RU2529958C1 - Strain of nitrogen-fixing bacteria pseudomonas sp for obtaining biological product against diseases of wheat caused by phytopathogenic fungi, and increase in productivity - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: bacterial strain Pseudomonas sp. IB-4 has high productivity, nitrogenase and growth-stimulating activities. The bacterial strain Pseudomonas sp. is deposited in the Russian National Collection of Microorganisms under the registration number of RNCM B-2830D and can be used in preparation of the biological product for reduction of the incidence of diseases of plants, caused by phytopathogenic fungi, and increase in plant productivity.

EFFECT: invention enables to increase the yield of wheat.

6 tbl, 5 ex

Description

The invention relates to agricultural microbiology and relates to a strain that has an antagonistic effect on pathogens of wheat diseases of smut and root rot, with nitrogenase (the ability to fix atmospheric nitrogen) and growth-promoting activity. The new strain can be implemented in the microbiological industry to obtain a biological product to protect agricultural plants from diseases and increase productivity.

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 ineffective for suppressing the root rot of cereals caused by a complex of fungi Fusarium spp. and Helminthosporium sativum.

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

Closest to the claimed are strains of Pseudomonas aureofaciens IB 51 and Pseudomonas putida IB 17 [4, 5], with the ability to exert an antagonistic effect on pathogens of wheat diseases of bunt and root rot. The disadvantage of the strains is their relatively low antagonistic activity and inability to fix atmospheric nitrogen and the synthesis of growth-promoting substances, i.e. lack of comprehensive effects on phytopathogens and plants.

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, including smut, with nitrogenase and growth-stimulating activity.

The task is achieved by the identification and use of the strain Pseudomonas sp. IB-4 isolated from arable soil sample of the Republic of Bashkortostan. The strain is deposited in the All-Russian collection of microorganisms with registration number VKM B - 2830D.

1. Cultural and morphological characteristics.

1.1. On an agarized nutrient medium having a composition, g / l: KH ₂ PO 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; sucrose - 20 g; agar - 1.5%; trace element mixture - 1 ml; distilled water up to 1000 ml, pH 6.8-7.2, the culture forms round colonies with a diameter of 3-4 mm, with a smooth shiny surface, convex, transparent, with smooth edges, soft viscous consistency. At 24 hours of cell growth, small sticks 0.5 × 1.5 μm, motile.

1.2. 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, growing at a temperature of 28 ° C and aeration-mixing mode of 180 rpm for 48 hours. Round colonies with a diameter of 0.5 cm, convex with smooth edges, mucous consistency.

1.3. On the KGA nutrient medium, colonies are round, 6 mm in diameter, convex, white, translucent in color, and have a viscous soft texture. They do not form pigment. At 24 hours of growth - small movable sticks.

2. Physiological and biochemical characteristics.

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

Attitude to carbon sources: metabolizes glucose, sucrose very well; well assimilates mannitol, maltose, xylose, sorbitol, rhamnose, lactose, dextrin, galactose, mannose, glycerin, fructose, ethanol; poorly assimilates arabinose, mesoinositol.

Attitude to nitrogen sources: grows on nitrogen-free media, maintaining high productivity of at least 8 · 10 ¹⁰ cells / ml.

The strain is stored on jambs with Fedorov’s medium in the refrigerator with reseeding to fresh jambs after 2-4 months, as well as in the lyophilized state.

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

Example 1. To compare the productivity of a new strain of Pseudomonas sp. IB-4 with known strains of Pseudomonas aureofaciens IB 51 and Pseudomonas putida IB 17 were grown in flasks on a rocking chair at a temperature of 28-30 ° C for 48 h under aeration conditions on a liquid nutrient medium LB of the following composition, g / l: peptone - 10 0; yeast extract - 5.0; sodium chloride - 10.0; tap water up to 1 liter. The results are shown in table. 1 and indicate a higher productivity of the proposed strain than the strains of the prototype. A high value of the productivity of the proposed strain was noted during cultivation on other nutrient media (table. 2).

Example 2. The study of the antagonistic activity of a new strain of Pseudomonas sp. 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 strain Pseudomonas sp. IB-4. Plates are incubated at 28 ° C for 3 days. Accounting is carried out in areas of absence or inhibition of fungal growth around the colony of the strain. The results are shown in table. 3 and indicate a wide spectrum and high antagonistic effect of the new strain.

Example 3. The antagonistic effect of the new strain against a complex of pathogens of root rot of wheat is tested in laboratory conditions on Zhnitsa wheat with a natural level of infection of seed obtained in the experimental field of the Institute of Biology, Ufa Scientific Center of the Russian Academy of Sciences. Seeds are additionally infected with spores of phytopathogenic fungi Fusarium oxysporum and Bipolaris sorokiniana (the spore suspension titer of both pathogens was 5 · 10 ⁴ CFU / ml). Wheat seeds are treated with a suspension of strain (with a titer of 10 ⁹ CFU / ml) at the rate of 1 ml of suspension per 1 kg of seeds. The seeds are transferred onto a strip of filter paper moistened to full moisture capacity, on top of which a strip of polyethylene is applied, rolled up, placed vertically in a beaker and left at room temperature. In control experiments, seed treatment is carried out with tap water. The experiment is performed in quadruplicate. Processing the results is carried out after 7 days. The effectiveness of the strain is determined by comparing the degree of manifestation of root rot of wheat seedlings in the experiment and control. The results are shown in table 4.

Example 4. The antagonistic effect of the strain against the causative agent of smut of wheat was tested on wheat cultivar "Reaper", artificially infected with Tilletia caries, in closed ground in a greenhouse. Disputes with solid smut spores were carried out at the rate of 1-2 g spores per 1 kg of grain. Wheat seeds were treated with a suspension of strain (with a titer of 10 ⁹ CFU / ml) at the rate of 1 ml of suspension per 50 g of seeds and were planted in vegetation vessels of 60 pieces, taking into account the germination of this wheat variety. In control experiments, seed treatment was carried out with tap water. The experiments were performed in triplicate.

The effectiveness of the strain was determined by comparing the degree of manifestation of plant diseases of hard smut in the experiment and control during the period of milk-wax ripeness of grain. The results are shown in table.5.

According to the results of the experiment, the biological effectiveness of the suppression of smut on the wheat in the strain Pseudomonas sp. IB-4 is 60.0%, with 52.7% for the selected analogue.

Example 5. Determination of growth-promoting activity of the claimed strain.

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, characterized by the ability to induce plant cell division and are the most active among open growth-regulating substances. The presence of cytokinins in the culture fluid determines the presence of growth-promoting activity of the microorganism. The formation of the claimed strain of cytokinins with a concentration of 300 ng / ml of culture fluid was detected by indirect competitive enzyme-linked immunosorbent assay [6].

Example 6. The strain Pseudomonas sp. IB-4 has nitrogenase activity (determined by the acetylene method), comparable in magnitude with the activities of nitrogen-fixing microorganisms of the genus Azotobacter (Table 6), proposed for use under various crops [7-10].

Thus, a new strain of Pseudomonas sp. IB-4 with a wide range and antagonistic effect on pathogens of fungal diseases of wheat, including against the pathogen of hard smut Tilletia caries, which has high productivity, nitrogenase and growth-stimulating activity, which generally allows to increase the yield by 1.84 times.

Table 1 The productivity of the strain Pseudomonas sp. IB-4 on LB Strain Fermentation time, h The number of viable cells (CFU), billion / ml Pseudomonas sp. IB-4 48 10 IB 51 (prototype) 48 one IB 17 (prototype) 48 one

table 2 The titer of microorganisms Pseudomonas sp. IB-4 during cultivation on various nutrient media (CFU), billion / ml Culture medium Fermentation time, h 24 36 48 KGA 0.1 2.2 21 Fedorova 0.4 2,8 33 S-40 0.4 5.5 95

Table 3 Antagonistic effect of strains of Pseudomonas sp. IB-4 and prototype strains for fungal phytopathogens Type of phytopathogenic fungi The diameter of the zone of inhibition of fungal growth, mm Strains Pseudomonas sp. IB-4 IB 51 IB 17 Fusarium culmorum 29th 9 fourteen Fusarium gibbosum 28 35 12 Fusarium graminesrum 28 7 26 Fusarium nivale 33 31 12 Fusarium semitectum 32 9 26 Fusarium solani eighteen 9 12 Bipolaris sorokiniana 49 55 22 Penicillium funiculosum 33 31 fourteen Fusarium oxysporum 32 29th - Alternaria altemata 48 19 eighteen

Table 4 The effectiveness of the new strain of Pseudomonas sp. IB-4 against root rot of wheat Experiment Options The development of wheat germ disease,% Altemaria + Penicillium Bipolaris sorokiniana Fusarium oxysporum The control 28.0 6.3 2.2 IB 51 (Prototype) 16,0 4.3 0,0 IB 17 (Prototype) 16,0 4.8 0.7 Paenibacillus sp. IB-1 12.0 4.0 0,0

Table 5 The effectiveness of the new strain of Pseudomonas sp. IB-4 against hard smut of wheat Experiment Options The prevalence of solid smut,% Biological effectiveness of suppressing the disease,% Gross crop weight in relation to control,% The control 55.0 - 100.00 IB 51 (Prototype) 26.0 52.7 173.5 Paenibacillus sp. IB-1 22.0 60.0 184.0

Table 6 Nitrogenase activity of the proposed strain of Pseudomonas sp. IB-4 and free-living nitrogen fixers Culture Nitrogenase activity, mcg N ₂ / ml / hour Pseudomonas sp. IB-4 0.68 Azotobacter chroococcum B 35 [8] 0.26 Azotobacter chroococcum W 25 [9] 0.45 Azotobacter chroococcum C 40 [10] 0.11 Azotobacter chroococcum B 1 [11] 0.22

Bibliography

1. Copyright certificate of the USSR 1464468, cl. A01N 63/00, 1988.

2. US patent 4714614, CL. 424-93, 1987.

3. Patent RU 1805849, cl. A01N 63/00, 1993.

4. Patent RU No. 2203945, cl. C12N 1/20, 2003.

5. Patent RU No. 2213774, cl. C12N 1/20, 2003.

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

7. Patent RU No. 2074159, cl. C05F 11/08, 1997.

8. Patent RU No. 2074158, cl. C05F 11/08, 1997.

9. Patent RU No. 2074157, cl. C05F 11/08, 1997.

10. Patent RU No. 2076088, cl. C05F 11/08, 1997.

Claims (1)

The bacterial strain Pseudomonas sp., Deposited in the All-Russian collection of microorganisms under registration number VKM B-2830D, to obtain a drug against wheat diseases caused by phytopathogenic fungi and increase productivity.