RU2568067C1 - STRAIN OF LEGUME BACTERIA Bradyrhizobium japonicum FOR PRODUCTION OF BACTERIAL FERTILISER FOR SOY - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain of legume bacteria Bradyrhizobium japonicum VKM B-2629D for production of bacterial fertiliser for soy is offered.

EFFECT: strain of Bradyrhizobium japonicum VKM B-2629D is characterised by resistance to high doses of ammonium nitrate, increases crop of soy seeds.

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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 Bradyrhizobium japonicum VKM B-2456D used for the preparation of soybean nitragin (RF Patent for the invention No. 2413762 of 03/10/11. Prototype).

The known strain has a relatively low level of amidase and catalase activity, is not resistant to high doses of ammonium nitrate, which limits its use in a production environment.

When creating the invention, the task was to obtain a strain with higher enzymatic activity, resistance to high doses of ammonium nitrate, which increases the seed productivity of the host plant.

The problem is solved by a new strain of nodule bacteria Bradyrhizobium japonicum MM - 117, which was isolated in 1996 from October-70 soybean nodules grown on brown forest soils of the Mazanovsky 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 VKM B-2629D and the GNU collection of the All-Russian Research Institute of Soybean under the number MM - 117.

The strain Bradyrhizobium japonicum VKM B-2629D is characterized by the following cultural-morphological and physiological-biochemical characteristics:

Cultural and morphological characters.

At a young age, the bacteria of the strain Bradyrhizobium japonicum VKM B-2629D are motile, they are elongated rods of size (2.3 ... 2.5) × 0.4 μm. Gram-negative. Slow-growing culture. In Petri dishes on mannitol-soy solid nutrient medium, these bacteria grow 10 days after sowing, forming convex, milky-white, mucous colonies with a diameter of 1.0 ... 2.0 mm. The bacteria Bradyrhizobium japonicum VKM B-2629D grow well on other nutrient media:

Norris's medium, g / l: K ₂ HPO 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 HPO ₄ - 0.5; KH ₂ PO 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 L.

When growing the Bradyrhizobium japonicum VKM B-2629D strain in test tubes on all of the above media (at a temperature of 28 ° C for 480 h), an abundant increase in the stroke of a paste-like milky-white 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 Bradyrhizobium japonicum VKM B-2629D does not change.

Physiologist-biochemical signs.

Aerobe.

The temperature range of growth is 27-30 ° C. The optimum growth temperature is 27-28 ° C.

The optimum pH is 7.0.

Relation to carbon sources. It is well absorbed and grows on sucrose, glucose, maltose, lactose, mannitol with the formation of alkaline products.

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.

Strain VKM B-2629D was identified by Bergie's “Qualifier” (1984) as a strain of the species Bradyrhizobium japonicum.

The strain Bradyrhizobium japonicum VKM B-2629D differs from the prototype strain (Bradyrhizobium japonicum VKM B-2456D) in its high amidase, catalase activity, and resistance to high doses of ammonium nitrate (gives rise to mannitol soy solid nutrient medium containing NH ₄ NO ₃ 40 g / l).

The invention is illustrated by the following specific example of the use of a strain of Bradyrhizobium japonicum VKM B-2629D.

Example

Inoculum is obtained by growing the strain in test tubes with agarized nutrient soybean medium with mannitol, 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; mannitol - 20.0; soy flour - 10.0; agar-agar - 15.0.

After seeding, tubes with bacterial culture are placed in thermostats with a temperature of 27-28 ° C for 20 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 molybdate) 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 Bradyrhizobium japonicum VKM B-2629D 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 Bradyrhizobium japonicum VKM B-2629D compared with the control (background - natural inoculation) stably increased the yield of soybean seeds during the three years of testing by an average of 1.9 c / ha.

The effectiveness of the strain Bradyrhizobium japonicum VKM B-2629D.

Claims (1)

A strain of soybean nodule bacteria of the species Bradyrhizobium japonicum VKM B-2629D for the manufacture of bacterial fertilizers for soybeans.