KR102354494B1 - Bacillus velezensis BM2021 STRAIN, COMPOSITION COMPRISING FOR BIOLOGICAL CONTROL OF PLANT OR INSECT PATHOGENS USING SAME AND METHOD FOR THE BIOLOGICAL CONTROL USING SAME - Google Patents

Abstract

The present invention relates to a Bacillus velegensis BM2021 strain deposited as KACC 92335P, which increases antifungal activity against strains inhibiting plant growth and is usefully used as a biological pesticide due to control efficacy against fungal pathogens, a plant diseases or pathogens control composition using the same, and a control method using the composition. In addition, the present invention relates to a composition for controlling plant diseases or pathogens including a Bacillus velezensis BM2021 strain (KACC 91394P), a culture solution, or a culture solution extract thereof. The plant disease is caused by Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, Metahizium anisopliae, and Beauveria bassiana. The pathogen is a rust disease occurring during breeding of white-spotted flower radish larvae. The method cultures the Bacillus velezensis BM2021 strain deposited as KACC 92335P at pH 6.

Description

Bacillus velegensis BM2021 strain, a composition for controlling plant diseases or pathogens using the same, and a control method using the same

The present invention relates to a Bacillus belegensis BM2021 strain, a composition for controlling plant diseases or pathogens using the same, and a control method using the same, and more specifically, increases antifungal activity against strains that inhibit plant growth, and prevents fungal pathogens It can be usefully used as a biological pesticide because it has a control effect against it.

In order to cultivate crops, the use of pesticides is inevitable to increase yield. Most commercially available pesticides are synthetic pesticides, and due to the misuse and abuse of synthetic pesticides, soil, water and agricultural products are contaminated, or various problems such as toxicity, ecosystem disturbance, and emergence of resistant strains are raised. Efforts to reduce the use of synthetic pesticides are increasing in order to solve these problems, and the demand for organic products that do not use synthetic pesticides is rapidly increasing.

Biological pesticides are products of pathogens, host-resistant microorganisms, natural pesticides and natural enemies separated and collected from the natural environment to control pests, pathogenic microorganisms and weeds of crops, and are divided into microbial pesticides, biochemical pesticides including pheromones, and plant pesticides. . Microbial pesticides include microorganisms such as bacteria, fungi, viruses and algae as active ingredients. The most widely known microbial pesticide is Bacillus thuringiensis (Bt), which is effective in controlling some insects that damage crops such as cabbage and potatoes.

Variety of secondary metabolic gram-positive bacteria of Bacillus (Bacillus) in bacteria is a well-researched and commercialized bacteria Pseudomonas (Pseudomonas) in the following, Bacillus Belle Zen sheath (Bacillus velezensis) strain is present large numbers in the soil, and that the biological activity It produces products and has the characteristics of forming endospores that are stable to heat and resistant to harsh environments.

The present invention relates to a composition comprising a culture medium of a Bacillus velezensis strain as an active ingredient. expected. In addition, it has been confirmed that there is a control effect against rust disease that occurs during breeding of the larvae of white-spotted flower larvae, so it can be usefully used as a biological pesticide.

Korean Patent Publication No. 10-2019-0053669

The present invention has been devised to solve the above problems, and an object of the present invention is to provide a novel Bacillus velezensis strain having an antagonistic action on plant diseases.

In addition, another object of the present invention is to provide a microbial preparation for controlling plant diseases using the novel strain.

Another object of the present invention is to provide a method for controlling plant diseases using the microbial agent.

The technical problems to be solved by the invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those of ordinary skill in the art to which the present invention belongs from the description below. will be able

The present invention is characterized in that the Bacillus velezensis BM2021 strain deposited as KACC 92335P.

The present invention is characterized in that it is a composition for controlling plant diseases comprising Bacillus velezensis BM2021 strain (KACC 91394P), its culture solution or a culture solution extract. The plant disease is Rhizoctonia solani ( Rhizoctonia solani ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Botrytis cinerea ), Metarhizium anisopliae ( Metarhizium anisopliae ), view It is characterized by being caused by Beauveria bassiana.

The present invention is characterized in that it is a composition for controlling pathogens comprising Bacillus velezensis BM2021 strain (KACC 91394P), its culture solution or a culture solution extract. The pathogen is characterized in that it is a rust disease that occurs during breeding of white spot larvae.

The present invention is a culture method of the Bacillus velezensis BM2021 strain deposited as KACC 92335P, characterized in that it is cultured at pH 6.

By means of solving the above problems, the present invention can be usefully used as a biological pesticide by increasing antifungal activity against strains that inhibit plant growth, and having control efficacy against fungal pathogens.

In addition, the Bacillus belegensis BM2021 strain of the present invention has been confirmed to have a control effect against rust disease that occurs during breeding of white spot flower larvae, so it can be usefully used as a biological pesticide.

1 is a graph showing the antifungal activity results according to the carbon source of the Bacillus belegensis BM2021 strain, according to an embodiment of the present invention.
Figure 2 is a graph showing the growth curve of the strain according to the initial pH conditions of the Bacillus belegensis BM2021 strain according to an embodiment of the present invention.
3 is a graph showing the results of antifungal activity according to the initial pH conditions of the Bacillus belegensis BM2021 strain, according to an embodiment of the present invention.
4 is a photograph showing the result of confirming the production of proteases of the Bacillus belegensis BM2021 strain, according to an embodiment of the present invention.
5 is a photograph showing the result of confirming cellulase secretion of the Bacillus belegensis BM2021 strain, according to an embodiment of the present invention.
6 is a photograph showing the results of antifungal activity against Rhizoctonia solani of Bacillus belegensis BM2021 strain according to an embodiment of the present invention.
7 is a photograph showing the results of antifungal activity against Sclerotinia sclerotiorum of Bacillus belegensis BM2021 strain according to an embodiment of the present invention.
8 is a photograph showing the results of antifungal activity against Bacillus belegensis BM2021 strain Botrytis cinerea according to an embodiment of the present invention.
9 is a photograph showing the results of antifungal activity against Metahizium anisopliae of the Bacillus belegensis BM2021 strain according to an embodiment of the present invention.
10 is a photograph showing the results of antifungal activity against Beauveria bassiana of the Bacillus belegensis BM2021 strain, according to an embodiment of the present invention.
11 is a lineage map of the Bacillus belegensis BM2021 strain, according to an embodiment of the present invention.
12 is a nucleotide sequence of the Bacillus belegensis BM2021 strain, according to an embodiment of the present invention.

Terms used in this specification will be briefly described, and the present invention will be described in detail.

The terms used in the present invention have been selected as currently widely used general terms as possible while considering the functions in the present invention, which may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

In the entire specification, when a part “includes” a certain element, it means that other elements may be further included, rather than excluding other elements, unless otherwise stated.

Hereinafter, with reference to the accompanying drawings, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

Specific details including the problem to be solved for the present invention, the means for solving the problem, and the effect of the invention are included in the embodiments and drawings to be described below. Advantages and features of the present invention, and a method for achieving them will become apparent with reference to the embodiments described below in detail in conjunction with the accompanying drawings.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

The present invention relates to a composition comprising the Bacillus velezensis BM2021 strain deposited with accession number KACC92335P and a culture solution of the strain as an active ingredient. Bacillus velezensis ( Bacillus velezensis ) BM2021 strain has been confirmed to have antifungal activity against strains that inhibit plant growth and control efficacy against fungal pathogens generated during insect breeding, thereby inhibiting the occurrence of plant pathogens, and of pesticides It can be used as a microbial agent that is safe for environmental pollution caused as a side effect. In addition, the present invention can be usefully used as a biological agrochemical as it has been confirmed that there is a control effect on rust disease that occurs during breeding of the larvae of the white-spotted plant larvae, which is an agrobacterium.

The present invention is a composition for controlling plant diseases comprising the Bacillus velezensis BM2021 strain deposited with accession number KACC92335P, a culture solution or extract thereof.

The plant disease is Rhizoctonia solani ( Rhizoctonia solani ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Botrytis cinerea ), Metarhizium anisopliae ( Metarhizium anisopliae ), view It is caused by Beria bassiana ( Beauveria bassiana ), and the present invention Bacillus velezensis BM2021 strain, a plant disease control composition comprising a culture solution or extract thereof can be used to control the plant disease.

The inventors Bacillus bell resin sheath (Bacillus velezensis) Culturing of the strain BM2021 (KACC 92335P) has exhibited a high bacterial count when put glucose into search conditions for the growth of the selected strain results carbon atoms. In addition, when cultured at a culture temperature of 34 ° C., pH 6, agitation speed of 100 rpm, and more than 48 hours, the OD value of 600 nm wavelength was 1.5 or more. The medium had the highest OD value at 0.36 g/L of anhydrous glucose, 0.4 g/L of yeast extract, 0.1 g/L of potassium phosphate (K ₂ HPO ₄ ), and 0.75 g/L of sodium chloride (NaCl). and high antifungal activity.

Example 1. Strain selection

After securing a soil sample to select antibacterial strains, 10 g of soil is mixed with 90 ml of sterile water, 1 ml is collected, diluted in 9 ml sterile water, and 100 μl of each is taken into TSA (tryptic soy agar) medium. It was cultured at 32 °C for more than 3 days by plate dilution. The isolated strain was inoculated into 10 ml of TSB (Tryptic Soy Broth) with platinum lice once, and the strain was first selected using the difference in the shape of the colony generated after culturing, and then the strain was separated by pure culture.

Example 2. Identification of selected strains and preparation of a phylogenetic tree

Among the strains were antibiotic activity is identified the best screening strains by 16s rDNA was sequenced by using the BLAST program microorganism, as a result, the Bacillus bell resin sheath (Bacillus velezensis) with 99% concordance for expressed Bacillus bell Resin Named cis ( Bacillus velezensis ) BM2021, this strain was deposited with the Korea Agricultural Microbiology Center (KACC) on February 25, 2021 and was given an accession number KACC 92335P.

Example 3. Exploration of growth conditions of antibacterial strains

(1) Proper medium conditions

Bacillus velezensis ( Bacillus velezensis ) In order to find out the proper culture medium conditions for the BM2021 strain, different carbon sources were tested. The experiment was divided into glucose, sucrose, corn starch, soy bean meal, soy peptone and peptone, and yeast extract 0.4 g /L, potassium phosphate (K ₂ HPO ₄ ) 0.1 g/L, sodium chloride (NaCl) 0.75 g/L was added and cultured.

(2) Proper pH conditions

Selected Bacillus velezensis ( Bacillus velezensis ) In order to find out the appropriate culture pH conditions for the growth of the BM2021 strain, using hydrogen chloride (HCl) and sodium chloride (NaOH) TSA (tryptic soy agar) medium pH 5.0 ~ pH After adjusting the interval from 11.0 to 1.0, a single colony of each strain was inoculated and cultured for 3 days at 37° C., and the growth of the bacteria was observed.

Example 4. Exploration of conditions for producing antibacterial substances

(1) Proper medium conditions

To examine the medium conditions for the selected Bacillus velezensis BM2021 strain, glucose, sucrose, corn starch, soy bean meal, soy peptone peptone) and peptone (peptone) were tested by varying the carbon source. A single colony was inoculated into a medium with a different carbon source, and the absorbance was measured at a wavelength of 600 nm with a spectrophotometer to measure the number of bacteria by sampling at intervals of 12 hours while culturing with shaking at 34 ° C. and 100 rpm for 48 hours. Each sample was centrifuged at 8000 x g for 10 minutes to separate the cells to prepare a culture filtrate. Antifungal activity was compared by an agar-well deffusion method using 100 μl of a 48-hour culture filtrate for each medium with a different carbon source. [Table 1] shows the number of cultures of Bacillus velezensis BM2021 according to the carbon source. 1 is a graph showing the antifungal activity results according to the carbon source.

carbon source vilable cell counts Log ₁₀ (cfu/ml) Bacillus velezensis glucose 8.57 ± 0.13 sucrose 8.44 ± 0.05 corn starch 8.48 ± 0.14 soy bean meal 7.94 ± 0.07 soy peptone 8.12 ± 0.09 peptone 8.15 ± 14

(2) Proper culture pH conditions

In order to examine the effect of the initial pH conditions on the growth and antifungal material production of the selected Bacillus velezensis BM2021 strain, the medium was prepared by adjusting the pH 5.0 ~ pH 11.0 of 200ml TSB medium at 1.0 intervals. A single colony was inoculated and sampled at 24 hour intervals while culturing with shaking at 34 °C and 100 rpm for 72 hours, and absorbance was measured at a spectrophotometer wavelength of 600 nm to measure the number of bacteria. Each sample was centrifuged at 8000 x g for 10 minutes to separate the cells to prepare a culture filtrate. Antifungal activity was compared by an agar-well deffusion method using 100 μl of a 48-hour culture filtrate for each pH.

Figure 2 is a growth curve of the strain according to the initial pH conditions, Figure 3 is a graph showing the antifungal activity results according to the pH conditions. As a result of the search for conditions for growth of the selected strain, a high number of bacteria was shown when glucose was added as a carbon source. And when cultured at a culture temperature of 34 ℃, pH 6, agitation speed of 100 rpm, and more than 48 hours, the OD value of 600 nm wavelength was 1.5 or more. The medium had the highest OD value at 0.36 g/L of anhydrous glucose, 0.4 g/L of yeast extract, 0.1 g/L of potassium phosphate (K ₂ HPO ₄ ), and 0.75 g/L of sodium chloride (NaCl). and high antifungal activity.

Example 5. Method for characterizing strains having antibacterial activity

(1) Proteases

In order to check the secretion of proteas of the selected Bacillus velezensis BM2021 strain, it was inoculated in TSA medium containing 0.5% skim milk and then cultured at 37 ℃ for 2 days while decomposing around the colony. The presence or absence of a halo zone was observed.

(2) Chitinase

To check whether the selected Bacillus velezensis BM2021 strain secretes chitinase, 10% colloidal chitinm Megazyme, USA) is inoculated into TSA medium containing During stationary culture, the presence or absence of chitin degradation rings around the colonies was observed.

(3) Cellulase

In order to check whether the selected Bacillus velezensis BM2021 strain secretes cellulase, 0.5% Carboxy Methyl Cellulose (CMC) is inoculated into TSA medium and cultured stationary at 37 ° C. After staining with 0.1% Congo Red, destaining with 1M NaCl and observing a clear zone.

(4) Phosphate solubilizing

In order to check the phosphoric acid solubilization of the selected Bacillus velezensis BM2021 strain, a single colony of the strain was inoculated with TSA medium containing 0.5% Tri-calcium phosphate and then inoculated at 37 ° C. The presence or absence of generation was observed.

(5) Measuring plant disease antifungal activity of the selected strain

Rhizoctonia solani ( Rhizoctonia solani ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Botrytis cinerea , Metarhizium anisopliae ( Metarhizium anisopliae ), Beauveria bassiana ( To test the antifungal activity against Beauveria bassiana ), the growth inhibitory activity was confirmed after replacement culture on a plate medium.

Characteristic results of strains with antibacterial activity

(1) Protease (Protease)

The selected Bacillus velezensis BM2021 strain was inoculated in 0.5% skim milk medium to check whether proteolytic enzymes were produced, and as shown in FIG. 4 , it was confirmed that protease was secreted.

(2) Chitinase (Chitinase)

As a result of confirming the secretion of chitinase that degrades chitin, the main component of the cell wall, the selected strain did not secrete chitinase.

(3) Cellulase

Cellulase is an enzyme that catalyzes the hydrolysis of β-1, 4 glycosidic bonds in fibrin, one of the main substances constituting the cell wall. As a result of confirming whether the selected strains secrete cellulase, as shown in FIG. 5 , a decomposition ring was generated.

(4) Phosphate solubilizing

As a result of confirming the presence or absence of phosphoric acid solubilization of the selected Bacillus velezensis BM2021 strain, the degradation ring around the colony was not observed.

(5) Plant pathogenic fungal growth inhibitory effect

As a result of confirming the phytopathogenic fungal growth inhibitory effect of the selected Bacillus velezensis BM2021 strain, Rhizoctonia solani ( FIG. 6 ), Sclerotinia sclerotiorum , FIG. 7 ), Botrytis cinerea ( Botrytis cinerea , FIG. 8 ), and Metarhizium anisopliae ( Metarhizium anisopliae , Rust bacillus, FIG. 9 ) were confirmed to have inhibitory activity.

Example 6. IAA (Indole 3-Acetic Acid) and ammonia production investigation

(1) IAA (Indole 3-Acetic Acid) production investigation

Indole-3-acetic acid (IAA), also known as a natural auxin, is a representative plant growth hormone and is a substance that can be used to promote root elongation and fruit formation in addition to promoting plant elongation. . In addition, it is a substance capable of inhibiting the growth of plant pathogens by inhibiting the absorption of iron ions by plant pathogens. In order to confirm the secretion of IAA (Indole 3-Acetic Acid), a single colony of Bacillus velezensis BM2021 of the present invention was inoculated into 100 ml of TSB medium and cultured with shaking at 34 ° C., 170 rpm conditions for 3 days. In order to remove the cells, the culture medium was centrifuged at 10,000 rpm for 10 minutes. By taking the culture filtrate from which the cells were removed, it was confirmed whether IAA was generated.

2 ml of Salkowsky's reagent (35% HClO4 50ml, 0.5M FeCl3 1ml) was added to 1ml of the culture filtrate to induce a reaction in the dark for 30 minutes. As a control, a TSB medium that was not inoculated with the strain was used. Whether or not IAA (Indole 3-Acetic Acid) was generated was confirmed by the presence or absence of pink color development.

(2) Ammonia production investigation

Ammonification is a process in which organic nitrogen is converted to inorganic nitrogen by microorganisms around the roots of crops. It can also inhibit the growth of harmful fungi. A single colony of Bacillus velezensis BM2021 was inoculated into 10 ml of peptone water (1% peptone, 0.5% NaCl, pH 7) and cultured with shaking at 34° C. and 170 rpm conditions for 3 days. In order to remove the cells, the culture medium was centrifuged at 10,000 rpm for 10 minutes. The culture filtrate from which the cells were removed was used for the reaction. As a control, a TSB medium that was not inoculated with the strain was used. 0.5 ml of Nessler's reagent was added to 1 ml of the culture filtrate, and the reaction was induced under dark conditions for 30 minutes. Then, ammonia formation was confirmed by the presence of dark yellow and brown colors.

IAA (Indole 3-Acetic Acid) and Ammonia Production Results

Below, the results of the production of IAA (Indole 3-Acetic Acid) and ammonia of Bacillus velezensis BM2021 of the present invention were confirmed. [Table 1] shows the reaction results. It was confirmed that IAA (Indole 3-Acetic Acid) and ammonia were generated.

reaction solution result Indole 3-Acetic Acid (IAA) (+) Ammonization (+)

By means of solving the above problems, the present invention can be usefully used as a biological pesticide by increasing antifungal activity against strains that inhibit plant growth, and having control efficacy against fungal pathogens.

In addition, the Bacillus belegensis BM2021 strain of the present invention has been confirmed to have a control effect against rust disease that occurs during breeding of white spot flower larvae, so it can be usefully used as a biological pesticide.

As such, those skilled in the art to which the present invention pertains will understand that the above-described technical configuration of the present invention may be implemented in other specific forms without changing the technical spirit or essential characteristics of the present invention.

Therefore, the embodiments described above are to be understood as illustrative and not restrictive in all respects, and the scope of the present invention is indicated by the following claims rather than the above detailed description, and the meaning and scope of the claims and their All changes or modifications derived from the concept of equivalents should be construed as being included in the scope of the present invention.

Claims (6)

Bacillus velezensis BM2021 strain deposited as KACC 92335P.
Bacillus velezensis BM2021 strain (KACC 91394P), including its culture solution or culture solution extract,
Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, Metahizium anisopliae or Beauveria bassiana ( A composition for controlling plant diseases, characterized in that it controls plant diseases caused by Beauveria bassiana).
Bacillus velezensis (Bacillus velezensis) BM2021 strain (KACC 91394P), a composition for controlling pathogens, comprising a culture solution or culture solution extract thereof, for controlling rust bacteria.
delete 4. The method of claim 3,
The pathogen is a composition for controlling pathogens, characterized in that it is generated during breeding of white spot larvae.
Bacillus velezensis BM2021, characterized in that the medium contains glucose as a carbon source, anhydrous glucose, yeast extract, potassium phosphate and sodium chloride, and cultured at a culture condition of pH 6.0 and a culture temperature of 34 ° C. (Accession No. KACC 92335P) A method of culturing the strain.

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