EA042519B1 - Bacillus mojavensis PS17 BACTERIAL STRAIN FOR INCREASING YIELD AND PROTECTING AGRICULTURAL PLANTS FROM PHYTOPATHOGENIC FUNGI - Google Patents

Description

The invention relates to the field of agricultural microbiology and biotechnology, in particular to the production of biological preparations for protecting plants from phytopathogenic fungi, and can be used to create biological fungicides to stimulate growth and increase crop yields and is a strain of the bacterium Bacillus mojavensis

PS17.

Many phytopathogenic fungi cause significant damage to the yield and quality characteristics of agricultural crops [1]. To protect plants from diseases caused by fungi, chemical and biological preparations - fungicides are used [2]. The use of chemical fungicides has a number of negative aspects associated with environmental problems and the development of resistance (resistance) to them in phytopathogens [3]. In this regard, effective biological protection of plants is of great importance [4]. To solve this problem, biological preparations based on various microorganisms are most often used. Bacteria of the genera Bacillus and Pseudomonas [5] are widely used as microorganisms of biological agents of biological products for protection against diseases, many of which are part of the natural microflora of plants, interacting with them mutually beneficially. Despite the fact that many microorganisms are able to stimulate plant growth and protect it from the effects of phytopathogenic organisms, from the point of view of ease of use, spore-forming bacteria of the genus Bacillus have an advantage, since their spores can be stored for a long time, which allows biological preparations based on them to maintain their activity for a long time and develop effective industrial technologies for their production [6]. A positive effect of strains of bacteria of the genus Bacillus on the growth and development of plants, a decrease in the development of diseases, and an increase in productivity was established [7]. In this regard, the search for effective strains of bacteria of the genus Bacillus is given close attention.

Known strains of bacteria of the genus Bacillus, which have the ability to suppress the growth of phytopathogenic fungi and stimulate the formation of crops. In particular, these are Bacillus subtilis VNIISKhM 128 and Fitosporin biopreparation created on the basis of this strain [8]; B. subtilis B 93 VIZR [9], B. subtilis BZR 517 [10], etc.

The disadvantage of all of the above strains is the lack of biological activity in relation to the suppression of phytopathogens and the insufficiently studied effect on crop yields.

One of the valuable sources for the search for biological agents for the creation of biological preparations for protection against diseases is endophytic bacteria [11].

Closest to the proposed invention is a strain of Bacillus mojavensis Lhv-97, which has fungicidal and bactericidal activities and isolated from rhizosphere soil [12].

The objective of the invention is to identify a strain suitable for obtaining a biological preparation with high activity in the suppression of phytopathogenic fungi and increasing crop yields, which allows expanding the range of biological plant protection against diseases.

The problem is solved by the fact that the selected strain of Bacillus mojavensis PS17, suitable for obtaining a biological product against phytopathogenic fungi and increasing crop yields.

The new strain was isolated from wheat seeds of the Sadokat variety (Republic of Tajikistan) and deposited at the National Bioresource Center All-Russian Collection of Industrial Microorganisms (BRC VKPM) National Research Center Kurchatov Institute - GosNIIgenetika, under the registration number VKPMV-13415.

The main selection criteria were the suppression of the growth of phytopathogenic fungi, a positive effect on plant productivity, the absence of pathogenicity to warm-blooded animals, and compatibility with other microorganisms. The species affiliation was determined using molecular genetic methods by the nucleotide sequence in 16S rRNA, as well as by amplification of a species-specific fragment characteristic of bacteria of the Bacillus mojavensis species at the National Research Center Kurchatov Institute - GosNIIgenetika.

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

The cells of the strain are Gram-positive aerobic spore-forming straight rods with rounded ends, 1.5-2.5x0.5-0.7 µm in size; arranged, as a rule, in pairs or singly, chains are less common. During sporulation, the cells do not swell, the spores are ellipsoid, located centrally.

On meat-peptone agar (MPA) after 2 days forms rounded colonies with a scalloped edge, 56 mm in diameter; with a predominantly granular surface, matte, opaque, color - white with greenish tints. The colonies have a convex base, raised folds, and a significantly raised central crater; have a viscous, viscous consistency, while they have an outer soft leathery layer, they do not grow into agar.

Bacillus mojavensis PS17 is an aerobic, chemoorganoheterotrophic bacterium that does not require

- 1 042519 in growth factors. It grows in the temperature range from 10 to 47°C with an optimal range of 28-32°C, at pH values from 4.5 to 8.5 with an optimum of 7.0-7.5, at a sodium chloride concentration of up to 5%.

Shows the activity of tryptophan deaminase and gelatinase. The activity of β-galactosidase (orthonitrophenyl-βD-galactopyranosidase), arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, and urease was not detected. Does not produce indole and hydrogen sulfide and restores nitrates. The Voges-Proskauer reaction (production of acetoin) is positive.

As a source of carbon and energy, it utilizes D-glucose, D-fructose, D-xylose, D-ribose, L-arabinose, D-mannose, D-mannitol, inositol, D-sorbitol, methyl-αD-mannopyranoside, methyl-aD-glucopyranoside, D -maltose, D-cellobiose, D-lactose, amygdalin, arbutin, esculin, salicin, D-melibiose, D-sucrose, D-trehalose, D-raffinose, gentibiose, glycerin, citrate.

Does not utilize erythritol, D-arabinose, D-adonitol, D-galactose, L-xylose, L-sorbose, L-rhamnose, methyl-βD-xylopyranoside, dulcitol, N-acetylglucosamine, inulin, D-melecytose, xylitol, D-turanose, D- lyxose, D-tagatose, D-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate.

The strain is stored at 4-6°C in test tubes with semi-liquid agar in a mineral medium (composition: potassium phosphate disubstituted 5.8 g/l, potassium phosphate monosubstituted 3 g/l, ammonium sulfate 1 g/l, glycerin 2 g/l with adding 0.7% agar, medium pH 7.0-7.2) under vaseline oil, which is poured into test tubes after 2 days of culture growth. Under such conditions, the shelf life of the strain without reseeding is at least 1 year.

The strain grows well on MPA, KingB, LB medium, wort agar (composition: unhopped beer wort concentrate, diluted with distilled water to a total sugar concentration of 10% (10° Balling), pH 7.0-7.2 with the addition of 2.0 % agar-agar), Gromyko's medium, Gauze's medium No. 2 (composition: tryptone - 2.5 g; peptone - 5.0 g; NaCl - 5.0 g; glucose - 10.0 g; agar-agar - 20 g ; tap water - 1000 ml; pH - 7.07.4) and a mineral medium (composition: potassium phosphate disubstituted 5.8 g / l, potassium phosphate monosubstituted 3 g / l, ammonium sulfate 1 g / l, glycerin 10 g / l with the addition of 0.7% agar, pH 7.0-7.2). Fermentation is carried out on a mixture of equal volumes of meat-peptone broth and 6°B unhopped beer wort (pH 6.9-7.2) at 30°C to 95% sporulation. The number of CFU is not less than 5x109 in 1 ml.

A study of the pathogenicity of the proposed strain for warm-blooded animals was carried out at Kazan Federal University, as a result of which it was concluded that, in terms of virulence, dissemination, toxicity and toxigenicity, the Bacillus mojavensis PS17 strain is not pathogenic for warm-blooded animals and meets the requirements for industrial microorganisms.

Example 1 Study of the antagonistic activity of the Bacillus mojavensis PS17 strain.

The study of the antagonistic activity of the Bacillus mojavensis PS17 strain was carried out using the confrontational method. A block with the mycelium of the test fungus was placed in the center of the Petri dishes with the KingB medium, a strain of Bacillus mojavensis PS17 was sown at a distance of 2-3 cm, a strain of Pseudomonas putida PCL1760 was used as a negative control, which was also sown by an injection that does not release any antagonistic substances that inhibit the development of micromycetes (Fig. 1-5). Petri dishes were placed for 48-72 hours in thermostats at a temperature of 25°C so that micromycetes and bacteria could develop. Observations were carried out on days 2-5, depending on the growth rate of the phytopathogen.

This method of determining antagonistic activity is considered to be qualitative, where the presence or absence of antagonistic properties is judged by the appearance of zones of inhibition of the growth of test cultures around the colonies of the tested microorganisms. This is due to the fact that with such a setting of the experiment, antagonism between microorganisms is realized due to the production of various antimicrobial metabolites by them. In turn, these metabolites diffuse into the nutrient medium; therefore, their accumulation at a particular point in the nutrient medium and, accordingly, the size of the area where antagonism manifestations are observed, depend on the volume of the medium, as well as on the quality and density of the agar.

The results of determining the antagonistic activity of the Bacillus mojavensis PS17 strain are shown in FIG. 1-5. Which presents as options: 1) Bacillus mojavensis PS17 strain, 2) Pseudomonas putida PCL1760. As a test culture of micromycetes used: Fig. 1 - Trichoderma asperellum RECB-74F, fig. 2 - Fusarium equisety, fig. 3 - Penicillum sp., fig. 4 - Fusarium sporotrichoides, fig. 5 - Fusarium oxysporum ZUM2407.

The results of the determination of antagonistic activity showed that all test cultures of micromycetes used in the study showed sensitivity to metabolites of the Bacillus mojavensis PS17 strain, which manifested itself as the appearance of growth inhibition zones of test cultures. According to the information provided, the Bacillus mojavensis PS17 strain is an antagonist and has a wide spectrum of activity.

Example 2. Laboratory tests of phytotoxicity and growth-stimulating activity of the Bacillus mojavensis PS17 strain.

To test the phytotoxicity and growth-promoting properties of the Bacillus mojavensis PS17 strain

- 2 042519 wheat grains of the Simbirpit variety were used. For this, the grains were soaked in a cell suspension grown on a rich LB medium, which consists of 10 g/l of bactotrypton, 5 g/l of yeast extract and 10 g/l of sodium chloride. The prepared medium was sterilized for 40 minutes at 110°С; after soaking, the seeds were placed in a cuvette on filter paper moistened with tap water. The seeds were germinated for 7 days at a temperature of 23°C with a day length of 16 hours. Plants were harvested and root length was measured. According to the results of the experiments, it was shown that the Bacillus mojavensis PS17 strain increased the length of wheat stems and roots (Table 1).

Table 1

Influence of Bacillus mojavensis PS17 on the biometric parameters of wheat plants cv. Simbirtsit

Option Experiment 1 Experiment 2 wheat germ Control 142.9±2.9 141.6±3.6 Bacillus mojavensis PS 17 146.5+14.9 151.6+6.3 wheat roots Control 100.9±5.8 102.5±2.4 Bacillus mojavensis PS 17 127.5±7.7 126.0±8.9

Example 3 Compatibility with beneficial microorganisms.

The compatibility of Bacillus mojavensis PS17 strain with useful bacteria Pseudomonas fluorescens RECB-14B, Streptomyces resistomycificus RECB-31B, Pseudomonas fluorescens RECB-44-B, Bacillus mojavensis RECB-50B, Trichoderma asperellum RECB-74F on rich and minimal media was carried out. As a result of confrontation tests, it was shown that from the indicated list of beneficial microorganisms, the strain Bacillus mojavensis PS17 inhibits the growth of the strain Trichoderma asperellum RECB-74B, Streptomyces resistomycificus RECB-31B and Bacillus amyloliquifaciens RECB-95B. At the same time, the growth of Bacillus mojavensis PS17 was suppressed by Streptomyces resistomycificus RECB-31B and Bacillus amyloliquifaciens RECB-95B. The absence of growth suppression of Pseudomonas fluorescens RECB-14B and Pseudomonas fluorescens RECB44B strains by Bacillus mojavensis PS17 and vice versa, the absence of growth suppression of Bacillus mojavensis PS17 by Pseudomonas fluorescens RECB-14B and RECB44B strains indicates the possibility of using this microorganism with other Pseudomonas-based biological preparations.

Example 4. Evaluation of the effectiveness of treatment of spring wheat plants with Bacillus mojavensis PS17 strain in the field.

The research was carried out on the experimental fields of the Kazan State Agrarian University. The object of the study was the spring wheat variety Ekada 66. The effectiveness of spraying plants during the growing season was studied. The experimental option was a biological product based on Bacillus mojavensis PS17 with a rate of 1.0 l/ha. The production of the biological product was carried out on a laboratory shaker Innova 40R (Eppendorf) at a temperature of 37°C, the incubation time was 17 hours with a rotation of 180 rpm. The medium for obtaining preparations is a liquid nutrient medium LB, Luria broth. The titer is not less than 2.5x10 ⁹ CFU / l. The untreated variant served as the control. The standard was the registered biological preparation Rizoplan based on Pseudomonas fluorescens strain AP-33 with a consumption rate of 1 l/ha. Agrotechnology of cultivation of spring wheat is generally accepted for the zone of the Pre-Kama region of the Republic of Tatarstan (Russia). Consumption of working fluid - 200 l/ha. The treatment was carried out twice - in the heading phase and in the flowering phase of wheat.

The use of various treatment options had an impact on the development of leaf mycoses (Table 2) and the incidence of ears (Table 3) with diseases.

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table 2

The development of leaf diseases and the biological effectiveness of their control when using bioagents on spring wheat (14 days after treatment),%, 2018

Option Development of the disease, % Biological efficiency, % leaf septoria true powdery mildew septori- oz leaf- ev true powdery mildew Control 13.0 2.5 Rizoplan 15.1 1.0 0 60.0 Bacillus mojavensis PS 17 10.0 0.5 23.1 80.0

In terms of foliar disease control, the use of Bacillus mojavensis PS17 had a more pronounced effect in reducing the development of leaf septoria and powdery mildew compared to the standard biofungicide Rizoplan.

Table 3

The prevalence of ear diseases and the biological effectiveness of their control when using bioagents on spring wheat in the phase of wax ripeness, %, 2018

Option Development of the disease, % Biological efficiency, %8 black ears Septoria black ear septoria Control 2.0 5.1 Rizoplan 1.5 2.1 25.0 58.8 Bacillus mojavensis PS 17 1.1 1.8 45.0 64.7

From the point of view of the control of ear diseases, the preparation based on Bacillus mojavensis PS17 significantly outperformed the standard biofungicide Rizoplan in terms of the effectiveness of mycosis control.

The treatment of plants with Bacillus mojavensis PS17 significantly increased the yield of spring wheat by 0.53 t/ha, which significantly exceeds the results obtained using the standard biofungicide Rizoplan (Table 4).

Table 4

Yield of spring wheat variety Ekada 66, t/ha, 2018

Option Productivity, t/ha Yield increase t/ha % Control 2.48 Rizoplan 2.57 0.09 3.8 Bacillus mojavensis PS 17 3.01 0.53 21.3 NSR05 0.08

Thus, the application twice during the growing season of a culture of endophytic bacteria Bacillus mojavensis PS17 at a rate of 1 l/ha has a pronounced effect on reducing the damage to spring wheat plants by major diseases, and leads to an increase in yield.

Example 5 Evaluation of the effectiveness of preplant treatment of potato tubers with Bacillus mojavensis PS17 strain in the field.

The object of the study is potatoes of the Veneta variety, a superelite reproduction. Options for processing tubers before planting were studied. Scheme of experience: 1. Control - without processing. 2. Standard chemical preparation - 290 ks (imidacloprid + pencicuron) Prestige (0.7 l/t). 3. Biological preparation - Rizoplan based on Pseudomonas fluorescens AP-33 (1 l/t). 4. Biological preparation based on the endophytic bacterium Bacillus mojavensis PS 17 (1.0 l/t). The repetition in the experiments is fourfold. The consumption rate of the working fluid is 20 l/t. The tubers were processed immediately before planting.

The results of evaluating the effect of treatment on the incidence of rhizoctoniosis sprouts are presented in table. 6.

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Table 6

The defeat of potato sprouts by rhizoctoniosis and the biological effectiveness of control when applying the treatment of tubers,%, 2018

Option Accounting indicator Lezni Biological efficiency, % R* R** R R Control 27.4 6.4 Prestige 5.1 0.6 81.4 90.6 Rizoplan 12.1 2.4 55.8 62.5 Bacillus mojavensis PS 17 5.5 0.7 79.9 89.1

Note: * P - prevalence of the disease, %; ** R - development of the disease, %. In terms of the effectiveness of rhizoctoniosis control, the preparation based on Bacillus mojavensis PS17 was superior to the standard biofungicide Rizoplan and slightly inferior to the indicators for the chemical preparation - Prestige.

The highest yield (Table 7) was obtained with the use of a preparation based on Bacillus mojavensis PS17. If, compared with the chemical standard, there were no significant differences in yield in this variant, then in comparison with the standard biofungicide Rizoplan, the increase in total yield was 3.1 t/ha.

Table 7

Potato yield of the Veneta variety when tubers are treated, t/ha, 2018

Option Productivity, t/ha Control Gain t/ha % Control 15.5 Prestige 23.3 7.8 50.2 Rizoplan 20.6 5.1 33.0 Bacillus mojavensis PS 17 23.7 8.2 53.0 NSR05 0.08

Thus, the bacterial strain Bacillus mojavensis PS17 has fungicidal activity against phytopathogenic fungi and increases crop yields, which will expand the range of biological plant protection against diseases.

List of used literature

1. Chumakov, A.E. Harmfulness of diseases of agricultural crops / A.E. Chumakov, T.I. Zakharov. - M.: Agropromizdat, 1990. - 127 p.

2. Shkalikov, V.A. Plant protection from diseases: a textbook / V.A. Shkalikov, O. O. Beloshapkina, D. D. Bukreev, and others; Ed. V.A. Shkalikova. M.: Colossus, 2010. - 404 p.

3. Tyuterev, S.L. Mechanisms of action of fungicides on phytopathogenic fungi / S. L. Tyuterev; Russian acad. s.-x. Sciences, All-Russian scientific, research. in-t plant protection. - St. Petersburg: Niva, 2010. - 170 p.

4. Shternshis, M.V. Biological plant protection: Textbook / M.V. Shternshis, I.V. Andreeva, O.G. Tomilova. - St. Petersburg: Publishing house "Lan", 2019.-332 p.

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5. Shternshis, M.V. Trends in the development of biotechnology of microbial plant protection products in Russia / M.V. Shternshis // Bulletin of the Tomsk State University. Biology. - 2012. - No. 2 (18). - S. 92-100.

6. Hamster, A.I. Optimal conditions for the cultivation of new bacteria of the genus Bacillus, promising for the development of biofungicides based on them / A.I. Khomyak, A.M. Asaturova, T.M. Sidorova // In the collection: Biological plant protection - the basis for the stabilization of agroecosystems. Materials of reports submitted to the 8th International Conference. - 2014. - S. 298301.

7. Asaturova A.M. Study of the effect of bacterization of seeds on the growth and development of winter wheat plants / A.M. Asaturova, V.D. Nadykta, V.Ya. Ismailov and others // Polythematic network electronic scientific journal of the Kuban State Agrarian University. - 2013. - No. 85. - S. 43-56.

8. Patent No. 2099947 Russia, A01N63/00, C12N1/20, C12N1/20, C12R1:125. Phytosporin biological product for plant protection against diseases / Smirnov V.V., Sorokulova I.B., Berezhnitskaya T.G., Vanyants G.M., Menlikiev M.Ya., Nedorezkov V.D., Mineev M.I., Vakhitov V.A., Baiguzina F.A. - No. 96121980/13; Appl. 11/15/1996; Published 12/27/1997.

9. Patent No. 2538157 Russia, C12N 1/20 A01N 63/00 C12R 1/125. The bacterial strain Bacillus subtilis B 93 VIZR for the protection of potatoes from diseases during storage / Novikova I.I., Boikova I.V., Pavlyushin V.A., Zeiruk V.N. -No. 2013152716/10; Appl. 11/27/2013; Published 01/10/2015.

10. Patent No. 2552146 Russia, C12N 1/20 A01N 63/02 C12R 1/125. The bacterial strain Bacillus subtilis BZR 517 for obtaining a biological product against phytopathogenic fungi/ Asaturova A.M., Dubyaga V.M. - No. 2013151375/10; Appl. 11/20/2013; Published 06/10/2015.

11. Patent No. 2648163 Russia, C12N 1/20. Bacillus mojavensis Lhv-97 strain with fungicidal and bactericidal activity/ Dunaytsev I.A., Lev I.O., Klykova M.V., Zhigletsova S.K., Sosna I.M., Torgonina I.V., Varlamova T.A., Zaitseva S.D. -No. 2017128916; Appl. 08/15/2017; Published 03/22/2018.

12. Chebotar V.K., Zaplatkin A.N., Shcherbakov A.V., et al. Microbial preparations based on endophytic and rhizobacteria, which are promising for increasing the productivity and efficiency of using mineral fertilizers in spring barley (Hordeum vulgare L.) and vegetable crops // Agricultural biology. - 2016. - V. 51 - No. 3. - S. 335-342.

Claims (1)

CLAIM The bacterial strain Bacillus mojavensis PS17, which has fungicidal activity against phytopathogenic fungi and increases the yield of crops, allows you to expand the range of biological plant protection against diseases, deposited at the National Bioresource Center VKPM V-13415.