biofertilizer on peat for non-leguminous plants is obtained, described in the invention “Bacteria strain Azosphillum lipoferum for the production of fertilizer for pasture ryegrass and boneless rump” according to patent No. 1806123 with priority dated 05.03.1991, IPC C05F 11/08, C12N 1 / 20, published March 30, 1993

The shelf life of preparations based on peat is up to 6 months.

The disadvantage of this method is the need to search and select the type of peat (different acidity, composition) suitable for the preparation of a biological product, since not all types of peat are suitable for this, and at present, criteria for assessing its suitability have not been found. In addition, peat reserves of the required quality in Russia are almost exhausted.

In addition, one of the problems in the production and practical application of legume bacteria-based preparations for legumes is their low viability during storage and use. Substances of a phenolic nature contained in a solid-phase carrier, peat, have an adverse effect on nodule bacteria, as a result of which the nitrogen fixation process is not effective enough.

The most technologically advanced and convenient in comparison with the form on solid-phase carriers is the liquid form of the drug, which represents a nutrient medium inoculated with bacteria. Biological products for various purposes in liquid form are also known in the art.

So, for example, the invention is known “A method for producing a microbiological preparation for protecting plants from harmful insects and ticks”, application No. 2004139258 of December 29, 2004, IPC A01N 63/00; C12N 1/20, published June 10, 2006. According to the invention, the preparation is a liquid mixture of cultures from epiphid microorganisms of bacteria and fungi, into which mineral salts (urea, ammonium phosphate disubstituted) are introduced, to enhance storage viability and effectiveness of the action on target objects, sodium humate and lignosulfonate; to give stability to the preparation during storage, increase the period of action on the treated surface, adherence, and spreadability, carboxymethyl cellulose 0.1-1.0%, surfactant, glycerol 5-10%, sodium humate are introduced.

The biggest disadvantage of the liquid form of biological products is the short shelf life, which, as a rule, is 10-15 days. After this, the bacterium titer in the preparation begins to decrease sharply, and therefore its effectiveness decreases. Meanwhile, modern agricultural production requires cheap, effective and convenient to use biological products that would remain effective for a longer time.

The invention is also known, "Biological product for cleaning soil and water from oil and oil products", patent No. 2428469, application No. 2010019723 with priority dated March 15, 2010, IPC C12N 1/20, C02F 3/34, C09K 3/32, B09C 1/10, published September 10, 2011

According to the invention, the biological product contains a water-containing culture fluid based on aerobic oil-oxidizing bacteria, normal C ₁₂ -C ₁₈ paraffins, ammonium oxalate, and potassium chloride, taken in a specific ratio. Moreover, an increase in the shelf life of the drug up to 3 months is achieved by the introduction of potassium chloride, which suppresses the harmful effects of the products of their vital activity on bacteria.

The invention is known "The drug" EXTRAGRAN "to stimulate growth and protect plants from diseases", application No. 2005130793 from 05.10.2005, IPC A01N 63/00; C12N 1/20, C12R 1/065, C12R 1/07, published April 10, 2007

According to the invention, the biological product is a liquid mixture of cultures from strains of microorganisms of the genus Bacillus mycoides and Azotobacter vineladii, taken in a certain ratio, in which additives are added in the form of humates with microelements in the following composition, wt.%:

culture fluid with microorganism strains 25-30 humates 8-12 trace elements 0.05-0.08 water rest

The disadvantage of the claimed drug "EXTRAGRAN" is the presence of problems during storage and transportation and the associated low stability of the drug.

The invention is also known, "Biological product in the form of an aqueous suspension to increase soil fertility" according to patent No. 2390518 with priority dated November 27, 2008, IPC C05F 11/08, published May 27, 2010 (prototype).

The biological product contains components in the following ratio, wt.%:

culture fluid based on Azotobacter vineladii strain 50-60 trace elements 0.2-0.3 salt 1.0-1.5 water up to 100%

The positive effect of the biological product on vegetables (tomatoes, cucumbers), potatoes, as well as barley and wheat has been established. Salt plays the role of a preservative, which allows to increase the shelf life of the drug up to 12 months

However, the preparations described above are not suitable for legumes, as the bacteria used in them are not nodule.

A unique feature of nodule bacteria is the ability to penetrate the roots of legumes and form nitrogen-fixing nodules on them, which can supply atmospheric nitrogen to plants. Without these nodules, atmospheric nitrogen is inaccessible to plants for nutrition.

Thus, in the current level of technology, a biological product (or a method for producing it) based on strains of nodule bacteria under legumes of liquid form with a sufficiently long shelf life has not been identified.

The objective of the invention is to obtain a liquid form of a biological product based on strains of nodule bacteria that increases the yield of legumes, with a long shelf life.

The problem is solved due to the fact that the biological product for legumes includes mixed in a ratio of 1: 1 to 1: 2 culture fluid containing a strain of nitrogen-fixing nodule bacteria with a titer of at least 10 ⁷ CFU / ml, and an aqueous solution containing, wt.% :

carboxymethyl cellulose sodium 2.0-5.0% potassium sorbate 0.5-3.0% glycerol 1.0-5.0%

The use of biological products based on strains of nitrogen-fixing nodule bacteria with a titer of at least 10 ⁷ CFU / ml, as you know, increases the yield of leguminous plants (for example, Khotyanovich A.V. Methods of cultivation of nitrogen-fixing bacteria, methods for producing and using drugs based on them. / Methodical recommendations Leningrad, 1991).

At the same time, glycerin in the preparation serves as a source of carbohydrate nutrition for bacteria and as an antistress.

Potassium sorbate (Potassium Sorbate, E202) - a potassium salt of sorbic acid, is a natural preservative and is widely used in food preservation, as prevents mold and foulness. It is used in the manufacture of many products to increase their shelf life, including potassium sorbate is widely used for preserving fruits and vegetables, egg and confectionery, meat and fish products, fruit and berry juices and soft drinks. Moreover, depending on the product, the potassium sorbate content norm varies in the range from 0.02% to 0.2%, i.e. the maximum allowable dosage in food products E202 is not more than 0.2%. allowed in all countries of the world. In most countries of the world, potassium sorbate E202 is permitted and widely used.

It was experimentally established that when potassium sorbate is included in a biological product, it not only inhibits extraneous microflora, but also does not adversely affect nodule bacteria at concentrations> 0.2%.

It is known that carboxymethyl cellulose (CMC) is a cellulose ether of the general formula: [C ₆ H ₇ O ₂ (OH) ₃ (OCH ₂ COOH) _x ] _n .

The main property of carboxymethyl cellulose is the ability to form a very viscous colloidal solution that does not lose its properties for a long time. In the chemical, food and medical industries, the most commonly used sodium salt is sodium carboxymethyl cellulose (Na-CMC).

Na-CMC is used as a plasticizer, thickener, and resorbent. It is used in the production of glue (for example, glue bustilate), to stabilize the clay slurries used in drilling oil and gas wells; as an additive to detergents, preventing the resorption of contaminants from the washing solution on the fabric; for sizing warp threads and as a thickener for printing inks; as flotation reagents; to increase the ductility of the ceramic mass and the strength of the product "raw".

Na-CMC has emulsifying, thickening, foaming, stabilizing, softening, gel-forming, film-forming properties. It can serve as a good substitute for gelatin, glycerin, starch. It is widely used in cosmetic production in toothpastes, creams and cosmetic masks, shampoos, shaving creams, hair conditioners, bath preparations. Non toxic

They are also used in the food industry (E-466) as a regulator and stabilizer of consistency, a thickener, and an encapsulation agent; it is used in the production of ice cream, mayonnaise, curd products, confectionery products (jelly, mousse, marmalade, jams, fruit and berry fillings, cream, pasta, pastries, pasta), sauces and meat products, in shells for fish, meat, is a part of encapsulation products.

It was experimentally found that sodium carboxymethyl cellulose (Na-CMC), which is included in a biological product based on strains of nodule bacteria, in the amount indicated above (2.0-5.0% in an aqueous solution) serves as a regulator and stabilizer of the consistency of the drug, without providing negative effects on bacteria.

Thus, the combined use of sodium carboxymethyl cellulose, potassium sorbate and glycerol in the indicated ratios in a liquid biological preparation based on nodule bacteria leads to an increase in the shelf life of the drug up to 6 months. All quantitative values and ratios to obtain the best result are established experimentally.

At the same time, it was experimentally established that the best result for increasing the yield of legumes is achieved using a bacterial preparation in which a culture liquid with a titer of at least 10 ⁷ CFU / ml nitrogen-fixing nodule bacteria and an aqueous solution are mixed, containing 2-5% Na-CMC, 0.5-3% potassium sorbate and 1-5% glycerol.

Moreover, upon receipt of the culture fluid, any known nutrient medium for growing nodule bacteria can be used, for example, a traditional legume medium of the following composition: peas - 70 g / l, sucrose - 2.5 g / l, glucose - 10 g / l, (NH ₄ ) ₂ SO ₄ - 1 g / l, KH ₂ PO ₄ - 0.5 g / l, K ₂ HPO ₄ - 0.5 g / l, MgSO ₄ · 7H ₂ O - 0.2 g / l, CaCO ₃ - 1 g / l, which is first inoculated with nodule bacteria, and then cultured in a fermenter at a certain temperature (usually 27-29 ° C) for a certain time (usually 72-96 hours) until a given bacterium titer is reached (see the aforementioned Methodical recommendations (Khotyanovich A.V., 1991)

Example 1

To obtain a liquid biological product, a bacterial strain Bradyrhizobium japonicum 634b from the Departmental Collection of Useful Agricultural Microorganisms of the Russian Agricultural Academy (RCAM) was used at the GNU VNIISHM Russian Agricultural Academy, where it was deposited on 04/20/1979 in a group of nodule bacteria under No.RCAM2490.

The bacterium Bradyrhizobium japonicum 634b was grown in a rocking flask for 72 hours at a temperature of 27 ° C on a traditional nutrient legume medium. At the end of the cultivation process, the titer of the bacterial strain 634b in the culture fluid was 9 · 10 ⁹ CFU / ml. After that, 100 ml of the obtained culture fluid was mixed in a ratio of 1: 1, i.e. with 100 ml of an aqueous solution containing 5 g of sodium carboxymethyl cellulose (5%), 3 g of potassium sorbate (3%) and 5 g of glycerol (5%). The concentration of bacteria in the finished liquid biological product was 4.5 · 10 ⁹ CFU in 1 ml of the drug.

Example 2

To obtain a liquid biological product, a bacterial strain Bradyrhizobium japonicum 640b was used from the Departmental Collection of Useful Microorganisms of Agricultural Purpose of the Russian Agricultural Academy (RCAM), at the GNU VNIISHM of the Russian Agricultural Academy, where it was deposited on June 24, 1981 in a group of nodule bacteria under No. RCAM24100.

The Bradyrhizobium japonicum 640b bacterium was grown in a rocking flask for 96 hours at a temperature of 29 ° C on a traditional nutrient legume medium. At the end of the cultivation process, the titer of the bacterial strain 640b in the culture fluid was 9.6 · 10 ⁹ CFU / ml. After that, 100 ml of the obtained culture fluid was mixed in a ratio of 1: 2, i.e. with 200 ml of an aqueous solution containing 4 g of sodium carboxymethyl cellulose (2%), 1 g of potassium sorbate (0.5%) and 2 g of glycerol (1%). The concentration of bacteria in the finished liquid biological product was 3.2 · 10 ⁹ CFU in 1 ml of the drug.

Experiments were conducted on the storage of biological products of various shapes, obtained on the basis of the same previously mentioned strains of nodule bacteria Bradyrhizobium japonicum 634b and Bradyrhizobium japonicum 640b. The preparations were tested on a solid support (peat inoculated with culture fluid), in liquid form without additives (culture liquid diluted with water in a ratio of 1: 1 to 1: 2) and in liquid form with additives in the form of Na-CMC, sorbate potassium and glycerol in the above proportions according to the invention, as described in examples 1 and 2.

All biological products involved in the experiments were stored at room temperature (20-22) ° C. At certain intervals, the titer of bacteria was measured in the preparations.

The measurement results are shown in table 1.

Table 1 Bacteria titers in various forms of drugs during storage Biological product Bacterium titer in the preparation, CFU in 1 ml The basis The form source in 14 days after 1 month after 3 months after 6 months Dry peat 3.7 · 10 ⁹ 7.210 ⁹ 6.410 ⁹ 4.210 ⁹ 3.4 · 10 ⁹ Bacterial strain Bradyrhizobium japonicum 634b Traditional liquid form without additives 4,510 ⁹ 3.4 · 10 ⁹ 1.2 · 10 ⁹ 0.00310 ⁹ 0 * Liquid form with additives according to the invention 4,510 ⁹ 4.210 ⁹ 3.8 · 10 ⁹ 2.9 · 10 ⁹ 2.810 ⁹ Dry peat 2.710 ⁹ 6.210 ⁹ 5.810 ⁹ 5.210 ⁹ 3.8 · 10 ⁹ Bradyrhizobium japonicum 640b bacteria strain Traditional liquid form without additives 3.210 ⁹ 2.410 ⁹ 1.610 ⁹ 0.04510 ⁹ 0 * Liquid form with additives according to the invention 3.210 ⁹ 3.210 ⁹ 3.110 ⁹ 2.9 · 10 ⁹ 2.610 ⁹

From the data shown in table 1, it follows that the titers of microorganisms in liquid form of biological products with additives according to the invention (Na-CMC, potassium sorbate and glycerin in the above ratios) even after 6 months are on par with the titers of these bacteria in dry peat form, which indicates the high efficiency of the preparations of the claimed liquid form with additives.

Thus, the introduction of a biological product based on nitrogen-fixing nodule bacteria and an aqueous solution with additives in the form of Na-CMC, potassium sorbate and glycerol in the above ratios allows to increase the shelf life of nodule bacteria in this preparation and, accordingly, increase the shelf life of the liquid form of the drug up to 6 months.

To evaluate the effectiveness of the claimed composition of a biological product based on nodule bacteria after a long shelf life (5 months), vegetative experiments were conducted on soybean plants of the Diva variety.

The preparations were obtained on the basis of the previously mentioned strains of nodule bacteria Bradyrhizobium japonicum 634b and Bradyrhizobium japonicum 640b grown on a traditional nutrient medium (bean broth with salts). The following drugs were tested:

- in liquid form without additives (culture fluid diluted with water), and 1 day has passed from the time the drug was received until its use, i.e. fresh biological product;

- in liquid form with additives according to the invention, as described above in examples 1 and 2, and the drug was used after 5 months of storage at a temperature of (20-22) ° C.

The bacteria titers in all variants of the experiment were values in the range (1.5-2) · 10 ⁹ CFU / ml of the drug.

In all variants of the experiment, soybean seeds were first soaked for 5-10 seconds in a biological product (inoculated), in the control, soybean seeds were inoculated with sterile water. Then seeds were sown in vegetation vessels with sod-podzolic soil (pH = 6.2-6.8; N _total = 0.26%; P ₂ O ₅ = 150 mg / kg; C = 4.7%; humus - 1.8%). The soil mass in each vessel was 5 kg, 5 inoculated soybean seeds were placed in each vessel. Repeat 6-fold.

The experiments, accounting and evaluation of the results were carried out in accordance with standard methods.

One part of the soybean was grown before the budding phase, after which the plants were extracted from the soil with roots and their biometric parameters were measured. The measurement results are shown in table 2.

Another part of soybeans was grown to the ripeness phase at the end of the growing season, after which ripened soybeans were collected, and the plants were also extracted from the soil with roots and their biometric parameters were measured.

The measurement results are shown in table 3.

table 2 Biometric parameters of soybean plants in the budding phase The average mass of the root, g The average height of the plant, cm The average mass of the plant, g Weight gain of the plant,% Number of nodules Variant of experience (inoculation of seeds with the drug) Control (water) 3.3 21,2 3.8 - - Traditional liquid form without additives (1 day storage) 4.2 32,4 7.1 85.75 32 The drug is based on the bacterial strain Bradyrhizobium japonicum 634b Liquid form with additives according to the invention (5 months of storage) 4.7 40.3 8.9 132.2 37 Traditional liquid form without additives (1 day storage) A drug based on the bacterial strain Bradyrhizobium japonicum 640b 4.9 38,4 9,4 136.8 39 Liquid form with additives according to the invention (5 months of storage) 3.4 39.7 8.4 121.1 43

Table 3 Biometric parameters of soybean plants in the ripening phase at the end of the growing season Variant of experience (inoculation of seeds with the drug) The average mass of the root, g The average height of the plant, cm The average mass of the plant, g Weight gain of the plant,% The average mass of beans on the plant, g Control (water) 3.9 24.3 8.3 - 5.2 Traditional liquid form without additives (1 day storage) 8.1 39.8 19.9 139.9 6.45 The drug is based on the bacterial strain Bradyrhizobium japonicum 634b Liquid form with additives according to the invention (5 months of storage) 8.7 40.3 22.1 166.3 6.6 Traditional liquid form without additives (1 day storage) 5,0 38,4 19.9 140.0 6.9 A drug based on the bacterial strain Bradyrhizobium japonicum 640b Liquid form with additives according to the invention (5 months of storage) 7.3 39.7 21,2 155.7 7.1

From the data shown in tables 2 and 3, it is seen that liquid preparations based on nitrogen-fixing nodule bacteria Bradyrhizobium japonicum 634b and Bradyrhizobium japonicum 640b, which include in their composition according to the invention an aqueous solution with the addition of Na-CMC, potassium sorbate and glycerol in the above ratios, even after 5 months of storage, have the same efficiency with respect to the biometric parameters of soy plants as fresh preparations without the named additives, obtained by traditional technology, and by the number of nodules formed on the roots in influencing power plant atmospheric nitrogen, as well as average weight taken from one plant beans characterize soybean yields, - surpass them.

Thus, by the example of two nitrogen-fixing nodule bacteria, it was experimentally confirmed that liquid biological preparations based on strains of nodule bacteria of the claimed composition that increase the yield of legumes are drugs that maintain their effectiveness over a long shelf life of 6 months.

Claims (1)

A biological product for a legume liquid culture based on nodule bacteria, consisting of mixed in a ratio of 1: 1 to 1: 2 culture fluid containing a strain of nitrogen-fixing nodule bacteria with a titer of at least 10 ⁷ CFU / ml, and an aqueous solution containing, wt. %:
carboxymethyl cellulose sodium 2.0-5.0% potassium sorbate 0.5-3.0% glycerol 1.0-5.0%