RU2495119C1 - STRAIN OF BACTERIA Bacillus subtilis 8A AS AGENT TO INCREASE PRODUCTIVITY OF PLANTS AND THEIR PROTECTION AGAINST PHYTOPATHOGENIC MICROORGANISMS - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain is deposited under the number RCAM 00876 in the collection of All-Russia Research Institute of Agricultural Microbiology of the Russian Agricultural Academy. The strain Bacillus subtilis 8A has high fungicide activity against phytopathogenic activity against phytopathogenic fungi and bactericide activity against phytopathogenic bacteria, and also growth-stimulating activity relative to various crops.

EFFECT: strain may find application in agriculture as an efficient agent to increase productivity of plants and their protection against phytopathogenic microorganisms.

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Description

The invention relates to biotechnology and relates to a new strain of endophytic bacteria Bacillus subtilis 8A for crop production as a means to increase the productivity of plants and their protection against phytopathogenic microorganisms.

The invention “Biological product Fitosporin for plant protection against diseases” is known and described according to the patent of the Russian Federation No. 2099947 with priority dated 11/15/1996 addressed to the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine (UA) and the Scientific and Production Association Bashkirskoye (RU), A01N 63/00; C12N 1/20; C12R 1: 125, publication December 27, 1997 (prototype).

The basis of the biological product "Fitosporin" is a bacterial strain Bacillus subtilis VNIISM 128 with a cell concentration of 10 ⁹ -10 ¹⁰ per 1 ml of saline in the amount of 92-98 vol.% And filler in the amount of 2-8 vol.%.

Moreover, the strain Bacillus subtilis VNIISKHM 128 is characterized by high antagonistic activity against phytopathogenic bacteria and fungi, which allows it to be used to protect various types of agricultural, ornamental, and woody plants.

The objective of the invention is to identify a strain suitable for use in agriculture as a means of increasing plant productivity and their protection against phytopathogenic microorganisms, allowing to expand the arsenal of such tools.

This problem is solved due to the fact that as a means of increasing plant productivity and protecting them from phytopathogenic microorganisms in agriculture, a strain of endophytic bacteria Bacillus subtilis 8A is used, which allows to expand the arsenal of such tools.

The strain of endophytic bacteria Bacillus subtilis 8A was isolated from winter wheat grain from Nota village, growing in Rostov Region, Veselovsky District, and deposited on 11/14/2011 in the Departmental Collection of Useful Agricultural Microorganisms of the Russian Agricultural Academy (RCAM) at GNU VNIISCHM under registration number RCAM 00876.

Cultivation medium: nutrient agar (ready-made nutrient medium produced by Biocompas-S LLC, TU 10-02-02-789-176-94). The optimum temperature for growth is 28 ° C. Cultivation for 48 hours.

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 (2.3-2.7) × (1.0-1.4) microns. The strain forms spores located in the center of the cell, positively stained by Gram. Growth in a liquid and semi-liquid nutrient medium is microaerophilic, respiratory metabolism.

On nutritious agar forms dry colonies of cream color, pasty consistency with uneven rugged edges. The diameter of the colonies is 10-15 mm. The optimum temperature for growth is 28 ° C. At temperatures above 45 ° C and less than 15 ° C, growth is slow. The optimal pH value of 7.1, growth also occurs at pH from 4.5 to 8.0.

Strain 8A uses arabinose, xylose, mannitol, glucose, galactose, fructose, maltose, sorbitol, glycerin, dextrin, starch and with the formation of alkali rhamnose and dulcite as the sole carbon source. It uses mineral forms of nitrogen - ammonium salts and nitrates, amino acids and proteins. The strain Bacillus subtilis 8A 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 45 ° C, 5% NaCl and 0.001% lysozyme.

It was revealed and experimentally established that the strain Bacillus subtilis 8A has fungicidal activity against phytopathogenic fungi Alternaria alternata, Fusarium culmorum, Fusarium graminearum, Fusarium sporotrichioides, phytopathogenic bacteria Clavibacter michiganens ss. sepedonicum, Erwinia carotovora ss. atroseptica, Pseudomonas syringae and phytostimulating effect in relation to various crops (for example, spring and winter wheat, flax, sunflower).

Table 1, 2 presents comparative results on the antagonistic activity of two bacterial strains: Bacillus subtilis VNIISKhM 128 - (producer of the biological product Fitosporin) - the prototype and the claimed strain of Bacillus subtilis 8A, in relation to phytopathogenic microorganisms.

Bactericidal activity was tested on 4 strains of phytopathogenic bacteria: Pseudomonas syringae (strain 213), Pseudomonas syringae (strain 8511), Erwinia carotovora var. atroseptica (strain 822), Clavibacter michiganense subs sepedonicus (strain 6028). First, bacteria were sown with a bacteriological loop with a continuous lawn on the surface of nutrient agar and cultured for 48 hours at 28 ° C. On the day of testing for bactericidal activity, strains of the indicated phytopathogenic bacteria were sown with a continuous lawn on hungry potato agar. Then, agar blocks carved with a sterile cork drill with a mature culture of the tested bacteria were transferred to the surface of the newly seeded medium. The transfer was carried out using a sterile scalpel and calcined tweezers. Cups with blocks were cultured for 24 hours at a temperature of 28 ° C, after which the diameter of the zone of inhibition around the blocks was measured.

The measurement results are presented in table 1.

 Table 1 Antagonistic activity of bacterial strains in relation to phytopathogenic bacteria Strain Zone of growth inhibition, mm Pseudomonas syringiae (strain 213) Pseudomonas syringiae (strain 8511) Erwinia carotovora var. atroseptica (strain 822) Clavibacter michiganense subs sepedonicus (strain 6028) Bacillus subtilis 128 (Fitosporin) - prototype 12.5 ± 1.0 13.1 ± 0.7 20.1 ± 1.6 10.9 ± 1.3 Bacillus subtilis 8A (claimed) 20.0 ± 1.5 21.7 ± 1.1 25.5 ± 1.5 23.5 ± 1.9

From the data shown in table 1, it is seen that the claimed bacterial strain Bacillus subtilis 8A has a greater antagonistic activity against phytopathogenic bacteria compared with the prototype strain Bacillus subtilis 128 VNIISCHM - producer of the biological product Fitosporin.

To determine the fungicidal activity of two strains of bacteria: Bacillus subtilis VNIISXM 128 - (producer of the biological product Fitosporin) - the prototype and the claimed strain of Bacillus subtilis 8A, the "wells" method was used. The following phytopathogenic fungi were selected for testing:

Fusarium sporotrichioides, Fusarium culmorum, Fusarium graminearum, Rhizoctonia solani. A fungal spore suspension (10 ⁵ CFU / ml) was added to a potato-dextrose agar preheated and cooled to a temperature of 37 ° C at the rate of 1 μl of suspension per 1 ml of medium. The resulting mixture was poured into Petri dishes, after hardening it was made by a calcined cork drill with 4 even through holes located in a square. 100 μl of bacterial suspension with a cell titer of 10 ⁸ CFU / ml was poured into these holes. One cup was left with empty "wells" as a control option. All cups with "wells" were cultured for 72 hours at a temperature of 28 ° C. Fungicidal activity was defined as the zone of inhibition of the growth of phytopathogenic fungus around the "wells".

The measurement results are presented in table 2.

table 2 Antagonistic activity of bacterial strains in relation to phytopathogenic fungi Strain Zone of growth inhibition, mm Fusarium culmorum Fusarium graminearum Fusarium sporotrichioides Rhizoctonia solani Bacillus subtilis 128 (Fitosporin) - prototype 11.3 ± 0.9 10.7 ± 0.5 13.5 ± 1.0 15.48 ± 1.1 Bacillus subtilis 8A (claimed) 16.5 ± 1.1 17.3 ± 1.0 21.7 ± 1.3 29.5 ± 1.9

From the data shown in table 2, it is seen that the claimed bacterial strain Bacillus subtilis 8A has a greater antagonistic activity against phytopathogenic fungi compared to the strain prototype Bacillus subtilis 128 VNIISKhM - producer of the biological product Fitosporin.

Evaluation of the growth-promoting activity of strains of Bacillus subtilis 8A and the prototype Bacillus subtilis VNIISKHM 128 was carried out on a cress of the Vitamin variety. For this, lettuce seeds were treated with suspensions of the indicated bacterial strains with a titer of 10 ⁵ CFU / ml and laid out on Petri dishes with cotton wool moistened to full moisture capacity and filter paper. The control was treated with sterile water. The experiment was carried out in 2-fold repetition, 50 seeds were used for each repetition. The cups were cultured for 72 hours at a temperature of 28 ° C, after which the length of the lettuce seedlings was measured.

The measurement results are presented in table 3.

Table 3 Growth-stimulating activity of bacteria, determined in relation to lettuce seedlings Option The average root length, mm The average length of the green part of the sprout, mm The control 4.4 ± 1.1 12.0 ± 1.0 Bacillus subtilis 128 (Fitosporin) - prototype 9.9 ± 2.2 10.3 ± 1.1 Bacillus subtilis 8A (claimed) 20.1 ± 2.1 15.3 ± 1.0

From the data shown in table 3, it is seen that the claimed bacterial strain Bacillus subtilis 8A has a stronger growth-promoting activity compared to the strain prototype Bacillus subtilis 128 VNIISCHM - producer of the biological product Fitosporin.

The high growth-promoting activity of the claimed strain of endophytic bacteria Bacillus subtilis 8A is caused by the ability to create a microbial-plant system with a plant by colonizing endophytic bacteria with the root system and internal tissues (rhizosphere) of the plant, which was experimentally confirmed. The following materials were used for research. As a plant material used radish varieties "Duro" and winter soft wheat Triticum aestivum varieties "Veda".

Four gram-positive bacteria from the laboratory were selected: two bacteria Bacillus subtilis 128 and two bacteria Bacillus subtilis 8A.

Bacteria were grown on LC medium (g / l) - tryptone 10.0; yeast extract - 5.0; NaCl - 8.0; MgSO ₄ · 7H ₂ O — 2.5; agar - 18, 1 ml of 1M Tris buffer.

Colonization of radish and wheat roots by bacteria was studied in gnotobiotic systems (Simon et. Al. 1996) containing sterile quartz sand with the addition of 15% nutrient solution (PNS) of the following composition (per 1 liter): 5 mM KNO ₃ , 5 mM Ca (NO ₃ ) ₂ , 2 mM MgSO ₄ , 1 mM KH ₂ PO ₄ .

Wheat seeds were sterilized for 30 seconds with a 70% solution of ethyl alcohol, then for 6 minutes 0.1% mercuric chloride (HgCl ₂ ).

Radish seeds were sterilized for 30 seconds with a 70% solution of ethyl alcohol, then for 5 minutes with a 3% solution of hypochlorite (HClO).

After sterilization, all seeds were thoroughly washed with sterile water, changing the water 5-6 times, and 30 minutes on a rocking chair.

Then the seeds were laid out in sterile Petri dishes on moistened filter papers (5 ml per cup) and germinated at a temperature of 28 ° C for a day.

For inoculation, an overnight (20-hour) culture of microorganisms was used, grown on a rocking chair on LC liquid at a temperature of 28 ° C. Bacterial cells were separated from the growth medium by centrifugation and washed with a 0.85% NaCl solution. For inoculation, sterile radish and wheat seedlings were soaked for 30 minutes in suspension with a bacterial cell titer of 10 ⁵ CFU / ml.

Inoculated plant seedlings were planted in quartz sand (to a depth of 5-6 mm) in gnotobiotic systems, 1 plant per system. The repetition of the experiment is six times.

Wheat and radish plants were grown in a phytotron at 21 ° C for 5 days, after which they were removed from the system and bacterial survival was determined by measuring their number in the rhizosphere and on the roots of plants.

The measurement results are shown in tables 4 and 5.

Table 4 Bacteria survival in the rhizosphere and on the roots of wheat Veda Bacteria strain Number of bacteria The ratio of the number of bacteria in the root to the number of bacteria in the rhizosphere,% Rhizosphere Root 10 ⁵ KOE / plant 10 ⁵ CFU / g rhizosphere 10 ⁵ CFU / root Bacillus subtilis 128 32 ± 8 27 ± 7 11 ± 2 34.3 Bacillus subtilis 8a 55 ± 14 38 ± 10 15 ± 4 27.3

Table 5 The survival rate of bacteria in the rhizosphere and on the roots of radish Duro Bacteria strain Number of bacteria The ratio of the number of bacteria in the root to the number of bacteria in the rhizosphere,% Rhizosphere Root 10 ⁵ CFU / plant 10 ⁵ KOE / g rhizosphere 10 ⁵ CFU / root Bacillus subtilis 128 183 ± 31 376 ± 32 5.1 ± 1.2 2,8 Bacillus subtilis 8a 144 ± 23 301 ± 48 14.5 ± 3.0 10.1

The results of experiments on the survival of endophytic bacteria isolated from winter wheat seeds in the rhizosphere and on the roots of wheat and radish plants, shown in Tables 4, 5, showed that endophyte strains are able to effectively colonize the rhizosphere and root system of both monocotyledonous (wheat) and and dicotyledonous plants (radish).

When using the inventive strain of the bacterium Bacillus subtilis 8A in biologics, it is also important that the bacteria form spores, which allows you to save these drugs without losing the viability of the bacteria for a long period, at least 12 months after receipt.

The concentration (number of viable cells and spores) of the strain of the bacterium Bacillus subtilis 8A for its effective use in various biological products should be 10 ⁶ -10 ⁸ cells in 1 ml of culture fluid, as it was experimentally established that the use of the strain in 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 and growth-promoting effect.

The following is an example of obtaining a liquid form of a biological product based on the claimed strain of endophytic bacteria Bacillus subtilis 8A.

Uterine culture

To obtain a uterine culture of the bacterial strain Bacillus subtilis 8A used liquid nutrient medium KSB prepared as follows. First, a potato broth was prepared by cutting 200 g of peeled potato into slices and boiling it in 800 ml of distilled water for 20 minutes, then the broth was filtered through a cotton-gauze filter and 10 g of sucrose was added to it. The pH of the mixture was adjusted to 7.0.

The resulting liquid nutrient medium was poured into 750 ml 100 ml rocking flasks and sterilized at 1 atm. 30 minutes. Then, the medium was inoculated in flasks with a sterile microbiological loop from a test tube with beveled nutrient agar (medium composition, see above) containing a pure Bacillus subtilis 8A culture. After that, the flasks were placed on a rocking chair (220 rpm) and cultured for 48 hours at a temperature of 28 ° C. Thus, in the flasks, a Bacillus subtilis 8A uterine culture with a bacterium titer of about 5 · 10 ⁹ CFU / ml was obtained, which was stored in the refrigerator for 1 month at a temperature of 4-6 ° C for subsequent inoculation with bottles or fermenters.

Work culture

For the industrial cultivation of the bacterial strain Bacillus subtilis 8A, the working culture was obtained in bottles or fermenters on a similar medium indicated above, and a 2-fold dilution of the nutrient medium is allowed. The norm of inoculation with uterine culture is 2-5%, the cultivation duration is 48 hours at a temperature of 28-30 ° C. When cultivating working crops, a temperature increase of up to 33 ° C is allowed. Thus, a bacterial suspension concentrate was obtained based on the Bacillus subtilis 8A strain with a bacterial titer of at least 10 CFU / ml.

The liquid form of the biological product based on the strain of Bacillus subtilis 8A

The resulting bacterial suspension concentrate based on the Bacillus subtilis 8A strain was diluted with sterile water in a ratio of 1:10 or 1:20. The resulting liquid form is kept for 3-5 days at a temperature of 20-25 ° C until a bacterium titer of at least 10 ⁸ CFU in 1 ml of the preparation, after which the preparation can be used for processing crops. The liquid preparation is sterilely poured into 0.25 polyethylene bottles or canisters; 0.5; 1.0 and 10.0 liters, which are pre-rinsed with alcohol.

Due to the relatively high biological activity of the strain used and its high growth rate, the liquid preparation in the finished form, as well as in production, is practically not infected by extraneous microflora. Nevertheless, constant monitoring of all stages of obtaining a biological product based on the Bacillus subtilis 8A strain is necessary.

The effectiveness of the biological product obtained on the basis of the bacterial strain Bacillus subtilis 8A in the aforementioned manner was tested on field experiments in the Ivanovo region in relation to the productivity of spring wheat of the regional variety Priokskaya. A field experiment with wheat was carried out on sod-medium podzolic medium loamy soil (pH - 5.6; humus - 1.7%; Hr -1.7 mEq / 100 g; P ₂ O ₅ - 190 mg / kg; K ₂ O - 156 mg / kg) of the experimental field of the Ivanovo State Agricultural Academy.

The field experiment was carried out according to the research methodology in the VIUA Geographic Network of Experiments (Evaluation of the effectiveness of microbial preparations in agriculture / Edited by A. Zavalin - M .: Russian Agricultural Academy, 2000 - 82 pp.). The sown area of the plot is 40 m ² , the accounting area is 30 m ² . The experiment was repeated four times. Sowing wheat was carried out on the site, the precursor of which was potato.

On the day of sowing, part of the seeds of wheat was not treated with anything (control), another part of the seeds was treated with Phytosporin liquid biological product based on the Bacillus subtilis 128 VNIISM prototype strain, and the last part of the seeds was treated with a liquid preparation based on the Bacillus subtilis 8A bacteria strain obtained by the method described above. In this case, wheat seeds were inoculated at the rate of 1 liter of the preparation per 9 liters of water with the addition of 20-30 g of Na-CMC - sodium salt of carboxymethyl cellulose. Spraying of plants in the bumping phase was carried out manually using a knapsack sprayer at the rate of 2 l of the drug per 200 l of water per hectare.

The agricultural technology of wheat cultivation corresponded to zonal technology. Harvesting was carried out by the Sampo combine.

The experimental options involved the sowing of wheat without additional application of mineral fertilizers in the soil and with the additional application of partially and fully nitrogen, phosphorus and potassium fertilizers.

Moreover, mineral fertilizers in the form of ammonium nitrate, double superphosphate and potassium chloride were introduced manually under pre-sowing cultivation according to the experimental scheme. The results are shown in tables 6, 7, 8.

Table 6 Crop of spring wheat s. Priokskaya on the background without fertilizers Experience Option - No Fertilizer Grain harvest, kg / ha Yield increase c % Control (no processing) 6.1 - - Bacillus subtilis 128 (Fitosporin) 7.4 1.3 21.3 Bacillus subtilis 8a 8.1 2.0 32.8

Table 7 Crop of spring wheat s. Priokskaya on the background of phosphorus and potash fertilizers Variant of experience - with RK (phosphorus + potassium) Grain harvest, kg / ha Yield increase c % RK + Control (without processing) 10,4 - - PK + Bacillus subtilis 128 (Fitosporin) 10.5 0.1 1,0 PK + Bacillus subtilis 8A 13.0 2.6 25.0

Table 8 Crop of spring wheat s. Priokskaya on the background of nitrogen, phosphorus and potash fertilizers Test Option - With NPK (Nitrogen + Phosphorus + Potassium) Grain harvest, kg / ha Yield increase c % NPK + Control (no processing) 14.3 - - NPK + Bacillus subtilis 128 (Fitosporin) 14.8 0.5 3,5 NPK + Bacillus subtilis 8A 16,4 2.1 14.7

Comparison of the wheat yield data given in tables 6, 7 and 8 allows us to conclude that the biological product obtained on the basis of the claimed bacterial strain Bacillus subtilis 8A has a stronger effect on the yield (productivity) of wheat in all variants of the experiment (as without mineral fertilizers, and with their additional application) compared with the exposure to the biological product Fitosporin based on a strain of the prototype Bacillus subtilis 128 VNIISKHM.

The effectiveness of the biological product obtained on the basis of the bacterial strain Bacillus subtilis 8A was tested on field experiments in Kazakhstan (Kostanay region, Karabalyk region) in relation to the yield of sunflower oilseed and flaxseed. Compared to the effectiveness of biological preparations, field experiments were carried out on chernozem soil, medium humus soil, heavy loam, 3-4% humus, and pH-7.1 of the Karabalyk SCO.

Agricultural technology: the precursor of spring soft wheat, sunflower and flax were sown in a four-field crop rotation with the last crop, pre-sowing cultivation of the soil to a depth of 8-10 cm; sowing was carried out with an SKS-610 precision seeder with a batch sowing unit.

The experimental options involved sowing flax and sunflower seeds without treatment (control) and with seed treatment with biological products: Fitosporin (based on the strain prototype Bacillus subtilis 128) and a preparation based on the inventive strain Bacillus subtilis 8A at a rate of 2.0 l / t, and with the processing of these biological products of plants during the growing season at the rate of 1.0 l / ha.

The experience is small plot, the plot area is 25 m ² in triplicate. Flax and sunflower seeds were harvested using the Wintersteiger small-sized combine and the yield was determined in all variants of the experiment.

The results of the experiments are shown in tables 9 and 10.

Table 9 Oil flax yield in Kazakhstan Variant of experience (strain of bacteria) Harvest of flax seeds, kg / ha Yield increase c % Control (no processing) 4,5 - - Bacillus subtilis 128 (Fitosporin) 5.2 0.7 15.6 Bacillus subtilis 8a 5,6 1,1 24.4

Table 10 Sunflower productivity in Kazakhstan Variant of experience (strain of bacteria) Harvest of sunflower seeds, t / ha Yield increase c % Control (no processing) 8.9 - - Bacillus subtilis 128 (Fitosporin) 10.3 1.4 15.7 Bacillus subtilis 8a 12.7 3.8 42.7

From the data shown in tables 9 and 10, it follows that the biological product obtained on the basis of the claimed bacterial strain Bacillus subtilis 8A has a stronger effect on the yield (productivity) of oil flax and oilseed sunflower in comparison with the biological product Fitosporin based on the strain prototype Bacillus subtilis 128 VNIISCHM.

That is, the results of the experiments confirm the high quality of the biological product based on the strain of Bacillus subtilis 8A and its corresponding activity in the cultivation of crops.

Thus, the strain of endophytic bacteria Bacillus subtilis 8A is suitable for use in agriculture as an effective means of increasing plant productivity and their protection against phytopathogenic microorganisms and expands the arsenal of such agents.

Claims (1)

The bacterial strain Bacillus subtilis 8A, deposited in the collection of the GNU All-Union Agricultural Research Institute of the Russian Academy of Agricultural Sciences under the number RCAM 00876, as a means of increasing plant productivity and their protection against phytopathogenic microorganisms.