RU2245918C1 - Strain of microorganism azotobacter vinelandii for preparing biopreparation for control of wheat root rots and enhancing quality and yield of harvest - Google Patents

Abstract

FIELD: agricultural microbiology.

SUBSTANCE: the strain Azotobacter vinelandii IB 4 is obtained by analytical selection of natural isolates by method for selection of producers eliciting rather high antagonistic activity with respect to fungal phytopathogens. The strain is deposited in collection of microorganisms of Biology Institution UNTS RAN at number Azotobacter vinelandii IB 4. For preparing the preparation the strain is grown in nitrogen-free nutrient medium at 28-30^oC for 60 h under aeration condition up to the titer value 10¹⁰ CFU/ml. The strain elicits high nitrogenase activity and ability for producing cytokinins that simulate the growth and development of plants.

EFFECT: valuable properties of microorganism strain.

7 tbl, 7 ex

Description

The invention relates to agricultural microbiology and relates to a strain that has an antagonistic effect on pathogens of root rot of wheat, increases wheat yield and protein content in grain. The new strain can be implemented in the microbiological industry to obtain a biological product to protect against diseases and increase the yield and grain quality of cereal crops.

A known strain of Pseudomonas sp. CMPM B-348, used to obtain the drug for the protection of cereals from the causative agent of ordinary root rot Helminthosporium sativum [1]. However, this strain is not effective in suppressing the root rot of cereals caused by a complex of fungi Fusarium sp. and Helminthosporium sativum.

Known strains of Pseudomonas putida, inhibiting the growth of the phytopathogenic fungus Fusarium oxysporum [2], and Pseudomonas putida BKM B-1743D, inhibiting the growth of fungi of the genus Fusarium [3]. A disadvantage of the known strains is a narrow spectrum of antagonistic action.

Known strain of Azotobacter chroococcum N B-3173, designed to obtain bacterial fertilizers [4], which increases the yield of barley. The disadvantage is the lack of antagonism towards phytopathogenic fungi.

Closest to the claimed strain is Azotobacter chroococcum 92 [5], which has the ability to inhibit the growth of some phytopathogenic fungi. The disadvantage of the strain is a narrow spectrum of antagonistic action and relatively low productivity.

An object of the invention is the selection of a new natural strain with a wide spectrum of antagonistic action on pathogens of fungal diseases of wheat, which has high productivity and nitrogenase activity, the ability to increase yield and grain quality.

The task is achieved by the identification and use of the Azotobacter vinelandii strain IB 4 isolated from arable land sample taken in the Salavat region of the Republic of Bashkortostan.

The strain is stored and maintained in the Collection of microorganisms of the Institute of Biology of the Ufa Scientific Center of the Russian Academy of Sciences, has a collection number Azotobacter vinelandii IB 4.

1. Cultural and morphological characteristics.

but. On an agarized nutrient medium (C-40), having the composition, g / l: KH ₂ PO ₄ - 0.2; People's Republic of China ₄ - 0.8; CaSO ₄ * 2H ₂ O - 0.1; MgSO ₄ * 7H ₂ O - 0.2; FeCl ₃ - 0.01; Na ₂ MoO ₄ - 0.01; yeast extract - 0.5; sucrose - 20 g; agar - 1.5%; distilled water up to 1000 ml, pH 7.4, the culture forms round colonies with a diameter of 3-4 mm, with a smooth shiny surface, convex, transparent, with smooth edges, soft mucous consistency. At 24 hours of cell growth, small sticks 0.5 * 0.7 μm, weakly mobile; at 48-72 hours - there are cysts.

b. On a nutrient agar medium Burke, having the composition, g / l: K ₂ HPO ₄ - 0.8; KH ₂ PO ₄ - 0.2; MgSO ₄ - 0.3; NaCl - 0.2; CaSO ₄ - 0.1; Fe ₂ (MoO ₄ ) - 0.01; glucose - 10 g; distilled water up to 1000 ml; agar-agar - 1.5%; pH 6.8-7.0, the culture forms round colonies with a diameter of 1-2 mm, with a smooth shiny surface, convex, transparent, colorless, uniform consistency.

from. On a solid nutrient medium with sodium benzoate as the only carbon source, there is no growth. When sodium benzoate is added to the medium in an amount of 0.15%, small colonies of 1-2 mm are observed.

d. On a liquid nutrient medium Fedorov with molasses, having the composition, g / l: K ₂ HPO ₄ - 0.3; CaHPO ₄ - 0.2; MgSO ₄ - 0.3; K ₂ SO ₄ 0.2; NaCl - 0.5; FeCl ₃ - 0.01; CaCO ₃ - 5.0; molasses - 40 g; trace element mixture - 1 ml; pH 6.8-7.0, the culture forms round colonies with a diameter of 4 mm, convex, with smooth edges, cream color, mucous consistency.

e. On the KGA nutrient medium, colonies are round with a diameter of 5 mm, flat, opaque, ivory, the texture is oily soft. They do not form pigment. At 24 hours of growth - small sedentary sticks with blunt ends, form cysts at 48 hours.

f. On Ashby's nutrient medium with mannitol as a source of carbon, the colonies are oily translucent with a diameter of 1-2 mm, and the texture is stretched. At 24-48 hours of cell growth, small rods of 0.6 μm, with rounded ends, are weakly mobile.

2. Physiological and biochemical characteristics.

The strain is an aerobic microorganism, the optimum growth temperature is 28 ° C.

Attitude to carbon sources: very well absorbs glucose, sucrose, molasses; well assimilates mannitol, maltose, xylose, galactose, mannose, glycerin, fructose, ethanol; poorly assimilates ramnose, dextrin, mesoinositol, lactose; does not absorb: sorbitol, arabinose. It grows weakly on MPA.

It grows well on a medium with sodium acetate as the only carbon source.

Attitude to nitrogen sources: grows on nitrogen-free media, maintaining high productivity of at least 1 * 10 ¹⁰ CFU / ml. Does not die when heated at 50 ° C for 15 minutes

The strain is resistant to ampicillin (700 μg / ml), kanamycin (400 μg / ml). Sensitive to rimfapicin (100 μg / ml), levomycetin (100 μg / ml).

The strain does not have phytopathogenic activity, as evidenced by the absence of maceration on the potato slices when applied to them with an injection of live cells of the strain.

The strain is stored on jambs with S-40 medium in the refrigerator with reseeding to fresh jambs after 2-4 months, as well as in a freeze-dried state.

The strain was identified by the Bergey Manual of Determinative Bacteriology.

Example 1. To compare the productivity of the new Azotobacter vinelandii IB 4 strain with the known Azotobacter chroococcum 92 strain, they were grown in flasks on a shaker at 180-200 rpm for 60 hours, at a temperature of 28 ° C in a liquid nutrient medium of the following composition,%: molasses - 3.0; To ₂ NRA ₄ - 0.03; CaHPO ₄ - 0.02; NaCl - 0.05; CaCO ₃ - 0.5; K ₂ SO ₄ - 0.02. The results are shown in table 1 and indicate a higher productivity of the proposed strain over the strain of the prototype. A high value of the productivity of the proposed strain was also noted when grown on other nutrient media used in the cultivation of microorganisms of the genus Azotobacter (Table 2).

Example 2. The study of the antagonistic activity of a new strain of Azotobacter vinelandii IB 4.

A suspension of fungal phytopathogen spores is sown with a continuous lawn on Petri dishes with agarized KGA medium (grated potato - 200 g; glucose - 20 g; agar - 20 g; tap water - 1 l). Then, bacteria of the Azotobacter vinelandii IB 4 strain are planted in the same plates with an injection. The plates are incubated at 28 ° C for 3 days. Accounting is carried out in areas of absence or inhibition of growth of the fungus around the colony of the strain. The results are shown in table 3 and indicate a wide spectrum of antagonistic action of the new strain.

Example 3. A new strain was tested in a small-plot experiment in the experimental field of the Institute of Biology of the Ufa Scientific Center of the Russian Academy of Sciences on the effectiveness of biological protection against a complex of pathogens of root rot of wheat and on improving the quality and quantity of yield on wheat of the Zhnitsa variety with a natural level of infection of seed. The day before sowing, wheat was treated with a culture fluid (with a titer of 4 * 10 ⁹ CFU / ml) of Azotobacter vinelandii strain IB 4, at the rate of 1 ml of culture fluid per 1 kg of grain, which corresponds to an average of 3.2 * 10 ⁴ living cells per 1 corn. The control was treated with sterile tap water. During the experiment, sections of 4 m ² in triplicate for each variant were used; all replicates of all variants were randomized among themselves. Famous commercial biological products Agat-25 in the amount of 0.04 kg / t and Fitosporin 0.5 kg / t were taken as standards. The protein content in the grain was evaluated according to GOST 10846-91. The experimental results are shown in Table 4 and indicate the greater efficiency of the new Azotobacter vinelandii IB 4 strain, which manifests itself in an increase in the degree of biological protection of wheat from root rot, an increase in its yield and an increase in the protein content in grain.

Example 4. The strain Azotobacter vinelandii IB 4 has a fairly high nitrogenase activity, which was determined by the acetylene method. The results are shown in table 5, in the same table shows the nitrogenase activity of some strains of bacteria of the genus Azotobacter, proposed for use under various crops [6-9].

Some microorganisms living in the rhizosphere of plants are able to synthesize cytokinins. Cytokinins are natural compounds involved in the hormonal regulation of plant ontogenesis, and are characterized by the ability to induce the division of plant cells and are the most active among open growth-regulating substances. The formation of the claimed strain of cytokinins in the amount of 118 ng in 1 ml of culture fluid was detected by indirect competitive enzyme-linked immunosorbent assay [10]. To confirm the presence of growth-promoting activity in the inventive strain, experiments were conducted on bean plants (examples 6 and 7).

Example 6. Haricot seeds of the Kharkiv white-seeded variety D-45 were soaked for 3 hours in 20 ml in a diluted culture fluid of Azotobacter vinelandii strain IB 4 with a titer of 10 ⁷ CFU / ml. The control was treated in sterile tap water. For each variant, 12 bean seeds were taken. Plants were grown 15 days after the appearance of the first seedlings. The results of the experiment are given in table.6.

Example 7. The ability of the claimed strain to stimulate root formation in bean cuttings was studied. For this, the cuttings of 15-day-old bean plants were placed in water with the addition of the culture fluid obtained by fermentation on C-40 nutrient medium, the titer of the resulting suspension was 10 ⁷ CFU / ml. Cuttings of control plants were placed in sterile tap water. The number of cuttings in each variant is 10, the duration of the experiment is 12 days. After the end of the experiment, the formed roots were counted. The results are shown in table.7.

The experimental results (Tables 6, 7) indicate the presence of a certain growth-stimulating activity of the new strain, expressed in both an improvement in the development of bean plants and a rather significant nodulation on the roots of these plants and stimulation of the formation of roots on cuttings.

Thus, a new strain of Azotobacter vinelandii IB 4 was isolated, which is characterized by high productivity, a wide range of antagonistic effects on fungal phytopathogens. Preplant seed bacterization of wheat by these microorganisms allows increasing its yield and protein content in grain. The strain has the ability to produce cytokinins, while exhibiting growth-promoting activity. The listed beneficial properties allow it to be used to combat wheat fungal diseases and increase its yield.

List of references

1. USSR copyright certificate No. 1464468, cl. A 01 N 63/00, 1988.

2. US patent No. 4714614, CL. 424-93, 1987.

3. Patent RU No. 1805849, cl. A 01 N 63/00, 1993.

4. Copyright certificate of the USSR No. 1359272, class C 05 F 11/08, 1985.

5. Copyright certificate of the USSR No. 922105, cl. C 05 F 11/08, 1982.

6. Patent RU No. 2074159, cl. C 05 F 11/08, 1997.

7. Patent RU No. 2074158, cl. C 05 F 11/08, 1997.

8. Patent RU No. 2074157, cl. C 05 F 11/08, 1997.

9. Patent RU No. 2076088, cl. C 05 F 11/08, 1997.

10. Kudoyarova G.R., Veselov S.Yu., Karavayko N.N., Guli-zade V.Z., Cheredova E.P., Mustafina A.R., Moshkov I.E., Kulaeva O.N. . An enzyme immunoassay system for determining cytokinins // Plant Physiology. - 1990. - T.37, Issue 1. - S.193-199.

Table 1 Strain Fermentation time, h The number of viable cells (CFU), billion / ml Azotobacter vinelandii ib 4 60 93 Azotobacter chroococcum 92 (prototype) 60 2,3

table 2
The titer of microorganisms Azotobacter vinelandii IB 4 when cultured on various nutrient media The number of viable cells (CFU) / ml Culture medium 24 h 48 h 60 h S-40 1 · 10 ⁷ 6.510 ⁸ 2.210 ¹¹ Fedorova 4 · 10 ⁷ 1 · 10 ⁹ 3 · 10 ¹¹ Ashby 6.510 ⁸ 2.210 ¹¹ 3.310 ⁹ KGA 2.610 ⁸ 6 · 10 ⁹ 9 · 10 ¹⁰

Table 3
Antagonistic effect of Azotobacter vinelandii IB 4 strains and prototype strain on fungal phytopathogens Type of phytopathogenic fungi The diameter of the zone of inhibition of fungal growth, mm Strains Azotobacter chroococcum 92 (prototype) Azotobacter vinelandii ib 4 Fusarium culmorum - twenty Fusarium gibbosum - 19 Fusarium graminesrum - fifteen Fusarium nivale - nine Fusarium semitectum - 14 Fusarium solani 2 nine Fusarium avenaceum - 19 Fusarium moniliforme - 12 Bipolaris sorokiniana 4 twenty Penicillium funiculosum - 14

Table 5
Nitrogenase activity of the proposed strain of Azotobacter vinelandii IB 4 and its analogues Culture Nitrogenase activity, Mkg N ₂ / ml / h Az IB 4 0.656 Az. chroococcum B 35 [6] 0.260 Az. chroococcum W 25 [7] 0.450 Az. chroococcum C 40 [8] 0,110 Az. chroococcum B 1 [9] 0.220

Table 6
The results of model experiments to study the effect of the new IB IB strain Az 4 on the development of bean plants Option The average height of plants, cm The average weight of the roots *, g The number of nodules formed, larger than 2 mm, pcs The control 21.81 9.60 2 Azotobacter vinelandii ib 4 23.95 15.60 46 * - figures are based on wet weight

Table 7
The results of the experiment on stimulating the formation of roots in bean cuttings Option The number of dead plants at the end of the experiment,% The number of cuttings that formed the roots, pcs The total number of roots on 10 cuttings of beans, pcs The control 8 2 17 Azotobacter vinelandii ib 4 1 7 93

Claims (1)

The bacterial strain Azotobacter vinelandii, deposited in the collection of microorganisms of the Institute of Biology of the Ufa Scientific Center of the Russian Academy of Sciences under the number Azotobacter vinelandii IB 4, to obtain a drug against wheat diseases caused by fungal phytopathogens, increasing the quantity and quality of the crop.