SU1316189A1 - Strain of bacteria azotobacter chroococcum as non-symbiotic nitrogen fixer for barley - Google Patents

Abstract

This invention relates to agricultural microbiology. It is a new Azotobacter strain. The strain has an affinity for barmen rizoplane and has a genetic label. It reproduces and functions on the barium rizoplan for at least 2 months. The strain causes a growth-stimulating effect, produces heteroauxin, B vitamins, gibberellin, forms a nitrogen-fixing association with barley, increases yield and plant productivity in terms of nitrogen content. The strain can be used to create bacterial fertilizer for barley. 5 tab. (O (L CO 05 00 co

Description

This invention relates to agricultural microbiology and is a new strain of azotobacter that can be used to develop and produce bacterial fertilizer that increases the yield and nitrogen content of barley on nitrogen-poor soil without mineral nitrogen fertilizers, and in scientific research work.

The aim of the invention is to obtain a new strain of azotobacter fixing atmospheric nitrogen, secreting growth substances and having an affinity for the root surface, i.e. entering into an associative symbiosis with barley, as well as having a genetic mark, allows you to easily distinguish it from natural strains.

The strain was obtained by multistep selection of bacteria on the surface of barley roots previously processed in the soil; 1 mm Ar.otc ijacter c: .rnocc ;; c-.j :. K (b :; carrying a transmissive plasmid allowing genetic exchange with a wide range of natural species of rhizoplap bacteria. Azob 1 4Ct, or hr:: .r dog.: -: K (p;) obtained from strain Ai ot C ler cl. i ooJccc-j. il, is stored in the Central Museum of Industrial Microorganisms under the number of CMPM B-2375. In the Central Museum of Industrial Micro (HOBbul strain of organisms is registered under N B-3173.

Strain A ;;. Jl:; .г1с1: er chr-vococcuri 3-3173 has the following morphological and physiological characteristics. Morphological signs. With the growth of the B-3173 strain on the agar Berca medium, a stroke of a two-day ivory culture, a mucosal consistency with wavy edges, opaque, a shiny surface, round colonies with a diameter of 1.5-2.5 mm margins, opaque, ivory, more mucous consistency than a stroke.

Cells of strain ii-3173 at a young age have: 1T form of a thick rod with rounded ends (3-7 x 1.5-2, b covered with a gelatinous capsule. There are burning types. Depending on age and environment, they can have a wide variety of Forms and sizes: cocci, sardines. Cells can be arranged singly, in pairs, short

g

50

5 0 5

0 5 Q

five

a chain and sarcin-like packets with g-kami surrounded by a common capsule. Calcium form - cyst. With aging, the culture acquires a brown occore. The pigment does not penetrate the medium. Gram cells can be dyed with aniline dyes.

Physiological and biochemical signs.

Strain b-3173 - aerobic. The optimum growth temperature is 28-32 0, minimum - 10 ° С, maximum - 45 ° С. The reaction of the strain on pH: optimum 7-7.8, maximum - 9, minimum - 6.

Strain B-3173 assimilates the following carbon sources; glucose, levulose, dextrin, galactose, sucrose, maltose, arabinose, xylose and mannose are from alcohols: mannitol, glycerol and ethyl alcohol.

 B-3173 does not assimilate salts of formate, salicylic, oleic acids, methyl alcohol, phenol, cellulose, helicellulose, lignin.

Strain B-3173 uses free nitrogen, nitrogen ammonium, nitrite and nitrate salts. Nitrate and nitrite nitrogen is first converted to ammonium, and then used by azotobacter.

Strain B-3173 needs magnesium, calcium, and small amounts of molybdenum and iron to develop.

Strain B-3173 on MPA grows poorly. Colonies of a two-day culture have a diameter of 0.5-1 mm and more intense color.

Strain B-3173, in contrast to strain K, has an increased ability to attach to the surface of barley roots and to function on them for at least 2 months. after inoculation.

Example I. Weekly seedlings of plant barley varieties Favorite growing on a liquid nutrient medium are treated with an equal number of cells of strains B-3173 (experiment) and K (control) and continue to grow in the light during the day. Then the roots are separated and sequentially washed in seven flasks with Burke's medium (the last two with sand), as in the selection of the strain. Equal weights of the roots are ground in a shredder and after dilution are sown on the indicator medium Burke. As a result of counting colonies, 26 ± 5 -1 O cells of strain B-3173 per 1 g of roots and 2 + 1.4-10 cells per schm were found in the rizoplan of barley

ma K per 1 g of roots. Thus, the ability of the strain cells to attach to the surface of the roots of cum varieties Favorite is 13 times higher than that of the known strain.

The presence of cells of strain B-3173 on the root surface during the barley vegetation after inoculation of the soil with weekly seedlings of plants was determined by fouling. After 1.2 months of vegetation, the roots, cleared of soil, are applied to the surface of the indicator medium. After two days, an intense and simultaneous fouling of the root surface with a continuous mass of cells of strain B-3173 B is noted when control is not detected during the treatment with strain K. Thus, cells of strain 5-3173 have the ability to colonize the surface of barley root varieties Favorite for at least 2 months. after inoculation.

Strain 3-3173 isolates a protein that is absent from strain K, and possesses increased synthesis of another protein as compared to strain K. These proteins, found when comparing electrophoreograms of exobelles of strains B-3173 and K (drawing, may be responsible for the affinity of cells of strain B-3173 for barium rhizoplane.

Strain B-3173 has the ability, under certain conditions, to form a nitrogen-fixing association with barley. Nitrogen fixation of barley plants treated with the strain B-3173 culture in vegetation vessels with sod-podzolic soil without nitrogen fertilizers, measured by the acetylene method, Bbmie, than nitrogen fixation in the natural association without inoculation.

Example 2. Vegetation vessels with sod-podzolic sandy soil (10 kg per vessel without mineral nitrogen fertilizers, 1) without plants, 2) with barley culture of the Favorite variety in the heading stage, 1. 1 and 2.1) inoculated with bacteria of strain B -3173 10 days after germination - 50 ml of cell suspension with a concentration of 10 cells / ml per vessel, 1.2 and 2.2) without inoculation) germination

Metically closed with an insulator of 10 l polyethylene film. 0.4 l of acetylene is introduced into the insulators. After 24 h, gas samples are taken and analyzed on a Chrom-5 gas chromatograph. The ethylene content in the samples are given in table. one.

Table 1

ten

15

0

Soil without

plants Strain-3173

Barley Grade Strain B-3173 Favorite

Traces

0.21 0.046

25

From the data table. 1, it follows that cells of strain B-3173 form a nitrogen-fixing association with barley, exceeding in association with nitrogenase activity barley association with wild soil diazotrophs.

Strain B-3173, when inoculating bacteria into the root zone of seedlings, has the ability to increase the productivity of barley plants in the content of total nitrogen and especially non-protein nitrogen of free amino acids.

Example 3. In a vegetative experiment (6 vessels with 60 plants in each variant of the experiment), sod-podzolic soil with weekly seedlings of barley variety Favorite is inoculated (25 ml per vessel) with a suspension of cells of strain B-3173 in concentration

about

10 cells / ml. After the growing season, the content of total and non-protein nitrogen in the grain is examined. In the variant of the experiment, without the addition of nitrogen fertilizers to the soil, the amount of nitrogen in the grain as a percentage of the absolutely dry mass is (Table 2).

Experience with inoculum

Control without

1.94

increase is reliable

The strain has the ability to stimulate the growth of barley during the processing of seeds.

Example 4. Barley varieties Favorite after sterilization of the surface with 70% ethanol and diacido are germinated in flasks with Burke's medium (control) and with suspension of strains K and B-3171 in Burke’s medium {10 ml each with a concentration of 10 cells / ml per 1 flask) and inoculated on the light system for 8 days (3 experience on 6 surfaces). Then the seedlings are removed and weighed. The biomass of seedlings, inoculated strains K and B-3173, pre BbmiaeT control, respectively, by 90% and 106%. Thus, the growth-stimulating ability of the B-RFR strain is 16 higher than that of the K strain, even under the condition of artificial saturation of the roots with bacteria of the known strain.

Strain B-3173 is resistant to ampicillin, kanamycin and tetracycline at concentrations of 100; 10 and 50 µg / ml, respectively.

The level of resistance is determined by the maximum concentration of antibiotics in Burke's environment, at which

K B-3173

6.65 + 0.2710.8j4-0.34l, 2jfO, 045

8, 4112, 61.3 + 0.016

table 2

0.17

1.77

ten,

0.05.

colonies of strain B-3173 does not inhibit s. The known strain is sensitive to these antibiotics and does not grow at the indicated concentrations. This property of Step B-3173 can be used to quickly identify a strain on the roots of plants in the field.

Example 5. The roots of plants inoculated with strain B-3173 are superimposed on the surface of the Burke agar medium containing ampicillin, kanamycin and tetracycline at concentrations of 100; 10 and 50 µg / ml, respectively. After 2 days, continuous fouling of the roots with the biomass of cells of strain B-3173 is observed in the absence of continuous fouling in strain K.

Example 6. The proposed strain increases the yield of barley and the assimilation of nitrogen. This is evident from the results presented in table. 3. In table. Figure 3 shows a comparison of the effect of the inoculation of seedlings of barley Favorite cultivar strains K and B-3173 on grain yield and nitrogen content in grain in the vegetation experiments on sod-podzolic soil (1985).

Table 3

The reliability of differences, P 0.05

Strain B-CRP can be used to prepare a bacterial fertilizer on a peat base, which we called Rizophil for the ability of bacteria to attach to plant roots.

Example 7. The strain is grown in a fermenter on a modified Burk medium of the following composition (see Table 4).

Table 4

Food Sugar 10.00 195.0

, 64 12.5

Kn ,, 163.1

MgS040.203,9

NaCl0,203,9

Case 4-2H, 0 0.051.0

, 001 0,002

Azotocacter chroococcum Azotobacter chroococcum B -3173

Continued table. 3

0.05

0.05

Continuation of table 4

20

, 003 0.006

Yeast

extract 0.5 9.8

Defoamer

(oleic

acid) 0.1 1.95

Water pipe

water1l 19.5 l

pH 6, 0 is adjusted to a concentration of 10 cells / ml with a 20% solution of NaOH or diluted CS1, and the culture fluid is inoculated in polystyrene - new packages with sterilized gamma rays at a dose of 2-10 g with ground peat. The ratio of the mass of inoculum and peat is 1: 3. After growing in a thermostat at 28 ° C for 3 days, the fertilizer is ready for use.

In tab. 5 presents comparative data on the affinity for rizoplane of barley and the presence of the genetic label of known and selected strains.

Table 5

2 + 1.4

None None

26 + 5

Complete

Is full

Claims (1)

The Azctobacter chroococcuin strain, which has an increased affinity for rozoplan barley and a genetic label, can be used in agriculture to create new bacterial fertilizers and develop rational methods for their use. Invention Formula The bacterial strain Azotobacter chroo-colony no. CMPM 3173 is a non-symbiotic nitrogen-fixer for barley. Stamps Azototoacter chroococcum K A2Qtobacter chroococcum B-3173 I I nm Loved keeping But & yi d elo / (