RU2800426C9 - Bacillus amyloliquefaciens p20 strain as an agent for combating potato rhizoctoniosis - Google Patents

Abstract

FIELD: biotechnology; agriculture.

SUBSTANCE: bacterial strain Bacillus amyloliquefaciens, which has the ability to suppress the development of rhizoctoniosis on potatoes due to the production of fungicidal substances in the nutrient medium during the cultivation of bacteria, was deposited at the All-Russian Research Institute of Agriculture under the registration number RCAM 05515.

EFFECT: extension of the range of agents for combating potato rhizoctoniosis.

1 cl, 5 tbl, 2 ex

Description

The invention relates to biotechnology and agriculture and represents the use of a new strainBacillus amyloliquefaciens P20 as a means to combat rhizoctonia on potatoes.

Known patent No. 2527291 for the invention “Fungicidal agent for treating potato tubers” with priority dated May 14, 2013, registered at Shchelkovo Agrokhim CJSC, MPK A01N 59/00, published on August 27, 2014.

The invention relates to agriculture and can be used when storing potato tubers for storage, as well as when processing them before planting to combat phytopathogens that cause various diseases. A fungicidal agent for treating potato tubers during storage and before planting contains benzoic acid or its amine salts (monoethanolamine, diethanolamine, triethanolamine, N,N-dimethylethanolamine and N,N-diethylethanolamine) as an active ingredient at a consumption rate of 0.030-0.6 kg d.v./t. The present invention is an effective and affordable means for treating potato tubers when storing and treating them during planting in the absence of a phytocidal effect.

Known patent No. 2761117 for the invention “Technology for the production of a liquid form of a combined biological product based on Bacillus subtilis and Bacillus megaterium var. phosphaticum" with priority dated 12/09/2020, registered at ECOS LLC, IPC A01N 63/22, C12N 1/20, C12R 1/11, C12R 1/125, published 12/06/2021.

The technology for the production of a liquid combined biological product based on strains of bacteria of the genus Bacillus is described, including separate, simultaneous cultivation of bacterial strains Bacillus subtilis VKPM B-13494 and Bacillus megaterium var. Phosphaticum. strain VKPM B-13497 with continuous aeration and stirring with a stirrer speed of 250-300 rpm in a liquid nutrient medium containing mannitol, enzymatic peptone, magnesium sulfate, potassium phosphate, sodium chloride and water in specified quantities and mixed in a ratio of 60:40 to 40:60. In this case, the medium is inoculated with a mother culture in the exponential growth phase with a concentration of (2-3)⋅10 ⁹ CFU/ml and cultivation is carried out until a stable level of cell titer in the bacterial suspension is established (2-3)⋅10 ⁹ CFU/ml. Then half of the contents are drained and disposed of from each fermenter, after which the volume of liquid in the fermenters is brought to the original level with a concentrated nutrient medium, mannitol, enzymatic peptone, magnesium sulfate, potassium phosphate, sodium chloride and water with a high concentration. First, it is cultivated until the titer is reached (5-10)⋅10 ⁹ CFU/ml. Then half the contents of each fermenter are poured into a storage tank, and the remaining contents of the fermenters are brought to the original volume with a concentrated nutrient medium and the cultivation cycle is repeated. At the end of the cultivation cycle, the resulting contents of the storage tank and fermenters are mixed together and poured into containers as a finished product for its intended use. The shelf life of the drug has been increased to 2 years, its adhesion to the treated surfaces and the uniformity of treatment of the leaf surface of plants have been improved.

Known patent No. 2538157 for the invention “Bacteria strain Bacillus subtilis B 93 VIZR for protecting potatoes from diseases during storage” with priority dated November 27, 2013, registered at the State Scientific Institution All-Russian Research Institute of Plant Protection of the Russian Academy of Agricultural Sciences, IPC C12N 1/20, A01N 63 /00, C12R 1/125, published 01/10/2015

The Bacillus subtilis strain - I5-12/23, deposited in the State Collection of Microorganisms of the State Scientific Institution VIZR under registration number Bacillus subtilis 93 VIZR, has antagonistic activity against phytopathogenic fungi and bacteria. The strain can be used to protect potatoes from diseases during storage. The invention improves the safety of tubers.

Known patent No. 2704859 for the invention “A strain of the fungus Beauveria bas-siana, used for the production of a biological product against the Colorado potato beetle, fungal pathogens and stimulation of potato growth during the growing season, a biological product based on it and a method for stimulating potato growth during the growing season, protecting it from the Colorado potato beetle and rhizoctoniosis" with priority dated 07/16/2019, registered to Lelyak A.I., IPC A01N 63/00, C12N 1/14, published 10/31/2019.

The invention is a strain of the entomopathogenic fungus Beauveria bassiana IC 1530-25-1, a method for pre-planting treatment of potato tubers with a biological product and a biological product. When pre-planting potato tubers, a 1-3% aqueous solution of spore-mycelial biomass of the entomopathogenic fungus strain Beauveria bassiana IC 1530-25-1 and nutrient medium components with a titer of at least 10 ⁵ CFU/ml is used as a liquid form of the biological product. Treatment of potato tubers with the specified aqueous solution of the biological product is carried out once with a consumption of the aqueous solution of the biological product at 10 liters per 1 ton of potatoes. When spraying potatoes during the growing season with an aqueous solution of a biological product, the spore-mycelial biomass of the entomopathogenic fungus strain Beauveria bassiana IC 1530-25-1 and components of the nutrient medium with a titer of at least 10 ⁶ CFU/ml and a consumption of an aqueous solution of a biological product of 300 liters per 1 ha are used. potato planting area. The invention allows to protect potatoes from the Colorado potato beetle, the fungal pathogen Thanatephorus cucumeris (imperfect stage of the fungus - Rhizoctonia solani), which causes rhizoctonia blight or black potato scab, and also stimulates potato growth during the growing season.

Known patent No. 2428008 for the invention “Method of combating blackleg, rhizoctonia, wrinkled and common mosaic of potatoes” with priority dated January 27, 2010, registered at the State Scientific Institution Yakutsk Research Institute of Agricultural Sciences of the Russian Agricultural Academy, IPC A01N 63/00, published on September 10, 2011 G.

The method involves pre-sowing treatment of potato tubers with a microbial preparation, which is a suspension of an equal combination of bacterial strains Bacillus subtilis TNP-3 and Bacillus subtilis TNP-5 containing 1×10 ⁹ CFU/ml in tap water (pH 7.8) for 30 minutes at the rate of 300 ml/kg of tubers. The invention allows to increase potato yields.

Known patent No. 2140138 for a group of inventions “Method of pre-sowing treatment of vegetable seeds and method of obtaining a preparation for pre-sowing treatment of vegetable seeds” with priority 11/13/98.

These methods are carried out using a biofungicidal preparation containing the bacterial strain Bacillus subtilis Ch-13 (deposited under registration number VNIISHM D-606), which has a pronounced antibiotic effect against all major phytopathogenic fungi: Fusarium, Rhizoctonia, Verticillium, living in the soil or on the surface seeds In this case, the bacteria form spores, which allows them to be preserved, the drug based on them, and the seeds treated with this drug without loss of bacterial viability for up to 6 months.

EFFECT: inventions make it possible to increase the efficiency of protecting vegetable crops from phytopathogenic fungi by pre-sowing seed treatment.

Known patent No. 2259397 for the invention “A product for protecting grain crops, sunflowers, grapes from phytopathogenic microorganisms, and vegetable crops from phytopathogenic bacteria” according to application No. 2003110469 with pr. 04/02/2003, published 08/17/2005, IPC: C 12 N 1/20 (C 12 N 1/20, C 12 R 1/25), A 23 K 3/00 (prototype).

In the previously known bacterial strain Bacillus subtilis Ch-13, antagonistic properties towards pathogens of agricultural crops were identified and experimentally established. In particular, for potatoes, the effectiveness of the strain against late blight ( Phytophtora infestans ), rhizoctonia ( Rhizoctonia solani ), and fusarium wilt ( Fusarium oxysporum ) has been confirmed. It has also been experimentally shown that the use of the bacterial strain Bacillus subtilis Ch-13, a producer of the biological product Extrasol, leads to an increase in the yield of grain crops and potatoes by an average of 30%.

The objective of the invention is to identify a new strain suitable for use in agriculture as a means to combat rhizoctonia on potatoes, and also to expand the arsenal of such means.

This problem is solved due to the fact that a new strain of bacteria Bacillus amyloliquefaciens P20 is used as a means to combat rhizoctonia on potatoes.

The strain of endophytic spore bacteria “ Bacillus amyloliquefaciens P20” was isolated from the internal tissues of potatoes ( Solanum tuberosum L.), variety “Sudarynya”, Leningrad region and deposited on April 7, 2022 in the Network Bioresource Collection in the Field of Genetic Technologies for Agriculture (RCAM) in FGBNU VNIISHM under registration number RCAM 05515. A certificate of deposit of the strain is attached.

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

The cells have the shape of regular rods with rounded ends and a monopolar peritrichous arrangement of flagella. Cell size 1.3 x 2.6 µm. The strain forms spores located in the center of the cell and stains positively for Gram.

On nutrient agar it forms wrinkled colonies of white color, stretchy consistency with uneven, jagged edges. The diameter of the colonies is 12-15 mm. The optimal temperature for growth is 36 ⁰ C. At temperatures more than 45 ⁰ C and less than 15 ⁰ C, growth is slow. The optimal pH value of the medium is 7.3; growth also occurs at a pH from 4.5 to 8.0.

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

The strain uses mineral forms of nitrogen - ammonium salts and nitrates, amino acids and proteins.

The bacterium strain Bacillus amyloliquefaciens P20 is grown on an accessible nutrient medium consisting of molasses and corn extract, which is an advantage compared, for example, with the cultivation of pseudomonads, which requires scarce and expensive nutrient media.

In accordance with the “Guidelines of the USSR Ministry of Health” No. 2620-82 and No. 4263-87, it was experimentally determined that the bacterial strain Bacillus amyloliquefaciens P20 is non-virulent, non-toxic and non-toxigenic, and belongs to the 4th hazard class.

The bacterium strain Bacillus amyloliquefaciens P20 has been identified and experimentally confirmed to suppress the development of rhizoctonia on potatoes by producing fungicidal substances into the nutrient medium during bacterial cultivation.

It has been experimentally established that the strainBacillus amyloliquefaciensP20 has fungicidal effect against phytopathogenic fungiFusarium oxysporum, Fusarium graminearum, Fusarium sporotrichioidesAndRhizoctonia solani.

To determine the fungicidal activity of the bacterium strain Bacillus subtilis Ch-13 (producer of the biological fungicide BisolbiSan, registered against rhizoctonia in the Ministry of Agriculture of the Russian Federation) - the prototype and the proposed strain Bacillus amyloliquefaciens P20, the “well” method was used. The following phytopathogenic fungi were selected for testing: Fusarium oxy-sporum, Fusarium graminearum, Fusarium sporotrichioides and Rhizoctonia solani. A suspension of spores of one of the phytopathogenic fungi listed above (10 ⁵ CFU/ml) was added to potato dextrose agar (PDA, Sigma-Aldrich, USA) heated and cooled to a temperature of 40°C at the rate of 1 μl of suspension per 1 ml of medium. The resulting mixture with each mushroom was poured into a separate Petri dish. After the mixture hardened, 3 even through holes were made in it with a calcined plug drill: 100 μl of a bacterial suspension based on the prototype strain of Bacillus subtilis Ch-13 with a cell titer of 10 ⁸ CFU/ml was poured into one, a suspension based on the claimed Bacillus strain was poured into the other amyloliquefaciens P20 with the same cell titer, and distilled water was poured into the third hole (control). All dishes with “wells” were cultivated for 72-96 hours at 25 ⁰ C. The fungicidal activity of the strains was determined by the zone of suppression of fungal growth around the “wells”.

The measurement results are presented in Table 1.

Table 1. Zones of suppression of the growth of various phytopathogenic fungi

Experience option with
"wells" Diameter of fungal growth suppression zone, mm F.oxysporum F.graminearum F. sporotrichioides Rhizoctonia solani Control (water) 0 0 0 0 Bacillus subtilis Ch-13
(prototype) 11.3±0.7 15.9±1.1 7.1±0.5 5.7±0.5 Bacillus amylolique-
faciens P20 (new) 18.3±1.5 34.5±2.3 13.7±1.0 11.5±0.9

From the data given in Table 1 it is clear that the claimed bacterial strain Bacillus amyloliquefaciens P20 has a stronger fungicidal effect against the phytopathogenic fungi Fusarium oxysporum, Fusarium graminearum, Fusarium sporotrichioides and Rhizoctonia solani compared to the prototype strain Bacillus subtilis P-13 (fungicide against rhizoctoniosis and fusarium, registered by the Ministry of Agriculture of the Russian Federation), increasing the diameter of the fungal growth suppression zone by 5.8-18.6 mm.

To determine the fungicidal activity of the prototype strain Bacillus subtilis Ch-13 and the claimed strain Bacillus amyloliquefaciens P20 depending on the concentration of their suspensions, the “well” method was used. A suspension of spores of the phytopathogenic fungus Phytophthora infestans 1841 with a titer (10 ⁵ CFU/ml) was added to potato dextrose agar (PDA, Sigma-Aldrich, USA) heated and cooled to a temperature of 40°C at the rate of 1 μl of suspension per 1 ml of medium. The resulting mixture was poured into two Petri dishes. In each Petri dish, after the mixture had hardened, 3 through holes were made in it with a calcined cork drill. In one cup, 100 μl of a suspension based on the prototype strain of Bacillus subtilis Ch-13 with a titer of 10 ⁸ CFU/ml in varying degrees of dilution (100%, 10% and 0.5%) was poured into these holes, and in the other cup - 100 μl of suspension based on the claimed strain Bacillus amyloliquefaciens P20 with the same cell titer of 10 ⁸ CFU/ml and with the same options for the degree of dilution of the suspension (100%, 10% and 0.5%). The dishes with “wells” were cultivated for 72 hours at 28 ⁰ C.

The fungicidal activity of the prototype strain Bacillus subtilis Ch-13 and the claimed strain Bacillus amyloliquefaciens P20, depending on the concentration of their suspensions, was determined by the size of the growth inhibition zone of the phytopathogenic fungus Phytophthora infestans 1841 around “wells” with a bacterial suspension of the appropriate concentration.

The measurement results are presented in Table 2.

Table 2. Zone of suppression of growth of phytopathogenic fungusPhytophthora

infestans 1841 depending on the concentration of the suspension

different strains of bacteria (prototype and claimed)

Experience option with
"wells" Zone of inhibition of growth of phytopathogenic fungus, mm Undiluted suspension (100%) Suspension diluted 1:10 (10%) Suspension diluted 1:200 (0.5%) Bacillus subtilis Ch-13
(prototype) 11.3±1.1 11.5±1.0 11.5±0.7 Bacillus amylolique-
faciens P20 (new) 19.5±1.3 18.5±1.1 18.5±0.9

From the data given in Table 2 it is clear that in all variants of diluting the bacterial suspension, the declared bacterial strain Bacillus amylo-liquefaciens P20 inhibits the growth of the mycelium of the phytopathogenic fungus Phytophthora infestans 1841 significantly more effectively in comparison with the prototype strain Bacillus subtilis Ch-13, increasing the zone oppression by 7.0-8.2 mm.

Below are examples of the production of a microbiological preparation based on the Bacillus amyloliquefaciens P20 strain.

Example 1.

A culture of Bacillus amyloliquefaciens P20 bacteria was grown in a liquid sterile nutrient medium containing:

4% wheat bran and 0.3% chalk,

on a shaker or in a fermenter at 34±1°C for 30-42 hours until the titer in the culture liquid is at least 10 ⁹ cells/ml and the spore content is at least 95%. Then a liquid form of the microbiological preparation was obtained in the following way: 40% of the culture liquid and 60% of water were mixed (the titer of the preparation was at least 3⋅10 ⁸ cells/ml). The resulting liquid form was kept for 3-5 days at 20-25°C until a bacterial titer of at least 3⋅10 ⁸ CFU per 1 ml of the drug was obtained, after which the drug was ready for use. The liquid preparation was sterilely poured into polyethylene bottles or canisters with a capacity of 0.25; 0.5; 1.0 and 10.0 liters, which were pre-rinsed with alcohol.

Example 2.

A culture of Bacillus amyloliquefaciens P20 bacteria was grown in a liquid sterile nutrient medium of the following composition:

Molasses - 25 g/l,

corn extract - 12.5 g/l,

MgSO ₄ x7H ₂ O - 0.25 g/l,

CaCl ₂ x2 H 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 34±1°C for 24-42 hours until the titer in the culture liquid is at least 3⋅10 ⁹ cells/ml and the spore content is at least 95%. After that, a liquid form of the microbiological preparation was obtained by mixing 20% of the culture liquid and 80% of water (the titer of the preparation was at least 3·10 ⁸ cells/ml). Next, the liquid preparation, ready for use, was poured into plastic bottles or canisters with a capacity of 0.25; 0.5; 1.0 and 10.0 liters, which were pre-rinsed with alcohol.

Due to the relatively high biological activity of the strain and its high growth rate, the liquid preparation in production and in finished form is practically not contaminated by foreign microflora. However, in any variant of obtaining a biological product based on the bacterial strain Bacillus amyloliquefaciens P20, constant monitoring of all stages is required.

The degree of biological protection of potatoes from rhizoctoniosis was studied in field experiments in the Leningrad region, Volosovsky district, on the fields of AgroInter LLC, when growing seed potatoes of high reproduction varieties Charoit and Gussar. The experiments were carried out using the fungicide BisolbiSan (biological standard against rhizoctonia (STATE CATALOG OF PESTICIDES AND AGROCHEMICALS, approved for use in the Russian Federation, 2022) - a producer of the Bacillus subtilis Ch-13 strain and a preparation based on the declared Bacillus amyloliquefaciens P20 strain.

Field experiments with potatoes of the Charoit and Gusar varieties were carried out under the same conditions, using the following options:

1) in the control variant, no treatment was carried out;

2) chemical fungicides and fungicidal protectants were used (tuber treatment - Celeste Top, KS + Uniform, consumption 0.4 l/t + 1.2 l/t; vegetation treatment - Infinito, KS, consumption 1.2 l/ha; Rapid Duet, SP, flow rate 2.0 kg/ha; Mancozeb, SP, flow rate 1.5 kg/ha; Zummer, KS, flow rate 0.3 l/ha; Sukhovey, VR + Infinito, KS, flow rate 2.0 kg /ha + 1.2 l/ha);

3) they were treated with the microbiological fungicide BisolbiSan - the producer of the prototype strain Bacillus subtilis Ch-13 (tuber treatment - BisolbiSan, F + Cruiser, KS, consumption 15 l/t + 0.2 l/t; vegetation treatment - BisolbiSan, F , consumption 10.0 l/ha – 5 times per season);

4) they were treated with a drug based on the declared bacterial strain Bacillus amyloliquefaciens P20 (all types of treatment are the same as in experiment 3) when treated with a biological standard).

Phytopathological records of the results obtained during the budding period of potato tops were carried out according to generally accepted methods (Guidelines for registration testing of fungicides in agriculture / edited by V.I. Dolzhenko. - St. Petersburg, 2009. - 377 p.).

The results of experiments with potatoes of the Charoit and Gusar varieties are in Tables 3, 4.

Table 3. Results of accounting for the degree of damage to potatoes of the Charoit variety

rhizoctoniasis during budding

Experience Option Development of the disease, score Biological effectiveness,% Stems Stolons Stems Stolons Control without treatment 1.3 1.5 - - Application of chemical fungicides and fungicidal protectants 1.2 1.13 7.7 24.6 Processing BisolbiSan
(biological standard),
PC. Bacillus subtilis Ch-13 1.2 1.33 7.7 11.3 Treatment with the drug for
based on new pcs. Bacillus
amyloliquefaciens P20 1.06 1.3 18.4 13.3

Table 4. Results of accounting for the degree of damage to potatoes of the Gusar variety

rhizoctoniasis during budding

Experience Option Development of the disease, score Biological effectiveness,% Stems Stolons Stems Stolons Control without treatment 1.23 2.26 - - Application of fungicides and fungicidal disinfectants 1.03 2.1 16.3 7.1 Processing BisolbiSan
(biological standard),
pcs . Bacillus subtilis Ch-13 1.1 1.9 10.5 15.9 Treatment with a drug based on a new piece. Bacillus amyloliquefaciens P20 1.03 1.7 18.4 24.8

From the data presented in Tables 3 and 4 it is clear that the treatment of tubers and potato plants of the Charoit and Gusar varieties with a preparation based on the declared strain of Bacillus amyloliquefaciens P20 is more effective compared to the use of the biological standard BisolbiSan based on the prototype strain of Bacillus subtilis Ch-13 and comparable to the effectiveness of using chemical fungicides and disinfectants.

Influence of the declared strainBacillus amyloliquefaciens P20 on the yield of potatoes of the Charoit variety was studied during a field experiment in the Leningrad region, Volosovsky district, on the fields of AgroInter LLC. The soil is soddy-podzolic, medium loamy, moderately cultivated, with the following agrochemical indicators of the arable layer: pH_Sol.– 6.5, content of mobile phosphorus P₂ABOUT₅ - 11.4 mg/100 g, exchangeable K₂O - 11.2 mg/100 g of soil. Organic matter content – 3.7%. The predecessor is barley, potato crop rotation. The soil preparation system for the experimental plot included the main, or fall, and pre-planting treatments. After harvesting the previous crop in the fall, autumn plowing was carried out to the full depth of the arable layer (18-20 cm). In the spring, when the soil became physically ripe, the plowed soil was plowed to the same depth, and then cultivated with simultaneous harrowing. Mineral fertilizers in the form of azophosphate (NPK 16:16:16) were applied during planting at a rate of 300 kg/ha in physical weight (46 kg/ha a.i.); Kalimag (300 kg/ha in physical weight) was applied for cultivation; ammonium sulfate (200 kg/ha in physical weight) - for hilling. Planting care consisted of two pre-emergence and two inter-row care (loosening and hilling). Three weeks before harvesting, chemical desiccation was carried out with Golden Ring. Harvesting was carried out manually and with a Grimme DR 1500 potato harvester, after preliminary chemical desiccation of the tops with Golden Ring, BP at a rate of 2.0 l/ha.

Tubers were treated with chemical and biological disinfectants on a stationary basis in order to improve the quality of their coating. The dressing installation was manufactured according to an individual project by the Institute of Agroengineering and Environmental Problems of Agricultural Production and consists of a chamber with nozzles mounted on a potato conveyor-loader (branch of the Federal State Budgetary Institution "Federal Scientific Agroengineering Center VIM"). The working fluid consumption was 14 l/t of seeds. After processing, the potato tubers were placed in nets and taken out into the open air to dry.

The following options for processing potato tubers were used in the experiment:

1. Control - without treatment.

2. Chemical protectant Emesto Silver is a translaminar fungicide against a complex of soil-dwelling fungi that cause rhizoctonia, fusarium, silver scab and anthracnose.

3. Treatment with BisolbiSan (biostandard) based on the Bacillus subtilis strain Ch-13 and its metabolic products with a cell titer. producer 5 10 ⁸ CFU/ml

(according to the “State Catalog of Pesticides and Agrochemicals Permitted for Use on the Territory of the Russian Federation”, registered for the fight against rhizoctonia, late blight and alternaria on potatoes as a fungicide and protectant for planting material).

4. Treatment with a preparation based on the claimed strain of Bacillus amyloliquefaciens P20 and its metabolic products with a cell titer of 5⋅10 ⁸ CFU/ml.

All treatments of potato tubers of the Charoit variety were carried out with a chemical disinfectant and biological products in the same way in all variants of the experiment.

The experimental results are presented in Table 5.

Table 5. Potato yield of the Charoit variety, depending on treatment

Option Productivity, avg. values, t/ha Increased control T % Control - no processing 20.34 - - Chemical treatment
disinfectant (Emesto Silver) 17.66
-2.68 -13.7 Processing BisolbiSan
(biological standard),
PC. Bacillus subtilis Ch-13 21.7 +1.36 +6.7 Treatment with a preparation based on pcs. Bacillus amyloliquefaciens P20
(new) 23.66 +3.32 +16.3

From the data given in Table 5 it is clear that as a result of using the drug based on the declared strain of Bacillus amyloliquefaciens P20, the yield of potato tubers of the Charoit variety increased by an average of 3.32 tons or 16.3% compared to the control and by 1.96 tons or 9.0% compared to the biological standard BisolbiSan based on the prototype strain of Bacillus subtilis Ch-13, not to mention the yield indicators when treating tubers with a chemical disinfectant (Emesto Silver).

That is, the treatment of potato tubers of the Charoit variety with a preparation based on the Bacillus amyloliquefaciens P20 strain has a better effect not only on suppressing the development of rhizoctonia, but also on increasing its yield in comparison with the chemical disinfectant and biological standard BisolbiSan based on the prototype strain Bacillus subtilis Ch-13 .

Thus, as a result of the experiments, the ability of the bacterial strain Bacillus amyloliquefaciens P20 to inhibit the development of potato rhizoctonia and increase the yield of its tubers was identified and experimentally confirmed.

It is also important that when using this strain as part of microbiological preparations, the bacteria Bacillus amyloliquefaciens P20 form spores, which makes it possible to preserve preparations based on them without loss of bacterial viability for a long period, at least 12 months.

The research results allow us to conclude that the titer (number of viable cells and spores) of the bacterial strain Bacillus amyloliquefaciens P20 for its effective use in various microbial preparations should be 10 ⁵ -10 ⁹ cells/ml of culture fluid. Since it has been experimentally established that the use of a strain with a titer of less than 10 ⁵ cells/ml leads to a decrease in the effect of inhibiting potato rhizoctonia and stimulating the growth of potato tubers, and an increase in titer over 10 ⁹ cells/ml does not lead to an increase in the effectiveness of the protective effect against rhizoctonia and an increase in yield potatoes.

Thus, it was experimentally established that the new strain of endophytic spore bacteria Bacillus amyloliquefaciens P20, which has been identified and experimentally confirmed to suppress the development of rhizoctonia on potatoes by producing fungicidal substances in the nutrient medium when cultivating bacteria, is suitable and promising for use in agriculture as a means to combat potato rhizoctonia and allows us to expand the arsenal of such means.

Claims (1)

The bacterial strain Bacillus amyloliquefaciens P20, deposited in the Federal State Budgetary Institution All-Russian Research Institute of Agriculture under registration number RCAM 05515 as a means to combat potato rhizoctonia, as well as to expand the arsenal of such means.