KR101535893B1 - New microorganism Bacillus amyloliquefaciens CC110 and Microbial agent biopesticide containing the same - Google Patents

Abstract

The present invention relates to a novel microorganism Bacillus amyloliquefaciens CC110, a microorganism preparation containing the same, and a microbial pesticide.
The novel microorganism Bacillus amyloliquefaciens of the present invention is a novel microorganism, The strain CC110 (KACC 91830P) is represented by SEQ ID NO: 1 and is characterized in that it has a controlling effect against a plant disease.
According to the present invention, Bacillus amyloliquefaciens CC110 strain, which is a novel microorganism having a controlling effect against a plant disease, and a microorganism preparation and a microbial pesticide are provided, whereby an environmentally friendly plant It is possible to control the disease and increase the number of crops.

Description

[0001] The present invention relates to a new microorganism Bacillus amyloliquefaciens CC110, a microorganism preparation containing the microorganism Bacillus amyloliquefaciens CC110 and a microbial agent biocide,

The present invention relates to a novel microorganism Bacillus amyloliquefaciens CC110, a microorganism preparation containing the same and a microbial pesticide containing the same, and more particularly, to a microbial agent and a microbial pesticide containing a microbial agent , And the microbial agent and microbial pesticide containing the strain as an active ingredient.

Today, agriculture has been devoted to increasing food production through methods such as breeding, improvement of soil fertility, pest control, and weed removal, but persistent pesticide use and abuse are causing serious pollution of soil and river.

In addition, since these pesticides remain in crops, they cause problems such as toxicity, environmental pollution, and toxicity to humans and livestock. Therefore, the use of pesticides is now being strengthened in the world.

As concerns about these pesticides and the quality of life have increased, recently, biological control using microorganisms and plant extracts has been of interest as a new alternative to controlling pests of crops.

In particular, as a biosurfactant using microorganisms, antibiotics or toxins produced from soil microorganisms are used to protect plants from infection with pathogens by using antagonistic action of microbial pesticides or microorganisms themselves that control pathogens or insects, And microbial fertilizers that promote microbial growth.

Such biological control can be used in place of chemical pesticides which are harmful to the environment, so that it is possible to provide safe agricultural products to consumers.

In particular, the control of botanicals has usually relied on the widespread use of toxic chemicals (pesticides).

These organic synthetic pesticide control methods often require toxic chemicals in the soil as well as the target plants and plant products, resulting in plant residue and soil residual toxicity problems, It is toxic to microorganisms and animals, and is known to be harmful to humans.

Therefore, much of the recent research on plant disease control is aimed at finding an environmentally friendly plant disease control method, and one of them is the development of biopesticides using bio-resources that are antagonistic to plant pathogenic microorganisms.

An antagonistic microorganism is a microorganism that inhibits the growth of another microorganism when cultured with another kind of microorganism. Antagonistic microorganisms are known to cause parasitism, predation, and antibiotic action.

Korean prior art 10-1260170 entitled " Pseudomonas CP236, a novel strain having antimicrobial activity, and a plant disease control composition containing the same, " or Korean Patent No. 10-1166853 " have.

An object of the present invention is to isolate and identify Bacillus amyloliquefaciens CC110, a novel microorganism having a controlling effect against a plant disease, and to provide a microorganism preparation and a microbial pesticide containing the strain.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

Novel microorganism Bacillus amyl quinone Lowry pension of the present invention for achieving the above object's (Bacillus amyloliquefaciens ) The strain CC110 (KACC 91830P) is represented by SEQ ID NO: 1 and is characterized in that it has a controlling effect against a plant disease.

The strain is characterized in that it is separated from cucumber leaves.

The plant disease is characterized in that it is any one of a necrotic disease, a powdery mildew, and a gray mold disease.

The strain is characterized in that it is used as an effective ingredient of any one of microbial pesticides, seed coating agents, soil conditioners, composting homeworks, foliar spraying agents, or traditional spraying agents.

The microbial agent for controlling plant diseases according to the present invention is a novel microorganism Bacillus amyloliquefaciens , CC110 (KACC 91830P) strain or a culture thereof and a culture dilution liquid thereof as an active ingredient.

The culture medium is characterized by being cultured in a TSB (Tryptic Soy broth) medium.

The culture medium is characterized by containing at least one of fructose, mannitol, sorbitol, sucrose and glucose as a carbon source.

The carbon source is contained in an amount of 5 to 10% by weight based on the weight of the culture liquid.

Examples of the nitrogen source include L-asparagine, yeast extract, casein, peptone, ammonium chloride (NH ₄ Cl), L-arginine, , Ammonium phosphate ((NH ₄ ) ₂ HPO ₄ ), and L-proline.

The nitrogen source is contained in an amount of 5 to 10% by weight based on the weight of the culture liquid.

The culture medium is characterized in that it is cultured at a pH of 4.5 to 9.5 and at a temperature of 7.5 to 55 ° C.

The culture broth contains Bacillus amyloliquefaciens (1 x 10 ^{5 to} 1 x 10 ⁸ spores per ml) CC110 strain is included.

The culture dilution is characterized by diluting the culture solution 2 to 5 times.

The microbial pesticide for controlling plant diseases according to the present invention is a microbial pesticide for controlling plant diseases, comprising the novel microorganism Bacillus amyloliquefaciens , CC110 (KACC 91830P) strain or a culture thereof and a culture dilution liquid thereof as an active ingredient.

The method for controlling the plant disease of the present invention is characterized in that the microorganism pesticide is sprayed on a plant in which the plant disease is present.

According to the present invention, Bacillus amyloliquefaciens CC110 strain, which is a novel microorganism having a controlling effect against a plant disease, and a microorganism preparation and a microbial pesticide are provided, whereby an environmentally friendly plant It is possible to control the disease and increase the number of crops.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the molecular biological identification of the novel strain Bacillus amyloliquefaciens CC110 isolated in Example 1. FIG.
2 is a view showing the nucleotide sequence of the novel strain Bacillus amyloliquefaciens CC110 isolated in Example 1. Fig.
3 is a diagram showing morphological characteristics of the novel strain Bacillus amyloliquefaciens CC110 isolated in Example 1. Fig.
4 is a graph showing the effect of the new strain Bacillus amyloliquefaciens CC110 isolated in Example 1 on the occurrence of cucumber mildew.
FIG. 5 is a graph showing the effect of inhibiting the occurrence of cucumber fungus in the new strain Bacillus amyloliquefaciens CC110 cell and the culture broth of the new strain isolated in Example 1 and the morphological change of the cucumber bacterium when cultured.
A: Cucumber leaf treated with culture solution of CC110 strain, cucumber leaf surface with no nociceptive disease after 7 days of inoculation
B: Three days after the treatment of the culture solution of CC110 strain on cucumber leaves with nociceptive disease, the sporangia of the nociceptive bacteria were transformed into the sporangia
C: Electron micrograph of CC110 cultured in sporangia of the pathogenic bacteria of cucumber
D: After 7 days from the culture of CC110 strain,
6 is a graph showing the growth ability of the novel strain Bacillus amyloliquefaciens CC110 strain isolated in Example 1 by pH.
7 is a graph showing the growth ability of the new strain Bacillus amyloliquefaciens CC110 strain isolated in Example 1 according to the kind of the medium.
FIG. 8 is a graph showing the growth ability according to the type of carbon source (0.5%) contained in the medium of the strain Bacillus amyloliquefaciens CC110 isolated in Example 1. FIG.
FIG. 9 is a graph showing the growth ability according to the type of carbon source (1.0%) contained in the culture medium of the new strain Bacillus amyloliquefaciens CC110 isolated in Example 1. FIG.
10 is a graph showing the growth ability according to the type of nitrogen source (0.5%) contained in the medium of the strain Bacillus amyloliquefaciens CC110 isolated in Example 1. Fig.
FIG. 11 is a graph showing the growth ability according to the type of nitrogen source (1.0%) contained in the culture medium of the novel strain Bacillus amyloliquefaciens CC110 isolated in Example 1. FIG.
12 is a view showing the effect of controlling the fresh strain Bacillus amyloliquefaciens CC110 strain isolated in Example 1 against cucumber gray mold.

The terms, techniques, and the like described in this specification are used in the meaning commonly used in the technical field to which the present invention belongs, unless otherwise specified.

The novel microorganism Bacillus amyloliquefaciens CC110 strain of the present invention is represented by SEQ ID NO: 1 and exhibits a controlling effect against a plant disease, and particularly exhibits a controlling effect against nosinosis, powdery mildew and gray mold The following Examples and Experimental Examples were used.

To illustrate, in order to select a new strain exhibiting a high control effect against a plant disease, a new strain was selected from soil.

The selected strain was inoculated in the form of an elliptical shape and observed to have characteristic physiological and biochemical characteristics after being inoculated and cultured in a medium (for example, as shown in Table 1 below).

Thus, this 16s rRNA analysis, Bacillus amyl Lori rake Pension's been identified as a novel strain (Bacillus amyloliquefaciens), which Bacillus amyl Lori rake Pension's (Bacillus amyloliquefaciens) called CC110 strain, and the strain of the National Academy of Agricultural Sciences Agricultural Genetic And deposited with the Support Center on July 10, 2013 to receive the accession number KACC91830P.

The Bacillus amyloliquefaciens CC110 strain consists of the entire nucleotide sequence shown in SEQ ID NO: 1 or a part thereof and is composed of an open reading frame (ORF) of 1,489 bp.

The new microorganism Bacillus amyloliquefaciens strain CC110 of the present invention can be industrially used because it can mass-produce the new strain in a short time when a culture solution is prepared through the following culture conditions.

As described above, the new microorganism grows well on a TSB (Tryptic Soy broth) medium as a culture medium and exhibits a lesion area ratio against cucumber mildew even when using a culture diluent diluted 2 to 5 times with the culture medium of the TSB medium (See example in Table 2 below, see FIG. 7).

In addition, the culture solution contains at least one of fructose, mannitol, sorbitol, sucrose and glucose as a carbon source, and L-asparagine (L- Asparagine, yeast extract, casein, peptone, ammonium chloride (NH ₄ Cl), L-arginine, ammonium phosphate ((NH ₄ ) ₂ HPO ₄ ) , L-proline (L-proline). Preferably, the carbon source and the nitrogen source are contained in an amount of 0.5 to 1.0 wt% based on the weight of the culture solution, respectively, to enable mass production of the strain (refer to Figs. 8, 9, 10 and 11).

When the carbon source is contained in an amount of less than 0.5% by weight, the energy-producing factor is too small to slow the growth rate of the strain. When the carbon source is contained in an amount exceeding 1.0% by weight, Slowing the growth rate of the strain.

If the amount of the nitrogen source is less than 0.5% by weight, the growth and reproduction of the strain may be delayed, thereby reducing the specificity of the substrate to be selectively catalyzed. When the amount of the nitrogen source is more than 1.0% by weight, excessive catalyst is added, And there is a possibility of causing deformation of the strain.

In addition, under the culturing conditions, the strain can grow at a temperature ranging from 7.5 to 55 ° C, particularly at 25 to 35 ° C (see Table 6 below).

The pH range of growth of the new strain is 4.5 to 9.5, and the optimum pH is 7.0 (see FIG. 6).

The novel microorganism of the present invention, Bacillus amyloliquefaciens , Cells of CC11 or culture and dilution thereof are used as an active ingredient of a microorganism preparation for controlling plant diseases or microbial pesticides.

In particular, the microbial agent has excellent control effects against nosocomial diseases, powdery mildew and gray mold fungi in plant diseases (see Tables 7, 8, and 12 below) , The antimicrobial activity is maintained, and the antimicrobial substance-producing ability is excellent, so that it can be used for microbial pesticide, seed coating agent, microbial nutrient, soil improving agent, composting homework, leaf surface spraying agent,

In addition, the above microbial agent may be added to crops or crops themselves suffering from mold disease, such as Bacillus amyloliquefaciens , The CC110 strain is concentrated and sprayed or treated, and the effective amount of the microorganism sprayed with about 1 × 10 ^{5 to} 1 × 10 ⁸ microorganisms per unit volume (ml) shows the best sterilization effect on the plant disease Tables 3 and 4 below).

The present invention will be described in more detail with reference to the following examples and experimental examples, but the scope of protection is not limited to the following examples and experimental examples.

<Example 1> Separation of a novel strain of the present invention

The cucumber leaves were surface-sterilized with a 10% sodium hypochlorite solution for 1 minute, ground, and heat-treated at 80 ° C for 10 minutes. Then, the cucumber leaves were spread on 1/10 TSA medium and cultured at 20 ° C for 2 days. Molecular biology identification, morphological characteristics, physiological biochemical characteristics, and nucleotide sequence analysis.

Example 2 Molecular Biological Identification, Morphological Characterization, Physiological Biochemical Characterization and Sequence Analysis of the Isolated New Strain

1) Molecular biological characteristics

16S rRNA gene sequencing analysis was used for phylogenetic analysis of CC110 strain.

In other words, the 16S rRNA gene was amplified by colony PCR using the universal primers 27F and 1492R primers.

The obtained PCR products were reacted with 27F and 1492R sequencing primers using a DNA sequencing kit (BigDye terminator Cycle Sequencing Ready Reactions v3.1; Applied Biosystem), and a nucleotide sequence of 1489 bp was determined using a 3700 Genetic Analyzer (Applied Biosystems) .

Nucleotide sequence similarity with the type strain is EzTaxon-e server (Kim et al ., 2012) and then sorts the base sequence using the CLUSTAL W program were calculated, in order to analyze the flexible relationship with MEGA version 5.1 program (Tamura et al. , 2011) was used to create an evolutionary tree.

The stability of the branch was investigated through 1,000 resampling (bootstrap values).

2) Morphology and physiological biochemical characteristics

The morphological characteristics were obtained by inoculating TSA medium for 1-2 days into a slide glass coated with 1.5% agarose and observing at a magnification of 1000 times using an optical microscope (DM2500, Leica). The strains which were not spores or already spores were observed with increasing or decreasing culture time.

Catalase activity was determined by the presence of 3% (v / v) hydrogen peroxide solution and the formation of bubble droplets. Oxidase activity was determined using 1% (w / v) tetramethyl- p- phenylenediamine (bioM).

Growth performance in anaerobic conditions was determined after incubation at 28 ° C for 2 weeks using AnaeroGen sachet pouches (Oxoid, England). Protein (10% skimmed milk, w / v), starch (1.0%, w / v), lipid (1.0% tributyrin), cellulose (0.1% The hydrolysis of v) was performed according to the method of Smibert & Krieg (1994) and was determined 7 days after culture.

Growth range of the pH (pH 0.5 increments) is _{citric acid / Na 2 HPO 4 (} pH5.06.0), NaH 2 PO 4 / Na 2 HPO 4 (pH6.58.0), Tris / HCl (pH8.5 and pH9.0 ), Na ₂ CO ₃ / NaHCO _{3 F} (pH 9.5 and pH 10.0) (Gomori, 1955).

Growth temperature ranged from 7.5 to 55 ℃ for 2 days in a temperature gradient incubator. The growth range of NaCl was adjusted to 0-16% at 1% intervals by adding NaCl to the medium.

2. Experimental results

(1) Morphological characteristics

The observed strain is shown in FIG. 3, and the physiological and biochemical characteristics of the strain are shown in Table 1 below.

Physiological biochemical characteristics
(Characteristics) CC110 Spore shape Ellipsoidal Spore position subterminal pH 5.0 to 10.0 NaCl (%) 1 to 15 Temperature
Temperature (° C) 10 to 55 Starch + Casein + CM-cellulose (CM-cellulose) + Gelatin + Lipid - Chitin - Nitrate reduction + Oxidase activity + Catalase activity + Glucose + Arabinose + Mannose + Mannitol + N-acetyl-glucosamine + Maltose + Gluconate + Caprate - Adipate - Malate + Citrate + Phenyl-acetate -

In addition, phylogenetic analysis showed that, as in Fig. 1, the strain was different from other similar species.

In order to support that the strain isolated in Example 1 was a new strain, the sequence analysis of ITS (internal transcribed spacer) region of 16S rDNA was performed, and the result was as shown in FIG.

This strain was named as Bacillus amyloliquefaciens CC110, a new microorganism, as described in the attached deposit certificate, and this strain was deposited with the National Agricultural Research Service Agricultural Genetic Resources Support Center and deposited on July 10, KACC91830P.

<Experimental Example 1> Confirmation of the effect of inhibiting the occurrence of cucumber mildew by dilution concentration of the culture medium of the novel strain of CC110 of the present invention

1. Experimental Method

The CC110 strain identified in Example 2 was shake cultured (28 DEG C, 150 rpm) in TSB medium, LB medium, NB medium and KB medium for 24 hours, and each culture medium was diluted 5 times and sprayed on cucumber. The sections were inoculated with 10 μl of cucumber noxious trophoblast (1 × 10 ⁵ / ㎖) at a temperature of 20 ° C for 12 hours a day, and examined for the occurrence of cucumber pollen on the 11th day after treatment.

The medium composition (/ l) is as follows.

TSB (Tryptic soy broth) medium; Casein 17 g, Soybean 3 g, Dextrose 2.5 g, NaCl 5 g, K ₂ HPO ₄ 2.5 g

NB (Nutrient broth) medium; Beef extract 3g, Peptone 5g

KB (King's broth) medium; Proteose peptone 10 g, Glycerol 15 g, K ₂ HPO ₄ 1.5 g, MgSO ₄ 5 g

LB (Luria Bertani broth) medium; 10 g of NaCl, 10 g of Tryptone, 5 g of yeast extract

2. Experimental results

The results of the experiment are shown in Table 2 below.

Processing contents Area Percentage of Cucumber Nutrient Disease by Medium (%) TSB LB NB KB No treatment Culture stock solution 4.0 0 11.0 0 22.0 Culture 5 times dilution 3.0 10.0 20.0 22.0

As shown in Table 2, the CC110 strains identified in Example 2 were lower than the untreated cucumber seedlings in the TSB medium, LB medium, NB medium, and KB medium, and the culture diluted with 5-fold dilution TSB medium, LB medium, and NB medium showed less than the untreated cucumber nodule area ratio. In particular, both the culture broth and the diluted solution in the TSB medium showed excellent effects on the area percentage of cucumber nodule lesion. In the present invention, it was found that culturing the new strain identified in Example 2 in the TSB medium is most suitable.

<Experimental Example 2> Confirmation of the inhibitory effect of the new strain on cucumber mildew

1. Experimental Method

(1) Confirmation of the inhibitory effect on cucumber mildew by treatment type of new strain

The CC110 strain identified in Example 2 was transplanted into TSB medium, cultured at 28 ° C for 2 days at 150 rpm, and centrifuged (8000 rpm, 10 ° C, 30 minutes).

Culture solution is undiluted, 2-fold, 5-fold, and the culture filtrate is undiluted, 2-fold, 5-fold, and the cells creating a leaf segment was treated cucumber leaves the pojanang of the downy mildew fungus 1 × 10 ⁶ ~ 1 × 10 different concentrations ⁸ / ml were inoculated at 10 ㎕ each, and fluorescence was irradiated in a thermostat at 20 캜 for 12 hours a day.

(2) Confirmation of the inhibitory effect of the new strains on cucumber mildew by volatile substances

TSA medium is transplanted CC110 strain to the dispensing plate I by incubation at 25 ℃, and the next day the diameter 25mm cucumber fluorescent pojanang of downy mildew fungus on leaf segment 1 × 10 ⁵ / in the ㎖ 10㎕ by inoculation point to a thermostat 20 ℃ Were irradiated for 12 hours a day, and the occurrence of cucumber pollen was examined 8 days after treatment.

2. Experimental results

The results of the experiment are shown in Tables 3 and 4 below.

Processing contents Dilution factor Percentage of cucumber nodule lesion area (%) Culture solution Undiluted solution 0.0 × 2 3.0 × 5 8.0 Culture filtrate Undiluted solution 0.0 × 2 4.0 × 5 7.0 Cell 1 x 10 ⁸ 9.4 5 × 10 ⁷ 10.0 5 × 10 ⁶ 20.0 1 x 10 ⁶ 19.0 No treatment - 21.0

Percentage of cucumber nodule lesion area (%) CC110 processing No treatment 0 13.2

As shown in Tables 3 and 4, it was confirmed that not only the undiluted solution but also the diluted solution diluted with 2 to 5 times of the culture solution and culture filtrate of the new strain identified in Example 2 exhibited excellent lesion area ratio against cucumber mildew disease, , It was confirmed that the lesion area ratio of 1 × 10 ⁵ to 1 × 10 ⁸ was superior to that of cucumber.

&Lt; Experimental Example 3 > The effect of the culture broth of the present invention on the control of cucumber mildew disease

1. Experimental Method

The culture of CC110 strain identified in Example 2 was cultivated at 28 ° C and 120 rpm for 24 hours using TSB tryptic soy agar medium (Difco, Laboratoris, Detroit) medium.

Cucumber seedlings were grown in a greenhouse and cucumber seedlings were grown in a 7 - cm - diameter vinyl pot at a ratio of 1: 1.

The microorganism cultured in the seedlings of the second cucumber seedlings was sprayed, and the microbial cells cultured after 5 hours were collected by using a brush and adjusted to 5 × 10 ⁴ ml ^-1 and left at room temperature for 2 hours.

Nutrient fungi were inoculated by spraying with 6 repetitions per 2 treatment plants in cucumber plants of 2 stage, and the inoculation area was irradiated with fluorescent lamps at 20 ℃ for 12 hours.

2. Experimental results

Percentage of cucumber nodule lesion area (%) CC110 processing No treatment 35.0 82.0

As shown in Table 5, it was confirmed that even in the culture medium of the novel strain identified in Example 2 of the present invention, the area ratio of cucumber fungus disease was far superior to that of the untreated control.

<Experimental Example 4> Confirmation of the mechanism of inhibiting the occurrence of cucumber mildew in the new strain CC110 of the present invention

1. Experimental Method

Transplant the CC110 strain identified in Example 2 on the TSA medium is busy I plate and incubated at 25 ℃, and the next day the diameter 25mm by the pojanang to 1 × 10 ⁵ / ㎖ of the downy mildew fungus of cucumber leaf segment 10㎕ point After inoculation, fluorescent lamps were irradiated in a thermostat at 20 ° C for 12 hours a day, and the occurrence of cucumber pollen was examined by a dissecting microscope 8 days after the treatment.

2. Experimental results

As shown in FIG. 4, there was no cucumber mildew in the CC110 strain on the plate, only the mature cucumber seedlings that were inoculated with the microscope (M205A, Leica) , And it was confirmed that many mycelium of cucumber mildew and mycelia were formed on a dissecting microscope.

<Experimental Example 5> Confirmation of inhibitory activity of cucumber mycobacteria in culture and cell of the novel strain of CC110 of the present invention

1. Experimental Method

The CC110 strain identified in Example 2 was transplanted into TSB medium, cultured at 28 ° C for 2 days at 150 rpm, and centrifuged (8000 rpm, 10 ° C, 30 minutes).

After culturing cucumber leaves at a concentration of 1 × 10 ⁸ / ㎖ in the culture medium, make leaf slices and inoculate 10 μl of 1 × 10 ⁵ / ml sporangia of the nosopharyngeal bacillus with a fluorescent lamp at 20 ° C. Disease development was investigated 8 days after treatment with 12 hours of irradiation per day.

2. Experimental results

As shown in FIG. 5, the cultivation of CC110 culture medium and cells inhibited the occurrence of cucumber mildew (top view in FIG. 5).

As shown in the optical and electron micrographs, it was confirmed that the shape of oocinosporin of oocyte gland was changed and the disease did not occur (FIG. 5, bottom view).

<Experimental Example 6> Confirmation of the culture characteristics of the novel strain of CC110 of the present invention

1. Experimental Method

TSB medium (Tryptic soy broth medium: pancreatic digest of casein 17 g, enzymatic) was used to establish large-scale culture conditions of CC110 strain identified in Example 2 using a temperature-gradient device (Bio-photo recorder, Advantec Toyo Kaisa, digest of soybean meal 3 g, dextrose 2.5 g, sodium chloride 5 g, dipotassium phosphate 2.5 g, distilled water 1 L) for 48 hours.

2. Experimental results

OD value per incubation temperature 7.5 ℃ 15.2 DEG C 19.6 ° C 23.1 DEG C 26.6 ° C 29.7 DEG C 33.1 DEG C 36.6 ° C 40.0 DEG C 43.5 DEG C 48.2 DEG C 55.0 0.015 1.506 2.410 2.468 2.519 2.645 2.475 2.980 2.181 2.014 1.418 0.352

As shown in Table 6, it was confirmed that the new strain CC110 identified in Example 2 of the present invention was able to grow at 7.5 to 55 ° C, and it was confirmed that the most growth occurred particularly at 25 to 35 ° C.

<Experimental Example 7> Determination of the growth rate of the novel strain of CC110 according to pH

1. Experimental Method

The CC110 strain identified in Example 2 was shake-cultured in TSB medium at 30 DEG C and 120 rpm for 24 hours, and the culture was diluted 10-fold.

The culture medium was prepared in 0.5 unit from pH 4.0 to 9.5 using a TSB medium sterilized in a microplate (100 well), and 350 쨉 l of the culture medium was divided into three replicates per pH medium. 10 쨉 l of the diluted CC110 strain was inoculated OD (optical density) was measured with an absorbance (600 nm) by culturing in Bioscreen C (Growth Curves AB Ltd) at 30 ° C for 48 hours.

2. Experimental results

As shown in FIG. 6, the strain CC110, which is a novel strain of the present invention, showed the best growth in a culture medium having a pH of 7.0 to 7.5.

<Experimental Example 8> The growth rate of the new strain of CC110 according to the kind of the medium was confirmed

1. Experimental Method

The CC110 strain identified in Example 2 was shake-cultured at 30 DEG C at 120 rpm for 24 hours, and the culture was diluted 10-fold. 350 .mu.l of the TSB medium, LB medium, KB medium and NB medium, which had been sterilized in a microplat (100 well), was subdivided into each well and 10 .mu.l of the diluted CC110 strain was inoculated in a Bioscreen C (Growth Curves AB Ltd) And the OD (optical density) was measured at an absorbance (600 nm) after culturing at 30 ° C for 48 hours.

2. Experimental results

As shown in FIG. 7, the strain CC110, which is a novel strain of the present invention, showed the best growth in the culture medium containing TSB medium.

<Experimental Example 9> The growth rate of the novel CC110 strain according to the kind and content of carbon source of the present invention

1. Experimental Method

Culturing was carried out by pre-incubating the CC110 strain identified in Example 2 above at 30 ° C for 24 hours in TSB medium, and adding 350 μl each of the sterilized medium (stock solution) to the microplat (100 well) Then, 10 μl of CC110 strain was inoculated, and plate was placed on Bioscreen C (Growth Curves AB Ltd). The computer was operated to measure the OD value.

2. Experimental results

As a result of the above experiment, as shown in FIGS. 8 and 9, the novel strain CC110 of the present invention can be produced by using fructose, mannitol, sorbitol, sucrose, glucose ) Contained 0.5% by weight to 1.0% by weight, the growth was confirmed to be good.

<Experimental Example 10> The growth rate of the novel strain of CC110 according to the kind and content of nitrogen source of the present invention

1. Experimental Method

Culturing was performed by pre-incubating the CC110 strain identified in Example 2 above at 30 ° C for 24 hours in TSB medium, adding 350 μl of each of the sterilized media (100 replicates) to the microplat (100 wells) After adding each 10 μl of CC110 strain to each kind of nitrogen source, a plate was put in Bioscreen C (Growth Curves AB Ltd.), and the computer was operated to measure the OD value.

2. Experimental results

As shown in FIGS. 10 and 11, the CC110 strain of the present invention is a strain of the present invention which is produced by adding L-asparagine, yeast extract, casein, peptone peptone, ammonium chloride (NH ₄ Cl), L-arginine, ammonium phosphate ((NH ₄ ) ₂ HPO ₄ ), and L- It was confirmed that those containing the% by weight were well grown.

<Experimental Example 11> Confirmation of the inhibitory effect of the novel CC110 strain of the present invention on cucumber powdery mildew

1. Experimental Method

In the experiment for cucumber powdery mildew, the CC110 strain identified in Example 2 was transplanted into the TSB medium and cultured at 28 ° C and 150 rpm for 2 days. The cultured solution was sprayed on the cucumber leaves where the powdery mildew was started, After incubation for 12 hours with a fluorescent lamp in a thermostat at a temperature of 0 ° C, the disease was examined 8 days after the treatment.

2. Experimental results

The results of the experiment are shown in Table 7 below.

Processing contents Area of powdery mildew disease (%) Control effect (%) CC110 0.0 100 No treatment 93.1 -

As shown in Table 7, it was confirmed that the novel strain CC110 of the present invention shows excellent control effect against powdery mildew.

<Experimental Example 12> Inhibitory effect of the novel strain of CC110 according to the present invention on the occurrence of cucumber gray mold

1. Experimental Method

Cucumber seedlings were mixed with TKS soil and long - term horticultural seedlings (1: 1) (V: V) to sow cucumber seeds and cultivated in the 2nd seedling stage. After 4 hours of spraying, as a nutrient source of the spore suspension v8 juice 10% solution and 0.1M KH ₂ PO ₄ was added.

The spore suspension (1 × 10 ⁶ cfu / ㎖ ^-1 ) of the gray mold with added nutrient source was inoculated into the cucumber seedlings and taken out 24 hours after inoculation on the inoculum controlled at 20 ° C and 100% humidity. After 6 days, Respectively.

2. Experimental results

The results of the experiment are shown in Table 8 and FIG.

Processing contents Area percentage of gray mold disease (%) Control effect (%) CC110 3.2 78.0 No treatment 81.2 -

As shown in Table 8 and FIG. 12, it was confirmed that the novel strain CC110 of the present invention shows excellent control effect against the gray mold disease.

As described above, the novel microorganism Bacillus amyloliquefaciens CC110 exhibits excellent control effect against nontoxic diseases, powdery mildew and gray mold diseases among plant diseases, and thus the microorganism preparation containing the microorganism preparation is excellent in environmental pollution and human body It can be used effectively as a microbial pesticide for environment-friendly plant disease control without toxicity.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. You can implement the examples. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

National Institute of Agricultural Science KACC91830P 20130710

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> New microorganism Bacillus amyloliquefaciens CC110 and Microbial          agent and biopesticide containing the same <130> p2013-0148 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1489 <212> DNA <213> Bacillus amyloliquefaciens CC110 <400> 1 tcaggacgaa cgctggcggc gtgcctaata catgcaagtc gagcggacag atgggagctt 60 gctccctgat gttagcggcg gacgggtgag taacacgtgg gtaacctgcc tgtaagactg 120 ggataactcc gggaaaccgg ggctaatacc ggatggttgt ttgaaccgca tggttcagac 180 ataaaaggtg gcttcggcta ccacttacag atggacccgc ggcgcattag ctagttggtg 240 aggtaacggc tcaccaaggc gacgatgcgt agccgacctg agagggtgat cggccacact 300 gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360 acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct 420 gttgttaggg aagaacaagt gccgttcaaa tagggcggca ccttgacggt acctaaccag 480 aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc 540 ggaattattg ggcgtaaagg gctcgcaggc ggtttcttaa gtctgatgtg aaagcccccg 600 gctcaaccgg ggagggtcat tggaaactgg ggaacttgag tgcagaagag gagagtggaa 660 ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcgact 720 ctctggtctg taactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc 780 ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag ggggtttccg ccccttagtg 840 ctgcagctaa cgcattaagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa 900 ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa 960 gaaccttacc aggtcttgac atcctctgac aatcctagag ataggacgtc cccttcgggg 1020 gcagagtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080 cccgcaacga gcgcaaccct tgatcttagt tgccagcatt cagttgggca ctctaaggtg 1140 actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200 cctgggctac acacgtgcta caatggacag aacaaagggc agcgaaaccg cgaggttaag 1260 ccaatcccac aaatctgttc tcagttcgga tcgcagtctg caactcgact gcgtgaagct 1320 ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca 1380 caccgcccgt cacaccacga gagtttgtaa cacccgaagt cggtgaggta accttttagg 1440 agccagccgc cgaaggtggg acagatgatt ggggtgaagt cgtaacagg 1489

Claims (16)

Having a nucleotide sequence represented by SEQ ID NO: 1, separated from cucumber leaves,
Which has an oxidase activity, catalase activity and salt tolerance and has a controlling effect against cucumber fungus or cucumber gray mold disease,
New microbial Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) CC110 (KACC 91830P) strain.
delete delete The method according to claim 1,
Wherein the strain is used as an active ingredient of any one of a microbial pesticide, a seed coating agent, a soil improving agent, a compostant homework, a leaf surface spraying agent,
New microbial Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) CC110 (KACC 91830P) strain.
A culture medium obtained by culturing the novel microorganism Bacillus amyloliquefaciens CC110 (KACC 91830P) according to any one of claims 1 to 4 in a TSB (Tryptic Soy broth) medium and a culture diluent thereof as an active ingredient However,
The culture solution contains 0.5 to 1.0% by weight of carbon source, which is composed of at least one of fructose, mannitol, sorbitol, sucrose, and glucose, In addition,
L-Asparagine, Yeast Extract, Casein, Peptone, Ammonium Chloride (NH ₄ Cl), L-Arginine, Ammonium Phosphate (NH ₄ ) ₂ HPO ₄ ) and L-proline is contained in an amount of 0.5 to 1.0 wt% based on the weight of the culture liquid.
Microorganism preparation for prevention of cucumber or cucumber gray mold.
delete delete delete delete delete 6. The method of claim 5,
Characterized in that the culture broth is cultivated at a pH of 4.5 to 9.5 and at a temperature of 7.5 to 55 ° C,
Microorganism preparation for prevention of cucumber or cucumber gray mold.
6. The method of claim 5,
The culture broth contains Bacillus amyloliquefaciens (1 x 10 ^{5 to} 1 x 10 ⁸ spores per ml) RTI ID = 0.0 &gt; CC110 &lt; / RTI &gt;
Microorganism preparation for prevention of cucumber or cucumber gray mold.
6. The method of claim 5,
Wherein the culture diluent is diluted 2 to 5 times with the culture solution.
Microorganism preparation for prevention of cucumber or cucumber gray mold.
delete delete Preparing a culture medium in which a new microorganism Bacillus amyloliquefaciens CC110 (KACC 91830P) strain of any one of claims 1 to 4 is cultured in TSB (Tryptic Soy broth) medium;
A carbon source of at least one of fructose, mannitol, sorbitol, sucrose and glucose is added to the culture solution in an amount of 0.5 to 1.0 wt% based on the weight of the culture liquid, L-asparagine, yeast extract, casein, peptone, ammonium chloride (NH ₄ Cl), L-arginine, ammonium phosphate ((NH ₄ ) ₂ HPO ₄ ), and L-proline to 0.5-1.0 wt% of the weight of the culture solution, and mixing them to prepare a culture medium for mass production;
Culturing the culture medium for mass production at a pH of 7.0 and a temperature of 25 to 35 ° C to mass-produce the new microorganism having a controlling effect against cucumber or cucumber gray mold disease
New microbial Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) Method for mass production of CC110 (KACC 91830P) strain.

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