KR100431614B1 - THE NOVEL ANTIFUNGAL BACTERIA Bacillus subtilis EBM 13 AND THE BIOPESTICIDES CONTAINING THEREOF - Google Patents

Abstract

The present invention relates to a novel microbial Bacillus subtilis EBM 13 having a wide range of antifungal activity and to a microbial pesticide containing the antifungal active substance and microorganism produced by the microorganism.

Bacillus subtilis EBM 13 is a plant pathogenic fungus Rice fever (Pyricularia oryzae),Receipt bottle (Pythium ultimum), Rice paddy disease (Rhizoctonia solani), Apple rot (Botryoshaeria dothidea), Sesame seed bottle (Bipolaris sorokniana), Gray Mold Disease (Botrytis cinerea), Pepper anthrax (Colletotrichum gloeosporioides), Rice fever (Pyricularia grisea), Watermelon blight (Mycosphaerella melonis), Pepper blight (Phytophthora capsici), Tomato round pattern disease (Alternaria solani), Root disease (Fusarium oxysporum), Mycobacterium tuberculosis (Sclerotinia sclerotiorum), Mushroom Fungus (Neurosporasp.), and candidiasis (specifically an animal pathogenic fungus)Candida albicansMicroorganisms having an antifungal activity spectrum are provided.

In addition, microbial pesticides for controlling pathogenic fungi containing antifungal active substances EBM13-P1, EBM13-P2, EBM13-P3, EBM13-P4, EBM13-P5 and Bacillus subtilis EBM 13 produced by Bacillus subtilis EBM 13 ( biopesticide).

Bacillus subtilis EBM 13 of the present invention has the ability to decompose organic matter, pesticide resistance and pesticide in addition to antifungal activity can be useful as a multifunctional pesticide with environmental conservation and purification.

Description

Novel microbial bacterium subtilis EBM 13 AND THE BIOPESTICIDES CONTAINING THEREOF

The present invention provides a novel bacterium Bacillus subtilis EBM 13 having a broad spectrum of antifungal activity against plant and animal pathogenic fungi and antifungal actives produced by the microorganism and microorganisms for controlling plant pathogenic fungi containing the microorganism. It is about a biopesticide.

In order to control pests, weeds and pathogenic microorganisms of crops, microbial pesticides are products of microorganisms separated or selected from nature or modified for use, or compounds secreted by microorganisms.

An essential element in the development of microbial pesticides is to select or modify useful microorganisms with excellent antagonism, and to mass produce the selected microorganisms or drugs secreted by the microorganisms and to efficiently deliver them to the crops.

Recently, research and development of microbial pesticides using microorganisms has been actively conducted.

Korean Patent Application No. 1990-0017551 (new Bacillus subtilis subspecies and the use of antifungal substance KRF-001 complex produced therefrom), Bacilus subtilis subspecies C. .Krictiensis ), but the antifungal activity spectrum has a narrow problem.

In Korean patent application 1993-0022037 (Method for producing microbial insecticide using bioencapsulation), a natural gel matrix rich in water is mixed with water to prepare a protein-rich natural gel matrix, and after autoclaving, Bacillus terringiensis spores are mixed. Thereafter, there is a method of preparing a bioencapsulated microbial insecticide by drying, but has a narrow antifungal activity spectrum and a complicated composition and composition of the biomatrix.

Korean Patent Application No. 1998-027468 (Improved Coating Pesticide Matrix, Method for Making and Composition Containing The Same) relates to a coated pesticide matrix comprising a pesticide, a polymer, a plasticizer, a sunscreen, an activity enhancer, and a suspending agent. Although known, the antifungal activity spectrum is narrow and the effect is limited.

In addition, Korean Patent Application No. 10-1998-0062437 (the microorganism having an antifungal activity against pathogenic fungi, an antifungal microbial pesticide containing the same, and a method for preparing the same), filed by the applicant, is filed with Bacillus subtilis variant amylolytica. Microorganisms showing antifungal activity such as Bacilius subtilis var. Amyloliquefaciens and Pseudomonas aeruginosa and bio matrix using the same have been described, but the composition of the bio matrix is complicated and the antifungal activity spectrum is narrow. .

The present invention provides a novel microorganism having a broad antifungal activity spectrum and excellent antifungal activity against plant pathogenic fungi, an antifungal active material from which the microorganism is produced, and an environmentally friendly fungal microorganism pesticide containing the microorganism. There is this.

Figure 1 is a comparison of the relationship between the flexibility of each EBM strain gene by RAPD

Figure 2 is a comparison of antifungal activity of Bacillus subtilis EBM 13 strain of the present invention

A: Rhizoctonia solani

B: Fusarium oxysporum

C: Botryoshaeria dothidea

D: Alternaria solani

E: Mushroom fungus ( Neurospora sp.)

3 is a reversed phase chromatogram of antifungal actives produced by Bacillus subtilis EBM 13 strain.

4 is a Maldi Mass spectrum of the antifungal active substance of the present invention.

Figure 5 is a Bacillus subtilis EBM 13 strain and microbial pesticide manufacturing using the bio matrix

Figure 6 is a photograph of the control effect against the fungus disease (Neurospora sp.) Of the microorganisms of the present invention

The present invention provides a novel microbial Bacillus subtilis EBM 13 (Accession No .: KCTC 0984BP) having broad antifungal activity spectrum and excellent antifungal activity against plant pathogenic fungi.

Also provided are novel antifungal actives produced by Bacillus subtilis EBM 13 of the present invention, EBM13-P1, EBM13-P2, EBM13-P3, EBP13-P4, and EBM13-P5.

Also provided are environmentally friendly microbial pesticides containing Bacillus subtilis EBM 13 of the present invention.

Bacillus subtilis EBM 13 of the present invention is a plant pathogenic fungi Pyricularia oryzae , Pythium ultimum , Rhizoctonia solani , Botryoshaeria dothidea , Sesame pattern disease ( Bipolaris sorokniana ) , Botrytis cinerea , Colletotrichum gloeosporioides , Pyricularia grisea , Watermelon vine disease ( Mycosphaerella melonis ), Red pepper blight ( Phytophthora capsici ), Tomato roundworm ( Alternaria solani ) It has antifungal activity against Fusarium oxysporum ), Sclerotinia sclerotiorum , Mushroom fungal disease ( Neurospora sp.), And Candida albicans , an animal pathogenic fungus.

In addition, Bacillus subtilis EBM 13 of the present invention has high ability to decompose organic matters such as glucan, starch, protein, cellulose and lignin, and is resistant to about 20 pesticides including herbicides, fungicides and insecticides, and decomposes pesticides. Therefore, it shows resistance to various kinds of pesticides.

Therefore, the antifungal active microorganism of the present invention can be used as an environmentally friendly multifunctional antifungal agent useful for environmental conservation and purification.

That is, the microorganism of the present invention may be used as an active ingredient such as soil improving agent, composting agent, foliar spraying agent or irrigation spraying agent, in addition to antifungal activity, having organic matter degrading ability, pesticide resistance and pesticide degrading ability.

In particular, it has an excellent effect on the inhibition of growth of Neurospora sp., Which is a problem when cultivating oyster mushrooms, and also for the preparation of microbial pesticides for controlling fungal diseases of mushrooms, as well as additives for producing non-sterile media for mushroom cultivation. Can be utilized.

In addition, Bacillus subtilis EBM 13 of the present invention does not appear to inhibit plant seed germination at all, and is useful for storage and preservation not only as a microbial pesticide but also as seed encapsulation.

Also provided is an antifungal active substance produced by Bacillus subtilis EBM 13 of the present invention.

The microbial Bacillus subtilis EBM 13 of the present invention was incubated under appropriate conditions, culture supernatants were obtained, and antifungal substances were isolated using gel filtering and Rotorper system.

Antifungal effect experiments were performed on the isolated materials, and materials showing excellent effects were analyzed using PAGE and MS Maldi spectrum.

The isolated antifungal actives are oligopeptides, which are different from those known in the prior art. (1044.8), EBM13-P3 (1045.83), EBM13-P4 (1058.73), and EBM13-P5 (1058.73).

The present invention also provides a microbial pesticide made by mixing the Bacillus subtilis EBM 13 of the present invention and a bio matrix that is a microbial carrier.

The biomatrix may be made by the applicant of Korean Patent Application No. 10-1998-0062437, which is filed by the applicant, but since the composition of the biomatrix of the patent application is complicated, it is used by simplifying the configuration as follows.

The biomatrix is composed of plant resources polymer materials, microbial origin biopolymer materials, nutrients, auxiliary materials and additives.

Plant resource polymer materials include potato flour, soy flour and starch flour.

Microbial biopolymers consist of mucopolysaccharides, poly-betahydroxy-alkanoates, flocculants.

Dietary supplements use glucose, peptone and inorganic salts.

Use it as vermiculite or charcoal powder.

As additives, extenders, UV blockers and surfactants are used.

A microbial pesticide is prepared by mixing Bacillus subtilis EBM 13 of the present invention into a biomatrix made by mixing such materials as follows.

The Bacillus subtilis of the present invention is cultured and concentrated, and then encapsulated with mucopolysaccharide.

The biomatrix made by mixing the biomatrix components is sterilized and then cooled to about 50 ° C.

Encapsulated Bacillus subtilis EBM 13 is mixed with the biomatrix, with the number of cells per gram of the biomatrix being 10 ^{8-10 9} and then homogeneously mixed in a homogenizer.

The microorganisms and bio-matrix complexes thus made are dried to form microbial pesticides in the form of powders of a suitable size.

Microbial pesticides prepared by encapsulating Bacillus subtilis EBM 13 of the present invention and then mixed with a biomatrix are excellent in terms of survival time, survival germination rate, antifungal activity, and antifungal substance generation ability of active bacteria.

This microbial pesticide can be used for antifungal microbial pesticides, seed coatings, microbial nutrients, soil improvers, composting agents, foliar sprays or irrigation sprays.

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

<Example 1> Selection of new strain Bacillus subtilis EBM 13

Several selection and identification experiments were conducted to select microorganisms with broad spectrum of activity against plant pathogenic fungi and excellent activity.

109 strains showing strong antifungal activity against plant pathogenic fungi were isolated from soil, jade, and compost in the Chungcheong area, and 45 strains were selected first.

Again, 12 strains, including EBM 1, 2, 3, 13, 31, 43, 67, 99, were selected on the basis of antifungal effect, strain activity, and the like.

The antifungal effect test was performed again, and excellent antifungal active bacteria EBM 3, EBM 13, EBM 31, and EBM 99 were selected.

These selected strains were subjected to API kits and additionally over 80 experiments to identify and identify their physicochemical properties (see Table 1).

As a result, EBM 3, EBM 13, EBM 31 strains were identified as Bacillus subtilis , EBM 99 was identified as Bacillus polymyxa .

Table 1. Physicochemical Properties of EBM Strains

Test substrate reaction / enzymes EBM13 EBM3 EBM31 EBM99 Gram Gram staining +/- + + + + Cell form rod / coccus rod rod rod rod Catalase hydrogen peroxide catalase + + + － OX tetramethyl-p-phenylene diamine cytocrome oxidase － － － － ONPGPNPG ortho-nitrophenyl-galactoside / p-nitro beta-galactosidase － － － + ADH arginine arginine dihydrolase + + + － CIT sodium citrate assimilation + + + － URE urea urease + + + － INDTRP tryptophane indol production － － － － GEL gelatine gelatinase (protease) + + + － ARA arabinose assimilation + + + + NO ₃ potassium nitrate reduction of nitrates to nitrites + + + + reduction of nitrates to nitrogen － － － － ESC esculin beta-glucosidase + + + + MNE mannose assimilation + + + ± NAG N-acetyl-glucosamine assimilation + + + － MAL maltose assimilation + + + ± GNT gluconate assimilation + + + + CAP caprate assimilation － － － － ADI adipate assimilation + + － － MLT malate assimilation + + + － PAC phenylacetate assimilation － ± － － PA proteolytic agar (gelatine) 1% tannic acid + + + + LA lipolytic agar (tween 80) opaque zone － － － + SA amylolytic agar (starch) Gram iodine + + + + CA cellulolytic agar (CMC) 0.5% congo red + + + + SPF spore staining spore formation + + + + Glucose acidification + + + + carbon dioxide (gas) － － － + Fructose acidification + + + + carbon dioxide (gas) － － － + Lactose acidification － － － + carbon dioxide (gas) － － － + Sucrose acidification + + + + carbon dioxide (gas) － － － + Mannitol acidification + + + + carbon dioxide (gas) － － － + VP Voges-Proskauer test + － + + MR-VP Methyl Red test － － － +

<Example 2> Comparison of Mutual Flexibility of EBM Strains

The comparative flexibility of the EBM strains selected in Example 1 was compared.

Patent applications of EBM strains EBM 13, EBM 3, EBM 31, EBM 43 with the same subname ( Bacillus subtilis) and KCTC 1660, a public strain of the Korea Biotechnology Research Institute Gene Bank, an international microorganism depositing institution, and a Korean patent filed by the applicant In the KCTC 8913P strain of the application 1998-0062437, using a random primer, the mutual flexibility relationship was compared through random amplified polymorphic DNA (RAPD) analysis of the gene (see Table 2, Figure 1).

As a result, each strain showed a unique DNA band, and each strain showed a different flexibility.

Table 2. Comparison of Mutual Flexibility between EBM Strains

Strain EBM 13 EBM 3 EBM 31 EBM 43 KCTC8913P KCTC1660 EBM 13 100 87.36 58.87 59.79 80.325 18.065 EBM 3 87.36 100 56.265 55.36 75.005 17.81 EBM 31 58.87 56.265 100 48.08 57.405 16.55 EBM 43 59.79 55.36 48.08 100 62.955 15.875 KCTC8913P 80.325 75.005 57.405 62.955 100 19.605 KCTC 1660 18.065 17.81 16.55 15.875 19.605 100

Example 3 Comparison of Antifungal Activity Spectrum of EBM Strains

14 kinds of plant pathogenic fungi Pyricularia oryzae , Pythium ultimum , Rhyzoctonia solani , Botryoshaeria dothidea , Bipolaris sorokniana , Blight fungus ( Botrytis cinerea ) anthracnose (Colletotrichum gloeosporioides), rice blast (Pyricularia grisea), watermelon blight (Mycosphaerella melonis), pepper blight (Phytophthora capsici), tomato-ply round blotch (Alternaria solani), Root Rot (Fusarium oxysporum), gyunhaekbyeong (Sclerotinia sclerotiorum), Antifungal activity was tested against fungal disease ( Neurospora sp.) And the animal pathogenic fungus Candida albicans .

Cell cultures precultured in nutrient medium were inoculated on PDA (Difco Co.) medium coated with pathogenic fungi using a paper disc (diameter 6 mm) and incubated at 30 ° C. for 2-3 days.

Table 3 shows the antifungal activity of the pathogenic fungi.

Table 3. Antifungal Activity Spectrum of EBM Strains

Antifungal active bacteria Antifungal activity C. A P. O P. U R. S B. D B. S B. C C. G P. G M. M P. C A. S F. O S. S N.s EBM 13 ◎ ● ◎ ● ● ● ● ● ● ● ● ● ● ● ● EBM 3 ● ● ○ ● ● ● ● ● ● ○ ● ● ● ● ● EBM 31 ● ● ● ● ● ◎ ● ● ● ● ● ◎ ● ● ● EBM 99 ● ● ● ◎ ● ● ● ● ● ● ● ● ● ● ●: very good; ◎: excellent; ○: slightly C. A: Candida albicans. P. O: Pyricularia oryzae (rice fever). U: Pythium ultimum . R. S: Rhizoctonia solani (Riceworm) B. D: Botryoshaeria dothidea (apple rot). B. S: Bipolaris sorokniana . B. C: Botrytis cinerea . C. G: Colletotrichum gloeosporioides (pepper anthrax). G: Pyricularia grisea (rice fever). M. M: Mycosphaerella melonis (watermelon blight) .P. C: Phytophthora capsici . A. S: Alternaria solani (tomato rounded disease). O: Fusarium oxysporum (locopathy, root disease). S. S: Sclerotinia sclerotiorum . s: Neurospora sp. Mushroom fungus

Bacillus subtilis EBM 13 of the present invention was very excellent in 13 plant pathogenic fungi, including rice fever, was excellent in one species of fungi and Candida fungi.

The excellent antifungal strain Bacillus subtilis EBM 13 of the present invention was deposited on April 13, 2001, with accession number KCTC 0984BP to the Genetic Bank of Korea Biotechnology Institute, an international microbial deposit institution for patent applications.

Example 4 Optimal Culture Conditions of Bacillus subtilis EBM 13

The bacterium subtilis EBM 13 strain of the present invention selected in Example 1 was examined for the optimal conditions for the production of antifungal actives.

The carbon, nitrogen, and phosphate sources of the medium composition used in the experiment are shown in Table 4, and the growth effect using this composition was confirmed by measuring the growth curve of the microorganisms at absorbance at 620 or 630 nm for 48 hours.

Table 4. Microbial medium composition

Badge Kinds Phosphoric Acid Concentration (mM) in Glu-MOPS Medium 0.01, 0.05, 0.1, 0.5, 1.0, 2.0 Nitrogen source Beef extract, cassamino acid, malt extract, peptone, tryptone, urea, yeast extract Carbon source Fructose, galactose, glycerol, lactose, maltose, mannitol, sucrose

(1) Growth amount by type of carbon source

Peptone (0.5%) was added to GM63 medium as a nitrogen source in a fixed amount, and 0.5% of each carbon source was added to investigate. Among the seven carbon sources except glucose, lactose and mannitol were effective as carbon sources.

(2) Growth amount by type of nitrogen source

It was measured by adding 0.5% of each nitrogen source to GM63 medium. Peptone and yeast extract were the best nitrogen sources for the growth of antifungal active strains, and urea was the lowest.

(3) Growth amount according to the concentration of phosphate

Phosphoric acid source was measured by varying the phosphoric acid concentration of Glu-MOPS medium growth of antifungal active strains. The growth rate was exactly proportional to the phosphoric acid concentration, and there was no difference in the growth at concentrations of 0.5 mM or more.

(4) Investigation of antifungal activity according to carbon source, nitrogen source type and phosphate concentration

The culture medium was taken from the antifungal active strain according to the culture time (6, 12, 24 hours), centrifuged to remove the microorganisms, and the antifungal active material was extracted using the same amount of butanol in the supernatant.

Only 1 ml of this was concentrated to measure the antifungal activity. Activity was measured by Candida (C. albicans) or Fusarium (Fusarium) is not inhibited growth ring diameter (cm Units). As a result, EBM 13 strain showed high activity without being affected by the medium composition.

(5) Investigation of antifungal activity according to amino acid, vitamin addition and pH change

EBM 13 strains were cultured with different temperature, pH, vitamins (B1, B2, B12) and added amino acids in order to compare the production ability of antifungal actives according to environmental factors.

The supernatant of the culture cultured in each section was extracted with the same amount of butanol and concentrated under reduced pressure, and the antifungal actives were measured from the concentrate, and the antifungal actives were maximally produced when vitamin B1 was added.

Amino acids had high antifungal activity in the treatment of arginine and lysine.

(6) ability to produce antifungal active substances according to temperature

Antifungal activity was measured by culturing the culture temperature for 15 days at 15, 25, 30, and 37 ° C, and the antifungal activity was excellent in the temperature range of 30 to 37 ° C.

Example 5 Purification and Analysis of Antifungal Active Substances

Bacillus subtilis EBM 13 of the present invention was incubated at 33-36 ℃ utilizing the optimum conditions investigated in Example 4.

The culture supernatant was saturated with (NH ₄ ) ₂ SO ₄ 70% and then gel filtered with a Sephadex G-50 (50 by 2.5 cm) column.

Then, antifungal proteins were separated and purified using disc gel electrophoresis, a gel eluter system and a Rotoper system.

Antifungal activity tests were performed on the isolated materials and good materials were selected.

PAGE and Maldi spectral analysis of the selected substances confirmed that the isolated antifungal actives were oligopeptides, which were different from those known in the art. According to the spectrum, EBM13-P1 (molecular weight: 1058.21), EBM13-P2 (1044.8), EBM13-P3 (1045.83), EBM13-P4 (1058.73), and EBM13-P5 (1058.73) were named (see FIGS. 4 and 5). .

Example 6 Preparation of Biomatrices for Preparation of Microbial Pesticides

In order to produce antifungal physiologically active substances while maintaining the antifungal active bacteria in the natural environment ecosystem or plants, it is necessary to supply nutrients (nutrients that can be used in the short and medium to long term), to protect the antifungal bacteria, to improve germination rates, and to maintain short and long term growth. It is necessary.

The bio-matrix (bio-matrix) having such a function was prepared.

The biomatrix is made from a mixture of plant-based macromolecules (biogels), microbial-based biopolymers, nutrients, supplements, and additives.

Potato powder and soy flour were used as the plant material polymer material.

As biopolymers of microbial origin, mucopolysaccharides (MPS), poly-beta-hydroxyalkanoates and flocculants were used.

For nutrition, glucose, peptone and inorganic salts were used.

As auxiliary materials, vermiculite and rice husk powder were used.

As additives, extenders, UV blockers and surfactants were used.

Table 5 shows the composition components and amount of components for producing the biomatrix.

Table 5. Bio Matrix Composition

ingredient Amount of ingredients Plant Resources Polymer Materials (Potato Powder, Soybean Powder) 400 g Microbial source biopolymer materials (mucopolysaccharides, poly-beta-hydroxyalkanoates, flocculants) 5-10g Nutrients glucose 2 g peptone 2 g MgCl ₂ 0.04g CaCO ₃ 2 g FeSO ₄ 7H ₂ O 120mg MnCl ₂ 4H ₂ O 20mg KH ₂ PO ₄ 0.1g Auxiliary Materials (Turquoise and Chaff) 5-10g Additives (extenders, UV blockers, surfactants) a very small amount H ₂ O 1ℓ

Example 7 Preparation of Microbial Pesticides Containing Bacillus Subtilis EBM 13

Bacillus subtilis EBM 13 of the present invention was cultured and concentrated.

Concentrated Bacillus subtilis EBM 13 was encapsulated with mucopolysaccharide (MPS).

The biomatrix made in Example 6 was sterilized and then cooled at about 50 ° C.

The number of encapsulated Bacillus subtilis EBM 13 cells was mixed to about 10 ⁸ -10 ⁹ per gram of the biomatrix, and then uniformly mixed in a homogenizer.

The Bacillus subtilis EBM 13 and the bio matrix composite thus prepared were dried and made into a powder of a suitable size to make a microbial pesticide.

Using the microbial pesticide of the present invention can also be made of a hydrate, granules, coating granules and the like.

Example 8 Preparation of Coated Seeds Using Microbial Pesticides

The coated seed was prepared using the microbial pesticide of the present invention.

A slurry was prepared by mixing a sticking agent, a filler, and the like with the microbial pesticide prepared in Example 7.

Seeds were pelleted into a slurry using a seed processing machine, or film coated.

Alternatively, various types of biopesticide-coated seeds were produced by applying both pellet coating and film coating simultaneously.

In the case of biopesticide-coated seeds, when sowing, microorganisms grow together with the roots of the plant to naturally settle in the root zone of the crop, and useful for invading pathogens. By secreting useful substances, it shows the effect of promoting the growth of crops.

Example 9 Preparation of Microbial Nutrient Using Microbial Pesticides

A microbial nutrient was prepared using the microbial pesticide of the present invention prepared in Example 7.

Powder and liquid form by mixing the microbial pesticide of the present invention, biopolymers (microgle), phytonutrients (nitrogen, phosphoric acid, potassium, amino acids, other inorganics, etc.), electrodeposition agent (sticker), filler (filler), UV blocker, etc. Made microbial nutritional supplements.

In the case of microbial nutrients, when applied to plants, not only the growth effect of the crop but also the bacillus subtilis EBM 13 of the present invention was excellent in controlling plant diseases.

Experimental Example 1 Investigation of Organic Matter Degradation and Pesticide Resistance of Bacillus subtilis EBM 13 Strain

(1) Environmental preservation and purification ability of Bacillus subtilis EBM 13

Antifungal Activity The activity of extracellular enzymes in liquid and solid media was investigated to determine whether the bacteria use biological waste resources to produce high value-added microbial pesticides and antifungal agents, as well as whether they are environmentally clean.

Bacillus subtilis EBM 13 of the present invention was excellent in the ability to decompose organic matter, such as glucan, starch, protein, cellulose, lignin.

(2) Pesticide Resistance and Resolution of Bacillus subtilis EBM 13

In order to analyze the resistance to pesticides of Bacillus subtilis EBM 13 of the present invention, 23 kinds of pesticides in NA medium, and fungicides include benomyl, flusilazole, tebuconazole and oxadisil (Oxadixyl), Edifenphos, Tricyclazole, Mancozeb, Chlorothalonil, Dichlofluanide, Pencycuron, Azoxystrobin (Azoxystrobin), Copper hydroxide, Isoprothiorane, Iprobenphos, Chlorfenapyr, Imidacloprid, Carbofuran, Carbofuran Fenpyroximate, Chlorpyrifos, BPMC, Diazinon, and herbicides were added Butachlor and Glyphosate, respectively. Observed.

For the analysis of pesticide resolution, strains were cultured at 30 ° C for 3 days in GM63 liquid medium containing pesticide instead of glucose as a carbon source, and in Glu-MOPS liquid medium containing pesticide as the only phosphorus (P) source for growth and resolution. UV spectra and TLC methods were used.

As a control, the strains were cultured in GM63 liquid medium containing glucose as a carbon source and Glu-MOPS 0.1 mM Pi liquid medium containing phosphoric acid, which was also the original phosphorus, in the same manner.

As a result, Bacillus subtilis EBM 13 of the present invention is resistant to various types of pesticides and the resolution was different for each pesticide.

Since most Bacillus subtilis EBM 13 of the present invention is resistant and degradable to most organic chemical pesticides that act as toxic and environmental pollutants, it can be used as an environmentally friendly multifunctional microbial pesticide which is very useful for environmental conservation and purification. Can be.

Experimental Example 2 Investigation of Stability and Performance of the Microbial Pesticide of the Present Invention

(1) Stability of Microbial Pesticides

It is important that microbial pesticides have at least one to two years of drug life.

The microbial pesticide of the present invention prepared in Example 7 was left for 0 months, 3 months, 6 months, 9 months, 12 months and then the number of living microorganisms regenerated was investigated.

Set the experimental section of the concentrated Bacillus subtilis EBM 13, each sample is plated on a NA plate medium and incubated for 24 hours at 30 ℃, and compared with the number of microorganisms initially put and the number of regenerated microorganisms for stability Measured.

As a result, the microbial pesticide of the present invention, which was encapsulated and dried compared to the control, was somewhat different but stable during the 12 month storage period.

In order to analyze the antifungal activity of the regenerated Bacillus subtilis EBM 13, an antifungal activity (AF) experiment was performed by randomly selecting 50 regenerated bacteria.

As a result, all of them possessed AF ⁺ antagonism and confirmed that all the regenerated strains retain their biological antifungal activity.

(2) Investigation of Leaf Surface and Soil Viability of Bacillus EBM 13 Strains in Microbial Pesticides

The bacterium of Bacillus subtilis EBM 13 in the microbial pesticides was examined at 1 and 2 weeks after the cabbage lobe, cabbage root soil, and general soil.

(3) Spray activity investigation of microbial pesticide

The microbial pesticide prototype (5,000 ppm, 10 ⁷ cfu / ml) prepared by encapsulating the Bacillus subtilis EBM 13 strain of the present invention had a control effect against cucumber gray mold compared to the control drug Fluazianam (200 ppm). Rather, it was higher (prototype: 83%, Fluazianam: 74%), and control effect for cucumber powdery mildew was very good (prototype: 90%, Hexaconazole: 87%).

(4) Investigation of germination rate of coated seeds using microbial pesticides

The germination rate of 100 seeds of cucumber, tomato, and pepper coated in Example 8 using the microbial pesticide of the present invention was investigated.

As a result, the germination rate of the seed and the germination of the seed did not show a difference or increase rather than the normal seed without coating.

(5) UV resistance investigation of microbial pesticide

The UV resistance of the microbial pesticides prepared in Example 7 was investigated.

Survival of Bacillus subtilis EBM 13 contained in microbial pesticides exposed to sun for 5 hours was not reduced.

Experimental Example 3 Investigation of Toxicity of Microbial Pesticides and Antifungal Agents of the Present Invention

Cytotoxicity test, skin irritation test and acute toxicity test were performed on microbial pesticides containing Bacillus subtilis EBM 13 and other commercialized microbial pesticides.

Acute toxicity experiments were performed by oral administration in both mice using the microbial pesticide of the present invention and other microbial pesticide prototypes.

As a result, the microbial pesticide of the present invention not only showed no toxicity even when administered 2,000 mg / kg higher than the general pesticide injection standard, but also showed the effect of increasing the weight compared to the control.

Bacillus subtilis EBM 13 of the present invention exhibits a broad spectrum of antifungal activity against plant pathogenic fungi, and is an environmentally friendly soil modifying agent as a multifunctional pesticide with environmental preservation and purification since it possesses organic degradation, pesticide tolerance and pesticide degradation. It can also be used for composting, environmental purifiers, etc.

In addition, the fungal control microbial pesticide made by coating the Bacillus subtilis EBM 13 of the present invention and then composited with a biomatrix has a high economical replacement effect and high performance using microorganisms native to our country, so the agricultural workforce is also innovative. Can be reduced.

Claims (7)

Bacillus subtilis EBM 13 (Accession No .: KCTC 0984BP) having antifungal activity against pathogenic fungi. According to claim 1, Pathogenic fungi are Pyricularia oryzae, Pythium ultimum, Rhizoctonia solani, Botryoshaeria dothidea , Bipolaris sorokniana , Blight fungus Botrytis cinerea , Colletotrichum gloeosporioides , Pyricularia grisea , Watermelon creeping mycophaerella melonis , Phytophthora capsici , Alternaria solani , Root rot disease ( Fusarium oxysporum) Sclerotinia sclerotiorum ), Bacillus subtilis EBM 13, characterized in that it is selected from fungal disease ( Neurospora sp) and candidiasis Candida albicans ). The method according to claim 1, Bacillus subtilis EBM 13 is a microbial pesticide, seed coating agent, microbial pesticide for controlling fungal diseases of mushrooms, additives for the production of sterilized non-sterile medium for mushroom cultivation, biotope, microbial nutrients, soil modifiers, composting agent Bacillus subtilis EBM, characterized in that it is used as an active ingredient of foliar spray or irrigation spray The antifungal active substance produced by the microorganism Bacillus subtilis EBM 13 of claim 1, comprising EBM13-P1 (molecular weight 1058.21), EBM13-P2 (1044.8), EBM13-P3 (1045.83), EBM13-P4 ( 1058.73), an antifungal active substance that is EBM13-P5 (1058.73). The Bacillus subtilis EBM 13 of claim 1 was coated with mucopolysaccharide; A microorganism pesticide prepared by making and drying a complex with a bio-matrix composed of plant-derived polymer materials, microbial-derived biopolymer materials, nutrients, auxiliary materials and additives. The method of claim 5, wherein the microbial pesticides, mushroom fungal disease control agent, additives for the preparation of sterile cultivation for mushroom cultivation, biotope, hydrating agent, granules, coating granules, microbial nutrients, encapsulating material of the microbial agent, foliar spray or irrigation spray Microbial pesticides, characterized in that used as zero. In the method for producing the antifungal active substance according to claim 4, using Bacillus subtilis EBM 13, lactose or mannitol as a carbon source, peptone or yeast extract as a nitrogen source, arginine or lysine is added, and vitamin B1 is added. In the culture medium, Bacillus subtilis EBM 13 was incubated at a temperature of 30 ~ 37 ℃, then obtained a culture supernatant, purified by gel filtration chromatography to prepare the antifungal active material of claim 4.