RU2757123C1 - Bacterial strain pantoea agglomerans f19 to increase the productivity of grain crops - Google Patents

Abstract

FIELD: biotechnology, microbiology and agriculture.

SUBSTANCE: invention relates to biotechnology, microbiology and agriculture. The bacterial strain Pantoea agglomerans F19, which possesses phosphate-solubilizing, phytoprotective and growth-stimulating activity, was deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under the registration number RNCIM B-13869.

EFFECT: bacterial strain Pantoea agglomerans RNCIM B-13869 can be used to improve the 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 phosphate-mobilizing, 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 Pseudomonas species 181a B-6646 (p. RF No. 2451069, IPC C12N 1/20, A01N 63/00, C12R 1/38, publ. 05/20/2012, bull. No. 14), which has the ability to release phosphates from insoluble mineral raw materials, active against fungi of the genus Fusarium, stimulates plant growth, increases productivity. The strain is not toxic to plants and can be recommended for creating a biophosphoric fertilizer with fungicidal properties on its basis.

The strain was isolated from decaying rock, which may be a limiting factor for its use in soils, in addition, it has a narrow spectrum of fungicidal activity.

Known phosphate-mobilizing strain of soil bacteria Lelliottia nimipressuralis CCM 32-3, producer of phytohormones, deposited in the All-Russian collection of industrial microorganisms FGBU GosNIIgenetika under registration number VKPM B-12783, used to obtain a biological product to optimize the mineral nutrition of plants, stimulate their growth and increase productivity. RF No. 2676926 C1, IPC C12N 1/20, A01N 63/02, C12R 1/01, publ. 11.01.2019, bul. No. 2).

The disadvantages of this strain are its limited ability to leach phosphorus and the lack of phytopathogenic activity.

Known bacterial strain Bacillus megaterium var. phosphaticum BKM B-2357D, mobilizing phosphorus and silicon from objects of the lithosphere (p. RF No. 2327737, IPC C12N 1/20, C05A 11/08, C12R 1/11, publ. 27.06.2008).

However, this strain does not possess growth-stimulating and phytoprotective activities.

Closest to the claimed is soil phosphate-dissolving bacteria Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3, which has the ability to suppress phytopathogenic fungi and bacteria. The Pseudomonas chlororaphis ssp chlororaphis Vsk-26a3 strain releases phosphates from insoluble mineral raw materials, has a broad spectrum of action against phytopathogenic fungi (suppresses 15 species) and bacteria, and also stimulates plant growth and increases their productivity. The strain was deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures "GKPM-Obolensk" under the number B-7427 (p. RF No. 2603281 C1, IPC C12N 1/20, A01N 63/00, publ. 27.11.2016, bull. No. 33) ...

The disadvantages of the known strain are its limited functional ability to leach phosphorus and a high susceptibility to lysis of colonies on a slant agar medium.

Thus, all known strains capable of mobilizing phosphorus from sparingly soluble phosphates 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 phosphate 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 phosphate solubilizing, phytoprotective and growth-stimulating activity.

The technical problem posed is solved by identifying a new natural strain Pantoea agglomerans F19 VKPM B-13869, which has a significant phosphate-solubilizing ability, as well as phytoprotective and growth-stimulating activity.

The Pantoea agglomerans F19 strain was isolated under natural conditions from soil (Primorsky Krai, Russia) by direct seeding on Pikovskaya agar medium at 22-25 ° C and deposited in the All-Russian collection of industrial microorganisms under the number VKPM B-13869.

The bacterial strain Pantoea agglomerans F19 VKPM B-13869 possesses phosphate solubilizing ability. The activity of phosphorus solubilization was determined by streaking microorganisms on plates with a dense agarized Pikovskaya medium. Activity is detected in the presence of a clearing zone (dissolution of sparingly soluble calcium phosphates) around the grown colonies of microorganisms.

The strain was identified by 16S PH analysis. The sequence analyzed was 589 base pairs 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 at 96 ° С for 1 min, 30 cycles including 96 ° С for 10 sec, 55 ° С for 20 sec, 72 ° С - 30 sec and final elongation at 55 ° С - 20 sec, 72 ° С - 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 medium Pikovskaya strain forms round, smooth, convex, shiny yellow colonies with smooth edges, the center is not expressed. In the smear, sticks measuring 0.5-1.0 × 1.0-3.0 μm, gram-negative, are found. Spores and cysts do not form. Catalas are positive. Oxidase negative. Indole is not formed. Carbohydrates are used: L-arabinose, galactose, xylose, maltose, mannitol, mannose, L-rhamnose, sucrose and trehalose. Glucose is fermented to form acid without gas. Hydrogen sulfide is not formed, urea is not hydrolyzed. Reduce nitrate. Dispose of sodium citrate.

For long-term storage, the culture of bacteria is inoculated into test tubes with semi-liquid medium of Pikovskaya with an injection with a loop. The composition of the medium: Ca ₃ (PO ₄ ) ₂ - 5.0 g, glucose - 20.0 g, NaCl - 0.2 g, MgSO ₄ - 0.1 g, MnSO ₄ - traces, FeSO ₄ - traces, agar- agar - 4.0 g. 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 agar slant tubes. Cultivated at room temperature. Pikovskaya's medium (for the isolation of microorganisms that dissolve sparingly soluble calcium phosphates): Ca ₃ (PO ₄ ) ₂ - 5.0 g, glucose - 20.0 g, NaCl - 0.2 g, MgSO ₄ - 0.1 g, MnSO ₄ - traces, FeSO ₄ - traces, agar-agar - 20.0 g.

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 phosphorus by a culture of bacteria Pantoea agglomerans F19 VKPM B-13869 (qualitative assessment).

A qualitative assessment of the ability of the Pantoea agglomerans F19 strain VKPM B-13869 to mobilize hard-to-reach phosphorus compounds was determined by streaking microorganisms on plates with a dense agarized Pikovskaya medium (composition: Ca ₃ (PO ₄ ) ₂ - 5.0 g, glucose - 20.0 g, NaCl - 0.2 g, MgSO ₄ - 0.1 g, MnSO ₄ - traces, FeSO ₄ - traces, agar-agar - 20.0 g). _{Ca 3} (PO ₄ ) ₂ served as a source of insoluble phosphorus. Activity was detected in the presence of a clearing zone (dissolution of sparingly soluble calcium phosphates) around the grown colonies of microorganisms. The diameter of the zones of dissolved calcium phosphates in experiments with the culture of bacteria Pantoea agglomerans F19 VKPM V-13869 was 16–21 cm. In this regard, it can be concluded that the Pantoea agglomerans F19 VKPM V-13869 strain is highly capable of mobilizing hardly soluble phosphates.

Example 2. Solubilization of phosphorus by a culture of bacteria Pantoea agglomerans F19 VKPM B-13869 (quantitative assessment).

The quantitative assessment of the ability of the Pantoea agglomerans F19 strain VKPM B-13869 to mobilize hard-to-reach phosphorus compounds was determined in Pikovskaya's liquid medium (without adding agar). Ca3 (PO4) 2 served as a source of insoluble phosphorus. 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 Pikovskaya agar medium. 1 ml of bacterial suspension at a concentration of 1 × 10 ⁷ CFU / ml was introduced into flasks with Pikovskaya's liquid medium. Control - Pikovskaya medium without inoculation. The studies were carried out in triplicate.

Bacteria Pantoea agglomerans F19 VKPM B-13869 were cultured for 3 days at 24 ° C under periodic conditions on shaking (230 rpm). After the end of the cultivation, the test samples were centrifuged for 10 minutes at 6000 rpm. The concentration of soluble phosphorus was determined colorimetrically using molybdic acid ammonium and ascorbic acid as a reducing agent. The content of water-soluble phosphorus accumulated by the culture of the studied batkeria was calculated from the difference in its concentration in the experimental (inoculated) and control (sterile) variants. The research results indicate that the bacteria Pantoea agglomerans F19 VKPM B-13869 actively mobilizes phosphates from sparingly soluble inorganic (Ca3 (PO4) 2) compounds. It was revealed that in the experimental samples the content of soluble phosphorus fluctuated within 161-204 mg / l, and in the control - 17.7-20.0 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 Pantoea agglomerans F19 strain VKPM B-13869 on the germination energy of cereal seeds

A working solution was prepared: Pantoea agglomerans F19 VKPM B-13869 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%, and of barley seeds by an average of 20% was observed in comparison with the untreated control.

Example 4. Evaluation of the effect of the Pantoea agglomerans F19 strain VKPM B-13869 on the laboratory germination of cereal seeds.

A working solution was prepared: the Pantoea agglomerans F19 strain VKPM B-13869 was grown in 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 16%, of spring wheat by 18% was observed.

Example 5. Evaluation of the effect of the strain Pantoea agglomerans F19 VKPM B-13869 on the length of the shoot and the length of the roots of seedlings of cereal crops.

A working solution was prepared: the Pantoea agglomerans F19 strain VKPM B-13869 was grown in 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 22%, of spring wheat by 7% was observed; an increase in the length of the root of spring barley by 30%, spring wheat - by 65%.

Example 6. Etching of seeds of barley and wheat before sowing with the bacterial strain Pantoea agglomerans F19 VKPM B-13869.

The seeds were sprayed from a spray bottle with a culture of bacteria Pantoea agglomerans F19 VKPM B-13869 at a concentration of 1 × 10 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 bacteria Pantoea agglomerans F19 VKPM B-13869 was observed.

Claims (1)

The bacterial strain Pantoea agglomerans F19 with phosphate solubilizing, phytoprotective and growth-stimulating activity and deposited in the All-Russian collection of industrial microorganisms (VKPM) under the registration number VKPM B-13869.