KR20100114746A - Bacillus subtilis kb-401 having antagonist activity against plant pathogenic bacteria and culture medium thereof - Google Patents

Abstract

PURPOSE: An antagonistic microorganism Bacillus subtilis KB-401 having antibacterial activity against plant pathogen is provided to prevent from various plant pathogens. CONSTITUTION: An antagonistic microorganism Bacillus subtilis KB-401(deposit number: KFCC 11388P) has an antibacterial activity against plant pathogen. An antagonistic microorganism Bacillus subtilis KB-401 or culture medium thereof has antibacterial activity for Botrytis cinerea, Colletotrichum sp., Fusarium sp., or Sphaerotheca fusca.

Description

Bacterial SUBTILIS KB-401 HAVING ANTAGONIST ACTIVITY AGAINST PLANT PATHOGENIC BACTERIA AND CULTURE MEDIUM THEREOF}

The present invention relates to antagonism microorganisms having antimicrobial activity against phytopathogens , and more particularly, citrus black spot disease ( Diaporthe citri ), grass brown spread disease ( Rhizoctonia solani ), gray mold disease ( Botrytis cinerea ), anthrax ( colletotrichum sp .), wilt (Fusarium sp.), cucumber powdery mildew (Sphaerotheca fusca), such as a valuable antagonistic microorganisms in the plant disease control Bacillus subtilis KB-401 (Bacillus subtilis KB- 401, KFCC 11388P) according to strains and microbial agent using the same. will be.

Damage to phytopathogens is also a major problem for all crops, including rice crops, horticultural crops, and fruit trees. Chemical pesticides are mainly used for the control of plant diseases, but as the global interest and awareness of the environment increases, environmental pollution, negative effects on the ecosystem and resistance to pathogens due to the use of chemical pesticides are increasing. Problems such as lowering of the control effect are gradually increasing. In addition, as the demand for eco-friendly agricultural products is increasing due to the increasing demand of eco-friendly agricultural products, the development of eco-friendly biopesticides that can replace chemical pesticides against plant diseases is highlighted.

In the case of foreign countries, control of potato beetle disease has already been attempted in the 1920s. Since 1960, more than 70 biopesticides have been commercialized in the United States. In Korea, since the research of biological control of ginseng root rot from the early 1970s, research on the biological control of various plant diseases has been ongoing, but the results are inadequate and have been difficult to be practical. In the 1990s, biological control of some plant diseases using microorganisms developed in Korea was effectively conducted. In the 2000s, microbial pesticides were registered in Korea based on continuous research and efforts.

To date, a total of 16 items of microbial pesticides using antagonistic microorganisms against plant diseases are registered, among which 12 are domestically produced. Among them, 12 microbial pesticides using Bacillus sp . Strains, and a total of 11 microbial pesticides using Bacillus subtilis . The target disease is only applied to some plant pathogens such as powdery mildew, gray mold, brown leaf blight and late blight.

Korean Patent Publication No. 2000-0038895 has a growth inhibitory effect against Botrytis cinerea , Fusarium sp. , Pythium sp. , And Grass brown spread bacterium Rhizoctonia solani . Bacillus subtilis GB-0365 and Bacillus subtilis GB-017, which are known to have been disclosed,

Korean Patent Registration No. 10-0732910 is a Bacillus subtilis that has antibacterial activity against Sphaerotheca macular , Botrytis cinerea , Rhizoctonia solani , and Fusarium solani . FBS-2 is disclosed.

Soil microorganisms, such as Bacillus subtilis, continue to be mutated and evolved to adapt to their soil and surrounding environment, thereby separating, identifying and testing antagonistic microorganisms with more effective antimicrobial activity against the target plant pathogens. Ongoing research is ongoing.

The present invention provides an antagonist microorganism for an environment-friendly microorganism by separating, identifying and cultivating antagonistic microorganisms having antimicrobial activity against phytopathogens for effective biological control of the various major plant diseases.

Hereinafter, the present invention will be described in detail.

The present invention isolates and identifies the antagonist microorganisms from the soil under crop cultivation all over the country to find antagonistic microorganisms having antimicrobial activity against phytopathogens from the soil microorganisms; Identifying antimicrobial activity against the isolated and identified antagonistic microorganisms; Finally, the best Bacillus subtilis KB-401 was selected through the step of confirming the control effect against plant disease after culturing the antagonist microorganisms.

The present invention provides Bacillus subtilis KB-401 (KFCC 11388P), an antagonistic microorganism having antibacterial activity against various phytopathogens.

Bacillus subtilis KB-401 of the present invention shows very good antimicrobial activity against grass brown spread disease ( Rhizoctonia solani ) or citrus black spot disease ( Diaporthe citri ).

In addition, Bacillus subtilis KB-401 of the present invention exhibits excellent antimicrobial activity against gray mold ( Botrytis cinerea ), anthrax ( colletotrichum sp. ), Wilting disease ( Fusarium sp. ), Cucumber powdery mildew ( Sphaerotheca fusca ).

Antagonist B. subtilis KB-401 of the present invention (Accession No .: KFCC 11388P) or a culture thereof is citrus citrus, Diaporthe citri , grass brown spread disease ( Rhizoctonia solani ), gray mold ( Botrytis cinerea ), anthrax ( Colletotrichum sp. ) And Fusarium sp. Show broad growth inhibition effect against pathogens such as Fusarium sp. And antagonistic microorganisms in biological control of various plant pathogens because they show antibacterial activity against cucumber powdery mildew ( Sphaerotheca fusca ). It is effective in providing eco-friendly microbial pesticides.

Hereinafter, the present invention will be described in detail with reference to Examples, and the scope of the present invention is not limited to these Examples.

Example 1 Isolation and Identification of Antagonist Microorganisms

In order to isolate the antagonistic microorganisms having antimicrobial activity against phytopathogens in the present invention, soil samples were collected from the soil under crop cultivation around the country. To isolate microorganisms from soil, TSA medium (Triptic Soy Agar; Trypton 17g, Soytone 3g, Glucose 2.5g, NaCl 5g, K ₂ HPO ₄ 2.5g, Agar 18g / L) was prepared, sterilized and incubated (Petri- about 20 ml each. 1 g of the collected soil sample is suspended in 100 ml of sterile physiological saline, and serially diluted to 10 ⁴ times, and 100 µl of the diluted solution is plated on a TSA medium. Only the single colonies of the Bacillus strains, which were grown while incubating for 48 hours in the incubator, were separately isolated. The isolated Bacillus strains were replaced with phytopathogens at 25 ° C. for 4-5 days in PDA medium (Potato Dextrose Agar), and the antimicrobial activity was checked by comparing the growth inhibition effect against phytopathogens and antagonistic microorganisms were selected. The phytopathogens used to identify the effect of growth inhibition were Alternaria kikuchiana , Citrus black spots , Diaporthe citri , Venturia nashicola , Apple Black Stars, Venturia inaequalis , Gray mold Botrytis cinerea , Tomato leaf fungus ( Cladosporium fulvum ), Capsicum anthrax ( Cetotohumhum coccodes ), Tomato wilting ( Fusarium oxysporum ), Potato wilting ( Fusarium solani ), Pepper rot ( Phytophthora capsici ) ( Phytophthora melonis ), Tomato Sclerotinia minor , Rice Fusarium moniliforme , Colletotrichum caudatum , Pythium sp. , Grass Brown Spread, Rhizoctonia solani 16 species were used.

 The main bacteriological characteristics of the selected antagonists are as follows.

Properties in TSA Medium Color Light pale yellow Flora circle Balance total surface Convex Balance total size 1.0-3.0 mm

Physiological Characteristics of Selected Antagonists Gram reaction positivity Resistant Spore Formation U Cell morphology Simplified Cell activity U Oxygen composition Aerobic Anaerobic growth Impossible Catalase production U VP test positivity
Oxidation glucose U Xylose U Mannitol U Arabinose U Glucose rotor gas generation U
Hydrolysis Casein positivity gelatin positivity starch positivity Citrate Availability U Propionate Availability radish Tyrosine resolution radish Nitrate reducing ability U Indole formation ability radish Salinity composition radish Growth at 7% salinity U Urea Hydrolysis radish
Growth by temperature lowest 15 ℃ optimal 30 ℃ Best 40 ℃

Strain identification for the selected antagonists was carried out using 16S rDNA sequencing. 16S chromosomal DNA sequencing of the isolated strain was performed in the following manner. First, the chromosomal DNA of the isolated strain was isolated, and then, 27F (5'-AGAGTTTGATCATGGCTCAG-3 ') and 1492R (5'-GGATACCTTGTTACGACTT-3') primers, which are universal primers used for 16S rRNA sequencing, were used. Amplified by PCR under conditions of denaturation at 94 ° C. for 1 minute, annealing at 60 ° C. for 1 minute, and polymerization at 72 ° C. for 1 minute and 30 seconds. The amplified PCR result was subjected to 0.8% agarose gel electrophoresis, and then purified by sequencing the 1S bp 16S chromosomal DNA using an ABI PRISM 3700 DNA analyzer (DNA Analyzer). The results were identified using the BLASTN program in comparison with the chromosomal RNA sequencing of GENEBANK and RDP (RNA database project). As a result, the selected strain was identified as Bacillus subtilis , therefore, the antagonistic microorganism of the present invention was named Bacillus subtilis KB-401, and the division was dated April 24, 2007. It was deposited with the Korean Federation of Culture Collections under the accession number of KFCC 11388P.

SEQ ID NO: 1 shows the 16S rDNA sequence (1400 bp) of Bacillus subtilis KB-401 of the present invention.

Example 2: Growth inhibitory effect on phytopathogenic bacteria of antagonistic microorganisms

As mentioned in Example 1, in order to confirm the effect of growth inhibition on phytopathogens , pear black pattern germ ( Alternaria kikuchiana ), citrus black spot germ (Diaporthe citri ), pear black star germ ( Venturia nashicola ), apple black star germ ( Venturia inaequalis ), Botrytis cinerea , Tomato leaf fungus ( Cladosporium fulvum ), Capsicum anthrax ( Colletotrichum coccodes ), Tomato withering fungus ( Fusarium oxysporum ), Potato withering fungus ( Fusarium solani ) Phytophthora capsici , Phytophthora melonis , Tomato Mycobacterium ( Sclerotinia minor ), Fusarium moniliforme , Colletotrichum caudatum , Grass Pisium leaf blight ( Pythium sp. ) Sixteen species of Rhizoctonia solani were used. First, PDA medium (Potato Dextrose Agar) was prepared, sterilized, and then dispensed by about 20 ml in a culture dish. One agar piece with phytopathogens is inoculated on one edge and the other is inoculated with a single colony of antagonist microorganisms. Thus prepared plate (plate) by replacing the incubation for 4 to 5 days at 25 ℃ to compare the growth inhibition effect on phytopathogens to investigate the antimicrobial activity.

Inhibitory Effects of Bacillus Subtilis KB-401 on Phytopathogenic Bacteria (Antibacterial Activity) Disease name (pathogen) Antimicrobial activity Pear Black Pattern Disease ( Alternaria kikuchiana ) + Citrus black spot disease ( Diaporthe citri ) +++ Pear Black Star Pattern ( Venturia nashicola ) + Apple Black Star Pattern ( Venturia inaequalis ) + Gray Mold Disease ( Botrytis cinerea ) ++ Tomato leaf fungal disease ( Cladosporium fulvum ) + Pepper Anthrax ( Colletotrichum coccodes ) ++ Tomato withering ( Fusarium oxysporum ) ++ Fusarium solani ++ Pepper Plague ( Phytophthora capsici ) ++ Melon plague ( Phytophthora melonis ) + Tomato Sclerotinia minor + Fusarium moniliforme ++ Turf anthrax ( Colletotrichum caudatum ) ++ Turfium leaf blight ( Pythium sp. ) + Grass brown spread disease ( Rhizoctonia solani ) +++

※ Antibacterial activity degree (㎜) 0 ~ 10: +, 10 ~ 20: ++, 20 or more: +++

1 is a photograph showing the growth inhibitory effect on grass brown spread disease ( Rhizoctonia solani ) of Bacillus subtilis KB-401 of the present invention.

Figure 2 is a photograph showing the growth inhibitory effect on citrus black spot disease ( Diaporthe citri ) of Bacillus subtilis KB-401 culture of the present invention. In FIG. 2, the Bacillus subtilis KB-401 culture medium was tested with the culture medium after the strain was removed.

As shown in Table 3 and Figures 1 and 2, Bacillus subtilis KB-401 of the present invention showed antimicrobial activity against various plant pathogens ( colletotrichum sp., Fusarium sp., Botrytis cinerea ), in particular It has been shown to exhibit high antimicrobial activity against citrus black spots ( Diaporthe citri ) and grass brown spread ( Rhizoctonia solani ).

Example 3: cultivation of antagonistic bacillus subtilis KB-401

The antagonistic B. subtilis KB-401 was cultured using M-2 medium (Table 4) and confirmed sporulation. The culture was incubated for 48 hours at 30 ℃, 250 L fermenter (Fermenter) at 150rpm, 1.0 ~ 1.5 vol / vol / min, pH 6.0 ~ 7.0, and confirmed the spore formation through a microscope (× 400). The number of spores in the culture resulted from 2.0 × 10 ⁹ to 1.0 × 10 ¹⁰ CFU (Colony Forming Unit) per ml.

M-2 medium composition for culturing Bacillus subtilis KB-401 Badge composition Content (/ ℓ) Glucose 20g Soybean flour 25 g KH ₂ PO ₄ 1 g K ₂ HPO ₄ 1 g CaCO ₃ 1 g MgSO _{4 7} H ₂ O 0.3 g CuSO ₄ · 5H ₂ O 0.01 g MnSO ₄ 0.02 g FeSO ₄ 7H ₂ O 0.02 g ZnSO ₄ · 7H ₂ O 0.02 g

Example 4 Control Effect of Bacillus Subtilis KB-401 Against Citrus Black Spot Pattern, Turf Brown Spread, Cucumber Powder

Antagonistic microorganism Bacillus subtilis KB-401 cultured in Example 3 was confirmed the control effect in the package of citrus black spot disease, grass brown spread disease, cucumber powdery mildew disease. In particular, cucumber powdery mildew is a biotic parasite (a bacterium that lives only on living organisms of dependent nutrients), so it is difficult to carry out a plate effect as in Example 2, and the control effect on direct packaging was tested. The control effect on the packaging was diluted 500 times with the culture medium, and citrus black spots were treated with four leaves every 14 days on the beginning of the plant. Grass brown spread disease was irrigated three times every 7 days on the beginning of the plant. The leaves were treated three times daily. The test plots were treated with three batches of complete randomization and egg mass, and compared with citrus black spot disease for dithianon hydration, turf brown spread disease for peniculon hydration, and cucumber powdery powder for phenarimol emulsion.

Control Effect of Bacillus Subtilis KB-401 on Citrus Black Spot Disease Test drug Foot disease degree (%) Control
(%) 1 repetition 2 repetitions 3 repetitions Average  KB-401 (500 times) 7.3 8.4 8.0 7.9 71.3  Dithianon Hydration (1000x) 1.2 1.8 2.6 1.9 93.1  No treatment 21.2 31.7 29.6 27.5 -

Control Effect of Bacillus Subtilis KB-401 on Grass Brown Spread Disease Test drug Foot disease degree (%) Control
(%) 1 repetition 2 repetitions 3 repetitions Average  KB-401 (500 times) 6.3 4.5 9.0 6.6 70.8  Pensicuron Hydrating (1000x) 1.0 0.7 3.5 1.7 92.5  No treatment 24.5 20.3 23.0 22.6 -

Control Effect of Bacillus Subtilis KB-401 on Cucumber Powdery mildew Disease Test drug Foot disease degree (%) Control
(%) 1 repetition 2 repetitions 3 repetitions Average  KB-401 (500 times) 5.5 6.7 7.0 6.4 69.2  Penarimol Emulsion (4000x) 0.8 1.1 1.5 1.1 94.7  No treatment 17.5 23.3 21.7 20.8 -

As shown in Tables 5 to 7, Bacillus subtilis KB-401 of the present invention showed a control effect of 71.3% of citrus black spot disease, grass brown spread disease of 70.8%, and cucumber powdery mildew disease. The control effect of 69.2% was shown to effectively inhibit each pathogen.

1 is a photograph showing the growth inhibitory effect on grass brown spread disease ( Rhizoctonia solani ) of Bacillus subtilis KB-401 of the present invention.

Figure 2 is a photograph showing the growth inhibitory effect on citrus black spot disease ( Diaporthe citri ) of Bacillus subtilis KB-401 culture of the present invention.

<110> KoreaBio Co., LTD <120> BACILLUS SUBTILIS KB-401 HAVING ANTAGONIST ACTIVITY AGAINST PLANT          PATHOGENIC BACTERIA AND CULTURE MEDIUM THEREOFF <130> SDP-290104 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1437 <212> DNA <213> Bacillus subtilis KB-401 <400> 1 ggagcgggct atctgcagtc gagcggacag atgggagctt gctccctgat gttagcggcg 60 gacgggtgag taacacgtgg gtaacctgcc tgtaagactg ggataactcc gggaaaccgg 120 ggctaatacc ggatggttgt ttgaaccgca tggttcagac ataaaaggtg gcttcggcta 180 ccacttacag atggacccgc ggcgcattag ctagttggtg aggtaacggc tcaccaaggc 240 aacgatgcgt agccgacctg agagggtgat cggccacact gggactgaga cacggcccag 300 actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct gacggagcaa 360 cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct gttgttaggg aagaacaagt 420 gccgttcaaa tagggcggca ccttgacggt acctaaccag aaagccacgg ctaactacgt 480 gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggaattattg ggcgtaaagg 540 gctcgcaggc ggtttcttaa gtctgatgtg aaagcccccg gctcaaccgg ggagggtcat 600 tggaaactgg ggaacttgag tgcagaagag gagagtggaa ttccacgtgt agcggtgaaa 660 tgcgtagaga tgtggaggaa caccagtggc gaaggcgact ctctggtctg taactgacgc 720 tgaggagcga aagcgtgggg agcgaacagg attagatacc ctggtagtcc acgccgtaaa 780 cgatgagtgc taagtgttag ggggtttccg ccccttagtg ctgcagctaa cgcattaagc 840 actccgcctg gggagtacgg tcgcaagact gaaactcaaa ggaattgacg ggggcccgca 900 caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac 960 atcctctgac aatcctagag ataggacgtc cccttcgggg gcagagtgac aggtggtgca 1020 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080 tgatcttagt tgccagcatt cagttgggca ctctaaggtg actgccggtg acaaaccgga 1140 ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta 1200 caatggacag aacaaagggc agcgaaaccg cgaggttaag ccaatcccac aaatctgttc 1260 tcagttcgga tcgcagtctg caactcgact gcgtgaagct ggaatcgcta gtaatcgcgg 1320 atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga 1380 gagtttgtaa cacccgaagt cggtgaggta acctttggag ccgcccgaag gacgggg 1437  

Claims (5)

Antagonistic microbial Bacillus subtilis KB-401 having antibacterial activity against phytopathogens (Accession No .: KFCC 11388P). The antagonist Bacillus subtilis KB-401, which has an antimicrobial activity against phytopathogens of claim 1, which is a grass brown spread disease ( Rhizoctonia solani ) or a citrus black spot disease ( Diaporthe citri ). According to claim 1, antagonistic bacillus subtilis having antimicrobial activity against phytopathogens of any one of Botrytis cinerea , Colletotrichum sp. , Fusarium sp. , Cucumber powdery mildew ( Sphaerotheca fusca ). S KB-401. Bacillus subtilis KB-401 (Accession No .: KFCC 11388P) medium having antibacterial activity against phytopathogens. According to claim 4, grass brown spread disease ( Rhizoctonia solani ), citrus black spot disease ( Diaporthe citri ), ash fungus ( Botrytis cinerea ), anthrax ( colletotrichum sp. ), Fusarium sp. , Cucumber powdery mildew ( Sphaerotheca fusca) Bacillus subtilis KB-401 (Accession No .: KFCC 11388P) culture having an antimicrobial activity against any one of the phytopathogens.

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