SU1698242A1 - Strain of tubercle bacteria bradyrhizobium sp - Google Patents

Abstract

The invention relates to agricultural microbiology and biotechnology, in particular, to obtaining microbiological means for increasing the yield of legumes (licorice). The aim of the invention is to obtain a strain of nodule bacteria Bradyrhizoblum sp. for the production of bacterial fertilizer for licorice naked. Strain of nodule bacteria Brodyrhlzoblum sp. (Glycyrrhiza) 3101 isolated from lichen lichen nodules, deposited with All-Union Scientific and Technical Information Institute under No. 3101. The strain allowed to increase the yield of green mass of licorice by 7.0 c / ha (61.4%) and the protein content in plants by 3.2%. 3 tab.

Description

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The invention relates to agricultural microbiology and biotechnology, in particular to the production of microbiological means of increasing the yield of legumes, namely licorice.

The purpose of this invention is to obtain a strain of the nodule bacteria Bradyrhizobium sp. for the production of bacterial fertilizer for licorice naked.

Bacteria strain Bradyrhizoblum sp. (Glycyrrhiza) 3101 were isolated from licorice nodules of the naked wild population. Identified by the determinant of bacteria Bergi.

Strain 2101 is deposited and stored in the collection of nodule bacteria of the All-Union Scientific Research Institute of Agricultural Microbiology under the number 3101.

Cultural and morphological features.

Strain 3101 grows on nutrient media of the following composition:

Mannitol-yeast agar, g / l: mannitol 10.0; K2HP04 0.5; MgSO 7H20 0.2; NaCI 0.1; yeast extract 0.4; agar 15.0; pH of 6.8-7.0.

Mannitol-yeast agar with bromopytimine, g / l: 10.0; K2HPO i 0: 5; MgS04x7H20 0.2; NaCl 0.1; yeast extract 0.4; agar 15.0; Bromthymol blue 5 ml 0.5% alcohol solution, pH 6.8-7.0.

Mannitol-yeast agar with amino peptide, g / l: mannitol 10.0; K2HP04 0.5; MgSOx7H20 0.2; NaCl 0.1; CaCO3 traces, yeast extract 0.4; aminopeptide 10 ml; agar 15.0; pH of 6.8-7.0.

On oblique mannitol-yeast agar at T 26-28 ° C and pH 6.8-7.0 in 5-day-old culture, the cells are in the form of short thick rods with rounded ends, size 1.5-2.0x0.7-0.8 um, gram Os

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mobile, up to 72 hours, moving, then as they age by the 5th day lose mobility, Slowly growing culture. Formation of specialized cells.

With aging culture on mannitol-yeast agar with a pH of 6.8-7.0 and at T 26-28 ° C at the age of 7 days or more, bacteroids of a branched form are formed.

Characteristics of growth in a dense environment.

1. String-Yeast Agar (MDA). T 26-28 ° C, pH 6.8-7.0. The stroke of the 5-day culture is moderate, colorless, slimy, flowing, slightly convex, brilliant. The colonies appeared on the 7th day, on the 10th day they were 2-2.5 mm in size, of the same type, whitish, round, slightly convex, spreading, mucous, S-shaped.

2. MDA + bromthymol blue, T 26-28 C, pH 6.8-7.0.

Bacteria growth on this medium is similar to growth on MDA, but the culture turns yellow and the medium turns greenish.

3. MDA + aminopeptide, T 26-28 ° C, pH 6.8-7.0.

The stroke of the 5-day culture is plentiful, colorless, slimy, flowing, flat. Colonies appear on the 4th day, on the 7th day they are 3-4 mm in size, of the same type, round, whitish, mucous, bulging, spreading, S-shaped. Growth in a liquid medium.

Milk with a litmus, T 26-28 ° C, does not peptonize, intensively alkalinizes. After 48 hours, the color of the milk changes towards the blue spectrum, the maximum color change is noted on the 15th day.

Physical and biochemical properties.

Symbiote. Macrosymbiont is a legume licorice plant.

Attitude to:

a) oxygen - aerobic

b) T ° is maximum 40 ° С; minimum 10 ° С: optimal 26-30 ° С.

c) pH is maximum 8L; minimum 6.0; 6.8-7.0 is optimal.

Attitude to carbohydrates. Intensive use of mannitol, rhamnose, xylose, maltose, sucrose, lactose, dextrin, weaker - glucose, galactose, sorbitol. The media with xylose, dextrin is acidified, with the remaining sugars it is alkalinized. Cellulose and starch do not digest.

Relation to nitrogen sources: it grows well on media with sodium nitrate, ammonium salts, and aminopeptide. On moc-peptone agar (MPA) bacteria do not grow.

Signs of strain resistant. Strain is not pathogenic

Strain 3101 is stored on mannitol agar at 4-5 ° C and pH 6.8-7.0 is transplanted once every 6 months.

Example 1 (assessments of symbiotic

properties of strain 3101).

Assessment of the symbiotic properties of strain 3103: virulence, nitrogen-fixing activity, and the effectiveness of symbiosis was carried out under conditions of microvegetation and propagation experiments.

Virulence is determined by the number and morphology of the nodules formed by inoculation of licorice naked by nodule bacteria. The nitrogen-fixing activity of the strains is determined by the magnitude of the activity of nitrogenase, the key enzyme in the process of binding molecular nitrogen. In turn, the nitro genase activity is measured by an acetyl method (by reducing acetylene) on intact plants.

The efficiency of symbiotic nitrogen fixation is determined by the total nitrogen accumulation of plants per vessel, as well as by

5, the yield of green mass of licorice and its nitrogen content, which is determined using the Kjeltek-auto Tecator system.

Microvegetation experience.

0V large tubes with 5 ml sterile

Vermiculite is added with 11 ml of Krasilikov-Koren medium, sterilized in an autoclave. Licorice seeds of the bare wild population are sterilized with concentrated sulfuric acid for 20 minutes, washed with sterile water and laid out on paper filters moistened with sterile water and Petri dishes for germination. After 2 days seedlings in

Aseptic conditions are laid out in tubes with medium (2 seedlings each). The experiments were set up in 10 fold replicates. The nodule bacteria were inoculated for inoculation of plants.

5 mannitol-yeast agar in test tubes for 5 days. at 28 ° C and suspended in a solution of trace elements (according to Fedorov's recipe). After 2 days. after seeding, 1 ml of inoculum is added to each tube,

0 having a concentration of 107 cells. Plants are grown in a phytotron at 24-26 ° C. After 45 days. The tubes are sealed and acetylene is injected with a syringe to a concentration of 10% by volume. The amount of ethylene

The 5 resulting from the reduction by acetylene with nitrogenase of nodule bacteria is determined after 24 hours on a gas chromatograph.

The magnitude of the nitrogenase activity of nodules, which measures the nitrogen-fixing activity of the strain, is expressed in micromole C2H4 for 24 hours per vessel.

The vegetation experiment was carried out in vessels with 6 kg of sod-podzolic soil with the addition of salts according to the recipe Prishnikova, but without nitrogen. Repetition of the experiment was 4-fold. Licorice seeds were used from a bare wild population.

For seed inoculation, a pure bacterial culture is grown on beveled mannitol-yeast agar. Culture in test tubes with a titer of 4-5 billion is washed off with tap water, then the suspension is applied to licorice seeds, laid out in wells, made in the soil of each vessel. 10 cells are introduced into the vessel.

Symbiotic properties: virulence and nitrogen-fixing activity of strain 3101 and known strains of Rhizobium meliloti 425a and Rhizobium meliloti 282 are shown in Table 1.

The comparative virulence and nitrogen-fixing activity of the strains 3101 and Rhizobium meliloti 425a and 282 upon inoculation of the licorice are given in Table 1.

As Table 1 shows, bacteria of the proposed strain 3101 are virulent on licorice, they form nodules typical of this plant on its roots; bacteria of strain 282 is poorly virulent on this plant, forms brown, isolated nodules not on all plants, and strain 425a is not virulent.

Strain 3101 is an active nitrogen fixing symbiote of licorice, the nitrogenase activity of nodules formed by the bacteria of this strain on licorice is higher than in control plants and plants inoculated with strains 425a and 282. Bacteria of strain 282 form inactive nodules on licorice lichen, their nitrogenase activity does not exceed the level of control plants.

Table 2 presents the data of the microvegetation experiment, the efficiency of symbiotic nitrogen fixation of the strains of Bradyrhlzobium sp. 3101, Rhizobium meliloti 425a and 282 during inoculation of licorice

According to the data obtained in the microvegetation experiment (Table 2), as a result of the active symbiotic fixation of atmospheric nitrogen by nodule bacteria of strain 3101, the total accumulation of nitrogen by licorice plants bacterized by this strain was 96.4 and 114.5% higher than when using strains 425a and 282, respectively. At the same time, the yield of green mass of plants inoculated with strain 3101 increased by 22.9%, and the nitrogen content in it was 1.69%. In the variants where strains 425a and 282 were used, nitrogen fixation of the atmosphere did not occur, the plants were not additionally supplied with this element, and the productivity of the plants did not increase. In contrast, inoculation of licorice with a bare strain of Rhizobium meliloti (Melilotus) 282 led to a significant decrease in the yield of green mass, which apparently can be explained by the fact that in this variant the bacteria were competitors of plants for the nitrogen they absorbed from the soil.

These materials indicate that the active symbiotic nitrogen fixation in licorice can only be obtained by inoculation with strains of nodule-specific bacteria specific to it. Therefore, prototype strains were not included in further experiments.

Example 2 (using strain 3101 to obtain bacterial fertilizer).

Sowing culture is grown on a rocking chair in flasks or 5 liter bottles for 3 days. on the medium of the following composition, wt.%: corn extract 0.7-1.0; baker's yeast (wet) 0.06; food sugar 1.0, (NH4) 2SCM 0.05; KH2PCto 0.025; K2HP04xZN20 0.025; MgSCMxTbbO 0.02; (sterile) 0.1; water piped to 100.0; pH 6.8-7.0. The titer of cells 4-5 billion / ml. The seed culture is used to prepare a liquid working culture. It is prepared in 5-liter bottles on the medium of the following composition, wt.%: Corn extract 0.7-1.0; Baker's yeast (wet) 0.05; molasses 1.0; glucose 0.5-1.0; (NH4) 2S04 0..05-0.1; KH2P04 0.025; K2HP04xZN20 0.025; MgS04 7H20 0.02: CAS02 0.02-0.1; tap water to 100.0, pH (after sterilization) 6.8-7.0.

- Bottles seeded with 5% of licorice nodule bacteria. The bottles are shaken on a rocking chair for 72-96 hours at a temperature of 28-30 ° C. Titer S-6 billion / ml. After microbiological control, the culture fluid from the bottles is used for the mixture with peat.

Preparation of peat Ground ground peat neutralized with chalk to a pH of 6.8-7.0, with a moisture content of 30-40% in plastic bags of 200-400 g each is sterilized by gamma irradiation at a dose of 2.5 Mrad. Sterile peat in packages is inoculated by injecting the mixture with culture liquid + molasses (6%) at the rate of the initial jHTpa of bacteria in peat 0 3-0.5 billion / g peat and humidity 45-50%. The bags are then loaded into special mixers, the rotation of which mixes their contents. Culture in peat is grown, keeping the packages for 3-5 days. at 15-20 ° C. Risotorphin is stored at 3-8 ° C in a dark, dry room, separate from the chemical precursors.

Characteristics of the finished product. Risotorphin for licorice on the proposed strain is in appearance a moist, dark, odorless bulk, insoluble in water. The humidity of the finished product is 50-55%. Risotorphin contains 3-4 billion bacteria in 1 g of peat. Shelf life of the drug 6 months. from the day of manufacture. Consumption of the drug 200 g per hectare rate of seeds.

 Statistical processing, P - 7.8%, HCPos-0.33, Background value of nitrogenase activity.

 Statistical processing, P - 1.8%, HCPos - 1.1 mg / vial.

Table 3 shows the data on the efficiency of licorice inoculation of the bare strain 3101.

Efficiency of licorice inoculation with goaty strain of Bradyrhizobium sp. (Glycyrrhiza) 3101 is presented in table 3.

From table 3 it follows that strain 3101 is an active nitrogen-fixing licorice symbiont naked and due to this 10 property it is able to increase the yield of green mass of the plant – farm by 7.0 centners / ha (61.4%) and increase the protein content in plants by 3.2%.

Claims (1)

15 claims Strain of nodule bacteria Bradyrhizobium sp. (Glycyrriza) VNIISHM-3101 for the production of bacterial fertilizer for licorice. 0 Table 1 table 2 Table 3