SU1458359A1 - Strain of bradyrhizobium sp. (lotus) bacteria for producing bacterial fertilizer - Google Patents

Abstract

The invention relates to agricultural microbiology and can be used for the production of bacterial fertilizers for legumes and horned twins. The aim of the invention is to obtain a new strain of nodule bacteria, which, when inoculated with horned hornet, increases the yield of green mass and the content of total nitrogen in it. Strain Bradyr- hizobium sp. (Lotus) isolated from the club .. 6eHbkoB l dventsa horned. The strain Bradyrhizobium sp, (Lotus) is deposited and stored in the ARRIAM collection under No. 476. A new strain of nodule bacteria Bradyrhizobium sp. (Lotus) ARRIAM No. 476, compared with non-inoculated control (spontaneous natural population) in vegetation experiments, increases the yield of green mass per hornet by 32.9-50.6% and the total nitrogen content by 0.47-1.35 %, in the field - yield of green mass by 25.1. -35.5%. 3 tab. (Ls

Description

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This invention relates to microbio logical means for increasing the yield of legumes and represents. is a strain of nodule bacteria for inoculation of the horned duck.

The purpose of the invention is to obtain a new strain of nodule bacteria, which, when inoculated with a hornet, yields a crop of green matter and co-v retains total nitrogen in it.

Strain Bradyrhizobium sp. (Lotus) 476 is derived from nodules and horned duck.

A strain of nodule bacteria L of the horned Bradyrhizobium sp, (Lotus) is deposited and stored in the VNIISM collection under No. 476,

Cultural and morphological characteristics of the strain Bradyrhizobium sp, (Lotus) 476.

The strain can be cultivated on pea, lupine and mannitol-yeast agar.

1. Pea agar, g / l: pea 100.0; sucrose 20.0; agar-agar 20.0; pH of 6.8-7.0.

The size of the cells of a 2-day culture on pea agar 1.2-1.9 x0.6-0.9 microns. Short daddy, slightly bent moving at a young age. Peritrichs Bacteroids have a fork shape. Gram-negative. Slowly growing culture. The bar is abundant, thin, colorless. The colonies are of the same type.

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whitish, round, mucous, with clear edges, 3-5 mm in diameter, appear on the 4th day.

2. Lupine agar g / l: Of5; KHjPO 0.5; MgS04 7H20 0.2; NaC10.2; CaS04 0.1; (NH4) 2Mo04 - traces; mannitol 20.0; lupine flour 10.6; agar-agar 20.0; pH of 6.8-7.0.

2.preparation of liquid working culture; .

3. peat preparation and its range of sterilizations;

4. infection of peat by the working culture;

5. Growing bacteria in peat. Preparation of a liquid seed culture.

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Stroke: Abundant, Mucus, Belova-10 tours. Liquid culture is absorbed in

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ty. The colonies of the same type, whitish, round, mucous, weakly spreading - etK, 4-5 mm in diameter, appear on the 4th day. ..

3. Mannitol yeast agar, g / l: mannitol 10.0; K NR040,5 | MgSO-7H-gO 0.2; NaCl yeast extract 0.4; agar-agar 15.0; pH of the medium is 6.8-7.0.

Stroke is plentiful, slimy, colorless, flowing down. Colonies of the same type, whitish, round, mucous, spreading, 4-6 mm in diameter, appear on the 4th day.

 Physiological and biochemical characteristics of the strain Bradyrhizobium sp. (Lotus) 476.

Milk with a litmus does not peptonize, alkalinize with the formation of a mucous zone on the surface. Gelatin I does not dilute Aerobe. The optimum temperature is 26-28 ° G.

Attitude to carbohydrates. Used by Many carbohydrates are acidified with medium. It grows well on media with glu I goat, galactose, mannitol, ram-j Zoya, less actively with dextrin, sucrose, maltose, lactose, weakly / with xylose and sorbitol. Cellulose and starch do not digest.

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Relation to nitrogen sources. It grows well on media with sodium nitrate, ammonium salts, and tyrosine. Bacteria do not grow on MPA. Flask flasks and in p-liter flasks for 2 sugok on the medium of the next composition, May. %: pea broth 10.0; sugar (food) 1.0;

15 (NH4) iS04 0.05; 0.05; .x X 0.5; MgS04 "7H, 0 0.2; CaCOj-0 water plumbing - the rest. pH after sterilization 6.8-7.0. Title 5-8 billion / ml. ..

20 The seed culture is used to prepare a liquid working culture in five liter bottles or an enzyme tori on the medium of the following composition, May. %: corn extract 1.0; me

25 lass 2.0; (NH) SO4; 0.1; 0.025 3H, jO 0.025; MgSO g7H 0 0.0 CAS02 0.1; water plumbing - the rest. The pH after sterilization is 6.8-7.0.

thirty . Bushi and fermenters are seeded with 5% seed culture of nodule bacteria. The bottles are shaken on a rocking chair for 40-48 hours. In fermenters, bacteria are grown.

35 18-36 h with aeration of 0.7-1.5 rpm under an overpressure of 0.3-0.5 at at 28-30 0. Titre not less than 5 billion / m After microbiological control, culture fluid from bottles

40 Or fermenters — an inoculum — are used for mixing with peat.

Peat preparation. Ground ground peat neutralized with chalk to a pH of 6.8-7.2 with a humidity of 30-40%

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Naki strain resistant. Strain not patato- plastic bags up to 200-400 g

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Gene.- Stored on pea agar at 4-5 ° C and subcultured once every 6 months. Strain Bradyrhigbbium sp. (Lotus) 476 identified by definition Bergey's Manual, of sistematic bacteriology, 198.4.

The method of preparation for the inoculation of a pair of horned on the basis of the strain of nodule bacteria Bradyrhizobium sp. (Lotus) 476.

Production technology includes the following operations:

1. preparation of Lidka sowing culture;

sterilized by gamma irradiation at a dose of 2.5 Mrad. Sterile TrF c. bags are inoculated with an injection of a mixture of culture fluid + molasses (6Z

gQ at the rate of initial titer, bacteria 0.3-0.5 billion, / g of peat and humidity of 45-60%. Then the packages are loaded into special mixers, the rotation of which mixes their contents.

gg Culture in peat is grown by keeping the bags for 3-5 days at 15–20, then the bags are stored at a temperature of 3–8 ° C in a dark, dry room, separately from the chemicals.

2.preparation of liquid working culture; .

3. preparation of peat and its gamma sterilization;

4. infection of peat by working culture;

5. Growing bacteria in peat. Cooking Liquid Sowing Kul

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tours. Liquid culture is absorbed in

tours. Liquid culture is absorbed in

katalochnyh flasks and in p-liter bugs for 2 sugok on medium of the following composition, May. %: pea broth 10.0; sugar (food) 1.0;

(NH4) iS04 0.05; 0.05; .x X 0.5; MgS04 "7H, 0 0.2; CaCOj-0,1; water plumbing - the rest. pH after sterilization 6.8-7.0. Title 5-8 billion / ml. ..

A seed culture is used to prepare a liquid working culture in five-liter bottles or enzyme-tori on a medium of the following composition, May. %: corn extract 1.0; molasses2, 0; (NH) SO4; 0.1; 0.025; 3H, jO 0.025; MgSO g7H 0 0.02; CAS0 0.1; water plumbing - the rest. The pH after sterilization is 6.8-7.0.

. Bushies and fermenters are seeded with 5% seed culture of nodule bacteria. The bottles are shaken on a rocking chair for 40-48 hours. In fermenters, bacteria are grown.

18-36 h with aeration of 0.7-1.5 rpm under an overpressure of 0.3-0.5 MPa at 28-30 0. The titer is at least 5 billion / ml. After microbiological control, culture liquid from bottles

Or fermenters — inoculum — are used to mix with peat.

Peat preparation. Ground ground peat neutralized with chalk to a pH of 6.8-7.2 with a humidity of 30-40%

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plastic bags up to 200-400 g

 g plastic bags up to 200-400 g

sterilized by gamma irradiation at a dose of 2.5 Mrad. Sterile TrF c. the packets are inoculated with an injection of the mixture: culture fluid + molasses (6Z

gQ at the rate of initial titer, bacteria 0.3-0.5 billion, / g of peat and humidity of 45-60%. The bags are then loaded into special mixers, the rotation of which mixes their contents.

gg Culture in peat is grown by keeping the bags for 3-5 days at 15–20 ° C, then the bags are stored at a temperature of 3–8 ° C in a dark, dry room, separately from dohimikatov.

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Characteristics of the finished product. Risotorphin for a week on the proposed strain is, in appearance, a moist, dark, odorless bulk, insoluble in water. The moisture content of the finished product 50-55%. Rizotorphin contains 4-6 billion nodule bacteria per gram of preparation. The shelf life of the drug is 6 months from the date of manufacture. Consumption of the drug 200 g per hectare rate of seeds. .

The technology of pre-sowing inoculation of seeds of a couple.

For the inoculation of seeds on a lane, in a vegetational experiment, a pure culture of the proposed strain was grown on beveled pea agar. The culture was dried with iodine and applied to the seeds of a double liter, laid out in the hole in the base of each vessel. 10 cells were inserted into the vessel.

In field trials, seeds of the twelve seeds were treated with peat preparation — rhisotorphine, produced on strain 476. For this, 200 g of the preparation was diluted in 1 liter of tap water, and without allowing the suspension to stand, it was applied to a hectare seed rate (10 kg / ha). Then the seeds were thoroughly mixed until the drug was evenly distributed. Risotrophin-treated seeds were sown on the same day in moist soil.

Methods of vegetation 35 and field experiments. Vegetation experiments were carried out in vessels with 6 kg of sod-podzolic soil with the addition of salts according to the prescription Prishnikova, but without nitrogen. The repetition of the experiment is 4-fold. A zoned cultivar of the crown of the horned Veselo-Podolsky was used;

Field experiments were performed on well cultivated sod-podzolic soils. The area of the division is 50 m. The rotation is 4 times.

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The use of the preparation based on the strain Bradyrhizobium sp. (Lotus) 476 for the inoculation of the horned couple. ; An example of a l.iB vegetation experiment with a double horned bird was used to compare the efficiency of Bradyrhizobium sp. (Lotus) 476 strain with the strain of nodule bacteria used to produce the Rhizpbium leguminosarum 250a pea preparation (strains of nodule bacteria

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to increase the yield of two horned horn has not yet been proposed). ,

The method of conducting experiments and preparation of the drug is similar to that described.

 - Data for example 1 are given in table. one.

Given in Table. 1, the data indicate that the nodule bacteria from the R. group, ie, guminosarum is not effective on a horn, a high yield increase was obtained only in the variant with the inoculation of this plant with specific nodule bacteria of a pair.

Example 2. The efficacy of symbiotic nitrogen fixation in Bradyrhizobium sp. (Ldtus) 476 strain was determined in vegetation and field experiments by the magnitude of the yield of green mass per hornet and the accumulation of nitrogen or protein in it compared to the spontaneous infection of plants with a natural population specific for the spring nodule bacteria found in the soil (variant without inoculation).

The results of vegetation experiments are given in table. 2

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As can be seen from the table. 2, the root nodule bacteria of the strain 476 strain actively fix atmospheric nitrogen in symbiosis with the horned pair, as a result of which the yield of the green mass of plants bacterized with this strain is 32–50% higher than in plants without inoculation. The content of total nitrogen in plants in the variant inoculated with strain 476 is increased by 0.47-0.99%. As a result, the total accumulation of nitrogen in plants bacterized with strain 476 is 1.6-2.8 times higher than that of non-inoculated ones.

Example 3. The efficacy of strain 476 was determined in field experiments on zoned varieties l dventsa.

The results of the experiments are presented in table. 3

As can be seen from the table. 3, the proposed strain provides for an increase in the yield of the green mass of a pair of horned on average by 37.4 tsUga (33.0%) and an increase in the content of protein in the crop by an average of 1.0%.

Thus, a new strain of nodule bacteria Bradyrhizobium sp. (Lotus) ARRIAM No. 476, which during inoculation yields a crop of green mass and the content of total nitrogen in it;

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rmula of invention

The strain of bacteria Bradyrhizobium J sp. (Lotus) ARRIAM No. 476 for npoH3t of bacterial fertilizer production under a double horn.

Table

Comparative effectiveness of Bradyrhizobium sp. 476 and Rhizobium leguminosarum 250a at. inoculation of l horned cattle

P 2.8%; HSPO, 5 1.0 g / soy.

Efficiency of symbiotic nitrogen fixation of the strain Bradyrhizobium sp. (Lorus) 476 in vegetation experiments

Merry Podolsky

Gelswies

No foreign 16.8 cool

PC. 47,625.8

No foreign 16.1 cuts

PC. 47621.4

5.3 32.9 2.47 529

table 2

1.9

1.8

1.4

1.2

Table 3. The effectiveness of the strain Bradyrhizobium sp. (Lotus) 476 in field trials

Moscow

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PC. 476 67.8 13.8 25.1 16.6 0.5

12.2

61.0 35.5 12.8 1.6 14.2

37.4 33.0 15.2 1.0

Beis ino-162

coolings

PC. 476 233

W / o 113.5 cuts

PC. 476 150.9

16,1

8.0 6.5

4.2 8.8

Claims (1)

Claim Bacterial strain Bradyrhizobium sp. (Lotus) VNIISHM No. 476 for production? of bacterial fertilizer under the 'horned lamb. Table 1 Comparative efficacy of strains of Bradyrhizobium sp. 476 and Rhizobium leguminosarum 250a upon inoculation of horned lamb Inoculation Option Harvest green mass, g / sos. Yield increase The total nitrogen content in plants,% g / sos. % Without inoku- rations 9.5 - 1.06 PCS. 250a 9.7 0.2 2.1 1.05 PCS. 576 13.3 3.8 40,0 2.41 P 2.8%; HCp0 _jS 1.0 g / sos. Table 2 ¹ Efficiency of symbiotic nitrogen fixation of Bradyrhizobium 'sp. (Lorus) 476 in growing experiments Baby variety ————————— Inoculation Option Harvest of aboveground mass, g / sos Yield increase The total nitrogen content in plants,% The accumulation of nitrogen on the vessel, mg. Statistical processing g / s % R, % HCP ^ s, g / s Fun-Pod- Without foreign 16.8 1.74 292 1.9 1.8 lansk cumulations PCS. 476 25.8 8.5 50.6 2.78 691 Gelsvis Without inoculation 16.1 2.00 322 1.4 1,2 PCS. 476 21,4 5.3 32.9 2.47 529 m I Moscow Carried inoculation 55.0 16.1 8.0 6.5 PCS. 476 67.8 13.8 25.1 16.6 0.5 Dedinovsky Without foreign 162 12,2 4.2 8.8 cumulations PCS. 476 233 61.0 35.5 12.8 1.6 Average Without inoculation 113.5 14.2 PCS. 476 150.9 37,4 33.0 _____ 1 ______’________ 15.2 1.0