KR101755325B1 - Microorganism mixture of Trichoderma harzianum and Bacillus subtilis having antimicrobial activity against plant pathogen and uses thereof - Google Patents

Abstract

The present invention relates to a mixture of Trichoderma marjorium and Bacillus subtilis strains having antimicrobial activity against a plant pathogenic bacterium, and uses thereof, wherein Trichoderma harzianum NB-1 according to the present invention and The mixture of Bacillus subtilis NB-23 strain can be used effectively as a microbial agent for biological control because it can control plant pathogenic harmful bacteria and fungi simultaneously by synergistic action. In addition, the microorganism preparation containing the strain of the present invention as an active ingredient can be used as an environmentally friendly biological pesticide free from the risk of environmental pollution and human toxicity due to residual pesticides caused by the side effects of pesticides, It will be possible.

Description

[0001] The present invention relates to a microorganism mixture of Trichoderma harzianum and Bacillus subtilis having antimicrobial activity against plant pathogenic bacteria,

The present invention relates to a mixture of Trichoderma marjorium and Bacillus subtilis strains having antimicrobial activity against plant pathogenic bacteria and their uses.

Growth and harvest damage caused by diseases, insects and weeds of major crops has reached 42%, reaching about $ 300 billion, of which about 15% are caused by the disease. There are about 1,535 diseases in Korea. Especially, the major diseases such as strawberry, tomato, and red pepper are repeatedly caused by the annual diseases, which are caused by soil infectious diseases such as gray mold, plague and anthrax, Has been reported to reach hundreds of billions of won to hundreds of billions of won.

Soil-mediated disease mediates more than 60% of the contagion mediators, and soil-infectious pathogens have a strong infertility ability, so they enter the dormant state immediately after separation and survive for a long time under unfavorable environmental conditions. It is difficult to effectively control microorganisms. Therefore, it is urgently required to develop a disinfectant using microorganisms.

Plague is known to be highly resistant pathogenic host specificity, when exchanged between the blood Saratov shoot cap thereof (Phytophthora capsici , Phytophthora infestans , Phytophthora ( Phytophthora infestans ), Phytophthora cactorum ) are occurring nationwide every year. In particular, the annual production of pepper in Korea is 1.45 trillion won, of which 20% and 15% are caused by plague and anthrax. In addition, foot rot disease is caused by bacterial pathogens, Ralstonia solanacearum , and damages domestic crops such as tomatoes, peppers and cucumbers. Able to survive. In summer, the disease spreads rapidly in high temperature. When the pathogens become infected, the plants die in a short period of time. Gray mold control is also difficult due to the chemical agents, tricot village in der (Trichoderma spp.) And Pseudomonas species (Pseudomonas microbial pesticides were developed using microbial spp., but the control effect was only about 60%, and the effect of control was different according to environmental conditions. Is urgently required.

In Korean Patent Laid-Open Publication No. 2015-0107220, there is disclosed a mixed microorganism preparation comprising a strain of Bacillus sp. Hong1001, a strain of Pseudomonas sp. Hong1002, a strain of Streptomyces sp. Hong1003, a strain of T. pneumoniae Hong-1004, Korean Patent No. 1535893 discloses a new microorganism Bacillus amyloliquefaciens CC110 and a microbial agent and a microbial pesticide containing the same. However, as in the present invention, the 'Tricorder' ≪ / RTI > and mixtures thereof and the use thereof.

The present invention has been made in view of the above-mentioned needs. In the present invention, in order to construct a technology for simultaneously controlling harmful bacteria and fungi of plant pathogenic bacteria using domestic indigenous microorganisms and microbial culture metabolites, ( Trichoderma harvianum NB-1 and Bacillus subtilis NB-23 strains were newly selected for the antimicrobial activity against plant pathogenic microorganisms, including Trichoderma marjorium NB-1 and Bacillus subtilis NB-23 The inventors of the present invention have completed the present invention by confirming that synergy is generated when the strain mixture is treated and the antimicrobial activity is remarkably increased as compared with the case where each of the strains is treated.

In order to achieve the object of the present invention, the present invention provides a method of producing Trichoderma harzianum and Bacillus subtilis strains.

In addition, the present invention provides a microbial preparation for antimicrobial activity against a plant pathogenic bacterium comprising the above-mentioned strain mixture or a culture solution thereof as an active ingredient.

The present invention also provides a method for controlling a plant disease comprising treating the plant or soil with an effective amount of the above-mentioned strain or a culture solution thereof.

The Trichoderma < RTI ID = 0.0 > harzianum NB-1 and Bacillus subtilis NB-23 can be effectively used as a microbial preparation for biological control because they can effectively control harmful bacteria and fungi of plant pathogenic bacteria simultaneously with synergistic action have. In addition, the microorganism preparation containing the strain of the present invention as an active ingredient can be used as an environmentally friendly biological pesticide free from the risk of environmental pollution and human toxicity due to residual pesticides caused by the side effects of pesticides, It will be possible.

FIG. 1 is a phylogenetic map showing the results of identifying the nucleotide sequence (SEQ ID NO: 1) amplified by the colony PCR method of the ITS region of the NB-1 strain selected in the present invention.
FIG. 2 shows phylogenetic positions of the 16s rRNA nucleotide sequence (SEQ ID NO: 2) and the Xylase B gene of the NB-23 strain selected in the present invention.
FIG. 3 shows the result of analysis of the antimicrobial active substance isolated and purified from a culture of the NB-23 strain selected in the present invention and analyzed using a GC-MS instrument.
FIG. 4 is a schematic diagram of a greenhouse port test method for investigating the antimicrobial effect of a mixture of NB-1 strain and NB-23 strain selected in the present invention for a causative agent of a plant disease.

In order to accomplish the object of the present invention, the present invention provides a method for producing an antimicrobial agent,Trichoderma harzianum) And Bacillus subtilisBacillus subtilis) ≪ / RTI >

In strain mixture according to one embodiment, the tricot der Haar town Jia num (Trichoderma harzianum) strains of tricot der town Har Jia num and NB-1 is deposited with number KACC93239P, Bacillus standing subtilis (Bacillus subtilis) strain benzoic acid (Benzoic acid) to produce Bacillus standing subtilis NB-23 accession number is KACC92152P that But is not limited thereto.

In the present invention, mycelial growth inhibition and bacterial growth inhibition tests were conducted using a plant pathogenic fungus and a bacterium standard strain, and NB-1 and NB-23 as the last two excellent strains were selected from the soil. As a result of identification of the two selected NB-1 and NB-23 strains, Trichoderma harzianum and Bacillus subtilis , respectively. The NB-1 strain is selected from the group consisting of Trichoderma The strain NB-23 was named as Bacillus subtilis strain NB-23 (hereinafter referred to as " NB-23 strain " (KACC92152P) on October 28, 2016 at the Institute of Agricultural Biotechnology (KACC).

In strain mixture according to one embodiment of the present invention, the mixture tricot der Haar town Jia num (Trichoderma harzianum strains of 10 ⁶ to 10 ⁸ cfu / g and Bacillus subtilis strains of 10 ⁷ to 10 ⁹ cfu / g, and preferably the strains of Tricoderma magazinum strain are 10 ⁷ cfu / g, and Bacillus subtilis strains may be mixed at 10 ⁸ cfu / g, but are not limited thereto.

In the strain mixture according to an embodiment of the present invention, the plant pathogenic bacteria may be a fungus causing a plant disease or a bacterium causing a plant disease, preferably Phytopthora capsici , Fusarium oxysporum , Sclerotinia < RTI ID = 0.0 > sclerotiorum , Streptomyces, scabiei , Clavibacter michiganensis ), collectotrichum goreosephioides ( collectotrichum gloeosprioides , Botrytis < RTI ID = 0.0 > cinerea , and Phytopthora capsici . However, the present invention is not limited thereto.

In addition, the present invention provides a microbial preparation for antimicrobial activity against a plant pathogenic bacterium comprising the above-mentioned strain mixture or a culture solution thereof as an active ingredient.

The antimicrobial microbial preparation for the plant pathogenic bacteria of the present invention may be a formulation in the form of a wettable powder or a suspension concentrate. The antimicrobial microbial preparation according to the present invention may be prepared in the form of a liquid sterilizing agent, and may be added in the form of a powdered powder, or may be granulated by formulating it into a powder. However, the formulation is not particularly limited. In other words, it can be formulated as a bio-disinfectant to overcome this in environment-friendly organic farming with limited supply of chemical disinfectants.

The wettable powder of the present invention may contain, as an active ingredient, Trichoderma < RTI ID = 0.0 > harzianum and Bacillus subtilis strain or a culture thereof, white carbon as a moisture absorbent, sodium bis [2-ethylhexyl] sulfosuccinate as a wetting agent, sodium lignosulfonate and an extender Preferably kaolin, and preferably 10% by weight Trichoderma < RTI ID = 0.0 > harzianum) and standing Bacillus subtilis (Bacillus subtilis) strain or the culture mixture thereof, 1% by weight of white carbon (white carbon), 1% by weight sodium bis [2-ethylhexyl] sulfosuccinate, 1 wt% sodium sulfo lignoceric Nitrate, and 87 wt% kaolin.

The liquid wettable powder of the present invention can be used as an active ingredient, as a mixture of Trichoderma harzianum and Bacillus subtilis strains or as a culture solution, a wetting agent and a dispersant thereof, of MBSC (nonylphenol, ethoxylated, monoether with sulfuric acid, sodium salt, sodium bis [20 ethylhexyl] sulfosuccinate Polyoxyethylene nonylphenol), isopropanol as an excipient and water as an extender. The liquid wettable powder preferably comprises 50% by weight of a mixture of Trichoderma harzianum and Bacillus subtilis strain or a culture thereof, 4% by weight MBSC, 30% by weight isopropanol and 16% by weight water But is not limited thereto.

In addition, the present invention provides a method for controlling a plant disease comprising the step of treating an effective amount of the above-mentioned strain or a culture thereof with a plant, a seed or a cultivation plant.

The plant bottle may be a Phytopthora capsici), Fusarium oxy Spokane Room (Fusarium oxysporum), Klee's Loti Nias Cle to room thio (Sclerotinia sclerotiorum), Streptomyces Scar sorrow (Streptomyces scabiei , Clavibacter michiganensis ), collectotrichum goreosephioides ( collectotrichum gloeosprioides , Botrytis < RTI ID = 0.0 > cinerea , Phytopthora capsici , and the like, but the present invention is not limited thereto.

As a method for controlling the plant disease, the Trichoderma harzianum ) and a Bacillus subtilis strain may be treated with an effective amount of a culture medium to a plant, plant seed or field.

The 'effective amount' of the present invention is an amount sufficient to produce a beneficial or desired result, in order to control a plant disease, a microorganism preparation having antimicrobial activity against plant pathogenic bacteria is uniformly diluted with water and then sprayed with a suitable spraying device such as a power disperser It can be applied to plants and cultivated land. When the wettable powder or the liquid wettable powder of the present invention is diluted in water, the concentration of the wettable powder or the liquid wettable powder is adjusted to about 10 ⁵ to 10 ¹⁰ cfu / ml, preferably about 10 ⁸ cfu / ml so that the effective ingredient can be in a biologically effective range But is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1. Ensure antimicrobial microorganisms

Chungcheongnam-do, Gangwon-do and Jeollanam-do were collected and 519 strains were isolated purely. Among these purely isolated strains, seven strains of phytopathogenic fungi and one strain of bacteria were used to inhibit mycelial growth and inhibition of bacterial growth, and the final two excellent strains were selected. As a result, the strain NB-1 showed excellent mycelial growth inhibition against the phytopathogenic fungus, and the strain NB-23 showed not only inhibition of pathogenic harmful bacteria but also inhibition of pathogenic fungal mycelial growth (Table 1). In the case of NB-23 strain, not only the strain itself but also the culture metabolism mainly showed excellent effect in inhibiting pathogenic bacteria and inhibiting mycelial growth of pathogenic fungi (Table 2).

Mycelial growth inhibition and bacterial inhibition test of plant pathogenic bacteria Strain
number Phytopthora capsici Fusarium oxysporum Collectotrichum gloeosprioides Botrytis
cinerea Sclerotinia
sclerotiorum Streptomyces scabiei Clavibacter
michiganensis NB-1 +++ +++ +++ +++ +++ + - NB-23 +++ +++ +++ ++ ++ + +++

Mycelial growth inhibition and bacterial inhibition test on plant pathogenic bacteria of the selected strain or supernatant Strain
number detach
area detach
badge Centrifugal
detach Phytopthora capsici Fusarium oxysporum Collectotrichum gloeosprioides NB-1 Cheonan, Chungnam PDA Mycelium +++ +++ +++ NB-1 Cheonan, Chungnam PDA Supernatant - - - NB-23 Cheonan, Chungnam NB Mycelium - - ++ NB-23 Cheonan, Chungnam NB Supernatant +++ +++ +++

Example  2. Antimicrobial activity of microorganisms Systematic  Location and identification

10 g of the culture of the antibacterial active microorganism NB-1 was added to 90 ml of sterilized distilled water, treated at 150 rpm for 1 hour, taken 1 ml, diluted to 10 ^-5 , and inoculated into PDA (Popato Dextrose Agar, Difco) After incubation for a day, fungal colonies were obtained. The ITS region was amplified by colony PCR to confirm the gene sequence. The amplified product was purified using a PCR product purification kit (Quagen) and sequenced using DNA sequencer 9791XI (Applied Biosystems). The nucleotide sequence of the NB-1 strain (SEQ ID NO: 1) was identified and identified as Trichoderma harzianum (Fig. 1).

10 g of the culture of the antibacterial active microorganism NB-23 was added to 90 sterilized distilled water and treated at 150 rpm for 1 hour. Then, 1 ml was diluted to 10 ^-6 , and 100 μl was inoculated into Nutrient Agar (Difco). After incubation at 28 ° C for 3 days, Respectively. The 16s rRNA region was amplified by colony PCR to confirm the nucleotide sequence of NB-23. The amplified product was purified using a PCR product purification kit (Quagen) and sequenced using DNA sequencer 9791XI (Applied Biosystems). As a result of analysis of the nucleotide sequence (SEQ ID NO: 2), it was confirmed that the gene belonged to the genus Bacillus. In order to identify the closely related species, an antimicrobial active microorganism was identified by analysis of gyrB gene. The gyrB gene was identified and identified as Bacillus subtilis subsp . subtilis (Fig. 2).

Example  3. Antimicrobial activity of microorganisms

Bacillus subtilis ( Bacillus < RTI ID = 0.0 > subtilis) < / RTI & subsp . subtilis ) NB-23 microbial cultures were isolated and purified to identify the components and the structure of the compounds. In order to separate and purify the active substance, substances were extracted using an organic solvent such as methanol and ethanol. And concentrated under reduced pressure using a rotary vacuum evaporator to obtain a concentrate. dH ₂ O and fractionated with n-hexane, methylene chloride, ethyl acetate (EtOAc), n-butanol (BuOH) and dH ₂ O sequentially. The obtained fractions were selected by MIC test using a paper disk method for bacterial pathogens (Tomato Pomarum pathogen). The fractions showing antimicrobial activity were analyzed by GC-MS instrument and the structure was confirmed to be benzoic acid (Fig. 3).

Example  4. Antimicrobial effect of antibacterial active microorganisms

The antimicrobial activity against the plant pathogenic fungi and bacterial pathogens was examined at various mixing ratios of the mixed microbial preparations described in Table 3 below. As a result, it was found that 10% of the cultivars of Trichoderma marjorium NB-1 + Bacillus subtilis The formulation containing 40% of the NB-23 cultivar as the main component exhibited the highest antimicrobial activity.

Antimicrobially active microorganisms and various mixing ratios Formulation NO. Name of input material (input ratio) T. harzianum NB-1 cultivar B. subtilis NB-23 culture Fungicide
(china clay) Anti-caking agent
(White carbon) NB-SP-1 100 0 0 0 NB-SP-2 90 10 0 0 NB-SP-3 80 20 0 0 NB-SP-4 70 30 0 0 NB-SP-5 60 40 0 0 NB-SP-6 40 30 20 10 NB-SP-7 20 40 40 0 NB-SP-8 10 40 45 5 NB-SP-9 0 100 0 0

In addition, the culture of Trichoderma marjorium NB-1 and Bacillus subtilis The antimicrobial activity of each of the NB-23 cultivars was compared with that of the NB-1 cultivars and Bacillus subtilis The antimicrobial activity of the mixture of NB-23 cultures was compared to determine whether the mixture of cultures had a synergistic effect. Antimicrobial activity against soil-infectious fungal pathogens and bacterial pathogens was examined by Jacques F. and Acar, MD (1980. In "Antibiotics in Laboratory Medicine." Williams and Wilkins, Baltimore / London. .

As a result, as shown in Table 4, the culture of Trichoderma marjorum NB-1 and Bacillus subtilis Compared with the antimicrobial activity of each of the NB-23 cultivars, the cultivars of Trichoderma marjorium NB-1 and Bacillus subtilis It was found that the mixture of the NB-23 cultivar had a remarkably excellent antimicrobial activity and showed a synergistic effect. For example, in the case of P. thiotharum capsaicin, NB-1 alone has 80% antibacterial effect and 75% when NB-23 alone is used. When mixed, the concentration of each strain is 50% , Respectively. The antimicrobial effect of about 77.5% is expected to be 40% and 37.5%, respectively. However, since the antibacterial effect of the mixture is 95%, the synergistic effect is remarkable.

Antibacterial effect of the mixed microorganism preparation prepared by mixing the antibacterial active microorganisms on the causative bacteria of plant diseases (Experiment NB-SP-8) NB-1
Single processing
(10 < ⁷ > cfu / g) NB-23
Single processing
(10 < ⁸ > cfu / g) T. harzianum NB-1 10% (10 < ⁷ > cfu / g)
+
B. subtilis NB-23 cultured 40% (10 ⁸ cfu / g) Phytopthora capsici 80% 75% 95% Fusarium oxysporum 82% 74% 88% Sclerotinia
sclerotiorum 80% 68% 87% Streptomyces
scabiei 65% 60% 81% Clavibacter
michiganensis 0% 75% 85% Collectotrichum gloeosprioides 85% 74% 96% Botrytis
cinerea 80% 69% 98%

Example  5. Antimicrobial effects of antimicrobial active microorganisms

In order to test the microorganisms that showed strong antimicrobial activity against soil infectious pathogens, antimicrobial active microorganisms and various mixing ratios were prepared by inoculating pathogens into tomato crops, and the control effect was tested under the following conditions (port test) .

end. Test crops (varieties): Tomatoes (jujube tomatoes)

I. Target pathogen: Fungi disease: Fusarium wilt of tomato oxysporum )

All. Bacterial Disease: Tomato Pomalum Pathogen ( Ralstonia solanacearum )

la. Test method:

As a result, 10% of the cultures of Trichoderma marjorium NB-1 + Bacillus subtilis The control ratio of NB-23 cultivar (40%) was 70% for Fusarium oxysporum and 78% for Ralstonia solanacearum . And showed high antimicrobial activity. In the port test, the control rate was somewhat lower than the control rate in the plate, but the control effect was remarkable.

Example  6. Safety of crops for antimicrobial active microbial formulation

We investigated the feasibility of NB-SP-8 treatment on major crops such as tomatoes and lettuce, where soil-infectious disease damage occurred frequently. The presence or absence of weakness was investigated by questionnaire survey according to treatment method.

0: No visual weakness

1: Very light weakness, slight minor weakness

2: Weakness is recognized in the cow parts of treated leaves

3: Approximately 50% of the treated leaves are vulnerable.

4: I am suffering considerable damage, but still have a good portion

Weak test results for major crops crops Processing method Test result pepper Processing 600g per 10a No Weak tomato No Weak cucumber No Weak Lettuce No Weak cabbage No Weak

Example  7. Demonstration test of farmhouse with antibacterial complex microbial agent

10% of the cultivars of Trichoderma marjorum NB-1 identified in Example 4 above and 10% of Bacillus subtilis A complex microbial preparation containing 40% of the NB-23 cultivar as a main component was prepared and the farm demonstration test was carried out as follows.

1. Test Method

end. According to the criteria and method of the registration standard and the phytotoxicity test of the pesticide listed in RDA.

I. Target disease: wilt ( Fusarium oxysporum )

All. Test crop (varieties): Tomato (Hibara)

la. Occurrence of the target disease: The rate of Lee Byungju in the non-treatment area was 13.8%, which was enough to examine the efficacy.

Processing contents Test agent Per 660m ²
usage Drug efficacy test Weak test Test year Requesting agency Dilution drainage and usage Treatment timing and method (treatment date) Standard amount
(Processing date) Dose (processing date) Trichoderma harzianum >
10 ⁶ CFU / g
Bacillus subtilis >
10 ⁶ CFU / g 400g 3,000 times
(1.8 L / m ² ) Immediately after the ceremony, it is treated 3 times every 10 days
(9/24, 10/4, 10/14) 3,000 times
(9/24) 1,500 times
(9/24) - Enviagro No treatment - - - - - - -

hemp. Overview of Seedlings: On September 23, 2016, it was officially established with a width of 100cm, a width of 40cm, and a planting distance of 90 ~ 50cm. No test drug or other drug was applied.

Placement and area of test strip (3 repetitions in single strip) division Number of treatment Repeatability Total number of cages Area per unit Required area Total area required Efficacy 3 3 9 20m ² 180m ² 225m ² Weak 3 3 9 5m ² 45m ²

bar. Meteorological conditions before and after spraying: Indoor test, no change of weather effect to affect the efficacy and weakness. (Meteorological Observation: Geumsan Unmanned Observatory)

Weather before and after spraying Month / day Rainfall (mm) High / Low Temperature (℃) Average temperature (℃) 9/24 * - 28.4 / 13.5 19.4 9/25 - 24.9 / 14.6 19.2 10/4 - 27.9 / 18.0 21.9 10/5 * 44.5 23.4 / 15.6 18.6 10/14 * - 24.0 / 6.6 13.2 10/15 - 24.8 / 8.0 14.6

2. Research Method

Investigation method division Survey item Number of investigations Date of investigation Investigation method Drug efficacy test Lee Byungju rate 1 time 11/14 After 31 days of final drug treatment,
Investigation of Lee, Byung-Ju on Jeonju Weak test Presence or absence of weakness 3rd time 6/17, 6/19, 6/21 The appearance of visible weakness after 3, 5, and 7 days of drug treatment

3. Test results

Drug test (31 days after final drug treatment) Test drug Lee Byungju (%) Significant difference
(DMRT) Control (%) Ⅰ repeat Ⅱ repeat Ⅲ repeat Average Trichoderma harzianum > 10 ⁶ CFU / g
Bacillus subtilis > 10 ⁶ CFU / g 10.0 12.0 9.0 10.3 b 70.6 No treatment 35.0 38.0 32.0 35.0 a -

c.v. (%): (14.9)

(3, 5 and 7 days after final drug treatment) Test agent Test crop Weak degree (0 ~ 5) Remarks Standard amount Quantity Trichoderma harzianum > 10 ⁶ CFU / g
Bacillus subtilis > 10 ⁶ CFU / g Tomato (Hibara) 0 0 No Weak

4. Summary of results

end. Efficacy

The test drug was found to be significant for controlling tomato wilt disease.

I. Weak

Test medication had no symptoms of weakness in the reference dose and the dose.

All. Test taker feedback

The test drug showed comparable efficacy to the reference drug, and the drug was judged to be practicable because no symptom was observed in the reference dose and the dose.

Agricultural Biotechnology Research Institute KACC93239P 20151014 Agricultural Biotechnology Research Institute KACC92152P 20161028

<110> NBagro CO., LTD. <120> Microorganism mixture of Trichoderma harzianum and Bacillus          subtilis having antimicrobial activity against plant pathogen and          uses thereof <130> PN17067 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 537 <212> DNA <213> Trichoderma harzianum <400> 1 tgcggaggga tcattaccga gtttacaact cccaaaccca atgtgaacgt taccaaactg 60 ttgcctcggc gggatctctg ccccgggtgc gtcgcagccc cggaccaagg cgcccgccgg 120 aggaccaacc taaaactctt attgtatacc ccctcgcggg tttttttata atctgagcct 180 ttctcggcgc ctctcgtagg cgtttcgaaa atgaatcaaa actttcaaca acggatctct 240 tggttctggc atcgatgaag aacgcagcga aatgcgataa gtaatgtgaa ttgcagaatt 300 cagtgaatca tcgaatcttt gaacgcacat tgcgcccgcc agtattctgg cgggcatgcc 360 tgtccgagcg tcatttcaac cctcgaaccc ctccgggggg tcggcgttgg ggatcggccc 420 tcccttagcg ggtggccgtc tccgaaatac agtggcggtc tcgccgcagc ctctcctgcg 480 cagtagtttg cacactcgca tcgggagcgc ggcgcgtcca cagccgttaa acaccca 537 <210> 2 <211> 1394 <212> DNA <213> Bacillus subtilis <400> 2 gtcgagcgga cggatgggag cttgctccct gaagtcagcg gcggacgggt gagtaacacg 60 tgggcaacct gcctgtaaga ctgggataac tccgggaaac cggggctaat accggataac 120 tcttttcctc acatgaggaa aagctgaaag atggtttcgg ctatcactta cagatgggcc 180 cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggccacgatg cgtagccgac 240 ctgagagggt gatcggccac actgggactg agacacggcc cagactccta cgggaggcag 300 cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgagtgatga 360 aggttttcgg atcgtaaaac tctgttgtca gggaagaaca agtaccggag taactgccgg 420 taccttgacg gtacctgacc agaaagccac ggctaactac gtgccagcag ccgcggtaat 480 acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa gcgcgcgcag gcggttcctt 540 aagtctgatg tgaaagcccc cggctcaacc ggggagggtc attggaaact ggggaacttg 600 agtgcagaag agaagagtgg aattccacgt gtagcggtga aatgcgtaga gatgtggagg 660 aacaccagtg gcgaaggcga ctctttggtc tgtaactgac gctgaggcgc gaaagcgtgg 720 ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgagt gctaagtgtt 780 agagggtttc cgccctttag tgctgcagca aacgcattaa gcactccgcc tggggagtac 840 ggccgcaagg ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 900 gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatctcctg acaaccctag 960 agatagggcg ttccccttcg ggggakhga tgacaggtgg tgcatggttg tcgtcagctc 1020 gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag 1080 cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac 1140 gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg atggtacaaa 1200 gggctgcaag accgcgaggt taagcgaatc ccataaaacc attctcagtt cggattgcag 1260 gctgcaactc gcctgcatga agccggaatc gctagtaatc gcggatcagc atgccgcggt 1320 gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg 1380 aagtcggtga ggta 1394

Claims (6)

A mixture of Bacillus subtilis NB-23 strain with an accession number of KACC93239P, Trichoderma harzianum NB-1, and accession number KACC92152P, which have antimicrobial activity against plant pathogenic bacteria. delete The plant disease pathogen as claimed in claim 1, wherein the plant pathogenic bacterium is selected from the group consisting of Phytopthora capsici , Fusarium oxysporum , Sclerotinia sclerotiorum , Characterized in that it is at least one selected from the group consisting of Streptomyces scabiei , Clavibacter michiganensis , Collectotrichum gloeosprioides and Botrytis cinerea . Lt; / RTI &gt; An antimicrobial microbial preparation for a plant pathogenic bacterium comprising the mixture of the strains of claims 1 or 3 or a culture thereof as an active ingredient. A method for controlling a plant disease comprising the step of treating an effective amount of a strain or a culture of the strain of claim 1 or 3 to a plant, plant seed or field. 6. The plant according to claim 5, wherein the plant is selected from the group consisting of Phytopthora capsici , Fusarium oxysporum , Sclerotinia sclerotiorum , Streptomyces scabiae Wherein the plant pathogen is selected from the group consisting of Streptomyces scabiei , Clavibacter michiganensis , Collectotrichum gloeosprioides and Botrytis cinerea . Wherein the plant disease is caused by the plant.

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