RU2130262C1 - Strain of bacterium pseudomonas species used for preparation against plant disease pathogens - Google Patents

Abstract

FIELD: microbiological industry, agriculture. SUBSTANCE: strain Pseudomonas species 7G is isolated from wild cereals rhizosphere and deposited in NII "Collection of microorganism cultures" GNC VB "Vector" at N B-694. For preparing the preparation against pathogens of fungal diseases in plants the strain is cultured in LB medium containing the following components, g/l: peptone, 10.0; yeast extract, 5.0; sodium chloride, 10.0; and water, up to 1 l for 16-18 h at 28-30 C to concentration 10¹⁰-10¹¹ cells/ml. Then cells are washed off with buffer from agarized medium and diluted to titer value 10⁶-10⁷ cells/ml. Strain shows antagonistic properties against fungi of genus Alternaria, Bipolaris, Fusarium, Rhizoctonia, Helmintosporium, Phytophthora. EFFECT: enhanced effectiveness of the preparation. 2 tbl

Description

 The invention relates to agricultural microbiology and can be used to protect plants from phytopathogens.

A known strain of Pseudomonas fluorescence biovar V MD-4f, inhibiting the reproduction of fungi of the genus Streptomyces, which are the causative agents of potato scab. Seed potatoes are treated with a suspension of cells of this strain with a concentration of 10 ⁹ cells / ml (Japanese Application N 1-19320 A, class A 01 N 63/00, publ. 03.08.89).

 The disadvantage of this strain is the limited spectrum of action (only on phytopathogenic fungi of the genus Streptomyces) and the high consumption of the suspension used for processing potatoes.

 A known strain of Pseudomonas putida, inhibiting the growth of the phytopathogenic fungus Fusarium oxysporum (US Patent N 4714614, class C 12 N 1/20, 1987). However, its effect on other phytopathogens is not described.

 A known strain of Pseudomonas cepacia AMMA, inhibiting the growth of phytopathogenic fungi of the species Aphanomyces (US Patent N 5244658 class. C 12 N 1/20, publ. 14.09.93). However, there is no information about its antagonistic activity against other phytopathogens.

The closest prototype to the claimed strain is the Pseudomonas aureofaciens strain, which inhibits the growth of virulent agrobacteria that cause bacterial cancer of grapes and other plants (RF Patent N 1482188 class C 12 N 1/20, publ. 15.03.94, BI N 5). A known strain deposited in VKPM under the number B-4117. Cells of this strain are grown for 24 hours at 30 ^° C. on Hottinger broth or potato agar to a cell titer of 10 ⁸ -10 ⁹ cells / ml. The disadvantage of this strain is a narrow spectrum of its antagonistic action. The aim of the invention is the selection of a natural strain of bacteria with a wide range of antagonistic effects on phytopathogenic fungi.

 A new strain of Pseudomonas species 7G was isolated from the rhizosphere of wild cereal plants in the Horde forest-steppe of the Novosibirsk region.

 The isolated strain of Pseudomonas species 7G was donated to the Research Institute “Collection of microorganism cultures of the SSC VB“ Vector ”under registration number B-694. The strain is stored in a freeze-dried state.

The strain Pseudomonas speciеs 7Г is characterized by the following features:
Morphological signs.

 These are gram-negative, mobile, short rods with polar flagella, they do not form spores, the size is 0.6 x (1.3-2.6) microns. Cells grow well on simple nutrient media: meat peptone agar (MPA), potato agar, minimal media with glucose or glycerin, on LB medium.

Colony morphology on agar media (at 28 ^o C for 2-3 days):
on the MPA, the colonies are convex, transparent with a matte tint, smooth, mucous consistency, diameter 5-7 mm;
on MPA with 2% glycerol, the colony is transparent-whitish, moderately mucous, convex, medium.

 The pigment on the MPA is yellow-green, diffusing into the medium.

 Pigmentation on King diagnostic media for fluorescent pseudomonads (King E.O. at all. J. Lab. Clin. Med., 1954, v. 44, p. 301).

 King A - no pigment.

 King B is a yellow-green, fluorescent, intense pigment.

Physiological and biochemical properties:
strict aerobic, oxidative glucose metabolism. It does not need growth factors. Starch and cellulose do not hydrolyze. The maximum growth temperature on nutrient media is 40 ^o C, the minimum is 4 ^o C, the optimal is 30 ^o C. It grows well at pH 6.5-7.5.

 As a sole source of carbon, it absorbs glucose and glycerin and does not absorb rhamnose, galactose, maltose, sorbitol, dulcite, and inositol.

The new strain is resistant to antibiotics:
rifampicin (50 μg / ml), ampicillin (200 μg / ml), novobiocin (50 μg / ml) and is sensitive to tetracycline (10 μg / ml).

 The strain is not phytotoxic, as evidenced by the absence of maceration on the slices of potatoes and necrotic spots on the leaves of tobacco when applied to them with an injection of a suspension of living cells of the strain.

 The strain is not virulent for laboratory animals: when twenty mice were injected intraperitoneally with a suspension of its cells of 1 billion cells in a volume of 0.5 ml, the animals did not die within 30 days.

 The new strain is stored on jambs of potato agar in the refrigerator with reseeding on fresh jambs after 2-4 months and in a freeze-dried state (in ampoules) for at least a year.

The strain is identified:
1. Source: H. Stolp, D. Dadkari in "The procaryotes", Berlinea, 1981, v. 1, cb. 61, p. 719-741.

 2. According to the qualifier "Bergey's Manual of Sistematic Bacteriology", v.1, ed Krieg N.R. et al., Baltimore / London, 1984.

For the cultivation of Pseudomonas specias Research Institute KKM (SSC WB "Vector) B-694, LB medium of the following composition is used, g / l: peptone 10.0, yeast extract 5.0, sodium chloride 10.0, water up to 1 L. Cultivation is carried out at 28-30 ^o C for 14-16 hours to achieve a cell titer of 1 • 10 ¹¹ cells / ml, then the suspension of cells is diluted to the desired concentration.

The defining difference of the new strain from the prototype is a wider spectrum of antagonistic action on pathogens of fungal diseases of plants. In addition, the new strain is characterized by high productivity and high metabolic rate. The properties of the new strain are illustrated by the following examples:
Example 1

The cells of the new strain are grown for 14 hours at 30 ^o C on plates with MPA on LB broth of the following composition, g / l:
Peptone - 10.0
Yeast Extract - 5.0
NaCl - 10.0
Distilled water - Up to 1 L
Cells are washed off with phosphate buffer (pH 7.2), centrifuged and suspended in the same buffer to a titer of 9 • 10 ¹⁰ cells / ml. The resulting suspension is used as a drug against phytopathogenic microorganisms.

 Example 2

We studied the effect of the new strain on phytopathogens. The bacterial culture was seeded with a continuous "lawn" on the surface of nutrient agar (MPA with 2% glycerol or King B medium) and grown at 28 ^° C for 2-3 days. Then discs of an agar medium with a culture of a new strain were cut out and placed on the surface of another agar plate (Chapek medium or potato agar), previously seeded with a suspension of phytopathogenic fungus in a deep way. Cups were placed in a thermostat at 28-30 ^o C for 3-6 days. Accounting was carried out for zones of absence or inhibition of fungal growth around a disk with a culture of a new strain. The antagonistic activity of the new strain with respect to 7 species of phytopathogenic fungi was studied. The results are shown in table 1
From the table. 1 shows that the new strain of Pseudomonas sp. 7G has an antagonistic effect against all studied species of phytopathogenic fungi.

 Example 3

In the production environment, Irmen CJSC, Ordynsky District, Novosibirsk Region, studied the effect of the drug on the basis of a suspension of cells of a new strain of Pseudomonas sp. 7G to reduce the degree of damage to soft spring wheat Novosibirsk 89 by root rot of cereals. For this, pre-sowing seed treatment was performed based on: 50 ml of a suspension of cells of the proposed strain (cell titer 1 • 10 ¹⁰ , cells / ml) per 1000 kg of grain. Control wheat seeds were treated with water. Seeds were sown in open ground in fields with a high mineral background (liquid fertilizers).

 As a result of processing, the results are obtained, which are given in table. 2.

 From the table. 2 shows that the treatment of wheat seeds Novosibirsk 89 with a suspension of cells of a new strain can reduce the incidence of plants by root rot by 7 times and increase wheat yield by 6 kg / ha.

 Thus, the proposed strain of Pseudomonas sp. KKM B-694 as part of a biological product allows you to suppress a wide range of phytopathogenic microorganisms and helps to increase the yield of cereal crops and other plants.

Claims (1)

 The bacterial strain Pseudomonas species Research Institute of CMC SSC VB "Vector" B-694 to obtain the drug against pathogens of plants.

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