RU2820245C1 - Strain of microbacterium sp. et2, stimulating growth of cereal crops - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Microbacterium sp. ET2, having the ability to stimulate the growth of wheat seeds and characterized by resistance to the action of grain dressing agents, is deposited under registration number “Ас-2212” in the Russian National Collection of Industrial Microorganisms. Invention can be used as a microbial preparation—a phytostimulator of growth and development of agricultural plants due to biosynthesis of auxins. Method of using the strain involves its cultivation in a medium with addition of 200–400 mcg/ml of L-tryptophan and use in form of initial or diluted liquid culture, including after drying in air. Strain is resistant to grain disinfectants, which ensures efficiency of its use due to a competitive advantage with respect to other bacteria, which are inhibited by the action of these substances.

EFFECT: invention makes it possible to accelerate wheat growth and increase plant biomass growth at early stages of plants development.

5 cl, 3 dwg, 5 tbl, 6 ex

Description

Field of technology to which the invention relates

The invention relates to the field of biotechnology, ecology and agricultural microbiology, and is a new strain of bacteria Microbacterium sp . ET2, effective for improving growth and increasing plant weight gain, especially in the initial stages of development, due to the strain’s ability to form auxins and stimulate the processes of root formation and growth. The method of cultivating the strain involves the addition of exogenous tryptophan on a nitrogen-free medium to increase the biosynthesis of auxins (the key compound is indolyl-3-acetic acid). The strain, which exhibits high resistance to treatment with disinfectants containing insecticides, fungicides and pesticides, can be used to obtain a biological product and phytostimulant for the germination of cereal seeds.

State of the art

Many of the bacteria that populate the rhizosphere of plants often belong to the PGPB group (short for plant growth-promoting bacteria). They provide the plant with cross-protection from various stress factors, increase resistance to phytopathogens and increase productivity by improving mineral nutrition or affecting the hormonal background of the plant. The indirect effects of PGPB are associated with the biological control and formation of extracellular enzymes and compounds that are toxic or inhibit the growth of pathogens. PGPBs are an alternative to chemicals in agriculture and are of particular interest as phytostimulants and biofertilizers for increasing crop yields and adaptive resistance of plants, as well as for normalizing the overall soil microbiome.

The choice of an effective PGPB strain, in addition to its own active growth-stimulating characteristics, is determined by its competitiveness, resistance to chemicals used in agriculture, as well as preservation of properties during storage and transportation.

Bacteria that stimulate plant growth and belong to the PGPB group synthesize indolyl-3-acetic acid (IAA), the most active compound among auxins (Tsavkelova E.A., Klimova S.Yu., Cherdyntseva T.A., Netrusov A. I. Microorganisms producing plant growth stimulants and their practical application (review) // Applied biochemistry and microbiology , (2006), 133-143. Biosynthesis pathways, the amount of IAA produced and the conditions for the optimal level of its biosynthesis can vary significantly among bacteria interacting with plants (Spaepen S., Vanderleyden J. Auxin and plant-microbe interactions // Cold Spring Harbor perspectives in biology , (2011) . 3 (4), a001438.). The formation of IAA was detected among representatives of the genera Azotobacter, Herbaspirillum, Azospirillum , Enterobacter , Rhizobium , Bradyrhizobium, Alcaligenes , Klebsiella, Sphingomonas . IAA biosynthesis is also found in bacilli, methylobacteria, pseudomonads and streptomycetes.

One of the modern strategies for isolating new strains producing biologically active compounds is to search for them among PGPB colonizing stress-tolerant plants growing in extreme environmental conditions. Of particular interest is the discovery of PGPB among previously unstudied or poorly studied bacteria in terms of phytostimulation. These include gram-positive, non-spore-forming representatives of the genus Microbacterium (family Microbacteriaceae, order Micrococcales , class Actinomycetia , phylum Actinomycetota).

Representatives of the genus Azotobacter are known to be used as nitrogen fixers, as well as producers of antimicrobial compounds and growth stimulants in biofertilizers, for example, strains of Azotobacter chroococcum (RU2286324, published 10.27.2006). The disadvantage of their use is sequential cultivation with subcultures on mineral-rich Ashby and Burke media with the addition of a significant amount of tryptophan (0.6 g/l), which leads to an increase in the cost of obtaining microorganism biomass.

A known strain of Pseudomonas chlororaphis (RU2588473 publ. 06.27.2016) for protecting plants from phytopathogenic fungi and bacteria, and stimulating plant growth, producing the heterocyclic antibiotic phenazine-1-carboxamide and auxins (11 μg/ml). The disadvantage of the strain is that its ability to fix nitrogen, as well as its potential survival when using grain treaters, has not been studied.

A known strain of the bacterium Azospirillum brasilense IMB B-7197 (UA85924C2, published on March 10, 2009) increases the yield of spring cereal crops by 26.4% due to associative nitrogen fixation, a positive effect on the photosynthetic apparatus and increased resistance to root rot pathogens. The strain, however, has not been studied for its ability to produce phytogrowth stimulants.

The Achromobacter album strain VNIISHM V-322D is known for producing a biological product that increases sugar content by 0.5-1.0% and sugar beet yield by 8-24% (RU2035442C1, published 05.20.1995). This strain has been shown to be resistant to the insecticide adifur and the contact fungicide TMTD. The biosynthesis of β-indolylacetic acid was studied using Muromtsev's glucose-aspartic medium with induction by tryptophan, which was added at a rate of 0.05 wt. The maximum values of IAA content were 68.3 μg/ml on the 10th day of cultivation. The disadvantage of this strain is that it only assimilates organic forms of nitrogen, so the effectiveness of its use in nitrogen-depleted conditions is difficult to predict. The stimulating activity of the strain against grain crops is not indicated, which also limits its use for cereal crops.

The known strain of Microbacterium NRRL B-50470 (RU2634386C2, published 10/26/2017). The strain has suppressor activity against fusarium (scab) of wheat and barley and, thus, significantly reduces yield losses caused by infection of fungi with Fusarium graminearum . The disadvantages of the invention include the lack of information on the growth-stimulating properties of the Microbacterium strain; there is no data on the biosynthesis of auxins and resistance to fungicides and other pesticides.

The closest analogue to the claimed invention is the bacterium strain Azospirillum zeae OPN-14 (VKPM B-12542) as a biological agent with growth-stimulating and plant productivity-enhancing activities (RU2678755C1, publ. 01/31/2019). The strain synthesizes auxin (IAA) at a concentration of 32-46 μg/ml and exhibits cytokinin-like activity, fixes atmospheric nitrogen (1269 nM C ₂ H ₄ / h × mg of dry cellular biomass), is resistant to a number of herbicides (Laren Pro, "Cortes SP", "Aminka"), as well as fungicides containing carbendazim. The growth-stimulating effect of A. zeae OPN-14 is demonstrated by stimulating the growth of pea plants, as well as by assessing the increase in the yield of sunflower and spring wheat. The main disadvantage of the known strain is that it is not resistant to fungicides containing tebuconazole and propiconazole, which are included in many of the most effective fungicides currently used in agriculture.

Thus, the technical problem solved by means of the claimed invention lies in the need to overcome the disadvantages inherent in analogues and the prototype by obtaining a bacterial strain that has growth-stimulating activity against cereal plants, including auxin biosynthesis, as well as resistance to modern fungicidal and insecticidal drugs used in agriculture as grain protectants.

Disclosure of the Invention

The technical result of the invention is the production of a new, resistant to drying in the range of 20-55 ° C, natural strain of non-spore-forming bacteria that retains viability when treated with grain protectants (at a level of at least 10 ⁷ CFU/ml), to stimulate germination and improve rhizogenesis of cereals crops (with an increase in root mass by no less than 40-50% for the example of wheat) due to microbial tryptophan-dependent auxin biosynthesis (at least 20-30 μg of indolyl-3-acetic acid / ml).

The technical result is achieved by a strain of bacteria Microbacterium sp. ET2, which has the ability to stimulate the growth of cereal seeds and is resistant to the action of grain disinfectants, deposited under registration number Ac-2212 in the All-Russian Collection of Industrial Microorganisms.

The technical result is achieved by a strain of bacteria Microbacterium sp. ET2, which has the ability to stimulate the growth of cereal seeds and is resistant to the action of grain disinfectants, deposited under registration number Ac-2212 in the All-Russian Collection of Industrial Microorganisms.

The technical result is also achieved by a microbial preparation - a phytostimulant for the growth and development of agricultural plants, including a strain of Microbacterium sp . ET2, the stimulating effect is achieved through the biosynthesis of the key auxin - indolyl-3-acetic acid. The use of the strain involves its cultivation in a medium supplemented with 200-400 μg/ml L-tryptophan, and use as an initial or diluted liquid culture, including after drying in air. The strain is resistant to grain disinfectants, which ensures the effectiveness of its use due to its competitive advantage over other bacteria inhibited by the action of these substances. The invention makes it possible to accelerate the growth of cereal crops and increase the growth of plant biomass in the early stages of plant development.

The technical result is also achieved by using a strain of Microbacterium sp. ET2 to stimulate the germination and growth of cereal plants.

Wheat, rye, oats, rice, corn, barley, and millet can be used as cereal crops.

The claimed bacterial strain Microbacterium sp . ET2 was isolated from the rhizoplane of the greenhouse epiphytic leafless orchid Chiloschista parishii Seidenf., cultivated in the Stock greenhouse of the Main Botanical Garden named after. N.V. Tsitsin RAS (GBS RAS, Moscow).

Strain Microbacteriumsp.ET2 deposited in the All-Russian Collection of Industrial Microorganisms under registration number Ac-2212. The strain was identified based on the study of its morphological, cultural, physiological and biochemical characteristics, as well as using sequencing of the 16S rRNA gene, including at the Federal State Unitary Enterprise GosNIIGenetika.

The resulting strain is characterized by the following characteristics:

Strain Microbacteriumsp. ET2 grows on the following media (g/l): TCA - tryptose-soy agar (tryptose-soy extract - 30.0, agar - 15.0); R2A medium (yeast extract - 0.5; peptone - 0.5; casamino acids - 0.5; dextrose - 0.5; soluble starch - 0.5; sodium pyruvate - 0.3; K₂NRO₄ - 0.3; MgSO₄ -0.05; agar - 15.0); modified Czapek medium (glucose - 20.0; K₂HPO₄ - 1.0; NaNO₃ - 2.0; MgSO₄ × 7H₂O - 0.5; KCl - 0.5; FeSO₄ × 7H₂O - 0.01; yeast extract - 0.5 g/l; pH 7.0-7.2).

Morphological and cultural characteristics

The cells are represented by gram-positive small round rods; coccoid cells may occur; in fixed and fuchsin-stained preparations they are located singly or in pairs. There is no dispute.

The morphology of colonies on solid (agar) nutrient media was determined after 5-10 days of growth at 28°C. Colonies on TCA medium are small, round, non-translucent, flatter, having a diameter of 3 mm, with a pinkish tint. Colonies on R2A medium are round, shiny, more convex, slimy, 3 mm in diameter; colonies are whitish, with a raspberry-violet tint of varying intensity; the pigment becomes more intense with age. The substrate is not pigmented.

Physiological and biochemical signs

Aerobic, mesophilic with a temperature optimum for growth of 30°C, grows well at 37°C, weak growth is also noted at 55°C.

In salt broth it grows well at 2% and 5% NaCl, growth was noted at 10% NaCl. The optimal pH value for growth is 6.8-7.2.

Chemoorganoheterotroph. Metabolism is mainly respiratory type. It uses glucose, sucrose, dextrose, galactose, D-mannose, D-maltose as the only carbon source; good growth is noted on mannitol, but weak on glycerol; it does not consume organic acids citrate and pyruvate. Does not hydrolyze milk casein and does not liquefy gelatin. On a medium with starch there is poor growth and insignificant amylolytic activity. Forms acid from carbohydrates: glucose, sucrose, galactose, D-mannose, D-maltose. Does not reduce nitrate.

Antibiotic resistance

The strain exhibits resistance to cephalexin (30 μg/ml) and ampicillin (10 μg/ml). Sensitive to tetracycline (50 mcg/ml).

Principal physiological properties

The strain exhibits low nitrogen-fixing activity on agar and liquid media. Grows diazotrophically on Ashby's medium with sucrose as a carbohydrate and microelements according to Fedorov. Nitrogenase activity was confirmed by the _acetylene method; the activity of this enzyme is 1.8 ± 0.2 ng _C2H4 /ml h.

The strain synthesizes indolyl-3-acetic acid (IAA, heteroauxin), a tryptophan-dependent synthesis.

Biosafety information

The strain is not pathogenic for warm-blooded animals and humans. It does not have phytopathogenic activity, which is confirmed by the absence of maceration on sections of potato tubers when applied to them by injection of living cells of the strain.

Storage conditions

The strain is maintained by subculture on fresh agar nutrient media (for example, R2A). The strain is stored in lyophilized form using skimmed milk powder as a protective medium at +4°C or in test tubes with short-cut medium under a layer of sterile petroleum jelly. The strain is stored at 4°C in test tubes under cotton-gauze stoppers.

Brief description of drawings

The invention is illustrated by the following drawings.

In fig. Figure 1 shows an example of growth stimulation according to the height parameter of wheat seedlings in vermiculite after their pre-sowing treatment with the proposed strain of the bacterium Microbacterium sp. ET2. On the left - wheat seeds soaked for 1 hour in a liquid culture of Microbacterium sp. ET2 grown on a nitrogen-free medium supplemented with L-tryptophan for 5 days and then diluted 100 times with tap water. On the right - wheat seeds were kept for 1 hour in a suspension of amorphous silicon dioxide in tap water (control), after which they were laid out in vermiculite. Plants are presented on the 13th day after sowing.

In fig. Figure 2 shows an example of stimulating the growth of wheat in vermiculite after bacterization of seeds with a strain of Microbacterium sp. ET2 after drying the culture at different temperatures. Where (a) are wheat seeds bacterized with the proposed strain of Microbacterium sp. ET2, grown on a nitrogen-free medium with the addition of 400 μg/ml L-tryptophan for 5 days, then dried at 55 ± 2°C for 24 hours on a carrier (amorphous silicon dioxide); after dilution in water to the initial concentration, the seeds were soaked in the resulting culture for 1 hour; (b) - wheat seeds soaked for 1 hour in water (control); (c) - wheat seeds bacterized with the proposed strain of Microbacterium sp. ET2, grown on a nitrogen-free medium with the addition of 400 µg/ml L-tryptophan for 5 days, then dried at 20±2°C for 24 hours on a carrier (amorphous silicon dioxide); after dilution in water to the initial concentration, the seeds were soaked in the resulting culture for 1 hour; (d) - wheat seeds bacterized with the proposed strain of Microbacterium sp. ET2, grown on a nitrogen-free medium with the addition of 400 μg/ml L-tryptophan for 5 days, then dried at 30°C for 24 hours on a carrier (amorphous silicon dioxide); after dilution in water to the initial concentration, the seeds were soaked in the resulting culture for 1 hour; e) - wheat seeds soaked for 1 hour in water (control).

In fig. Figure 3 shows an example of stimulation of wheat growth at the early stages of development in terms of the shoot length parameter by a strain of Microbacterium sp. ET2 after drying the crop at different temperatures, where (a) - wheat seeds soaked for 1 hour in water (control); (b) - wheat seeds bacterized with the proposed strain of Microbacterium sp. ET2, grown on a nitrogen-free medium supplemented with L-tryptophan for 5 days, then dried at 30°C for 24 hours; after dilution in water to the initial concentration, the seeds were soaked in the culture for 1 hour; (c) - wheat seeds bacterized with the proposed strain of Microbacterium sp. ET2, grown on a nitrogen-free medium supplemented with L-tryptophan for 5 days, then dried at room temperature (20°C) for 24 hours; after dilution in water to the initial concentration, the seeds were soaked in the culture for 1 hour; (d) - wheat seeds bacterized with the proposed strain of Microbacterium sp. ET2, grown on a nitrogen-free medium with the addition of L-tryptophan for 5 days, then dried at 55±2°C for 24 hours; After dilution in water to the initial concentration, the seeds were soaked in the culture for 1 hour.

Carrying out the invention

The possibility of implementing the proposed invention is confirmed by the following examples, but is not limited to them.

Example 1: DNA Extraction and Strain Identification

The nucleotide sequence of the 16S rRNA region was determined by nanopore sequencing using Oxford Nanopore Technologies technology using a MinION instrument (Oxford Nanopore Technologies) and cell R9.4.1. For this purpose, DNA was isolated from the biomass of the strain culture grown in R2A medium using the Monarch kit (New England BioLabs) in accordance with the manufacturer’s protocol. DNA quality was assessed in a 1% agarose gel and also using a Nanodrop spectrophotometer (Thermo Scientific). DNA concentration was measured using a Qubit 3.0 fluorimeter (Invitrogen, Thermo Fisher Scientific corporation); sample preparation was carried out using the Qudye dsDNA HS Assay kit (Lumiprobe) according to the manufacturer’s instructions. Genomic DNA sequencing was performed according to the Native barcoding genomic DNA protocol (with EXP-NBD104, EXP-NBD-114, and SQK-LSK109). When conducting bioinformatics analysis, current signals recorded in the FAST5 format were converted to the FASTQ format; the Guppy basecaller 6.4.6 program was used for conversion. The genome assembly was carried out using the Flye 2.9.1 and Medaka 1.4.2 programs.

Identification was carried out by comparative analysis of the nucleotide sequence corresponding to the 16S rRNA fragment with homologous sequences from the NCBI database (National Center for Biotechnology Information website; www.ncbi.nlm.nih.gov/blast).

The results of the sequence analysis revealed that the strain belongs to the genus Microbacterium and has 99.6% similarity with Microbacterium saccharophilum strain RS59 (gene fragment sequence; accession number MH715208.1) and 98.49% similarity with the species Microbacterium caowuchunii strain ST-M6 (sequence complete genome; accession number CP044231.1).

Example 2. Study of the nitrogen-fixing activity of a strain of Microbacterium sp. ET2

The strain was grown on Ashby's dense medium containing no bound forms of nitrogen (g/l): sucrose - 20.0; K ₂ HPO ₄ - 0.2; MgSO ₄ × 7H ₂ O - 0.2; NaCl - 0.2; K ₂ SO ₄ - 0.1; CaCO ₃ - 5.0; FeСl ₃ - 0.1; microelements solution according to Fedorov - 1 ml/l; agar-agar (Oxoid) -15 g/l, distilled water. Composition of microelements according to Fedorov, g/l: H ₃ BO ₃ - 5.0; (NH ₄ ) ₂ MoO ₄ × 2H ₂ O - 5.0; ZnSO ₄ × 7H ₂ O - 0.2; KI - 0.5; NaBr - 0.5; Al ₂ (SO ₄ ) ₃ × 18H ₂ O - 0.3; distilled water. Seeding was carried out with a bacteriological loop and streaks. The grown biomass was used as an inoculum, sown in a liquid medium of the same composition and cultivated at 28°C for 4 days in 15 ml penicillin vials with 5 ml of medium.

To determine the activity of nitrogenase, the key enzyme of nitrogen fixation, the analytical method of gas chromatography of acetylene reduction was used. To do this, the cotton plugs were replaced with sealed rubber ones, rolled up with aluminum caps, 1 ml of acetylene was introduced and incubated for 3 hours at 28°C. Then, 1 ml of sample was taken from the bottles and the content of the resulting ethylene was measured on a Crystal 2000M gas chromatograph (Khromatek, Russia). Distilled water and medium without inoculum served as negative control samples. The determination was carried out in triplicate and the arithmetic mean was calculated.

Under the tested conditions _, the potential nitrogen-fixing activity was 1.8 ± 0.2 ng _C2H4 /ml per 1 hour of incubation. Despite the low levels of nitrogenase activity, the strain retains the ability to grow under conditions limited by the content of nitrate and ammonium nitrogen in the environment, which is an important characteristic of biological activity during bacterization of plants and the formation of microbial-plant consortia.

Example 3. Cultivation of a strain of Microbacterium sp. ET2 and determination of auxin content (indolyl-3-acetic acid) in the culture medium.

The strain was cultivated on solid (agar) R2A medium for 3-4 days, then the biomass was removed with a bacteriological loop and added to flasks with 50 ml of liquid medium of the following composition (g/l): K ₂ HPO ₄ - 0.5; KH ₂ PO ₄ - 0.3; MgSO ₄ 7H ₂ O - 0.1; NaCl - 0.5; CaCl ₂ 6H ₂ O - 0.03; yeast extract - 0.5; glucose - 5.0; water - tap. As an inducer of IAA biosynthesis, L-tryptophan was added to the medium in amounts of 100, 200 and 400 μg/ml. They were cultured at 28°C on a rocking chair (100 rpm) for 5 days.

Quantitative analysis of auxin was carried out colorimetrically (Gordon SA, Weber RP Colorimetric estimation of indole-acetic acid // Plant Physiol. - 1951. - V. 26. - P. 192-195) on a spectrophotometer (KFK-3-01-"ZOMZ" ) after 5 days of cultivation using Salkovsky's reagent (0.05 M FeCl3 in 35% HClO4), which was added at a rate of 1:1 (by volume) to the supernatant obtained after centrifugation of the culture (10 min at 10,000 g ). Samples were incubated in the dark at 30°C for 15 min. The control was a non-inoculated medium with the addition of a reagent. The presence of IAA manifested itself in a pink (crimson) color. The colored samples were photocolorimetered at 530 nm, in a cuvette with an optical path length of 0.5 cm. The optical density (OD) of the culture was measured at 540 nm, in a cuvette with an optical path length of 0.5 cm. The IAA concentration was determined using a calibration graph plotted in the IAA concentration range from 1 up to 50 mcg/ml.

Table 1. Auxin production by the proposed strain of the bacterium Microbacterium sp. ET2 under conditions of indolyl-3-acetic acid biosynthesis induction by tryptophan. L-tryptophan content in the medium, µg/ml Auxins, µg/ml Optical density (OD) of the culture IAA production, µg/OD (ratio of auxin biosynthesis to crop OD) 0 0.3 ± 0.01 2.2 ± 0.18 0.14 100 9.5 ± 0.7 1.6 ± 0.12 5.9 200 21.9 ± 1.2 1.8 ± 0.10 12.9 400 31.8 ± 3.5 1.1 ± 0.12 28.9

Table 1 shows that auxin biosynthesis by Microbacterium sp. ET2 is 22 μg/ml when using 200 μg/ml exogenous tryptophan and 32 μg/ml when adding 400 μg/ml L-tryptophan. It is known that pseudo-monon strains, for example, Pseudomonas Chlororaphis BKM B-2956D and Pseudomonas Aureofaciens BS1393, which is used in the commercial preparation of Psevodobacterin-2, form 11.2 and 16.4 μg/ml of Auxin (Patent RU 2588473 6.2016) respectively . Thus, the use of exogenous tryptophan in an amount of 200 μg/ml is sufficient for the biosynthesis of the necessary growth-stimulating amounts of auxin by the proposed strain of Microbacterium sp. ET2.

Example 4. Assessment of the survival rate of a strain of Microbacterium sp. ET2 when exposed to grain disinfectants.

For effective colonization by bacteria of the root system of cereals sown into the soil with seeds undergoing mandatory pre-sowing treatment with disinfectants, it is necessary to determine the resistance of the proposed strain to the effects of herbicides, fungicides and pesticides. For this purpose, combined working solutions of disinfectants were used, where the active ingredients are difenoconazole (67 g/l), imidacloprid (333 g/l), tebuconazole (17 g/l) and fludioxonil (75 g/l). The strain culture grown on R2A medium was added to test tubes with a working solution of disinfectants, mixed on a vortex and left for 2 hours, after which an aliquot (50 μl) was sown on the surface of the agar medium, rubbing with a spatula, directly from the original solutions, as well as from their decimal dilutions obtained by dilution in sterile tap water (1:9 by volume). Sterile tap water was used as a control. Colony-forming units were counted 3-5 days after incubation of the inoculated dishes at 28°C. The results are presented in Table 2.

Table 2. Assessment of cell viability of Microbacterium sp. strain. ET2 in solutions of disinfectants. Working solution Number of colony forming units (CFU) after treatment / ml of working solution Protectant
(pesticide, fungicide, insecticide) 1.1 × 10 ⁷ Control (water) 1.6 × 10 ⁹

According to the results obtained, the used disinfectants, despite a decrease in the number of viable cells in the population by two orders of magnitude, did not have a significant inhibitory effect on the culture of the strain Microbacterium sp. ET2 and did not cause a toxic effect that completely suppressed its development. The culture maintained viability at a level of 10 ⁷ CFU/ml after treatment with a disinfectant. Resistance of Microbacterium sp. ET2 to neonicotinoids (imidacloprid) and triazoles (tebuconazole and difenoconazole) ensures the effectiveness of this crop due to its competitive advantage over other bacteria inhibited by these substances. For example, the previously described strain of Azospirillum zeae OPN-14, which stimulates plant growth, turned out to be unstable to tebuconazole (RF Patent RU2678755C1, publ. 01/31/2019). Considering that the studied substances are included in the main disinfectant preparations used in agriculture, the resistance of the Microbacterium sp. strain to them. ET2 makes it a priority for wide and large-scale applications.

Example 5. Assessment of the survival rate of a strain of Microbacterium sp. ET2 after drying the culture in a liquid medium with the addition of a carrier (amorphous silicon dioxide).

To use the strain, it is important to maintain the viability of the culture when drying. This factor determines the safety of the drug itself, as well as the resistance of the microorganism to drying out while in the soil. For these purposes, a culture of the strain Microbacterium sp. ET2, grown in liquid medium (composition indicated in example 3) with tryptophan, was dried at different temperatures (20°C, 30°C and 55°C). To do this, the strain was grown in a liquid medium for 4-5 days, an aliquot (4 ml) was poured into open Petri dishes (diameter 40 mm), 1.4 g of amorphous silicon dioxide was added to the consistency of thick sour cream and mixed, covering the top with sterile gauze, and left open for a day. Amorphous silicon dioxide is added at the rate that 0.2 - 0.5 g of amorphous silicon dioxide is used per 1 ml of culture liquid.

After drying and for subsequent use, a solution was prepared by adding sterile tap water (4 ml) to the original volume, thus obtaining an initial suspension, from which successive decimal dilutions were then prepared for plating on the surface of the nutrient medium and counting CFU. The dishes were incubated until individually visible colonies appeared and were counted. The results are presented in Table 3.

Table 3. Survival of Microbacterium sp. strain. ET2 when drying a liquid culture in air on a carrier at different temperatures. Air temperature, °C Number of colony forming units (CFU) after drying/ml inoculum 20 ± 2 4.7 × 10 ⁷ 30 ±2 5.1 × 10 ⁷ 55 ± 2 8.2 × 10 ⁶ Control (without drying) 7.1 × 10 ⁸

According to the data presented, the culture of Microbacterium sp. ET2 remains viable when dried in air in the temperature range from 20 to 55°C. At temperatures exceeding 55 ± 2°C, the survival rate of the microbial population decreases, however, remaining at a fairly high level. Thus, the proposed strain is resistant to drying in liquid culture, which facilitates its transportation and subsequent use at the site of seed bacterization, and also promotes survival in conditions of lack of moisture.

Example 6 . Bacterization of wheat seeds to stimulate germination.

Wheat seeds were soaked for 1 hour in different cultures of Microbacterium sp. ET2 grown in a liquid medium (composition indicated in example 2). For this purpose, a nutrient medium with the addition of L-tryptophan was used. We also used variants of the culture mixed with a carrier (amorphous silicon dioxide), for which 4 ml of the culture was added to cups (40 mm in diameter), to which the powder carrier was added and mixed evenly until the consistency of thick sour cream. Seeds were soaked in liquid culture or placed in culture with carrier and left for 1 hour. The incubation time, limited to 1 hour, was chosen in order not to increase the additional time for pre-sowing treatment of grain seeds, which is usually spent in agricultural technology, despite the fact that longer bacterization may contribute to a more effective stimulating effect of the strain.

After the crop treatment, the seeds were sown in vermiculite pots to avoid any additional influence on seed growth due to biota and nutrient compounds contained in the soil substrate. Nine seeds were sown in one pot, and the experiment was performed in triplicate. The controls were seeds soaked in tap water, as well as seeds placed in a carrier (amorphous silicon dioxide in water). Plants were grown at a temperature of 20-22°C and a light duration of 14 hours for two weeks. Watering was carried out with settled tap water as needed. Analysis of seed growth and the effect of the strain on stimulating seed germination and development was carried out by measuring the length of the shoot, as well as determining the mass of roots and shoots. The roots of the plant were washed in water and dried between layers of filter paper, and then weighed.

The strain showed its effectiveness in stimulating rhizogenesis and development of wheat in the early stages of growth during the first two weeks. Several experimental designs were used:

1. We used a strain of Microbacterium sp. ET2 grown for 5 days in a nitrogen-free medium supplemented with 200 μg/ml tryptophan. The culture was tested in two versions: not mixed (initial culture in a liquid medium) and mixed with amorphous silicon dioxide (carrier). In addition, the culture was used in the original concentration, as well as diluted 100 times with tap water. Wheat seeds were placed in experimental solutions for 1 hour. As a control, the seeds were soaked in tap water and also placed for the same time in a medium for cultivating bacteria and a suspension of amorphous silicon dioxide, also prepared in tap water. The results are presented by measurements on the 13th day after bacterization and are displayed in table 4 and in Fig. 1.

Table 4. Effect of Microbacterium sp. strain. ET2 on the early development of wheat seeds in liquid culture and on a carrier (amorphous silicon dioxide). Options
experience
Indicators CONTROL EXPERIENCE Culture without a carrier Culture with a carrier Water Wednesday Carrier + water Source culture Culture diluted 100 times with water Source culture + carrier Culture diluted 100 times with water + carrier Number of germinated plants, % 100 100 78 100 100 89 90 Plant height, cm 20.9
±0.83 19.0
±0.60 20.2
± 2.07 24.2
± 2.50 25.3
±2.67 20.8
± 1.25 19.8
± 3.63 Weight of the above-ground part, g 0.14
±0.02 0.13
±0.02 0.13
±0.03 0.17
±0.03 0.21
±0.007 0.15
±0.02 0.14
±0.04 Root mass, g 0.09
±0.03 0.10
±0.01 0.11
±0.01 0.11
±0.02 0.16
±0.12 0.14
±0.03 0.11
±0.03

According to the data presented, the strain Microbacterium sp. ET2 stimulates the development of wheat in the early stages of its development. When using the original undiluted culture, stimulation of plant growth is expressed in an increase in the mass of roots by 11.1%, the mass of the aerial parts (shoots, leaves) by 21.4% and plant height by 15.8%. The stimulating effect is maximally manifested when using a liquid culture diluted 100 times with water: the increase in plant height is 21.05% (of the water control), the increase in the mass of leaves and roots is 50% and 61.6%, respectively. The data obtained also show that the nutrient medium for bacteria in its original form, due to the excess content of minerals, somewhat inhibits plant growth. This may also be related to the more effective effect of the diluted culture. In addition, the carrier also has some inhibitory effect on the germination of wheat seeds, which is expressed in a decrease in the number of sprouted seeds, both in the control variant with the carrier and when it is used together with a culture of the Microbacterium sp strain. ET2. Seeds treated with amorphous silicon dioxide are practically the same or slightly smaller than the control (water) in terms of the height and weight of the above-ground parts of the plants. Despite the fact that the use of Microbacterium sp. ET2 allows to overcome this negative effect to some extent; it is obvious that the use of a culture involves its use without additional mixing with a given carrier.

Considering that the culture showed the ability to survive desiccation at temperatures above 50°C (Example 5) and an increase in IAA yield when using 400 μg/ml L-tryptophan (Example 3), the use of a Microbacterium sp. strain was tested. ET2 grown in a nitrogen-free medium with the addition of 400 μg/ml tryptophan as an inducer of IAA biosynthesis for 5 days, followed by drying. The culture was mixed with amorphous silicon dioxide to the consistency of thick sour cream and left in open containers to dry for a day at different temperatures (20°C, 30°C and 55°C). After drying and for subsequent use, a working solution was prepared by adding sterile tap water to the original volume, into which, after stirring, wheat seeds were placed for 1 hour. As a control, seeds were soaked in tap water and also placed in a suspension of amorphous silica in tap water for the same time. The results are presented in Table 5 and FIG. 2 and 3.

Table 5. Effect of Microbacterium sp. strain. ET2 on the development of wheat seeds after drying the crop on a carrier on the 16th day after bacterization. Options
experience
Indicators Control (water) Drying at 20±2°C Drying at 30±2°C Drying at 55±2°C Number of germinated plants, % 100 100 90 100 Plant height, cm 21.6 ± 0.5 22.4 ± 1.8 22.5 ± 0.7 23.3 ± 1.1 Weight of the above-ground part, g 0.15 ± 0.02 0.19 ± 0.03 0.2 ± 0.03 0.2 ± 0.02 Root mass, g 0.07 ± 0.01 0.1 ± 0.01 0.1 ± 0.01 0.1 ± 0.02

According to the results obtained, the use of 400 μg/ml exogenous tryptophan allows the Microbacterium sp. ET2 remains effective in stimulating the development of germinating wheat seeds after drying the crop at different temperatures. The height of bacterized plants is 4-8% greater than the control ones, the weight of their aerial parts is by 26.7% and 33.3%, and the weight of roots is by 42.8%.

In fig. 2 and 3 show examples of wheat plants on the 16th day after bacterization with the proposed strain of Microbacterium sp. ET2 after drying the culture on a carrier (amorphous silica). Unlike the control, inoculated plants have taller seedlings and a more developed root system.

Thus, the technical result of the claimed invention is stimulation of the growth and development of agricultural plants using the example of grain crops (wheat). It is achieved by using a strain of the bacterium Microbacterium sp. to treat plants. ET2, which has pronounced biological activity due to the biosynthesis of the phytohormone auxin; the strain is resistant to chemical disinfectants (pesticides) used in agriculture, as well as stability and retention of viability and growth-stimulating activity when dried in air, including at a temperature of 55±2°C. The use of the proposed strain Microbacterium sp. ET2 as a phytostimulant is possible both in a liquid culture grown using 200-400 μg/ml L-tryptophan, and using a carrier when cultivated in a medium with 400 μg/ml L-tryptophan.

Claims (5)

1. Bacterial strain Microbacterium sp. ET2 , deposited in the All-Russian Collection of Industrial Microorganisms under registration number VKPM Ac-2212, which has the property of stimulating the germination and growth of wheat plants. 2. A drug having a property that stimulates the growth of wheat plants, including the strain according to claim 1, grown on a nitrogen-free medium with the addition of L-tryptophan. 3. The drug according to claim 2, characterized in that it additionally contains a carrier. 4. The drug according to claim 3, characterized in that amorphous silicon dioxide is used as a carrier. 5. Use of the strain according to claim 1 to stimulate the germination and growth of wheat plants.