Biological preparation for leguminous crop of liquid form based on nodule bacteria

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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The invention relates to agricultural biotechnology and can be used to produce liquid forms of microbiological preparations based on nodule bacteria, increasing the yield of legumes.

Known invention "Strain of bacteria Bradyrhizobium sp. (Lotus) for the production of bacterial fertilizers under ladines horned" copyright certificate №1458359 priority from 27.02.1987,, MKI C05F 11/08 published 15.02.1989,

In the materials for this author's testimony described the technology of production of a biological product on the basis of peat - Isotropin, which includes the preparation of the culture fluid from the strain of nodule bacteria Bradyrhizobium sp. (Lotus) 476, preparation of peat (grinding, packaging, sterilization) with subsequent contamination of the culture fluid with risø-bacterial and growing bacteria in peat.

In a similar way we obtain a biofertilizer on peat for nabibovich plants described in the invention, a Strain of bacteria Azosphillum lipoferum for the production of fertilizer for perennial ryegrass and brome awnless" in patent No. 1806123 priority from 05.03.1991,, IPC C05F 11/08, C12N 1/20, published 30.03.1993,

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

The disadvantage of this method is the need of search and selection of the type of peat (different acidity, with the Tav), suitable for preparation of a biological product, as it is suitable, not all types of peat, and currently, the criteria for assessing its suitability was not found. Besides peat reserves the right quality in Russia is almost exhausted.

In addition, one of the problems of production and practical application under legumes drugs on the basis of nodule bacteria is their low viability during storage and use. Contained in the solid-phase carrier - peat substances of phenolic nature have adverse effects on nodule bacteria, resulting in the process of isotopically is not efficient enough.

The most technologically advanced and comfortable compared with the form on the solid phase carrier is a liquid form of the drug, which is a nutrient medium inoculated with bacteria. The prior art also biological products for different purposes in liquid form.

So, for example, is known for the invention "Method of obtaining microbiological preparation for protection of plants from harmful insects and mites", application No. 2004139258 from 29.12.2004,, IPC A01N 63/00; C12N 1/20, published 10.06.2006, According to the invention the product is a liquid mixture of cultures from epipheny microorganisms bacteria and fungi that enter mineral salts (urea, ammonium FOS is ornately disubstituted), to enhance viability during storage and the effectiveness of actions on the target objects enter HUMATE and sodium lignosulfonate; to impart stability to the drug during storage, increasing the validity of the treated surface, adhesion, effective flowing property enter carboxymethylcellulose 0.1 to 1.0%, surfactant, glycerin 5-10%, sodium HUMATE.

The biggest disadvantage of liquid forms of biologics is a short shelf life, which usually is 10-15 days. After that, the titer of bacteria in the product begins to decline sharply, and therefore, falls and its effectiveness. Meanwhile, modern agricultural production requires a cheap, effective and easy to use biologics that would preserve its effectiveness for a longer time.

It is also known invention "biological product for cleaning soil and water from oil and oil products", patent No. 2428469, application No. 2010109723 priority from 15.03.2010, IPC C12N 1/20, C02F 3/34, SC 3/32, VC 1/10, published on 10.09.2011,

According to the invention the biological product contains aqueous culture liquid-based oxidizing aerobic bacteria, normal paraffins C₁₂-C₁₈, oxalic ammonium, potassium chloride, taken in a certain ratio. Moreover, the increase of the shelf life of the preparation is the same up to 3 months is achieved by injection of potassium chloride, which suppresses the harmful effects on bacteria, their metabolic products.

You know the invention of "Drug "EXTROGEN" 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 10.04.2007,

According to the invention the biological product is a liquid mixture of cultures of strains of microorganisms of the genus Bacillus mycoides and Azotobacter vineladii taken in a certain ratio, which put an additive in the form of humates with minerals in the following composition, wt.%:

The disadvantage of the requested drug, "EXTROGEN is having problems with storage and transportation and related low stability of the drug.

It is also known invention "biological product in the form of an aqueous suspension to improve soil fertility" in patent No. 2390518 with priority dated 27.11.2008,, IPC C05F 11/08 published 27.05.2010, (prototype).

Biological product contains the following components when the ratio wt.%:

The positive influence of a biological product for vegetables (tomatoes, cucumbers, potatoes, and barley and wheat. Salt plays the role of preservative that allows you to increase the shelf life of the drug up to 12 months

However, the above drugs are not suitable for legumes, as used in these bacteria are not a nodule.

A unique feature of nodule bacteria is the ability to penetrate into the roots of leguminous plants and form them nitrogen-fixing nodules, can provide atmospheric nitrogen plants. Without these nodules atmospheric nitrogen available to plants for food.

Thus, in the existing level of technology not identified biological product (or the method of its production) on the basis of strains of root nodule bacteria under legumes liquid form with a fairly long shelf life.

The task of the invention to provide a liquid form, bio is Reparata-based strains of root nodule bacteria, increasing the yield of legumes, with a long shelf life.

The problem is solved due to the fact that the biological product under legumes culture includes mixed in a ratio of from 1:1 to 1:2 culture fluid containing the strain of nitrogen-fixing nodule bacteria titer not less than 10⁷CFU/ml, and an aqueous solution containing, wt.%:

Application of biological preparations on the basis of strains of nitrogen-fixing nodule bacteria titer not less than 10⁷CFU/ml, as is known, increases the yield of leguminous plants (e.g., Hotyanovich A.V. Methods of cultivation of nitrogen-fixing bacteria, methods of preparation and use of drugs based on them. / Guidelines. Leningrad, 1991).

While the glycerin in the product is a source of carbohydrate food for bacteria and antistress agent.

Potassium sorbate (Potassium Sorbate, E202) - potassium salt of sorbic acid is a natural preservative and is widely used in the canning of food products because it prevents mold and protogen the E. It is used in the manufacture of many products to increase their shelf life, including potassium sorbate is widely used for canning fruits and vegetables, egg and confectionery products, meat and fish products, fruit juices and soft drinks. However, depending on the product norm content of potassium sorbate is changed in the range from 0.02% to 0.2%, i.e. the maximum dosage in food E202 is not more than 0.2%. allowed in all countries of the world. In most countries of the world, potassium sorbate E202, they are widely used.

An experimental apparatus was found that the inclusion of potassium sorbate in biologic he not only suppresses extraneous microflora, but has no adverse effect on nodule bacteria at concentrations of >0.2 percent.

It is known that the carboxymethyl cellulose (CMC) is a simple cellulose ether of the General formula: [(C₆H₇O₂(OH)₃(Och₂COOH)_x]_n.

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

Na-CMC is used as a plasticizer, zag is Stites, resorbent. Used in the manufacture of adhesive (e.g. glue Bustilat), to stabilize the clay suspensions used for drilling oil and gas wells, and as an additive to cleaning agents that prevent resorbtive contaminants from the washing solution on the fabric; for smoothing of the warp and as a thickener for printing inks; as flotation agents; to increase the plasticity of the ceramic mass and the strength of the product "raw".

Na-CMC has emulsifying, thickening, foaming, stability, softening, gelling, film forming properties. Can serve as a good substitute for gelatin, glycerin, starch. Widely used in cosmetic production in toothpastes, creams and cosmetic masks, shampoos, shaving creams, hair conditioners, products for bathing. Non-toxic.

Are also used in the food industry (E-466) as a regulator and stabilizer consistency, a thickener, a means for encapsulation, used in the manufacture of ice cream, mayonnaise, cheese products, confectionery products (gel, mousse, jelly, jams, fruit fillings, cream, pasta, pastries, pasta, sauces and meat products, shell fish, meat, part of the funds for encapsulation.

An experimental apparatus was installed that included the biological product on the basis of strains of root nodule bacteria sodium carboxymethylcellulose (Na-CMC) in the above number (2,0-5,0% in the composition of the aqueous solution) is the regulator and stabilizer consistency of the drug, without adverse effect on the bacteria.

Thus, sharing in a liquid biological product on the basis of nodule bacteria of sodium-carboxymethyl cellulose, potassium sorbate and glycerol in the specified proportions increases the shelf life of the drug for up to 6 months. All quantitative values and ratios to obtain the best result is established experimentally.

Thus it is found experimentally that the best results for increasing the yield of legumes is achieved by use of the bacterial preparation, which are mixed in a ratio of from 1:1 to 1:2 culture liquid with a titer of not less than 10⁷CFU/ml of nitrogen-fixing nodule bacteria and an aqueous solution containing 2-5% Na-CMC, 0.5 to 3% of potassium sorbate and 1-5% glycerol.

Moreover, when receiving the culture fluid may be used any known nutrient medium for growing root nodule bacteria, such as traditional bean environment the following composition: pea - 70 g/l sucrose and 2.5 g/l, glucose 10 g/l (NH₄)₂SO₄1 g/l, KH₂PO₄- 0.5 g/l, K₂HPO₄- 0.5 g/l, MgSO₄·7H₂O - 0.2 g/l, caso₃1 g/l, which first inoculant rhizobia, and then cultivated in a fermenter at a certain temperature (usually 27-29°is) for a certain period of time (usually 72-96 hours) to achieve a given titer of bacteria (see the above guidelines. Hotyanovich A.V., 1991).

Example 1.

To obtain biological liquid forms used bacterial strain of Bradyrhizobium japonicum b from the Departmental collection of useful microorganisms for agricultural purposes RAAS (RCAM), wildebeest ARRIAM RAAS, where he was deposited 20.04.1979, group nodule bacteria under No. RCAM2490.

The bacterium Bradyrhizobium japonicum b were grown in chachalacas flask for 72 hours at 27°C in traditional nutritious legumes environment. At the end of the cultivation process titer strain of bacteria b in the culture fluid was 9·10⁹CFU/ml then 100 ml of the obtained culture liquid were mixed at a ratio of 1:1, i.e. with 100 ml of an aqueous solution containing 5 g of sodium salt of carboxymethyl cellulose (5%), 3 g of potassium sorbate (3%) and 5 g of glycerol (5%). The concentration of bacteria in the finished biological liquid form was 4.5·10⁹CFU in 1 ml of the drug.

Example 2.

To obtain biological liquid forms used bacterial strain of Bradyrhizobium japonicum 640 from the Departmental collection of useful microorganisms for agricultural purposes RAAS (RCAM), wildebeest ARRIAM RAAS, where he was deposited 24.06.1981, group nodule bacteria under No. RCAM24100.

The bacterium Bradyrhizobium japonicum 640 virus the Wali in chachalacas flask for 96 hours at a temperature of 29°C in traditional nutritious legumes environment. At the end of the cultivation process titer strain of bacteria 640 in the culture fluid was 9.6·10⁹CFU/ml then 100 ml of the obtained culture liquid was mixed in the ratio 1:2, i.e. with 200 ml of an aqueous solution containing 4 g of sodium salt of carboxymethyl cellulose (2%), 1 g of potassium sorbate (0.5%) and 2 g of glycerin (1%). The concentration of bacteria in the finished biological liquid form was 3.2·10⁹CFU in 1 ml of the drug.

There were experiments on the storage of biological products of various shapes, obtained on the basis of the same previously mentioned strains of nodule bacteria Bradyrhizobium japonicum b and Bradyrhizobium japonicum 640. Test were subjected to drugs on a solid medium (peat inoculated culture fluid), in liquid form without additives (culture liquid, diluted with water in the ratio from 1:1 to 1:2) and in liquid form with additives in the form of Na-CMC, potassium sorbate and glycerol in the above ratio according to the invention as described in examples 1 and 2.

All biologics involved in the experiments were kept at room temperature (20-22)°C. After a certain period of time in the preparations was measured value of the titer of bacteria.

The measurement results are shown in table 1.

From the data presented in table 1, it follows that the values of the titers of microorganisms in liquid form biologics with additives according to the invention (Na-CMC, potassium sorbate and glycerin, in the above ratio) even after 6 months are on the same level with the titles of these bacteria in dry peat form, indicating a high efficacy of the inventive liquid form with additives.

Thus, the introduction of the biological product on the basis of nitrogen-fixing nodule bacteria and water solution with additives in the form of Na-CMC, potassium sorbate and glycerol in the above ratio can improve the life safety of this drug nodule bacteria and, accordingly, to increase the shelf life of the liquid form of the drug for up to 6 months.

To evaluate the effectiveness of the proposed structure of a biological product on the basis of nodule bacteria after long term storage (5 months) were conducted vegetation experiments on plants of soybean cultivar "Diva".

Drugs were based on the earlier strains of nodule bacteria Bradyrhizobium japonicum b and Bradyrhizobium japonicum 640 grown in conventional nutrient medium (bean decoction of the salts). Test were subjected drugs:

in liquid form without additives (culture liquid, diluted with water), and after receiving the drug prior to its use has been 1 day, 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 is used after 5 months of storage at a temperature (20-22)°C.

Titers of bacteria in all variants of the experience was value in range (1,5-2)·10⁹CFU/ml of the drug.

In all variants of the experience first, the soybean seeds were soaked for 5-10 seconds in a biological product (inoculable), in control - inoculation of soybean seeds was carried out with sterile water. Then produced seed in pots with sod-podzolic soil (pH of 6.2 to 6.8; N_total=0,26%; P₂O₅=150 mg/kg; C=4.7%; humus - 1.8 percent). Mass of soil in each vessel - 5 kg in each vessel were placed on 5 of inoculated soybean seeds. Repeated 6 times.

, Conducting experiments, recording and evaluation of the obtained results was carried out according to standard methods.

One part soy grew up phase of budding, after which the plants were removed from soil by roots and measured their biometric parameters. The measurement results are shown in table 2.

Another part of the soybeans were grown to stage of ripeness at the end of the vegetation period, after which Sobir is whether the mellow soy beans, and plants are also extracted from the soil with roots and measured their biometric parameters.

The measurement results are shown in table 3.

From the data presented in table 2 and 3, it is seen that the biological liquid forms on the basis of nitrogen-fixing nodule bacteria Bradyrhizobium japonicum b and Bradyrhizobium japonicum 640, including in the composition according to the invention the aqueous solution with the addition of Na-CMC, potassium sorbate and glycerol in the above ratio, even after 5 months of storage have the same effectiveness in terms of biometric parameters of soybean as fresh drugs called without additives produced by traditional technology, and the number of formed nodules on the roots, affecting power plants atmospheric nitrogen, and average the mass taken from the same plant beans, characterizing soybean yields, is superior to them.

Thus, for example, two nitrogen-fixing nodule bacteria empirically confirmed that the biological liquid forms on the basis of strains of root nodule bacteria of the proposed structure, increasing the yield of legumes, are drugs that retains its effectiveness over a long period of storage is 6 months.

The biological product under legumes culture liquid forms on the basis of nodule bacteria, consisting of mixed in a ratio of from 1:1 to 1:2 culture liquid, which contains Asa strain of nitrogen-fixing nodule bacteria titer not less than 10 ⁷CFU/ml, and an aqueous solution containing, wt.%: