RU2259397C2 - Agent for protection of cereal agricultural crops, sunflower and grape against phytopathogenic microorganisms and vegetable crops against phytopathogenic bacteria - Google Patents

Abstract

FIELD: agriculture, biotechnology, microbiology.

SUBSTANCE: invention represents the strain of bacterium Bacillus subtilis CH-13 that is used for protection of cereal agricultural crops, sunflower and grape against phytopathogenic microorganisms and vegetable crops against phytopathogenic bacteria also. The strain Bacillus subtilis CH-13 is isolated from chernozem (black) soil in Republic Moldova and deposited at registration number VNIISCHM D-606 from 10 September 1998 in the group of epiphyte microorganisms. Ability of the strain to inhibit different pathogenic microflora results to enhancing productivity of different species of agricultural crops.

EFFECT: valuable antibacterial properties of agent.

4 tbl, 2 ex

Description

The invention relates to biotechnology and is the use of a bacterial strain Bacillus Subtilis Ch-13 as a means to protect cereal crops, sunflower, grapes from phytopathogenic microorganisms, and vegetable crops from phytopathogenic bacteria.

In Russia, in order to protect agricultural plants from phytopathogenic microflora and increase productivity, biological preparations based on bacterial strains from the Pseudomonas group have been used. The drug under the commercial name Rizoplan, the active principle of which is the living cells of the bacteria Pseudomonas sp., Selected for their ability to suppress phytopathogens - root rot pathogens, is used for presowing treatment of seeds mainly of grain crops. The use of Rizoplan protects plants from defeat by the causative agents of late blight, downy mildew, and bacteriosis (Collection of articles “Genetics of economically valuable traits of higher plants”, editors-in-chief A. Shchapov, L. Kolosova, Institute of Cytology and Genetics, SB VOGiS H.I.Vavilova, Novosibirsk, 1990, pp. 194-198).

The well-known "Method of seed treatment" described in the application No. 2170987 of Great Britain with a priority of 02/14/85, MKI 4 A 01 C 1/06, published in ISM No. 10 for 1987. This method involves treating the seeds with a composition consisting of microorganisms, a carrier, for example bran, and an adhesive such as gatti resin.

The most favorable results are obtained when processing wheat seeds. Microorganisms can stimulate their growth (Azospirillum), bind nitrogen (Rhizobium), prevent plant diseases (Pseudomonas or Bacillus), or kill insects (Streptomyces).

Also known is the "Method of protecting cabbage from bacterial diseases" according to patent No. 1793878 with priority dated 12/10/90, published on 02/07/93 in the Bulletin of inventions of the Russian Federation No. 5.

According to the named method, to protect cabbage from bacteriosis, cabbage seeds after hydrothermal treatment are treated with a suspension of the bacterial strain "Pseudomonas sp. VKPM B-3481 "at a concentration of 10 ⁶ -10 ⁹ cells / ml at the rate of 20 ml per 1 kg of seeds. The use of a bacterial suspension "Pseudomonas sp. VKPM B-3481 ”reduces the development of vascular and mucous bacterioses and increases the yield of cabbage.

The well-known "Bacillus subtilis bacterial strain No. 11 (VNIISKHM), which has antagonistic and stimulating properties and the method of its use for protecting barley from helminthosporiosis" according to the patent of the Russian Federation No. 93001490 with a priority dated 01.11.93, author Popova Zh.P.

The bacterial strain Bacillus subtilis No. 11, isolated from a soil sample taken from under cotton in Egypt, has antagonistic properties against phytopathogenic soil microorganisms, in particular Helminthosporium sativun, and it also stimulates the growth of barley plants. The proposed method of using the strain optimizes its effect due to the simultaneous application of green fertilizers (siderates) to the soil along with bacterization of seeds.

A known group of inventions "Method of presowing treatment of seeds of vegetable crops and a method for producing a preparation for presowing treatment of seeds of vegetable crops" according to the patent of the Russian Federation No. 2140138 with a priority of 11/13/98, authors Chebotar V.K., Bykova N.V., Temnova .V., Orlova N.A. and Khotanovich A.V.

These methods are carried out using a biofungicidal preparation containing the bacterial strain Bacillus subtilis Ch-13 (deposited under the registration number VNIISCHM D-606 in the group of epiphytic microorganisms). EFFECT: inventions make it possible to increase the efficiency of protecting vegetable crops from phytopathogenic fungi by pre-sowing seed treatment (the closest analogue).

The task of the invention is to identify means in the form of a bacterial strain suitable for use in agriculture to protect cereal crops, sunflower, grapes from phytopathogenic microorganisms, and vegetable crops from phytopathogenic bacteria.

This problem is solved due to the fact that as a means of protecting cereal crops, sunflowers, grapes from phytopathogenic microorganisms, and vegetables from phytopathogenic bacteria, the bacterial strain Bacillus subtilis Ch-13 was used, in which the ability to suppress various pathogenic microflora (phytopathogenic microorganisms: phytopathogenic bacteria and phytopathogenic fungi), which leads to an increase in the yield of different types of crops: grain ovyh crops, vegetables, sunflower and grapes.

The strain of rhizospheric spore bacteria "Bacillus subtilis Ch-13" was isolated from chernozem soil of the Republic of Moldova and deposited under the registration number VNIISKHM D-606 dated 09/10/1998 in the group of epiphytic microorganisms.

The strain is characterized by the following morphological-cultural and physiological-biochemical characteristics.

The cells are in the form of regular rods with rounded ends and a monopolar peritrichous arrangement of flagella. The cell size is 1.0 × 2.0 μm. The strain forms spores located in the center of the cell, positively stained by Gram. After 24 hours of growth, an accumulation of poly-β-hydroxybutyrate was observed in the liquid nutrient medium. Growth in a liquid and semi-liquid nutrient medium is microaerophilic, respiratory and fermentative metabolism. On meat and peptone agar forms dry colonies of a creamy color, pasty consistency with uneven rugged edges. The diameter of the colonies is 20-40 mm. The optimum temperature for growth is 37 ° C. At temperatures above 45 ° C and less than 15 ° C, growth is slow. The optimal pH is 6.8; growth also occurs at pH from 4.5 to 8.0.

The strain Bacillus subtilis Ch-13 hydrolyzes casein, gelatin, starch and litmus milk. Litmus was discolored. The strain has strong catalase activity and does not have lipase activity. The strain is able to grow at a temperature of 40 ° C, the presence of 7% NaCl and 0.001% lysozyme.

The strain uses the arabinose, xylose, mannitol, glucose, galactose, fructose, maltose, sorbitol, glycerin, dextrin, starch and with the formation of alkali rhamnose and dulcite with the formation of acid as the sole carbon source.

The strain of the bacterium "Bacillus subtilis Ch-13" is grown on an accessible nutrient medium consisting of bran and chalk, which is a significant advantage compared, for example, with the cultivation method of pseudomonas, which use scarce and expensive nutrient media.

In accordance with the "Methodological instructions of the Ministry of Health of the USSR" No. 2620-82 and No. 423-87, it was experimentally determined that the strain Bacillus subtilis Ch-13 is non-virulent, non-toxic and non-toxic, which characterizes it as non-pathogenic for warm-blooded animals and it refers to the 4th hazard class.

The antagonistic properties of the strain Bacillus subtilis Ch-13 in relation to pathogens of agricultural crops were revealed and experimentally established:

- for winter and spring wheat - the strain is effective against powdery mildew (Erysiphe graminis), brown rust (Puccinia recondita), Fusarium root rot (Fusarium culmorum);

- for spring barley - effective against moldy seeds (Penicillium, Alternaria), root rot (Bipolaris sorokiniana), dark brown spotting (Drechlera sorokiana);

- for white cabbage and cauliflower - effective against vascular bacteriosis Xanthomonas campestris), blackleg (Rhizoctonia solani), pitious root rot (Pythium irregulare);

- for potatoes - it is effective against figoftorosis (Phytophtora infestans), rhizoctonia (Rhizoctonia solani), fusarium wilt (Fusarium oxysporum);

- for sunflower - effective against white rot (Sclerotinia sclerotiorum), phomopsis (Phomopsis helianthi);

- for sugar beets - effective against root eater (Pythium debarianum, Phoma betae), cercosporosis (Cercospora beticola);

- for grapes - the strain is effective against mildew (Plasmopara viticola), oidium (Uncinula necator), gray rot (Botrytis cinerea).

Comparative antagonistic activity of rhizospheric bacteria, producers of biological products Planriz (Pseudomonas fluorescens AP-33), Phytosporin (Bacillus subtilis VNIIXM 128) and Extrasol (Bacillus subtilis Ch-13) in relation to phytopathogenic bacteria are presented in table 1.

Table 1.
Antagonistic activity of rhizospheric bacteria that are part of various biological products in relation to phytopathogenic bacteria. Drug (Strain) Zone of growth inhibition, mm Erwinia carotovora A-1 Pseudomonas syringiae 8300 Pseudomonas syringiae 2314 Erwinia carotovora 3391 Clavibacter michiganense 17-1 Planriz (Pseudomonas fluorescens AP-33) 8.0 ± 0.6 12.6 ± 1.1 14.2 ± 1.3 15.5 ± 1.9 20.6 ± 1.9 Fitosporin (Bacillus subtilis VNIISCHM 128) 14.2 ± 1.3 18.9 ± 1.6 11.8 ± 0.9 28.8 ± 2.1 16.0 ± 1.2 Extrasol (Bacillus subtilis Ch-13) 28.4 ± 2.0 32.0 ± 2.6 48.0 ± 3.3 49.5 ± 4.1 36.0 ± 2.7

From the data given in table 1 it is seen that the preparation Extrasol based on the bacterial strain Bacillus subtilis Ch-13 has the highest antagonistic activity against phytopathogenic bacteria compared with biological products based on other known bacterial strains.

Comparative antagonistic activity of rhizospheric bacteria, producers of biological products Planriz (Pseudomonas fluorescens AP-33), Phytosporin (Bacillus subtilis VNIIXM 128) and Extrasol (Bacillus subtilis Ch-13) in relation to phytopathogenic fungi are presented in table 2.

Table 2.
Antagonistic activity of rhizospheric bacteria that are part of various biological products in relation to phytopathogenic fungi Drug (Strain) Zone of growth inhibition, mm Phytophtora capsici Rhizoctonia solani Fusarium culmorum Fusarium solani Pythium spp. Planriz (Pseudomonas fluorescens AP-33) 12.5 ± 1.0 13.3 ± 1.1 8.0 ± 0.7 10.8 ± 0.8 10.1 ± 0.9 Fitosporin (Bacillus subtilis VNIISCHM 128) 26.5 ± 2.2 16.7 ± 1.8 16.2 ± 1.3 16.4 ± 1.3 22.1 ± 2.5 Extrasol (Bacillus subtilis Ch-13) 48.1 ± 3.2 29.6 ± 1.9 33.0 ± 2.4 23.8 ± 1.9 38.1 ± 2.9

From the data given in table 2, it can be seen that the preparation Extrasol, based on the bacterial strain Bacillus subtilis Ch-13, has the highest antagonistic activity against phytopathogenic fungi compared to biological preparations based on other known bacterial strains.

Experiments were conducted on the effect of a biological product containing the bacterial strain Bacillus subtilis Ch-13 on the yield of spring crops and potatoes. The results of the experiments are shown in table 3 and 4, respectively.

Table 3.
The effect of a biological product based on the strain of Bacillus subtilis Ch-13 on the yield of grain crops. Household Kulturf, grade Area of experience, ha Grain harvest, kg / ha Increase Strain Ch-13 The control c / ha % CJSC Agrobalt oats, s. Borrus fifty 39.0 35.2 3.8 10.8 CJSC Torosovo spring wheat, s. Irgina fifty 28.0 21.0 7.0 33.3 CJSC Sumino spring barley, s. Krinichny 80 28.0 23.0 5.0 21.7 CJSC Kotelskoye spring barley, s. Suzdal 65 41.4 35.0 6.4 18.3 CJSC Kotelskoye oats, s. Argamak 22 42.0 30.0 12.0 40.0 CJSC Seltso spring wheat, s. Fortress 32 25.0 18.0 7.0 38.9 CJSC Gomontovo spring barley, s. Ipari 64 29.0 22.0 7.0 31.8 Region average 51.9 33.7 26.6 8.4 32,7

Table 4.
The effect of a biological product based on a strain of Bacillus subtilis Ch-13 on potato productivity. Household Grade Area of experience, ha Harvest tubers, t / ha Increase Extrasol The control c / ha % CJSC Torosovo Nevsky 28 241 211 thirty 14.2 CJSC Seltso Nevsky thirty 250 190 60 31.6 CJSC Gomontovo Nevsky 53 252 205 47 22.9 Region average 37 247.7 202 45.7 28

The results of the experiments, shown in tables 3 and 4, show that the use of the bacterial strain Bacillus subtilis Ch-13, the producer of the biological product Extrasol, leads to an increase in the yield of grain crops and potatoes by an average of 30%.

It is also important that when using the specified strain in the composition of biological products, the bacteria “Bacillus subtilis Ch-13” form spores, which allows you to save these drugs without loss of bacterial viability for a long period, at least 6 months from the date of receipt.

The concentration (number of viable cells and spores) of the strain of the bacterium "Bacillus sobtilis Ch-13" for its effective use in various biological products should be 10 ⁶ -10 ⁸ cells per 1 ml of culture fluid. It was experimentally established that the use of the strain at a concentration of less than 10 ⁶ cells / ml leads to a decrease in the antagonistic effect, and an increase in concentration above 10 ⁹ cells / ml does not increase the effectiveness of the protective effect.

The following are examples of the manufacture of biological products based on the inventive strain "Bacillus subtilis Ch-13".

Example 1

The bacterial culture "Bacillus subtilis Ch-13" is grown in a sterile liquid nutrient medium containing 4% wheat bran and 0.3% chalk, on a rocking chair or in a fermenter at 37 ± 1 ° C for 24-36 hours. 5 · 10 ⁸ cells / ml are accumulated in the culture fluid. Then get the biological product in the following way. First mix:

70 ml of culture fluid

230 g - defecate

after which the resulting mass is dried at 60 ° C for 12 hours and a fine powder is prepared from it by grinding (strain titer - a little more than 10 ⁶ cells / g).

Example 2

The bacterial culture "Bacillus subtilis Ch-13" is grown in a liquid sterile nutrient medium of the following composition:

peptone - 20 g / l

glycerin - 10 g / l

K ₂ NRA ₄ - 1.5 g / l

MgSO ₄ - 1.5 g / l

on a shaker or in a fermenter at 37 ° C for 24-36 hours until a titer of 10 ⁹ cells / ml is reached in the culture fluid.

Then get a liquid form of the biological product, mixing 20% of the culture fluid and 80% water (titer of the biological product - 2 · 10 ⁸ cells / ml).

Claims (1)

The use of the bacterial strain Bacillus subtilis Ch-13 VNIISKHM D-606 as a means to protect cereal crops, sunflower, grapes from phytopathogenic microorganisms, and vegetable crops from phytopathogenic bacteria.