RU2081905C1 - Strain of bacterium pseudomonas maltophilia for preparing the preparation against plant disease pathogen - Google Patents

Abstract

FIELD: agriculture microbiology. SUBSTANCE: strain of bacterium Pseudomonas maltophilia VKM CR-332D is isolated from the soil and deposited in All-Union Collection of industrial microorganisms at N CR-332D. Strain is grown on medium containing chitin (carbon source), nitrogen and phosphorus at 28 C for 7 days. Then cells were separated from medium and used for plant protection against phytopathogens as suspension at cell titer = 5 x 10⁶--5 x 10⁹.. Agent is used against phytopathogenic fungi. EFFECT: enhanced effectiveness of agent. 2 tbl

Description

 The invention relates to the field of agricultural microbiology, biotechnology and can find application in agriculture in protecting plants from pathogens of their diseases.

 A known bacterial strain Pseudomonas putida, used for the preparation of a preparation against fungal diseases of plants, as well as for their protection against soil nematodes (US patent N 4751081, CL 424-33, 1987) prototype.

 However, this known strain is not a natural bacterial strain, it is designed in the laboratory, which can lead to its degeneration in natural conditions.

 A known strain of Pseudomonas putida, suppressing the growth of the phytopathogenic fungus Fusarium rkysporum (US patent N 4714614, CL 424-93, 1987).

 However, there is no information on the spectrum of action of this known strain.

 The technical result achieved by the implementation of the present invention is the ability to prepare a preparation against plant pathogens based on a natural strain of bacteria.

 This is achieved by the fact that the bacterial strain P. maltophilia BKMCR-332D is used to prepare the drug against plant pathogens.

 The new strain was isolated from sod-podzolic soil under a birch forest (Moscow region).

 The new strain is characterized by the following features.

These are non-spore-forming gram-negative, straight or slightly curved short rods, mobile, with a bundle of polar flagella. The colony morphology on nutrient media was determined after 4-5 days of growth at 28 ^o C. On meat-peptone agar (MPA), the colonies were mucous, convex to the center, yellowish-gray, more yellow in the center. The pigment is dark yellow or brown, sometimes weak. On organic media with tyrosine, the stroke is bluish-black with a dark brown pigment diffusing into the medium.

 On King A medium, pigmentation is absent, on King B medium it forms a dark brown, almost black pigment, diffusing into the medium belonging to the melanin group. No green fluorescent pigment.

Physiological and biochemical characteristics of the strain
It does not grow on a mineral medium without growth factors (methionine). With some carbon sources on a Hug-Lifeson medium under aerobic conditions it produces a strongly alkaline reaction. The oxidase reaction (according to Kovacs) is negative. It has a strong proteolytic activity: it quickly and completely decompresses gelatin (every other day at 28 ^o C). Peptones and often curdles milk. Not capable of true denitrification. Does not accumulate poly-β-hydroxybutyrate. Does not form Levan from sucrose, does not hydrolyze starch. Produces catalase. On MPA with cysteine forms hydrogen sulfide. Actively forms lysine decarboxylase and is weaker than arginine dehydrolase. It does not have urease activity.

It does not grow at +4 ^o C and +41 ^o C. The optimum temperature for growth is 28-30 ^o C. Does not grow at pH 4.5. It grows on MPA with 3% NaCl.

 A characteristic feature is the relatively limited range of sources of carbohydrates suitable for growth. It utilizes sodium citrate and malonate. It grows well on a mineral medium with ammonium salts. Nitrate salts are not used as the sole source of nitrogen.

 The new strain is resistant to ampicillin (200 μg / ml), tetracycline (10 μg / ml), sensitive to chloramphenicol (20-50 μg / ml), streptomycin (100 μg / ml).

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

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

 To obtain a freeze-dried preparation, strain crates were grown for 24 hours on Hottinger agar broth and washed with the following composition: 1 part of physiological solution + 1 part of a mixture prepared from 10 parts of cattle blood serum without preservative and 1 part of sucrose solution 1 g / ml.

The strain is identified:
1) from the original source Hugh R. // Int. J. Syst. Bact. 1981, V. 31, N 2, p. 195;
2) from the original source Komagata K et all. // Int. J. Syst. Bact. 1974, V. 24, p. 242;
3) by the determinant Krieg NR et all. // Bergey's manual of Systematic Bacteriology, 1981, V. 1.

Example 1. The strain Pseudomonas maltophilia VKM CR-332D was grown on a medium of the following composition (g / l):
Yeast extract 0.5
(NH ₄ ) ₂ SO ₄ 1
MgSO ₄ x 7H ₂ O 0.3
KH ₂ PO ₄ 1.36
Before sterilization, the pH of the medium was adjusted to 7.0 with a 4% NaOH solution. As the sole carbon source, colloidal chitin obtained by treating chitin from a crab exoskeleton with concentrated HCl was introduced into the medium (Enthomophage, 1975, V. 20, No. 1, p. 43-48). An aqueous suspension of colloidal chitin (1: 1) was prepared and added to the medium at a final concentration of 2% (v / v). The substrate and salts were sterilized separately.

The strain was grown for 7 days at +28 ^o C, after which the cells were separated by filtration. The filtrate was used to obtain the preparation of chitinolytic enzymes of ferchitis, and the cells of the strain in the form of a suspension either in lyophilized form (with or without filler) were used as a preparation for protecting plants from phytopathogens. The working cell suspension contains 5 • 10 ⁶ 5 • 10 ⁹ cells / ml.

Example 2. The method of agar blocks studied the antagonistic effect of the new strain on phytopathogenic fungi. The cell culture of the strain was sown with a "continuous lawn" on the surface of nutrient agar (MPA with 2% glycerol or King B sulfur) and grown at 28 ^o C for 2-3 days. Then, “blocks” were cut out with a cork drill (diameter 8–10 mm) and transferred to the surface of another nutrient medium (Chapek’s medium or potato agar), which was previously seeded with a deep test method using a phytopathogenic fungus test culture (about 100 spores per 1 ml of medium). The cups were placed in a thermostat at 28-30 ^o C. Accounting was carried out for zones of absence or suppression of growth of the fungus around the disk with the culture of a new strain after 3-6 days (depending on the growth rate of the test culture). The results are presented in table 1 and show the high antagonistic activity of the new strain against phytopathogenic fungi.

Example 3. Growing uterine plantings of carnation for repair at the Orangery Complex state farm in the Moscow Region in order to obtain elite planting material of rooted cuttings of M ² generation cloves had previously led to mass infection of cultivated uterine plants M ^{1 with the} Fusarium oxysporum carnation withering agent by the end of the third month and to fall up to 35-40% of plants by this time. Moreover, in the remaining plants, latent Fusareous infection reached 70%, which made it completely impossible to use such uterine plants for the production of elite planting material.

When clove generation of M ¹ cells of the Pseudomonas maltophilia strain VKM CR-332D was added to the carnation plant substrate, plants were grown for 8-10 months. Table 2 shows the data on the loss of plants and their infection with a latent oil infection, indicating the effectiveness of the new strain.

 Thus, a preparation based on a bacterial strain according to the invention is an effective means of protecting plants from pathogens of their diseases.

Claims (1)

 The bacterial strain Pseudomonas maltophilia VKM CR-332D for the preparation of the drug against plant pathogens.

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