RU2586521C1 - Sinorhizobium fredii NODULE BACTERIAL STRAIN FOR PRODUCTION OF BACTERIAL FERTILISER FOR SOY - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and can be used for production of bacterial fertiliser. Sinorhizobium fredii nodule bacterial strain TB-467, having resistance to high doses of ammonium nitrate, is deposited in Russian National Collection of Microorganisms of G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of Russian Academy of Sciences, registration No. VKM B-2632D.

EFFECT: invention provides higher yield of soya seeds.

1 cl, 1 tbl, 1 ex

Description

The invention relates to agricultural microbiology and can be used for the production of bacterial fertilizers for soybeans.

Closest to the invention is a strain of Sinorhizobium fredii KB-11 VKM B-2457D used for the preparation of soya nitragin (RF Patent for the invention No. 2413707 of 03/10/11, prototype).

The disadvantage of this strain is that it is not resistant to high doses of ammonium nitrate, which limits its use in a production environment.

The objective of the invention is to obtain a strain that is resistant to high doses of ammonium nitrate.

The problem is solved by a new strain of nodule bacteria Sinorhizobium fredii TB-467, which was isolated in 1996 from October-70 soybean nodules grown on meadow chernozem-like soil in the Tambov district of the Amur Region.

The resulting strain is deposited and stored in the All-Russian collection of microorganisms of the Institute of Biochemistry and Physiology of Microorganisms (IBPM) named after G.K. Scriabin of the Russian Academy of Sciences under registration number BKM B-2632D and the collection of the Federal State Budget Scientific Institution of the All-Russian Research Institute of Soybean under the number TB-467.

The strain Sinorhizobium fredii VKM B-2632D is characterized by the following cultural-morphological and physiological-biochemical characteristics.

Cultural and morphological features

At a young age, the bacteria of the strain Sinorhizobium fredii VKM B-2632D are motile, are elongated rods of size (2.3 ... 2.5) × 0.4 microns. Gram-negative. Fast growing culture. In Petri dishes on mannitol-soy solid nutrient medium, these bacteria grow 3-4 days after sowing, forming convex transparent mucous colonies with a diameter of 1.0 ... 3.1 mm. The bacteria Sinorhizobium fredii VKM B-2632D grow well on other nutrient media:

Meat-peptone agar, g / l: dry nutritious agar - 20.0; agar-agar - 10.0.

Norris Wednesday, g / l: K ₂ NRA ₄ - 0.5; MgSO ₄ - 0.8; NaCl - 0.2; FeCl ₃ - 0.01; yeast extract - 2.0; mannitol - 10.0.

Soybean medium with mannitol, g / l: K ₂ NRA ₄ - 0.5; KH ₂ RO ₄ - 0.5; NaCl - 0.1; MgSO ₄ - 0.1; CaSO ₄ - 0.1; molybdenum salt - traces; mannitol - 20; soy flour - 10.0; agar-agar - 15.0.

Soya medium with glucose, g / l: K ₂ HPO ₄ - 0.5; KH ₂ PO ₄ - 0.5; NaCl - 0.1; MgSO ₄ - 0.1; CaSO ₄ - 0.1; molybdenum salt - traces; glucose - 20.0; soy flour - 10.0; agar-agar - 15.0.

Soybean medium with sucrose, g / l: K ₂ HPO ₄ - 0.5; KH ₂ PO ₄ - 0.5; NaCl - 0.1; MgSO ₄ - 0.1; CaSO ₄ - 0.1; molybdenum salt - traces; sucrose - 20.0; soy flour - 10.0; agar-agar - 15.0.

Soybean medium with maltose, g / l: K ₂ HPO ₄ - 0.5; KH ₂ PO ₄ - 0.5; NaCl - 0.1; MgSO ₄ - 0.1; CaSO ₄ - 0.1; molybdenum salt - traces; maltose - 20.0; soy flour - 10.0; agar-agar - 15.0.

Soybean medium with lactose, g / l: K ₂ HPO ₄ - 0.5; KH ₂ PO ₄ - 0.5; NaCl - 0.1; MgSO ₄ - 0.1; CaSO ₄ - 0.1; molybdenum salt - traces; lactose - 20.0; soy flour - 10.0; agar-agar - 15.0.

A portion of soy flour is obtained by grinding germinated soy seeds, pour 1 liter of water and boil for 30 minutes. Salts are added to the filtrate, adjusted to 1 liter.

When growing the Sinorhizobium fredii VKM B-2632D strain in test tubes on all of the above media (at a temperature of 28 ° C for 72 hours), an abundant increase in the size of the gel-shaped transparent consistency was noted. During long-term storage of pure culture in a refrigerator at a temperature of 4 ° C, the consistency of the stroke of the strain Sinorhizobium fredii VKM B-2632D does not change.

Physiological and biochemical characteristics

Aerobe.

The temperature range of growth is 27-39 ° C. The optimum growth temperature is 27-28 ° C.

Optimum pH 7.0; pH range 6.0-8.0.

Attitude towards carbon sources

It is well absorbed and grown on sucrose by glucose, maltose, lactose, mannitol with the formation of acidic products.

Osmostable, gives good growth on media with a NaCl concentration of 0.4µ. The signs of the strain are resistant. The strain is not pathogenic. Replanted once every 6 months. It is stored in test tubes on mannitol-soy agar at a temperature of 4-5 ° C.

The strain of Sinorhizobium fredii VKM B-2632D differs from the prototype strain (strain Sinorhizobium fredii KB-11 VKM B-2457D) in resistance to high doses of ammonium nitrate (gives growth on mannitol-soy solid nutrient medium with a content of NH ₄ NO _{3 of} 40 g / l).

The invention is illustrated by the following specific example of the use of a strain of Sinorhizobium fredii VKM B-2632D.

Example

Inoculum is obtained by growing the strain in test tubes with agarized nutrient soybean medium with mannitol, g / l: K ₂ NRA ₄ - 0.5; KH ₂ RO ₄ - 0.5; NaCl - 0.1; MgSO ₄ - 0.1; CaSO ₄ - 0.1; molybdenum salt - traces; mannitol - 20.0; soy flour - 10.0; agar-agar - 15.0.

After seeding, tubes with a bacterial culture are placed in thermostats with a temperature of 27-28 ° C for 5 days. During this time, in tubes on a jamb of a dense nutrient medium, an abundant increase in the bacterial mass of the test strain is noted.

To inoculate seeds on the day of soybean sowing, a bacterial-molybdenum mixture is prepared. To do this, 2.5 ml of a pre-prepared 1.25% solution of molybdenum (25 g / l ammonium molybdenum) are added to the tubes with a stroke of the test strain of soybean nodule bacteria. The bar from the jamb of the nutrient medium is washed off with a molybdenum solution. By shaking the tubes, a homogeneous bacterial-molybdenum mixture is obtained, which is immediately treated with soybean seeds. The resulting suspension volume (2.5 ml) is used for presowing treatment of 250 g of soybean seeds. After applying the suspension, the soybean seeds are thoroughly mixed, dried in the shade and sown in the prepared soil.

The table provides data on the effectiveness of the strain VKM B-2632D, depending on the conditions of the year, obtained as a result of field experiments. The experiments were carried out on meadow chernozem-like soils with a variety of soybean Harmony. Bacterialized seeds were sown on the day of treatment. The strain Sinorhizobium fredii VKM B-2632D compared with the control (background - natural inoculation) stably increased the yield of soybean seeds for 3 years from 4 years of testing, an average of 3.0 c / ha.

The effectiveness of the strain Sinorhizobium fredii VKM B-2632D

Claims (1)

A strain of soybean nodule bacteria of the species Sinorhizobium fredii VKM B-2632D for the manufacture of bacterial fertilizers for soybeans.