Phosphate-dissolving strain pseudomonas species 181a with fungicidal properties

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.

3 tbl, 6 ex

 

The invention relates to biotechnology and the new bacterial strain Pseudomonas species a 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.S., R.J. Soper, G.J. Racz Reactions of phosphate fertilizers in soils // The role of phosphorus in agriculture / Ed. F.E. Khasawneh, E.C. Sample, and E.J. Kamprath Madison, WI: American Society of Agronomy, 1980. P.263-310; Muromtsev G.S., Marchukova T.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 the products and will arousal 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., Kim, 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). 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 crops, primarily pathogens of fusariosis, it will significantly reduce the use of chemical fungicides, increasing the it 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, Avigen, F.T.Shen, 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 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 VKM B-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 in agar containing insoluble phosphate rock. It is known that the determination results of the dissolution of fosfatdegidrogenaza, received 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 and increase yields.

The problem is solved in that a strain of Pseudomonas species a isolated from adresas rock Karelia using the 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 Pseudomonas species a 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 deposited in the Public collections of pathogenic microorganisms and cell cultures "GKM-Obolensk" (poblems Serpukhov district, Moscow region) under number B-6646.

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

Cultural morphological traits

Cells are motile, gram-negative, short oval sticks, do not form spores. The size of the cells of 1-1 .5×1.5 to 2.5 μm, there is a cellular polymorphism.

During growth on agar containing hydrolyzate, fish meal (RM-agar) (fsri GNORPM, poblems)), the strain forms a convex, mucoid colonies beige-yellow color with a diameter of 5-7 mm During growth on minimal medium containing CA3(RHO4)2as the sole source of phosphorus, forms a convex, mucoid colonies beige-yellow color with a diameter of 3-5 mm with zones of dissolution of phosphate in diameter up to 7 mm

The strain grows well on the following environments: hydrolyzed fish meal (RM)-broth (fsri GNORPM, poblems) + 2% glucose, at a minimum synthetic medium composition (g/l): K2NRA4- 1,5; KN2RHO4- 3,0; NH4CI is 0.5; glucose to 20.0; yeast extract - 2,0; MgSO4- 0,4; NaCl - 3.0, trace element solution 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).

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, urease and hydrogen sulfide, has a b-galactosidase, b-glucosidase, phosphatase, N-acetyl-bD-glucosaminidase,-glutamyltransferase not decompose phenylalanine, adonitol, arginine, raffinose, sorbitol, lactose, lysine, ornithine, raffinose, cellobiose, dulcitol. Oxidase positive.

The sources of carbon

Learns acetoin, galactose, glucose, Inositol, xylose, starch, malonate, maltose, mannitol, melibiose, rhamnose, sah is a rose, trehalose, fructose, citrate Simmons.

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 bacterial strain Pseudomonas species I keep 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 Pseudomonas species a no phytotoxicity. Indicators of phytotoxicity strain Pseudomonas species a have negative values (table 1), indicating the presence of stimulating plant growth effect.

The strain Pseudomonas species a 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 bacteria Pseudomonas species a

Cell culture strain Pseudomonas species a to test th ovat as follows: in a flask with a volume 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 the strain Pseudomonas species a. The cultivation is carried out with aeration (160-250 rpm) at 28°C for 10-15 hours until the titer of the culture fluid of 5-10×109CL/ml

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

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-Dabout/Dto×100)%,

where And is an indicator of phytotoxicity;

Dabout- the average of the parameter in the experience;

Dto- the average of the parameter in the control.

Negative values of the indicator And indicate a stimulating effect.

Table 1
Indicator of phytotoxicity strain Pseudomonas species a for seedlings of wheat varieties Oriole.
Option processing seedsThe germination of seeds, % of controlIndicator of phytotoxicity, %
RootsStem
A cell suspension of Pseudomonas species a100-23,5-16,8

Example 3. Demonstration phosphotransferase activity of the strain Pseudomonas species a

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 Pseudomonas species a 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 measuring the intensity of the color of its molybdenum complex with Tween 80 at 350 nm (Pupyshev A.B. Stable reagent for one-step determination of inorganic phosphate // L is the responsibility deal. - 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 2620±150 µg/ml, which is more than 30 times higher than that of the prototype.

Example 4. Demonstration of the antagonistic strain Pseudomonas species a 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 pathogen 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 a culture liquid of Pseudomonas species a obtained in example 1. Cup incubated at 28°C for 4 days. For F. frost zone of inhibition is 1.0 mm, F. culmorum - 1.5 mm for F. oxysporum - 2.5 mm

Example 5 Demonstration of the stimulation of plant growth as a result of application of the strain Pseudomonas species a

As the test culture use marigolds rejected (Tagetes patula), which is convenient for cultivation in laboratory conditions and is sensitive to the presence of phosphorus. In each pot 1.5 litre placed 1550 g of sandy loam soil deficient in available phosphorus (<5 mg P2About5/100 g soil Kirsanov), pH 6.8. The soil is sifted through a sieve (2 mm) and sterilized before use (2 ATM, 2 h). soil pH of 6.8. 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 (P2About5) 19,6%). Ore make the full rate only once, just prior to planting, mixing with the soil sample pot. In embodiments using a strain of Pseudomonas species a ore is first mixed with the biomass, and then applied to the soil at the rate of 106cells in 1 g of soil. In embodiments without the use of ore biomass is mixed directly with soil from the same 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) minus 0.1; P2About5- 0,1.

All variants reproduce in 4 replications. In each pot seed is t for 10 seeds. Shoots thinned out within 3-5 days after their occurrence with the purpose of alignment in height, leaving in each pot 1 plant. Plants grow 3.5 months, then dig them up, dried in air and weighed. The results are shown in table 2.

Table 2
The influence of the strain Pseudomonas species a on the weight of the plant marigolds
No. Varian-TAFeature optionsThe total dry weight of the plant, gDry weight of flowers and buds, g
The introduction of phosphate oreThe introduction of strain Pseudomonas species a
13,76±0,180,28±0,01
2+br4.61±0,320,34±0,03
3+ 4,46±0,290,46±0,04
4++5,15±0,370,55±0,04

In the variants with the application of strain Pseudomonas species I noted earlier, the appearance of colors on average 7-10 days. Also in the variants with the application of strain Pseudomonas species a the total weight of the plants increased by 22%and 15%, and the weight of the flowers and buds by 21 and 20% compared with the corresponding options without bacteria.

Example 6. Demonstration of increased crop production resulting from the application of the strain Pseudomonas species a

As a culture test using spring wheat varieties "Lada".

Bookmark experience have on dark-gray forest loam soil with the following agrochemical parameters: pH of soil extract 5,3; humus content in the layer 0-40 cm (Tyurin) - 4,6%, available phosphorus - 0.25 g/kg soil, rolling potassium to 0.13 g/kg of soil. Additionally fertilizers do not contribute.

In the experience of using the basic methods and schemes, common in breeding, research institutions and State testing.

Sowing is carried out in the optimal agronomy time technology recommended for growing spring wheat, taking into account weather conditions.

Pic is b making cells of the strain Pseudomonas species a: seed treatment before sowing. To do this, from the culture fluid obtained in example 1, to prepare 500 ml of a 3%cell suspension in sterile distilled water (1,5-3,0×108cells/ml). In the resulting suspension soaked 1000 g of wheat seeds varieties Lada for 30 min, thoroughly stirring the mixture. In the control variant (without processing) seeds soaked in distilled water. Then the seeds are dried in air and spend sowing plots drill SSPC-7M. The application rate of 250 g of seeds per plot 10 m2. Repeated 4 times. Harvesting combine SAMPO-130". The results are shown in table 3.

Table 3
The influence of the strain Pseudomonas species a on the wheat harvest
No.OptionYield, t/ha
1The control without treatment1,03±0,22
2Treatment of seeds with Pseudomonas species a1,37±0,26

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

Thus, the strain Pseudomonas species a has a high ability to release phosphate from the technology Department is Voronovo mineral raw materials, that improves their availability to plants. In addition, it inhibits the growth of phytopathogenic fungi of the genus Fusarium, diseases, 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 bacterial strain Pseudomonas species a for protection of plants from 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-6646.



 

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3 dwg, 2 tbl, 1 ex

FIELD: agriculture.

SUBSTANCE: method to produce vegetable-microbal associations for phytoremediation based on micro-propagating plants of tomato, rape and arabidopsis and plasmid-containing rhizosphere bacteria, having antimicrobial activity against bacteria Erwinia carotovora and fungi Phytophthora infestans, includes colonization of planting stock of plants cultivated in vitro with the strain Pseudomonas aureofaciens VKM V-2500 D, which carries plasmids pBS216, pKSl, imparting it resistance to naphthalene and arsenic, or Pseudomonas aureofaciens VKM V-2501 D, which carries plasmids pBS216, pBS501, imparting it resistance to naphthalene and nickel.

EFFECT: invention makes it possible to increase protection of plants against toxic effect of naphthalene and heavy metals, and also growth and resistance of plants to phytopathogenic microorganisms.

5 dwg, 3 tbl, 13 ex

FIELD: medicine.

SUBSTANCE: invention relates to strain of microorganism, ensuring recovery of soil and animal gastrointestinal tract (GIT) microbiocenosis, possessing antibacterial and fungicidal activity, and to based on them preparation form and can be used in biotechnology, veterinary medicine and plant protection. Strains Bacillus licheniformis IC-831-1-2, Bacillus licheniformis IC-832-1-2, Bacillus licheniformis IC-833-1-2, Bacillus licheniformis IC-834-1-2 are obtained by method of directed selection and are deposited in All-Russian Collection of Industrial Microorganisms FSUE GosNIIGenetika under registration numbers All-Russian collection of industrial microorganisms respectively: B-10561, B-10562, B-10563 and B-10564. Preparation is characterised by containing filling agent with bacteria biomass in spore form Bacillus licheniformis All-Russian collection of industrial microorganisms B-10561, or All-Russian collection of industrial microorganisms B-10562, or All-Russian collection of industrial microorganisms B-10563, or All-Russian collection of industrial microorganisms B-10564, or their mixture with titre of each bacteria strain not less than 1·103 CFU/g or 1·103 CFU/ml. As filling agent it contains water or powder-like sorbent.

EFFECT: invention makes it possible to increase efficiency of restoring soil, animal GIT microbiocenosis and extend spectrum of antibacterial and fungicidal activity.

9 cl, 9 tbl, 10 ex

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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