RU2451069C1 - PHOSPHATE-DISSOLVING STRAIN PSEUDOMONAS SPECIES 181a WITH FUNGICIDAL PROPERTIES - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: strain Pseudomonas species 181a recovered from Karelian breaking rock with using a selective minimal mineral medium containing insoluble calcium phosphate; it is deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures GKPM-Obolensk (Obolensk settlement, Serpukhovsky area of Moscow region) No. B-6646. The strain shows high ability to release phosphates from insoluble mineral raw material; it is active against fungi Fusarium, stimulates plant growth, provides higher crop yield. The strain is non-toxic for plants and may be recommended for making a biophosphoric fertiliser with fungicidal properties on its basis.

EFFECT: invention provides higher efficiency of struggle with Fusarium blight control, and providing higher crop yield.

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Description

The invention relates to biotechnology and relates to a new bacterial strain Pseudomonas species 181a, which has the ability to release phosphates from insoluble mineral raw materials and inhibit the growth of phytopathogenic fungi of the genus Fusarium, which can be used to produce biophosphoric fertilizers with fungicidal properties.

The problem of phosphorus nutrition of plants remains one of the most acute in agriculture, which is explained by two main reasons: a limited supply of phosphate ores and the rapid binding of this element in the soil when applied with fertilizers. Only 5-25% of phosphorus introduced with chemical fertilizers is assimilated by plants (Sample E.S., Soper RJ, Racz GJ Reactions of phosphate fertilizers in soils // The role of phosphorus in agriculture / Ed. Khasawneh FE, Sample EC, Kamprath EJ Madison , WI: American Society of Agronomy, 1980. P.263-310; Muromtsev GS, Marshunova TN, Pavlov VF, Zolnikova NV role of soil microorganisms in the soil plant nutrition // successes mikrobiol . 1985. V.20. S.174-198). The rest of the phosphate is washed out or passes into the soil in an insoluble form and becomes inaccessible to plants. At the same time, the production of phosphorus-containing chemical fertilizers is an energy-intensive and expensive process, extremely polluting the environment. An increase in fertilizer application rates upsets the nutritional balance of plants, reduces product quality and disrupts the ecosystem of soils, groundwater and water bodies. In this regard, currently in many countries intensive studies are being conducted on the possibility of replacing the industrial chemical production of phosphorus fertilizers with microbiological (Vassilev N., Vassileva M. Biotechnological solubilization of rock phosphate on media containing agro-industrial wastes // Appl. Microbiol. Biotechnol. 2003 . - V.61 - P.435-440;. Anandham R., Choi KH, Gandhi PI, Yim WJ, Park SJ, Kim KA, Madhaiyan M., Sa TM Evaluation of shelf life and rock phosphate solubilization of Burkholderia sp. in nutrient-recommended clay, rice bran and rock phosphate-based granular formulation // World J. Microbiol. Biotechnol. 2007. - V.23. - No. 8. - P.1121-1129).

At the same time, in modern grain production, the problem of combating the defeat of crops of cereals and products of their harvest with fusarium rot is very urgent (Sokolov M.S., Kolombet L.V. Agrotechnogenic factors to minimize the harmfulness of fusarium wheat ears // Agrochemistry. 2007. - No. 12 . - S.63-80). The causative agents of Fusarium cereal ears are mainly fungi of the genus Fusarium, which not only reduce the yield, but also contaminate the grain and the food products made from it with mycotoxins, making these products dangerous to human and animal health.

If antagonists of pathogens of agricultural crops, and first of all causative agents of Fusarium infections, are used as microorganisms that dissolve phosphate ore in the field, this will significantly reduce the use of chemical fungicides and increase environmental and food safety. The creation of a complex biophosphorus fertilizer with fungicidal properties will further improve environmental safety, including by reducing environmental pollution that accompanies the production and use of traditional chemical phosphorus fertilizers.

Currently known phosphorus dissolving microorganisms (e.g., U.S. Patent №5026417; YPChen, PDRekha, A.V.Arum, FTShen, W.-A. Lai, C / C / Young Phophate solubilising bacteria from subtropical soil and their. .. tricalcium phosphate solubilizing abilities // Applied Soil Ecology - 2006. - V.34 - P.34-41) and micro-organisms on which to create a drug for controlling plant diseases (eg, RF patent №2019966; VA Pavlyushin, S.L.Tyuterev, E.V.Popova, I.I.Novikova, G.A.Bykova, N.S.Domnina new complex biologics to protect vegetable crops from fungal and bacterial diseases // Biotechnology -.. 2010 . - No. 4. - S.69-80).

Known strain of Pseudomonas aureofaciens VKM B-2390 is designed to protect plants from fungal pathogens, including those from fungi of the genus of Fusarium, a soil purification from arsenic and dissolved phosphates (RF patent №2323967).

However, the antagonistic and phosphate-dissolving activity of this strain is characterized only qualitatively, the latter being ascertained by the presence of a clarification zone in agar containing insoluble phosphate raw materials. It is known that the results of determination of phosphate dissolution by microorganisms obtained on solid and liquid media often do not coincide, and studies in a liquid medium are necessary for the quantitative assessment of phosphate-dissolving activity (Gupta R., Singal R., Shankar A., Kuhad RC, Saxena RK A modified plate assay for screening phosphate solubilizing microorganisms // J. Gen. Appl. Microbiol. 1994. V.40. P.255-260; Deubel A., Fankem H., Nwaga D., Antoun Y., Merbach W. In vitro mobilization of calcium, iron and aluminum phosphate by rhizosphere bacteria of African oil palm // Proceedings of 3 ^rd International Symposium on Phosphorus Dynamics in the Soil-Plant Continuum / Ed. Alves VMC et al. Uberlandia, Brazil: Embrapa Milho e Sorgo, 2006. P.232-233). He also confirmed the effect of the claimed strain on increasing yields.

Closest to the claimed invention is a strain of Pseudomonas sp. NJ-101, which gave zones of inhibition of up to 1.8 mm with cultures of phytopathogens such as Fusarium oxysporum, F. solani and F. udum, and transferred into solution up to 74.6 μg / ml of phosphate from Ca ₃ (PO ₄ ) ₂ (Bano N., Musarrat J. Characterization of a novel carbofuran degrading Pseudomonas sp.with collateral biocontrol and plant growth promoting potential // FEMS Microbiol. Lett. 2004. V.231. P.13-17).

However, the activity of this strain is low and its effect on increasing yields is also not confirmed.

The objective of the invention is to obtain a new strain with high phosphate-dissolving activity and the ability to suppress phytopathogenic fungi of the genus Fusarium to combat fusarium and increase productivity.

The problem is solved by the fact that the proposed strain Pseudomonas species 181a, isolated from collapsing rock of Karelia using a selective minimum mineral medium containing insoluble calcium phosphate (Ca ₃ (PO ₄ ) ₂ ). The main selection criteria were the degree of dissolution of Ca ₃ (PO ₄ ) ₂ , inhibition of the growth of phytopathogenic fungi of the genus Fusarium, stimulation of plant growth, and harmlessness to warm-blooded animals.

The proposed strain Pseudomonas species 181a has a high ability to dissolve phosphates, inhibits the growth of phytopathogenic fungi of the genus Fusarium and stimulates plant growth.

The proposed strain was deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk" (p. Obolensk, Serpukhov district of Moscow region) under the number B-6646.

The bacterial strain Pseudomonas species 181a is characterized by the following properties.

Cultural and morphological features

The cells are mobile, gram-negative, short oval rods, do not form spores. The cell size is 1-1.5 × 1.5-2.5 μm, cell polymorphism is observed.

When growing on agar containing a fish meal hydrolyzate (GRM agar) (FGUN SSCPMB, p. Obolensk)), the strain forms convex, mucous colonies of beige-yellow color with a diameter of 5-7 mm. When growing on a minimal medium containing Ca ₃ (PO ₄ ) ₂ as the sole source of phosphorus, it forms convex, mucous colonies of beige-yellow color with a diameter of 3-5 mm with zones of dissolution of phosphate up to 7 mm in diameter.

The strain grows well in the following environments: fish meal hydrolyzate (GRM) broth (FGUN SSCPMB, p. Obolensk) + 2% glucose, on a minimal synthetic medium of the composition (g / l): K ₂ НРО ₄ - 1.5; KH ₂ PO ₄ - 3.0; NH ₄ CI - 0.5; glucose - 20.0; yeast extract - 2.0; MgSO ₄ 0.4; NaCl - 3.0, trace element solution - 10 ml / l (trace element composition,%: iron sulfate (FeSO ₄ × 7H ₂ O) - 0.01; copper sulfate (CuSO ₄ × 5H ₂ O) - 0.1; manganese sulfate (MnSO ₄ × 2H ₂ O) - 0.1; zinc sulfate (ZnSO ₄ × 7H ₂ O) - 0.01).

Physiological and biochemical characteristics

Obligatory aerob, temperature optimum of growth 28-30 ° С, does not grow at 41 ° С; grows within the pH range from 3.5 to 8.0.

It does not need additional growth factors (prototroph). Oxidative glucose metabolism. Molecular nitrogen does not fix. Starch hydrolyzes. Fiber does not decompose. The strain does not produce indole, urease and hydrogen sulfide, possesses b-galactosidase, b-glucosidase, phosphatase, N-acetyl-bD-glucosaminadase, γ-glutamyltransferase, does not decompose phenylalanine, adonitol, arginine, raffinose, lorinose, sorbitol, lorinose, sorbitol, sorbite, sorbitol raffinose, cellobiosis, dulcitol. The oxidase reaction is positive.

Attitude towards carbon sources

Absorbs acetoin, galactose, glucose, inositol, xylose, starch, malonate, maltose, mannitol, mellibiosis, ramnose, sucrose, trehalose, fructose, Simmons citrate.

Attitude to nitrogen sources

Uses ammonia and nitrate salts of nitrogen as the sole source of nitrogen.

Saprophyte. 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 living cells of the strain.

The strain is not pathogenic for warm-blooded animals: it does not cause the death of CBA mice by subcutaneous administration of 10 ⁸ CFU.

Storage conditions

The bacterial strain Pseudomonas species 181a is stored on plates or jambs with timing agar at 5-7 ° C. Reseeding on fresh environments - once a month. Long-term freeze-dried storage in a lactose-polyglucin protective environment. Store at a temperature of (4-8) ° C, re-laying after 3 years.

The strain Pseudomonas species 181a lacks phytotoxicity. Indicators of phytotoxicity of the strain Pseudomonas species 181a have negative values (table 1), indicating the presence of a stimulating plant growth effect.

The strain Pseudomonas species 181a inhibits the growth of the following phytopathogenic fungi: Fusarium oxysporum, Fusarium graminearum, Fusarium culmorum.

The possibility of carrying out the invention is illustrated by the following examples, but is not limited to.

Example 1. Obtaining a bacterial culture of Pseudomonas species 181a

A cell culture of the strain Pseudomonas species 181a for testing is prepared as follows: 150 ml of a sterile GRM broth medium is poured into a 750 ml flask, 6 ml of a sterile 50% glucose solution is added and 1-2 ml of a stationary culture of the Pseudomonas species 181a strain is added. Cultivation is carried out with aeration (160-250 rpm) at 28 ° C for 10-15 hours to a titer of culture fluid 5-10 × 10 ⁹ cells / ml.

Example 2. Demonstration of the absence of phytotoxicity in the strain Pseudomonas species 181a

From the culture fluid obtained in example 1, prepare 1% (v / v) suspension of cells in sterile distilled water (0.5-1.0 × 10 ⁸ cells / ml). In the resulting suspension, wheat seeds of the Oriole variety are soaked for 2 hours. In a control experiment, the seeds are soaked in distilled water. 50 seeds of each variant are laid out on filter paper moistened with distilled water, which is placed in Petri dishes and incubated in a thermostat at 28 ° C for 4 days, constantly moisturizing. After a day, seed germination is assessed (table 1), and after 4 days, the seedlings are morphometric, measuring the length of all roots and stem.

Phytotoxic activity of the strain is calculated by the formula:

A = (100-D _o / D _to × 100)%,

where A is an indicator of phytotoxicity;

D _about - the average parameter in the experiment;

D _to - the average parameter in the control.

Negative values of indicator A indicate a stimulating effect.

Table 1 The phytotoxicity index of the strain Pseudomonas species 181a for wheat seedlings of the Oriole variety. Seed treatment option Seed germination,% to control Phytotoxicity indicator,% Roots Stem Pseudomonas species 181a cell suspension one hundred -23.5 -16.8

Example 3. Demonstration of phosphate-dissolving activity of strain Pseudomonas species 181a

To determine the phosphate-dissolving activity in flasks with a capacity of 750 ml make 100 ml of sterile mineral medium of the following composition (g / l): NH ₄ Cl, 0.16; MgSO ₄ × 7H ₂ O, 0.2; Ca ₃ (PO ₄ ) ₂ × 2H ₂ O, 5.0; glucose, 10. 2-3 ml of the culture of Pseudomonas species 181a grown in Example 1 are added to the medium. Incubation is carried out on a New Brunswick rocking chair at 160-180 rpm at 28 ° C.

To determine the amount of phosphate transferred to the solution, a one-step method is used, based on measuring the color intensity of its molybdenum complex with Tween 80 at 350 nm (AB Pupyshev, A stable reagent for one-step determination of inorganic phosphate // Laboratory work. - 1991. - No. 9. - S.12-16). The measurement of phosphate in solution is carried out in triplicate. After two days, the concentration of phosphate in the solution is 2620 ± 150 μg / ml, which is more than 30 times higher than that of the prototype.

Example 4. Demonstration of the antagonism of the strain Pseudomonas species 181a against phytopathogenic fungi of the genus Fusarium

The following strains of phytopathogenic fungi of the genus Fusarium are used as test objects (the pathogen-induced disease is indicated in parentheses): F. graminearum Schwabe 1838 K-33 (spike and grain fusarium), F. culmorum (WGSmith, 1884) Saccardo 1895 (root rot cereal), F. oxysporum Schlecht No. 6 (fusarium).

To determine the suppressive properties using the method of holes. Suspensions of phytopathogenic fungi (10 ⁵ CFU / ml) are prepared by washing off the mycelial and spore mass from mushroom lawns on potato-glucose agar (KGA) (Egorov N.S. Guide to practical studies in microbiology. - M.: Moscow State University, 1995. - 217 p.) With a sterile solution of 0.9% sodium chloride. The resulting suspension in an amount of 0.1 ml is seeded on the surface of the KGA in Petri dishes and rubbed with a spatula. Wells 4–5 mm in diameter are made in the center of pathogen-seeded Petri dishes with KHA, into which 0.1 ml of Pseudomonas species 181a culture fluid obtained in Example 1 is added. The dishes are incubated at 28 ° C. for 4 days. For F. graminearum, the inhibition zone is 1.0 mm, F. culmorum is 1.5 mm, for F. oxysporum is 2.5 mm.

Example 5. Demonstration of stimulation of plant growth as a result of the use of strain Pseudomonas species 181a

Tagetes patula, which is convenient for growing in the laboratory and sensitive to the presence of phosphorus, is used as a test culture. In each pot was placed 1.5 liter 1550 g of sandy loam soil deficient in available phosphorus (<5 mg P ₂ O _5/100 g soil Kirsanov), pH 6.8. The soil is sieved through a sieve (2 mm) and sterilized before use (2 atm, 2 hours). soil pH 6.8. Sources of mineral nutrition of nitrogen and potassium contribute fractionally during the growing season in the form of salt solutions. As a source of phosphorus, crushed phosphorite ore of the Yegoryevsky deposit is used (phosphorus oxide (P ₂ O ₅ ) content of 19.6%). Ore is brought in full once once, immediately before sowing, mixing with a soil sample of the pot. In embodiments using the Pseudomonas species 181a strain, the ore is first mixed with biomass and then introduced into the soil at the rate of 10 ⁶ cells per 1 g of soil. In variants without the use of ore, biomass is mixed directly with soil from the same calculation of 10 ⁶ cells per 1 g of soil. The total introduction of nutrients in terms of NPK for the entire growing season is, g per 1 kg of dry soil: nitrogen (N) - 0.15; potassium oxide (K ₂ O) -0.1; P ₂ O ₅ - 0.1.

All options are reproduced in 4 replicates. 10 seeds are sown in each pot. Seedlings are thinned out for 3-5 days after they appear in order to level them in height, leaving 1 plant in each pot. Plants are grown for 3.5 months, then they are dug up, dried in air and weighed. The results are shown in table 2.

table 2 The effect of the strain Pseudomonas species 181a on the weight of marigold plants Option No. Option Description The total dry weight of the plant, g Dry weight of flowers and buds, g The introduction of phosphate ore The introduction of strain Pseudomonas species 181a one 3.76 ± 0.18 0.28 ± 0.01 2 + 4.61 ± 0.32 0.34 ± 0.03 3 + 4.46 ± 0.29 0.46 ± 0.04 four + + 5.15 ± 0.37 0.55 ± 0.04

In the variants using the strain Pseudomonas species 181a, an earlier appearance of flowers is observed on average for 7-10 days. Also, in variants using the Pseudomonas species 181a strain, the total weight of plants increased by 22 and 15%, and the weight of flowers and buds increased by 21 and 20% compared with the corresponding variants without bacteria.

Example 6. Demonstration of increasing plant productivity as a result of the use of strain Pseudomonas species 181a

As a test culture, spring wheat of the Lada variety is used.

The experiment is carried out on dark gray forest loamy soil with the following agrochemical parameters: pH of the salt extract 5.3; the humus content in the layer is 0-40 cm (according to Tyurin) - 4.6%, mobile phosphorus - 0.25 g / kg of soil, mobile potassium - 0.13 g / kg of soil. Additionally, fertilizers are not introduced.

The experience uses the basic methods and schemes generally accepted in breeding, research institutions and the State variety testing.

Sowing is carried out in the optimal time for the agricultural zone according to the technology recommended for the cultivation of spring wheat, taking into account weather conditions.

The method of introducing cells of the strain Pseudomonas species 181a: seed treatment before sowing. For this, 500 ml of a 3% suspension of cells in sterile distilled water (1.5-3.0 × 10 ⁸ cells / ml) are prepared from the culture fluid obtained in Example 1. In the resulting suspension, 1000 g of Lada wheat seeds are soaked for 30 minutes, thoroughly mixing the mixture. In the control variant (without treatment), the seeds are soaked in distilled water. Then the seeds are dried in air and the plots are sown with the SSFK-7M seeder. The seeding rate of 250 g of seeds per plot of 10 m ² . Repeat 4-fold. Harvesting is carried out by the SAMPO-130 combine. The results are shown in table 3.

Table 3 The influence of the strain Pseudomonas species 181a on the wheat crop No. Option Productivity, t / ha one Control without processing 1.03 ± 0.22 2 Seed treatment for Pseudomonas species 181a 1.37 ± 0.26

When using the strain Pseudomonas species 181a, the yield of spring wheat increased by 33%.

Thus, the strain Pseudomonas species 181a has a high ability to release phosphates from insoluble mineral raw materials, which improves their availability to plants. In addition, it inhibits the growth of phytopathogenic fungi of the genus Fusarium, pathogens of cereal diseases and increases productivity. The strain is not toxic to plants, stimulates their growth and can be recommended for creating biophosphoric fertilizers with fungicidal properties on its basis.

Claims (1)

A phosphate-soluble bacterial strain Pseudomonas species 181a has been deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures "GKPM-Obolensk" under the number B-6646 to protect plants from diseases caused by fungi of the genus Fusarium and to increase productivity.