RU2634387C2 - Method for plant growth and development stimulation, increase of yield and protection against phytopathogenic fungi in arid zone - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method involves the treatment of seeds and plants with a suspension of cyanobacteria Anabaena constricta IPPAS B-2020 for 1 hour before planting. The treatment is also provided by spilling under the root in phase 2-4 of true leaf and in the flowering and budding phase. The suspension contains 5 g of dry cyanobacteria Anabaena constricta IPPAS B-2020 biomass per 1 l of water.

EFFECT: invention allows to increase seed germination and plant productivity and their resistance to phytopathogens.

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Description

The invention relates to agriculture, agricultural microbiology and biotechnology, and is intended to stimulate plant growth, increase productivity and protect against phytopathogenic fungi in the arid zone.

The most modern ways to increase productivity and protect plants from diseases are based on the use of microorganisms: bacteria, micromycetes and microalgae. Microalgae and cyanobacteria are used in the form of axenic or algologically pure cultures.

The Strain of cyanobacteria Nostoc muscorum f. linckia (Roth) Born et. Flash 28-1 106 - active non-symbiotic nitrogen fixer from the collection of the All-Russian Research Institute of Agricultural Microbiology to increase the yield of rice plants [RF patent No. 1180366]. Rice seeds were inoculated with a known strain, grown under the conditions of a micro-plot experiment, and plant productivity was determined.

The disadvantages of this method are:

- a narrow range of properties of cyanobacteria - has only stimulating properties;

- the use of one type of plant - rice;

- low yield of 18%.

The closest, in fact, to the claimed invention (prototype) is a method of stimulating the development, growth and productivity of plants using an algologically pure culture of Chlorella vulgaris [RF patent No. 2448453].

The disadvantages of this method are:

- lack of fungicidal and antioxidant activity of the culture;

- complicated periodic strait of plants throughout the entire cycle of growing plants.

The main objective of the invention is the creation of an effective technology for growing plants, providing stimulation of growth and development, increasing productivity and protection against phytopathogenic fungi in arid conditions.

The technical result from the use of the invention is to increase seed germination, increase growth energy, increase plant productivity, protect against phytopathogens, improve the health of plants and soils, due to the presence of antioxidant properties of cyanobacteria in an arid climate.

L., произрастающего в аридной зоне на территории Астраханского региона. Культура цианобактерий хранится на среде BG-11. Состав среды: (г/л): K₂HPO₄ - 0,04; MgSO₄⋅7H₂O - 0,075; CaCl₂⋅2Н₂О - 0,036; Na₂CO₃ - 0,02; ЭДТА - 0,001; лимонная кислота - 0,006; железо лимонно-аммиачное-0,006; микроэлементы - 1 мл [Нетрусов, А.И. Практикум по микробиологии [Текст]: учеб. пособие для высших учеб. заведений / А.И. Нетрусов, М.А. Егорова, Л.М. Захарчук [и др.] / Под ред. А.И. Нетрусова. - М.: Академия, 2005. - 352 с.].The technical result is ensured by the fact that before planting the seeds and plants are treated with a suspension of cyanobacteria Anabaena constricta IPPAS B-2020 with a concentration of 5 g of dry biomass of cyanobacteria per 1 liter of water for 1 h and provide spilling under the root in phase 2-4 of this leaf and in the phase of flowering and budding of plants. An algologically pure culture of cyanobacteria Anabaena constricta IPPAS B-2020 was isolated using the accumulative culture method from the rhizosphere of the arboreal plant Yasen Pencilvan

L. growing in the arid zone on the territory of the Astrakhan region. A cyanobacterial culture is stored on BG-11 medium. The composition of the medium: (g / l): K ₂ HPO ₄ - 0.04; MgSO ₄ ⋅ 7H ₂ O - 0.075; CaCl ₂ ⋅ 2Н ₂ О - 0.036; Na ₂ CO ₃ - 0.02; EDTA - 0.001; citric acid - 0.006; lemon-ammonia iron-0.006; trace elements - 1 ml [Netrusov, A.I. Workshop on Microbiology [Text]: textbook. allowance for higher education. institutions / A.I. Netrusov, M.A. Egorova, L.M. Zakharchuk [et al.] / Ed. A.I. Netrusova. - M.: Academy, 2005. - 352 p.].

Raw and dry biomass of cyanobacteria for the preparation of the suspension was obtained as follows. The cyanobacteria Anabaena constricta IPPAS B-2020 were cultured in a vortex bioreactor at a temperature of 28 ° C for 5 days at 60 rpm with a fluorescent lamp (4000 kelvin). Seeds were added in an amount of 20% of its total volume. During this time, there was a maximum increase in biomass - 47 mg / m ² / h dry matter. Then the biomass was dried in a thermostat at a temperature of 37 ° C to constant weight and crushed.

The cyanobacteria culture Anabaena constricta IPPAS B-2020 is deposited in the Collection of microalgae cultures (IPPAS) of the Federal State Budgetary Institution of Science of the Institute of Plant Physiology K.A. Timiryazev RAS under the number IPPAS B-2020. Certificate of deposit is attached.

Cells of cyanobacteria are blue-green, cylindrical, usually in the middle, laced. The width of the cells is 5.0-6.5 microns; the length of the cells is 3.0-4.4 microns, a length of 5-7 microns is found. Heterocysts are round spherical, 5-7 microns in diameter. Disputes are unknown.

A method of stimulating plant growth and development, increasing productivity and protecting against phytopathogenic fungi using cyanobacteria has been tested in the biotechnology laboratory of the FSBEI HPE Astrakhan State University, field experiments on open ground in the territory of the FSBEI HPE Technological Park Astrakhan State University, the beginning of the Astrakhan region and on the territory of the All-Russian Research Institute of Irrigated Vegetable and Gourd FGBNU VNIIOB, Kamyzyak, Astrakhan Region. Below are the results of testing.

Example 1. The study of the fungicidal properties of the culture of cyanobacteria Anabaena constricta IPPAS B-2020.

The fungicidal activity of the cyanobacteria culture was studied using the method of holes on phytopathogenic fungi Fusarium graminearum, Fusarium sporotrichioides, Fusarium culmorum, Fusarium poae, Alternaria tenuissima, Pythium ultimum, provided by the GNU Research Institute of Agricultural Microbiology of the Russian Agricultural Academy. A suspension of cyanobacteria in a concentration of 5 g of absolutely dry biomass of cyanobacteria per 1 liter of sterile distilled water was placed in the wells; distilled water was added as a control. Also in the experiment used a water-alcohol extract of cyanobacteria in a concentration of 100 mg of absolutely dry biomass of cyanobacteria per 1 ml of liquid (dilution of alcohol with water 1/1). A dilution of alcohol with water 1/1 was used as a control. The experiments were performed in four replicates. Table 1 shows the diameter of the zone of growth inhibition of fungi by cyanobacteria.

An analysis of the data showed that the spectrum of zones of inhibition of growth of phytopathogenic fungi ranges from 6.0 to 33.0 mm. The largest diameter of the fungal growth inhibition zone was noted when using a suspension of cyanobacteria in relation to Fusarium culmorum - 33.0 mm and Fusarium poae - 28.2 mm. The aqueous-alcoholic extract to a greater extent suppressed the development of Fusarium sporotrichioides with a zone of 27.3 mm (mycelial lysis was noted during microscopy).

Example 2. The study of the antioxidant properties of cyanobacteria Anabaena constricta IPPAS B-2020 photometric method.

Antioxidant activity (AOA) was determined by evaluating the colorimetry of free stable radicals based on the DPPH DPPH reaction (2,2-diphenyl-1-picrylhydrazyl (C ₁₈ H ₁₂ N ₅ O ₆ , M = 394.33) dissolved in ethanol, s a sample of antioxidant (AN) according to the scheme:

DPPH * + AN → DPPH-H + A *

DPPH (DPPH) - the test was performed by the photometric method with a 0.5 mM alcohol solution of DPPH (Sigma-Aldrich) at a wavelength of 510 nm on a PromEcoLab spectrophotometer PE-5300. 96% ethanol was used to prepare working solutions. Measurements were performed after 60 minutes. The following solutions were prepared for measurement:

- as a control (without the presence of an antioxidant): 1500 μl of 96% ethanol + 500 μl of a 0.5 mM DPPH solution;

- with the presence of an antioxidant (test liquids): 1480 μl of 96% ethanol + 500 μl of a 0.5 mM DPPH solution + 20 μl of the test substance;

- with the presence of an antioxidant (test liquids): 1400 μl of 96% ethanol + 500 μl of a 0.5 mM DPPH solution + 100 μl of the test substance.

To study the antioxidant properties of cyanobacteria, the following solutions were prepared:

- culture fluid of cyanobacteria;

- water extract at a concentration of 100 mg of absolutely dry biomass of cyanobacteria per 1 ml of water;

- water-alcohol extract in a concentration of 100 mg of absolutely dry biomass of cyanobacteria per 1 ml of liquid (dilution of alcohol with water 1/1). Antioxidant activity (I,%) was calculated by the formula:

I (%) = (A ₀ -A ₁ ) / A ₀ × 100,

where A ₀ is the optical density in the control,

A ₁ - the optical density of the studied liquids.

Research data on antioxidant activity are presented in table 2.

A pronounced antioxidant activity is possessed by an aqueous extract of cyanobacteria in a volume of 20 μl of 39.34%, as well as aqueous and aqueous-alcoholic extracts in a volume of 100 μl. The solutions prepared on the basis of the specificity of the method (culture fluid and extracts) revealed the antioxidant properties of cyanobacteria.

Example 3. The study of phytotoxicity and phytostimulation of cyanobacteria Anabaena constricta IPPAS B-2020

The phytotoxicity and phytostimulating properties of cyanobacteria were investigated using a test on watercress seeds according to GOST 12038-84. Watercress seeds were placed in Petri dishes with filter paper and cotton (wet chambers) in triplicate. The seeds were preliminarily sterilized by treatment with 70% ethanol for 3-5 min, after which they were washed 3 times with sterile distilled water. 50 seeds were placed in each chamber, which were moistened with a suspension with a concentration of 5 g of absolutely dry biomass of cyanobacteria per 1 liter of sterile distilled water. Control seeds were soaked in sterile distilled water. Seeds treated with suspension and distilled water were germinated for three days in a luminostat and at a temperature of 25 ° C. The results are presented in table 3.

Germination of treated seeds is 12% greater than in control. The length of the root during treatment with cyanobacteria exceeds the control by an average of 1.4 cm. The length of the stem during bacterialization with cyanobacteria is 0.5 cm higher than in the control.

Example 4. The study of phytostimulating activity of cyanobacteria Anabaena constricta IPPAS B-2020 in open ground on bell pepper

The experiments were carried out in open ground at the territory of the Technopark FSBEI HPE Astrakhan State University, the village of Beginning of the Astrakhan Region on seeds of the California Miracle pepper variety. Plants were planted in 3-row plots. The distance between plants is 40 cm. In each variant, 50 plants were planted in triplicate. The duration of the experiment is 4 months. The experimental design included the following options: double treatment with cyanobacteria — seed bacterization and spillage into phase 2-4 of this leaf (without fertilizing); control - without fertilizing. The temperature during the experiment ranged from 10 to 40 ° C.

An algologically pure culture of cyanobacteria of the species Anabaena constricta IPPAS B-2020 in the form of a suspension (5 g of absolutely dry biomass of cyanobacteria per 1 liter of sterile distilled water) was used to process seeds and plants of pepper. Before sowing, bacterization of seeds was carried out, which was kept for 1 hour in suspension with cyanobacteria, and control seeds in water.

During the experiment, at the fruiting stage, the length of the root, the height of the plants, the mass of one fruit, the number of fruits and yield were measured.

The results are presented in table 4.

Plants treated with suspensions of cyanobacteria significantly exceeded the control in terms of root length and plant height.

Accelerated fruit ripening was observed in the treated plants for 5–8 days compared with the control. From table 5 it is seen that the mass of one fruit of pepper and the number of fruits when treated with a suspension of cyanobacteria exceeds the control mass. The yield of pepper in the control was 0.143 ± 0.27 kg / bush, and when processed with a suspension, it was 0.272 ± 0.12 kg / bush.

Table 6 shows that the number of plants treated with a suspension of cyanobacteria was 0%, and in the control 9%, which confirms the high fungicidal activity of the culture of cyanobacteria.

Example 5. An example of a method of stimulating growth and development, increasing productivity and protection against phytopathogenic fungi of tomato plants in the open ground.

The experiments were performed in open ground on the territory of the All-Russian Research Institute of Irrigated Vegetable and Gourd FGBNU VNIIOB in the city of Kamyzyak in the Astrakhan Region on seeds of tomato variety of the Gift of the Trans-Volga Region. Plants were planted in rows - 10 plants / row, the row spacing was 40 cm. The distance between plants was 40 cm. In each variant, 50 plants were planted in triplicate. The duration of the experiment is 3 months. The experimental design included the following options: triple treatment with cyanobacteria - seed bacterization, spilling in the phase 2-4 of this leaf, spilling in the flowering and budding phase of plants (without fertilizing); control - without fertilizing. The temperature during the experiment ranged from 10 to 40 ° C.

Before sowing, tomato seeds were bacterized, which were kept in suspension with cyanobacteria for 1 hour, and control seeds in water. To obtain a suspension of 5 g of absolutely dry biomass of an algologically pure culture of cyanobacteria of the species Anabaena constricta IPPAS B-2020, they were added to 1 liter of sterile distilled water and mixed thoroughly. In the phase 2-4 of this leaf and in the phase of flowering and budding of plants, spilling under the root was carried out in the amount of 10-15 ml of suspension per plant.

During the experiment at the fruiting stage, the yield of plants and the number of productive bushes were determined. Acceleration of fruit formation of treated plants was noted 5-8 days earlier than without treatment.

As a result, the number of productive bushes in the control amounted to 31.73%, in the experiment it was 38.82% (Table 7). The yield of tomatoes in the control was 0.31 kg / bush, and when processing the suspension was 1.14 kg / bush.

Table 8 shows that the number of plants treated with a suspension of cyanobacteria was 0%, and in the control 12%, which confirms the high fungicidal activity of the culture of cyanobacteria.

Thus, the claimed method provides stimulation of the growth and development of plants, increasing productivity, protection against phytopathogenic fungi and improving agricultural crops.

Claims (1)

A method of stimulating plant growth, increasing yield and protecting against phytopathogenic fungi in the arid zone, namely, that seeds and plants are treated with a suspension of Anabaena constricta IPPAS B-2020 cyanobacteria with a concentration of 5 g of dry biomass of cyanobacteria per 1 liter of water for 1 hour and provide spilling under the root in the phase 2-4 of this leaf and in the phase of flowering and budding of plants.