RU2850691C1 - Bacterial composition for stimulating plant growth and stress resistance under conditions of short-term drought - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology. The following are proposed: a strain of Bacillus velezensis MGMM173 bacteria, deposited under registration number RCAM06912, for stimulating plant growth and protecting against phytopathogenic fungi; a bacterial composition for stimulating plant growth and stress resistance under conditions of short-term drought, consisting of the claimed strain and strains of bacteria Curtobacterium oceanosedimentum MGMM171, Stenotrophomonas rhizophila MGMM118, and Pseudomonas cedrina MGMM106, mixed in equal volume ratios with a titre of each bacterial strain of at least 1x10⁷ CFU/ml, and a method for increasing plant productivity under conditions of short-term drought, including the treatment of plant seeds with the declared bacterial composition in a specified amount.

EFFECT: expansion of the arsenal of means and methods capable of stimulating the growth of agricultural plants under conditions of short-term drought.

4 cl, 10 dwg, 6 tbl, 21 ex

Description

This group of inventions relates to agricultural microbiology and biotechnology and represents new means and methods for preventing the effects of drought on crops. The invention can be used in agricultural production and for personal use in growing plants on private farms.

Providing the population with agricultural products remains one of the most pressing global challenges. Due to climate change and the increasing prevalence of drought in regions, the need for products that can mitigate the impact of drought on plants is constantly growing.

In light of global environmental concerns, an equally important criterion for the selection of products is their safety for the environment and humans. Therefore, non-toxic compounds, materials, and microorganisms are promising ways to combat drought.

Microorganisms are increasingly being used to combat drought as an environmentally friendly alternative.

The patent (CN108004185B dated 08/05/2018) usedBacillus velezensisstrain E6 CGMCC No. 9665 as a bioprotective agent against phytopathogens, a drought stress regulator, and a growth stimulant. The bacteria, at concentrations of up to 10⁸CFU/ml inhibited the growth of phytopathogens, but showed a relatively limited reduction in the impact of drought on plants. Biometric parameters in the experimental plants were 22.5 ± 1.43 cm compared to the control (21.2 ± 1.46 cm), with fresh weight increasing by 0.07 g and dry weight by 0.005 g.

(US 20230332168 A1, dated 10.19.2023) describes a consortium consisting of Curtobacterium oceanosedimentum and Pantoea dispersa that improved resistance to sudden soybean death syndrome. Curtobacterium oceanosedimentum was shown to increase cotton root length and, in combination with Bacillus amyloliquefaciens and Pseudomonas oryzihabitans, increased germination.

CN 101497542 A from 07.06.2011 Pseudomonas cedrina UW3 and Pseudomonas putida UW4 together with chemical fertilizers increased plant biomass by 13% (stems) and 25% (roots) compared to the control (chemical fertilizers).

The endophytic bacteria Agrobacterium sp. FA13, Pantoea sp. FF34, Sphingobium sp. FC42, Pseudomonas sp. FB12, Enterobacter sp. FD17, Micrococcus sp. S2, Bacillus sp. S4, Pantoea ananatis S6, Actinobacter sp. S9 and Paenibacillus sp. S10 are known to increase plant biomass under drought and other abiotic stress conditions when combined with a formulation consisting of an agriculturally compatible carrier, a stickiness enhancer, a microbial stabilizer, a fungicide, an antibacterial agent, a herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide and a nutrient (US 9364005 B2, 10.01.2015). However, the patent specifies not only environmentally friendly bacteria, but also the plant pathogen Pantoea ananatis , which is a potentially dangerous phytopathogen that can cause losses in production (Coutinho TA, Venter SN Pantoea ananatis : an unconventional plant pathogen //Molecular plant pathology. - 2009. - Vol. 10. - No. 3. - Pp. 325-335).

The patent (RU 2666370 C1, September 7, 2018) uses a biopreparation that is highly active against various phytopathogenic fungi and has growth-promoting activity. The microorganisms include combinations of fungal and/or bacterial strains with different properties, providing a broad spectrum of activity: fungi of the genus Trichoderma , bacteria of the genus Bacillus and Pseudomonas - fungicidal/bactericidal properties; fungi of the genus Beauveria , bacteria of the genus Bacillus - insecticidal properties; fungi of the genus Glomus , bacteria of the genus Azospirillum , Agrobacterium and Bacillus - inoculants that improve plant mineral nutrition and stimulate their growth. The authors mention that the preparation has the ability to reduce the effects of drought on plants, but examples demonstrating these properties are not described.

The technical problem that the claimed group of inventions aims to solve is the expansion of the arsenal of means and methods capable of stimulating the growth of agricultural plants in conditions of short-term drought.

The technical result is the implementation of the specified purpose - new products that expand the range of means for regulating plant growth during short-term drought, due to the simultaneous presence of various enzymatic activities and the production of indole-3-acetic acid, as well as a high degree of colonizing properties together with antagonistic activity in relation to phytopathogens, as well as a new method that increases the adaptation of agricultural plants to changing conditions, which stimulates their growth, including in conditions of short-term drought.

The stated technical problem is solved, and the technical result is achieved by the claimed objects:

1. Bacillus velezensis MGMM173 strain of bacteria to stimulate plant growth and protect against phytopathogenic fungi;

2. Curtobacterium oceanosedimentum MGMM171 bacterial strain to stimulate plant growth;

3. Pseudomonas cedrina MGMM106 bacterial strain to stimulate plant growth;

4. a bacterial consortium consisting of the bacterial strains Curtobacterium oceanosedimentum MGMM171, Stenotrophomonas rhizophila MGMM118, Bacillus velezensis MGMM173, and Pseudomonas cedrina MGMM106, in equal volume ratios with a titer of each bacterial strain of at least 1×10 ⁷ CFU/ml;

5. a method for increasing plant productivity under short-term drought conditions, characterized by the fact that plant seeds are treated with a consortium of microorganisms consisting of bacterial strainsCurtobacterium oceanosedimentumMGMM171, Stenotrophomonas rhizophilaMGMM118, Bacillus velezensisMGMM173 AndPseudomonas cedrinaMGMM106, in equal volume ratios with a titer of each bacterial strain of at least 1×10⁷CFU/ml.

The inventions are carried out as follows.

The species affiliation of the strains is determined by determining the nucleotide sequence of the 16S rRNA gene fragment with subsequent comparison of nucleotide identity with the sequences deposited in the international GenBank database (Lane D. J. 16S/23 SrRNA sequencing. // Nucleicacid techniques in bacterial systematics. - 1991. - P. 115-175; Altschul, S. F. Basic local alignment search tool. / Altschul, S. F., W. Gish, W. Miller, E. W. Myers, D. J. Lipman // J Mol Biol. - 1990. - V. 215. - P. 403-410). The results of sequencing the variable regions of the genes encoding 16S rRNA are presented in Table 1.

The method for obtaining bacterial strains, species identification and determination of enzymatic activities is carried out according to the methods described in (RU2822893C1, 15.07.2024).

Bacillus velezensis MGMM173

The Bacillus velezensis MGMM173 strain has been deposited in the Network Bioresource Collection in the Field of Genetic Technologies for Agriculture (RCAM) of the All-Russian Research Institute of Agricultural Microbiology under registration number RCAM06912 (strain deposition certificate No. 246/10 dated October 30, 2024).

The strain was isolated from the rhizosphere of the Dokuchaevsky 190 SV maize variety (included in the State Register of Breeding Achievements Approved for Use in 2018). Samples were collected in the Republic of Tatarstan near the village of Bol'shiye Kabany, 55°62'13.62", 49°32'68.39".

According to the results of 16S gene sequencing, the Bacillus velezensis MGMM173 strain was 98.67% identical to the Bacillus velezensis FZB42 strain (Table 1).

The strain grows on LB, KB, and PDA media at 30°C. Bacillus velezensis MGMM173 grows as dirty-white, irregularly shaped, convex colonies with a fringed edge and fold formation at 30°C after 2 days on LB medium. Bacillus velezensis MGMM173 exhibits cellulolytic and proteolytic activity.

Bacillus velezensis MGMM173 is an aerobic, Gram-positive, endospore-forming motile bacterium. This rod-shaped bacterium measures 1.8-2.7 × 0.63-0.64 µm and is capable of metabolizing compounds such as adipate, arabinose, cellobiose, esculin, fructose, glucose, glycerol, glycogen, histidine, lactate, lactose, malate, maltose, mannitol, mannose, melibiose, raffinose, rhamnose, ribose, salicin, sorbitol, sucrose, trehalose, and xylose.

Recommended storage conditions and methods: at -80°C in a 30% glycerol solution in phosphate-buffered saline.

Pseudomonas cedrina MGMM106

The Pseudomonas cedrina strain MGMM106 has been deposited in the Network Bioresource Collection in the Field of Genetic Technologies for Agriculture (RCAM) of the Federal State Budgetary Scientific University

All-Russian Research Institute of Agricultural Microbiology under registration number RCAM06909 (strain deposition certificate No. 243/10 dated October 30, 2024).

Pseudomonas cedrina MGMM106 was isolated from the rhizosphere of the Yoldyz soft spring wheat variety (Russian Federation patent for breeding achievement No. 7665, published January 13, 2015). Samples were collected in the Republic of Tatarstan near the village of Bol'shiye Kabany, 55°62'13.62", latitude 49°32'68.39".

According to the results of 16S gene sequencing, the Pseudomonas cedrina MGMM106 strain was 99.86% identical to the Pseudomonas cedrina T0a85 strain (Table 1).

The strain grows on LB, KB, and PDA media at 30°C. Colonies of Pseudomonas cedrina MGMM106 grow as off-white, glossy, convex, round colonies with smooth edges at 30°C after 2 days on LB medium. Pseudomonas cedrina MGMM106 exhibits proteolytic, phytase, and lipase activity.

Pseudomonas cedrina MGMM106 is an aerobic, Gram-negative, motile bacterium. This rod-shaped bacterium measures 1-5 µm x 0.5-1.0 µm and is capable of metabolizing compounds such as arabinose, glucose, malate, and mannose.

Recommended storage conditions and methods: at -80°C in a 30% glycerol solution in phosphate-buffered saline.

Curtobacterium oceanosedimentum MGMM171

The Curtobacterium oceanosedimentum MGMM171 strain has been deposited in the Network Bioresource Collection in the Field of Genetic Technologies for Agriculture (RCAM) of the All-Russian Research Institute of Agricultural Microbiology under registration number RCAM06905 (strain deposition certificate No. 239/10 dated October 30, 2024).

Curtobacterium oceanosedimentum MGMM171 was isolated from seeds of the Universiade winter wheat variety (Russian Federation Patent for Breeding Achievement No. 9591, published April 5, 2018). Samples were collected in the Republic of Tatarstan near the village of Bol'shiye Kabany, 55°62'13.6", latitude 49°32'68.3".

According to the results of 16S gene sequencing, the Curtobacterium oceanosedimentum MGMM171 strain was 98.35% identical to the Curtobacterium oceanosedimentum 1257 strain (Table 1).

The strain grows on LB, KB, PDA media at 30°C. Colonies of the Curtobacterium oceanosedimentum MGMM171 strain grow as pinkish, smooth, slightly convex, glossy, round colonies with a smooth edge at 30°C after 2 days on LB medium. Strain

Curtobacterium oceanosedimentum MGMM171 has proteolytic and lipase enzymatic activity.

Curtobacterium oceanosedimentum MGMM171 is an aerobic, Gram-positive bacterium. This rod-shaped bacterium, measuring 0.6-3.0 × 0.3-0.5 µm, is capable of metabolizing compounds such as glucose and sucrose.

Recommended storage conditions and methods: at -80°C in a 30% glycerol solution in phosphate-buffered saline.

Stenotrophomonas rhizophila MGMM118

The Stenotrophomonas rhizophila strain MGMM118 has been deposited in the Network Bioresource Collection in the Field of Genetic Technologies for Agriculture (RCAM) of the All-Russian Research Institute of Agricultural Microbiology under registration number RCAM06905 (strain deposition certificate No. 310/11 dated November 28, 2023).

Stenotrophomonas rhizophila MGMM118 was isolated from the rhizosphere soil of the Universiade winter wheat variety (Russian Federation Patent for Breeding Achievement No. 9591, published April 5, 2018). Samples were collected in the Republic of Tatarstan near the village of Bol'shiye Kabany, 55°62'13.62", latitude 49°32'68.39".

According to the results of 16S gene sequencing, the Stenotrophomonas rhizophila strain MGMM118 was 98.61% identical to the Stenotrophomonas rhizophila strain D81_CV1R (Table 1).

The strain grows on LB, KB, and PDA media at 30°C. Colonies of Stenotrophomonas rhizophila strain MGMM118 grow as light-yellow, semi-matte, slightly convex, round colonies with smooth edges at 30°C after 2 days on KingB medium. Stenotrophomonas rhizophila strain MGMM118 exhibits amylase, cellulose, and proteolytic activity.

Stenotrophomonas rhizophila MGMM118 is an aerobic, gram-negative bacterium. These rod-shaped bacteria are 0.45–0.65 (0.92–1.55 µm) in size and are capable of metabolizing compounds such as glucose, fructose, lactose, maltose, and cellobiose.

Recommended storage conditions and methods: at -80°C in a 30% glycerol solution in phosphate-buffered saline.

The bacteria included in the consortium possess enzymatic activities important for growth and development, such as proteolytic, lipase, cellulase, amylase and phytase.

Consortium of microorganisms

The compatibility of the bacterial strains Curtobacterium oceanosedimentum MGMM171, Stenotrophomonas rhizophila MGMM118, Bacillus velezensis MGMM173 and Pseudomonas cedrina MGMM106, included in the declared consortium, is confirmed by a compatibility test (Xu X. M. et al. Combined use of biocontrol agents to manage plant diseases in theory and practice // Phytopathology. - 2011. - Vol. 101. - No. 9. - P. 1024-1031). For this purpose, the bacterial strains are plated on LB and KB media in lines perpendicular to each other and incubated at 30°C for 2 days. The incompatibility of the strains is judged by the growth inhibition zones at the intersections of the seeding lines. In Fig. 1. Compatibility tests of the specified strains Curtobacterium oceanosedimentum MGMM171, Stenotrophomonas rhizophila MGMM118, Bacillus velezensis MGMM173, and Pseudomonas cedrina MGMM106 in KB and LB media were demonstrated (Figs. 1A and 1B). The compatibility test did not reveal growth inhibition at the sites of their contact with each other, which indicates the compatibility of the claimed strains Curtobacterium oceanosedimentum MGMM171, Stenotrophomonas rhizophila MGMM118, Bacillus velezensis MGMM173, and Pseudomonas cedrina MGMM106, which allows them to be grown together and determines the effectiveness of the claimed consortium.

The consortium is obtained as follows. The above-mentioned strains Curtobacterium oceanosedimentum MGMM171, Stenotrophomonas rhizophila MGMM118, Bacillus velezensis MGMM173 and Pseudomonas cedrina MGMM106 are grown in liquid LB medium. The resulting bacterial suspension is applied through a series of dilutions to an agarized LB medium using a Drygalski spatula and incubated for 24 hours at 30°C. The concentration of each strain is adjusted to 10 ⁷ CFU/ml using liquid LB medium. The resulting bacterial suspensions of the strains are mixed in equal proportions. The claimed consortium is obtained with a titer of each bacterial strain of at least 1×10 ⁷ CFU/ml. The consortium of microorganisms has the appearance of a liquid bacterial culture. It is cloudy yellow in color and has a specific odor. The consortium of microorganisms is stored for 3 months in a 0.3% sodium chloride solution at a temperature of +3-5°C.

Determination of the antagonistic activity of Bacillus velezensis MGMM173, Pseudomonas cedrina MGMM106, Stenotrophomonas rhizophila MGMM118 and Curtobacterium oceanosedimentum MGMM171 strains against the phytopathogenic fungus Fusarium oxysporum was carried out according to the method (Fan, Ze-Yan, et al. ''Diversity, distribution, and antagonistic activities of rhizobacteria of Panax notoginseng.'' Journal of ginseng research - 2016, Vol. 40.2, Pp. 97-104). During testing of antagonistic properties, it was observed that the bacterial strains Bacillus velezensis MGMM173 and Pseudomonas cedrina MGMM106 inhibit the growth of the phytopathogen Fusarium oxysporum.

The growth-promoting properties of a bacterial consortium comprising Bacillus velezensis MGMM173, Pseudomonas cedrina MGMM106, Stenotrophomonas rhizophila MGMM118, and Curtobacterium oceanosedimentum MGMM171 were tested on Universiade winter wheat seeds and Svetlana KLP sunflower seeds (included in the State Register of Breeding Achievements Approved for Use in 2020). These crops were selected as belonging to the third group of agricultural crops based on drought tolerance (slightly arid zones)—winter wheat as a grain crop and sunflower as an oilseed. Winter wheat and sunflower seeds untreated with the microbial consortium served as controls. The biometric parameters of winter wheat plants, untreated and treated with the claimed microbial consortium, were compared. Similarly, the biometric parameters of sunflower plants, untreated and treated with the claimed microbial consortium, were compared. The results of determining the growth-promoting properties of the claimed microbial consortium are presented in Tables 2-3 and Figures 2-3 for winter wheat and sunflower, respectively.

Table 2. Parameters of biometric indicators of winter wheat plants after treatment with a consortium of microorganisms

Winter wheat Indicators Control Consortium Shoot length, cm 27.5±3.5* 32.5±2.05* Root length, cm 17.5±1.6* 18.8±1.8* Dry weight of shoots, g 0.052±0.006 0.06±0.001* Dry weight of roots, g 0.047±0.002* 0.067±0.003*

* - p<0.05, significantly significant difference

The biometric parameters of winter wheat plants treated with the claimed microbial consortium were higher than those in the control experiments: shoot length increased by 17% (from 27.5 cm in the control to 32.5 cm in the test), and root length by 7.5% (from 17.5 cm in the control to 18.8 cm in the test). Weight indicators also exceeded those in the test sample compared to the control: shoot weight increased by 31% (from 0.052 g in the control to 0.06 g in the test sample), and root weight by 43% (from 0.047 g in the control to 0.067 g in the test sample). The biometric parameters of sunflower plants treated with the proposed microbial consortium were higher than those in the control experiments: shoot length increased by 16% (from 10.9 cm in the control to 12.6 cm in the test sample), root length by 13% (from 7.5 cm in the control to 8.5 cm in the test sample). Shoot dry weight increased by 13.8% (from 0.063 g in the control to 0.068 g in the test sample), and root weight by 73% (from 0.02 g in the control to 0.04 g in the test sample).

Table 3. Parameters of biometric indicators of sunflower plants after treatment with a consortium of microorganisms

Sunflower Indicators Control Consortium Shoot length, cm 10.9±0.04* 12.6±1.05* Root length, cm 7.5±1.6* 8.5±1.6* Dry weight of shoots, g 0.063±0.001* 0.068±0.002* Dry weight of roots, g 0.02±0.003* 0.04±0.002*

* - p<0.05, significantly significant difference

The drought resistance of plants treated with the consortium was tested using Universiade winter wheat seeds and Svetlana KLP sunflower seeds. Winter wheat and sunflower seeds untreated with the proposed microbial consortium served as controls. The experimental and control plants were grown for two weeks in a closed climate chamber at a temperature of 25°C, 70% humidity, and a 16-hour daylight period (nighttime temperature of 20°C, humidity of 50%). After two weeks of cultivation in quartz sand, the plants were left without water for 14 days. The consortium-treated plants demonstrated high drought tolerance compared to the control plants. The plants were then carefully removed from the sand along with their root systems, and biometric parameters (root and shoot length) were measured. To measure dry matter, shoot and root samples are then dried in a dry-heat oven at 40°C for 3 days. The biometric parameters of untreated and treated winter wheat plants with the claimed microbial consortium are compared, as are the biometric parameters of untreated and treated sunflower plants with the claimed microbial consortium. The results of the comparison—determining the drought-tolerance properties of plants with the claimed microbial consortium—are presented in Tables 4-5 and as histograms in Figs. 4-5.

Table 4. Parameters of biometric indicators of winter wheat plants after treatment with a consortium of microorganisms and exposure to short-term drought

Winter wheat Indicators Control Consortium Shoot length, cm 28.9±2.9* 33.25±1.69* Root length, cm 43.39±2.1* 49.2±3.1* Dry weight of shoots, g 0.105±0.005* 0.112±0.002* Dry weight of roots, g 0.115±0.004* 0.193±0.003*

* - p<0.05, significantly significant difference

Table 5. Parameters of biometric indicators of sunflower plants after treatment with a consortium of microorganisms and exposure to short-term drought

Sunflower Indicators Control Consortium Shoot length, cm 10.5±1.5* 11.4±1.6* Root length, cm 10.1±1.1* 11.1±1.3* Dry weight of shoots, g 0.088±0.004* 0.103±0.005* Dry weight of roots, g 0.012±0.001* 0.017±0.001*

* - p<0.05, significantly significant difference

The biometric parameters of winter wheat plants treated with the claimed microbial consortium were higher than in the control experiments, particularly in shoot length by 16.7% (from 28.9 cm in the control to 33.25 cm in the experiment) and root length by 12.4% (from 43.39 cm in the control to 49.2 cm in the experimental sample). The weight indicators also exceeded those in the experimental sample compared to the control: shoot weight increased by 6.7% (from 0.105 g in the control to 0.112 g in the experimental sample), and root weight by 67.4% (from 0.115 g in the control to 0.193 g in the experimental sample). The biometric parameters of sunflower plants treated with the proposed microbial consortium were also higher than those in the control experiments, particularly shoot length by 8.2% (from 10.5 cm in the control to 11.4 cm in the test sample) and root length by 7.7% (from 10.1 cm in the control to 11.1 cm in the test sample). Shoot dry weight increased by 16.7% (from 0.088 g in the control to 0.103 g in the test sample), and root weight by 36.4% (from 0.012 g in the control to 0.017 g in the test sample).

Statistical analysis was performed using the OriginLab pro SR1 b9.5.1.195 statistical software package. Significant differences between groups were analyzed using one-way ANOVA and Tukey's post hoc test for significant differences at p<0.05.

Thus, the claimed bacterial consortium, consisting of Curtobacterium oceanosedimentum MGMM171, Stenotrophomonas rhizophila MGMM118, Bacillus velezensis MGMM173 and Pseudomonas cedrina MGMM106, MGMM106 in equal volume ratios with a titer of each bacterial strain of at least 1×10 ⁷ CFU/ml, is capable of stimulating plant growth both under normal conditions and in the case of temporary drought.

The claimed method for increasing plant productivity under short-term drought conditions consists of treating plant seeds with the claimed consortium - microorganisms Curtobacterium oceanosedimentum MGMM171, Stenotrophomonas rhizophila MGMM118, Bacillus velezensis MGMM173 and Pseudomonas cedrina MGMM106 in equal volume ratios (1:1:1:1) with a titer of each bacterial strain of at least 1×10 ⁷ CFU/ml. The treatment of plant seeds with the declared consortium of microorganisms is carried out in an amount of 1-5 liters of pure consortium, diluted in 10 liters of water, per 1 ton of seeds in accordance with the State Catalog of Pesticides and Agrochemicals as of October 2, 2024. URL: https://mcx.gov.ru/upload/iblock/e83/1oe7m7myxe0fyawn5kata6qna8mgeb21.zip (Accessed: November 15, 2024). To evenly distribute the consortium throughout the seed biomass, an auger seed treater is used.

The proposed method, which expands the range of methods for increasing crop productivity and survival in dry conditions, has a positive effect on plant biometric indicators, which are higher than in control experiments.

The following reagents and equipment are used in the implementation of inventions:

- dNTP, concentration 100 mM (Eurogen, Russian Federation);

- L-asparagine monohydrate, purity 99.0% (PanReac Applichem, Spain);

- L-tryptophan, 100% (PanReac Applichem, Spain);

- Taq polymerase (Eurogen, Russian Federation);

- Microbiological agar (Diam, Russian Federation);

- Agarose (Diam, Russian Federation);

- Buffer for Taq polymerase (Eurogen, Russia);

- Sodium hydroxide, 98-100.5% (PanReac Applichem, Spain);

- Sodium hypochloride, 5% (PanReac Applichem, Spain);

- Glycerin, 99-101% (PanReac Applichem, Spain);

- Glucose monohydrate, 98.5-100.5% (PanReac Applichem, Spain);

- Laboratory water distiller (distiller) Liston A 1104 (AlmaMed, Russian Federation);

- Sodium dodecyl sulfate (SDS), 85% (PanReac Applichem, Spain);

- Ferric ammonium alum 12-aqueous, for analytics ACS, ISO (PanReac Applichem, Spain);

- Isopropanol, 99.8% (PanReac Applichem, Spain);

- Isotonic solution (DPBS) (Gibco (Thermo Scientific), USA);

- Carboxymethylcellulase sodium salt, 98% (PanReac Applichem, Spain);

- Carboxymethyl cellulose, 99% (Look chemical, China);

- Calcium carbonate, 99-100% (PanReac Applichem, Spain);

- Congo red, for analytics (PanReac Applichem, Spain);

- Starch, analytical grade (LenReaktiv, Russian Federation);

- Sodium molybdate 2-aqueous, for analytics (PanReac Applichem, Spain);

- Nystatin, 85% (Sisco Research Laboratories, India);

- Ammonium nitrate, 99% (PanReacApplichem, Spain);

- Yeast peptone (Diam, Russian Federation);

- Lugol's solution 0.33% iodine (PanReac Applichem, Spain);

- Sucrose, 90% (Diam, Russian Federation);

- Sulfuric acid, 95% (PanReac Applichem, Spain);

- Hydrochloric acid, 37% (PanReac Applichem, Spain);

- Ammonium sulfate, for analytics (PanReac Applichem, Spain);

- Iron sulfate heptahydrate, 99.5% (PanReac Applichem, Spain);

- Magnesium sulfate heptahydrate, 99% (PanReac Applichem, Spain);

- Manganese sulfate 1-aqueous, for analytics (PanReac Applichem, Spain);

- Dry skim milk (Central Drug House, India);

- Twin 80, pharma grade (Diam, Russian Federation);

- Trypan blue, 0.4% (Sigma aldrich, USA);

- Tryptone casein (Diam, Russian Federation);

- Phenol, 99.4% (Component-Reactive, Russian Federation);

- Sodium phytate, 90% (Sigma aldrich, USA);

- Anhydrous disubstituted potassium phosphate, 98% (PanReac Applichem, Spain);

- Monopotassium phosphate, 98% (PanReac Applichem, Spain);

- Phosphate buffered saline (Diam, Russian Federation);

- Potassium chloride, 99% (PanReac Applichem, Spain);

- Calcium chloride dihydrate, 99% (PanReac Applichem, Spain);

- Sodium chloride, 99% (PanReac Applichem, Spain);

- Chloroform, 99.9% (Component-Reactive, Russian Federation);

- Ethanol, 95% (BIOMEDKHIM-PHARM, Russian Federation);

Equipment and materials:

- 6-well plate (NEST, People's Republic of China);

- _CO2- incubator S-Bt Smart Biotherm (Binder, Germany);

- Vortex MX-E (DLab, People's Republic of China);

- DNA amplifier Veriti (Thermo Scientific, USA);

- Mechanical single-channel dispensers Sartorius Proline with a volume of 100-1000 µl, 20-200 µl, 0.5-10 µl (Sartorius, Germany);

- Mechanical 8-channel dispensers Sartorius Proline with a volume of 5-50 µl, 10-100 µl (Sartorius, Germany);

- Goryaev chamber 4-grid, version 3 (Diam, Russian Federation);

- Culture HEK 293 (Human Embryonic Kidney 293);

- Culture plates (Corning, USA);

- Laminar flow cabinet BMB-II-Laminar-S-1.5 (class II type A2) (Neoteric lamsystems, Russian Federation);

- Stereo microscope SZM-45 (Biobase, People's Republic of China);

- Low-temperature freezer MNT-80 (Pharm-invest, Russian Federation);

- Gel Extraction Kit (Eurogen, Russian Federation);

- MilliporeSigma(Chemicon(MTT) cell growth assay kit (Sigma aldrich, USA);

- Tips for 1000 µl, 200 µl, 10 µl (Corning, USA);

- Optical microscope MF23 (MShot, Russian Federation);

- Microbiological loop Biologix, People's Republic of China);

- PCR plates (Nest, People's Republic of China);

- Eppendorf tubes with a volume of 1.5 ml (Sovtech, Russian Federation);

- Falcon type test tubes with a volume of 50 ml (Nest, People's Republic of China);

- Gel documentation system Gel Doc XR+ (Bio-rad, USA);

- Spectrophotometer Spectro StarNano (BMG Labtech, Germany);

- Microbiological alcohol lamp (Diam, Russian Federation);

- Steam sterilizer VKa-75 R PZ (AlmaMed, Russian Federation);

- Dry heat oven GP-10 (Kasimov Instrument Plant, Russian Federation);

- Laboratory dry-air thermostat TV-80 (JSC GRPZ branch of Kasimov Instrument Plant, Russian Federation);

- Filter paper (Diam, Russian Federation);

- Syringe filters 0.22 nm (Sartorius, Germany);

- Sterile Petri dishes d=90 mm (Perint, Russian Federation);

- Shaker-incubator ZQZY-78ES Zhichu (Zhichu, People's Republic of China);

- Microbiological spatula (Diam, Russian Federation);

- Sterile syringes 10 ml (Diam, Russian Federation);

- Electrophoresis horizontal chamber with power source (Bio-rad, USA).

The invention is illustrated by examples of specific implementation.

Example 1. Isolation of strains

Bacteria isolation method: 9 ml (in a 1:9 ratio) of sterile phosphate buffer (pH 7.0) are added to 1 g of sample and the mixture is vigorously shaken on a vortex for 1 minute. The wash is used to obtain serial dilutions up to 10 ^-4 , which are plated on Petri dishes on a non-selective King B medium (peptone - 10 g / l, glycerol - 10 g / l, microbiological agar - 18 g / l, magnesium sulfate - 12.5 mM, dipotassium phosphate - 8.6 mM) with the antibiotic nystatin (32 μg / ml). The cultures are incubated at a temperature of + 30 ° C for 24 hours. After incubation, bacterial colonies are selected from the dishes.

Example 1A. Bacillus velezensis MGMM173

Bacillus velezensis MGMM173 was isolated from the rhizosphere of the Dokuchaevsky 190 SV corn variety. The colony surface of Bacillus velezensis MGMM173 is opaque, beige, and folded.

Example 1B. Stenotrophomonas rhizophila MGMM118

Stenotrophomonas rhizophila MGMM118 was isolated from the rhizosphere of the Universiade winter wheat variety. The Stenotrophomonas rhizophilia MGMM118 bacterial strain appeared as light-yellow, semi-matte, slightly convex, round colonies with smooth edges.

Example 1B. Pseudomonas cedrina MGMM106

Pseudomonas cedrina MGMM106 is isolated from the rhizosphere of the Yoldyz spring wheat variety. Colonies of the Pseudomonas cedrina MGMM106 strain grow as dirty-white, glossy, convex, round colonies with smooth edges.

Example 1D. Curtobacterium oceanosedimentum MGMM171

Curtobacterium oceanosedimentum MGMM171 is isolated from the seeds of the Universiade winter wheat variety. The Curtobacterium oceanosedimentum MGMM171 strain grows as pinkish, smooth, slightly convex, glossy, round colonies with a smooth edge.

Example 2. Identification of strains

Identification method: genomic DNA of the strains is isolated from the biomass of the test strains using a standard method (Gautam, Akash. "Phenol-Chloroform DNA Isolation Method." DNA and RNA Isolation Techniques for Non-Experts. Cham: Springer International Publishing, - 2022. 33-39). Next, a fragment of the 16S rRNA gene is amplified using standard primers 27F (5'-gagtttgatcctggctcag-3') and 1492R (5'-taccttgttacgactt-3').

PCR program for amplification:

1) Initial DNA denaturation - 94°C, 4 min

2) Amplification - 35 cycles:

a) DNA denaturation - 95°C, 45 sec

b) annealing of primers - 55°C, 45 sec

c) DNA synthesis - 72°C, 2 min

3) Final elongation - 72°C, 3 min

4) Storage - 12°C.

The resulting PCR products are purified from agarose gel using the Gel Extraction Kit (Thermo Scientific) and subjected to Sanger sequencing (sequencing is performed by Eurogen).

The consensus sequence of the forward and reverse sequencing chromatographies was obtained using Clone Manager Professional 9.5 and uploaded to the NCBI database for the nucleotide blast set (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC). The results of identification using the 16S rRNA method are presented in Table 1.

Example 2A. Bacillus velezensis MGMM173

According to the results of 16S gene sequencing, the Bacillus velezensis MGMM173 strain was 98.67% identical to the Bacillus velezensis FZB42 strain.

Example 2B. Stenotrophomonas rhizophila MGMM118

According to the results of 16S gene sequencing, the Stenotrophomonas rhizophila strain MGMM118 was 98.61% identical to the Stenotrophomonas rhizophila strain D81_CV1R.

Example 2B. Pseudomonas cedrina MGMM106

According to the results of 16S gene sequencing, the Pseudomonas cedrina MGMM106 strain was 99.86% identical to the Pseudomonas cedrina T0a85 strain.

Example 2G. Curtobacterium oceanosedimentum MGMM171

According to the results of 16S gene sequencing, the Curtobacterium oceanosedimentum MGMM171 strain was 98.35% identical to the Curtobacterium oceanosedimentum 1257 strain.

Example 3. Determination of enzymatic activities

Cellulase

Cellulolytic activity of bacterial strains is identified by inoculating 5 μl of each strain onto a Petri dish with BM medium supplemented with sodium carboxymethylcellulose (5.8 g/l dibasic potassium phosphate, 3 g/l monobasic potassium phosphate, 1 g/l ammonium sulfate, 12.5 mM magnesium sulfate heptahydrate, 10 g/l sodium carboxymethylcellulose, 18 g/l agar). Dishes are incubated for 3 days at +30°C. Cellulolytic activity is identified by detecting yellow zones or clearing around the colony after staining with Congo red.

Protease

To determine proteolytic activity, 5 μl of bacterial strains are plated on a Petri dish with BM medium supplemented with milk protein (dipotassium phosphate - 5.8 g/l, monopotassium phosphate - 3 g/l, ammonium sulfate - 1 g/l, magnesium sulfate heptahydrate - 12.5 mM, dry skim milk - 10 g/l, agar - 18 g/l) and incubated for 24 hours at +30°C. Clear zones are observed around the colonies of strains exhibiting proteolytic activity.

Phytase

The phytase activity of the strains is determined on the phytase screening medium (PSM) (glucose - 20 g/l, sodium phytate - 5 g/l, ammonium nitrate - 5 g/l, magnesium sulfate heptahydrate - 0.5 g/l, potassium chloride - 0.5 g/l, ferrous sulfate heptahydrate - 0.01 g/l, manganese sulfate tetrahydrate - 1 g/l, agar - 18 g/l, calcium chloride - 2 g/l, pH adjusted to 6.3). For this, 5 μl of each strain are plated on a Petri dish with the medium and incubated for 1-2 days at +30°C. Strains showing activity form a clearing zone around the colonies.

Lipase

To determine the presence of extracellular lipases, 5 µl of the strains are plated on a medium supplemented with Tween 80 (polyoxyethylene sorbitan monooleate) as an analogue of high-molecular fatty acids (peptone - 10 g/l, sodium chloride - 5 g/l, calcium chloride dihydrate - 1 g/l, agar - 20 g/l, Twin 80 - 10 g/l). The dishes are incubated for 3 days at +30°C. Extracellular lipase activity in bacteria is detected by the presence of an opaque zone of calcium salts of fatty acids around the colonies.

Example 3A. Bacillus velezensis MGMM173

Bacillus velezensis MGMM173 exhibits cellulolytic and proteolytic activity. Figure 6 shows the results of cellulolytic activity testing. After staining with Congo red, a yellow zone formed around Bacillus velezensis MGMM173, confirming cellulolytic activity. Figure 7 shows the results of proteolytic activity testing. After incubating the plates at 30°C for 24 hours, a clear zone formed around Bacillus velezensis MGMM173, confirming proteolytic activity.

Example 3B. Stenotrophomonas rhizophila MGMM118

Stenotrophomonas rhizophila strain MGMM118 exhibits cellulolytic and proteolytic activity. Figure 6 shows the results of cellulolytic activity testing. After staining with Congo red, a yellow zone formed around the Stenotrophomonas rhizophila strain MGMM118, confirming cellulolytic activity. Figure 7 shows the results of proteolytic activity testing. After incubating the plates at 30°C for 24 hours, a clear zone formed around the Stenotrophomonas rhizophila strain MGMM118, confirming proteolytic activity.

Example 3 B. Pseudomonas cedrina MGMM106

The Pseudomonas cedrina MGMM106 strain has proteolytic, phytase, and lipase activity. Fig. 7 shows the results of proteolytic activity testing. After incubating the plates at +30°C for 24 hours, a clear zone formed around the Pseudomonas cedrina MGMM106 strain, confirming the presence of proteolytic activity. Fig. 8 shows the results of phytase activity testing. After incubating the plates at +30°C for 48 hours, a clear zone formed around the Pseudomonas cedrina MGMM106 strain, confirming the presence of phytase activity. Fig. 9 shows the results of lipase activity testing. After incubating the plates at +30°C for 48 hours, an opaque zone formed around the Pseudomonas cedrina MGMM106 strain, confirming the presence of lipase activity.

Example 3G. Curtobacterium oceanosedimentum MGMM171

The Curtobacterium oceanosedimentum MGMM171 strain exhibits proteolytic and lipase enzymatic activity. Figure 7 shows the results of proteolytic activity testing. After incubating the plates at 30°C for 24 hours, a clear zone formed around the Curtobacterium oceanosedimentum MGMM171 strain, confirming its proteolytic activity. Figure 9 shows the results of lipase activity testing. After incubating the plates at 30°C for 48 hours, an opaque zone formed around the Curtobacterium oceanosedimentum MGMM171 strain, confirming its lipase activity.

Example 4. Determination of biocompatibility of strains.

Strain compatibility is tested as follows: bacterial strains are plated on LB, King B, and PDA media in perpendicular lines and incubated at 30°C for 2 days. Incompatibility is assessed by zones of growth inhibition at the intersection of the inoculation lines. Based on the test results, Bacillus velezensis MGMM173, Pseudomonas cedrina MGMM106, Stenotrophomonas rhizophila MGMM118, and Curtobacterium oceanosedimentum MGMM171 are compatible (Fig. 1).

Example 5. Determination of antagonistic activity of strains against the phytopathogenic fungus Fusarium oxysporum.

The antagonistic activity of the strains Bacillus velezensis MGMM173, Pseudomonas cedrina MGMM106, Stenotrophomonas rhizophila MGMM118 and Curtobacterium oceanosedimentum MGMM171 against the phytopathogenic fungus Fusarium oxysporum was carried out according to the method (Fan, Ze-Yan, et al. ''Diversity, distribution, and antagonistic activities of rhizobacteria of Panax notoginseng.'' Journal of ginseng research - 2016, Vol. 40.2, Pp. 97-104). In a Petri dish with PDA (Potato Dextrose Agar) medium (potato broth - 1 l, dextrose - 20 g/l, agar - 20 g/l), 5 μl of the bacterial strain suspension and 5 μl of the phytopathogenic fungus suspension are added to the center at equal distances from each other and kept for 5 days at a temperature of +30°C. The presence of antagonistic activity is judged by the zones of phytopathogen growth inhibition.

During testing of the antagonistic activity of the strains, it was observed that the Bacillus velezensis MGMM173 strain inhibits the growth of the phytopathogen Fusarium oxysporum (Fig. 10). In Fig. 10, the absence of Fusarium oxysporum growth is noticeable around the growth zone of Bacillus velezensis MGMM173.

Example 6. Determination of the amount of indole-3-acetic acid (IAA) produced

The amount of indole-3-acetic acid (IAA) is determined by a colorimetric method. For this purpose, each strain is grown in LB medium with the addition of 0.1% of total L-tryptophan as an IAA precursor at 28 (1°C) with constant shaking (180 rpm) for 3 days. After incubation, the bacterial cultures are centrifuged at 10,000 rpm for 10 min at 4°C. 1 ml of each supernatant is mixed with Salkowski's reagent (1:4) (0.1 g of iron ammonium alum is dissolved in 100 ml of 50% sulfuric acid). The mixture is incubated in a dark place at 28 (1°C for 30 min, then the optical density is measured at 530 nm. The concentration of IAA produced is estimated using the IAA standard curve (0-1000 μg/ml). As a blank experiment, 1 ml of sterile LB mixed with Salkovsky's reagent.

The data presented in Table 6 demonstrate that the strains Pseudomonas cedrina MGMM106 (61.62±2.3 μg/ml) and Bacillus velezensis MGMM173 (23.50±0.5 μg/ml) produce IAA in relatively high concentrations, while other studied strains produce IAA in low concentrations: 6.91±1.37 μg/ml Curtobacterium oceanosedimentum MGMM171, and 14.56±0.09 μg/ml Stenotrophomonas rhizophila MGMM118 (Table 6).

Table 6. Results of determining the amount of IUK production

No. Name of the species Strain name Amount of IAA, μg/ml 1 Bacillus velezensis FZB42 MGMM173 23.50±0.5 2 Stenotrophomonas rhizophila D81_CV1R MGMM118 14.56±0.09 3 Pseudomonas cedrina T0a85 MGMM106 61.62±2.3 4 Curtobacterium oceanosedimentum 1257 MGMM171 6.91±1.37

Example 7. Method of obtaining a consortium

Curtobacterium oceanosedimentum MGMM171, Stenotrophomonas rhizophila MGMM118, Bacillus velezensis MGMM173, and Pseudomonas cedrina MGMM106 strains were grown for 12 hours in liquid LB medium (sodium chloride - 10.0 g/l, tryptone - 10.0 g/l, yeast extract - 5.0 g/l). The resulting bacterial culture was applied through a series of dilutions to LB medium (sodium chloride - 10.0 g/l, tryptone - 10.0 g/l, yeast extract - 5.0 g/l, bacteriological agar - 18 g/l), smearing it over the surface with a Drygalski spatula, and incubated for 24 hours at 30°C. The CFU titer in the initial culture is determined by the average colony count, taking into account the dilution rate. The concentration of each strain is adjusted to 1×10 ⁷ CFU/ml using liquid LB medium. The resulting 5 ml bacterial suspensions of the strains are mixed in equal volumetric proportions. The resulting consortium has a titer of at least 1×10 ⁷ CFU/ml for each bacterial strain. The microbial consortium appears as a liquid bacterial culture. It is cloudy yellow in color and has a specific odor. The microbial consortium is stored for 3 months in a 0.3% sodium chloride solution at 3-5°C.

Example 8. Growth-promoting properties of a consortium of microorganisms.

Prepare a bacterial suspension in a quantity of 3.75 ml (at _OD595 = 1.3) and add 0.5% sodium chloride solution (up to 10 ml). The cell count is adjusted to 1× ¹⁰⁷ CFU/ml. Separately add 20 seeds of winter wheat variety Universiade and sunflower variety Svetlana KLP to the microbial consortium until completely immersed and held for 15 minutes. The seeds are dried at room temperature. Quartz sand (fraction 0.6-1.2 mm) is moistened with a liquid mineral medium PRR (1.25 mM Ca(NO ₃ ) ₂ , 1.25 mM KNO ₃ , 0.50 mM MgSO ₄ , 0.25 mM KH ₂ PO ₄ and a solution of microelements in the amount of 10% of the total volume (0.75 mg / l KI, 3.0 mg / l H ₃ BO ₃ , 10.0 mg / l MnSO ₄ ×H ₂ O, 2.0 mg / l ZnSO ₄ ×5H ₂ O, 0.25 mg / l Na ₂ MoO ₄ ×2 H ₂ O, 0.025 mg / l CuSO ₄ ×5 H ₂ O, 0.025 mg / l CoCl ₂ ×6H ₂ O) 240 ml (8% of the total sand volume) are added to the sand, holes are made, and the treated seeds are planted. Seeds not treated with the claimed microbial consortium are used as a control. The plants are grown for two weeks in a closed climate chamber at a temperature of 25°C, 70% humidity, and a 16-hour daylight period (nighttime temperature 20°C, humidity 50%).

After two weeks of growing winter wheat and sunflower with and without a microbial consortium, the plants are carefully removed from the sand along with their root systems, and biometric measurements (root and shoot length) are taken. Then, to measure dry mass, shoot and root samples are dried in a dry-heat oven at 40°C for three days.

Example 9. Testing the drought resistance of plants treated with the consortium.

A bacterial suspension of the claimed consortium is prepared in an amount of 3.75 ml (at OD ₅₉₅ = 1.3) and 0.5% sodium chloride solution is added (up to 10 ml), the cell count is adjusted to 1×10 ⁷ CFU/ml. Twenty seeds of the Universiade winter wheat variety and the Svetlana KLP sunflower variety are separately added to the microbial consortium until completely immersed and kept for 15 minutes. The seeds are dried at room temperature. Expanded clay is placed at the bottom of the growing container to a height of 2 cm. Quartz sand (fraction 0.6-1.2 mm) is moistened with 60 ml of PRR liquid mineral medium (2% of the total sand volume), holes are made in the sand, and the treated sunflower and winter wheat seeds are planted. Sunflower and winter wheat seeds untreated with the proposed microbial consortium were used as controls. The plants were grown for two weeks in a closed climate chamber at a temperature of 25°C, 70% humidity, and a 16-hour daylight period (nighttime temperature 20°C, humidity 50%).

After two weeks of growing winter wheat and sunflower seeds treated with and without the bacterial consortium in quartz sand, the plants were left unwatered for 14 days. The consortium-treated plants demonstrated high drought tolerance compared to control plants. The plants were then carefully removed from the sand along with their root systems, and biometric parameters (root and shoot length) were measured. To measure dry mass, shoot and root samples were dried in a dry-heat oven at 40°C for 3 days. The biometric parameters of untreated and untreated winter wheat plants treated with the claimed microbial consortium were compared, as were the biometric parameters of untreated and untreated sunflower plants treated with the claimed microbial consortium. The comparison results are presented in Tables 4-5 and as histograms in Figs.

Thus, new strains of bacteria Curtobacterium oceanosedimentum MGMM171, Bacillus velezensis MGMM173, and Pseudomonas cedrina MGMM106, possessing enzymatic activities important for the growth and development of plants, a bacterial consortium consisting of the said strains and the strain Stenotrophomonas rhizophila MGMM118 in equal volume ratios with a titer of each bacterial strain of at least 1×10 ⁷ CFU/ml, as well as a method expanding the arsenal of methods for increasing the adaptation of agricultural plants in conditions of short-term drought, are declared.

Claims (4)

1. The bacterial strain Bacillus velezensis MGMM173, deposited in the Network Bioresource Collection in the Field of Genetic Technologies for Agriculture (RCAM) of the All-Russian Research Institute of Agricultural Microbiology under registration number RCAM06912, for stimulating plant growth and protecting against phytopathogenic fungi. 2. A bacterial composition for stimulating growth and stress resistance of plants under conditions of short-term drought, consisting of bacterial strains Curtobacterium oceanosedimentum MGMM171, Stenotrophomonas rhizophila MGMM118, Bacillus velezensis MGMM173 and Pseudomonas cedrina MGMM106 in equal volume ratios with a titer of each bacterial strain of at least 1×10 ⁷ CFU/ml. 3. A method for increasing plant productivity under short-term drought conditions, characterized in that plant seeds are treated with a bacterial composition consisting of the bacterial strains Curtobacterium oceanosedimentum MGMM171, Stenotrophomonas rhizophila MGMM118, Bacillus velezensis MGMM173 and Pseudomonas cedrina MGMM106 in equal volume ratios with a titer of each bacterial strain of at least 1×10 ⁷ CFU/ml. 4. A method for increasing plant productivity under short-term drought conditions according to paragraph 3, characterized in that the treatment of plant seeds with a bacterial composition is carried out in an amount of 1-5 liters of bacterial composition diluted in 10 liters of water per 1 ton of seeds.