RU2213774C1 - Strain of bacterium pseudomonas putida for preparing preparation against wheat diseases caused by fungal phytopathogens - Google Patents

Abstract

FIELD: agriculture microbiology. SUBSTANCE: invention relates to the novel strain Pseudomonas putida 17 that is isolated from aerotanks sewage of biological cleansing equipment in Orsk town. The strain is deposited in collection of cultures of Biology Institute of Ufa scientific center of Russian Academy Science at = 17. For preparing the preparation strain is cultured in medium LB containing the following components, g/l: peptone, 10.0; yeast autolysate, 5.0; sodium chloride, 10.0; water, up to 1 l, for 48 h at 28-30 C under aeration up to titer 10⁹ CFU/ml and prepared suspension is used for treatment of wheat seeds. The preparation prepared on basis of this strain inhibits the development of wheat diseases caused by kernel smut and root rots. EFFECT: valuable properties of strain. 3 tbl, 4 ex

Description

 The invention relates to agricultural microbiology and relates to a strain that exerts an antagonistic effect on pathogens of wheat diseases with hard smut and root rot and therefore can be used to protect agricultural plants from them.

 A known strain of Pseudomonas sp. CMPM B-348, used to obtain the drug for the protection of cereals from the pathogen 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 H. sativum.

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

 As a prototype, a bacterial strain Pseudomonas species was selected that exhibited antagonistic activity against phytopathogenic fungi Alemaria spp., Bipolaris sorokiniana, Fusarium oxysporum, F. gibbosum, F. sumbucinum [4]. However, there is no information on the effect of this strain on such widespread pathogens of plant diseases as Fusarium culmorum, F. gramineamm, F. nivale, F. semitectum, F. solani.

 In addition, in all of the listed analogues and prototype there is no information about the activity of the isolated microorganism strains with respect to the causative agent of the most harmful wheat disease - smut (Tilletia caries), losses from which lead to a shortfall of 10-15% of the crop.

 An object of the invention is the selection of a new natural strain with a wide spectrum of action on pathogens of fungal diseases of plants, including smut.

 The task is achieved by identifying and using the strain Pseudomonas putida IB 17, isolated from wastewater in aeration tanks of biological treatment facilities in Orsk. The strain is deposited in the culture collection of the Institute of Biology of the Ufa Scientific Center of the Russian Academy of Sciences under IB 17. It is characterized by the following features.

Morphological signs. Short movable sticks, gram-negative, do not form a spore. The colony morphology on nutrient media was determined after 4-5 days of growth at 28 ^° C. On meat and peptone agar (MPA) and MPA with 2% glycerol, round with smooth edges, smooth, slightly convex, opaque, grayish-white colonies with a diameter of 6 -7 mm, pigment yellow-green, fluorescent, diffusing into the medium.

 Pigmentation on King diagnostic media for fluorescent pseudomonads: King A - no pigment; King B - a yellow-green fluorescent pigment is formed, diffusing into the medium.

 In liquid nutrient media (BCH) an abundant, dense precipitate is formed. On MPA-jambs, the stroke is plentiful, continuous with a smooth edge.

Physiological and biochemical characteristics. In relation to oxygen - aerob. The oxidase reaction is positive. Growth on a synthetic medium is moderate. Does not thin the gelatin. Does not form indole, levan from sucrose. Does not hydrolyze starch, lecithin, tween-80. Does not reduce nitrates to nitrites. Produces catalase. Grows at 4 ^o C. Resistant to NaCl: growth at 6% concentration.

 As sources of carbon, it uses glucose, lactose, maltose, sucrose, xylose, galactose, raffinose, arabinose, rhamnose, propanol, butanol, hexanol, pyruvate, citrate, acetate, propionate, lactate, formic acid, isobutyric acid, succinic acid, α ketoglutaric acid, alanine, valine, leucine, methionine, tryptophan, glycerin. Does not absorb oxalate, adipate, maleic acid, anthranilic acid, acrylic acid, aminobenzoic acid, mannitol, sorbitol, inositol.

 The strain is stored on the jambs of the MPA in the refrigerator with reseeding to fresh jambs after 2-4 months.

 The strain was identified by Bergey's Manual of Determinative Bacteriology.

 Example 1

 Getting biomass.

The strain is grown for 48 hours at 28-30 ^o With aeration to a titer of 10 ⁹ CFU / ml on LB medium of the following composition, g / l: peptone 10.0; yeast extract 5.0; sodium chloride 10.0; tap water up to 1 liter. If necessary, the cell suspension is diluted with water to the desired titer.

 The defining difference of the proposed strain from the prototype is a wider spectrum of action on pathogens of fungal diseases of plants.

 Example 2. The study of the antagonistic activity of a new strain of Ps. putida IB 17.

On plates of glucose-peptone agar (glucose 5 g; peptone 5 g; potassium phosphate disubstituted 4 g; potassium phosphate monosubstituted 2 g; distilled water 1 l), a suspension of fungal phytopathogen is sown with a continuous lawn. Then, bacteria of the Ps strain are planted on the same plates with an injection. putida IB 17. The 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 strain. The results are shown in table. 1 and indicate a wide spectrum of antagonistic action of the new strain.

Example 3. The antagonistic effect of the new strain against a complex of root rot pathogens of wheat seedlings is tested in laboratory conditions on Zhnitsa wheat with a natural level of infection of seed obtained in the experimental field of the Bashkir Agrarian University. Wheat seeds are treated with a suspension of strain (with a titer of 10 ⁹ CFU / ml) at the rate of 1 • 10 ⁶ cells per grain, transferred to filter paper, covered with plastic wrap to prevent liquid evaporation and placed in a beaker. In control experiments, seed treatment is carried out with tap water. As standards, Dividend Star, Planriz and Fitosporin are used. 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 experiments are performed in triplicate, the results are shown in table. 2.

The same table also shows the results of laboratory experiments carried out by a similar method, but with the addition of phytopathogenic fungi Fu-sarium oxysporum and Bipolaris sorokiniana into the seed material (spore suspension titer of both pathogens was 5 • 10 ⁴ CFU / ml).

 From table 2 it follows that the new strain, both under the conditions of a natural infectious background of infection and in conditions of additional infection of the seed with phytopathogens, surpasses the well-known biological preparations Phytosporin and Planriz in the effect on phytopathogens.

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 field trials. 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. In control experiments, seed treatment was carried out with tap water. As a reference, the biological preparation Fitosporin was used. The experiments were performed in triplicate. The effectiveness of the strain was determined by comparing the degree of manifestation of plant diseases with solid smut, as well as root rot during harvest. The results are shown in table.3.

 From the table. 3 it follows that pre-sowing treatment of wheat with a suspension of the inventive strain of bacteria allowed to increase grain yield (66.02%). The biological effectiveness of the claimed strain in this case exceeds the effectiveness of the well-known commercial drug Fitosporin.

 Thus, the use of the proposed strain allows for a wider antagonistic effect on phytopathogens, including the causative agent of durum wheat.

Sources of information
1. Copyright certificate of the USSR 1464468, cl. A 01 N 63/00, 1988.

 2. US patent 4714614, CL. 424-93.1987.

 3. Patent RU 1805849, cl. A 01 N 63/00, 1993.

 4. Patent RU 2130261, cl. A 01 N 63/00, C 12 N 1/20, 1999.

Claims (1)

 The bacterial strain Pseudomonas putida, deposited in the collection of microorganisms of the Institute of Biology of the Ufa Scientific Center of the Russian Academy of Sciences under the number IB 17, to obtain a drug against wheat diseases caused by fungal plant pathogens.

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