RU2795906C1 - Consortium of psychrotolerant bacterial strains for biological protection and stimulation of growth of agricultural plants - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: basis of a biological protection agent and a growth stimulant for agricultural plants. The consortium contains bacterial strains Bacillus simplex 948 P-1TS RNCIM B-12245 and Bacillus megaterium 312 TS RNCIM B-12244, taken in a ratio of 1:1 and having a total cell titer of at least 108 CFU/ml.

EFFECT: increasing crop yields, their adaptive capabilities by stimulating plant growth, as well as stress resistance to abiotic environmental factors.

1 cl, 1 dwg, 1 tbl

Description

SUBSTANCE: invention relates to agricultural microbiology and biotechnology, concerns biological agents for stimulating growth and increasing plant productivity, and represents a consortium of Bacillus simplex 948 P-1TS and Bacillus megaterium 312 TS strains.

The basis of the invention is psychrotolerant strains of bacteria Bacillus simplex 948 P-1TS and Bacillus megaterium 312 TS, deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) of the Federal State Unitary Enterprise GosNIIgenetika under registration numbers B-12245 and I-12244, respectively.

Biological farming is of great importance for the greening of agriculture. One of the elements of biological farming is the use of microbial preparations created on the basis of microorganisms that are safe for humans, do not pollute the environment, heal the soil and restore its fertility, as well as providing a beneficial aftereffect for subsequent crops in the crop rotation.

Known and patented are many different strains of microorganisms proposed for use in the production of biological products as the main active agents of biopesticides and/or biofertilizers and biostimulants. A significant part of these microorganisms are representatives of the genera Bacillus and Pseudomonas. In addition, some of the beneficial bacteria, as proved by the example of bacteria of the genus Bacillus, are fixed and winter in the rhizosphere of the plant, creating a positive aftereffect, manifested in the sanitation of the soil and crop residues against pathogenic fungi and bacteria, as well as enriching the arable microbiocenosis with beneficial microflora. All other drugs, both chemical and biological nature, act in contact. In other words, their effectiveness depends on the chemical formula, time and area of contact with interacting or diseased plant tissues, and the accuracy of selecting the concentration of active substances.

From a technological point of view, it is the bacteria r. Bacillus are promising for the creation of microbiological preparations based on them, due to their spore-forming ability, resistance to drying (osmotolerance), which makes it possible to obtain stable products.

Known strains of Bacillus subtilis ARRIAM 128 [Author's certificate No. 1717156, publ. 03/07/1992] and the biological product Fitosporin, created on the basis of this strain [RF Patent No. 2099947, Appl. 11/15/1996, publ. 12/27/1997], B. subtilis No. 10, which is the basis of the Alirin B biological product [RF Patent No. 2081167, Appl. 04/08/1994, publ. 06/10/1997], B. subtilis B-40 [RF Patent No. 2094990, Appl. 07/08/1993, publ. 11/10/1997], B. subtilis 11 V (VKM V-2218D) [RF Patent No. 2182172, Appl. 04/05/2001, publ. 05/10/2002], Bacillus pumilus BKM CR-333D [RF Patent No. 1817875, publ. 05/20/1995], B. pumilus A1.5 [RF Patent No. 2551968, Appl. 08/01/2013, publ. 06/10/2015], Brevibacillus laterosporus VKPM B-7767 [RF Patent No. 2242125, Appl. 03/12/2003, publ. December 20, 2004], Bacillus atrophaeus VKPM-11474 [RF Patent No. 2570624, Appl. 03/06/2014, publ. 12/10/2015].

The disadvantage of all of the above strains is insufficient biological activity, manifested in antagonism to phytopathogenic and not only microorganisms and protection of plants from certain diseases, while their effect on plant yield remains unknown.

A known strain of Bacillus subtilis M1 [RF Patent No. 2307158, Appl. 08/31/2005, publ. September 27, 2007]. The specified microorganism increases the germination of spring wheat seeds, stimulates the growth of seedlings, increases their weight, reduces the infection of seedlings with root rot pathogens, but practically does not contribute to an increase in yield.

The strains used in the composition of biological products are mesophilic and work at a temperature of +10 degrees. Which makes them ineffective when used in the autumn-spring period.

Known drug for protecting plants from diseases based on the strain of Bacillus subtilis IPM-215 [RF Patent No. 2019966, IPC A01N 63/00, Appl. 07/24/1991, publ. 1994]. The disadvantages of this drug are the limited scope, the lack of available sources of information on the healing effect on plants with bacterial damage, the effective protective effect against diseases in open ground conditions and the ability to nitrogen fixation and phosphate mobilization. In addition, the composition of the drug is multicomponent, the technology for obtaining the drug is multi-stage and complex.

A biological product is known for autumn, spring and summer tillage, root and root dressing during the growing season of plants, as well as pre-sowing seed treatment, which is a mixture of suspensions of thirteen different strains: Agrobacterium tumefaciens B-4116, Agrobacterium radiobacter B-956, Azotobacter chroococcum B- 2375, Bacillus thuringiensis B-2918, Bacillus subtilis B-6554, Bacillus subtilis B-4419, Bacillus megaterium B-4440, Bacillus megaterium B-200, Bradyrhizobium japonicum B-1978, Erwinia ananas B-5292, Lactobacillus casei B-3961, Pseudomonas fluorescens B-l138, Rhodopseudomonas palustris B-1620. [RF Patent No. 2322061, IPC A01N 63/00, C12N 1/20, Appl. 04/19/2006, publ. 2008].

A significant disadvantage of preparations based on several bacterial strains of different taxonomic affiliation is the multicomponent species and generic composition of microorganisms with different nutritional needs, requiring both separate cultivation conditions and various environmental conditions for maximum manifestation of properties.

The technical problem to be solved by the claimed invention is the creation of an effective microbial consortium that promotes biological protection, as well as stimulating the growth and increasing the yield of agricultural plants.

The technical result is achieved through the use of a consortium of bacterial strains, including strains of bacteria Bacillus simplex 948 R-1TS VKPM B-12245 and Bacillus megaterium 312 TS VKPM B-12244. It is provided that bacterial strains are taken in a ratio of 1:1 and have a total cell titer of at least 10 ⁸ CFU/ml.

The strains were isolated from permafrost in the vicinity of Tarko-Sale, YNAO. The species affiliation of the culture was determined based on the results of sequencing of the 16S rRNA gene at the Federal State Unitary Enterprise GosNIIGenetika through repeated sequencing and identification using species-specific primers.

Properties of strains-producers.

The claimed strain of Bacillus simplex 948 P-1TS deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) Federal State Unitary Enterprise GosNIIgenetika under registration number VKPM B-12245, has the following characteristics. Daily culture cells form endospores. Optimal growth conditions are 20-25°C. On dense nutrient media they form colonies 3-4 mm in diameter, round, shiny, with a smooth edge, slightly convex, translucent.

Bacillus megaterium 312 TS was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) of the Federal State Unitary Enterprise GosNIIgenetika under the registration number VKPM B-12244 - cells are gram-positive, motile, spore-forming rods, single or connected in long chains; colonies are rounded, smooth, white, convex, oily shiny, wavy-fringed edge, optimum growth temperature is 20-25℃.

For cultivation, LB medium (Luria-Bertani) is used. The culture is maintained by reseeding on solid media, stored in a glycerol solution at -70°C or in a freeze-dried state.

biochemical signs.

Bacillus simplex 948 P-1TS ferments L-lysine, urea, 5-bromo-3-indoxylnonanoate.

Bacillus megaterium 312 TS ferments L-ornithine, L-arginine, L-lysine, utilizes galacturic acid, potassium 5-ketogluconate, D-maltose, adonitol, palatinose, D-glucose, L-arabinose, D-arabitol, L-rhamnose, inositol, D-cellobiose.

To obtain a consortium, monocultures were grown separately during the day, then suspensions of monocultures were prepared with a cell concentration of at least ¹⁰⁴ CFU/ml and mixed in equal ratios (1:1), then they were grown by submerged cultivation at a temperature of 28°C for 12 -16 hours, at 200 rpm. Culture growth was monitored by sampling every 2 hours and the concentration of microbial cells was determined on a Thermo spectrophotometer at a wavelength of 540 nm. The resulting culture fluid contains at least 10 ⁸ CFU/ml (60-100% spores) and secondary metabolites (antibiotic substances, proteolytic enzymes, hormones, etc.), due to which the specific activity of the liquid preparation based on a consortium of microorganisms is enhanced.

The claimed consortium has auxin-producing, chitinase activity, growth at low positive temperatures.

Phosphorus is one of the most important mineral elements in the life of plants, which are able to absorb it only in the form of inorganic phosphate anions, mainly in the form of H ₂ PO ^4- . Despite the high content of total phosphorus in the soil, its bioavailability, as a rule, is a limiting factor for plant growth, development and productivity. Representatives of the genera Bacillus are considered to be one of the most active phosphate mobilizers.

The mobilization activity of the Bacillus simplex 948 P-1TS and Bacillus megaterium 312 TS strains of sparingly soluble mineral phosphates in the NBRIP liquid medium was studied under sterile experimental conditions. The source of phosphorus was Ca ₃ (PO ₄ )2, which was added to the medium at the rate of 5 g/L. The studies were carried out in triplicate. The inoculum was prepared by growing bacteria for 24 hours in test tubes on LB agar medium. An aqueous suspension of bacteria was prepared and added to flasks with liquid NBRIP medium. The bacteria Bacillus simplex 948 P-1TS and Bacillus megaterium 312 TS were cultivated for 10 days at 15°C under periodic conditions on shakers (190 rpm). After the end of cultivation, the test samples were centrifuged for 10 minutes at 6000 rpm. The concentration of soluble phosphorus (PO ₄ )-3 in the centrate culture fluid was determined calorimetrically using ammonium molybdate. The content of water-soluble phosphorus accumulated by the Bacillus simplex 948 P-1TS and Bacillus megaterium 312 TS strains was estimated from the calibration curve, from the difference in its concentration in the experimental (inoculated) and control (sterile) variants. The research results indicate that Bacillus simplex 948 P-1TS and Bacillus megaterium 312 TS actively mobilize phosphates from sparingly soluble inorganic (Ca ₃ (PO ₄ ) ² ) compounds. It was found that in the test samples the content of soluble phosphorus varied within mg/l, and in the control - mg/l (table 1).

Table 1. Dissolution of phosphates by strains of Bacillus sp. at different cultivation temperatures Strain Cultivation temperature, °C 15 28 Bacillus simplex 948P-1TS 0.62 mg/ml 0.93 mg/ml Bacillus megaterium 312TS 0.33 mg/ml 0.73 mg/ml Control 0.27 mg/ml 0.25 mg/ml

At the next stage of the research, the ability of Bacillus simplex 948P-1 TS and Bacillus megaterium 312 TS strains to produce the phytohormones auxin and gibberellin was evaluated.

Phytohormones contribute to the awakening and rapid germination of seeds, stimulate the growth of the apical bud and inhibit the development of axillary shoots. At high concentrations, auxins increase the production of the phytohormone ethylene, which inhibits growth processes, but at the same time switches the metabolism to the production of enzymes that activate the protective functions of the plant organism and increase its resistance to phytopathogens. Gibberellins actively influence the flowering of plants, the formation and development of ovaries. A high concentration of these phytohormones gives plants parthenocarpic properties (the ability to self-pollinate). Even such a pronounced plant growth inhibitor as abscisic acid plays a positive role in the ontogeny of winter crops, causing the timely transition of plants to a dormant state, gradually stopping all growth processes, thereby increasing the resistance of winter crops to low temperatures, frosty and little snowy winters. These microorganisms Bacillus simplex 948P-1 TS and Bacillus megaterium 312 TS, consuming plant root exudates, release growth hormones in response, which stimulate root formation and plant growth processes.

Quantification of phytohormones was determined using a spectrophotometer. The strains were cultured in LB medium supplemented with L-tryptophan (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 1 g/l as a precursor of indole-3-acetic acid (IAA) (Indolyl-3-acetic acid - chemical auxin formula). Cultivation was carried out in a periodic mode with stirring at a speed of 140 rpm at a temperature of 5 and 22 °C. The content of IAA in the culture suspension was determined using Salkovsky's reagent. All studies were performed in triplicate. The results are presented in table. 2 and tab. 3.

Table 2 The content of IAA in the culture liquid of the studied bacterial strains, μg/ml. Strains Growing temperature 5°C 22°C Bacillus simplex 948P-1TS 16.0 ± 3.6 95.2 ± 10.8 Bacillus megaterium 312TS 7.7 ± 0.8 17.0 ± 2.9 Table 3 The content of gibberellin in the culture liquid of the studied bacterial strains, µg/mL Strains Growing temperature 15°C 27°С Bacillus simplex 948P-1TS 0.39±0.03 0.44±0.01 Bacillus megaterium 312TS 0.37±0.00 0.41±0.00

The antifungal activity of the claimed consortium of strains was tested against 4 phytopathogenic fungi: Alternaria sp., Fusarium avenaceum (Fr.) Sacc., Fusarium graminearum (Schwabe), Microdochium nivale (Fr) Samuels and IC Hallet.

Bacteria were sown with a bacteriological loop in a continuous lawn on the surface of nutrient agar and cultivated for 24 hours at 28°C. On the day of the experiment, strains of phytopathogenic fungi were sown in a continuous lawn on potato-glucose agar for antifungal activity. Then, holes were cut out with a cork drill and 100 μl of bacterial suspension was added - bacterial strains Bacillus simplex 948 P-1TS VKPM B-12245 and Bacillus megaterium 312 TS VKPM B-12244 taken in a ratio of 1: 1 and having a total cell titer of at least 108 CFU /ml One plate was left with empty wells as a control. All cups were cultured for 72 h at 28°C. Antifungal activity was determined by the zone of inhibition of the growth of a phytopathogenic fungus around the wells. The measurement results are presented in Table 4.

Table 4 Antifungal activity of strains of Bacillus spp. depending on the cultivation temperature. Strain t°C Growth retardation zones, mm Alternaria sp. F. avenaceum F. graminearum M. nivale Bacillus simplex 948P-1TS 5 0 0 0 0 22 0 0 0 0 Bacillus megaterium 312TS 5 21.0 ± 2.12 17.0 ± 1.41 13.0 ± 1.06 23.0 ± 0.14 22 0 0 0 0

The synthesis of antifungal substances (antibiotics, hydrolytic enzymes, toxins) is one of the most common effects of microbial antagonism.

Determination of the enzymatic activity of strains of Bacillus simplex 948P-1 TS and Bacillus megaterium 312 TS "antagonist bacteria" was carried out using standard tests for lipase, chitinase and protease (table 5).

Table 5 Production of hydrolytic enzymes by Bacillus simplex 948P-1 TS and Bacillus megaterium 312 TS strains Strain Lipase (µmol/(ml min)) protease
(u/(ml 30 min)) chitinase
mcg/ml Bacillus simplex 948P-1TS 2.63±0.28
0.36±0.04 0.20±0.02
0.09±0.01 114.27
150.18 Bacillus megaterium 312TS 1.46±0.13
1.33±0.12 0.01± 0.13
1.18±0.02 97.76
76.55

The invention is illustrated by the following examples.

Example 1. To assess the growth-stimulating activity of a suspension of the consortium Bacillus simplex 948 P-1TS VKPM B-12245 and Bacillus megaterium 312 TS VKPM B-12244, taken in a 1:1 ratio and having a total cell titer of at least 108 CFU/ml. on the growth and development of seeds of wheat (Triticum aestivum L.), laboratory experiments were carried out, for comparison, the commercial drug Raxil Ultra (Bayer, Germany) and the biopesticide Fitosporin-M Universal (NVP BashIncom, Russia) were taken. The experiment was carried out both with monocultures of microorganisms and with a consortium.

Before sowing, the surface of the seeds was sterilized, then inoculated with suspensions for 2 hours and dried in air. Seeds were germinated in filter paper rolls at 22°C. Control seeds inoculated with sterile water served as negative controls. Commercially available preparations served as positive controls. The seeds were treated according to the manufacturer's instructions.

Length (a) and biomass (b) of wheat seedlings were measured by measurements on the 14th day of treatment. Treatments not related by the same letter in each column are significantly different from each other (p   < 0.05; means ± standard deviation).

The results of laboratory experiments showed that in all experimental groups, with the exception of Raxil-Ultra, the treatment significantly increased the length of the root and shoot compared to the control (Fig. 1). Thus, the shoot length was most favorably affected by the treatment with Bacillus simplex 948P-1 TS, while the treatment with Raxil-Ultra reduced the recorded values by more than 30%. Root biomass accumulation was most stimulated by seed treatment with Bacillus simplex 948P-1 TS and Bacillus megaterium 312 TS consortium, while shoot biomass was most stimulated by phytosporin-M treatment compared with the control group. (See Fig.1. Influence of strains of the genus Bacillus spp. on the morphometric parameters of wheat seedlings (Triticum aestivum L.).

In general, it should be noted that the treatment of seeds with the claimed consortium of bacteria leads to an increase in the main parameters of plant growth.

Example 2. Evaluation of wheat yield in the field.

Seed treatment was carried out according to the same scheme as in Example 1. The experiment was carried out both with monocultures of microorganisms and with a consortium. To assess the biological efficiency, winter wheat seeds were treated with a suspension of a consortium of Bacillus simplex 948P-1 TS and Bacillus megaterium 312 TS strains, taken in a 1:1 ratio and having a total cell titer of at least 10 ⁸ CFU/ml. Wheat seeds were soaked in bacterial suspension for 2 hours. Seeds were treated with commercial preparations Raxil Ultra (Bayer, Leverkusen, North Rhine-Westphalia, Germany), which is a fungicide containing tebuconazole and Fitosporin-M (Russian Federation), containing Bacillus subtilis 26D spores, according to the manufacturer's recommendations. Control seeds were soaked in sterile water.

Sowing was carried out in the third decade of August. The field experience was laid on plots with an accounting area of 1 m ² , the distance between rows was 20 cm. The seeding rate was 400 seeds per plot. The number of sowing rows - 4. Seeding depth - 5 cm.

Winter wheat was harvested in the phase of full ripeness. The effects of various treatments were evaluated after harvest. We took into account such characteristics as the weight of 1000 grains, plant height, the number of productive stems, the length of the main ear, the number of grains per ear, and the mass of grain per ear. The results are presented in Table 6. The results obtained were statistically processed using Origin 2021 software (OriginLab Corporation, Northampton, Massachusetts, USA).

The yield of winter wheat is largely determined by the nature of plant growth and development during the growing season. A significant contribution to the formation of the crop is made by the stem as a photosynthetically active organ, as well as an organ of a temporary supply of substances.

The results of the study showed that pre-sowing treatment of seeds with the chemical fungicide Raxil Ultra, the biological product Fitosporin-M and the consortium of bacterial strains Bacillus simplex 948P-1 TS and Bacillus megaterium 312 TS had a different effect on the yield of winter wheat.

An analysis of the grain yield showed that seed dressing with the chemical fungicide "Raxil Ultra" did not significantly affect the values of the indicator (table 6). Interestingly, in the Fitosporin-M, Bacillus simplex 948P-1 TS and Bacillus megaterium 312 TS groups, the grain yield was slightly lower than in the control. In turn, the yield increase was recorded when seeds were treated with a suspension of a consortium of Bacillus simplex 948P-1 TS and Bacillus megaterium 312 TS strains, which made it possible to obtain a yield increase of 50.2% compared to the control group (Table 6).

Table 6. Effect of treatment of winter wheat seeds with various preparations and bacterial strains on productivity indicators. Experiment The number of grains in the ear, pcs. Weight of 1000 grains, g Yield, g/m ² Control 45.8 ± 11.70 38.31 395.23 Raxil-Ultra 42.73 ± 9.63 35.9 400.40 Fitosporin-M 42.67 ± 9.66 35.72 331.20 Bacillus megaterium312 TS 38.26 ± 9.74 38.93 357.86 Bacillus simplex 948P-1TS 41.27±2.46* 34.51 285.12 Consortium of Bacillus simplex 948P-1 TS and Bacillus megaterium 312 TS 46.13 ± 8.57 39.73 593.74

Asterisks indicate statistically significant differences from control at p < 0.05 (n = 400).

Example 3 Effect of seed pretreatment with a consortium of bacteria Bacillus simplex 948P-1 TS and Bacillus megaterium 312 TS on the mitotic index.

In this experiment, the method of calculating the mitotic index was used to evaluate growth processes at the cellular level. The effect of a consortium of Bacillus simplex 948P-1 TS and Bacillus megaterium 312 TS bacteria, taken in a 1:1 ratio and having a total cell titer of at least 108 CFU/ml, was evaluated.

The studied bacterial strains were cultivated at 5°C and 25°C in LB medium. Barley seeds (n = 30) were soaked in a bacterial suspension for 2 h, then placed in sterile Petri dishes (10 seeds per dish) on the surface of wet filter paper in a checkerboard pattern with embryos in one direction. Petri dishes were kept at 20 ± 2°C for 3 days. Control seeds were soaked in distilled water. Root tips were fixed in Carnoy's solution [(ethanol (99%): glacial acetic acid (3:1)] for 24 h and stained with acetocarmine dye. Plant cells were examined under a microscope and the mitotic index, phase indices (I) of dividing cells were calculated. .

Mitotic index (%), calculated by formula (1)

MI = (Number of Dividing Cells/Total Cells) × 100 (1)

The effect of seed bacterization on the mitotic index and the frequency of mitotic phases are shown in Table 7.

Table 7. Mitotic index of oat seedling root meristem cells affected by treatment with the genus Bacillus. Strain 5°C 22°C 45°С N n MI±m N n MI±m N n MI±m Bacillus megaterium312 TS 1411 241 17.08±0.56 815 166 20.36±0.76 506 74 16.62±0.48* Bacillus simplex 948P-1TS 3201 830 25.93±0.72* 3084 597 19.36±0.42 1326 167 12.59±0.53* control 3454 351 10.16±0.89 3454 351 10.16±0.89 3454 351 10.16±0.89

Note: N is the total number of microscopic cells; n is the number of investigated phases; MI ± m - mitotic index. *differences with control are significant at P<0.05

Treatment of oat seeds with a consortium of Bacillus simplex 948P-1 TS and Bacillus mega-terium 312 TS grown at +5°C led to the maximum increase in the intensity of cell divisions, especially in Bacillus simplex 948P-1 TS (25.93%). The intensity of cell divisions compared with the control increased by an average of 1.5 times.

Thus, a significantly positive effect was found when seeds were treated with bacterial strains grown at 5 and 22°C on the mitotic activity of the cells of the apical meristem of the roots of oat seedlings, which in turn confirms the growth-stimulating and protective effects of the consortium on the plant.

EFFECT: invention allows to increase crop yields, their adaptive capabilities by stimulating plant growth, as well as stress resistance to abiotic environmental factors.

Claims (1)

A consortium of psychrotolerant bacterial strains for biological protection and growth stimulation of agricultural plants, characterized in that it includes bacterial strains Bacillus simplex 948 P-1TS VKPM B-12245 and Bacillus megaterium 312 TS VKPM B-12244.

Images