KR20180001716A - Antimicrobial composition for preventing bacterial blight containing extracts of Bacillus subtilis culture medium - Google Patents

Abstract

The present invention relates to an antimicrobial composition for preventing bacterial blight. The antimicrobial composition comprises an extract of Bacillus subtilis culture medium. The antimicrobial composition according to the present invention has effects of preventing and treating rice blight disease by inhibiting the growth of rice blight disease causative bacteria. Further, the antimicrobial composition according to the present invention has an effect that the composition can be used as natural biological agricultural chemicals replacing chemical fertilizers and agricultural chemicals that have conventionally been used. The antimicrobial composition according to the present invention has an effect of providing a method of controlling bacterial diseases of plants including rice blight disease, rice sheath blight and root rot by using the antimicrobial composition in biological control. Further, the antimicrobial composition according to the present invention can be used as a composition for promoting the growth of the plants.

Description

[0001] The present invention relates to an antimicrobial composition for preventing bacterial blight caused by Bacillus subtilis culture medium extracts,

The present invention relates to an antimicrobial composition for controlling bacterial blight disease.

Rice Blight disease is a disease caused by infection of bacteria through wound of rice leaves. Rice blight blight appears as a gray or white lesion on the leaf vein representing the vascular disease, and the edge of the leaf turns yellow and the leaf dries. Blight of rice blight spreads rapidly at the onset, and about 50% of the total rice yield is lost in the affected area. In order to prevent the blight of rice blast, leaf is washed with water or sprayed with medicines, dryness of the leaf should be dried before the secondary infection can be prevented.

Xanthomonas orzae pv. orzae ( Xoo ) is known to be a major cause of blight of rice blight, and Xanthomonas is mostly composed of plant pathogenic bacteria. Xanthomonas oryzae pv. Oryzae is a Gram-negative, aerobic bacterium that has unipolar hairs and is rod-shaped. Bacteria dried in diseased lesions or diseased Bacteria remaining in dead plants can live for more than 8 months, but die quickly in high temperature and high humidity environments.

In the cultivation of crops, a large amount of chemical fertilizers and pesticides are used to reduce the damage caused by bacterial diseases and to secure the quality and production of agricultural products. Misuse and abuse of chemical fertilizers and pesticides cause side effects such as reduction of arable land, loss of essential microorganisms in the soil, and environmental pollution. Therefore, there is a need to develop a composition or method that replaces chemical fertilizers and pesticides.

It is an object of the present invention to provide an antimicrobial composition for controlling bacterial blight disease that exhibits an antibacterial effect by including an extract of Bacillus subtilis culture solution.

In order to accomplish the above object, the present invention provides an antibacterial composition for controlling bacterial blight comprising a culture extract of Bacillus subtilis culture.

The Bacillus subtilis may include a 16S rRNA sequence of SEQ ID NO: 1 and may be Bacillus subtilis MJMP2. The Bacillus subtilis culture extract may contain Iturin A have.

The present invention also provides a method for producing an antimicrobial composition for controlling bacterial blight disease. The antimicrobial composition for controlling bacterial blight disease comprises the steps of: culturing Bacillus subtilis; Isolating a fraction comprising a lipopeptide from a Bacillus subtilis culture; And a step of purifying the fraction comprising the isolated lipid peptide.

The step of separating the fractions comprises performing size exclusion chromatography. The step of purifying the fractions also includes performing reverse phase High Performance Liquid Chromatography (HPLC).

In addition, the present invention provides a plant growth promoting composition comprising a Bacillus subtilis culture solution extract. The Bacillus subtilis may include a 16S rRNA sequence of SEQ ID NO: 1 and may be Bacillus subtilis MJMP2. The Bacillus subtilis culture extract may contain Iturin A have.

The present invention also provides a method of controlling bacterial diseases in plants using the Bacillus subtilis culture solution extract. The Bacillus subtilis may include a 16S rRNA sequence of SEQ ID NO: 1 and may be Bacillus subtilis MJMP2. The Bacillus subtilis culture extract may contain Iturin A have. The bacterial diseases of the plant may be selected from the group consisting of rice blight blight disease, rice sheath blight disease and root rot disease.

According to the present invention, the antimicrobial composition for controlling bacterial blight disease contains Bacillus subtilis culture liquid extract to inhibit the growth of causative bacteria of the blight of rice blight, thereby preventing and treating blight of blight of rice blast. In addition, there is an effect that can be used as a natural biological pesticide that replaces chemical fertilizers and pesticides conventionally used.

By using the antimicrobial composition for controlling bacterial blight disease in a biological control, it is possible to provide a method for controlling bacterial diseases of plants such as blight of blight of rice blast, rice sheath blight blight and root rot disease.

In addition, the composition for controlling bacterial blight disease, which comprises the Bacillus subtilis culture solution extract, can be used as a composition for promoting plant growth.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing the relationship between strains based on the 16S rRNA sequence (Neighbor-joining tree).
Figure 2 shows the structure of Inturin A isolated from Bacillus subtilis MJMP2.
Fig. 3 shows the inhibitory effect of Bacillus subtilis culture liquid extract on rice blossom blight of rice. (A: Inhibitory effect of Bacillus subtilis culture liquid extract on rice blast fungus inhibition in rice dumplings, B: Inhibitory effect of Bacillus subtilis culture extract on bacterial blight of rice blast)
Figure 4 shows the effect of the extract of Bacillus subtilis extract on plant growth. (A: Measurement of root germination and root growth of Arabidopsis seed germs of Bacillus subtilis culture extract, B: Measurement of root length of Arabidopsis after 7 days of culture)

Hereinafter, the present invention will be described in detail.

According to one aspect of the present invention, Bacillus  Subtilis ( Bacillus subtilis ) Bacterial < RTI ID = 0.0 > blight The present invention provides an antibacterial composition for controlling bacterial blight. remind Bacillus Subtilis  16S < / RTI > rRNA  Sequence.

The genus Bacillus (Bacillus) to produce a variety of antimicrobial agents according to the species. In particular, Bacillus subtilis has been reported to contain antifungal active substances such as subtilin and surfaction, and antifungal active substances such as alboleutin, bacillomycin, fengycin, iturin and mycosubtilin.

Iturin is a cyclic lipopeptide secreted by Bacillus subtilis . Iturin is known to have antibacterial activity against plant pathogens. In addition, iturine A produced by Bacillus subtilis is a product of microbial fermentation and does not cause accumulation and toxicity problems in the body.

The antimicrobial composition for controlling bacterial blight disease preferably contains 10 μg / ml to 100 μg / ml of Bacillus subtilis culture solution extract, more preferably 50 μg / ml to 100 μg / ml.

In addition, the Bacillus subtilis culture solution extract may contain Iturin A.

Further, according to another aspect of the present invention, blight  A method for producing an antimicrobial composition for controlling antimicrobial activity is provided. The bacterial blight  The antimicrobial composition for controlling Bacillus  Bacillus subtilis ); Bacillus Subtilis From the culture medium  Lipid peptides ( Lipopeptide ) The fraction  Separating; And separate Lipid peptides  Included The fraction  And purifying.

The step of separating the fractions containing the lipid peptides is preferably carried out by size exclusion chromatography. In addition, the step of purifying the fractions containing the lipid peptide is preferably performed by reverse phase high performance liquid chromatography (HPLC).

Further, according to another aspect of the present invention, Bacillus Subtilis  The present invention provides a method for controlling bacterial diseases of plants using a culture extract.

The Bacillus subtilis may comprise a 16S rRNA sequence of SEQ ID NO: 1.

The antimicrobial composition for controlling bacterial blight disease preferably contains 10 μg / ml to 100 μg / ml of Bacillus subtilis culture solution extract, more preferably 50 μg / ml to 100 μg / ml.

In addition, the Bacillus subtilis culture solution extract may contain Iturin A.

The bacterial disease of the plant may be at least one disease in the group consisting of rice blast fungus disease, rice sheath blight disease and root rot disease.

According to still another aspect of the present invention, Bacillus Subtilis  The present invention provides a plant growth promoting composition comprising a culture extract.

The Bacillus subtilis may comprise a 16S rRNA sequence of SEQ ID NO: 1.

The antimicrobial composition for controlling bacterial blight disease preferably contains 10 μg / ml to 100 μg / ml of Bacillus subtilis culture solution extract, more preferably 50 μg / ml to 100 μg / ml.

In addition, the Bacillus subtilis culture solution extract may contain Iturin A.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  1. From Kimchi Bacillus  Subtilis ( Bacillus subtilis ) MJMP2 Isolation and Identification of.

1-1 Isolation of microorganisms from kimchi

The microorganisms were isolated from kimchi. The used kimchi was aged for a month and purchased at local market. The purchased kimchi was homogenized with a stainless steel blender. 1 g of homogenized kimchi was mixed with 10 ml of 0.8% saline to prepare a kimchi suspension. 100 μl of the kimchi suspension was applied to Luria-Bertany agar medium and incubated at 37 ° C. for about 24 hours until colonies appeared. One colony of the formed colonies was plated on another LB agar medium and cultured.

As a result, more than 200 bacterial strains were identified in bacterial isolates isolated from kimchi.

1-2 in kimchi Isolated  bacteria Separated  Determination of antimicrobial activity and identification of bacteria having antimicrobial activity

The antimicrobial activity of bacterial isolates isolated from kimchi was determined by Xanthomonas oryzae pv. oryzae ( Xoo ) as an indicator.

Phytopathogenic bacteria, Xanthomonas oryzae pv. oryzae KACC10331 was obtained from the Korean Agricultural Culture Collection (KACC) and cultivated on a PDA (potato dextrose agar) medium at 25 to 28 ° C.

Fusarium oxysporum KACC 40037 and Rhizoctonia solani AG-2-2 (IIIB) KACC 40151 was obtained from the Korean Agricultural Culture Collection (KACC) and cultured on a PDA (potato dextrose agar) medium at 25 to 28 ° C.

To analyze the antimicrobial activity of the bacterial isolate isolated from kimchi, the bacterial isolate was plated on LB medium and cultured at 37 ° C for 24 hours. After culture, the culture broth was loaded onto a paper disk and plated on a PDA plate inoculated with the pathogen. Mycelial growth inhibition activity of bacterial isolates isolated from kimchi was measured 3 days after culture. For the next experiment, bacterial fractions with strong mycelial growth inhibitory activity were selected.

In order to identify the microorganisms contained in the bacterial fraction, the 16S rRNA sequence was analyzed. Bacterial genomic DNA extracts were obtained using phenol. PCR was performed using primers fD1 (SEQ ID NO: 2) and rP2 (SEQ ID NO: 3) to amplify the 16S rRNA sequence region of bacteria.

After PCR, the PCR product was purified using QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA) and sequenced using ABI PRISM 310 Genetic Analyzer (Perkin Elmer, Wellesley, MA, USA).

As a result of measuring the antibacterial activity of the bacterial isolate isolated from kimchi, a strain showing strong antimicrobial activity among the bacterial isolates isolated from kimchi was identified and named MJMP2. The MJMP2 strain morphologically showed bacteria of the genus Bacillus. The colonies of Bacillus subtilis MJMP2 were irregularly rounded in the nutrient medium and showed off-white or white.

The 16S rRNA sequence was used to identify MJMP2. As a result, it was confirmed that the MJMP2 strain was close to the Bacillus subtilis and the similarity was 99% or more. The schematic diagram is based on the Neighbor-joining method and is shown in FIG.

From the above results, it was confirmed that MJMP2 isolated from kimchi was Bacillus subtilis .

Example  2. Bacillus  Subtilis ( Bacillus subtilis ) MJMP2 Lt; / RTI > Of lipid peptides  Extraction and purification.

2-1 Bacillus Subtilis MJMP2 Of culture medium extract

The bacterial fraction having mycelial growth inhibitory activity selected in Example 1-2 was inoculated into M9 broth and cultured at 30 ° C. The culture was centrifuged at 6000 x g for 20 minutes and then aseptically filtered (0.45 mu m) to obtain fractions containing lipid peptides.

A supernatant was obtained by mixing the same volume of butanol as the fraction containing the lipid peptide. The supernatant was vacuum dried to obtain a dried Bacillus subtilis MJMP2 culture broth extract.

2-2 Bacillus Subtilis MJMP2 Analysis of culture extract

The dried culture extract was dissolved in butanol and filtered through a 0.2 μm nonpyrogenic hydrophilic membrane (Sartorius AG, Goettingen, Germany). The filtrate was subjected to size exclusion chromatography to separate the components. The isolated extracts were concentrated, and the Xanthomonas oryzae pv. oryzae ( Xoo ) was used to measure the antimicrobial activity.

As a result of the above size exclusion chromatography, the extract isolated from the 5th to 8th rows was identified as Xanthomonas oryzae pv. showed strong antimicrobial activity against oryzae ( Xoo ).

Among the isolated extracts, the 5th to 8th extracts with antimicrobial activity were selected and purified by reverse phase high performance liquid chromatography (HPLC). Further, the purified extract was subjected to mass spectrometry using an LC-MS-8030 system (Shimadzu, Kyoto, Japan).

ESI-MS analysis of the purified extract confirmed major signals at 1042.6 m / z, 1056.6 m / z, 1072.7 m / z and 1083.8 m / z in negative and positive full scan modes. As a result, it was confirmed that the molecular weights coincide with that of iturin A having an acyl chain at C14, C15, C16 and C17. In the positive mode, the main signals of 1066 m / z, 1080.6 m / z and 1160.8 m / z were confirmed. The results indicate that the fatty acyl chains are bound to C14, C15 and C17 of this Turin A (Fig. 2).

Example  3. Bacillus  Subtilis ( Bacillus subtilis ) MJMP2 Evaluation of Antioxidant Activity against the Blight of Rice Blot.

3-1 Rice Eateries ( explants )in Bacillus  Subtilis ( Bacillus subtilis ) MJMP2 Evaluation of Inhibitory Effect of Cultured Extracts on Blight Bacterial Blight of Rice

The inhibitory activity of rice blight blight was measured using X. oryzae KACC 10331.

X. oryzae KACC 10331 was cultured and diluted to an OD ₆₀₀ value of 0.5 in sterilized MES buffer (3 mM, pH 6.0) Xoo A suspension was prepared. Xoo The suspension was inoculated into 96-well plates in 200 μl increments.

The rice leaves were cut to a length of 15 cm, and the surface of the prepared rice leaves was sterilized with sterilized solution and washed three times with distilled water. (4 x ⁴ mm ² ) was prepared with the prepared rice leaves. The rice meal pieces were placed in a 96-well plate inoculated with the Xoo .

The culture extract of the dried Bacillus subtilis MJMP2 prepared in Example 2-1 was dissolved in 50% DMSO to prepare an extract of Bacillus subtilis MJMP2 culture. The Bacillus subtilis MJMP2 culture supernatant was treated to a concentration of 10, 50 or 100 μg / ml on a 96-well plate inoculated with Xoo . The 96-well plate was incubated at 28 ° C for 5 days.

The results of the antimicrobial activity measurement of the Bacillus subtilis MJMP2 culture broth extract in rice meal were shown in FIG. 3-A. The control group treated with Xoo (Fig. 3 Aa) showed a white lesion, which is a symptom of rice blotch blight. In the experimental group treated with Bacillus subtilis MJMP2 (Fig. 3 (A) -b, c, and d), symptoms of blight of blight were reduced. Particularly, white lesions were completely removed from the 50 μg / ml treatment group (FIG. 3 (A) -c) and the 100 μg / ml treatment group (FIG. 3 (A) -d).

From the above results, it can be seen that the extract of Bacillus subtilis MJMP2 culture liquid has a treatment and alleviation effect on rice blast fungus.

3-2 In rice Bacillus  Subtilis ( Bacillus subtilis ) MJMP2 Evaluation of Antioxidant Activity against the Blight of Rice Blot.

Milliang, a susceptible rice variety, was cultivated in a 40x40 cm pot for 6 weeks. Six seedlings were planted in the pots, and each treatment was repeated three times. Bacillus subtilis MJMP2 culture extract (50 μg / ml) was sprayed on rice leaves. Two days after spraying, Xoo suspension (OD ₆₀₀ = 0.5) was inoculated with a brush on the rice leaves. The pollen treated with Xoo suspension was placed in a greenhouse according to a randomized block design and cultured for 7 days.

The results of measuring the inhibitory effect of the culture extract of Bacillus subtilis MJMP2 on rice blight of rice blast in rice were shown in Fig. 3-B. A negative control (Fig. 3 Be) without Xoo treatment showed no symptoms of rice blight blight. A positive control (Fig. 3 Bf) treated with Xoo induced blight of rice blight. On the other hand, the experimental group (Fig. 3 Bg) treated with Bacillus subtilis MJMP2 culture medium (50 μg / ml) and treated with Xoo did not exhibit the symptoms of blight of rice blight.

From the above results, it can be seen that the extract of Bacillus subtilis MJMP2 culture liquid has an effect of preventing blight of rice blast.

Example  4. Bacillus  Subtilis ( Bacillus subtilis ) MJMP2 Evaluation of Plant Growth Promoting Activity of Cultured Extracts of.

Arabidopsis Col-0, an ecotype of the thaliana , was used for the growth promotion experiment.

The control group was cultured on Murashige & Skoog (MS) agar medium with surface sterilized Arabidopsis seeds. In the experimental group, surface-sterilized Arabidopsis seeds were cultured on MS agar medium with Bacillus subtilis MJMP2 medium extract (50 μg / ml). The control and experimental groups were incubated in a growth chamber at 22 ° C for 4 days. In addition, the MS agar medium in the control and experimental groups was sealed with parafilm and cultured in a growth chamber at 22 ° C. After 7 days of culture, root lengths of control and experimental groups were measured.

The results of measuring the plant growth-promoting activity of the culture extract of Bacillus subtilis MJMP2 are shown in Fig. The root length of Arabidopsis was improved in the experimental group treated with Bacillus subtilis MJMP2 culture extract (Fig. 4-A). The root length of the Arabidopsis leaf was measured after 7 days of cultivation, and the root length of the control group was 15 mm. On the other hand, the root length of the experimental group treated with Bacillus subtilis MJMP2 culture extract was 25 mm (Fig. 4-B).

From the above results, it can be seen that the extract of Bacillus subtilis MJMP2 culture liquid has an effect of promoting plant growth.

Example  5. Bacillus  Subtilis ( Bacillus subtilis ) MJMP2 Antimicrobial spectrum of culture extracts of.

R. solani of Bacillus subtilis MJMP2 culture broth extract And F. oxysporum were measured.

Plant pathogen Fusarium oxysporum KACC 40037 and Rhizoctonia solani AG-2-2 (IIIB) KACC 40151 was obtained from the Korean Agricultural Culture Collection (KACC) and cultured on a PDA (potato dextrose agar) medium at 25 to 28 ° C.

The R. solani Or Bacillus subtilis MJMP2 culture extract was inoculated 2 cm away from the fungal disc of PDA (potato dextrose agar) agar medium containing F. oxysporum . The antimicrobial effect of the Bacillus subtilis MJMP2 culture extract was measured after incubation at 25 ° C for 3 to 7 days. The antimicrobial effect was quantified by measuring the diameter of the area where R. solani and F. oxysporum were inhibited.

The results of the antimicrobial spectrum measurement of the extract of the Bacillus subtilis MJMP2 culture medium are shown in Table 1. The extract of Bacillus subtilis MJMP2 culture broth was obtained from Rhizoctonia solani KACC 40151 and Fusarium oxysporum KACC 40037. The Rhizoctonia solani is a pathogen of rice sheath blight, Fusarium Oxysporum is a pathogen of root rot.

From the above results, it can be seen that the extract of Bacillus subtilis MJMP2 culture has the effect of inhibiting rice sheath blight and root rot disease in addition to rice blight blight.

Antimicrobial Activity of Bacillus subtilis MJMP2 Culture Extracts on Various Pathogens of Rice. Strain name Activity Pathogen Xanthomonas oryzae +++ Rice blight disease Rhizotonia solani ++ Sheath blight of rice Fusarium oxysporum ++ Root rot

* +++: strong inhibitory activity with inhibition zone of more than 10 mm.

* ++: moderate inhibitory activity with inhibition zone of 5-10 mm.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Myongji University Industry and Academia Cooperation Foundation <120> Antimicrobial composition for preventing bacterial blight          containing extracts of Bacillus subtilis culture medium <130> P16-E382 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1425 <212> DNA <213> Bacillus subtilis <220> <221> rRNA &Lt; 222 > (1) .. (1425) <223> Bacillus subtilis MJMP2, 16S rRNA sequence <400> 1 tgcagtcgag cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa 60 cacgtgggta acctgcctgt aagactggga taactccggg aaaccggggc taataccgga 120 tggttgtttg aaccgcatgg ttcaaacata aaaggtggct tcggctacca cttacagatg 180 gacccgcggc gcattagcta gttggtgagg taacggctcca ccaaggcgac gatgcgtagc 240 cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag 300 gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg 360 atgaaggttt tcggatcgta aagctctgtt gttagggaag aacaagtacc gttcgaatag 420 ggcggtacct tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg 480 gt; ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcattgg aaactgggga 600 acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt 660 ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggagcgaaag 720 cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa 780 gtgttagggg gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg 840 agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900 atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaat 960 cctagagata ggacgtcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag 1020 ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga tcttagttgc 1080 cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga aggtggggat 1140 gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggacagaac 1200 aaagggcagc gaaaccgcga ggttaagcca atcccacaaa tctgttctca gttcggatcg 1260 cagtctgcaa ctcgactgcg tgaagctgga atcgctagta atcgcggatc agcatgccgc 1320 ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac 1380 ccgaagtcgg tgaggtaacc ttttaggagc cagccgccga agtga 1425 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> fD1 primer <400> 2 agagtttgat cctccctctc ag 22 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rP2 primer <400> 3 acggctacct tgttacgact t 21

Claims (10)

An antimicrobial composition for controlling bacterial blight comprising a culture extract of Bacillus subtilis .
The method according to claim 1,
Wherein the Bacillus subtilis is Bacillus subtilis &lt; RTI ID = 0.0 &gt; MJMP2. &Lt; / RTI &gt;
The method according to claim 1,
Wherein said Bacillus subtilis has the 16S rRNA sequence of SEQ ID NO: 1.
The method according to claim 1,
Wherein the culture extract of Bacillus subtilis contains Iturin A. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;

A plant growth promoting composition comprising the Bacillus subtilis culture liquid extract according to claim 1.
Culturing Bacillus subtilis;
Isolating a fraction comprising a lipopeptide from a Bacillus subtilis culture; And
Purifying a fraction comprising an isolated lipid peptide;
Wherein the antimicrobial composition has an antibacterial activity.
The method according to claim 6,
Wherein the step of separating the fractions is carried out by size exclusion chromatography.
The method according to claim 6,
Wherein the step of purifying the fraction is carried out by reversed phase high performance liquid chromatography (HPLC).
A method for controlling bacterial disease in a plant using the Bacillus subtilis culture solution extract according to claim 1.
10. The method of claim 9,
Wherein the bacterial disease of the plant is selected from the group consisting of Rice blight disease, Sheath blight of rice and Root rot. .

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