KR101379708B1 - Bacillus amyloliquefaciens EML-CAP3 strain, composition for control plant diseases and control method of plant diseases with same - Google Patents

Abstract

The present invention relates to a novel Bacillus amyloliquisions EML-CAP3, a composition for controlling plant diseases and a method for controlling plant diseases, containing the same, which are isolated from endogenous bacteria in red pepper. By spraying or irrigating the EML-CAP3 strain, its culture or its extracts on the plant, it is possible to control various plant diseases biologically, not only applicable to several crops at the same time, but also to the effects on several diseases in one crop at the same time. There is an effect that can increase the marketability and productivity of the crop.

Description

Bacillus amyloliquefaction EML-CAP3, a composition for controlling plant diseases and a method for controlling plant diseases comprising the same {Bacillus amyloliquefaciens EML-CAP3 strain, composition for control plant diseases and control method of plant diseases with same}

The present invention relates to a novel Bacillus amyloliquefactions EML-CAP3 isolated from red pepper as endogenous bacteria, a composition for controlling plant diseases and a method for controlling plant diseases containing the same.

Pesticides and fertilizers, the products of modern science, have greatly contributed to the liberation of humankind from hunger by increasing agricultural productivity. Recently, however, there has been a great deal of social interest in the negative aspects of human pesticides and fertilizers such as phosphorus and livestock toxicity and environmental pollution due to misuse and residue of pesticides and fertilizers. With the advent of the well-being era, there is an increasing demand for eco-friendly agricultural products that have reduced the use of synthetic pesticides or grown crops without using pesticides. In addition, farmers are increasingly importing cheaper imported agricultural products due to import liberalization. As a preference for organic farming with high income per unit area, the area of cultivating environmentally friendly agricultural products is gradually increasing.

However, it is very difficult to harvest crops without controlling plant diseases when crops are grown with environmentally friendly farming methods in actual packaging. Studies have shown that peaches and apples lose 100% and 97%, respectively, and vegetables such as cucumbers, cabbage, and tomatoes have a yield reduction of 39-63% when cultivated without crop protection. Therefore, in order to produce eco-friendly agricultural products, there is a need for biopesticides having excellent effects that can replace synthetic pesticides.

Biological pesticides include microbial pesticides that use microorganisms to control plant diseases, pests and weeds, and biochemical pesticides that use natural products produced by plants or microorganisms. In the case of microbial pesticides, it is difficult to mass-produce and formulate products, and the preservation stability of the products is low, and thus the market is not greatly expanded because standardization, standardization, and ease of distribution are not secured like chemical pesticides. In addition, when the microbial pesticide is used for the actual packaging, the plant disease can be controlled only after the microorganisms have grown in a significant amount after treatment, and thus there is a difficulty in controlling the plant disease in a short time. In addition, since the spectrum of applied diseases is narrow, various bio-pesticides must be treated in order to control other plant diseases occurring in the crops, which realistically puts an economic burden on the farmers.

On the other hand, Bacillus bacteria, which are mainly present in the root zone of the soil, produce various secondary metabolites that exhibit antimicrobial activities such as suctinine and iturin, and are Gram-positive, characterized by forming endogenous spores that are resistant to adverse environments such as heat. It is a bacterium. In addition, except for a few species, most of the strains of the genus Bacillus are safe for phosphorus and axial toxicity, and recently, many studies have been conducted to develop biological control agents using them.

In Korea, Bacillus amyloliquations LP03 strain (Korean Patent No. 0807403) and Bacillus atropeas CNU05-1 strain (Korean Patent No. 0773091), Bacillus for controlling plant diseases as strains for control of gray mold among Bacillus microorganisms. Subtilis KCCM10639 or KCCM10640 strain (Korean Patent No. 0767437) has been registered.

A number of microbial control agents exhibiting antifungal activity as strains of the Bacillus genus have been disclosed, but biopesticides that can effectively control various plant diseases have not yet been established, and thus, development of new biological control agents is required. .

KR 10-0773091 B1 2007.10.29 KR 10-0767437 B1 2007.10.09 KR 10-0807403 B1 2008.02.19

Accordingly, the present inventors can biologically control various plant diseases by immersing or irrigating Bacillus amyloliquefaciens EML-CAP3, which is an endogenous bacterium isolated from the leaves and stems of red pepper, in plant seeds, and also In addition to inhibiting spore germination and mold growth inhibition effect on phytopathogenic fungi, it was confirmed that there is an excellent plant germination promoting effect and completed the present invention.

An object of the present invention is a novel Bacillus amylolytics ( Bacillus) having excellent control against various plant pathogens amyloliquefaciens ) to provide a method for producing EML-CAP3 and its culture or extract thereof.

Still another object of the present invention is to provide a bioassay for phytopathogenic fungi using a culture extract of Bacillus amyloliquefaciens EML-CAP3.

Still another object of the present invention is the Bacillus amylolytics ( Bacillus Amyloliquefaciens ) To provide a composition for controlling plant diseases comprising EML-CAP3 and its culture or extract.

Still another object of the present invention is the Bacillus amylolytics ( Bacillus amyloliquefaciens ) To provide a method for controlling phytopathogenic microorganisms or pathogenic microorganisms using EML-CAP3 and its culture or extract.

In order to achieve the above object, the present invention provides a Bacillus amyloliquefaciens EML-CAP3 (Accession No .: KCTC 11971BP) having a control activity against plant pathogens.

Hereinafter, the present invention will be described in detail.

Bacillus amyl Lowry query Patience (Bacillus amyloliquefaciens) EML-CAP3 according to the invention in my isolated from the pepper leaves and stems of plants of viable cells, analysis of the nucleotide sequence of 16S rDNA of the strain, Bacillus amyl Lowry query Patience (Bacillus amyloliquefaciens ) and the near- failure . The novel strain was named Bacillus amyloliquefaciens EML-CAP3, and the Bacillus amyloliquefaction EML-CAP3 strain was identified on June 28, 2011 in the Korea Research Institute of Biotechnology. It was deposited with the Microbial Resource Center and received accession number KCTC 11971BP. See FIGS. 1 and 2.

The present invention provides a method for producing Bacillus amyloliquefaciens EML-CAP3 and its culture or its culture extract.

Bacillus amylolytics of the present invention ( Bacillus amyloliquefaciens ) EML-CAP3 contains LB medium (Luria-Bertani, DifcoTM, USA), YMB medium (Yeast Malt Broth, yeast extract 3g, malt extract 3g, peptone 5g, dextrose 10g, distilled water 1000ml), potato agar medium (Potato Dextrose) Agar, Difco ™, USA) can be cultured in a common medium for bacterial growth. In addition, the culture pH is preferably 4 to 6, the culture time is 24 to 48 hours, more preferably within 48 hours.

In addition, while general bacteria grow well at relatively high temperatures of about 30 ° C. to 40 ° C., and do not grow well when the temperature is low, the growth temperature of Bacillus amyloliquefaciens EML-CAP3 according to the present invention is 20 to 20 ° C. It is well cultured at 35 ℃, considering that the onset temperature of the main plant disease is 20 to 25 ℃ has a great advantage as a biological control for plant diseases control.

Bacillus amylolytics according to the present invention ( Bacillus amyloliquefaciens ) EML-CAP3 may be cultured further comprising a carbon source, a nitrogen source or a mixture thereof in a general medium for bacterial growth. The carbon source may be one or more selected from the group consisting of sucrose, dextrose, lactose, lactose, mannitol, soluble starch, and sorbitol. In addition, the amount to be added may preferably be added from 0.05 to 1.0% by weight (w / v). In particular, the use of water-soluble starch as the carbon source is preferable to increase the growth and antifungal activity of the strain.

In addition, the nitrogen source may be any material capable of supplying nitrogen to the culture, for example, sodium nitrate (NaNO ₃ ), ammonium nitrate (NH ₄ NO ₃ ), ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), malt extract (Malt extract), peptone (Peptone) and tryptone (Tryptone) may be used at least one selected from the group consisting of, the amount added may be preferably added in 0.05 to 1.0% by weight (w / v).

In more detail, it is preferable to add malt extract and peptone as a nitrogen source in the medium containing the said carbon source. The content of the preferred nitrogen source in the medium is 0.05 to 1.0 wt% (w / v), more preferably 0.5 to 1.0 wt%. If the content of the carbon source or nitrogen source in the medium is more than the above range may result in a deficiency of other nutrients, if less than the above range, the growth effect of the strain due to the separate supply of the carbon source or nitrogen source is insignificant.

In addition, in the case of using the general medium for bacterial growth, that is, the medium of LB, adding the carbon source and the nitrogen source to the concentration of the LB medium, that is, 1% or 2.4%, does not significantly affect the growth of the strain. The use of a small amount of media in the process also has the advantage of reducing production costs. See FIGS. 8 and 9.

The present invention is Bacillus amylolytics ( Bacillus amyloliquefaciens ) Provides a composition for controlling plant diseases comprising EML-CAP3 and its culture or extract.

In addition, Bacillus amylolytics according to the present invention ( Bacillus amyloliquefaciens ) EML-CAP3 has no pathogenicity against crops such as cucumbers, sesame seeds, red peppers, tomatoes, strawberries, rice, barley, wheat, and grass.

More specifically, Bacillus amylolytics ( Bacillus amyloliquefaciens ) EML-CAP3 (Accession No .: KCTC 11971BP), a plant disease control composition comprising a culture solution or a culture extract thereof is a pepper blight (pathogen: Phytophthora capsici ), pepper anthrax (pathogen: Colletotrichum gloeosporioides ), pepper bacterial spot pattern disease (pathogen: Xanthomonas campestris ), rice blast (pathogen: Magnaporthe grisea ), Bipolaris oryzae ), Fusarium proliferatum ), tomato late blight (pathogen: Phytophthora infestans ), tomato ash fungus (pathogen: Botrytis) cinerea ) exhibited a control effect against at least one plant disease selected from the group consisting of, among them, it was confirmed that effectively control plant diseases, such as pepper anthrax, rice blast, tomato gray mold disease.

Bacillus amylolytics according to the present invention ( Bacillus amyloliquefaciens ) EML-CAP3 (Accession No .: KCTC 11971BP) Strain itself, its culture, or a composition comprising the extract of the culture, to inhibit the plant disease on the environment in which the plant is growing (eg soil, water) or on the surface of the growing plant. Various plant diseases can be biologically controlled by spraying or irrigation in amounts that can be achieved.

In addition, plant seeds may be Bacillus amylolytics (Bacillus amyloliquefaciens) EML-CAP3 (Accession No .: KCTC 11971BP) By immersing in a culture solution or a culture filtrate, phytopathogenic fungal growth inhibition or plant germination can be promoted.

The plant can be used for one or more seeds selected from the group consisting of cucumbers, sesame seeds, peppers, tomatoes, strawberries, rice, barley and grass, which in turn provides the effect of reducing the use of synthetic fertilizers.

The culture may further contain a biopolymer when culturing the strain. The biopolymer is used as a delivery medium for formulation in the manufacture of a liquid hydration type, and is transparent and does not leave scars, and maintains proper viscosity, and is selected from the group consisting of bran, oatmeal, rice bran, soybean meal, and cornmeal. One or more types can be used. At this time, the soybean meal is preferably used as the biopolymer material added during the inoculation after inoculation of the Bacillus amyloliquefaction EML-CAP3 strain.

In addition, if necessary, it can be used as a composition for controlling plant diseases by mixing with a carrier commonly used in the field of pesticides and formulated into powder, pellets, granules or solutions. The carrier may be water, white carbon, kaolin, and the like, and the culture may be a liquid culture or a solid culture.

In addition, the water extract of the strain culture may sequentially obtained through extraction with an organic solvent, preferably a C _{1 -4} alcohol, hexane, acetone, using an organic solvent selected from chloroform and ethyl acetate, alone or in combination of two or more Extraction can be performed using.

More specifically, the supernatant obtained by centrifuging the Bacillus amyloliquefaciens EML-CAP3 strain culture is an extract extracted with one or more extraction solvents selected from ethyl acetate and chloroform, and preferably, the supernatant is ethyl acetate. Partition extraction was carried out, and the aqueous layer was again obtained by partition extraction with ethyl acetate in the same volume. In the extraction, it is preferable to mix the same volume of the extraction solvent with the supernatant.

The present invention Bacillus amylolytics on a slide glass ( Bacillus amyloliquefaciens ) dropping EML-CAP3 (Accession No .: KCTC 11971BP) culture filtrate and oidium of phytopathogenic fungi; And

It provides a bioassay method for phytopathogenic fungi comprising the step of: moving the slide glass to a petri plate maintained in saturation humidity to investigate the spore germination inhibition rate of phytopathogenic fungi at each time period.

The phytopathogenic fungi are characterized by powdery mildew.

The bioassay according to the present invention is more effective in terms of time and cost than other conventional antimicrobial assays, and it can be confirmed that it reacts very sensitively at very low concentrations of the cultures to be assayed. When the amount is low, the bioassay is very useful and shows an inhibitory activity in a concentration-dependent manner, which may be useful for high-speed screening technology (HTS).

The present invention is Bacillus amylolytics ( Bacillus Amyloliquefaciens ) EML-CAP3 (Accession No .: KCTC 11971BP), and a culture method or a control method for the control of the phytopathogenic microorganism or pathogenic microorganism comprising the step of immersion treatment or irrigation treatment before sowing.

In the present invention, the phytopathogenic microorganism is Phytophthora capsaish ( Phytophthora capsici ), Colletotrichum gloetosporioides gloeosporioides ), Xanthomonas campestris ), Pyricularia grisea , Bipolaris oryzae ), Fusarium proliferatum proliferatum ), Phytophthora infestans ) and Botrytis cinerea , and at least one member selected from the group consisting of the pathogenic microorganisms Escherichia coli ( Esherichia coli) coli ) or Staphyloccocus aureus .

The control method is Bacillus amylolytics of the present invention ( Bacillus amyloliquefaciens ) EML-CAP3 (Accession No .: KCTC 11971BP), its culture or culture extracts were treated with a suspension containing the strain at a concentration of 1 × 10 ³ cfu / ml to 1 × 10 ¹² cfu / ml when immersed or irrigated. Characterized in that.

Bacillus amylolytics according to the present invention in a specific method ( Bacillus amyloliquefaciens ) EML-CAP3 (Accession No .: KCTC 11971BP), its culture or its extract is sprayed on soil, leaves, stems, flowers or fruits of crops, can be used by diluting the composition in water, the microorganism of the present invention In order to add the antimicrobial activity of the strain may further comprise a foliar spray or irrigation spray. In addition, during the spraying season, the preventive treatment before plant pathogens can increase and increase the control effect.

Bacillus amyloliquilation EML-CAP3 strain according to the present invention is a microorganism native to Korea and has a high economical replacement effect, has a strong antibacterial activity against pathogens causing various plant diseases including anthrax and environmentally friendly Can be.

In addition, the Bacillus amyloliquilation EML-CAP3 strain according to the present invention has a high economical replacement effect according to the use of microorganisms and has a high performance has the effect of increasing the marketability and productivity of the crop.

Figure 1 shows the nucleotide sequence of 16S rDNA of novel Bacillus amyloliquefaciens EML-CAP3 (KCTC 11971BP) according to the present invention,
Figure 2 is a result of confirming the phylogenetic position through 16S rDNA of the novel Bacillus amyloliquefaciens EML-CAP3 (KCTC 11971BP) according to the present invention (the tree is Bacillus atrophaeus JCM 9070 and Bacillus licheniformis DSM 13 strain Is used as an out-group and the scale bar shows 0.01 substitutions per nucleotide),
Figure 3 shows a culture photograph (A) of a novel Bacillus amyloliquefaciens EML-CAP3 (KCTC 11971BP) according to the present invention and a photograph (B) observed with a scanning electron micrograph (B). ,
4 is a result confirming the inhibitory activity effect of various Bacillus amyloliquefaciens EML-CAP3 (KCTC 11971BP) according to the present invention against various phytopathogens,
Figure 5 shows the growth curve according to the medium and temperature during the culture of novel Bacillus amyloliquefaciens EML-CAP3 (KCTC 11971BP) according to the present invention,
(A: LB medium, B: YMB medium, C: PDB medium)
Figure 6 shows the growth curve according to pH in the culture of novel Bacillus amyloliquefaciens EML-CAP3 (KCTC 11971BP) according to the present invention,
Figure 7 shows the growth curve according to the carbon source (A) and nitrogen source (B) in the culture of novel Bacillus amyloliquefaciens EML-CAP3 (KCTC 11971BP) according to the present invention,
Figure 8 shows the growth curve according to the composition of the basic medium and the carbon source and nitrogen source in the culture of novel Bacillus amyloliquefaciens EML-CAP3 (KCTC 11971BP) according to the present invention,
Figure 9 shows the OD (Optical density) and CFU (colony forming units) according to the composition of the basic medium and the carbon source and nitrogen source in the culture of novel Bacillus amyloliquefaciens EML-CAP3 (KCTC 11971BP) according to the present invention Shows the growth curve compared to
Figure 10 is a schematic diagram showing the culture separation process of novel Bacillus amyloliquefaciens EML-CAP3 (KCTC 11971BP) according to the present invention,
Figure 11 is an antibacterial against the bacillus bacterium (A) and the antibiotic resistance strain staphylococcus (B) as an ethyl acetate extract of the Bacillus amyloliquefaciens EML-CAP3 (KCTC 11971BP) culture medium according to the present invention Activity,
12 is a result confirming the effect of spore germination on powdery mildew bacteria of the novel Bacillus amyloliquefaciens EML-CAP3 (KCTC 11971BP) filtrate according to the present invention,
Figure 13 is a result confirming the growth inhibitory effect of the fungus occurring on the rice seed of Bacillus amyloliquefaciens EML-CAP3 (KCTC 11971BP) culture medium according to the present invention.
(A: distilled water, B: added 31.25 ppm of Spotag, C: 50-fold dilution of LB, D: 100-fold dilution of LB, E: 50-fold dilution of Bacillus amylolytics EML-CAP3 culture, F: Bacillus amyloliquiparation EML 100-fold dilution of CAP3 culture)

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by the following examples, and various modifications or changes can be made within the spirit and scope of the present invention to those skilled in the art.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

Example 1 Isolation and Identification of Bacillus Amylolytics EML-CAP3

(1) Isolation of Strains

The leaves and stems of red pepper ( Capsicum annuum L.) were collected from November 2008 (Winter) to October 2010 (Autumn) in Nampyeong-eup, Naju-si, Jeollanam-do. The leaves and stems of the pepper were stored at 4 ° C. and used within 24 hours to isolate plant endogenous strains.

In the case of leaves, 4 pieces cut into 1 cm 2 (1 cm length 1 cm) size per leaf were immersed in 2% NaOCl for 1 minute and then surface sterilized and washed three times with sterile water and dried. The separation method was divided into direct plating method and dilution plating method.

First, in the case of the dilution plating method, the surface sterilized leaves were put in a 1.5 ml eppendorf tube and ground at a ratio of 1: 1 (g: ㎖). After grinding, 100 μl of the stock solution and the 10-fold dilutions were taken and dispensed into PDA (Potato dextrose agar, Difco ™, USA) medium and plated.

Direct plating method was applied to PDA medium by 3 pieces of surface sterilized leaves. Stems were surface sterilized using the same method and cut into three pieces of 1 cm in length, respectively, by direct plating method and dilution plating method. Each medium was incubated in a multi-chamber incubator (HT-103-4, Hanbaek, Seoul, Korea) at 23 ° C. for 14 days to measure bacterial colony forming unit (CFU), and LB agar (Luria-Bertani, Difco ™). , USA) was transferred to the medium and purified.

For preservation of the purely isolated strains, the aliquots were collected in 1.5 ml eppendorf tubes containing 20% glycerol (-80 ° C) and used for necessary experiments.

As a result, as can be seen in Table 1, it was possible to isolate 216 purely isolated endogenous bacteria that are distinguished from the naked eye observation, form and growth pattern among the strains isolated from the leaves and stems of the pepper.

In addition, at 25 ℃ for 7 days anthrax (Colletotrichum gloeosporioides) in a culture and then, a sterile 8 mm borer to starting the gilhanggyun weeks for the various plant pathogens, including anthrax (Colletotrichum gloeosporioides) EML-CG01 to cause anthrax A hole was cut and placed in the middle of the potato agar medium. The culture medium of strains shaken at 150 rpm for 24 hours at 32 ° C was drawn at a position 2 cm away from the pathogen, and then grown at 25 ° C for 7 days to measure growth refrigeration. Strains with good growth and the highest antimicrobial activity against Colletotrichum gloeosporioides were selected and finally selected, which was named EML-CAP3. The growth curve of the selected EML-CAP3 strain was good at 24 hours at 35 ° C., but growth was low at 24 hours, and growth was low even at 25 ° C., which was low.

In addition, as a result of observing the selected EML-CAP3 strain by electron microscopy, as shown in Figure 3, the gram-positive long rod-like spores were formed, and the formed colonies showed a smooth gloss surface.

(2) Molecular systematic analysis of strain

The sequencing was performed for the final identification of the strain isolated and selected in (1). After separating the DNA of the EML-CAP3 strain, 27f (5'-AGA GTT TGA TCM GGC TCA G-3 ') and 1492r (5'-GGT TAC CTT GTT ACG ACT T-3') were used for sequencing 16S rDNA. Polymerase chain reaction (PCR) was carried out using two oligonucleotides as primers, which were denatured at 95 ° C for 1 minute, annealing at 53 ° C for 40 seconds, and at 72 ° C. The DNA was amplified by enzymatic method under the conditions of extension for 1 minute.

The amplified DNA was purified after nucleotide sequence analysis (Fig. 1). As a result of comparison of nucleotide sequences with standard strains using EzTaxon (Chun et al., 2007), as shown in FIG. 2, it was confirmed that Bacillus amyloliquefaciens had a close relationship with Bacillus amyloliquefaciens . It was named Loriquations EML-CAP3 strain ( Bacillus amyloliquefaciens EML-CAP3).

The Bacillus amyloliquiation EML-CAP3 strain was deposited on June 28, 2011 to the microbial resource center in the Korea Biotechnology Research Institute of Korea's patent strain deposit institution was given the accession number KCTC 11971BP.

Example 2 Investigation of Antimicrobial Activity of Phytopathogenic Bacteria of Bacillus Amyloliquisation EML-CAP3

The antibacterial activity against the following phytopathogens was investigated using the selected Bacillus amyloliquilysis EML-CAP3.

Phytopathogens include Red pepper bacterium ( Phytophthora capsici EML-PHC01), Red pepper anthrax bacterium ( Colletotrichum gloeosporioides EML-CG01), Pyricularia grisea EML-PGR01, Rice seed germ ( Bipolaris oryzae EML-ORY15) Fusarium The results are shown in Table 2 below using proliferatum EMLGYP2).

As can be seen in the results of Table 2 and the results of Figure 4, Bacillus amylolytics EML-CAP3 is antibacterial activity against all of the pepper bacterium, pepper anthrax, rice blast, rice sesame seed germ, fog grass In particular, it was confirmed that it has excellent antimicrobial activity against pepper anthrax and rice blast.

The above results further confirm that Bacillus amylolytics EML-CAP3 has a control effect against pepper blight, pepper anthrax, rice blast, rice sesame seed disease, and mist wilting disease.

Example 3 Mass Production Method of Bacillus Amylolytics EML-CAP3

(1) Culture Characterization

The culture characteristics of the Bacillus amyloliquilysis EML-CAP3 was investigated.

As medium, LB medium (Luria-Bertani, DifcoTM, USA), YMB medium (Yeast Malt Broth, yeast extract 3g, malt extract 3g, peptone 5g, dextrose 10g, distilled water 1000 ml), potato agar medium (Potato Dextrose Agar, Difco ™, USA) was used by autoclaving at 121 ° C. for 15 minutes. Pre-culture was prepared by inoculating a single colony of the active strain in LB culture and shaking culture at 32 ℃ for 24 hours at 150 rpm. After inoculating 0.2% of the preculture solution into the prepared culture solution, the culture temperature was set at 25 ° C, 27 ° C, 30 ° C, 32 ° C, and 35 ° C at 3, 6, 12, 24, 36, 48, 72, 96 at 150 rpm. After shaking for 120 hours, the absorbance (OD) at 660 nm was measured using a Microplate reader (Bio-Tek Powerwave XS, US).

As a result, as can be seen in Figure 5, the medium was confirmed that the growth of the strain in the YMB medium, the temperature is 35 ℃ temperature conditions.

In addition, to investigate the effects of the initial pH change of Bacillus amyloliquidation EML-CAP3, the absorbance was adjusted for each time by adjusting the pH to 3, 4, 5, 6, 7, 8, and 9 in the basic medium of 2.4% LB. Measured. As a result, as can be seen in Figure 6, it was confirmed that the growth of the strain excellent under the conditions of pH 4 to 6.

In addition, the availability of the carbon source and nitrogen source of Bacillus amylolytics EML-CAP3 was investigated.

Sucrose, dextrose, lactose, mannitol, mannitol, soluble starch and sorbitol were used as carbon sources, and sodium nitrate (nitrogen) was used as a nitrogen source. Nitrogen sources such as NaNO ₃ ), ammonium nitrate (NH ₄ NO ₃ ), ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), malt extract, peptone and tryptone were used. Carbon sources and nitrogen sources were added 1% each to 1% LB medium, and the absorbance was measured at each time.

As a result, as can be seen in Figure 7, the carbon source was water-soluble starch, nitrogen source was confirmed that the excellent growth of the strain in malt extract and peptone.

In addition, to confirm the growth difference of the strain according to the concentration of the basic medium was prepared 1% and 2.4% LB as the basic medium, and the absorbance and CFU was measured by time.

As a result, as can be seen from Figure 8, when the carbon source and the nitrogen source was not added, it was confirmed that the growth was good at 2.4% of the concentration of LB medium than 1%.

In order to confirm the absorbance and CFU difference when combining the basic medium (1% LB) and the active strain optimal carbon source and nitrogen source, 1% of the optimal carbon source and nitrogen source of the active strain obtained as a result of the experiment were added to the basic medium by hour Absorbance and CFU were measured. As a result, as shown in FIGS. 8 and 9, when the carbon source and the nitrogen source were not added, the growth of LB medium was better at 2.4% than the 1%, but when the carbon source and the nitrogen source were added. The growth of the strain according to the basic medium concentration was confirmed that there is no significant difference.

From the above results, the mass culture of Bacillus amyloliquidation EML-CAP3 was prepared under conditions of pH 4 to 6 by adding water-soluble starch as carbon source, malt extract or peptone as nitrogen source to YMB medium (or LB medium). It is preferable to use, and by incubating at a temperature of 35 ℃ after inoculation of the strain for 24 hours, it was confirmed that it is possible to mass-produce a strain excellent in growth.

Example 4 Investigation of Spore Germination Inhibition Effect of Bacillus Amyloliquisation EML-CAP3 in Powdery mild Pathogens

Bacillus amyloliquisions EML-CAP3 was carried out the following experiment to investigate the spore germination inhibition against various powdery pathogens.

After inoculating Bacillus amyloliquisation EML-CAP3 strain into LB medium, incubated at 120 rpm for 24 hours at 32 ° C, centrifuged to remove the cells, and then with a 0.1 μm syringe filter (Pall corporation, USA). Filtration gave a culture filtrate.

Dropping the culture filtrate obtained by diluting 50 times and 100 times on the slide glass drop by drop, and Chinese powder ( Acer) caused powdery mildew buergerianum ), cedar ( Euonymus japonica ), Lagerstroemia indica ), Zinnia elegans , Quercus phillyraeoides ), green pepper ( Capsicum annuum There is L. Angulosum ) leaves were collected and lightly shaken the spores of powdery mildew pathogen , put into a Petri plate in a saturated humidity state after 24 hours to measure the germination rate of the spores through an optical microscope is shown in Table 3 below.

As a result, as shown in Table 3 and Figure 12, the germination rate of Chinese maple leaf powdery mildew bacteria in distilled water treatment group was 27.26 ± 4.90 (100% standard), but 19.99% in the treatment group diluted 50-fold with Bacillus amylolytics EML-CAP3 It can be confirmed that the germination rate of, and when the control (germination rate on distilled water) is calculated as 100%, it was confirmed that the spore germination inhibition rate is about 5 times, that is, 80%.

The above results show that Bacillus amyloliquefactions EML-CAP3 is effective in controlling a variety of fungal diseases, and in particular, shows an excellent inhibitory effect against various powdery mildew bacteria can be developed as a powdery mildew control agent.

In addition, from the above results, the activity of the bacterial culture against Bacillus amyloliquefaction EML-CAP3 against powdery mildew was measured by the slide culture method.

It is then mixed with Bacillus amyloliquefaction EML-CAP3 culture filtrate in distilled water at various dilution ratios, dropping the diluted culture filtrate on a slide glass, and then dropping the spores (oidium) of powdery mildew on top of it, and then saturated Inhibition of spore germination of powdery mildew pathogens was investigated by moving to a Petri plate kept in humidity.

As a result, it was more effective in terms of time and cost than other antimicrobial assays, and it could be confirmed that the spores of other white powdery pathogens could be used in addition to Chinese maple powdery mildew. In addition, it can be confirmed that the reaction is very sensitive at a very low concentration of the target culture of the assay, the bioassay is very useful and concentration-dependent inhibitory activity when the amount of the active substance or culture to be assayed is low, high-speed screening technology (HTS) has the advantage of being useful.

Example 5 Antimicrobial Activity According to the Biopolymer Addition

In order to develop the product using the Bacillus amyloliquilation EML-CAP3 strain, various biopolymers were added to the culture medium, and then cultured and the antibacterial activity of the culture strains was changed.

A medium was prepared by mixing 10% by weight of bran, oatmeal, rice bran, soybean meal, and cornmeal as the main ingredients (biopolymers) in 50% by weight of tertiary distilled water, and sterilized for 15 minutes at 121 ° C and 1 atmosphere. After inoculation, 1 ml of Bacillus amylolytics EML-CAP3 (1 × 10 ⁶ CFU concentration) was inoculated and incubated in a 32 ° C. stirred incubator.

In order to measure the number of viable cells after the stationary culture, after incubation for the last 20 days, 1 g of each solid medium was taken and placed in 9 ml of sterile distilled water and diluted to 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ times and smeared on LB medium. After culturing at 32 ° C. for 2 days, the number of viable cells (CFU, colony forming unit) was measured using a digital colony counter (KT0074A, Kastech, Korea).

Take 1 g each of the solid medium, add 10 ml of sterile distilled water, stir to dissolve the solid medium well, centrifuge at 1500 rpm for 15 minutes, and place 50 μl of the supernatant on a paper filter (diameter 8 mm). , 7 days incubation at 25 ℃ to measure the growth inhibition ring to evaluate the antimicrobial activity against heat-resistant bacteria.

As a result, as shown in Table 4, when the bran, rice bran, oatmeal, and corn medium medium were cultured as the main component, the antimicrobial activity against the germ bacterium showed moderate antimicrobial activity, and the soybean meal medium was somewhat different from the other main ingredients. It was confirmed that the low antimicrobial activity.

In addition, the number of viable cells of Bacillus amylolytics EML-CAP3 cultured by varying the composition of the basic medium and the biopolymer, as shown in Table 5 below, Bacillus amylolytics EML-CAP3 added soybean meal as a main component In case of culturing, the number of viable cells increased by 10 times or more by 30 times (average 20 times) by less than 10 times more than the case of using bran as the main ingredient.

[ Example  6] Bacillus Amylorium quaternization EML - CAP3  Investigation of Antimicrobial Activity of Extracts against Phytopathogenic and Pathogenic Microorganisms

The composition for controlling plant diseases comprising the Bacillus amyloliquefaction EML-CAP3 was prepared by the following method.

First, the extract of Bacillus amyloliquelation EML-CAP3 was prepared by the method of FIG. In more detail, Bacillus amyloliquelation EML-CAP3 was inoculated (1 × 10 ⁶ ) in LB medium and incubated for 48 hours at 32 rpm at 120 rpm. The culture solution was centrifuged at 6,000 rpm for 15 minutes to remove the cells, and the recovered supernatant was the same volume of chloroform (CHCl ₃ , OCI, 67-66-3) and ethyl acetate (EtOAc, OCI, 141-78-6) Two times each was extracted. The recovered organic solvent layer was concentrated under reduced pressure with a concentrator (N-100, Tokyo Rikakikai Co., Ltd) and used as a crude extract for antimicrobial activity test.

The antimicrobial activity against phytopathogenic microorganisms or pathogenic microorganisms was investigated using the extract of Bacillus amyloliquidation EML-CAP3 extracted by the above method.

The phytopathogenic microorganism is Pyricularia grisea EML-PGR01) and vegetable antibacterial bacterium ( Xanthomonas campestris ) were used, and pathogenic microorganisms were Escherichia coli and multidrug-resistant staphyloccocus. aureus ) was used.

As a result, as can be seen in Table 6 and FIG. 11, the control composition obtained by recovering the ethyl acetate (EtOAc) layer and the chloroform (CHCl ₃ ) layer has high antibacterial activity against phytopathogenic microorganisms as well as pathogenic microorganisms, and also has an active range. Broadly, it was confirmed that Bacillus amyloliquilysis EML-CAP3, its culture solution and extract of the culture solution can all be usefully used for the control of pathogenic microorganisms.

[ Example  7] Bacillus Amylorium quaternization EML - CAP3 Inhibition of phytopathogenic fungi of Plant germination  Promotion effect investigation

The bacteriostatic fungal growth inhibition and plant germination promoting effect of the Bacillus amyloliquisions EML-CAP3 was investigated.

In order to confirm the effect of inhibiting the fungi generated on rice seeds, incubated at 120 rpm for 24 hours at 32 ° C. in LB broth, the cells were centrifuged to remove the cells, and then a 0.1 μm syringe filter (Pall corporation, USA) was used. Filtration gave a filtrate.

Rice seeds were immersed in culture (stock solution, diluent) and culture filtrate (stock solution, diluent) of Bacillus amyloliquefaction EML-CAP3 for 24 hours, respectively, followed by ampicillin, neomycin, streptomycin ( Ten rice seeds were plated on a medium containing antibiotics such as Streptomycin) and a humidified solid Petri plate, respectively, and the number of molds and germination of seeds during culture were measured. Sterilized water, culture broth LB stock solution and diluent were used as a control, and positive control (Sporta (prochloraz 25%, Bayer) used for seed sterilization) was used under the same conditions.

As a result, as can be seen in Table 7 and Figure 13, even when diluting Bacillus amyloliquefaction EML-CAP3 culture and culture filtrate 100 times when immersion treatment of rice seed is used to inhibit the growth of fungi superior to the control In addition, it was confirmed that it is effective in promoting germination of rice.

The above results are also confirmed that the Bacillus amyloliquilation EML-CAP3 culture and culture filtrate is effective in promoting plant germination as well as inhibiting the growth of phytopathogenic fungi.

Korea Biotechnology Research Institute KCTC11971BP 20110628

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Claims (12)

delete At least one plant disease selected from the group consisting of Bacillus amyloliquefaciens EML-CAP3 (Accession No .: KCTC 11971BP), rice sesame seed disease disease comprising its culture solution or culture extract thereof, Chinese maple powdery mildew, and crape powdery mildew Control composition. delete 3. The method of claim 2,
The plant disease is characterized in that caused by at least one phytopathogenic microorganism selected from the group consisting of Bipolaris oryzae , Chinese maple powder ( Sawadaea nankinensis ) and Crape myrtle ( Golovinomyces cichoracearum ) Bottle control composition. 3. The method of claim 2,
The culture extract is obtained by using an organic solvent selected from C _1-4 alcohol, hexane, acetone, chloroform and ethyl acetate alone or by extracting the culture solution using two or more kinds sequentially, for controlling plant diseases. Composition. delete delete Plant seeds from Bacillus amyloliquefaciens EML-CAP3 (Accession No .: KCTC 11971BP) from the group consisting of rice sesame seed disease, Chinese maple powder and crape powder At least one phytopathogenic fungal growth inhibition or plant germination method selected. 9. The method of claim 8,
The plant is characterized in that at least one selected from the group consisting of red pepper, rice, Chinese maple and crape myrtle. Bacillus amyloliquefaciens Bacillus amyloliquefaciens EML-CAP3 (Accession Number: KCTC 11971BP), rice sesame seed disease, Chinese maple powdery mildew and powdered blue powdery mildew, comprising immersion or irrigation of the culture or its extracts before sowing. Control method for controlling one or more phytopathogenic microorganisms selected from the group consisting of. delete 11. The method of claim 10,
The control method is characterized in that the concentration of Bacillus amyloliquefaciens EML-CAP3 (Accession No .: KCTC 11971BP) during immersion treatment or irrigation treatment is 1 × 10 ³ cfu / ㎖ to 1 × 10 ¹² cfu / ㎖ Control method.

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