RU2736340C9 - Agricultural growth stimulant - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the biotechnology. Disclosed is an agent based on the strain of bacteria Bacillus subtilis Ch-13, deposited under registration number of the All-Russian Scientific Research Institute of Agricultural Meteorology VNIISKhM D-606 and having the ability to produce plant growth hormones - auxins and B vitamins.

EFFECT: invention provides wider range of biopreparations stimulating growth of crops.

1 cl, 9 tbl, 2 ex

Description

The invention relates to biotechnology and agriculture and is the use of a known bacterial strain Bacillus subtilis Ch-13 as a means to stimulate the growth of crops.

Known RF patent №2327737 with pr. 01.08.2006 for the invention "Strain of bacteria Bacillus megaterium, mobilizing phosphorus and silicon from objects of the lithosphere and resistant to polyhexamethylene guanidine", IPC C12N 1/20, C05F 11/08, C12R 1/11, publ. June 27, 2008

The invention relates to agricultural microbiology and geochemistry. Strain Bacillus megaterium var. phosphaticum BKM B-2357D is capable of leaching phosphorus and silicon from objects of the lithosphere and is resistant to polyhexamethylene guanidine. The strain can be used to obtain a bacterial preparation that increases the survival rate in the soil and the yield of agricultural crops, for the reclamation of technologically contaminated lands.

Known RF patent No. 2558291 with pr. 24.12.2013, for the invention "Multifunctional means for crop production", IPC C12N 1/20, A01N 63/00, publ .: application 27.062015, publ. 27.07.2015

The described strain of spore bacteria Bacillus megaterium 501 GR has a growth-stimulating effect, improves phosphorus nutrition of plants, and also contributes to bioremediation of soils contaminated with pesticides. The use of the Bacillus megaterium 501 GR strain allows increasing the productivity of plants, as well as protecting them from phytopathogenic fungi and microorganisms that cause root diseases.

Known RF patent No. 2649359 with a priority of 12/13/2016 for the invention "Strain of endophytic bacteria Bacillus megaterium V3 as a means to improve nutrition, accelerate growth and increase productivity of grain, vegetable, industrial and wood crops" IPC A01N 63/00, C12N 1/20, publ. 02.04.2018

The high growth-stimulating activity of the Bacillus megaterium V3 strain is caused by the ability to create a plant-microbial system with a plant by colonizing the root system and internal tissues of the plant with endophytic bacteria.

The strain can be used as part of various microbial preparations, for the effective use of which the titer of the bacteria Bacillus megaterium V3 should be 10 ⁵ -10 ⁹ cells / ml of culture fluid.

There are also known foreign publications that describe the use of bacteria of the species Bacillus megaterium as a means of stimulating the growth of plants and / or their roots (for example, in patent KR 20100963774 B1 dated 06/14/2010 for the Bacillus megaterium strain KNUC 251 or in the US application 20130014434 published 01/17/2013, in which the nutrient composition for the roots of the transplanted plant contains, among other microorganisms, the bacteria Bacillus megaterium).

Known group of inventions "A method of pre-sowing treatment of vegetable seeds and a method of obtaining a preparation for pre-sowing treatment of vegetable seeds" according to RF patent No. 2140138 with a priority of 11/13/98, the authors Chebotar V.K., Bykova N.V., Temnova O V., Orlovoy N.A. and Khotyanovich A.The. These methods are carried out using a biofungicidal preparation containing the bacterial strain Bacillus subtilis Ch-13. The inventions make it possible to increase the efficiency of protecting vegetable crops from phytopathogenic fungi during pre-sowing seed treatment.

Also known is the invention "Means for the protection of grain crops, sunflowers, grapes from phytopathogenic microorganisms, and vegetable crops from phytopathogenic bacteria" according to RF patent No. 2259397 from 02.04.2003, authors Chebotar V.K., Bykova N. V., Temnova O.V., Orlova N.A., Khotyanovich A.V., IPC C12N 1/20, C12R 1: 125, А01С 1/06, publ. August 27, 2005

The bacterial strain Bacillus subtilis Ch-13 is used in the production of microbiological fertilizers to increase the productivity of vegetable, grain and industrial crops in peat form "Bisolbi", dry form "BisolbiFit" and liquid form "Extrasol" (Certificate of state registration No. 0680-07- 2018-216-0-0-0-1 dated March 29, 2007).

Known invention "Method for producing biofertilizers" by RF patent No. 2241692 with a priority of 11.10.2002, the authors Chebotar V.K., Kozakov A.E., Erofeeva S.V., IPC C05G 1/0, C12N 1/20, C12R 1: 125, publ. 10.12.2004

This method consists in combining granular mineral fertilizers with microbial biomass in the form of a bacterial preparation based on the bacterial strain "Bacillus subtilis Ch-13", which has antagonistic properties to phytopathogenic microflora in the soil. Combination is performed by dusting or spraying the surface of mineral fertilizer granules with a bacterial preparation, or in the form of a liquid fraction in the composition of moisture-protective compositions of mineral fertilizers.

At the same time, in addition to the total sanitizing effect of phytopathogenic microflora, the granules of mineral fertilizers are enriched with a biological product, which contributes to a more efficient assimilation of mineral substances from these fertilizers by plants, because the mineral elements in the granules treated with the biological product are in a more stable physicochemical state, less susceptible to leaching and transition to a gaseous state (most of all, this concerns mineral nitrogen).

Known invention "Method of obtaining biomineral fertilizers and ameliorants (options)" by RF patent No. 2512277 with a priority of 10.10.2012, the authors Chebotar V.K. and Erofeeva SV., IPC C05F 11/08, publ. 10.04.2014

The method provides for the combination of dry or liquid mineral fertilizers (ameliorants) with a microbiological preparation based on agronomically beneficial microorganisms: in the 1st variant - in the form of a dry fine powder with a titer of 10 ² -10 ⁵ cells / g at the rate of 2-6 kg / t, and in the 2nd variant - in liquid form with a titer of 10 ⁴ -10 ⁷ cells / g, which is applied to a dry mineral fertilizer or ameliorant by fine spraying, or applied to a liquid mineral fertilizer, at the rate of 1-4 kg / t

This increases the utilization rate of nutrients from fertilizers and ameliorants (KIA) by 10-50%, which leads to an increase in the productivity of agricultural plants by 12-40%.

Known invention "Strain of bacteria Bacillus subtilis 8A as a means of increasing the productivity of plants and their protection against phytopathogenic microorganisms" by RF patent No. 2495119 with priority from 29.11.2012, the authors Chebotar V.K. et al., IPC C12N 1/20, C12N 1/125, A01N 63/00, publ. 10.10.2013 (prototype).

The specified strain has high fungicidal and bactericidal activity. A high growth-stimulating activity of the Bacillus subtilis 8A strain and a biological product based on it was also established, which leads to an increase in plant productivity.

The objective of the invention is to identify a new strain or new properties in a known strain suitable for use in agriculture as a means to stimulate the growth and increase the productivity of vegetable and grain crops, as well as to expand the arsenal of such means.

This problem is solved due to the fact that the bacterial strain Bacillus subtilis Ch-13 is used as a means to stimulate the growth of agricultural crops, in which the ability to stimulate the growth of agricultural crops has been identified and experimentally confirmed by producing plant growth hormones and vitamins on the roots of plants.

The strain of rhizospheric spore bacteria "Bacillus subtilis Ch-13" was isolated from the chernozem soil of the Republic of Moldova and deposited under the registration number VNIISKhM D-606 from 10.09.1998 in the group of epiphytic microorganisms.

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

The cells are in the form of regular rods with rounded ends and a monopolar peritrichous arrangement of flagella. Cell size 1.0 × 2.0 μm. The strain forms spores located in the center of the cell, positively stains according to Gram. After 24 hours of growth on liquid nutrient medium, the accumulation of poly-β-hydroxybutyrate was observed. Growth in a liquid and semi-liquid nutrient medium is microaerophilic, the metabolism is respiratory and fermentative. On meat-peptone agar, it forms dry colonies of creamy color, pasty consistency with uneven jagged 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 optimum pH of the medium is 6.8; growth also occurs at a pH of 4.5 to 8.0.

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

As the only source of carbon, the strain uses arabinose, xylose, mannitol, glucose, galactose, fructose, maltose, sorbitol, glycerin, dextrin, starch to form acid, and rhamnose and dulcite to form alkali.

The bacterial strain Bacillus subtilis Ch-13 is grown on an available nutrient medium consisting of molasses and corn extract, which is an advantage over, for example, the method of cultivating pseudomonads, which uses scarce and expensive nutrient media.

In accordance with the "Methodological instructions of the Ministry of Health of the USSR" No. 2620-82 and No. 4263-87, it was experimentally determined that the Bacillus subtilis strain Ch-13 is non-virulent, non-toxic and non-toxic, belongs to the 4th hazard class.

The strain of rhizospheric spore bacteria "Bacillus subtilis Ch-13" on the basis of modern methods can also be identified as Bacillus amiloliqufaciens (Borriss R., Chen XH, Rueckert C, Blom J., Becker A., et al. 2011. Relationship of Bacillus amyloliquefaciens clades associated with strains DSM 7T and FZB42T: a proposal for Bacillus amyloliquefaciens subsp.amyloliquefaciens subsp.nov. and Bacillus amyloliquefaciens subsp.plantarum subsp.nov. based on complete genome sequence comparisons. Int J Syst Evol Microbiol 61: 1786-1801) and Bacillus velezensis (Christopher A. Dunlap, Soo-Jin Kim, Soon-Wo Kwon and Alejandro P. Rooney. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens; Bacillus methylotrophic Bacillus laterylotrophicus or later heterotypic synonyms of Bacillus velezensis based on phylogenomics.International Journal of Systematic and Evolutionary Microbiology / 2016, 66, 1212-1217 DOI 10.1099 / ijse m. 0.000858).

The ability of the Bacillus subtilis Ch-13 strain to produce plant growth hormones - auxins and vitamins - in a nutrient medium for growing bacteria has been identified and experimentally established.

The production of phytohormones is one of the main mechanisms for stimulating plant growth by various associated bacteria, including species of the genus Bacillus (Perez-Garcia A, Romero D, de Vicente A. 2011. Plant protection and growth stimulation by microorganisms: biotechnological applications of Bacilli in agriculture. Curr Opin Biotechnol.: 22:187-193). HPLC studies have shown that the supernatant (the supernatant is the liquid phase that remains after insoluble matter is precipitated during centrifugation or sedimentation).

Bacillus subtilis Ch-13, grown on a medium with tryptophan, contains indole-3-acetic acid (IAA) and several related compounds V.K. Chebotar, N.M. Makarova, A.I. Shaposhnikov, L.V. Kravchenko. Antifungal and phytostimulating properties of the rhizosphere strain Bacillus subtilis Ch-13 - a producer of biological products. 2009. Applied Biochemistry and Microbiology, vol. 45, no. 4, p. 465-469). IAA is the most biologically active member of the auxin family. It is assumed that the effect of bacterial IAA is based on an increase in the total absorbing area of the roots, which leads to an increase in the absorption of water and nutrients and stimulates plant growth (Spaepen S, Vanderleyden J, Remans R. 2007. Indole-3-acetic acid in microbial and microorganism- plant signaling FEMS Microbiol Rev 31: 425-448).

Using the Salkovsky reaction (Brick, JM, Bostock, RM, and Silvers tone, SE 1991. Rapid in situ assay for indole acetic acid production by bacteria immobilized on nitrocellulose membrane. Applied Environment Microbiology, 57, 535-538), the auxin content in culture fluid of the Bacillus subtilis strain Ch-13. For this, the strain was grown in a liquid medium 1/20 TSB (manufacturer Sigma, USA), with the addition of L-tryptophan 500 kg / ml, at 200 rpm, at room temperature for 2 days. The resulting suspension was centrifuged at 4000 rpm for 2 minutes. The supernatant was used for analysis. For preliminary determination of the content of indole compounds (including bacterial auxins), the Salkovsky reaction was used. The test sample was mixed 1: 1 with Salkovsky's reagent (50 ml of 35% perchloric acid and 1 ml of 0.5 M ferric sulfate), left for 30-40 minutes, after which the optical density was measured at 530 nm. 1/20 TSB was used as a control, with the addition of L-tryptophan 500 μg / ml, as a positive control - 1/20 TSB, with the addition of heteroauxin 100 μg / ml. The production of bacterial auxins in the nutrient medium for growing bacteria in the bacterial strain Bacillus subtilis Ch-13 was 3-5 μg / ml.

Vitamins from group B have a significant effect on plants, accelerate metabolism, strengthen roots, under their influence, plants grow and develop better. Vitamins are important micronutrients produced by many plants and bacteria (Palacios OA., Bashan Y, de-Bashan LE. 2014. Proven and potential involvement of vitamins in interactions of plants with plant growth-promoting bacteria: an overview. Biol. Fertil. Soils 50: 415-432).

The main physiological role of vitamins is that they serve as cofactors in numerous metabolic pathways and are also antioxidants.

Vitamin B1 (thiamine) helps plants cope with stressful conditions and also stimulates their growth. Vitamin B3 (nicotinic acid and its second form, nicotinamide) is excellent for plant resuscitation. Vitamin B6 (or pyridoxine) stimulates the immune system of plants. Vitamin B12 (cyanocobalamin) is involved in the production of chlorophyll, activates plant growth and metabolism.

Analysis of the culture fluid of the Bacillus subtilis Ch-13 bacterial strain grown in 1/20 TSB liquid medium (72 hours at 28 ° C) showed the presence of vitamin B3 in it (nicotinic acid in an amount of 0.0475 μg / ml, nicotinamide - 0, 0146 μg / ml) and vitamin B6 (pyridoxine) in an amount of 0.072 μg / ml.

Analysis of the culture fluid of the bacterial strain Bacillus subtilis Ch-13 grown on potato-dextrose medium (PDB, Sigma, USA) for 7 days at 30 ° C in the stationary phase, revealed the presence of vitamin B1 (thiamine) in it in the amount of 0.0091 μg / ml and vitamin B12 (cyanocobalamin) in the amount of 0.0045 μg / ml.

Thus, the results of experiments with the culture liquid of the bacterial strain Bacillus subtilis Ch-13 confirm the presence of B vitamins in it, namely: B1, B3, B6 and B12, which are capable of influencing plant immunity, their resistance to stress factors and ultimately - on the productivity of plants.

It has been experimentally established that the bacterial strain Bacillus subtilis Ch-13 has a growth-stimulating effect in relation to various agricultural crops

The analysis of the growth-stimulating activity of bacteria: the Bacillus subtilis Ch-13 strain and the prototype Bacillus subtilis 8A strain was carried out in a laboratory experiment according to the original method using lettuce plants of the Yeralash variety and spring wheat of the Trizo variety. For this, plant seeds were first sterilized for 2 minutes in 70% ethanol and washed in sterile tap water. Then the seeds were soaked for 20 min in a suspension of bacteria with a cell titer of 10 ⁷ CFU / ml and placed in sterile humid chambers, 25 pieces each. in 4-fold repetition. In the control, the seeds were soaked in sterile tap water. Then the seeds were incubated in a phytotron for 96 h at a temperature of (25 + 1) ° C. After incubation, the length of roots and shoots was measured, and the growth-stimulating activity of the tested bacterial strains was calculated in relation to control seedlings. The results are presented in tables 1, 2.

From the data shown in Table 1, it can be seen that the studied bacterial strain Bacillus subtilis Ch-13 has a stronger growth-stimulating activity in relation to lettuce seedlings in comparison with the prototype Bacillus subtilis 8A strain, increasing the average root length by 1.2 times, and the average the length of the green part of the sprout is 1.18 times.

From the data shown in Table 2, it can be seen that the studied bacterial strain Bacillus subtilis Ch-13 has a stronger growth-stimulating activity in relation to wheat seedlings in comparison with the prototype Bacillus subtilis 8A strain, increasing the average root length by 1.4 times, and the average the length of the green part of the sprout is 1.37 times.

It was also experimentally established that the Bacillus subtilis strain Ch-13 has a phytostimulating effect in relation to various agricultural crops, in particular, to barley, watercress and corn.

Microvegetation experiments were carried out in which the influence of Bacillus subtilis bacteria on the development of the root system and green mass of spring barley plants of the "Vakula" variety, on the biomass of watercress of the "Spring" variety and on the biomass of corn varieties "Dobrynya" was determined.

Experiments on the effect of bacteria of the Bacillus subtilis strain Ch-13 and bacteria of the prototype Bacillus subtilis 8A strain on the development of roots and biomass of barley, depending on the concentration of the studied strains, were carried out in a vegetation box with 16 hours of daylight hours, a temperature of 22 ° C and a light intensity of 5000- 6000 lx. Quartz sand with a fraction of 0.6-1.0 mm, pre-sieved and washed from clay and other impurities, was used as a substrate. The sand was sterilized for an hour at 1.5 atm, cooled, and the vessels were filled by weight. For irrigation and nutrition of plants, a mineral solution PNS was used containing (mg / l): Ca (NO ₃ ) × 4H ₂ O - 972; MgSO ₄ × 7H ₂ O - 616; NaNO ₃ 140; KH ₂ PO ₄ 170; NH ₄ NO ₃ 55; Fe-EDTA - 20; ZnSO ₄ × 7H ₂ O - 1.7; B4Na ₂ O ₇ × 7H ₂ O 3.35; CuSO ₄ × 5H ₂ O - 0.25 and Na ₂ MoO ₄ × 2H ₂ O - 0.12 mg / l.

Seedlings were inoculated with an overnight (20-hour) culture of bacterial strains grown on a liquid LB medium of the following composition (g / l): tryptone - 10.0, yeast extract - 5.0, Mg ₂ SO ₄ - 2.5, pH = 7.2. For this, barley seeds were placed in a Petri dish and soaked for 30 min in a working solution of a bacterial suspension with a known titer, after which they were planted in sand. 7 barley seeds were planted on the vessel, soaked in a suspension of the studied strains for 30 minutes, without prior germination. Barley plants were grown for 16 days, after which the root system was washed from sand and the total root length and plant biomass were analyzed using the Image J 1.50b software. For analysis, 5 typical plants were selected from each variant. The results of the experiments are presented in Table 3.

Thus, from the data in Table 3, it can be seen that solutions of the bacterial suspension of Bacillus subtilis Ch-13 with a titer of 105-107 CFU / ml have a significant effect on the total length of germinal roots of barley c. "Vakula", increasing it by 35-54% compared to the control, as well as the biomass of plants, increasing it by 20-32% compared to the control. At the same time, the Bacillus subtilis Ch-13 strain has a greater stimulating effect on the total length of barley embryonic roots in comparison with the prototype Bacillus subtilis 8A strain used in plant growing as a means to increase plant productivity.

That is, the data of Table 3 confirm, using the example of spring barley plants of the "Vakula" variety, a more effective growth-stimulating effect of the Bacillus subtilis strain Ch-13 in comparison with the prototype Bacillus subtilis 8A strain.

To determine the growth-stimulating activity of bacteria of the Bacillus subtilis Ch-13 strain and the prototype Bacillus subtilis 8A strain, 0.1%, 1% and 10% solutions of bacterial suspensions of the studied strains with an initial bacterial titer of 1 were prepared on plants of the spring watercress variety. 0 × 10 ⁹ CFU / ml. Watercress seeds were placed in sterile test tubes, filled with prepared solutions of the studied strains, and soaked for 10 min. In the control, the seeds were soaked in sterile tap water. Wet chambers were prepared: filter paper was laid out in sterile Petri dishes and moistened with sterile tap water with a volume of 5 ml per chamber. Watercress seeds were placed with sterile tweezers on the surface of wet chambers, 25 seeds per dish (at least 3 times). The humid chambers were placed in a phytotron and incubated for 5 days at t ° 28 ° C, after which the length of the shoot and root of the seedlings was measured. The data obtained were processed using the Excel program.

The results of the experiment are presented in Table 4.

From the data presented in Table 4, it can be seen that 0.1%, 1% and 10% solutions of the bacterial suspension of Bacillus subtilis Ch-13 with an initial bacterial titer of 1.0x109 CFU / ml had an effective effect on the length of cress roots, increasing them by 40.9-73.1% compared to the control, as well as a greater stimulating effect on the length of the roots of watercress in comparison with the influence of the prototype strain Bacillus subtilis 8A (an increase of 13.3-46.1%). In addition, 0.1-10% solutions of the bacterial suspension of Bacillus subtilis Ch-13 influenced the length of the shoots of watercress, the length of which increased by 9.1-13.4% compared to the control, which is greater than the effect of the bacterial suspension of the strain -prototype Bacillus subtilis 8A (increase by 0.5-6.6%).

Thus, the data in Table 4 confirm the more effective growth-stimulating effect of the Bacillus subtilis strain Ch-13 in comparison with the prototype Bacillus subtilis 8A strain using the example of the Vesenny cress cultivar.

To determine the growth-stimulating activity of bacteria of the Bacillus subtilis Ch-13 strain and the prototype Bacillus subtilis 8A strain on corn plants of the Dobrynya variety, 0.1%, 1% and 10% solutions of bacterial suspensions of the studied strains were prepared with an initial bacterial titer of 1.0 × 10 ⁹ CFU / ml. Corn seeds were placed in sterile cups, filled with prepared solutions of the studied strains, and soaked for 10 min. In the control, the seeds were soaked in sterile tap water. Wet chambers were prepared: filter paper was laid out in sterile Petri dishes and moistened with sterile tap water with a volume of 5 ml per chamber. With sterile tweezers, corn seeds were placed on the surface of the wet chambers, 10 pieces per dish (at least 3 times). The humid chambers were placed in a phytotron and incubated for 5 days at t ° 28 ° C, after which the length of the shoot and root of the seedlings was measured. The data obtained were processed using the Excel program.

The results of the experiment are presented in Table 5.

From the data presented in Table 5, it can be seen that 0.1%, 1% and 10% solutions of the bacterial suspension of Bacillus subtilis Ch-13 with an initial bacterial titer of 1.0x10 ⁹ CFU / ml had a significant effect on the length of corn shoots, increasing their by 26-36.8% compared to control, and also had a greater stimulating effect on the length of corn shoots compared to the influence of the prototype Bacillus subtilis 8A strain (an increase of 13.3-23.5%). The results of the experiments showed that 0.1-10% solutions of the bacterial suspension of Bacillus subtilis Ch-13 significantly affected the length of corn roots, the length of which increased by 17.7-33.2% compared to the control, which was greater than the effect of bacterial suspensions of the prototype strain Bacillus subtilis 8A (an increase of 12.1-16.6%).

Thus, the data of Table 5 confirm, using the example of corn variety "Dobrynya", a more effective growth-stimulating effect of the Bacillus subtilis strain Ch-13 in comparison with the prototype Bacillus subtilis 8A strain.

Field experiments on the effect of the microbiological preparation Extrasol containing the bacterial strain Bacillus subtilis Ch-13 on the yield of spring barley, winter wheat, sunflower, peas and potatoes were carried out.

Field production experience to study the comparative effect of the microbiological fertilizer Extrasol based on the Bacillus subtilis Ch-13 strain with a standard seed dressing agent Vial Trust was carried out in the field of OJSC Agroindustrial Association Elekom, Romodanovskiy district of the Republic of Mordovia. The culture under study is spring barley, variety "Zazersky 85", a valuable brewing variety of Belarusian selection.

The experiment was carried out according to the following scheme: in the control - the use of the disinfectant Vial Trust, and in the experiment - in addition to the disinfectant, the barley seeds were treated with Extrasol at a dose of 0.25 l / t + spraying of barley crops with Extrasol at a dose of 1 l / ha.

The day before sowing, the barley seeds were treated with Extrasol according to the officially recommended standards. The seeds were treated in a shaded place with their full wetting with the working solution. The working solution was prepared on the day of treatment.

The treated seeds were dried in the shade and the next day sowing was carried out at a rate of 5 million seeds / ha and or 230 kg / ha in physical weight.

Spraying of barley crops with Extrasol was carried out at the recommended time in the evening in calm weather.

Fertilization with a general background was applied in spring for cultivation at a dose of N34, i.e. 0.1 t / ha of ammonium nitrate in physical weight.

The results of the experiment are presented in Table 6.

From the data presented in Table 6, it follows that the microbiological fertilizer Extrasol based on the Bacillus subtilis Ch-13 strain has an effect both on increasing the productivity of straw biomass and on increasing the yield of spring barley grain of the "Zazersky 85" variety. With the double application of Extrasol, the grain yield increased by 16.4%, and the straw biomass increased by 20.6%. A higher increase in the yield of spring barley was noted in the variant with double treatment of seeds and crops with microbiological fertilizer Extrasol - 0.48 t / ha.

Field production experiments to study the effectiveness of the Extrasol fertilizer based on the Bacillus subtilis strain Ch-13 on the yield of winter wheat varieties Rostovchanka-5, Vita, Prikumskaya 141, Ukrainka Odessa, Doka, Odessa 200, Irishka, Batko were carried out in farms located in various agricultural zones of the Stavropol Territory: extremely arid, arid and unstable moisture, where Extrasol treated seeds and plants of winter wheat during the growing season. A variant where Extrasol treatment was not carried out was used as a control.

The results of the experiments are presented in Table 7.

From the data presented in Table 7, it follows that the use of the microbiological fertilizer Extrasol based on the Bacillus subtilis strain Ch-13 for the treatment of seeds and plants of winter wheat during the growing season in arid agricultural zones of the Stavropol Territory and the city of Krasnodar has an effect on increasing the grain yield of winter wheat by 2 , 5-6.2 c / ha, or 5.4-23% compared to the control.

Field production experiments to study the effectiveness of the Extrasol fertilizer based on the Bacillus subtilis strain Ch-13 on the yield of peas of the Eifel, Multik, Madonna, Phoenix varieties and the PR63A90 hybrid and PR64A15 hybrid sunflower were carried out in farms located in various agricultural zones of the Stavropol Territory: unstable, arid moisture and sufficient moisture. A variant where Extrasol treatment was not used was used as a control. The results of the experiments are presented in Table 8.

From the data presented in Table 8, it follows that the use of the microbiological fertilizer Extrasol based on the Bacillus subtilis strain Ch-13 for the treatment of seeds and plants during the growing season of peas and sunflowers in various agricultural zones of the Stavropol Territory increased the grain yield of peas by 2-7 c / ha or per 7.1-33.3% in comparison with the control and the yield of sunflower grain by 2-3 centners / ha or by 10.8-14%.

Field experience of testing the microbiological preparation Extrasol based on the Bacillus subtilis Ch-13 strain was carried out on potatoes, the Udacha variety, at Bagaevsk-Agro LLC, Bagaevsky district, Rostov region. Tillage: after the predecessor, non-moldboard plowing to a depth of 33 cm, leveling; in the spring - cultivation, loosening. Irrigation: on average after 5-6 days (based on 4 m ³ / m ² per season). Application of mineral fertilizers: in autumn - azofoska 500 kg / ha; in spring - azofoska 100 kg / ha, ammonium nitrate 100 kg / ha; during the growing season, simultaneously with watering, foliar dressing with ammonium nitrate 100 kg / ha.

The use of Extrasol in the experimental version took place against the background of all the protective measures against diseases, pests and weeds, which were carried out by the farm in this field of potatoes.

Treatment of tubers: a disinfectant with fungicidal and insecticidal properties Prestige 1 l / t + humic fertilizer Lignohumate 0.2 kg / ha (Agronomist's Guide to protecting crops from pests, diseases and weeds for 2016. - Azov: AzovPechat LLC, 2016 , 260 p.) When planting to protect tubers from soil-dwelling diseases and pests.

Weed control: Hurricane Forte, BP (500 g / l glyphosate to-you) - 2.5-3 l / ha in the autumn after its predecessor, Titus CTC (250 g / kg rimsulfuron) twice: 30 g / ha 1 -th wave of weeds, 20 g / ha - on the 2nd wave.

Disease control: Ridomil Gold MC, VDG (640 g / kg mancozeb + 40 g / kg mefenoxam) 2.5 kg / ha spraying (three times): 1st prophylactic at a leaf height of 10-12 cm, then spraying Shirlan, SK (500 g / l of fluazinam) 0.3-0.4 l / ha, in the phase of closing the rows, interval of 7-10 days.

Pest control: Aktara, VDG (250 g / kg thiamethoxam) 60 g / ha, spraying in the inflorescence phase against larvae of the Colorado potato beetle I-IV instars - once.

Experiment scheme: Treatment of tubers in a potato planter when planting with a consumption rate of Extrasol 1 l / t (10% working solution). Spraying potatoes during the growing season in tank mixtures with fungicides with a consumption rate of Extrasol 2 l / ha (1% working solution): the first spraying in the budding phase - the beginning of flowering - (Shirlan 0.4 l / ha + Extrasol 2 l / ha); the second spraying before wilting (dying off) of the tops - (Ridomil Gold MC, VDG 2.5 kg / ha + Extrasol 2 l / ha);

Reference. Measures to protect potatoes from weeds, diseases and pests according to the scheme adopted in the farm.

The results of the experiment are presented in Table 9.

Thus, from the data presented in Table 9, it follows that the three-time use of the biological preparation Extrasol based on the Bacillus subtilis strain Ch-13 when growing potatoes (treatment of seed tubers and two treatments during the growing season during the period of active growth of the aerial part of plants) in combination with protective measures made it possible to increase the yield of the crop by 26 c / ha and obtain an increase in yield of 10% in comparison with the control option (the scheme of treatments adopted in the farm).

During visual observations, it was noted that shoots appeared 1-2 days earlier on the experimental plot with the use of Extrasol; flowering completed faster; the average height of the bushes was 5.6% less; there was no lodging of stems at the base of the bush as in the standard; tubers were formed by 27% less, but they were on average 42% larger than in the standard; the tops began to dry out 5 days later. On a good agricultural background, in combination with protective measures, the Extrasol preparation based on the Bacillus subtilis Ch-13 strain provided an increase in potato yield due to an increase in plant resistance to unfavorable weather conditions.

Below are examples of the manufacture of microbiological preparations based on the Bacillus subtilis strain Ch-13.

Example 1

A culture of bacteria 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 33 ± 1 ° C for 24-36 hours until a titer of at least 1 × 10 ⁹ cells / ml and a spore content of at least 90% are reached in the culture liquid. Then, a liquid form of a microbiological preparation is obtained in the following way: 20% of the culture liquid and 80% of water are mixed (the titer of the preparation is at least 1.5 × 108 cells / ml).

Example 2

A culture of bacteria Bacillus subtilis Ch-13 is grown in a sterile liquid nutrient medium of the following composition:

Molasses - 25 g / l,

corn extract - 12.5 g / l,

MgSO ₄ × 7H ₂ O - 0.25 g / l,

CaCl ₂ × 2H ₂ O - 1 g / l,

MnSO ₄ - 0.1 g / l,

FeSO ₄ - 0.0001 g / l,

CuSO ₄ - 0.0001 g / l

on a rocking chair or in a fermenter at 33 ° C for 24-36 hours until the culture liquid reaches a titer of at least 1 × 10 ⁹ cells / ml and a spore content of at least 90%. After that, a liquid form of a microbiological preparation is obtained by mixing 20% of the culture liquid and 80% of water (the titer of the preparation is not less than 1.5 × 10 ⁸ cells / ml).

Thus, as a result of the experiments, the ability of the bacterial strain Bacillus subtilis Ch-13 to stimulate the growth of agricultural crops due to the production of plant growth hormones on their roots - auxins and B vitamins was revealed and experimentally confirmed.

At the same time, on the basis of the bacterial strain Bacillus subtilis Ch-13, various biological preparations can be obtained, which makes it possible to expand the arsenal of existing similar means.

It is also important that when using the specified strain in the composition of microbiological preparations, the bacteria Bacillus subtilis Ch-13 form spores, which allows you to preserve these preparations without losing the viability of bacteria for a long time, not less than 12 months.

The number of viable cells and spores of the bacterial strain Bacillus subtilis Ch-13 for its effective use in the composition of various microbiological preparations should be 10 ⁴ -10 ⁹ cells per 1 ml of culture fluid. It has been experimentally established that the use of a strain at a concentration of less than 10 ⁴ cells / ml leads to a decrease in the effect of stimulating the growth of agricultural crops, and an increase in the concentration above 10 ¹⁰ cells / ml does not lead to an increase in the efficiency of their growth stimulation.

Claims (1)

The use of the bacterial strain Bacillus subtilis Ch-13, deposited at the All-Russian Research Institute of Agriculture under the number D-606, as a means to stimulate the growth of agricultural crops.