RU2757506C1 - Pseudarthrobacter equi s2 bacterial strain for increasing the productivity of grain crops - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, microbiology and agriculture. The bacterial strain Pseudarthrobacter equi S2, possessing potassium-solubilizing, phytoprotective and growth-stimulating activity, was deposited in the Russian national collection of industrial microorganisms (RNCIM) under the registration number RNCIM B-13868.

EFFECT: bacterial strain Pseudarthrobacter equi RNCIM B-13868 can be used to improve plant nutrition, improve soil biocenosis and increase the yield of grain crops.

1 cl, 6 ex

Description

The invention relates to biotechnology, microbiology and agriculture and relates to the production of bacterial strains with potassium-solubilizing, phytoprotective and growth-stimulating activity. The strain is promising for obtaining bacterial preparations for improving plant nutrition, improving biocenosis and increasing the yield of grain crops.

One of the aspects of the positive influence of microorganisms on plants is the improvement of their mineral nutrition. Therefore, the ability to convert sparingly soluble compounds of macro- and microelements into a soluble form suitable for absorption by roots is an important factor in stimulating plant growth and development.

Known strain Bacillus mucilaginosus Bac-10 (p. RF No. 2289621, IPC C12N 1/20, A01N 63/00, C12R 1/07, publ. 20.12.2006), compatible with some chemical pesticides and capable of transforming silicates, apatites, tricalcium phosphate , sodium nucleonate.

However, the patent does not provide information on the possibility of accumulation of potassium in dissolved form.

A fertilizer based on the bacterial strain Bacillus mucilaginosus - Bac 1208, capable of producing an exopolysaccharide that creates a useful microflora of the rhizosphere, is known from the prior art (p. RF No. 2081867, IPC C05F 11/08, C12N 1/20, publ. 20.06.1997), and fertilizer based on the bacterial strain Bacillus mucilaginosus, capable of transforming poorly soluble compounds of phosphorus and potassium into a form accessible to plants, with phytopathogenic activity (p. RF No. 2408722, IPC C12N 1/20, C05F 11/08, publ. 10.01.2011).

However, the authors did not provide any information on the qualitative or quantitative release of potassium, in addition, the growth-stimulating and phytoprotective role was not described.

It is known to use as a fertilizer (p. RF No. 2148571, IPC C05F 11/08, A01N 63/00, publ. 05/10/2000) a dietary supplement consisting of associations of nitrogen-fixing bacteria Azotobacter chroococcum and Beijerinckia fluminensis, taken in the ratio (1-0, 5) :( 1-0.5) and decompose phosphorus- and potassium-containing compounds of bacteria Bacillus megaterium and Bacillus mucilaginosus, taken in the ratio (1-5: 0.5-2).

The disadvantage of this solution is the multicomponent composition and a small amount of liberated potassium. Not described, also phytoprotective and growth-stimulating role of these microorganisms.

The closest is the bacterial strain Paenibacillus mucilaginosus Pm 2906 VKPM B-12259 (p. RF No. 2626543, IPC C12N 1/20, C05F 11/08, publ. 28.07.2017), which promotes the formation of metabolic products that accelerate the growth of the root and vegetative systems of plants and stimulating the production of plant hormones. This strain has the ability to dissolve water-insoluble compounds of phosphorus and potassium in the soil, and also actively participates in the nitrification process. The bacterial strain Paenibacillus mucilaginosus Pm 2906 has an increased ability to synthesize bacteria with a wide range of suppression of phytopathogenic microflora and contributes to an increase in stress resistance, survival and productivity of a wide range of agricultural and ornamental crops.

However, the authors did not provide any information on the qualitative or quantitative release of potassium.

Thus, all known strains capable of mobilizing potassium from poorly soluble compounds or possessing phytoprotective and / or growth-stimulating activity have a number of disadvantages due to insufficiently high efficiency and laboriousness of the biomass production method, low level of potassium solubilization and plant growth stimulation. In addition, most of the known strains, to achieve these properties, are used mainly as part of consortia and do not have an independent phytoprotective and growth-stimulating activity.

The technical problem posed to the invention is the expansion of the range of strains with potassium-solubilizing, phytoprotective and growth-stimulating activity.

The technical problem posed is solved by identifying a new natural strain Pseudarthrobacter equi S2 VKPM B-13868, which has a significant potassium-solubilizing ability, as well as phytoprotective and growth-stimulating activity.

The Pseudarthrobacter equi S2 strain was isolated under natural conditions from soil (Primorsky Krai, Russia) by direct plating on GRM agar at 22-25 ° C and deposited in the All-Russian collection of industrial microorganisms under the number VKPM B-13868.

The bacterial strain Pseudarthrobacter equi S2 VKPM B-13868 has a potassium-solubilizing ability. The activity of potassium solubilization was determined by inoculation of microorganisms on a modified Alexandrov medium of the following composition (per 1000 ml): agar - 20 g; powder of aluminosilicates 3 g; glucose - 5 g; MgSo ₄ * 7 H ₂ O - 0.5 g; CaCO ₃ - 0.1 g; FeCl ₃ - 0.006 g; Ca ₃ (PO ₄ ) - 2 g; + bromothymol blue - 2.5 g. Activity is detected in the presence of a clearing zone (dissolution of potassium aluminosilicates) around the grown colonies of microorganisms.

The strain was identified by 16S RNA analysis. The sequence analyzed was 588 bp long. To amplify the 16S rRNA region of the samples, a pair of primers BAK11w 5'-AGTTTGATCMTGGCTCAG-3 'and BAK2 5'-GGACTACHAGGGTATCTAAT-3' were used, which are optimized for identifying a wide range of bacterial DNA (Zucol et al., 2006). The amplification reaction was carried out using an iCycler thermal cycler (Bio-Rad Laboratories, USA), programmed for the following reaction conditions: preliminary denaturation 96 ° C - 1 min, 30 cycles including 96 ° C - 10 sec, 55 ° C - 20 sec, 72 ° C - 30 sec and final elongation 55 ° C - 20 sec, 72 ° C - 3 min. The volume of the reaction mixture was 25 μl containing 0.3 mM of primers BAK11w and BAK2, 0.2 mM of each nucleotide, 2.5 μl of 10X Encyclo buffer, 0.5 μl of 50X Encyclo polymerase, and 50 ng of DNA sample. The amplification result was analyzed in 1% agarose gel with the addition of ethidium bromide. The resulting specific product with a size of 780 bp. was excised from the gel, purified and used in the sequencing reaction using the BigDye Terminator v3.1 according to the manufacturer's recommendations (Thermo Fisher Scientific, USA).

Cultural, morphological and biochemical characteristics of the strain.

On agar nutrient agar, the strain forms uniform, dotted, glossy, flat, smooth colonies of pale white color with even edges, the center is not pronounced. In a smear of a daily culture, short single rods measuring 0.4-0.7 microns are found, in a smear of a 3-day culture of cocci are gram-positive. Spores and cysts do not form. Aerobes. Cultivated at a temperature of 24-27 ° C. Optimum pH 6.0-6.4. Catalase-positive, oxidase-negative. Casein, tyrosine, gelatin and starch are hydrolyzed (slowly, 3 days). They do not reduce nitrate, do not produce acid from glucose, glycogen, lactose, sucrose, xylose, ribose, mannitol, maltose. Transfer up to 2% NaCl.

For long-term storage, the culture of bacteria is inoculated into tubes with semi-liquid nutrient agar. After 3 days, it is poured with sterile liquid paraffin with a layer of 0.5-1 cm. Then the culture is placed in a refrigerator at 4 ° C. (Storage method under vaseline oil).

For propagation, the strains are inoculated into tubes with slant nutrient agar. Cultivated at a temperature of 24-27 ° C.

Fermentation of the strain was carried out in the following way: sterile nutrient broth was added to test tubes with a daily culture of bacteria so that it completely covered the surface of the dense nutrient medium. After 1 hour, the thus obtained bacterial washes are used to inoculate the nutrient broth flasks. Inoculated flasks are incubated at 24 ° C for 7 days with periodic (2 times a day) shaking by hand until the bacterial ^{count is at least 1 × 10 7} μl / ml.

Example 1. Solubilization of potassium by a culture of bacteria Pseudarthrobacter equi S2 VKPM B-13868 (qualitative assessment).

The ability of the Pseudarthrobacter equi S2 strain VKPM B-13868 to mobilize inorganic potassium was assessed by streaking microorganisms on plates with Aleksandrov's solid agar medium of the following composition (per 1000 ml): agar - 20 g; aluminosilicate powder (KAlSiO ₄ ) - 3 g; glucose - 5 g; MgSO ₄ * 7 H ₂ O - 0.5 g; CaCO ₃ - 0.1 g; FeCl ₃ - 0.006 g; Ca ₃ (PO ₄ ) - 2 g; + bromothymol blue - 2.5 g. Activity is detected in the presence of a clearing zone (dissolution of potassium aluminosilicates) around the grown colonies of microorganisms for 7-10 days. The potassium-mobilizing activity of bacteria was assessed quantitatively as the area of the phosphate dissolution zones. As a result, the diameter of the dissolution zones of aluminosilicates was observed - 15-20 mm.

Example 2. Solubilization of potassium by a culture of bacteria Pseudarthrobacter equi S2 VKPM B-13868 (quantitative assessment).

The quantitative assessment of the ability of the Pseudarthrobacter equi S2 strain VKPM B-13868 to mobilize hard-to-reach potassium compounds was determined in Aleksandrov's liquid medium (without the addition of agar). _{Potassium aluminosilicate (KAlSiO 4} ) was used as a source of insoluble potassium. The medium was poured into 100 ml Erlenmeyer flasks (volume 250 ml). The inoculum was prepared by growing bacteria for 24 hours in test tubes on Aleksandrov's agar medium. 1 ml of a suspension of bacteria at a concentration of 1X10 ⁷ CFU / ml was introduced into flasks with liquid Alexandrov's medium. Control - Alexandrov's medium without inoculation. The studies were carried out in triplicate.

The bacteria Pseudarthrobacter equi S2 VKPM B-13868 were cultivated for 5 days at 24 ° C under periodic conditions on shaking chairs (230 rpm). After the end of the cultivation, the test samples were centrifuged for 10 minutes at 6000 rpm. The concentration of soluble potassium was determined by the tetraphenylborate method. The content of water-soluble potassium accumulated by the culture of the studied bacteria was calculated from the difference in its concentration in the experimental (inoculated) and control (sterile) variants. The research results indicate that the bacteria Pseudarthrobacter equi S2 VKPM B-13868 actively mobilizes potassium from hardly soluble inorganic (KAlSiO ₄ ) compounds. It was revealed that in the experimental samples the content of soluble potassium fluctuated within 167-184 mg / l, and in the control - 5.1-7.0 mg / l.

The growth-stimulating and phytoprotective effect was studied by identifying the potential effect of the studied bacteria when acting on the germination of seeds of cereal plants.

Example 3. Evaluation of the effect of the Pseudarthrobacter equi S2 strain VKPM B-13868 on the germination energy of cereal seeds

A working solution was prepared: Pseudarthrobacter equi S2 VKPM B-13868 was grown on a liquid nutrient medium at 23-25 ° C for 5-7 days until a concentration of 1 * 10 ⁹ CFU / ml was reached. Plant seeds, in the amount of 100 pieces of each variety, were soaked in the working solution, the exposure time was 30 minutes. Then all the seeds were laid out in 10 pieces on the surface of filter paper moistened with sterile water in sterile Petri dishes for germination. The seeds were germinated at 23 ° C. On the 3rd day, the germination energy was determined. As a result of the action of the drug, an increase in the germination energy of wheat seeds by an average of 18% was observed, and of barley seeds - by an average of 18% compared to the untreated control.

Example 4. Evaluation of the effect of the Pseudarthrobacter equi S2 strain VKPM B-13868 on the laboratory germination of cereal seeds.

A working solution was prepared: the Pseudarthrobacter equi S2 strain VKPM B-13868 was grown on a liquid nutrient medium at 23-25 ° C for 5-7 days until a concentration of 1 * 10 ⁹ CFU / ml was reached. Plant seeds, in the amount of 100 pieces of each variety, were soaked in the working solution, the exposure time was 30 minutes. Then all the seeds were laid out in 10 pieces on the surface of filter paper moistened with sterile water in sterile Petri dishes for germination. The seeds were germinated at 23 ° C. On the 7th day, the laboratory germination of seeds was assessed. As a result of the action of the drug, an increase in laboratory germination of seeds of spring barley by 15%, of spring wheat by 22% was observed.

Example 5. Evaluation of the effect of the strain Pseudarthrobacter equi S2 VKPM B-13868 on the length of the shoot and the length of the roots of seedlings of cereal crops.

A working solution was prepared: the Pseudarthrobacter equi S2 strain VKPM B-13868 was grown on a liquid nutrient medium at 23-25 ° C for 5-7 days until a concentration of 1 * 10 ⁹ CFU / ml was reached. Plant seeds, in the amount of 100 pieces of each variety, were soaked in the working solution, the exposure time was 30 minutes. Then all the seeds were laid out in 10 pieces on the surface of filter paper moistened with sterile water in sterile Petri dishes for germination. The seeds were germinated at 23 ° C. On the 7th, the shoot length and the length of the roots of the seedlings were assessed. As a result of the action of the preparation, an increase in the length of the aboveground part of spring barley by 21%, of spring wheat by 9% was observed; an increase in the root length of spring barley by 40%, spring wheat by 60%.

Example 6. Etching of seeds of barley and wheat before sowing with the bacterial strain Pseudarthrobacter equi S2 VKPM B-13868.

The seeds were sprayed from a spray bottle with a culture of bacteria Pseudarthrobacter equi S2 VKPM B-13868 at a concentration of 1x10 ⁴ CFU / ml at the rate of 100 ml per 1 kg of seeds. The seeds of barley and wheat treated in this way were laid out, observing sterility, on Petri dishes with a dense Czapek medium. 10 seeds were spread on one cup. In total, 100 barley seeds and 100 wheat seeds were taken into the experiment. Control - untreated seeds. As a result of the experiment, 100% fungicidal and bactericidal activity of the bacteria Pseudarthrobacter equi S2 VKPM B-13868 was observed.

Claims (1)

The bacterial strain Pseudarthrobacter equi S2, possessing potassium-solubilizing, phytoprotective and growth-stimulating activity and deposited in the All-Russian collection of industrial microorganisms (VKPM) under the registration number VKPM B-13868.