RU2603281C1 - PHOSPHATE-DISSOLVING STRAIN Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3, WITH FUNGICIDAL AND BACTERICIDAL ACTIVITY - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and can be used for production of biphosphate fertilizer. Strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 exhibiting fungicidal and bactericidal activity for protecting plants from diseases caused by fungi and bacteria is deposited in State Collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk", No. B-7427.

EFFECT: invention provides higher yield of agricultural plants.

1 cl, 5 tbl, 7 ex

Description

The invention relates to biotechnology and relates to a new bacterial strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3, which has the ability to release phosphates from insoluble minerals and inhibit the growth of phytopathogenic fungi, as well as pathogens of human, animal and plant diseases. The strain can be used to create biophosphorus fertilizers based on it with fungicidal and bactericidal properties, as well as to develop antimicrobial agents against bacterial pathogens in humans and animals.

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 G.S., Marshunova T.N., Pavlova V.F., Zolnikova N.V. Role of soil microorganisms in soil nutrition of plants // Successes microbiol. 1985.T. 20.P. 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 research is being carried out on the possibility of replacing the industrial chemical production of phosphorus fertilizers with microbiological (N. Antoun. Beneficial microorganisms for the sustainable use of phosphates in agriculture // Procedia Engineering. 2012. Vol. 46, pp. 62 -67; P. Jain, DS Khichi. Phosphate solubilizing microorganism (PSM): an ecorriendly biofertilizer and pollution manager // J. of Dynamics in Agricultural Research. 2014 .-- Vol. 1 (4), pp. 23-28).

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 cereals are mainly fungi of the genus Fusarium.

In addition, one of the most common and harmful diseases of winter crops is the so-called snow mold (V.F. Peresypkin, S.L. Tyuterev, T.S. Batalova. Diseases of crops with intensive cultivation technologies. - M.: Agropromizdat, 1991 .-- 272 p .; S. Chakraborty, A.S. Newton. Climate change, plant diseases and food security: an overview // Plant Pathology. 2011. - No. 60. - Pp. 2-14). The main causative agents of the disease are Microdochium nivale fungi, as well as the Fusarium genus: F. culmorum (WG Sm.) Sacc., F. avenaceum (Fr.) and others (T.I. Ishkova, L.I. Berestetskaya, E.L. Gasich, Diagnostics of the main fungal diseases of cereals. - St. Petersburg: VIZR. - 2002. - 76 p .; BC Gorkovenko, L.A. Oberyukhtina, E.A. Kurkina. Harmfulness of the Microdochium nivale fungus in winter wheat agrocenosis // Protection Plant Quarantine. 2009, No. 1, pp. 34-36). Snow mold develops in early spring immediately after snow melts and even under snow. The defeat of winter wheat crops in some years reaches 100%, which leads to a complete loss of crop. To combat the pathogens of snow mold, it is necessary to use psychrophilic microorganisms that can exhibit antagonistic activity at low temperatures.

Although phytopathogenic fungi occupy the first place in terms of damage to crop production among other pathogens, phytopathogenic bacteria also lead to a significant decrease in yield and its further damage during storage. Plant diseases are caused by bacteria of the genera Erwinia, Xantomonas, etc.

If antagonists of crop pathogens are used as microorganisms that dissolve phosphate ore in the field, this will significantly reduce the use of chemical pesticides, and will increase environmental and food safety. The creation of complex biophosphorus fertilizers with fungicidal and bactericidal properties will further improve environmental safety, including by reducing environmental pollution, accompanying the production and use of traditional chemical phosphorus fertilizers.

Phosphate-soluble microorganisms are currently known (e.g., US Pat. No. 5026417; YP Chen, PD Rekha, A.V. Arum, FT Shen, W. - A. Lai, CC Young. Phophate solubilising bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities // Applied Soil Ecology. - 2006. - V. 34. - P. 34-41) and microorganisms on the basis of which preparations for the control of plant diseases were created (for example, RF patents No. 1805849, No. 2019966, No. 2099947, No. 2213774; V.A. Pavlyushin, S.L. Tyuterev, E.V. Popova, I.I. Novikova, G.A. Bykova, N.S. Domnina.New complex biological products for the protection of vegetables from fungal and bacterial diseases / / Biotechnology. - 2010. - No. 4. - S. 69-80).

Known strain Pseudomonas aureofaciens VKM B-2390, designed to protect plants from fungal pathogens, to clean the soil from arsenic and dissolving phosphates (RF Patent No. 2323967). The strain has a narrow spectrum of antagonistic action, and its phosphate-dissolving activity is characterized only qualitatively 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).

A known 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. Vol. 231. pp. 13-17). This strain also has a narrow spectrum of action, its activity is low and its effect on increasing yields has not been confirmed.

Н., Fraga R. Phosphate solubilizing bacteria and their role in plant growth promotion // Biotechnol. Adv. 1999. Vol.17, pp.319-339). Кроме того, не подтверждена активность штамма при пониженных температурах против Microdochium nivale.A known strain of Pseudomonas sp., Exhibiting antagonistic activity against phytopathogenic fungi and bacteria and dissolving phosphates (WO 2010037072 A1). However, its phosphate-dissolving ability is characterized only qualitatively by the diameter of the enlightenment zone in agar containing Ca ₃ (PO ₄ ) ₂ and a decrease in pH in the medium. It is known, however, that a decrease in pH in the culture medium of a microorganism with insoluble phosphate, although it indicates the ability to transfer phosphate to solution, is not directly related to this ability (

N., Fraga R. Phosphate solubilizing bacteria and their role in plant growth promotion // Biotechnol. Adv. 1999. Vol.17, pp. 319-339). In addition, the activity of the strain at low temperatures against Microdochium nivale was not confirmed.

Closest to the claimed invention is the previously isolated phosphate-soluble strain of Pseudomonas species 181a with fungicidal properties (RF Patent 2451069). The strain has high phosphate-dissolving properties: converts up to 2620 μg / ml phosphate into solution and exhibits antagonistic activity against fungal phytopathogens of the genus Fusarium: for F. graminearum, the inhibition zone is 1.0 mm, for F. culmorum - 1.5 mm, and for F. oxysporum - 2.5 mm. However, the strain has a narrow spectrum of activity, develops poorly at low temperatures and is not active against Microdochium nivale.

The technical result is to obtain a new strain with high phosphate-dissolving activity, growth-promoting properties and the ability to suppress phytopathogenic fungi and bacteria, including the inhibition of Microdochium nivale at low temperatures, to combat plant diseases and increase productivity, as well as to develop drugs against pathogens diseases of humans and animals.

The technical result is achieved by the fact that the proposed strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3, isolated from rhizospheric soil in the territory of the plant for the production of phosphorus fertilizers in Voskresensk (Moscow region) in 2005 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 pathogenic fungi and bacteria, stimulation of plant growth and harmlessness to warm-blooded animals.

The proposed strain of Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 has the ability to dissolve phosphates, inhibits the growth of phytopathogenic fungi, including Microdochium nivale at low temperatures, and bacterial pathogens, stimulates plant growth, increases productivity.

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-7427.

The bacterial strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 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 fish meal hydrolyzate (GRM agar) (FBUN SSCMB, Obolensk), after 24 hours of cultivation at 28 ° C, colonies with light yellow pigmentation with a diameter of 1-2 mm grow. The colonies are round, with smooth edges, flat, transparent.

The strain grows well in the following environments: fish meal hydrolyzate (GRM) broth (FBUN SSCMB, item Obolensk) + 2% glucose, on a minimal synthetic medium of the composition (g / l): K ₂ HRO ₄ - 1.5; KH ₂ PO ₄ - 3.0; NH ₄ Cl - 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 of 20-30 ° С, grows at 4 ° С and does not grow at 41 ° С; grows within the pH range from 3.0 to 8.5.

It does not need additional growth factors (prototroph). Oxidative glucose metabolism. Molecular nitrogen does not fix. Starch hydrolyzes. Fiber does not decompose. The strain hydrolyzes arginine, with the help of glucosidases and galactosidases, some oligosaccharides are cleaved, but not raffinose and sorbose. Ferments mannitol, sucrose, glycerin, maltose, salicin, melibiosis, does not have urease activity. Oxytest is positive. The oxidase reaction is positive.

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

Uses ammonia and nitrate salts 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.

Antibiotic resistance: resistant to carbenicillin, cefalexin, clarithromycin, fusidine, amoxicillin, furagin, cefaclor, roxithromycin, itraconazole, cephalotin, benzylpenicillin, streptomycin, furazolidinufufufenefuinonefufenefuinonefufenefuinonefuinfuizonefuinonefuinfuizonefuinfuinonefuinonefuinonefuinonefuinonefuinfuinfuizonefuinfuizonefuinfuizonefuinfuizonefuinufinufinufinufinufinufinucin

The bacterial strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 is stored on plates or jambs with timing agar at 4-8 ° C. Reseeding on fresh environments - once a month. Long-term freeze-dried storage in a lactose-polyglucin protective environment. Store at 4-8 ° C, re-laying after 5 years.

The strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 lacks phytotoxicity. Indicators of phytotoxicity of the strain have negative values (table 1), indicating the presence of a stimulating plant growth effect.

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 chlororaphis ssp chlororaphis Vsk-26a3

A cell culture of the strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 for the tests is prepared as follows: 150 ml of sterile GRM broth medium is poured into a 750 ml flask, 6 ml of sterile 50% glucose solution is added and 1-2 ml of the stationary culture of the Pseudomonas chlororaphis strain is added ssp chlororaphis Vsk-26a3. The cultivation is carried out with aeration (160-250 rpm) at 28 ° C for 17-20 hours to a titer of culture fluid 5-10 × 10 ⁹ CFU / ml.

Example 2. Obtaining a dry preparation based on a strain of Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 for field trials

The culture fluid obtained in Example 1 is mixed with a protective medium containing 2% polyglucin and 7% lactose and dried on a Virtis BT-4k lyophilizer (USA). The result is a dry powder with a content of 1-25 × 10 ⁹ CFU / g

Example 3. Demonstration of the absence of phytotoxicity in the strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3

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 CFU / ml). In the resulting suspension, wheat seeds of the Moskovskaya-56 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 kept 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

,

,

where A is an indicator of phytotoxicity;

D _o - the average parameter in the experiment;

D _to - the average parameter in the control.

Negative values of indicator A indicate a stimulating effect.

Example 4. Demonstration of phosphate-soluble activity of the strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3

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. On Wednesday, add 2-3 ml of a culture of Pseudomonas chlororaphis ssp chlororaphis grown in example 1. 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-stage method is used, based on measuring the color intensity of its molybdenum complex with Tween 80 at 350 nm (Pupyshev, A.B. Stable reagent for one-stage determination of inorganic phosphate // Laboratory work. - 1991. - No. 9. - S. 12-16). The measurement of phosphate in solution is carried out in triplicate.

As a result of incubation of the culture of Pseudomonas chlororaphis ssp chlororaphis, a decrease in pH in the medium from 7.2 to 4.6 is observed, and the phosphate concentration in the solution is 2580 ± 190 μg / ml, which indicates the high phosphate-soluble properties of the strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 on prototype level Pseudomonas species 181a.

Example 5. Demonstration of the antagonism of the strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 against fungi and bacteria

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), and bacterial suspensions (10 ⁸ CFU / ml) from bacterial lawns on GRM agar (Egorov N .С. Guide to practical exercises in microbiology. - M .: Publishing House of 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 KHA (in the case of fungi) and timing agar (in the case of bacteria) in Petri dishes and triturated with a spatula. Wells 5 mm in diameter are made in the center of pathogen-seeded Petri dishes with agar, into which 0.1 ml of Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 culture fluid obtained according to Example 1 is added. The plates are incubated at 28 ° C for 2-4 days . In the case of antagonism of Microdochium nivale fungi (snow mold), the plates are incubated in the refrigerator at a temperature of 4-8 ° C for 7-10 days. The results are presented in tables 2-3.

The data of table 2 demonstrate the suppression of the claimed strain of 15 species of phytopathogenic fungi, including two strains of Microdochium nivale at low temperatures (4-8 ° C).

The data in table 3 confirm the antagonistic activity of the claimed strain against phytopathogenic bacteria and bacterial pathogens of humans and animals.

Example 6. Demonstration of increased soybean yield as a result of the use of strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 with a natural infectious background

As a test culture, soybean 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) - the humus content (according to Tyurin) is 5.3%. The content of mobile phosphorus (according to Kirsanov) is 34.0 mg / 100 g of soil, the content of mobile potassium (according to Kirsanov) is 19.2 mg / 100 g of soil. Additionally, fertilizers are not introduced.

The experiments use the basic techniques 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 soybean cultivation, taking into account weather conditions.

Use a dry preparation based on the strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3, prepared according to example 2, with a titer of 1 × 10 ⁹ . Method of application: treatment of seeds before sowing. Soybean seeds are initially moistened with water (at the rate of 2% of the weight of the seeds), then dusted with the required amount of dry preparation and thoroughly mixed.

Infectious background - natural. The repetition in the experiments is 4-fold, the plot area is 25.6 m ² , the accounting plot area is 16.3 m ² . The results are shown in table 4.

When using the strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3, soybean yield significantly increased by 15.8%.

Example 7. Demonstration of increasing winter wheat yield as a result of the use of the strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 with artificial infectious background

Test culture: Viola winter soft wheat.

The experiment is carried out in a crop rotation on gray forest loamy soil with the following agrochemical parameters: pH of salt extract - 5.13; the humus content in the layer is 0-20 cm (according to Tyurin) - 3.1%, mobile phosphorus (according to Kirsanov) - 139 mg / kg of soil, mobile potassium - 162 mg / kg of soil. Additionally, fertilizers are not introduced. The experiment 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 winter wheat, taking into account weather conditions. The accounting area of the plot is 5 m ² , the repetition is three times.

Use a dry preparation based on the strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3, prepared according to example 2, with a titer of 1.2 × 10 ¹⁰ . Method of application: treatment of seeds before sowing, as well as treatment of plants in the tillering phase. 2 hours before sowing, seeds moistened with distilled water are dusted with a dry preparation based on a strain of Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 at a dose of 0.25 kg / t. Processing of plants in the tillering phase is carried out using a manual sprayer with an aqueous suspension of a dry preparation based on a strain of Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 at a dose of 1.5 kg / ha at a flow rate of 200 l / ha.

Infectious background: artificial. To prepare the inoculum in order to create an infectious background, the wheat grain is placed in flasks and moistened with water at a water: grain ratio (1: 1). Next, the flasks are placed in an autoclave and sterilized at a pressure of 1 atm for 40 minutes. After autoclaving, the infection of flasks with grain is carried out separately by two strains of Microdochium nivale, one of which is isolated from the leaves of affected winter wheat plants in the Ryazan region, and the other is obtained from K.A. Timiryazev. Inoculated flasks are placed in a refrigerator for 3 months, maintaining the temperature not higher than 10 ° C. During the cultivation of the pathogen, the flasks are periodically shaken. For infection, the finished inoculum is introduced into plots of 30 g of infected grain per 1 m ² .

The test results are shown in table 5.

As follows from table 5, infection with pathogens of snow mold significantly reduced the yield compared with uninfected control by 22.1%. When using the strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3, the yield of winter wheat more than doubled compared with the infected control. In the variant with the use of the strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3, the best wintering of winter soft wheat plants, a greater plant height and a larger number of grains from an ear are also noted in comparison with the infected and uninfected controls.

Thus, the strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 has a high ability to release phosphates from insoluble mineral raw materials. The strain has a wide spectrum of action against pathogenic fungi and bacteria, suppresses 15 types of phytopathogenic fungi, including Microdochium nivale at low temperatures. The proposed strain stimulates plant growth, increases productivity. The strain is not toxic to plants and animals and can be used to create biophosphorus fertilizers based on it with fungicidal and bactericidal properties, as well as to develop antimicrobial agents against fungal and bacterial pathogens of humans and animals.

Claims (1)

Phosphate-soluble bacterial strain Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3, with fungicidal and bactericidal activity, to protect plants from diseases caused by fungi and bacteria, deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk" under the number B-7427.