RU2413707C2 - STRAIN OF Sinorhizobium fredii KB-11 TYPE LEGUME BACTERIA FOR PRODUCING BACTERIAL FERTILISER FOR SOYA BEAN - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to production of bacterial fertiliser for soya bean. The Sinorhizobium fredii KB-11 legume bacteria strain is extracted from a natural population of legume bacteria common in the south of Amur region and deposited in the All-Russian collection of microorganisms under number B-2457D.

EFFECT: invention increases soya seed production and improves quality.

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Description

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

Known strains of nodule bacteria of soybean 639A, 648A, used for the preparation of soybean nitragin related to the slow-growing species Bradyrhizobium japonicum.

The disadvantages of these strains is that they grow only at pH 7 and a temperature of 27-28 ° C (optimal conditions for growing soybean nodule bacteria) and only mannitol is used as a source of carbon nutrition.

The objective of the invention is to obtain a more technologically advanced strain with high adaptive capabilities, the rapid growth of biomass in conditions other than optimal.

The problem is solved by a new strain of nodule bacteria Sinorhizobium fredii KB-11, which was isolated in 1989 from nodules of a mid-ripening soybean variety VNIIS-1, grown on meadow-chernozem-like soil of the Konstantinovsky 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.Skryabin of the Russian Academy of Sciences under the registration number VKM B-2457D and the collection of the GNU of the All-Russian Scientific and Research Institute of Soybeans under the number KB-11.

The strain Sinorhizobium fredii BKM B-2457D 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 KB-11 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 KB-11 grow well on other nutrient media:

Wednesday Norris, g / l: K ₂ NRO 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.

Soybean 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 strain KB-11 in test tubes on all of the above media (at a temperature of 28 ° C for 72 hours), an abundant increase in the stroke of a gel-like 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 strain KB-11 does not change.

Physiological and biochemical characteristics.

Aerobe.

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

Optimum pH 7.0; pH range 6.0-8.0.

Relation to 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 VKM strain B-2457D was identified by Bergie's “Qualifier” (1980) as a strain of the Sinorhizobium fredii species.

The strain Sinorhizobium fredii VKM B-2457D differs from the previously known strains of soybean nodule bacteria in its ability to use various sources of carbon nutrition and grow well in a wide temperature and pH range.

The invention is illustrated by the following specific example of the use of a strain of Sinorhizobium fredii VKM B-2457D.

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 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 bacterium is obtained - a molybdenum mixture, 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-2457D depending on the conditions of the year, obtained as a result of field experiments. The experiments were carried out on meadow chernozem soils, with a variety of soybean Sonata. Bacterialized seeds are sown on the day of treatment. The strain Sinorhizobium fredii KB-11, VKM B-2457D compared with the control (background - natural inoculation) stably increased the yield of soybean seeds for 8 years

Strain Soybean seed yield by years Average yield 1992 2001 2002 2003 2004 2005 2006 2007 2008 2009 Control without processing 18.8 32,0 26.7 16.5 11.9 26.0 18.8 18.8 27.3 22.0 21.9 KB-11 VKMV-2457D 18.8 32,7 30,2 16.5 14.7 26.8 21,4 21,4 28.1 24.2 23.5

The strain of soybean nodule bacteria of the species Sinorhizobium fredii KB-11, VKM B-2457D for the manufacture of bacterial fertilizers for soybeans is high-tech, has accelerated growth on various sources of carbon nutrition.

Claims (1)

A strain of soybean nodule bacteria of the species Sinorhizobium fredii KB-11, VKM B-2457D for the manufacture of bacterial fertilizers for soybeans.