Phosphate-dissolving strain acinetobacter species with fungicidal properties

FIELD: medicine.

SUBSTANCE: strain Acinetobacter species the State Collection of Pathogenic Microorganisms (GKPM) - Obolensk B-6645 showing high ability to release phosphates from insoluble mineral raw material, to stimulate plant growth, to provide higher crop yield; it is active against fungi Fusarium and deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures GKPM-Obolensk (Obolensk settlement, Serpukhovsky area of Moscow region) No. B-6645 and may be used for creating a biophosphoric fertiliser with fungicidal properties.

EFFECT: invention allows inhibiting growth of phytopathogenic fungi Fusarium.

2 tbl, 5 ex

 

The invention relates to biotechnology and the new bacterial strain Acinetobacter species 305, with the ability to release phosphate from insoluble minerals and inhibit the growth of phytopathogenic fungi of the genus Fusarium, which can be used to obtain bitstrea fertilizer with fungicidal properties.

The problem of phosphate nutrition of plants remains one of the most acute in agriculture, because of two main reasons: a limited supply of phosphate ores and rapid binding of this element in the soil when making fertilizer. Only 5-25% of applied chemical fertilizers phosphorus is assimilated by plants (Sample E.C., R.J. Soper, G.J. Racz Reactions of phosphate fertilizers in soils // The role of phosphorus in agriculture / Ed. Khasawneh F.E, Sample E.C, E.J. Kamprath Madison, WI: American Society of Agronomy, 1980. P. 263-310; Muromtsev G.S., Marchukova G.N., Pavlov V.F., Zolnikova N.V. the Role of soil microorganisms in soil and plant nutrition // Success microbiol. 1985. T.20. S-198). The rest of phosphate washed out or goes to ground in an insoluble form and becomes unavailable to plants. The production of phosphate-based chemical fertilizers is energy intensive and expensive process, it is extremely polluting. Increased rates of fertilizer upsets the balance of plant nutrition, reduces the quality of products and n will reset ecosystem soils, groundwater and water bodies. In this regard, at present, in many countries, the intensive research the possibility of replacing the industrial chemical production of phosphate fertilizers microbiological (Vassilev n, Vassileva m Biotechnological solubilization of rock phosphate on media containing agro-industrial wastes // Appl. Environ. Biotechnol. 2003. V.61. P.435-440; R. Anandham, Choi K.H., Gandhi P.I., Yim W.J., Park, S.J., Kirn K.A., M. Madhaiyan, Sa T.M. Evaluation of shelf life and rock phosphate solubilization of Burkholderia sp.in nutrient-amended clay, rice bran and rock phosphate-based granular formulation // World J. Environ. Biotechnol. 2007. V.23 supported. No. 8. P.1121-1129).

At the same time modern zernoproizvodstve problem is urgent struggle with lesions sown to cereals and products of their harvest by Fusarium rot (Sokolov, MS, particularly, to the L.V. Agrotechnology factors minimize the severity of Fusarium head blight of wheat // Agrochemistry. 2007. No. 12. P.63-80). Wheat, corn and barley that make up two thirds of the world's cereal production, the most vulnerable to infection by fusariose. Pathogens of fusariosis ears of grain are mainly fungi of the genus Fusarium, which not only reduce the yield but also contaminate the grain and manufacturing of food by mycotoxins, making these products dangerous to animal and human health.

If the microorganisms, dissolving the phosphate ore in the field, will be used antagonists of pathogens of agricultural ku is tour, and first of all pathogens fusariosis, it will significantly reduce the use of chemical fungicides, will improve environmental security and food security. The establishment of a comprehensive bitstrea fertilizer with fungicidal properties will allow to improve environmental safety, including by reducing pollution associated with the production and use of traditional chemical phosphate fertilizers.

Currently known phosphotransferase microorganisms (for example, U.S. patent No. 5026417; Y.P.Chen, P.D.Rekha, A.B.Arum, F.T.Shen, W.-A. Lai, C/C/ Young. Phophate solubilising bacteria from subtropical soil and their tricalcium phosphate solu-bilizing abilities // Applied Soil Ecology. - 2006. - V.34. - P.34-41) and microorganisms, on the basis of which drugs to combat plant diseases (for example, patent RF №2019966; Waspalloy, Slautered, Avimova, You, Gaskova, Nsidomnode. New integrated biologics to protect vegetable crops from fungal and bacterial diseases // Biotechnology. - 2010. No. 4 - P.69-80).

A known strain Pseudomonas aureofaciens BKM-2390 designed to protect plants from fungal pathogens, including fungi of the genus Fusarium, clean soils from arsenic and dissolving phosphate (RF Patent No. 2323967).

However, antagonistic and phosphotransferase activity of this strain are characterized by only qualitatively,the latter is ascertained by the presence of zones of enlightenment, agar, containing insoluble phosphate rock. It is known that the results of the dissolution of phosphate by microorganisms obtained on solid and liquid media, often do not coincide, and to quantify phosphotransferase activity research is required in a liquid medium (Gupta R., Singal R., Shankar, A., Kuhad R.C., Saxena R.K. A modified plate assay for screening phosphate solubilizing microorganisms // J. Gen. Appl. Environ. 1994. V.40. P.255-260; Deubel, A., Fankem H., D. Nwaga, Antoun Y., W. Merbach In vitro mobilization of calcium, iron and aluminum phosphate by rhizosphere bacteria of African oil palm // Proceedings of the 3rdInternational Symposium on Phosphorus Dynamics in the Soil-Plant Continuum / Ed. Alves V.M.C. et al. Uberlandia, Brazil: Embrapa Milho e Sorgo, 2006. P. 232-233). He also confirmed the influence of the claimed strain on increasing yields.

Closest to the claimed invention is a strain of Pseudomonas sp.NJ-101, which gave zone of inhibition to 1.8 mm with cultures of pathogenic fungi such as Fusarium oxysporum, F. solani and F. udum, and translated into the solution to 74,6 μg/ml of phosphate of CA3(RHO4)2(N. Bano, Musarrat J. Characterization of a novel carbofuran degrading Pseudomonas sp.with collateral biocontrol and plant growth promoting potential // FEMS Environ. Lett. 2004. V.231. P.13-17). However, the activity of this strain is low and its impact on the increase of productivity is also not confirmed.

The task of the invention to provide a new strain of high phosphotransferase activity and ability to suppress phytopathogenic fungi of the genus Fusarium to fight fusariose the increased yields.

The problem is solved in that a strain of Acinetobacter species 305, isolated from soil of Krasnodar region with the use of selective minimal mineral medium containing insoluble calcium phosphate (CA3(RHO4)2). The main selection criteria were the degree of dissolution of CA3(RHO4)2the growth inhibition of phytopathogenic fungi of the genus Fusarium, stimulation of plant growth and harmless to warm-blooded.

The proposed strain Acinetobacter species 305 has a high ability to dissolve the phosphate, inhibits the growth of phytopathogenic fungi of the genus Fusarium and stimulates plant growth.

The proposed strain Acinetobacter species 305 deposited in the Public collections of pathogenic microorganisms and cell cultures "GKM-Obolensk" (p. Obolensk. Serpukhov district, Moscow region) under number B-6645.

The strain of bacteria Acinetobacter species 305 is characterized by the following properties.

Cultural morphological traits

Cells are fixed, short, rod-shaped, gram-negative, do not form spores.

The strain grows well on the following environments: hydrolyzed fish meal (RM)-broth (fsri GNORPM, poblems)+2% glucose mineral medium composition (g/l): K2NRA41,5; KN2RHO43,0; NH4Cl of 0.5; glucose of 20.0; yeast extract 2,0; MgSO40,4; NaCl, 3.0, trace element solution is 10 ml/l (the composition of trace element solution, %: ferrous sulfate (FeSO4×7H2O) 0,01; copper sulfate (CuSO4×5H2O) 0,1; manganese sulfate (MnSO4×2H2O) 0,1; zinc sulfate (ZnSO4×7H2O) 0,01).

The morphology of colonies on nutrient media was determined after 1-2 days of growth at 28°C. Colonies on medium RM-agar (fsri GNORPM, p. Obolensk) opaque, round, convex, smooth, slimy, white color, diameter 3-4 mm, Strain builds on agar Endo mucous bright crimson colony without metallic luster on agar Levin - pink mucoid colonies on McConkey agar - purple colonies on TSI agar - allocation of gas and changes the color of the agar from crimson to yellow-red.

Physiological and biochemical characteristics

Obligate aerobe, temperature optimum growth of 28-30°C, does not grow at 41°C; grow within a pH of from 3.5 to 8.0.

Additional growth factors does not need (prototroph). The metabolism of glucose oxidation. Molecular nitrogen does not fix. Starch hydrolyses. Fiber does not decompose. The strain does not produce indole, phosphatase and hydrogen sulfide, is urease and b-galactosidase, b-glucosidase,-glutamyltransferase not decompose ornithine, arginine, phenylalanine.

The sources of carbon

Assimilates with the formation of acid adonitol, acetoin, galactose, glucose, dulcitol, Inositol, xylose, lactose, lysine, malonate, maltose, Manni is ol, melibiose, rhamnose, raffinose, sucrose, sorbitol, trehalose, fructose, cellobiose, Simmons citrate, esculin.

The sources of nitrogen

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

Saprophyte. The strain does not possess phytopathogenic activity, as evidenced by the absence of maceration on the slices of potatoes when applied to them by the injection of living cells of strain.

The strain is not pathogenic for warm-blooded: it does not cause the death of the mice of CBA after subcutaneous administration of 108SOME.

Storage conditions

The strain of bacteria Acinetobacter species 305 is stored on cups or jambs with RM-agar at 5-7°C. Subcultures to fresh medium once a month. Long-term storage in the lyophilized state in lactose-polyglycidol protective environment. Store at temperatures (4-8°C, Peratallada after 3 years.

The strain Acinetobacter species 305 no phytotoxicity. Indicators Phi-motociclete strain Acinetobacter species 305 has a negative value (table 1), indicating the presence of stimulating plant growth effect.

Strain Acinetobacter species 305 inhibits growth following phytopathogenic fungi: Fusarium oxysporum, Fusarium frost, Fusarium culmorum.

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

Example 1. Culture of bacteria Acietobacter species 305.

Cell culture strain Acinetobacter species 305 for testing is prepared as follows: into a flask with a capacity of 750 ml pour 150 ml of sterile medium RM-broth, add 6 ml of sterile 50%glucose solution and introduce 1-2 ml of a stationary culture of a strain of Acinetobacter species 305. The cultivation is carried out with aeration (160-250 rpm) at 28°C for 12-15 h to the final titer of the culture fluid of 5-10×109cells/ml

Example 2. Demonstration of the absence of phytotoxicity in strain Acinetobacter species 305.

From the culture fluid obtained in example 1, to prepare a 1% (v/v) cell suspension in sterile distilled water (0.5 to 1.0×108cells/ml). In the resulting suspension soaked seeds of wheat cultivar "Golden Oriole" within 2 hours In the control experiment the seeds are soaked in distilled water. 50 seeds of each option are laid on moistened with distilled water filter paper, which is placed in Petri dishes, and incubated at 28°C for 4 days, constantly wetting. A day to evaluate the germination of seeds (table 1), and after 4 days the seedlings morphometric, measuring the length of all roots and stems.

Fetotoksicheskoe activity of strain calculated by the formula:

A=(100-D0/Dto×100)%,

where And is an indicator of phytotoxicity;

D0- the average of the parameter in the experience;

Dto- the average PA is ametra in control.

Negative values of the indicator And indicate a stimulating effect.

Table 1
Indicator of phytotoxicity strain Acinetobacter species 305 seedlings of wheat varieties Oriole
Option processing seedsThe germination of seeds, % of controlIndicator of phytotoxicity, %
RootsStem
Suspension cells of Acinetobacter species 305100-29,4-19,8

Example 3. Demonstration phosphotransferase activity of the strain Acinetobacter species 305.

To determine phosphotransferase activity in a flask with a capacity of 750 ml make 100 ml of sterile mineral medium of the following composition (g/l): NH4Cl 0,16; MgSO4×7H2O 0,2; CA3(RHO4)2×2H2O 5,0; glucose 10. On Wednesday contribute 2-3 ml of culture Acinetobacter species 305 grown as in example 1. Incubation lead to the rocking New Brunswick at 160-180 rpm at 28°C.

To determine the amount of phosphate that is passed into the solution, use one-step method, based on measure the intensity of colouring of its molybdenum complex with Tween 80 at 350 nm (Pupyshev, A.B. Stable reagent for one-step determination of inorganic phosphate // Laboratory work. - 1991. No. 9. - P.12-16). Measurement of phosphate in solution is carried out in three replications. Two days later the concentration of phosphate in solution is 2560±140 µg/ml, which is more than 30 times higher than that of the prototype.

Example 4. Demonstration of the antagonistic strain of Acinetobacter species 305 against phytopathogenic fungi of the genus Fusarium.

As test objects using the following strains of phytopathogenic fungi of the genus Fusarium (in parentheses are caused by pathogenic disease): F. frost Schwabe 1838 K-33 (Fusarium head blight and grain), F. culmorum (W.G.Smith 1884) Saccardo 1895 (root rot of cereals), F. oxysporum Schlecht No. 6 (Fusarium).

To determine the suppressive properties use the holes. Suspensions of pathogenic fungi (105CFU/ml) is prepared by removing the mycelial and spore mass with lawns fungi on potato-glucose agar (CCA) (Egorov NS Guide to practical exercises in Microbiology. - M.: Moscow state University press, 1995. - 217 C.) sterile solution of 0.9% sodium chloride. The resulting suspension in an amount of 0.1 ml are seeded on the surface of the CCA in Petri dishes and rubbed with a spatula. In the centre, planted pathogens Petri dishes with the CCA make holes with a diameter of 4-5 mm, which contribute 0.1 ml of the culture fluid Acinetobacter species 305 obtained in example 1. Cup incubated at 28°C in those who tell 4 days. For F. frost and F. culmorum area oppression is 1.5 mm for F. oxysporum 2 mm.

Example 5. Demonstration of increasing wheat yields as a result of application of the strain Acinetobacter species 305.

As a culture test using spring wheat cultivar "Golden Oriole".

Substrate for plants is sandy loam soil with low content of available phosphorus (less than 3 mg of P2About5/100 g soil Kirsanov) and pH 6.8. Previously the soil is thoroughly mixed and sieved through a sieve with a mesh size of 2.5 mm, and then sterilized by autoclaving for 2 hours at a pressure of 2 ATM.

As vessels for growing use pots with a volume of 1.5 l For artificial infection of seeds treated with the suspension of phytopathogen F. frost Schwabe 1838 To-33. For 100 g of seeds using 50 ml of a suspension of conidia of the fungus F. frost with a title 3x105CFU/ml, grown on the CCA.

Seeds are sown in 10 pieces per each pot. After germination of the plants vypilivayut the purpose of alignment in height, leaving in each pot for 6 plants. For each option used by 4 of the pot.

Mineral nutrition of nitrogen and potassium contribute fractionally during the vegetation period in the form of salt solutions. As a source of phosphorus use powdered phosphorite ore mark field (the content of the oxide of phosphorus(P 2O5) 19,6%). Ore make the full rate only once, just prior to planting, mixing with the soil sample of each pot. In embodiments using a strain of Acinetobacter species 305 ore is first mixed with the biomass, and then applied to the soil at the rate of 106cells in 1 g of soil. Total making batteries in terms of NPK for the entire growing season is, g kg-1 dry soil: nitrogen (N) 0,15; potassium oxide (K2O) of 0.1; P2O50,1.

Plants grown for 3 months. The results for the mean mass of grains are shown in table 2.

Table 2
The influence of strain Acinetobacter species 305 on yield of wheat under different treatment options
no versionFeature optionsThe average grain weight, g
Infection with F. frostThe introduction of strain Acinetobacter species 305
1+4,95±0,20
2++ 5,68±0,43
35,80±0,35
4+6,94±0,55

In the variants with the application of strain Acinetobacter species 305 noted earlier onset timing of major developmental phases of plants on average 5-7 days. Also available with the use of a strain of Acinetobacter species 305 grain weight increased by 15-20% compared with the corresponding infected and uninfected options.

Thus, a strain of Acinetobacter species 305 has a high ability to release phosphate from insoluble minerals, inhibit the growth of phytopathogenic fungi of the genus Fusarium, the main pathogens of cereal crops, and increases productivity. The strain is not toxic to plants, stimulates their growth and can be recommended to create on its basis bitstrea fertilizer with fungicidal properties.

Phosphotransferase strain of bacteria Acinetobacter species with antifungal properties for protecting plants against diseases caused by fungi of the genus Fusarium, and increase yields deposited in the Public collections of pathogenic microorganisms and cell cultures "GKM-Obolensk" number In-6645.



 

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9 cl, 9 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: invention relates to microbiological fertilisers for plants, specifically to a microbiological composition based on Rhizobium (symbiotic nitrogen fixer) legume bacteria. The composition contains Rhizobium lupini bacteria and associative nitrogen-fixing bacteria Flavobakterium, with components in weight ratio of 1.0-1.5:1.5-2.0. Due to synergetic interaction of the components, the composition has high physiological activity and has not phytotoxic effect on crop plants. The invention enables to efficiently increase plant biomass in mixed legume-grass seeds by approximately 7.1-7.8% compared to inoculation of seeds with legume bacteria, and by 8.9-9.9% compared to inoculation of seeds with associative bacteria. The degree of inhibition of crop plants or damage falls 1.4-8-fold compared to control crop plants.

EFFECT: microbiological composition increases accumulation of green material by 87-91 centner per hectare or 20-23% compared to control plants - yield in mixed lupine seeds and barley without inoculation of seeds with microbiological preparations.

2 tbl

FIELD: plant production.

SUBSTANCE: method includes spraying of vegetative solanaceous plants with Steinermena feltiae suspension in combination as antidesiccant with agent obtained from biomass of Mortierella jenkinii micromycete according to claimed technology.

EFFECT: insect pest control with improved effect.

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