RU2595405C1 - STRAINS OF BACTERIA Bacillus, Pseudomonas, Rahnella, Serratia HAVING PHYTOPROTECTIVE AND GROWTH-STIMULATING ACTIVITY, AND PREPARATION BASED ON SAID STRAINS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions includes a preparation and strains of microorganisms for production thereof, providing phytoprotective and growth-stimulating properties, and relates to biotechnology and agricultural microbiology. Bacillus amyloliquefaciens VKPM V-11986 strain, having phytoprotective activity, Bacillus amyloliquefaciens VKPM V-11987 strain, having phytoprotective activity, strain of bacteria Pseudomonas brassicacearum VKPM V-11984 having growth-stimulating activity, strain of bacteria Rahnella aquatilis VKPM V-11985 having growth-stimulating activity, strain of bacteria Serratia plymuthica V-12008 having growth-stimulating activity, are used to produce said preparation. Preparation contains cultural liquid of strains of bacteria Bacillus amyloliquefaciens VKPM V-11986, Bacillus amyloliquefaciens VKPM V-11987, Pseudomonas brassicacearum VKPM V-11984, Rahnella aquatilis VKPM V-11985, Serratia plymuthica VKPM V-12008 at a ratio of 1:1:1:1:1 with titre of each strain of bacteria of not less than 1×10⁹ CFU per 1 cm³.

EFFECT: group of inventions provides restoration of soil biocenosis and higher quality of crop yield.

6 cl, 12 tbl, 6 ex

Description

The invention relates to biotechnology and agricultural microbiology and relates to the production of microbial preparations based on a mixture of bacterial strains for improving the biocenosis, accelerating the decomposition of lignocellulose, increasing the yield of agricultural crops and the quality of the products obtained.

The basis of the invention is the bacteria Bacillus amyloliquefaciens VKPM B-11986, Bacillus amyloliquefaciens VKPM B-11987, Pseudomonas brassicaceamm VKPM B-11984, Rahnella aquatilis VKPM B-11985, Serratia plymuthica VKPM B-12008, possessing phytostatic, and phytolithic, possessing phytotoxic, and medicinal, the basis of these strains, which reduces the infectious background during the vegetation period of the plants, ensures the improvement of the biocenosis, accelerates the decomposition of straw and crop residues, stimulates the growth and development of plants.

A well-known consortium of lactic acid, nitrogen-fixing and phosphate-mobilizing bacteria that contributes to the improvement of the soil and allows to increase productivity by 25-35%, plant survival by 20-40% and the quality of standard planting material (US Pat. RF No. 2127509). The consortium is used for pre-sowing seed treatment, for direct application to the soil before sowing or planting plants, for processing the vegetative part of plants or to be applied to the soil during the growing period. However, there is no data on the ability of the consortium to reduce the number of pathogenic microorganisms and decompose plant debris in the soil.

A known consortium of nitrogen-fixing and phosphate-mobilizing bacteria Azospirillum sp. and a Bacillus polymixa antagonist capable of decomposing cellulose-containing materials (straw, xylan, hemicellulose, lignin) (US Pat. No. 5,147,441 A). However, the properties of the consortium were studied only in laboratory conditions; there is no data on the effectiveness of its action in the field.

The following bacterial fungicides based on bacteria of the genus Bacillus against diseases of various cultures are registered and included in the List of the State Chemical Commission of the Ministry of Agriculture of the Russian Federation: the drug Integral is a liquid based on a strain of Bacillus subtilis 24D, the drug Fitosporin-M is a powder based on a strain of Bacillus subtilis 24D (RF patent No. 21931975 , 2129004), the drug Bactofit wettable powder based on a strain of Bacillus subtilis IPM 215 (US Pat. RF No.2019966). The drug Bactophosphine (based on Bacillus mucilaginosus) is intended to enrich the soil, as well as restore its fertility, increase the germination and yield of many types of crops in open and protected ground. Microbiological fertilizer Baikal Em-1 contains a consortium of representatives of various groups of microorganisms, including fungi, which, developing in the soil when introduced as a symbiotic community, are designed to increase the number of soil microflora and, accordingly, fertility and productivity. However, the above drugs do not have a wide enough spectrum of action.

Known consortia of bacteria Bacillus subtilis IC-1435-1-1, Bacillus amyloliquefaciens IC-1436-1-23, Bacillus amyloliquefaciens IC-1437-1-23, Bacillus licheniformis VKPM B-10561 (US Pat. RF No. 2482174) and bacteria Bacillus licheniformis IC-831-1-2, Bacillus licheniformis IC-832-1-2, Bacillus licheniformis IC-833-1-2, Bacillus licheniformis IC-834-1-2 (US Pat. RF 2440413) possessing antibacterial and fungicidal activity and providing restoration of soil microbiocenosis. However, the spectrum of antimicrobial activity of the consortium against plant pathogens is not wide enough.

Known biological product, consisting of a mixture of suspensions of the following strains deposited in VKPM: Agrobacterium tumefaciens B-4116, Agrobacterium radiobacter B-956, Azotobacter chroococcum B-2375, Bacillus thurengiensis B-2918, Bacillus subtillis Bisillus subtillis Bisillis Bisillus Bisillus subtilis-Bisillus subtillis Bisillus subtillis Bisillus subtillis Bisillus subtillis Bisillus subtillis Bisillus subtillis Bisillus subtillis Bisillus subtillis Bisillus subtillis Bisillus subtillis Bisillus subtillis Bisillus subtillis Bisillus subtillis Bisillus subtillis Bacillus megaterium B-4440, Bacillus megaterium B-200, Bradyrhizobium japonicum B-1978, Ervinia ananas B-5292, Lactobacillus casei B-3961, Pseudomonas fluorescens B-1138, Rhodopseudomonas palustris B-1620. The invention allows to restore soil fertility and improve its structure, increase seed germination, strengthen the immune system of plants, increase resistance to diseases and pests, which significantly increases the yield and quality of food (Pat. RF №2322061). However, the method for producing a preparation based on 10–13 strains is rather laborious: initially, bacterial strains are grown separately for (36 ± 2) hours at a temperature of 37 ° C on a nutrient medium optimal for each strain; the grown cultures are washed off with a 9% sodium chloride solution and calculated according to the optical turbidity standard (OSO 45-28-59-85-P) the number of microbial cells in 1 ml of suspension, then a suspension of each strain is prepared in a protective medium, for example, in a sugar gelatin mixture, bringing the concentration of microbial cells of each culture to 1.0-1 5 × ¹⁰ September 1 ml, after which the mixture was prepared suspensions strains in a certain ratio.

Known drug (US Pat. RF No. 2313941) for protecting plants from pathogens of agricultural crops with growth-promoting effect, including the culture fluid of microorganism strains, characterized in that as the culture fluid of microorganism strains, it contains culture fluid of the bacterial strain Bacillus subtilis BAG-65 and strain actinomycete Streptomyces sindenensis BAG-55, as well as auxiliary additives.

The closest technical solution is the invention (US Pat. RF No. 2482174), which relates to biotechnology and plant protection. The bacterial strain Bacillus subtilis IC-1435-1-1 was deposited in VKPM under registration number B-10641. The bacterial strain Bacillus amyloliquefaciens IC-1436-1-23 was deposited in VKPM under registration number B-10642. The bacterial strain Bacillus amyloliquefaciens IC-1437-1-23 was deposited in VKPM under registration number B-10643. The strains obtained by selection and provide restoration of soil microbiocenoses, have bactericidal, fungicidal and virucidal activity. The strain-based preparation contains a filler or water with bacterial biomass in the spore form of Bacillus subtilis VKPM B-10641, or Bacillus amyloliquefaciens VKPM B-10642, or Bacillus amyloliquefaciens VKPM B-10643, or a mixture thereof in a 1: 1: 1 ratio with titer each bacterial strain is not less than 1-10 ⁴ CFU / g or 1 · 10 ⁴ CFU / ml. The drug has bactericidal and fungicidal activity.

However, these strains also have a limited effect aimed at individual pathogens of agricultural diseases, the drug based on them does not have growth-promoting activity, does not have a wide enough spectrum of antagonistic activity and the ability to restore soil microbiocenosis to evolutionarily normal. The proposed biological product is not intended for use during the growing season of plants and is recommended only for application to the soil.

The objective of the present invention is to obtain a preparation based on a mixture of bacterial strains with complex - phytoprotective, cellulolytic and growth-promoting - activity, designed to reduce the infectious background during the growing season of plants, as well as accelerate the decomposition of straw and crop residues, activate soil microbiocenosis and increase crop yields and quality of products.

The problem is solved by using the drug and the following strains:

1. The strain of bacteria Bacillus. amyloliquefaciens 133, with phytoprotective activity and deposited in the All-Russian collection of industrial microorganisms (VKPM) under registration number VKPM B-11986.

2. The bacterial strain Bacillus amyloliquefaciens 67, having phytoprotective activity and deposited in the All-Russian collection of industrial microorganisms (VKPM) under registration number VKPM B-11987.

3. The bacterial strain Pseudomonas brassicacearum S-1, with growth-promoting activity and deposited in the All-Russian collection of industrial microorganisms (VKPM) under registration number VKPM B-11984.

4. The bacterial strain Rahnella aquatilis E 101, with growth-promoting activity and deposited in the All-Russian collection of industrial microorganisms (VKPM) under registration number VKPM B-11985.

5. The bacterial strain Serratia plymuthica 53, with growth-promoting activity and deposited in the All-Russian collection of industrial microorganisms (VKPM) under registration number VKPM B-12008.

6. A preparation for protecting plants from pathogens of agricultural crops, which has phytoprotective, cellulolytic and growth-promoting properties, including the culture fluid of a mixture of bacterial strains. Moreover, as the culture fluid of the bacterial strains, it contains the culture fluids of the bacterial strains Bacillus amyloliquefaciens 133 VKPM B-11986, Bacillus amyloliquefaciens 67 VKPM B-11987, Pseudomonas brassicacearum S-1 VKPM B-11984, Rahnella aquatilis E 101 VKPM B-MP-B plymuthica VKPM B-12008, in a ratio of 1: 1: 1: 1: 1 with a titer of each bacterial strain of at least 1 × 10 ⁹ CFU in 1 cm ³ .

The technical result that can be obtained using the proposed facilities is to improve the biocenosis, accelerate the decomposition of lignocellulose, increase crop yields and the quality of the products.

The cultures that make up the mixture of bacterial strains were isolated from the soil, as well as from samples taken from compost pits and from the rumen of a domestic goat, identified using microscopic, biochemical, molecular genetic research methods and transferred for storage to VKPM under the numbers: B. amyloliquefaciens VKPM B-11986, B. amyloliquefaciens VKPM B-11987, P. brassicasearum VKPM B-11984, R. aquatilis VKPM B-11985, S. plymuthica VKPM B-12008.

Cultural and morphological characteristics of the strains included in the claimed composition

Cultural characters during surface cultivation on ΜΠΑ: strains of B. amyloliquefaciens VKPM B-11986 and B. amyloliquefaciens VKPM B-11987 form round grayish colonies with a smooth edge, 3-5 mm in diameter, mucous, raised in a young culture and rough, flat in aging; strain P. brassicasearum VKPM B-11984 - small, convex, transparent colonies with a smooth edge, fluorescent under UV light; strain R. aquatilis VKPM B-11985 - smooth, grayish-white, convex, translucent colonies with smooth edges, the diameter of the colonies is 2-4 mm, the profile is convex; strain S. plymuthica VKPM B-12008 - cream-colored, round mucous, opaque, shiny, convex, the edge of the colonies is even. All of these strains are not zoopathogenic and phytopathogenic.

Morphological features

Vegetative cells of B. amyloliquefaciens VKPM B-11986 are motile, they are bacillary rods 0.8 × 1.2-1.5 μm in size with rounded ends. Spores are ellipsoidal, located centrally or subterminally. Sporangium is not inflated. Cells are located singly, in pairs or short chains. Gram stain is positive.

Vegetative cells of B. amyloliquefaciens VKPM B-11987 are motile, they are bacillary rods 0.8 × 1.2-1.5 μm in size with rounded ends. Spores are ellipsoidal, located centrally or subterminally. The sporangium is not bloated. Cells are located singly, in pairs or short chains. Gram stain is positive.

Cells of P. brassicasearum VKPM B-11984: straight or slightly curved rods 0.6-0.7 * 1.3-2.3 mm, single, in pairs, less often in short chains. Gram-negative.

Cells of S. plymuthica VKPM B-12008 gram-negative straight rods, 0.5-0.8 × 0.9-2.0 μm in size. Asporogenous.

Cells of R. aquatilis VKPM B-11985 gram-negative bacilli 0.3-0.4 × 0.6-1.2 μm in size. Asporogenous. The nature of flagellation is peritrichous.

The main property of the mixture of strains is the presence of phytoprotective, cellulolytic and growth-promoting activities.

Antimicrobial activity was established against phytopathogenic fungi of the genera Fusarium, Alternaria, Botrytis, Sclerotinia, Rhizoctonia and phytopathogenic bacteria of the genera Pseudomonas, Agrobacterium, Xanthomonas, Erwinia by the method of holes in Petri dishes, while the zones of suppression of growth of phytopathogens are 18-27 mm in diameter.

A mixture of bacterial strains is characterized by the production of hydrolytic enzymes: endo-1,4-β-glucanase (0.8-1.5 u / ml), xylanase (1.5-3.0 u / ml), protease (10.0- 12.0 units / ml).

The use of a mixture of bacterial strains ensures the fixation of molecular nitrogen in the range of 81-120.2 nM C ₂ H _{4 /} vial / day, accelerates the solubilization of calcium phosphates (the diameter of the dissolution zones of Ca ₃ (PO ₄ ) ₂ is 10-16 mm).

Thanks to the listed properties, the growth-promoting activity of the mixture of strains is provided, for example: the germination of lettuce seeds when treated with a 2% working solution increases by 5-9%, the weight of the wet mass by 9%, the length of the aerial parts of plants by 10-20%, the length of the root by 10-25%.

The producer strains that make up the drug are non-pathogenic and harmless to warm-blooded animals, do not have toxicity, allergenicity and toxigenic properties.

Example 1. Getting the drug

The cultivation of each strain of bacteria B. amyloliquefaciens VKPM B-11986, Bacillus amyloliquefaciens VKPM B-11987, P. brassicacearum VKPM B-11984, R. aquatilis VKPM B-11985 and S. plymuthica VKPM B-12008 is carried out separately on nutrient media, the composition which are shown in table 1. The cultivation conditions are as follows: aeration intensity of 1.0 l of air / l of medium / min, stirrer rotation speed (200 ± 20) rpm, temperature 30 ° C. The cultivation duration is 48 hours.

Bacterial culture liquids containing live bacterial cells, their spores and metabolites obtained by separate cultivation of producer strains are mixed in equal proportions (1: 1: 1: 1: 1).

The main indicators of the finished product are shown in table 2.

Example 2. The effect of the drug on the cellulolytic activity of the soil and the degree of decomposition of straw and stubble in model and model field conditions

The study of cellulolytic activity of the soil under the influence of the inventive preparation was carried out for one month by the application method. For this, 1.2 kg of sod-podzolic sandy loam soil (pH 5.7-6.0, humus - 2.20-2.56%, mobile forms of P ₂ O ₅ - 133-160 mg / kg, K ₂ O - 135-161 mg / kg) containing flaxen plates were treated with 100 ml of a 2% working solution of the preparation. As a control, soil treated with water was used. The introduction of the drug into the soil provides for 1 month decomposition of flax web by 93.67% and increases the cellulolytic activity of the soil by 33.67% compared with the control.

To analyze the destruction of cellulose under the influence of the preparation, 5 g of straw was placed in a container, moistened with sterile distilled water in an amount of 5 ml, covered with a lid with holes for aeration, and left for a day. Next, straw was treated from the sprayer with a working solution of the drug with a volume of 390 μl per container, 300 g of sifted land without plant residues was added, mixed and moistened again with 10 ml of sterile distilled water. The working solution of the claimed preparation is 500 μl per 100 ml of sterile distilled water, and the standard (preparation based on the Trichoderma fungus, mycelial form) is 100 μl per 100 ml of sterile distilled water. (When calculating the concentration of the working solution of the preparations, we proceeded from the amount of dry biomass in the preparations). The conditions of the experiment: temperature - 22-24 ° C, humidity 60%. After 30 days, the results were processed. To do this, the earth was sifted through sieves of different diameter cells - No. 2, 5 mm - 1 mm. The straw was collected in kraft bags and dried in an oven at 50 ° C for 8 hours (to a constant residual dry weight). The results are shown in table 3.

As a result of experience in the decomposition of straw, the best indicators were obtained in the variant with the claimed preparation, the use of which increases the decomposition of straw by 4 times compared to the control and 2 times compared to the standard.

Model field experiments to study the effect of the drug on the rate of decomposition of winter triticale straw and crop-root residues of corn were carried out on sod-podzolic sandy loamy soil (pH - 5.87, humus - 2.39%, 145 mg / kg soil, P ₂ O ₅ and 147 mg / kg of soil) as follows: 1.0 kg of soil (in terms of air-dry) and 25 g (in terms of standard humidity - 16%) of winter triticale straw and crop-root residues of corn were placed in a container thoroughly mixed the soil with plant debris, after which the preparation was treated with 2% working solutions a, they were transferred to 25 × 40 cm fiberglass bags. The depth of bagging was 10-15 cm. The experiment was carried out for 2 months. Processing of the results was carried out similarly to the previous experiment.

Based on the results of studies (see table 4), it was found that the use of the drug positively affects the increase in the rate of destruction processes, providing an increase in the degree of mineralization of straw by 7% and stubble by 5% relative to the control, while the mineralization rate reaches 0.32% / day and 0.46% / day, which is 1.7 and 1.3 times higher than the same indicator for straw and stubble in the control.

Thus, the ability of the drug to increase the cellulolytic activity of the soil and accelerate the decomposition of straw and stubble in laboratory and model field conditions has been established.

Example 3. The effect of the drug on the microbiological activity of the soil

The use of the drug for tillage after plowing straw stimulates the vital activity of soil microorganisms, increasing soil biogenicity 2.2 times (44.86 million CFU / g) compared with the control (19.77 million CFU / g).

One of the indicators of the intensification of mineralization processes in the soil may be the ratio of bacteria assimilating organic and mineral nitrogen. In soils with a more vigorous mineralization process, microorganisms that assimilate mineral nitrogen usually outnumber the microflora that develops due to organic nitrogen. We found that the mineralization coefficient when using the inventive preparation is 2 times higher than the control indicators, which is similar to the standard, the mineralization coefficient of which is 2.2 times higher than the control (table 5).

Thus, the claimed drug when applied to the soil containing fresh organic matter, positively affects the increase in the total microbial number of the soil and leads to the intensification of mineralization processes.

Example 4. Phytoprotective and growth-promoting effects of the drug in the processing of sugar beet seeds, Portland hybrid.

During the last growing seasons, the following diseases of sugar beet were widespread and developed: vascular bacteriosis, causing a complete loss of root crop turgor; viral jaundice; viral mosaic that developed at the end of the growing season. To study the phytoprotective and growth-promoting effects of the drug, beet processing was carried out according to the scheme shown in table 6. The experiment was laid down in 3 repetitions on experimental plots with an area of 600.0 m ² . The results are shown in tables 7, 8.

The inventive drug reduces the prevalence of bacteriosis in crops of sugar beet to 56%, the biological effectiveness of the drug is 44.4%, which slightly exceeds the performance of the chemical drug "Kagatnik" and almost 2 times exceeds the biological standard.

Along with a high phytoprotective effect, the claimed drug has a positive effect on the standing density of sugar beets, increasing it to 56.3 thousand units / ha, which is 39% higher than in the control.

In addition, when using the inventive preparation, there is an improvement in the qualitative and quantitative characteristics of the sugar beet crop. So, under the influence of the drug, the sugar content of root crops increases, which increases sugar collection from 1 ha of crops by 4%, from 5.35 tons in the control to 5.56 tons in the processed version. The yield in this case increases by 13% compared with the control and by 4% compared with chemical and biological standards.

Thus, the claimed preparation has a high phytoprotective effect against sugar beet bacterioses during the growing season and improves the qualitative and quantitative characteristics of the crop.

Example 5. Disinfection of spring wheat seeds

The fungicidal activity of the claimed preparation against phytopathogenic fungi Bipolaris sp., Fusarium sp., Alternaria sp. and other pathogens of rot of spring wheat. For this, spring wheat seeds c. Tuleyevskaya was treated at the rate of 0.25 L of the drug per 1 ton of seeds. The initial seed contamination established in the laboratory of phytoimmunology and plant protection of Kurgan State University (Kurgan) is shown in Table 9. Laboratory tests showed that seed treatment leads to almost complete suppression of pathogens of fusarium-helminthosporious root rot (Table 9).

An energy assessment of wheat cultivation under conditions of seed disinfection with biological preparations is given in table 10.

Thus, a high phytoprotective effect of the claimed preparation is shown for seed treatment of spring wheat.

Example 6. Processing vegetative plants of winter wheat

Location of field research: the second agroclimatic region - the central part of the Ryazan region. Culture: winter wheat, grade Galina. The soil in the experimental plot: dark gray forest heavy loam. Humus content 3.2%; hydrolytic acidity 2.5-4.7 mg · equiv; The pH of the salt extract is 5.2. The predecessor: barley.

Method of application - spraying plants. Fluid flow rate: 300 l / ha. Multiplicity of treatments: single. The experimental design is shown in table 11.

Economic efficiency (stored yield in kg / ha,% of control and benchmark) for the options is presented in table 12.

It has been established that the use of the claimed preparation contributes to an increase of 4.2 kg / ha to the crop, which is 17% higher than the control indicators, 10.4% higher than the reference indicators and corresponds to the action of the prototype used for tillage together with starting doses of nitrogen fertilizers.

Claims (6)

1. A preparation for protecting plants from pathogens of agricultural crops, having phytoprotective, cellulolytic and growth-promoting properties, including the culture fluid of microorganism strains, characterized in that it contains culture fluids of the bacterial strains Bacillus amyloliquefaciens VKPM B-11986, Bacillus amyloliquefaciens VKPM B-11987, Pseudomonas brassicacearum VKPM B-11984, Rahnella aquatilis VKPM B-11985, Serratia plymuthica VKPM B-12008 in a ratio of 1: 1: 1: 1: 1 with a titer of each bacterial strain not less than its 1 × ¹⁰ September CFU in 1 cm ^3. 2. The bacterial strain Bacillus amyloliquefaciens with phytoprotective activity and deposited in the All-Russian collection of industrial microorganisms (VKPM) under registration number VKPM B-11986 used for the drug according to claim 1. 3. The bacterial strain Bacillus amyloliquefaciens with phytoprotective activity and deposited in the All-Russian collection of industrial microorganisms (VKPM) under registration number VKPM B-11987 used for the drug according to claim 1. 4. The bacterial strain Pseudomonas brassicacearum, with growth-promoting activity and deposited in the All-Russian collection of industrial microorganisms (VKPM) under registration number VKPM B-11984, used for the drug according to claim 1. 5. The bacterial strain Rahnella aquatilis, with growth-promoting activity and deposited in the All-Russian collection of industrial microorganisms (VKPM) under registration number VKPM B-11985, used for the drug according to claim 1. 6. The bacterial strain Serratia plymuthica, with growth-promoting activity and deposited in the All-Russian collection of industrial microorganisms (VKPM) under registration number VKPM B-12008 used for the drug according to claim 1.