RU2081167C1 - Strain of bacterium bacillus subtilis for preparing the preparation against phytopathogenic fungi - Google Patents

Abstract

FIELD: agriculture microbiology. SUBSTANCE: strain of bacterium Bacillus subtilis is isolated from compost and deposited in Collection pure cultures VIZR at N 10. Preparation is obtained by culturing the above indicated strain on liquid medium containing 3% cultural extract, 1.5% molasses and water for 3 days at 27-29 C followed by lyophilization of cultural fluid by drying. Yield of dry biomass is 40 g/l at titer 5-10⁹ spores/g. EFFECT: enhanced productivity of strain indicated above. 3 tbl

Description

 The invention relates to agricultural microbiology and is a new strain of the bacterium, which is an antagonist in relation to a number of phytopathogenic fungi. The strain B.subtilis was deposited in the collection VIZR under N 10. Identification of the strain was carried out according to "Multiple identifier of bacteria Bergi", M. Mir, 1980

 In recent years, the amount of fungicides used has been severely limited due to their high toxicity.

 Currently, microbiological preparations based on antagonist strains are used to protect plants from mycoses: phytolavin based on Streptomyces Lavendulae and trichodermin based on Trichoderma Lignorum / "List of chemical and biological agents for controlling pests, plant and weed diseases, and plant growth regulator approved for use in agricultural for 1986-1990 M. 1987, S. 95-96 /.

 The main disadvantage of these drugs is low biological effectiveness and a narrow spectrum of diseases.

 The closest to the proposed is the Bacillus subtilis strain IPM-15, which is a producer of bactofit / baciphyte / preparation for protection against powdery mildew of cucumbers, cotton verticillosis wilt, root rot and vegetable and grain crops / Problems of creating and using microbiological plant protection products. M. 1989, p. 253, 257-295 /.

 The disadvantage of this strain is its insufficiently high efficiency.

 The strain Bacillus subtilis N 10 has a high antagonistic activity against a wide range of pathogens of plant mycoses and is a producer of the biological product Alirin B. The strain is isolated from zoocompost in the Leningrad region.

 Cultural and morphological characters.

The proposed strain B.subtilis 10-VIZR has the following morphological and cultural characteristics: rods have a width of 0.7-1.0 μm, a length of 1.7-3.0 μm, spores 0.7-0.9 μm wide, 1 1-1.9 microns. The form of the spores is elliptical, the prevailing position of the spores is central, there is no bloating sporangia. To study the cultural and morphological properties of the strain, the culture is grown at 28 ± 2 ^o C for 3-5 days on solid nutrient media of the following composition:
Potato agar:
Peeled potatoes 200 g
Water 1 l / after 20 minutes of boiling, the broth is filtered, the volume is adjusted to 1 l, agar is added /
Agar 20 g
sterilized at 1 atm. 20 minutes.

Agarized corn and molasses medium:
Corn Extract 30 g
Molasses 15 g
Agar 20 g
Tap water Up to 1 l
pH 7.2
sterilized at 1 atm. 20 minutes.

Meat peptone agar with glucose:
Meat peptone broth 1 l
Glucose 10 g
Agar 20 g
sterilized at 0.5 atm. 20 minutes.

 On all solid nutrient media, at the beginning of growth, small colonies of irregular whitish color are observed. By 48 hours of growth, large matte flat colonies with irregular whitish edges grow on potato agar, which later become shiny, slightly thicken and become beige. On an agarized corn-molasses medium, growing colonies with uneven edges are observed, flat, the middle of the colony with small folds, the color is beige. After a week, the edges of the colony become shiny, the color darkens a little. On meat and peptone agar with glucose, flat, whitish colonies grow with uneven edges, a dull surface, the middle of the colony is shiny, almost transparent. After a week, the colonies become brilliant, acquire a beige color, slightly thicken.

Physiological characteristics: hydrolyzes starch, does not hydrolyze hippurate, has nitrotreductase activity, does not grow under anaerobic conditions, the maximum growth temperature is 45-50 ^o C, the minimum is 10-15 ^o C.

 Like most bacteria, it grows well on artificial nutrient media. MPB, Saburo, MPA, media with various nitrogen sources: peptone, casein, amino acids, proteins and their hydrolysates, yeast hydrolysates, etc. Utilize glucose, sucrose, arabinose, dextrose, xylose, mannitol, starch.

 The biosynthesis method.

A. Sowing medium:
Molasses 1.5%
Corn Extract 3.0%
pH neutral
B. Fermentation medium is similar to inoculation.

Cultivation is carried out in deep conditions at 26-25 ^o C for 3 days. The inoculum was grown for 24 hours. The culture fluid was dried freeze-dried or spray-dried.

 The method of storage of the strain: lyophilization in ampoules of culture on the environment of Chapek. Remapping into ampoules after 5 years.

 Antagonistic properties: inhibits the growth of phytopathogenic fungi, does not inhibit the growth of bacteria. To determine the antagonistic spectrum of action, the strain was grown for 72 hours on a medium containing 3.0% corn extract and 1.5% molasses. Antagonistic activity was determined in the culture fluid, biomass extract and filtrate of the culture fluid by the methods of wells, blocks and paper discs. Zones of growth retardation were determined after 3-5 days depending on the growth rate of the test cultures.

Growth retardation zones / radius in mm / test germs
Fusarium graminearum 25-30
F.oxysporum 20-30
F.culmorum 25-30
F.sporotrichiella 25-30
F.sambucium 20-25
Alternaria brassicicolae 10-15
Colletotrichum atrawenta 10
Verticillium dahliae 8-10
Sclerotinia sclerotiorum 5-7
Bipolaris sorokiniana 5-7
Ascochyta melonis 10-12
Example 1. The strain Bacillus subtilis 10-VIZR was grown on a medium of the following composition: corn extract 3% molasses 1.5% the rest tap water, pH before sterilization of 7.0. The cultivation duration of 3 days on a rocking chair at 28 ± 1 ^o C. the Culture fluid was dried lyophilized. A yield of dry biomass of 40 g / l with a titer of 5 • 10 ⁹ spores / g was obtained. The resulting biological product / Alirin B / based on the proposed strain was tested in protection against fusarium root rot of cucumber and verticillous wilt. The powder was added to the wells when planting seedlings in the film production greenhouse of the Detskoselsky state farm in the amount of 0.3 g / l / 0.1 g per plant /. Bactofit prototype-biological product based on the Bacsllus subilis strain was also introduced in the recommended amount (0.2 g per plant). In addition, Trichoderma lignorum-based biological product, widely used in protecting vegetables from root rot, was used as a reference. The rate of application of 1 g under the plant. During the growing season, we observed the development of plants, yield, and also the development of diseases. The experience was taken into account at the end of the growing season. Taking into account the prevalence and development of cucumber root rot was assessed during plant elimination on a 5-point scale:
0 healthy root
1 separately deep brown spots or slight browning of the central root,
2 maceration of the affected tissue,
3 central root, root neck, brown roots, maceration covered up to 1/2 root,
4 the root is brown, there are no lateral roots, more than 1/2 root is soaked, the plant dies.

,
где
R развитие болезни,
(a^ob) сумма произведений числа больных растений /а/ на соответствующий балл (b) поражения;
n общее число учтенных растений, шт.The development of the disease / in / was calculated by the formula

,
Where
R development of the disease,
(a ^o b) the sum of the products of the number of diseased plants / a / by the corresponding score (b) of the lesion;
n total number of registered plants, pcs.

 with the highest score of the scale.

где
P распространенность,
n число больных растений, шт.The prevalence of the disease / in / was calculated by the formula

Where
P prevalence,
n number of diseased plants, pcs.

 N total number of registered plants, pcs.

The data are presented in table. 1 and indicate that the introduction of a biological product on the basis of the proposed strain into the soil reduces the development of verticillosis wilting by 2.5 times, the prevalence and development of fusarium root rot by 3-3.5 times compared with the control. At that time, the introduction of bactofite on the basis of the prototype strain, as well as trichodermin, was much less effective, despite the significantly higher application rates / 0.6 and 3.0 g / m compared to 0.3 g / m /. Wilting decreased by 30-40% and root rot by 40-50%
Example 2. A biological product based on B.subtilis was applied to the soil when planting astra seedlings in an amount of 1 g / m. Bactofit / prototype / and trichodermin were added in the same amount. Accounting for wilting caused by fungi from the genera Fusarium Verticillium and Botrytis was carried out during July and August. The data are presented in table. 2.

Example 3. A biological product based on the B. subtilis strain was produced according to example 1. The grown biomass was spray dried, and a dry biomass yield of 35.9 g / l with a titer of 1.2 • 10 ¹⁰ cf / g was obtained.

Evaluation of the effectiveness of the strain against fusarium spike was carried out on plots of spring wheat, artificially infected with F.graminearum. Infection was carried out with a suspension of conidia of the fungus / 10 ⁶ conidia / ml / by spraying the ears / 0.2 ml / ear / in the stage of formation of the caryopsis / water ripeness /. Treatment with biological products based on the proposed strain / Alirin B / and the prototype strain / bactofit / was carried out by spraying with a 0.1% suspension for 24 hours of infection. Folicure was used as a chemical standard. The data are presented in table. 3.

 As can be seen from the table, the use of a biological product based on the proposed strain is more effective in comparison with the prototype and can reduce the development of the disease by the main indicators by 2 times.

 Thus, the above data indicate that the proposed strain of B. subtilis has a high antagonistic activity against a complex of phytopathogenic fungi and can be a producer of a biological product to protect agricultural crops from fungal infections.

Claims (1)

 The bacterial strain Bacillus Subtilis VIZR N 10 to obtain a drug against phytopathogenic fungi.

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