WO2014065589A1 - Lysinibacillus sphaericus tc1 strain, microbial preparation for controlling plant diseases comprising said strain, and plant disease control method using same - Google Patents

Lysinibacillus sphaericus tc1 strain, microbial preparation for controlling plant diseases comprising said strain, and plant disease control method using same Download PDF

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WO2014065589A1
WO2014065589A1 PCT/KR2013/009480 KR2013009480W WO2014065589A1 WO 2014065589 A1 WO2014065589 A1 WO 2014065589A1 KR 2013009480 W KR2013009480 W KR 2013009480W WO 2014065589 A1 WO2014065589 A1 WO 2014065589A1
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strain
culture
group
plant diseases
microbial agent
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PCT/KR2013/009480
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French (fr)
Korean (ko)
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김진철
최경자
최용호
장경수
윤미영
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한국화학연구원
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Priority claimed from KR1020120117986A external-priority patent/KR101956833B1/en
Priority claimed from KR1020120118043A external-priority patent/KR102015051B1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom

Definitions

  • Lysinibacillus spalicus TC1 strain a microbial agent for controlling plant diseases comprising the strain and a plant disease control method using the same
  • the present invention provides a novel Ricinibacillus spalicus ys // 2 / bac /// is sphaericus) TCI strain that exhibits inhibitory activity in the growth of various pathogenic bacteria and fungi; Microbial preparations for controlling plant diseases comprising the same; And to a plant disease control method using the microbial agent.
  • the bacterium of the genus Bacillus a Gram-positive bacterium, is the second most widely researched and commercialized bacterium after Pseudomonas genus 0 euio3 ⁇ 4 as), which produces a variety of secondary metabolites that exist in the soil and are biologically active. Has the characteristic of forming endospores that exhibit resistance in
  • Bacillus Velensis strain was developed by Gustafson in 1994 under the trade name Kodiak as a seed and foot sugar treatment agent for cotton and peanuts (Backman et al., Improving Plant Productivity with Rhizosphere).
  • Bacteria, pp. 3-8 (1994)) and in 2001, Bacillus Velensis and Bacillus amyloliquifaciens (Bacillus amyIoIiquefac) iens): a microbial disinfectant called BioYield was introduced. There is a bar.
  • Bacillus spp. Bacteria are widely used in South Korea as a bacterium for improving yield, while Bayer Germany and Taensa Co., Ltd. use Bacillus FZB 24 bacteria to control soil infectious diseases and microbial fungicides, respectively. It was developed.
  • Korean Patent No. 1163986 discloses a plant endogenous microorganism, Bacillus Velensis 03 ⁇ 4c /// "s velezensis) G341 strain (KCTC 11497BP) isolated from ginseng roots.
  • the present inventors have diligently researched to develop microbial agents exhibiting control effects on various plant diseases, etc.
  • Ricinibacillus spalicus TC1 strains, tomato foot blight, pepper In addition to showing a very good control effect against plant diseases such as late blight and Chinese cabbage root gall disease, it was found that the volatile substances produced by the strain inhibit the growth of various pathogenic bacteria and fungi.
  • Another object of the present invention for controlling plant diseases comprising the novel strain It is to provide a microbial agent.
  • Still another object of the present invention is to provide a plant disease control method using the microbial agent.
  • the present invention provides a Ricinibacillus spalicus TC1 strain (KCTC 12287BP) to produce a volatile substance exhibiting antimicrobial activity.
  • the present invention is a plant containing any one or more selected from the group consisting of Lysinibacillus spalicus TC1 strain, spores of the strain, culture of the strain and extract of the culture as an active ingredient It provides a microbial preparation for disease control.
  • the present invention provides a method for controlling a plant disease comprising treating the microbial agent to a plant or the surrounding environment.
  • the present invention also relates to tetrachloroethylene, 3-methyl-1-butane, methylpyrazine, cyclonucleanone, 2,5-dimethylpyrazine, ⁇ , ⁇ -dimethylformamide, and 2- (2-ethoxyethoxy
  • a composition for controlling plant diseases comprising any one or more selected from the group consisting of ethane.
  • Figures 2 to 4 show the control effect against tomato foot blight, pepper blight, and Chinese cabbage root gall disease in the culture of Ricinibacillus spalicus TC1 strain, respectively.
  • Figure 5 shows the control effect on the tomato foot blight of spray-dried samples prepared with Ricinibacillus spalicus TC1 strain culture medium.
  • 6 and 7 show the control effect against tomato foot blight and pepper blight following treatment with a single culture and a mixed culture of the Bacillus velensis G341 strain and Ricinibacillus spalicus TC1 strain, respectively.
  • Figure 8 shows the control effect on the tomato foot blight of the mixed culture spray spray of the Bacillus Velensis G341 strain and Ricinibacillus spalicus TC1 strain.
  • FIG. 9 shows antibacterial activity against Ralstonia solana serum following treatment with a single culture and a mixed culture of Bacillus velogenis G341 strain and Ricinibacillus spalicus TC1 strain.
  • Figure 10 shows the control effect of the mixed culture solution, bacteria and supernatant of tomato Bacillus blight strains of Bacillus Velensis G341 strain and Lysinibacilli sp.
  • Figure 12 shows the control effect against tomato foot blight according to the treatment concentration of the ethyl acetate extract of Bacillus Velensis G341 strain culture.
  • FIG. 13 is a UV spectrum of Compounds 1 and 2, which are antimicrobial active substances isolated from Bacillus velogenis G341 strain culture.
  • FIG. 13 is a UV spectrum of Compounds 1 and 2, which are antimicrobial active substances isolated from Bacillus velogenis G341 strain culture.
  • FIG. 14 shows the structure of an antimicrobial active material isolated from Bacillus balensis G341 strain culture medium, where R is H and dipicidine, and when R is 0H.
  • the present invention provides a Ricinibacillus spalicus TC1 strain (KCTC 12287BP) that produces volatiles exhibiting antimicrobial activity. Strains of the invention have been shown to have the following morphological biochemical and genetic characteristics:
  • Morphological and physiological characteristics Gram-positive bacteria, mesophilic strains, rod-shaped strains. It also forms endospores and is a typical facultative anaerobic strain.
  • the strain of the present invention was identified as a novel strain belonging to Ricinibacillus sp. Liqueur (KS // bac /// ws sp? Aer / cus). On October 10, 2012, it was deposited with the Korea Institute of Biotechnology and Gene Bank as Accession No. KCTC 12287BP. Ricinibacillus spalicus TC1 strain according to the present invention is characterized by showing excellent control against various plant diseases of tomato foot blight pepper blight and cabbage root gall disease (Examples 4 to 6).
  • the ricinibacillus spalicus TC1 strain of the present invention can produce a volatile substance exhibiting antimicrobial activity, such as tetrachloroethylene (tetrachloroethylene),
  • the culture medium or extract of the culture medium of the present invention contains the volatile substance.
  • the present invention is a plant disease control containing any one or more selected from the group consisting of Lysinibacillus spalicus TC1 strain (KCTC 12287BP) ( spores of the strain, culture of the strain and extract of the culture) as an active ingredient It provides a microbial preparation for.
  • Lysinibacillus spalicus TC1 strain KCTC 12287BP
  • spores of the strain, culture of the strain and extract of the culture spores of the strain, culture of the strain and extract of the culture
  • the microbial preparations comprising the strain of Ricinibacillus spalicus TC1 according to the present invention is Ralstonia solanacearum ( ⁇ / 072/3 solanacearum), the subspecies of Ashdoborax avene ⁇ - ⁇ ⁇ ° fl (Acidovorax avenae subsp.cattlyae), Agrobacterium tumefaciens, Burkholderia Written by BurkhoMeria glumae, Pectobacterium chrysanthemi 0 CO ) aC i er / 1 ⁇ 2; chrysanthemi), Pseudomonas syringae strain Actinide (/ 3 ⁇ 4ei / G3 ⁇ 4Z70 / 7as syringae v.
  • mi ch iganens is ⁇ ; Rhizoctonia solan /), Magna Forte o ⁇ ⁇ iagnaporthe oryzae), Botrytis Cinerea (5o / y / s cinerea), Colletotricum Coco ⁇ ⁇ (Collet otri chum coccodes), Scleroti Nia sclerotiorum (5c / ro / 72 / a sclerotioru), phytophthora capsaici (/ 3 ⁇ 4 ⁇ ⁇ / 3 ⁇ 43 ⁇ 4 ⁇ / ⁇ 3 capsici), phytophthora inhyeans and plasmodio It is possible to inhibit the growth of pathogenic mycobacteria (fungi) such as fora brassica (/ as / 70i / pAora brass / cae) (Examples 8 and 9).
  • pathogenic mycobacteria fungi
  • fungi such as fora brassica (/ as / 70i
  • the microbial agent according to the present invention further comprising any one or more selected from the group consisting of Bacillus Velensis G341 strain (KCTC 11497BP), spores of the strain, culture of the strain and extract of the culture solution. It features.
  • the microbial agent may include at least one selected from the group consisting of Bacillus vegetosis G341 strain (KCTC 11497BP), spores of the strain, cultures of the strains, and extracts of the cultures; And at least one selected from the group consisting of Ricinibacillus sparycus TC1 strain (KCTC 12287BP), spores of the strain, cultures of the strains, and extracts of the cultures, with a weight ratio of 1: 9 to 9: 1, preferably May be included in a weight ratio of 1: 1 .
  • KCTC 11497BP Bacillus vegetosis G341 strain
  • Ricinibacillus sparycus TC1 strain KCTC 12287BP
  • the microbial preparations comprising the Bacillus Velensis G341 strain and the Ricinibacillus spalicus TC1 strain are more resistant to the Bacillus Velensis G341 strain and Lys than when the culture medium of each strain is used alone.
  • Synibacillus spalicus TC1 strains when used in combination at a weight ratio of 1: 1 showed better control activity against tomato foot blight and pepper blight (Examples 12 and 13).
  • the characteristics of the Bacillus Velensis G341 strain is described in detail in Korean Patent Registration No. 1163986.
  • the ethyl acetate extract of the Bacillus bellensis G341 strain culture medium exhibits a concentration-dependent control effect against tomato foot blight and oxydipidine, a nonvolatile antibacterial active material produced from the strain.
  • (oxydifficidin) and dificidin show excellent antimicrobial activity against various Gram-negative bacteria and Gram-positive bacteria, especially Ralstonia solanacerum.
  • the Bacillus Velensis G341 strain can produce volatiles that exhibit antimicrobial activity, such as dimethyl disulfide, 1-butanol and 3-hydroxy-2-butanone (3-hydroxy). -2-butanone) (Examples 18-20).
  • the microbial agent of the present invention simultaneously comprising the G341 strain is Ralstonia solanacearum 03 ⁇ 4 / sio a solanacearum), subspecies of Ashidoborax avene ° fl iAcidovorax a venae subsp. cattlyae), Igrobacterium Grob ce "/ w tumefaciens, Berkholderia
  • Antimicrobial activity may be exhibited against any one or more pathogens selected from the group consisting of (Example 19, Table 5).
  • Microbial preparations according to the present invention Ricinibacillus spalicus TC1 strain alone; Alternatively, the Bacillus velensis G341 strain and Ricinibacillus spalicus TC1 strain mixture may be mixed with a carrier, and then formulated into powder, pellet granules, or a solution.
  • Preferred carriers may be water, white carbon, kaolin, dextrin, or the like.
  • the microbial agent of the present invention comprises a culture medium of the strain of Lysinibacillus sp. Liqueur TC1, or Bacillus velogenis G341 strain culture and a strain of Ricinibacilli sp. in Mixed mixed cultures can be prepared by mixing a with a carrier such as white carbon and dextrin and then spray drying it (see Examples 7 and 14).
  • strain culture may include all of the culture solution containing the strain or culture medium filtered strain.
  • culture liquid extract is preferably extracted from the culture medium of the strain with an appropriate extraction solvent, for example, ethyl acetate, butanol, water or a mixed solvent thereof, more preferably the culture supernatant is the same amount of ethyl acetate or butanol Ethyl acetate layer or butanol layer fractionated by, or the culture supernatant may be the same amount of ethyl acetate and butane fraction and remaining aqueous solution layer.
  • an appropriate extraction solvent for example, ethyl acetate, butanol, water or a mixed solvent thereof
  • the culture supernatant is the same amount of ethyl acetate or butanol Ethyl acetate layer or butanol layer fractionated by, or the culture supernatant may be the same amount of ethyl acetate and butane fraction and remaining aqueous solution layer.
  • the present invention provides a method for controlling plant diseases, comprising treating the m
  • the plant disease may be any one or more selected from the group consisting of tomato green blight, pepper blight, cabbage root gall disease and tomato root black layer.
  • the method is Ralstonia solanacearum? A / sic / a solanacearum), Ashidoborax avenae subspecies Carlia (// w ⁇ avenae subs.
  • Agrobacterium tumefaciens (4gTobacie / " / iOT tumefaciens), Berkholderia Glumae ( ⁇ / 1 ⁇ 2o / flfe / a glimae), Pectobacterium chrysantemi (fec otec er / i / zz? chrysanthemi), Pseudomonas syringe strain Actinide (/ 3 ⁇ 4e fc / w?
  • the method for controlling plant diseases according to the present invention comprises treating the microbial preparation according to the present invention by i) treating the soil, ii) treating the surface of the plant, iii) coating the seed, or iv) combining them. It can be carried out, through which it is possible to prevent the suppression or death of plant growth by plant diseases.
  • composition for controlling plant diseases comprising any one or more selected from the group consisting of 2- (2-ethoxyethoxy) -ethane.
  • Example 1 Isolation of TC1 Strains from the Carrier
  • the test strain was isolated from the carrier used during the tomato root nematode experiment.
  • the microbial carrier prepared by the inorganic material prepared by the Korea Research Institute of Chemical Technology was immersed in Tryptic Soy Broth (TSB), Difco, USA for 30 minutes, then dried and treated in tomato roots. .
  • TSA Tryptic soy agar
  • Example 2 Identification of strain 1C1 isolated in Example 1 Morphological and biochemical characteristics of the strains were analyzed, and genetic characteristics were analyzed through the gry A gene sequence.
  • Colony polymerase to obtain the nucleotide sequence of the gyr A gene of the genetic characteristic strain Colony PCR was performed. Collect colonies using toothpicks from a single colony and mix them with PCR-premix (PCR-premix: iNtRON Biotechnology, South Korea) and then p-gyr Af (5'— CAG TAC GGA AAT GCG TAC GTC CTT-3 Gyr A gene was amplified using primers SEQ ID NO: 1) and p-gyr Ar (5'-CAA GGT AAT GCT CCa GGC ATT GCT-3 '; SEQ ID NO: 2).
  • the amplified PCR product was purified using a Wizard PCR prep kit (Promega, Medison, WI, USA), and then subjected to macrogen (Daejeon, South Korea) to obtain a base sequence.
  • the base sequence was compared with GenBank database and base sequence using BLAST of NCBI.
  • the sequences were sorted by CLUSTAL Z, and a neighbor-joining tree was created using PHYDIT program version 3.0.
  • the strain was Lysinibacilli sp.
  • the novel strain showing a homology of about 98% with C3-41 (Fig. 1). From the above results, the strain obtained in the present invention was identified as Ricinibacillus spalicus strain, which was named as Ricinibacillus spalicus TC1 and deposited on October 10, 2012 to the Korea Institute of Bioscience and Biotechnology. Deposited as KCTC 12287BP.
  • Example 3 Confirmation of Nonvolatile Antimicrobial Active Substance Production Capacity of Ricinibacillus spalicus TC1 Strain
  • tomato foot Antimicrobial activity was measured in Ralstonia solanacearum / so; 7 / a solanacearum ⁇ .
  • the Ricinibacillus spalicus TC1 strain of the present invention is inoculated in tryptic soy liquid medium and 30 ° C. 12, 24, 36, 48 and 72 hours incubation in the incubator was used as an experimental group.
  • Example 4 Control Effect of the Lysinibacilli Sparicus TC1 Strain Culture on Tomato Foot Blight Inhibitory effect on the development of tomato foot blight on the culture of Cinibacilli sp. Liqueur TC1 strain was assayed by soil irrigation method.
  • the cultivated clay soil was filled and seeded in pots with a diameter of 4.5 cm using the cultivated tomato varieties, and then grown in a greenhouse of 25 ⁇ 5T. 3 weeks later, transplanted into a 10 cm diameter pot.
  • 20 ml of Ralstonia solanacerum suspension, a pathogen adjusted to 0.D value 0.5 (5X 10 8 CFU / ml) was irrigated and incubated in a 30 ° C constant temperature and humidity room to develop tomato foot blight. Induced.
  • the incidence of foot blight is 0 when the plant is very fresh, 1 when the leaf is slightly soaked (less than 25%), 2 when it is moderately soaked (less than 50%) and 3 when it is severely soaked (about 75%).
  • the death rate was rated as 4 and based on this, the incidence inhibition rate was calculated as ⁇ (control-to-control-to-treatment rate) / control-to-control rate ⁇ x 100. The results are shown in FIG.
  • Example 6 Control Effect of Ricinibacillus spalicus TC1 Strain Culture on Chinese Cabbage Root Black Disease Plasmodiophora Brassica of Cabbage Root Disease of Ricinibacillus spalicus TC1 Strain Culture Solution (/ Vas / TOi / Inhibitory effect on cipAara brass icae) was tested by soil irrigation method.
  • bokbok crossover cabbage was sown in the horticultural soil.
  • Chinese cabbage root black (10 g / field soil, finely ground with a blender) was inoculated into the field soil after incubation in a greenhouse for 2 weeks, and then mixed with well mixed soil.
  • 10 ml of the ricinibacillus spalicus TC1 strain culture of the invention was irrigated, and the experiment was repeated five times, and after 3 weeks of inoculation, the plants were removed to remove the soil from the roots and the incidence was examined.
  • the control no treatment
  • the incidence of cabbage root gall disease is 0: healthy, 1: root gall formed in the roots 2: root gall formed in the roots, 3: root gall formed in the roots and roots as a whole, 4: The root nodules were largely formed in the roots and main roots, and the control value was calculated according to the formula (No treatment incidence-treatment incidence) / no treatment incidence X 100.
  • the results are shown in Table 1 and FIG. 4. Table 1 Experimental results showed that the oily cultures were the most effective, but they significantly inhibited the growth of Chinese cabbage (Table 1).
  • Example 8 Determination of the antimicrobial activity of the volatiles produced by the Ricinibacillus sp. Liqueur TC1 strain In order to examine whether the volatiles produced by the strain of Ricinibacillus sp. Liqueur TC1 show antimicrobial activity against phytopathogens. Ten representative Gram-negative and Gram-positive bacteria were examined by using a medium prepared by completely removing the central part with a width of 1 cm of the Petri dish medium so that one substance or strain did not go to the other side.
  • tryptic soy agar medium was dispensed into Petri dishes, and then 1 cm of medium was completely removed from the center of the medium to prevent growth of pathogenic bacteria by diffusion.
  • Ralstonia EI three of3 ⁇ 4 (Ralstonia solanacearum), subspecies of Ashidovorax avenae avenae subsp. cattlyae), Agrobacterium tu efaciens (Burkholderia glumae), Pectobacterium chrysanthemi) Pseudomonas syringe strain Actinade O ⁇ ei / ato / Boyias syringae v. actinidiae), Pseudomonas syringe strain Lakrimmans (3 ⁇ 4ei ⁇ ra (was syringae v.
  • the TC1 strain of the present invention is pectobacterium It was confirmed that a volatile substance showing antimicrobial activity was produced against all pathogenic bacteria except Carotobora subspecies Carotobora.
  • Example 9 Determination of Antifungal Activity of Volatile Substances Produced from Ricinibacillus sparycus TC1 Strains Culture Effect of Volatile Substances Produced by Ricinivacillus sparycus TC1 Strains on the Mycelial Growth of Plant Disease Bears In order to examine, it was confirmed by a mycofumigation plate assay (Strobel et al., 2001, Microbiology, 147: 2943-2950) for representative plant diseases.
  • the agar medium containing the cells was inoculated using a cork bore with a diameter of 8 mm from the edge of the strain growing in PDA medium.
  • the plate was sealed with a para film and then cultured at 28 ° C. for 3-5 days, and the degree of mycelial growth of the plant disease bear was measured. The results are shown in Table 3 below.
  • the mycelial growth inhibition rate was calculated according to the formula of (mycelial diameter of untreated pathogen-mycelial diameter of treated pathogen) / mycelial diameter of untreated pathogen X 100.
  • Table 3 As shown in Table 3, Fujium oxysporum f. S p. The mycelial growth of all pathogenic bears except Raphani ()) was inhibited by the volatiles produced by the Lysinibacilli sparycus TC1 strain.
  • Example 10 Component Analysis of Volatile Substances Produced by Ricinivacillus spalicus TC1 Strains In order to analyze the components of volatiles produced by the Ricinibacillus sparycus TC1 strain of the present invention, DeMilo et al. (1996) was modified to capture volatiles.
  • GC / MS Component analysis of the obtained volatiles was measured as follows using GC / MS.
  • the GC / MS instrument was a GC / MS instrument equipped with an HP 7890A mass spectrometer (Mass selective deterctor, Agilent Technologies Inc., Chandler, AZ, USA).
  • the separation column was DB-WAX (30 mX 0.25 mm, Id, 0.25 mm film thickness, J & W, Folsom, CA, USA).
  • the temperature of Aubon was maintained at 30 ° C. for 5 minutes and then increased to 250 ° C. at a rate of 10 ° C./min, and the flow rate of carrier gas was maintained at 1 ml / min (He).
  • the silverization of the compounds was carried out by electron impact ionization (EI-mode) method, and the ionization voltage and ion source temperature were set at 70 eV and 230 ° C, respectively.
  • the molecular weight range was set to 40-350 (m / z) and the temperature of the GC injector was set to 250 ° C.
  • Volatile component analysis of each peak separated in the TIC includes the mass spectrum library (Wieley 275 & N, NBS 75K), the spectra of the mass spectral data book, and the retention index in the literature. Based on the results, the material identification results according to the retention time and area ratio are shown in Table 4 below.
  • the volatiles produced from the Ricinibacillus sparycus TC1 strain of the present invention are tetrachloroethylene, 3-methyl-1-butanol, methinepyrazine, cyclonuxanone, 2,5-dimethylpyrazine, It was found that ⁇ , ⁇ -dimethylformamide and 2- (2-especially) -ethanol.
  • Example 11 Preparation of a Mixed Liquid Culture of Bacillus Velensis G341 Strains and Ricinibacillus Sparicus TC1 Strains 3 ⁇ 4 To prepare liquid cultures of two strains, Bacillus Velensis G341 strains and lis stored at ⁇ 80 ° C.
  • Cinibacillus spalicus TC1 strain was inoculated in tryptic agar medium (TSA) and then incubated at 30 ° C for 1 day.
  • Single colony from each strain was inoculated in tryptic soy liquid medium (TSB) and cultured for 24 hours at 150 rpm at 30 ° C as a source of inoculum using 200 ml of TSB in a 500 ml Erlenmeyer flask Was inoculated simultaneously at the final 1%.
  • TSA tryptic agar medium
  • TAB tryptic soy liquid medium
  • G341 and TC1 strains were halved three times each before treatment of the pathogen, and each strain alone (G341 and TC1 groups), and G341 and TC1 strains were 2: 1.
  • the incidence of foot blight was measured in the same manner as described in Example 4, except that the mixed cultures were mixed in different amounts of 1: 1 and 1: 2 in the soil by 20 ml per pot, respectively. The results are shown in FIG. W
  • the Bacillus velensis G341 strain culture (1 / 2-fold dilution) alone treatment group showed a 47% control effect
  • the treatment group showed 80% control effect.
  • the mixed group of two strains (1 / 2-fold dilution) at the ratios of 2: 1, 1: 1 and 1: 2 showed a high control effect of more than 80%, especially Bacillus Velensis
  • the mixture of the G341 strain and the Lysinibacillus sp. Liqueur TC1 strain was treated in a 1: 1 ratio, it was found that the highest control, that is, 100% control effect was obtained.
  • Example 13 Effect of controlling pepper late blight of mixed culture medium The inhibitory effect of pepper late blight onset by phytophthora capsaici (/ 3 ⁇ 4 ⁇ ⁇ ⁇ 3) was assayed using the mixed culture medium prepared in Example 11 by soil irrigation method.
  • the control (no treatment) was performed in the same manner as above by treating only distilled water instead of the culture, and repeated 10 experiments for each treatment.
  • the incidence of pepper blight is 0 when the plant is very fresh, 1 when the leaf is slightly soaked (less than 253 ⁇ 4 »), 2 when it is moderately soaked (less than 50%) and 3 when it is severely soaked (about 75%).
  • the death rate was rated as 4, and on this basis, the inhibition rate was calculated as ⁇ (onset control-onset of treatment) / onset of control ⁇ x 100.
  • Example 14 Effect of Controlling Tomato Foot Blight on Mixed Culture Spray Spray Drying
  • 10 g of white carbon and 10 g of dextrin were added to 1 L of each culture of the two strains, followed by inlet. Spray dried samples were prepared by spray drying at a temperature of 165 ° C. and an outlet temperature of 94 ° C.
  • the dilution solution 50 times diluted with the spray dried sample thus prepared was examined in the same manner as described in Example 12 to investigate the control effect by the single treatment and the mixed treatment on tomato foot blight, and the results are shown in FIG. 8.
  • the single culture medium of the Lysinibacillus spalicus TC1 strain was also excellent in the control effect, but the mixture with the Caribbeansis G341 strain culture medium showed a stronger control effect.
  • Example 15 Antibacterial Activity Effect of the Mixed Culture Solution In order to confirm the production of the antibacterial active material in the single culture and the mixed culture of the two strains, the in vitro antibacterial activity was measured by Ralstonia. Investigations were carried out using Solanaserum strains.
  • the bacterium B. regensis G341 strain up to about 1.05 cm
  • the mixed cultures (G & T) of the two strains showed strong antibacterial activity.
  • the stronger antibacterial activity in the mixed cultures of the two strains Up to about 1.55 cm).
  • Example 16 Control effect of tomato foot blight on mixed culture medium, cells and supernatant The control effect of the mixed culture of two strains cultured in the same manner as described in Example 15 on the tomato foot blight was investigated.
  • the experiment was carried out by comparing the mixed culture medium, the cells from which the mixed culture medium was removed, and the supernatant from which the cells were removed.
  • the sample treatment method for confirming the control effect was performed in the same manner as described in Example 12.
  • Example 17 Treatment or Preventive Effect of Mixed Culture Spray Spray Drying Sample on Tomato Foot Blight Control Sprayed according to the method described in Example 14 above using a mixed culture of two strains prepared in the same manner as described in Example 15 above. A dry sample was prepared and used to investigate the control effect on tomato green blight.
  • Spray dried samples were diluted 100- or 50-fold with distilled water and then irrigated 7 days, 3 days, 1 day and 1 day after inoculation of tomato fusiform bacillus (Ralstonia solanacerum). The incidence was examined 10 days after inoculation of the pathogen, and the results were shown in FIG. 11 by repeating the experiment twice with 5 repetitions per treatment.
  • Example 18 Tomato Foot Blight Control Effect of Ethyl Acetate Extract from Bacillus Velensis G341 Strain Culture As confirmed in Example 15 above, Ricinibacillus spalicus TC1 strain did not produce nonvolatile antibacterial activity , Bacillus Velensis G341 strain produces a nonvolatile antibacterial active material.
  • the culture supernatant of the Bacillus Velensis G341 strain was extracted twice using the same amount of ethyl acetate.
  • the ethyl acetate layer obtained above was concentrated under reduced pressure, dissolved in methanol, diluted in 20 solution (250 ppm) of 3, and 33.3 yg / ml, 1000 yg / ml and 2000 yg / ml three days before inoculation
  • the tomatoes were irrigated with sheep. The incidence was examined 10 days after inoculation of the pathogen and repeated twice with 5 replicates per treatment.
  • Example 19 Isolation and Identification of Nonvolatile Antibacterial Active Ingredients from Bacillus Velensis G341 Strain
  • the strain was 200 ml of tryptic soy. After inoculating liquid medium,
  • Shake culture was carried out at 150 rpm for 3 days at 30 ° C.
  • the culture solution obtained above was 8,000 After centrifugation at g for 10 minutes to obtain the culture supernatant (3 L), it was fractionated twice in sequence with the same amount of ethyl acetate.
  • the ethyl acetate layer was concentrated under reduced pressure to obtain 550 mg of a fraction. Using this was added to the reverse phase sep-pak eluted by adjusting the concentration to 100% methane in 100% water.
  • Fractions showing antimicrobial activity against Ralstonia solanacerum were collected and concentrated under reduced pressure to obtain 240 mg of eluate.
  • the fraction obtained through the Sephadex column was dissolved in methanol at the level of 10 mg / ml, then dipped in a 30 ⁇ 1 drop on a paper disk, and dried, and then killed in a tryptic soy agar medium inoculated with Ralstonia solanacerum.
  • the diameter of the part showing antimicrobial activity was measured while incubating at 30 ° C. In the untreated section, only 30 ⁇ l of methanol was treated.
  • UV-Vis spectra and EI / MS were used. After dissolving Compound 1 in methanol, absorption spectra were measured to show maximum absorbance at 233 nmCband I) and 270 nm (band II) (FIG. 13). The molecular weight of Compound 1 was measured using EI / MS, and the molecular ion peak of z 559 [ ⁇ - ⁇ was observed as a result of mass spectrometry to determine the molecular weight of 560. In addition, NMR results of compound 1 was identified as oxydipicidin by confirming that the results are consistent with the results of oxydifficidin, a material produced from Bacillus subtilis Siyb / 7 / s).
  • Example 20 Antimicrobial Activity of Compounds Isolated from Bacillus Velensis G341 Strain Culture Medium To determine whether the antimicrobial material isolated in Example 19 exhibited antimicrobial activity against plant disease bacteria, representative Gram-negative bacteria and Gram-positive bacteria were Antibacterial activity was examined using 96-well plates.
  • a single colony of each plant disease bacterium as described in Table 5 below was inoculated in tryptic soy liquid medium, and then prepared by shaking culture for 1 to 2 days at 30 ° C and 150 rpm.
  • Prepared plant disease bacteria were adjusted to IX 10 8 CFU / ml to prepare a suspension and diluted 1,000-fold with sterile TSB medium.
  • the two compounds (oxydipicidine and dipidine) isolated in Example 19 were dissolved in a mixture of methane and water, and the final concentration was 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 yg / ml.
  • tryptic soy agar medium is dispensed into Petri dishes, and then 1 cm of medium is completely removed from the center of the medium to spread the pathogen. Growth was not inhibited.
  • mice were dispensed potato agar medium (Potato Dextrose Agar, Bee ton and Dickinson Co., Farnklins Lakes, NJ, USA), and the other side In cotton, the strain dispensed tryptic agar medium.
  • Phytopathogenic fungi used in the experiments were Lyactonia solani (RS), Magna Forte duck (M0), Botrytis cinerea (BC), Colletotricum cocodes (CC), Fuzarium oxysporum f . sp.
  • Raphani F0
  • Sclerotinia sclerothiorum SS
  • phytoptotora capsaish PC
  • phytophthora infestans PI
  • the mycelial growth inhibition rate was calculated according to the formula of (mycelial diameter of untreated pathogen-mycelial diameter of treated pathogen) / mycelial diameter of untreated pathogen X 100.
  • MO Magna Forte ⁇ ⁇ (iagnapor ie oryzae),
  • PC phytophthora capcici (/ 3 ⁇ 4 (3 ⁇ 4 ⁇ 3 capsici),
  • a single colony of Bacillus Velensis G341 strain After inoculating Trypt ic Soy Broth (TSB) and shaking culture at 150 rpm at 30 ° C for 1 day, 100 ml of TSB medium was added to a 500 ml Erlenmeyer flask and 1% of the strain was inoculated. .
  • TSB Trypt ic Soy Broth
  • one line was set at a height of 1 cm from the culture medium and connected to a glass tube 1 cm in diameter and 15 cm in height to collect volatiles.
  • the line was set to a height of 10 cm from the Erlenmeyer flask culture and set to allow air to be injected from the outside.
  • the glass tube was filled with 150 mg of charcoal to collect volatile substances. After incubation at 30 ° C. and 150 rpm for 3 days, the charcoal was harvested, dissolved in methanol, and filtered to obtain only pure volatiles.
  • GC / MS Component analysis of the obtained volatiles was measured as follows using GC / MS.
  • the GC / MS instrument was a GC / MS instrument equipped with an HP 7890A mass spectrometer (Mass selective deterctor, Agilent Technologies Inc., Chandler, AZ, USA).
  • the column for separation was DB—WAX (30 mX 0.25 mm, Id, 0.25 mm film thickness, Folsom, CA, USA).
  • the temperature of the oven was maintained at 30 ° C for 5 minutes and then raised to 250 ° C at a rate of 10 ° C / min, and the carrier gas flow rate was maintained at 1 ml / min (He).
  • Ionization of the prepared compounds was carried out by electron impact ionization (EI-mode) method, and the ionization voltage and ion source temperature were set at 70 eV and 230 ° C, respectively.
  • the molecular weight range was set to 40-350 (m / z) and the temperature of the GC injector was set to 250 ° C.
  • Volatile component analysis of each peak separated in a total ionization chromatogram (TIC) by GC / MS includes a mass spectrum library (Wieley 275 & N, NBS 75K) and a mass spectral data book. ), And the retention index in the literature, and the results of material identification according to retention time and area ratio are shown in Table 8 below.
  • Table 8 As shown in Table 8, it can be seen that the volatiles produced from the Bacillus bellensis G341 strain are dimethyldisulfide, 1-butanol and 3-hydroxy-2-butanone.

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Abstract

The present invention relates to: a novel Lysinibacillus sphaericus TC1 strain exhibiting an activity in suppressing the growth of various pathogenic bacteria and fungi; a microbial preparation for controlling plant diseases comprising same; and a plant disease control method using the microbial preparation. The microbial preparation according to the present invention is environmentally friendly and can exhibit an outstanding plant disease controlling effect even in a small amount and can enhance the commercial potential and productivity of crops, and prevents problems caused by persistence of agrichemicals, and is useful in agricultural ecosystem conservation and integrated disease control.

Description

리시니바실러스 스패리쿠스 TC1 균주， 상기 균주를 포함하는 식물병 방제용미생물 제제 및 이를 이용한식물병 방제 방법 발명의 분야  Lysinibacillus spalicus TC1 strain, a microbial agent for controlling plant diseases comprising the strain and a plant disease control method using the same
|，  | ，
본 발명은 다양한 병원성 세균 및 진균의 생장의 억제 활성을 나타내는 신규한 리시니바실러스 스패리쿠스 ys//2/bac///is sphaericus) TCI 균주; 이를 포함하는 식물병 방제용 미생물 제제; 및 상기 미생물 제제를 이용한 식물병 방제방법에 관한 것이다. 배경기술 그람양성 세균인 바실러스 속 세균은 슈도모나스 0 euio¾ as)속 다음으로 널리 연구되고 상용화된 세균으로서, 토양 내에 다수 존재하고 생물학적 활성이 있는 다양한 2차 대사산물을 생산하며., 열에 안정하고 열악한 환경에서 저항성을 나타내는 내생포자 (endospore)를 형성하는 특징을 갖는다.  The present invention provides a novel Ricinibacillus spalicus ys // 2 / bac /// is sphaericus) TCI strain that exhibits inhibitory activity in the growth of various pathogenic bacteria and fungi; Microbial preparations for controlling plant diseases comprising the same; And to a plant disease control method using the microbial agent. The bacterium of the genus Bacillus, a Gram-positive bacterium, is the second most widely researched and commercialized bacterium after Pseudomonas genus 0 euio¾ as), which produces a variety of secondary metabolites that exist in the soil and are biologically active. Has the characteristic of forming endospores that exhibit resistance in
바실러스 속 미생물 중 바실러스 벨레젠시스 균주는 1994년에 구스타프손 (Gustafson)사에서 코디악 (Kodiak)이라는 상품명으로 목화와 땅콩의 종자 및 발고탕 처리제로서 개발된 적 있으며 (Backman 등, Improving Plant Productivity with Rhizosphere Bacteria, pp. 3-8 (1994)), 2001년에는 바실러스 벨레젠시스균과 바실러스 아밀로리퀴파시엔스 {Bacillus amyIoIiquefac]iens)^ : 흔합하여 제조한 바이오일드 (BioYield)라는 미생물 살균제가 출시된 바 있다. 또한， 증국에서는 바실러스 속의 많은 균들을 수확량 증진을 위한 세균이라 하여 널리 사용하고 있으며， 독일 바이엘 (Bayer)사 및 미국 탠사 (Taensa)사에서는 각각 바실러스 FZB 24균을 이용한 토양전염병 방제제 및 미생물 살균제를 개발한 바 있다. Among the Bacillus microorganisms, the Bacillus Velensis strain was developed by Gustafson in 1994 under the trade name Kodiak as a seed and foot sugar treatment agent for cotton and peanuts (Backman et al., Improving Plant Productivity with Rhizosphere). Bacteria, pp. 3-8 (1994)), and in 2001, Bacillus Velensis and Bacillus amyloliquifaciens (Bacillus amyIoIiquefac) iens): a microbial disinfectant called BioYield was introduced. There is a bar. In addition, many Bacillus spp. Bacteria are widely used in South Korea as a bacterium for improving yield, while Bayer Germany and Taensa Co., Ltd. use Bacillus FZB 24 bacteria to control soil infectious diseases and microbial fungicides, respectively. It was developed.
국내에서는 지금까지 바실러스 속 미생물을 이용한 미생물 살균제로서 '테라스'， '홀인원', '그린올'， '씰러스'， '슈팅스타' 및 '재노탄' 등의 제품이 등록되어 있는데， 이들의 경우 제품에 따라 흰가루병과 잿빛곰광이병， 잔디 갈색잎마름병 또는 잔디 피시움마름병 등에 효과가 있다. 그러나， 난방제 식물병인 토마토 풋마름병， 토마토 뿌리흑선층병， 배추 뿌리혹병， 고추 역병에 대해서는 거의 효과가 없어 사용에 많은 제한이 따른다.  In Korea, products such as 'terrace', 'hole in one', 'greenol', 'sealus', 'shooting star' and 'jaetan' are registered as microbial disinfectants using microorganisms of Bacillus. Depending on the product, it is effective in powdery mildew, gray bear disease, grass brown leaf blight or grass pistil blight. However, it is almost ineffective against tomato plant blight, tomato root blight disease, cabbage root gall disease, and pepper blight, which is a heating plant disease.
한편, 대한민국 특허 제 1163986호는 인삼 뿌리에서 분리한 식물내생미생물인 바실러스 벨레젠시스 0¾c///"s velezensis) G341 균주 (KCTC 11497BP)가토마토 풋마름병， 고추 역병， 배추 뿌리혹병 및 토마토 뿌리혹선충 등 다양한 식물병에 방제효과를 나타냄을 개시하고 있다. 본 발명자들은 다양한 식물병에 방제효과를 나타내는 미생물 제제를 개발하기 위하여 예의 연구한 결과， 리시니바실러스 스패리쿠스 TC1 균주가 토마토 풋마름병， 고추 역병 및 배추 뿌리혹병 등의 식물병에 대하여 매우 우수한 방제효과를 나타낼 뿐만 아니라， 상기 균주가 생산하는 휘발성 물질이 다양한 병원성 세균 및 곰광이균의 생장을 억제한다는 사실을 발견하였다. 또한, 기존의 바실러스 벨레젠시스 G341 균주와 리시니바실러스 스패리쿠스 TC1 균주의 흔합 배양액을 이용하여 미생물 제제를 제조할 경우 다양한 식물병에 대한 방제효과가 증가하고， 바실러스 벨레젠시스 G341 균주를 리시니바실러스 스패리쿠스 TC1 균주와 함께 배양할 때 항세균 활성물질을 더 많이 생산한다는 사실을 발견하였다. 발명의 요약 본 발명의 목적은， 다양한 식물병에 대하여 방제 효과가 우수한 신규 리시니바실러스 스패리쿠스 균주를 제공하는 것이다.  On the other hand, Korean Patent No. 1163986 discloses a plant endogenous microorganism, Bacillus Velensis 0¾c /// "s velezensis) G341 strain (KCTC 11497BP) isolated from ginseng roots. The present inventors have diligently researched to develop microbial agents exhibiting control effects on various plant diseases, etc. As a result of Ricinibacillus spalicus TC1 strains, tomato foot blight, pepper In addition to showing a very good control effect against plant diseases such as late blight and Chinese cabbage root gall disease, it was found that the volatile substances produced by the strain inhibit the growth of various pathogenic bacteria and fungi. Using a mixed culture of Jensis G341 strain and Ricinibacillus sparycus TC1 strain Biologic preparations have been shown to increase control effects against various plant diseases and produce more antibacterial actives when incubated with the bacillus B. genus G341 strain with Lysinib. Sp. Summary of the Invention It is an object of the present invention to provide a novel Ricinibacillus spalicus strain with excellent control against various plant diseases.
본 발명의 다른 목적은 상기 신규 균주를 포함하는 식물병 방제용 미생물 제제를 제공하는 것이다. Another object of the present invention for controlling plant diseases comprising the novel strain It is to provide a microbial agent.
본 발명의 또 다른 목적은 상기 미생물 제제를 이용한 식물병 방제방법을 제공하는 것이다. 상기 목적을 달성하기 위하여， 본 발명은 항균 활성을 나타내는 휘발성 물질을 생산하는 리시니바실러스 스패리쿠스 TC1 균주 (KCTC 12287BP)를 제공한다.  Still another object of the present invention is to provide a plant disease control method using the microbial agent. In order to achieve the above object, the present invention provides a Ricinibacillus spalicus TC1 strain (KCTC 12287BP) to produce a volatile substance exhibiting antimicrobial activity.
상기 다른 목적을 달성하기 위하여， 본 발명은 리시니바실러스 스패리쿠스 TC1균주, 상기 균주의 포자, 상기 균주의 배양액 및 상기 배양액의 추출물로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 함유하는 식물병 방제용 미생물 제제를 제공한다.  In order to achieve the above another object, the present invention is a plant containing any one or more selected from the group consisting of Lysinibacillus spalicus TC1 strain, spores of the strain, culture of the strain and extract of the culture as an active ingredient It provides a microbial preparation for disease control.
상기 또 다른 목적을 달성하기 위하여， 본 발명은 상기 미생물 제제를 식물 또는 주변환경에 처리하는 것을 포함하는 식물병의 방제방법을 제공한다. 또한， 본 발명은 테트라클로로에틸렌， 3—메틸 -1-부탄을， 메틸피라진, 사이클로핵사논, 2，5-디메틸피라진， Ν,Ν-디메틸포름아미드， 및 2-(2-에특시에톡시) -에탄을로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는, 식물병 방제용 조성물을 제공한다. 도면의 간단한설명 본 발명의 상기 및 다른 목적과 특징들은 첨부된 도면과 함께 하기 본 발명의 설명으로부터 명확해질 것이다: 도 1은 리시니바실러스 스패리쿠스 TC1 균주의 gyrase A 유전자 염기서열에 의한 계통수 (phytogenetic tree)를 나타낸 것이다.  In order to achieve the above another object, the present invention provides a method for controlling a plant disease comprising treating the microbial agent to a plant or the surrounding environment. The present invention also relates to tetrachloroethylene, 3-methyl-1-butane, methylpyrazine, cyclonucleanone, 2,5-dimethylpyrazine, Ν, Ν-dimethylformamide, and 2- (2-ethoxyethoxy Provides a composition for controlling plant diseases, comprising any one or more selected from the group consisting of ethane. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects and features of the present invention will become apparent from the following description of the invention, taken in conjunction with the accompanying drawings, in which: FIG. 1 is a phylogenetic tree derived from the gyrase A gene sequence of a strain of Ricinibacillus sp. phytogenetic tree).
도 2 내지 4는 각각 리시니바실러스 스패리쿠스 TC1 균주 배양액의 토마토 풋마름병, 고추 역병 및 배추 뿌리혹병에 대한 방제효과를 나타낸 것이다. 도 5는 리시니바실러스 스패리쿠스 TC1 균주 배양액으로 제조한 분무 건조 시료의 토마토 풋마름병에 대한 방제효과를 나타낸 것이다. Figures 2 to 4 show the control effect against tomato foot blight, pepper blight, and Chinese cabbage root gall disease in the culture of Ricinibacillus spalicus TC1 strain, respectively. Figure 5 shows the control effect on the tomato foot blight of spray-dried samples prepared with Ricinibacillus spalicus TC1 strain culture medium.
도 6 및 7은 각각 바실러스 벨레젠시스 G341 균주 및 리시니바실러스 스패리쿠스 TC1 균주의 단독 배양액 및 흔합 배양액 처리에 따른 토마토 풋마름병 및 고추 역병에 대한 방제효과를 나타낸 것이다.  6 and 7 show the control effect against tomato foot blight and pepper blight following treatment with a single culture and a mixed culture of the Bacillus velensis G341 strain and Ricinibacillus spalicus TC1 strain, respectively.
도 8은 바실러스 벨레젠시스 G341 균주 및 리시니바실러스 스패리쿠스 TC1 균주의 흔합 배양액 분무건조물의 토마토 풋마름병에 대한 방제효과를 나타낸 것이다.  Figure 8 shows the control effect on the tomato foot blight of the mixed culture spray spray of the Bacillus Velensis G341 strain and Ricinibacillus spalicus TC1 strain.
도 9는 바실러스 벨레젠시스 G341 균주 및 리시니바실러스 스패리쿠스 TC1 균주의 단독 배양액 및 흔합 배양액 처리에 따른 랄스토니아 솔라나세럼에 대한 항세균활성을 나타낸 것이다.  FIG. 9 shows antibacterial activity against Ralstonia solana serum following treatment with a single culture and a mixed culture of Bacillus velogenis G341 strain and Ricinibacillus spalicus TC1 strain.
도 10은 바실러스 벨레젠시스 G341 균주 및 리시니바실러스 스패리쿠스 TC1균주의 흔합 배양액， 균체 및 상층액의 토마토 풋마름병에 대한 방제효과를 나타낸 것이다.  Figure 10 shows the control effect of the mixed culture solution, bacteria and supernatant of tomato Bacillus blight strains of Bacillus Velensis G341 strain and Lysinibacilli sp.
도 11은 바실러스 벨레젠시스 G341 균주 및 리시니바실러스 스패리쿠스 FIG. 11 shows Bacillus bellensis G341 strain and Ricinibacillus spalicus
TC1 균주의 흔합 배양액 분무건조 시료의 토마토 풋마름병에 대한 예방 및 방제효과를 나타낸 것이다. It shows the prevention and control effect of the mixed culture liquid spray-dried samples of TC1 strain against tomato foot blight.
도 12는 바실러스 벨레젠시스 G341 균주 배양액의 에틸아세테이트 추출물의 처리 농도에 따른 토마토 풋마름병에 대한 방제효과를 나타낸 것이다.  Figure 12 shows the control effect against tomato foot blight according to the treatment concentration of the ethyl acetate extract of Bacillus Velensis G341 strain culture.
도 13은 바실러스 벨레젠시스 G341 균주 배양액으로부터 분리한 항균활성 물질인 화합물 1 및 2의 UV 스펙트럼이다.  FIG. 13 is a UV spectrum of Compounds 1 and 2, which are antimicrobial active substances isolated from Bacillus velogenis G341 strain culture. FIG.
도 14는 바실러스 밸레젠시스 G341 균주 배양액으로부터 분리한 항균활성 물질의 구조로서， R이 H인 경우 디피시딘을 나타내고 R이 0H인 경우 옥시디피시딘을 나타낸다.  FIG. 14 shows the structure of an antimicrobial active material isolated from Bacillus balensis G341 strain culture medium, where R is H and dipicidine, and when R is 0H. FIG.
발명의 상세한설명 이하， 본 발명을 상세히 설명한다. Detailed description of the invention Hereinafter, the present invention will be described in detail.
• 본 발명은 항균 활성을 나타내는 휘발성 물질을 생산하는 리시니바실러스 스패리쿠스 TC1 균주 (KCTC 12287BP)를 제공한다. 본 발명의 균주는 하기와 같은 형태학적 생화학적 및 유전학적 특징을 갖는 것으로 나타났다:  The present invention provides a Ricinibacillus spalicus TC1 strain (KCTC 12287BP) that produces volatiles exhibiting antimicrobial activity. Strains of the invention have been shown to have the following morphological biochemical and genetic characteristics:
1)형태학적 및 생리학적 특성: 그람 양성균이며， 중온성 균주이며， 간상 모양의 균주이다. 또한 내생포자 (endospore)를 형성하며， 전형적인 조건적 (facultative) 혐기성균주이다.  1) Morphological and physiological characteristics: Gram-positive bacteria, mesophilic strains, rod-shaped strains. It also forms endospores and is a typical facultative anaerobic strain.
2) 유전학적 특성: gyr A 유전자의 염기서열 (서열번호: 3) 분석 결과, 리시니바실러스 스패리쿠스 C3-41 균주와 98%정도의 상동성을 나타낸다.  2) Genetic characteristics: The nucleotide sequence of the gyr A gene (SEQ ID NO: 3) shows 98% homology with Ricinibacillus sp. Liqueur C3-41.
상기 특성들로부터, 본 발명의 균주는 리시니바실러스 스패리쿠스 ( KS/ / bac///ws sp?aer/cus)에 속하는 신규 균주로 확인되었는 바, 이를 리시니바실러스 스패리쿠스 TC1 균주로 명명하고 2012년 10월 10일자로 한국생명공학연구원 유전자은행에 기탁번호 제 KCTC 12287BP호로서 기탁하였다. 본 발명에 따른 리시니바실러스 스패리쿠스 TC1균주는 토마토 풋마름병 고추 역병 및 배추 뿌리혹병 둥의 다양한 식물병에 대하여 우수한 방제 효과를 나타내는 것을 특징으로 한다 (실시예 4내지 6).  From the above characteristics, the strain of the present invention was identified as a novel strain belonging to Ricinibacillus sp. Liqueur (KS // bac /// ws sp? Aer / cus). On October 10, 2012, it was deposited with the Korea Institute of Biotechnology and Gene Bank as Accession No. KCTC 12287BP. Ricinibacillus spalicus TC1 strain according to the present invention is characterized by showing excellent control against various plant diseases of tomato foot blight pepper blight and cabbage root gall disease (Examples 4 to 6).
또한, 본 발명의 리시니바실러스 스패리쿠스 TC1 균주는 항균 활성을 나타내는 휘발성 물질을 생산해 낼 수 있는데， 이러한 물질로는 테트라클로로에틸렌 (tetrachloroethylene),  In addition, the ricinibacillus spalicus TC1 strain of the present invention can produce a volatile substance exhibiting antimicrobial activity, such as tetrachloroethylene (tetrachloroethylene),
3-메틸 - 1-부탄올 ( 3-met hy 1-1-but anol), 메틸피라진 (me t hy 1 pyr az i ne )， 사이클로핵사논 (cyclohexanone), 2,5—디메틸피라진 (2，5— dimethylpyrazine)， Ν,Ν-디메틸포름아미드 (N,N-dimethylformamide) 및 2-(2-에톡시에톡시) -에탄올 (2-(2— ethoxyethoxy)-ethanol)이 포함된다 (실시예 10). 이에 본 발명의 균주의 배양액 또는 배양액의 추출물은 상기 휘발성 물질을 포함한다. 따라서 , 본 발명은 리시니바실러스 스패리쿠스 TC1균주 (KCTC 12287BP) ( 상기 균주의 포자， 상기 균주의 배양액 및 상기 배양액의 추출물로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 함유하는 식물병 방제용 미생물 제제를 제공한다. 3-methyl- 1-butanol (3-met hy 1-1-but anol), methylpyrazine (me t hy 1 pyr az i ne), cyclohexanone, 2,5—dimethylpyrazine (2，5 — Dimethylpyrazine), Ν, Ν-dimethylformamide and 2- (2-ethoxyethoxy) -ethanol (2- (2—ethoxyethoxy) -ethanol) (Example 10) . Thus, the culture medium or extract of the culture medium of the present invention contains the volatile substance. Therefore, the present invention is a plant disease control containing any one or more selected from the group consisting of Lysinibacillus spalicus TC1 strain (KCTC 12287BP) ( spores of the strain, culture of the strain and extract of the culture) as an active ingredient It provides a microbial preparation for.
또한， 본 발명에 따른 리시니바실러스 스패리쿠스 TC1 균주를 포함하는 미생물 제제는 랄스토니아 솔라나세아럼 (^ / 072/3 solanacearum) , 아시도보락스 아베내 아종 τΊΊ-^Ι °fl (Acidovorax avenae subsp . cattlyae) , 아그로박테리움 투메파시엔스 (Agwbacienu/n tiunefaciens)， 버크홀더리아 글^^ BurkhoMeria glumae) , 펙토박테리움 크리산테미 0 C O)aCier/½; chrysanthemi) , 슈도모나스 시린개 변종 액티니데 (/¾ei/G¾Z70/7as syringae v. actinidiae) , 슈도모나스 시린개 변종 라크리만스 (/^se fcffOTas syringae pv. lachry ans) , 산토모나스 악소노포디스 Λί¾»ω235 axonopodis) 및 클라비박터 미시가넨시스 아종 口 1시 7]·넨시스 ( 7aw'bac er michiganensis subs . mi ch iganens is)^- 같은 병원성 세균; 및 리족토니아 솔라니 Rhizoctonia solan/) ,마그나포르테 오 ^^ iagnaporthe oryzae) , 보트리티스 씨네레아 (5o /y /s cinerea) , 콜레토트리쿰 코코^ ^{Collet otri chum coccodes) , 스클레로티니아 스클레로티오룸 (5c/ ro /72/a sclerotioru ) , 파이토프토라 캡사이시 (/¾κ σ/¾¾ Λο/·3 capsici) , 파이토프토라 인혜^한스 {Phytophthora infestans) 및 플라즈모디오포라 브래시캐 (/ as/70i/pAora brass/cae)와 같은 병원성 곰광이균 (진균)의 생장을 억제할 수 있다 (실시예 8 및 9). 또한， 본 발명에 따른 미생물 제제는， 바실러스 벨레젠시스 G341 균주 (KCTC 11497BP), 상기 균주의 포자， 상기 균주의 배양액 및 상기 배양액의 추출물로 이루어지는 군으로부터 선택되는 어느 하나 이상을 추가로 함유하는 것을 특징으로 한다. 상기 미생물 제제는， 바실러스 벨레젠시스 G341 균주 (KCTC 11497BP) , 상기 균주의 포자, 상기 균주의 배양액 및 상기 배양액의 추출물로 이루어지는 군으로부터 선택되는 어느 하나 이상; 및 리시니바실러스 스패리쿠스 TC1 균주 (KCTC 12287BP) , 상기 균주의 포자， 상기 균주의 배양액 및 상기 배양액의 추출물로 이루어지는 군으로부터 선택되는 어느 하나 이상을 1:9 내지 9:1의 중량비， 바람직하게는 1:1의 중량비로 포함할 수 있다. In addition, the microbial preparations comprising the strain of Ricinibacillus spalicus TC1 according to the present invention is Ralstonia solanacearum (^ / 072/3 solanacearum), the subspecies of Ashdoborax avene τΊΊ- ^ Ι ° fl (Acidovorax avenae subsp.cattlyae), Agrobacterium tumefaciens, Burkholderia Written by BurkhoMeria glumae, Pectobacterium chrysanthemi 0 CO ) aC i er / ½; chrysanthemi), Pseudomonas syringae strain Actinide (/ ¾ei / G¾Z70 / 7as syringae v. actinidiae), Pseudomonas syringae strain Lacrimans (/ ^ se fcffOTas syringae pv. lachry ans), Santomonas axonopodis Λί¾ » ω235 axonopodis) and Clavibacter myganigansis subspecies 1: 7; pathogens such as 7aw ' bac er michiganensis subs. mi ch iganens is ^^; Rhizoctonia solan /), Magna Forte o ^ ^ iagnaporthe oryzae), Botrytis Cinerea (5o / y / s cinerea), Colletotricum Coco ^ ^ (Collet otri chum coccodes), Scleroti Nia sclerotiorum (5c / ro / 72 / a sclerotioru), phytophthora capsaici (/ ¾κ σ / ¾¾ Λο / · 3 capsici), phytophthora inhyeans and plasmodio It is possible to inhibit the growth of pathogenic mycobacteria (fungi) such as fora brassica (/ as / 70i / pAora brass / cae) (Examples 8 and 9). In addition, the microbial agent according to the present invention, further comprising any one or more selected from the group consisting of Bacillus Velensis G341 strain (KCTC 11497BP), spores of the strain, culture of the strain and extract of the culture solution. It features. The microbial agent may include at least one selected from the group consisting of Bacillus vegetosis G341 strain (KCTC 11497BP), spores of the strain, cultures of the strains, and extracts of the cultures; And at least one selected from the group consisting of Ricinibacillus sparycus TC1 strain (KCTC 12287BP), spores of the strain, cultures of the strains, and extracts of the cultures, with a weight ratio of 1: 9 to 9: 1, preferably May be included in a weight ratio of 1: 1 .
본 발명의 일 실시양태에 따르면, 바실러스 벨레젠시스 G341 균주 및 리시니바실러스 스패리쿠스 TC1 균주를 포함하는 미생물 제제는 상기 각각의 균주의 배양액을 단독으로 사용할 때보다 바실러스 벨레젠시스 G341 균주 및 리시니바실러스 스패리쿠스 TC1 균주를 1:1의 중량비로 흔합하여 사용하였을 때 토마토 풋마름병 및 고추 역병에 대하여 보다 더 우수한 방제 활성을 나타내었다 (실시예 12 및 13). 상기 바실러스 벨레젠시스 G341 균주의 특성은 대한민국 특허등록 제 1163986호에 상세히 기재되어 있다.  According to one embodiment of the invention, the microbial preparations comprising the Bacillus Velensis G341 strain and the Ricinibacillus spalicus TC1 strain are more resistant to the Bacillus Velensis G341 strain and Lys than when the culture medium of each strain is used alone. Synibacillus spalicus TC1 strains when used in combination at a weight ratio of 1: 1 showed better control activity against tomato foot blight and pepper blight (Examples 12 and 13). The characteristics of the Bacillus Velensis G341 strain is described in detail in Korean Patent Registration No. 1163986.
본 발명의 일 실시양태에 따르면， 상기 바실러스 벨레젠시스 G341 균주 배양액의 에틸아세테이트 추출물은 토마토 풋마름병에 대하여 농도 의존적인 방제 효과를 나타내며 상기 균주로부터 생산되는 비휘발성 항세균활성 물질인 옥시디피시딘 (oxydifficidin) 및 디피시딘 (dif f icidin)은 다양한 그람음성세균 및 그람양성세균, 특히 랄스토니아 솔라나세아럼에 대하여 우수한 항균 활성을 나타낸다. 또한， 바실러스 벨레젠시스 G341 균주는 항균 활성을 나타내는 휘발성 물질을 생산해 낼 수 있는데， 이러한 물질로는 디메틸디설파이드， 1-부탄올 (1-butanol) 및 3-히드록시 -2-부타논 (3-hydroxy-2-butanone)이 포함된다 (실시예 18 내지 20).  According to an embodiment of the present invention, the ethyl acetate extract of the Bacillus bellensis G341 strain culture medium exhibits a concentration-dependent control effect against tomato foot blight and oxydipidine, a nonvolatile antibacterial active material produced from the strain. (oxydifficidin) and dificidin show excellent antimicrobial activity against various Gram-negative bacteria and Gram-positive bacteria, especially Ralstonia solanacerum. In addition, the Bacillus Velensis G341 strain can produce volatiles that exhibit antimicrobial activity, such as dimethyl disulfide, 1-butanol and 3-hydroxy-2-butanone (3-hydroxy). -2-butanone) (Examples 18-20).
따라세 리시니바실러스 스패리쿠스 TC1 균주 및 바실러스 벨레젠시스 According to Lysinibacilli sp. Liqueur TC1 strain and Bacillus velogenis
G341 균주를 동시에 포함하는 본 발명의 미생물 제제는 랄스토니아 솔라나세아럼 0¾/sio a solanacearum) , 아시도보락스 아베내 아종 °fl iAcidovorax a venae subsp. cattlyae) , 이그로박테리움 투메파시엔스 ( grob c e"/ w tumefaciens) , 버크홀더리아The microbial agent of the present invention simultaneously comprising the G341 strain is Ralstonia solanacearum 0¾ / sio a solanacearum), subspecies of Ashidoborax avene ° fl iAcidovorax a venae subsp. cattlyae), Igrobacterium Grob ce "/ w tumefaciens, Berkholderia
^^^ Burkholderia glumae) , 펙토박테리움 크리산테미 (/¾cio?ac e/"/½? chrysanthemi) , 슈도모나스 시린개 변종 액티니데 0 «;(/(¾ Mas syringae pv. actinidiae) , 슈도모나스 시린개 변종 라크리만스 (/¾e ifewcwas syringae pv. lachrymans) , 산토모나스 악소노포디스 U 72 j¾OTOTas axonopodis) , 클라비박터 미시가넨시스 아종 口 1시 7]·넨시스 ( Clavibacter ichiganensis subsp . michiganensis)， 리족토니아 솔라니 iRhizoctonia solani) , 마그나포르테^^^ Burkholderia glumae), Pectobacterium chrysanthemi (/ ¾cio? Ac e / "/ ½? Chrysanthemi), Pseudomonas syringe strain Actinide 0«; (/ (¾ Mas syringae pv. Actinidiae), Pseudomonas Syringe varieties Lachrymans (/ ¾e ifewcwas syringae pv. ， Littoonia solani iRhizoctonia solani, Magnaporte
^리^ Magnaporihe oryzae) , 보트리티스 씨네레아 ( o /yi/s cinerea) , 콜레토트리쿰 코코댁 ^Col let otri chum coccodes) , 스클레로티니아 스클레로티오룸 (5Wero //j/a sclerotiorum) , 파이토프토라 캡사이시 iPhytophthora capsici)， 파이토프토라 인페스탄스 (/¾ op2 ?ora infestans) , 플라즈모디오포라 1 ?1] {Plas odiphora brass icae) , 아시도보락스 콘자시 (Acidovorax konjaci), 펙토박테리움 카로토보라 아종 ？ᅳ로토보라 (Pectobacter m carotovora subsp . carotovora) , 산토모나스 유베지 7]·토리 ο]· (Xanthomonas euvesicatoria) 및 산토모나스 프르니 (Xanthomonas pruni)S. 이루어진 군으로부터 선택되는 어느 하나 이상의 병원균에 대해서 항균 활성을 나타낼 수 있다 (실시예 19, 표 5). 본 발명에 따른 미생물 제제는， 리시니바실러스 스패리쿠스 TC1 균주 단독; 또는 바실러스 벨레젠시스 G341 균주 및 리시니바실러스 스패리쿠스 TC1 균주 흔합물을 담체와 흔합한 후， 분말， 펠랫 과립 또는 용액 등으로 제형화하여 사용할 수 있다. 바람직한 담체로는 물， 화이트 카본， 카올린， 덱스트린 등이 사용될 수 있다. ^ Lina Magnaporihe oryzae), Botrytis Cinerea (Collet otri chum coccodes), Sclerotinia sclerotium (5Wero // j / a sclerotiorum), iPhytophthora capsici, phytophthora infestans (/ ¾ op2? ora infestans), plasmodiophora 1? 1] {Plas odiphora brass icae) Acidovorax konjaci), Pectobacter m carotovora subsp.carotovora, Xanthomonas euvesicatoria, and Xanthomonas pruni . Antimicrobial activity may be exhibited against any one or more pathogens selected from the group consisting of (Example 19, Table 5). Microbial preparations according to the present invention, Ricinibacillus spalicus TC1 strain alone; Alternatively, the Bacillus velensis G341 strain and Ricinibacillus spalicus TC1 strain mixture may be mixed with a carrier, and then formulated into powder, pellet granules, or a solution. Preferred carriers may be water, white carbon, kaolin, dextrin, or the like.
본 발명의 일 실시양태에 따르면， 본 발명의 미생물 제제는 리시니바실러스 스패리쿠스 TC1균주의 배양액，또는 바실러스 벨레젠시스 G341 균주 배양액 및 리시니바실러스 스패리쿠스 TC1 균주 배양액을 1:1의 중량비로 흔합한 흔합 배양액을 을 화이트 카본 및 덱스트린과 같은 담체와 흔합한 후 이를 분무 건조함으로써 제조될 수 있다 (실시예 7 및 14 참고). 본 발명에서 "균주의 배양액" 은 균주를 포함하는 배양액 또는 균주를 여과한 배양액을 모두 포함할 수 있다. According to one embodiment of the present invention, the microbial agent of the present invention comprises a culture medium of the strain of Lysinibacillus sp. Liqueur TC1, or Bacillus velogenis G341 strain culture and a strain of Ricinibacilli sp. in Mixed mixed cultures can be prepared by mixing a with a carrier such as white carbon and dextrin and then spray drying it (see Examples 7 and 14). In the present invention, "strain culture" may include all of the culture solution containing the strain or culture medium filtered strain.
본 발명에서 "배양액 추출물" 은 균주의 배양액을 적절한 추출 용매, 예를 들어， 에틸아세테이트， 부탄올， 물 또는 이들의 흔합용매로 추출한 것이 바람직하며， 더욱 바람직하게는 배양 상징액을 동량의 에틸아세테이트 또는 부탄올로 분획한 에틸아세테이트층 또는 부탄올층, 또는 배양 상징액을 동량의 에틸아세테이트와 부탄을로 분획하고 남은 수용액층일 수 있다. 나아가， 본 발명은 본 발명에 따른 미생물 제제를 식물 또는 주변 환경에 처리하는 것을 포함하는， 식물병의 방제방법을 제공한다.  In the present invention, "culture liquid extract" is preferably extracted from the culture medium of the strain with an appropriate extraction solvent, for example, ethyl acetate, butanol, water or a mixed solvent thereof, more preferably the culture supernatant is the same amount of ethyl acetate or butanol Ethyl acetate layer or butanol layer fractionated by, or the culture supernatant may be the same amount of ethyl acetate and butane fraction and remaining aqueous solution layer. Furthermore, the present invention provides a method for controlling plant diseases, comprising treating the microbial agent according to the present invention to a plant or the surrounding environment.
상기 식물병은 토마토 풋마름병， 고추 역병， 배추 뿌리혹병 및 토마토 뿌리흑선층으로 이투어진 군으로부터 선택되는 어느 하나 이상일 수 있다. 또한,상기 방법은 랄스토니아솔라나세아럼 ?a/sic /a solanacearum) , 아시도보락스 아베내 아종 카를리애 ( // w^ avenae subs . cattlyae) , 아그로박테리움 투메파시엔스 (4gTobacie/"/iOT tumefaciens) , 버크홀더리아 글루매 (^/½o/flfe/ a glimae) , 펙토박테리움 크리산테미 ( fec otec er/i/zz? chrysanthemi) , 슈도모나스 시린개 변종 액티니데 (/¾e fc/w?/2as syringae pv. actinidiae), 슈도모나스 시린개 변종 라크리만스 (/¾ei/i¾7?cwas syringae pv. lachrymans) , 산토모나스 악소노포디스 ( a^ wiwas axonopodis) , 클라비박터 미시가넨시스 아종 미시가넨시스 ( Clavibacter michiganensis subs . michiganensis) , 리족토니아 솔라니 Rhizoctonia solani) , 마그나포르테 오리재 ( g7?apor /je oryzae) , 보트리티스 씨네레아 (及) ryi/s cinerea) , 콜레토트리쿰 코코혜 ^ Co U e t ot r i chum coccodes) , 스클레로티니아 스클레로티오룸 (6Wero /^/a sclerot ioruw) , 파이토프토라 캡사이시 Phytophthora capsici)， 파이토프토라 인페스탄스 (/¾yiopA 2ora infestans) , 플라즈모디오포라 브래시캐 (jD/as/woiZ/p ora brass icae) , 아시도보락스 콘자시 (Acidovorax konjaci), 펙토박테리움 카로토보라 아종 ^로토보라 (Pectobacterium carotovora subsp. carotovora) , 산토모나스 유베 7ΐ7ΐ·토리 (Xanthomonas euvesicatoria) 및 산토모나스 프르니 (Xanthomonas pruni)로 이루어진 군에서 선택되는 어느 하나의 생장을 억제하는 것을 특징으로 한다. 본 발명에 따른 식물병의 방제방법은,본 발명에 따른 미생물 제제를 i) 토양에 처리하거나， ii)식물 표면에 처리하거나, iii)종자에 코팅하거나， 또는 iv) 이들을 조합한 방법으로 처리함으로써 수행될 수 있으며, 이를 통해 식물병에 의한 식물 성장의 억제 또는 고사를 방지할 수 있다. The plant disease may be any one or more selected from the group consisting of tomato green blight, pepper blight, cabbage root gall disease and tomato root black layer. In addition, the method is Ralstonia solanacearum? A / sic / a solanacearum), Ashidoborax avenae subspecies Carlia (// w ^ avenae subs. Cattlyae), Agrobacterium tumefaciens (4gTobacie / " / iOT tumefaciens), Berkholderia Glumae (^ / ½o / flfe / a glimae), Pectobacterium chrysantemi (fec otec er / i / zz? chrysanthemi), Pseudomonas syringe strain Actinide (/ ¾e fc / w? / 2as syringae pv.actinidiae), Pseudomonas syringae strain Lacrimans (/ ¾ei / i¾7? cwas syringae pv.lachrymans), Santomonas axonopodis (a ^ wiwas axonopodis) Clavibacter michiganensis subs. Michiganensis, Rhizoctonia solani, Magna Forte duck, g7? Apor / je oryzae, Botrytis cinei ryi / s cinerea ^ Co U et ot ri chum coccodes), Sclerotini Sclerothiorum (6Wero / ^ / a sclerot ioruw), Phytophthora capsici, Phytophthora infestans (/ ¾yiopA 2ora infestans), Plasmophora brassica (jD / as / woiZ / p ora brass icae, Acidovorax konjaci, Pectobacterium carotobora subspecies ^ Potoboacterium carotovora subsp. It is characterized by inhibiting any growth selected from the group consisting of Xanthomonas pruni. The method for controlling plant diseases according to the present invention comprises treating the microbial preparation according to the present invention by i) treating the soil, ii) treating the surface of the plant, iii) coating the seed, or iv) combining them. It can be carried out, through which it is possible to prevent the suppression or death of plant growth by plant diseases.
상기 제제 중 본 발명의 리시니바실러스 스패리쿠스 TC1 균주, 또는 바실러스 벨레젠시스 G341 균주 및 리시니바실러스 스패리쿠스 TC1 균주의 흔합물은， 예를 들어， IX 104세포 /i 내지 IX 1010세포 / ， 바람직하게는 IX 105 세포 / 내지 IX 108 세포 /II 의 농도로 상기와 같이 제제화된 용액 등을 대상식물에 대해 흘러내릴 정도의 양으로 처리할 수 있으나， 이에 제한되는 것은 아니다. 또한， 본 발명은 테트라클로로에틸렌， 3-메틸 -1-부탄을, 메틸피라진， 사이클로핵사논, 2，5-디메틸피라진， Ν,Ν-디메틸포름아미드， 및In the above formulations, the combination of the Ricinibacillus spalicus TC1 strain of the present invention, or the Bacillus bellensis G341 strain and the Ricinibacillus spalicus TC1 strain, is, for example, IX 10 4 cells / i to IX 10 10 The cells /, preferably IX 10 5 cells / to IX 10 8 cells / II and the like can be treated in such an amount as to flow down to the target plant, but is not limited thereto. The present invention also relates to tetrachloroethylene, 3-methyl-1-butane, methylpyrazine, cyclonucleanone, 2,5-dimethylpyrazine, Ν, Ν-dimethylformamide, and
2-(2-에록시에톡시) -에탄을로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는， 식물병 방제용 조성물을 제공한다. Provided is a composition for controlling plant diseases, comprising any one or more selected from the group consisting of 2- (2-ethoxyethoxy) -ethane.
[실시예] EXAMPLE
이하, 본 발명을 하기 실시예에 의하여 더욱 상세하게 설명한다. 단， 하기 실시예는 본 발명을 예시하기 위한 것일 뿐， 본 발명의 범위가 이들만으로 한정되는 것은 아니다. 실시예 1: 담체로부터의 TC1균주의 분리 시험균주는 토마토 뿌리흑선충 실험 중 사용되었던 담체로부터 분리되었다. 구체적으로， 한국화학연구원에서 제조한무기물질로 제조한 미생물 담체를 트립틱소이액체배지 (Tryptic Soy Broth(TSB) , Difco사, 미국)에 30분간 침지한 후 음건한 다음 토마토 뿌리 부분에 처리하였다. 토마토를 이식한 다음 1개월 후에 미생물 담체를 마쇄하여 트립틱 소이 한천 배지 (Tryptic soy agar(TSA), Difco사， 미국)에 도말하였다. 30°C에서 3일 동안 배양한 후， 세균 콜로니가 나타나면 영양 한천 배지 (Nutrient agar)에서 3-4회 계대 배양하여 단일콜로니를 분리하였다ᅳ 실시예 2: 1C1균주의 동정 실시예 1에서 분리한 균주에 대하여 형태학적 및 생화학적 특성을 분석하고, gry A유전자 염기서열을 통해 유전학적 특성을 분석하였다. Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, the scope of the present invention It is not limited only to these. Example 1 Isolation of TC1 Strains from the Carrier The test strain was isolated from the carrier used during the tomato root nematode experiment. Specifically, the microbial carrier prepared by the inorganic material prepared by the Korea Research Institute of Chemical Technology was immersed in Tryptic Soy Broth (TSB), Difco, USA for 30 minutes, then dried and treated in tomato roots. . One month after the tomato transplant, the microbial carrier was crushed and plated in Tryptic soy agar (TSA), Difco, USA. After incubation for 3 days at 30 ° C, when bacterial colonies appear, single colonies were isolated by passage 3-4 times in nutrient agar medium (Nutrient agar) 2: Example 2: Identification of strain 1C1 isolated in Example 1 Morphological and biochemical characteristics of the strains were analyzed, and genetic characteristics were analyzed through the gry A gene sequence.
(1) 형태학적 및 생화학적 특성 그람 염색결과 양성으로 나타났으며， 간상 (rod)의 균주였다. 30°C 내치 37°C에서 잘 생장하는 중온성 균주이몌 영양분이 고갈될 때 내생포자를 형성하였다. 그리고 혐기성인 상태에서도 잘 자라는 전형적인 조건성 혐기성균주이다. (1) Morphological and biochemical characteristics Gram staining was positive and rod strain. Endogenous spores formed when nutrients were depleted in mesophilic strains growing well at 30 ° C and 37 ° C. It is a typical conditional anaerobic strain that grows well even in anaerobic conditions.
(2) 유전학적 특성 균주의 gyr A 유전자의 염기서열을 얻기 위하여 콜로니 중합효소 연쇄반응 (colony PCR)을 수행하였다. 단일 콜로니로부터 이쑤시개를 이용하여 콜로니를 채취하여 PCR-프리믹스 (PCR-premix: 인트론 바이오테크놀로지 (iNtRON Biotechnology), 대한민국)와 흔합 후 p-gyr A-f (5'— CAG TAC GGA AAT GCG TAC GTC CTT-3'； 서열번호: 1) 및 p-gyr A-r(5'-CAA GGT AAT GCT CCa GGC ATT GCT-3'； 서열번호 2) 프라이머를 이용하여 gyr A 유전자를 증폭하였다. 증폭된 PCR 산물을 위저드 PCR프렙 키트 (Wizard PCR prep kit:프로메가 (Promega)， Medison, WI, USA)를 사용하여 정제한 후， 마크로젠 (대전， 대한민국)에 의뢰하여 염기서열을 얻었다. 염기서열은 NCBI의 BLAST를 이용하여 GenBank 데이터 베이스와 염기서열을 비교하였다. CLUSTAL Z로 염기서열을 정렬하고， PHYDIT 프로그램 버전 3.0을 사용하여 neighbour- joining tree를 작성하였다. (2) Colony polymerase to obtain the nucleotide sequence of the gyr A gene of the genetic characteristic strain Colony PCR was performed. Collect colonies using toothpicks from a single colony and mix them with PCR-premix (PCR-premix: iNtRON Biotechnology, South Korea) and then p-gyr Af (5'— CAG TAC GGA AAT GCG TAC GTC CTT-3 Gyr A gene was amplified using primers SEQ ID NO: 1) and p-gyr Ar (5'-CAA GGT AAT GCT CCa GGC ATT GCT-3 '; SEQ ID NO: 2). The amplified PCR product was purified using a Wizard PCR prep kit (Promega, Medison, WI, USA), and then subjected to macrogen (Daejeon, South Korea) to obtain a base sequence. The base sequence was compared with GenBank database and base sequence using BLAST of NCBI. The sequences were sorted by CLUSTAL Z, and a neighbor-joining tree was created using PHYDIT program version 3.0.
균주의 gyr A 유전자의 염기서열 (서열번호: 3)을 neighbour-joining 알고리즘을 바탕으로 분석한 결과, 본 균주는 리시니바실러스 스패리쿠스 As a result of analyzing the nucleotide sequence of the gyr A gene of the strain (SEQ ID NO: 3) based on the neighbor-joining algorithm, the strain was Lysinibacilli sp.
C3-41과 98% 정도의 상동성을 나타내는 신규한 균주임을 알 수 있었다 (도 1). 이상의 결과로부터 본 발명에서 수득된 균주는 리시니바실러스 스패리쿠스 균주로서 동정되었으며， 상기 균주를 리시니바실러스 스패리쿠스 TC1로 명명하고 한국생명공학연구원 생물자원센터에 2012년 10월 10일자 기탁번호 제 KCTC 12287BP호로서 기탁하였다. 실시예 3: 리시니바실러스 스패리쿠스 TC1 균주의 비휘발성 항균 활성 물질 생산능 확인 실시예 1에서 분리한 리시니바실러스 스패리쿠스 TC1 균주가 비휘발성 항균활성 물질을 생산하는지 조사하기 위하여, 토마토 풋마름병균인 랄스토니아 솔라나세아럼 /s o;7/a solanacearum^ 대상으로 항균 활성을 측정하였다. It can be seen that the novel strain showing a homology of about 98% with C3-41 (Fig. 1). From the above results, the strain obtained in the present invention was identified as Ricinibacillus spalicus strain, which was named as Ricinibacillus spalicus TC1 and deposited on October 10, 2012 to the Korea Institute of Bioscience and Biotechnology. Deposited as KCTC 12287BP. Example 3 Confirmation of Nonvolatile Antimicrobial Active Substance Production Capacity of Ricinibacillus spalicus TC1 Strain In order to investigate whether the Ricinibacillus sparycus TC1 strain isolated in Example 1 produces a nonvolatile antimicrobial active substance, tomato foot Antimicrobial activity was measured in Ralstonia solanacearum / so; 7 / a solanacearum ^.
먼저, 랄스토니아 솔라나세아럼을 트립틱 소이 액체배지에 접종하고 First, inoculate Ralstonia Solanasearum into Tryptic Soy Liquid Medium.
30 °C 배양기에서 24시간 배양하여 접종 균주로 사용하였다. 또한， 본 발명의 리시니바실러스 스패리쿠스 TC1 균주는 트립틱 소이 액체배지에 접종하고 30°C 배양기에서 12, 24, 36, 48 및 72시간 배양하여 실험군으로 사용하였다. 24 hours incubation in a 30 ° C. incubator was used as the inoculation strain. In addition, the Ricinibacillus spalicus TC1 strain of the present invention is inoculated in tryptic soy liquid medium and 30 ° C. 12, 24, 36, 48 and 72 hours incubation in the incubator was used as an experimental group.
페트리 디쉬에 랄스토니아 솔라나세아럼이 1% 접종된 트립틱 소이 한천 배지를 플레이트 높이의 2/3 정도 부어서 굳혔고， 리시니바실러스 스패리쿠스 TC1균주 배양액으로부터 8，000 rpm으로 15분간 원심분리한 후， 0.2 μιη막으로 여과하여 균체를 제거한 상징액을 제조하였다. 상기에서 배양 시간별로 수득한 배양 여액을 각각 30 /^씩 취하여 페이퍼 디스크 (paper disc; 지름 =8 mm)에 흡수시킨 다음 배지 위에 올린 뒤 30°C 배양기에서 1-2일간 배양하여 페이퍼 디스크 주위의 클리어 존 (clear zone)의 크기로 항균 활성을 관찰하였다. 이때， 비교를 위해 대조군으로 멸균한 트립틱 소이 액체배지만을 사용하였다. Trytic Soy agar medium in which Petri dishes were inoculated with Ralstonia solanacearum in 1% was poured and hardened by about two-thirds of the plate height, and centrifuged at 8,000 rpm for 15 minutes from the culture medium of Lysinibacilli sp. after separating, to thereby prepare a supernatant by removing the cells it was filtered with a 0.2 membrane μι η. Each of the culture filtrates obtained by the incubation time above was taken 30 / ^, absorbed in a paper disc (diameter = 8 mm) and then placed on a medium and incubated for 1-2 days in a 30 ° C incubator around the paper disc The antimicrobial activity was observed by the size of the clear zone. At this time, only sterile tryptic soy liquid medium was used as a control for comparison.
그 결과， 배지만을 처리한 무처리 대조군과 마찬가지로 리시니바실러스 스패리쿠스 TC1 균주 배양액 처리구에서는 배양기간에 상관없이 전혀 항균 활성을 보이지 않는 것으로 나타났다. 따라서， 본 발명의 리시니바실러스 스패리쿠스 TC1 균주는 비휘발성 항균 활성 물질을 생산하지 않는 것을 알 수 있었다■ 실시예 4: 리시니바실러스 스패리쿠스 TC1 균주 배양액의 토마토 풋마름병에 대한방제 효과 리시니바실러스 스패리쿠스 TC1 균주 배양액의 토마토 풋마름병 발병 억제효과를 토양관주 방법으로 검정하였다.  As a result, as in the untreated control treated with only the medium, the treatment of the Lysinibacillus sp. Liqueur TC1 strain culture medium showed no antimicrobial activity at all regardless of the culture period. Therefore, it could be seen that the Lysinibacillus spalicus TC1 strain of the present invention did not produce a nonvolatile antimicrobial active substance. Example 4 Control Effect of the Lysinibacilli Sparicus TC1 Strain Culture on Tomato Foot Blight Inhibitory effect on the development of tomato foot blight on the culture of Cinibacilli sp. Liqueur TC1 strain was assayed by soil irrigation method.
구체적으로， 서광 토마토 품종을 사용하여 지름 4.5 cm 포트에 원예용 상토를 채우고 파종한 후 25土 5T의 온실에서 재배하였다. 3주 후 지름 10 cm 포트에 이식하고，. 4엽기가 되었을 때 0.D값 0.5(5X 108 CFU/ml)로 조절한 병원균인 랄스토니아 솔라나세아럼 현탁액을 20 ml씩 관주 처리하여 30°C 항온항습실에서 배양하여 토마토 풋마름병 발병을 유도하였다. 이때 병원균을 처리하기 3일 전에 본 발명의 리시니바실러스 스패리쿠스 TC1 균주 배양액 원액 및 1/2배 희석액을 포트당 20 ml씩 토양에 관주처리하였다. 토마토 풋마름병의 발병 억제 효과는 12일간 관찰하였다. 이때， 비교를 위해 대조구 (무처리구)는 리시니바실러스 스패리쿠스 TC1 균주 배양액 대신 증류수만을 처리하여 상기와 동일한 방식으로 수행하였으며， 각 처리당 5본씩 3회 반복 실험하였다. Specifically, the cultivated clay soil was filled and seeded in pots with a diameter of 4.5 cm using the cultivated tomato varieties, and then grown in a greenhouse of 25 土 5T. 3 weeks later, transplanted into a 10 cm diameter pot. At the 4-leaf stage, 20 ml of Ralstonia solanacerum suspension, a pathogen adjusted to 0.D value 0.5 (5X 10 8 CFU / ml), was irrigated and incubated in a 30 ° C constant temperature and humidity room to develop tomato foot blight. Induced. At this time, three days prior to the treatment of the pathogen, the stock solution and the fold dilution of the Lysinibacillus spalicus TC1 strain culture of the present invention was irrigated with 20 ml of soil per pot. Inhibitory effect of tomato foot blight was observed for 12 days. At this time, for comparison The control (no treatment) was carried out in the same manner as described above by treating only distilled water instead of the culture medium of Ricinibacillus sp. Liqueur TC1 strain, and repeated three experiments of 5 copies per treatment.
풋마름병 발병률은 식물이 아주 싱싱한 경우는 0， 잎이 약간 시든 경우 (25%미만)는 1，중간 정도 시든 경우 (50%미만)는 2，심하게 시든 경우 (75% 정도)는 3， 완전히 시들어 고사한 경우를 4로 등급을 정하였으며， 이를 근거로 발병 억제율은 { (대조구의 발병를-처리구의 발병률 )/대조구의 발병률 }x 100으로 계산하였다. 그 결과는 도 2에 나타내었다.  The incidence of foot blight is 0 when the plant is very fresh, 1 when the leaf is slightly soaked (less than 25%), 2 when it is moderately soaked (less than 50%) and 3 when it is severely soaked (about 75%). The death rate was rated as 4 and based on this, the incidence inhibition rate was calculated as {(control-to-control-to-treatment rate) / control-to-control rate} x 100. The results are shown in FIG.
도 2에서 보는 바와 같이， 본 발명의 리시니바실러스 스패리쿠스 TC1 균주의 배양액 원액을 처리한 경우 접종 12일 동안 토마토 풋마름병이 전혀 발병하지 않아 100%방제효과를 보였으며， 1/2희석액을 처리한 경우에서도 67% 내지 80%의 방제효과를 보였다. 또한, 1/2 회석액 처리구에서는 5개의 식물체 중 단 1개의 식물체에서만 발병한 반면 대조구 (무처리구)의 경우 대부분의 식물체가 병이 들어 고사하여 본 발명의 균주의 방제효과가 우수함을 알 수 있었다. 실시예 5: 리시니바실러스 스패리쿠스 TC1 균주 배양액의 고추 역병에 대한 방제 효과 리시니바실러스 스패리쿠스 TC1 균주 배양액의 고추역병 발병 억제효과를 토양관주 방법으로 검정하였다.  As shown in Figure 2, when treated with the culture solution of the Lysinibcillus sp. Liqueur TC1 strain of the present invention did not develop tomato foot blight at 12 days of inoculation showed 100% control effect, 1/2 diluted solution In the case of treatment, the control effect was 67% to 80%. In addition, in the half-dilution treatment group, only one of the five plants was developed, whereas in the control group (untreated group), most of the plants died of the disease, and the control effect of the strain of the present invention was found to be excellent. Example 5 Control Effect of Lysinivacillus spalicus TC1 Strain Culture on Pepper Blight Disease The inhibition of the development of pepper blight disease on the culture of pepper blight was tested by soil irrigation method.
구체적으로， 부강 고추 유묘를 사용하여 지름 7.0 cm인 플라스틱 포트 내의 원예용 상토에 이식하여 6엽기까지 키운 후， 본 발명의 리시니바실러스 스패리쿠스 TC1 균주 배양액 20 ml를 포트당 20 ml씩 토양에 관주처리하였다. 배양액 처리 3일 후에 고추 ^^균 Uhytophthora caps/c/)의 유주자낭 현탁액 (IX if)4유주자낭 / )을 각각의 포트에 20 ^씩 관주 처리함으로써 식물 병원균을 접종하였다.고추 역병의 발병 억제 효과는 10일간 관찰하였다. 이때， 비교를 위해 대조구 (무처리구)는 리시니바실러스 스패리쿠스 TC1 균주 배양액 대신 증류수만을 처리하여 상기와 동일한 방식으로 수행하였으며， 각 처리당Specifically, after transplanting to gardening topsoil in plastic pots with a diameter of 7.0 cm using Bujin pepper seedlings to grow up to 6 leaves, 20 ml of the Lysinibacillus spalicus TC1 strain culture medium of the present invention in soil 20 ml per pot Irrigation. After 3 days of treatment, the plant pathogens were inoculated by irrigation of the capsicum sac suspension (IX if) 4 capsular sac /) of pepper ^^ bacterium Uhytophthora caps / c /) in each port by 20 ^. Observation was carried out for 10 days. At this time, for comparison, the control (untreated) was a culture medium of Ricinibacillus sparycus TC1 strain. Instead, only distilled water was treated and performed in the same manner as above.
5본씩 3회 반복 실험하였다. Five experiments were repeated three times.
고추 역병 발병를은 식물이 아주 싱싱한 경우는 0， 잎이 약간 시든 경우 (25%미만)는 1，증간 정도 시든 경우 (50%미만)는 2，심하게 시든 경우 (75% 정도)는 3， 완전히 시들어 고사한 경우를 4로 등급을 정하였으며, 이를 근거로 발병 억제율은 { (대조구의 발병를-처리구의 발병률 )/대조구의 발병를 }χ 100으로 계산하였다. 그 결과는 도 3에 나타내었다.  0, if the plant is very fresh with pepper blight, 1 with slightly weakened leaves (less than 25%), 2 with less severe (less than 50%) 2, 3 with severely withered (about 75%) The death rate was rated as 4 and based on this, the incidence of inhibition was calculated as {(induction of control-incidence of control) / control of incidence as} χ 100. The results are shown in FIG.
도 3에서 보는 바와 같이 본 발명의 리시니바실러스 스패리쿠스 TC1 균주의 배양액을 처리한 군은 접종 7일 후에 약 43%의 고추 역병 방제효과를 나타낸 반면， 대조구는 대부분의 식물체가 병이 들어 고사하여 본 발명의 균주의 방제효과가 우수함을 알 수 있었다. 실시예 6: 리시니바실러스 스패리쿠스 TC1 균주 배양액의 배추 뿌리흑병에 대한방제 효과 리시니바실러스 스패리쿠스 TC1 균주 배양액의 배추 뿌리혹병을 발생시키는 플라즈모디오포라 브래시캐 (/Vas/TOi/cipAara brass icae) 균에 대한 발병 억제효과는 토양관주 방법으로 검정하였다.  As shown in Figure 3, the group treated with the culture medium of the ricinibacillus spalicus TC1 strain of the present invention showed about 43% pepper blight control effect after 7 days of inoculation, while the control group was killed by most plants It was found that the control effect of the strain of the present invention is excellent. Example 6 Control Effect of Ricinibacillus spalicus TC1 Strain Culture on Chinese Cabbage Root Black Disease Plasmodiophora Brassica of Cabbage Root Disease of Ricinibacillus spalicus TC1 Strain Culture Solution (/ Vas / TOi / Inhibitory effect on cipAara brass icae) was tested by soil irrigation method.
구체적으로, 원예용 상토에 삼복 엇갈이 배추를 파종하였다. 2주간 온실에서 키운 다음 포장에서 채취한 배추 뿌리흑 (10 g/밭 흙 을 믹서기로 곱게 갈아 거즈로 걸러 얻어진 액을 밭 흙에 접종한 후 잘 섞어 제조한 이병토에 이식하였다. 이식 후 바로 본 발명의 리시니바실러스 스패리쿠스 TC1 균주 배양액 10 ml을 관주 처리하였다. 실험은 동일한 실험을 5회 반복 수행하였다. 이어, 접종 3주 후에 식물체를 뽑아 뿌리에 묻어 있는 흙을 제거한 후 발병도를 조사하였다. 이때, 비교를 위해 대조구 (무처리구)는 리시니바실러스 스패리쿠스 TC1 균주 배양액 대신 증류수만을 처리하였다.  Specifically, bokbok crossover cabbage was sown in the horticultural soil. Chinese cabbage root black (10 g / field soil, finely ground with a blender) was inoculated into the field soil after incubation in a greenhouse for 2 weeks, and then mixed with well mixed soil. 10 ml of the ricinibacillus spalicus TC1 strain culture of the invention was irrigated, and the experiment was repeated five times, and after 3 weeks of inoculation, the plants were removed to remove the soil from the roots and the incidence was examined. At this time, the control (no treatment) was treated only with distilled water instead of the culture medium of Ricinibacillus spalicus TC1 strain.
배추 뿌리혹병 발병률은 0: 건전， 1: 세근에 뿌리혹이 형성된 것 2: 주근에 뿌리혹이 형성된 것, 3:세근 및 주근에 뿌리혹이 전체적으로 형성된 것, 4： 세근 및 주근에 뿌리혹이 전체적으로 크게 형성된 것으로 정의하였으며, 이를 근거로 방제가를 (무처리 발병율 - 처리구발병률 )/무처리발병율 X 100의 식에 따라 계산하였다. 그 결과를 하기 표 1 및 도 4에 나타내었다. 【표 1】
Figure imgf000017_0001
실험 결과 방제효과는 유박 배양체가 가장 우수한 것으로 나타났지만， 배추의 생장을 크게 억제하는 것으로 나타났다 (표 1). The incidence of cabbage root gall disease is 0: healthy, 1: root gall formed in the roots 2: root gall formed in the roots, 3: root gall formed in the roots and roots as a whole, 4: The root nodules were largely formed in the roots and main roots, and the control value was calculated according to the formula (No treatment incidence-treatment incidence) / no treatment incidence X 100. The results are shown in Table 1 and FIG. 4. Table 1
Figure imgf000017_0001
Experimental results showed that the oily cultures were the most effective, but they significantly inhibited the growth of Chinese cabbage (Table 1).
또한, 도 4에서 나타난 바와 같이， 무처리구의 경우 뿌리에 혹이 발생하거나 식물 병원균에 의하여 일부 식물은 완전히 고사한 반면, 본 발명의 리시니바실러스 스패리쿠스 TC1 배양액 처리구는 흑이 적게 발생하고 또한 식물체도 잘 자란 것을 확인할 수 있었다. 실시예 7: 리시니바실러스스패리쿠스 TC1균주 배양액으로 제조한분무 건조시료의 토마토풋마름병에 대한방제효과  In addition, as shown in FIG. 4, in the case of the untreated group, some plants were completely killed by the roots or plant pathogens, whereas the treatment group of the Ricinibacillus spalicus TC1 culture solution of the present invention produced less black and also the plant. It was confirmed that well grew. Example 7 Control Effects of Spray Drying Samples Prepared from Ricinibacillus sparycus TC1 Strain Culture on Tomato Foot Blight
7-1： 분무 건조 시료의 제조 본 발명의 리시니바실러스 스패리쿠스 TC1 균주 50 ml를 트립틱 소이 액체배지 (Difco)에 접종하여 30°C에서 일간 진탕 배양하여 접종원으로 사용하였다. 2 L의 삼각플라스크에 500 ml TSB 배지를 제조하여 멸균한 후 전배양액 5 ml를 접종하였다. 이어， 30°C에서 150 rpm으로 3일 배양하였으며, 배양액 1 L를 수확하여 10 g의 화이트 카본과 10 g의 덱스트린을 첨가하여 섞은 후 주입구 (inlet) 온도 165°C와 배출구 (outlet) 온도 94°C로 분무 건조하여 본 발명의 균주의 분무 건조 시료를 제조하였다. 7-2： 분무 건조 시료의 토마토 풋마름병에 대한 방제효과 상기에서 제조한 분무 건조 시료를 이용하여 토마토 풋마름병에 대한 방제효과를 확인하였다. 7-1: Preparation of spray dried sample 50 ml of the strain of Ricinibacillus sparycus TC1 of the present invention was inoculated in Tryptic Soy Liquid Medium (Difco) and shaken at 30 ° C for 1 day to use as an inoculum. 500 ml TSB medium was prepared in a 2 L Erlenmeyer flask and sterilized, followed by inoculation of 5 ml of the preculture. Followed by, 30 ° at C and cultured 3 days with 150 rpm, and then mixed by harvesting the culture broth 1 L by the addition of dextrin 10 g of white carbon and 10 g of the injection port (inlet) temperature of 165 ° C and a discharge port (outlet) temperature 94 Spray dried to ° C to prepare a spray dried sample of the strain of the present invention. 7-2: Control effect of the spray-dried sample on the tomato foot blight disease The spray-drying sample prepared above was confirmed for the control effect on the tomato foot blight disease.
상기 실시예 4에 기재된 것과 동일한 방식으로 토마토 풋마름병의 발병을 유도한 후， 본 발명의 균주의 분무 건조 시료를 50배 회석한 회석액을 포트당 20 ml씩 토양에 관주 처리한 후 방제 효과를 확인하였다. 그 결과를 도 5에 나타내었다.도 5에 나타난 바와 같이，본 발명의 균주의 분무 건조 시료의 50배 희석액은 12일이 경과한 후에 44%의 방제효과를 나타내었다. 실시예 8: 리시니바실러스 스패리쿠스 TC1 균주가 생산하는 휘발성 물질의 항균 활성 측정 본 발명의 리시니바실러스 스패리쿠스 TC1 균주가 생산하는 휘발성 물질이 식물병원균에 항균활성을 나타내는지 살펴보기 위하여， 대표적인 10가지 그람음성 및 그람양성균을 대상으로 페트리디쉬 내 배지의 너비 1 cm로 중앙부위를 완전히 제거하여 한 쪽에 있는 물질이나 균주가 반대쪽에 가지 않도록 제조한 배지를 이용하여 조사하였다.  After inducing the onset of tomato foot blight in the same manner as described in Example 4, and after irrigation treatment with soil 20 ml per pot of distilled liquor 50 times a spin-drying sample of the spray dried sample of the present invention in soil Confirmed. The results are shown in Fig. 5. As shown in Fig. 5, the 50-fold dilution of the spray dried sample of the strain of the present invention exhibited 44% control effect after 12 days. Example 8: Determination of the antimicrobial activity of the volatiles produced by the Ricinibacillus sp. Liqueur TC1 strain In order to examine whether the volatiles produced by the strain of Ricinibacillus sp. Liqueur TC1 show antimicrobial activity against phytopathogens. Ten representative Gram-negative and Gram-positive bacteria were examined by using a medium prepared by completely removing the central part with a width of 1 cm of the Petri dish medium so that one substance or strain did not go to the other side.
구체적으로，트립틱 소이 한천 배지를 페트리디쉬에 분주한 다음 배지의 중앙으로부터 1 cm의 배지를 완전히 제거하여 확산에 의하여 병원 세균의 생장이 억제되지 않도록 하였다.  Specifically, tryptic soy agar medium was dispensed into Petri dishes, and then 1 cm of medium was completely removed from the center of the medium to prevent growth of pathogenic bacteria by diffusion.
실험에 사용하기 위하여， 랄스토니아 会 E I 세 of¾ (Ralstonia solanacearum)， 아시도보락스 아베내 아종
Figure imgf000018_0001
avenae subsp . cattlyae)， 아그로박테리움 투메파시엔스 {Agrobacterium tu efaciens) 버크흘더리아 글루매 {Burkholderia glumae)， 펙토박테리움 크리산테미 Pectobacterium chrysanthemi) 슈도모나人 시린개 변종 액티나데 O^ei/ato/Boyias syringae v. actinidiae) , 슈도모나스 시린개 변종 라크리만스 ( ¾ei<±ra(was syringae v. lachrywans) , 산토모나스 악소노포디스 ?σΒ0 35 axonopodis) 및 클라비박터 미시가넨시스 아종 π] Λ| 7\^\^}^{Clavibacter michiganensis subsp. michiganensis) 균주는 트립틱 소이 액체배지에서 30°C 조건에서 1일간 배양하였으며 2X 106 eel Is/ml로 조절하여 사용하였다. For use in experiments, Ralstonia EI three of¾ (Ralstonia solanacearum), subspecies of Ashidovorax avenae
Figure imgf000018_0001
avenae subsp. cattlyae), Agrobacterium tu efaciens (Burkholderia glumae), Pectobacterium chrysanthemi) Pseudomonas syringe strain Actinade O ^ ei / ato / Boyias syringae v. actinidiae), Pseudomonas syringe strain Lakrimmans (¾ei <± ra (was syringae v. Lachrywans), Santomonas axonopodis? ΣΒ0 35 axonopodis) and Clavibacter myganigansis subspecies π] Λ | 7 \ ^ \ ^} ^ {Clavibacter michiganensis subsp. michiganensis strain was incubated for 1 day at 30 ° C in tryptic soy liquid medium and used to adjust to 2X 10 6 eel Is / ml.
배지의 한쪽 면에， 상기 병원균의 현탁액 3 ml을 각각 취하여 약 1 cm 간격으로 점적 접종하였다. 반대편 배지에는 리시니바실러스 스패리쿠스 TC1 균주 배양액을 50 μ ΐ씩 플레이트의 표면에 균질하게 도말하였다. 휘발성 물질이 플레이트 밖으로 나가지 않도록 플레이트를 파라필름으로 밀봉한 다음 30°C에서 1일간 배양하였다. 모든 실험은 3회 실시하였으며 병원성 세균의 생장정도를 무처리구와 비교하여 저해활성을 측정하였다. 실험결과는 하기 표 2에 나타내었다. On one side of the medium, 3 ml of each suspension of the pathogen was taken and inoculated at about 1 cm intervals. In the opposite medium, 50 μl of Ricinibacillus spalicus TC1 strain culture was plated homogeneously on the surface of the plate. The plate was sealed with parafilm to prevent volatiles from leaving the plate and then incubated at 30 ° C. for 1 day. All experiments were performed three times and the inhibitory activity was measured by comparing the growth of pathogenic bacteria with that of no treatment. The experimental results are shown in Table 2 below.
【표 2】 Table 2
Figure imgf000019_0002
Figure imgf000019_0001
나타낸 바와 같이 본 발명의 TC1 균주는 펙토박테리움 카로토보라 아종 카로토보라를 제외한 모든 병원성 세균에 대하여 항균 활성을 나타내는 휘발성 물질을 생산한다는 것을 확인할 수 있었다. 실시예 9: 리시니바실러스 스패리쿠스 TC1 균주 배양액으로부터 생산되는휘발성 물질의 항진균활성 측정 본 발명의 리시니바실러스 스패리쿠스 TC1 균주가 생산하는 휘발성 물질이 식물병 곰광이의 균사 생육에 미치는 영향을 살펴보기 위하여， 대표적인 식물병 곰광이균을 대상으로 곰광이 훈증 플레이트 분석 (mycofumigation plate assay) (Strobel et al. , 2001, Microbiology, 147 :2943-2950)을 통해 확인하였다.
Figure imgf000019_0002
Figure imgf000019_0001
As shown the TC1 strain of the present invention is pectobacterium It was confirmed that a volatile substance showing antimicrobial activity was produced against all pathogenic bacteria except Carotobora subspecies Carotobora. Example 9 Determination of Antifungal Activity of Volatile Substances Produced from Ricinibacillus sparycus TC1 Strains Culture Effect of Volatile Substances Produced by Ricinivacillus sparycus TC1 Strains on the Mycelial Growth of Plant Disease Bears In order to examine, it was confirmed by a mycofumigation plate assay (Strobel et al., 2001, Microbiology, 147: 2943-2950) for representative plant diseases.
실험 방법은 한 플레이트가 반으로 나누어진 I플레이트를 이용하였으며， 상기 플레이트의 한쪽 면에는 감자한천배지 (potato dextrose agar)(Becton and Dickinson Co., Far nkl ins Lakes, NJ, USA)를 분주하였고, 다른 한쪽 면에는 TSA 배지를 분주하였다. 실험에 사용된 식물병 곰팡이균은 리족토니아 솔라니 {Rhizoctonia solani RS) , 마그나포르테 오리재 ( ¾g p ri ze oryzae; M0), 보트리티스 씨네레아 (^ y /s cinerea; BC), 콜레토트리쿰 Experimental method used an I plate divided in half, one side of the plate was dispensed with potato agar medium (potato dextrose agar) (Becton and Dickinson Co., Far nkl ins Lakes, NJ, USA), On the other side, TSA medium was dispensed. Phytopathogenic fungi used in the experiments were Rhizoctonia solani RS, Magna Forte duck (¾ g p ri ze oryzae; M0), Botrytis cinerea (BC), Choleto Tricum
3—3 ^^ CoUetotrichuin coccodes) CO, 푸자리움 옥시스포름 f. sp. 라파니 (j sar/uw oxysporum f . sp. raphani', F0) , 스클레로티니아 스클레로티오룸 (5c/ero //3/a sclerot iorum SS), 파이토프토라 캡사이시 0¾yop? ?ora capsici; PC) 및 파이토프토라 인페스탄스 (/¾yo /? Aora infest ans\ PI)이며， 각각 PDA 배지에서 25°C에서 3일내지 7일간 배양한 후 사용하였다. 3—3 ^^ CoUetotrichuin coccodes) CO, fujium oxysporum f. sp. Raphani (j sar / uw oxysporum f. Sp.raphani ' , F0), sclerotinia sclerotiorum (5c / ero // 3 / a sclerot iorum SS), phytophthora capsaici 0¾yop? ? ora capsici; PC) and phytophthora infestans (/ ¾yo /? Aora infest ans \ PI), and were used after incubation in PDA medium at 25 ° C. for 3 to 7 days, respectively.
구체적으로， 1일 동안 TSB배지에서 30°C 및 150 rpm으로 전배양한 리시니바실러스 스패리쿠스 TC1 균주 배양액 50 ml을 트립틱 소이 한천 배지를 분주한 플레이트에 균질하게 도말하였다. 그리고 플레이트의 다른 한쪽 면에는，Specifically, 50 ml of Ricinibacillus spalicus TC1 strain culture precultured in TSB medium at 30 ° C. and 150 rpm for 1 day were homogenously plated on a plate dispensed with Tryptic Soy Agar Medium. And on the other side of the plate,
PDA 배지에서 왕성하게 자라고 있는 균주의 가장자리에서 지름 8 mm의 코르크 보어러를 이용하여 균체를 포함하고 있는 한천배지를 떼어낸 것을 접종하였다. 상기 플레이트를 파라 필름으로 밀봉한 다음 28°C에서 3-5일간 배양하면서 식물병 곰광이의 균사 생육저해 정도를 측정하였고 그 결과를 하기 표 3에 나타내었다. The agar medium containing the cells was inoculated using a cork bore with a diameter of 8 mm from the edge of the strain growing in PDA medium. The plate was sealed with a para film and then cultured at 28 ° C. for 3-5 days, and the degree of mycelial growth of the plant disease bear was measured. The results are shown in Table 3 below.
균사 생육 저해율은 (무처리구 병원균의 균사 지름 - 처리구 병원균의 균사 지름) /무처리구 병원균의 균사 지름 X 100의 식에 따라 계산하였다.  The mycelial growth inhibition rate was calculated according to the formula of (mycelial diameter of untreated pathogen-mycelial diameter of treated pathogen) / mycelial diameter of untreated pathogen X 100.
【표 3】
Figure imgf000021_0001
표 3에서 나타낸 바와 같이， 푸자리움 옥시스포룸 f. Sp. 라파니 ( ))을 제외한 모든 병원성 곰광이의 균사 생육이 리시니바실러스 스패리쿠스 TC1 균주가 생성하는 휘발성물질에 의해 저해됨을 알 수 있었다. 실시예 10: 리시니바실러스 스패리쿠스 TC1 균주가 생산하는 휘발성 물질의 성분 분석 본 발명의 리시니바실러스 스패리쿠스 TC1 균주가 생산하는 휘발성 물질의 성분을 분석하기 위하여， DeMilo et al. (1996)의 방법을 변형하여 휘발성 물질을 포집하였다. Table 3
Figure imgf000021_0001
As shown in Table 3, Fujium oxysporum f. S p. The mycelial growth of all pathogenic bears except Raphani ()) was inhibited by the volatiles produced by the Lysinibacilli sparycus TC1 strain. Example 10 Component Analysis of Volatile Substances Produced by Ricinivacillus spalicus TC1 Strains In order to analyze the components of volatiles produced by the Ricinibacillus sparycus TC1 strain of the present invention, DeMilo et al. (1996) was modified to capture volatiles.
리시니바실러스 스패리쿠스 TC1 균주의 단일 콜로니를 트립틱 소이 액체배지에 접종한 후 30°C에서 150 rpm으로 1일 진탕배양한 후, 500 ml 삼각플라스크에 100 ml의 TSB배지를 넣고 상기 균주를 1%접종하였다. 투 -웨이 고무 코르크 (Two-way rubber cork)를 이용하여 하나의 선에는 배양액으로부터 lcm 높이의 위치로 설정하였으며 휘발성 물질을 포집할 수 있도록 지름 1cm 및 높이 15cm의 유리관과 연결하였고， 다른 하나의 선은 삼각플라스크 배양액으로부터 10cm의 높이로 설정하였으며 외부로부터 공기가 주입될 수 있도록 설정하였다. 이때 유리관 안에는 150 mg의 차콜로 채운 후 휘발성 물질을 포집하도톡 하였다. 30 및 150 rpm으로 3일간 배양한 후 차콜을 수확하였으며， 메탄올에 용해한 후 여과하여 순수한 휘발성 물질만을 획득하였다. Lee shinny Bacillus sphaericus were inoculated single colonies of TC1 strain in Tryptic Soy Broth and incubated 1 days of shaking at 30 ° C with 150 rpm, into a TSB culture medium of 100 ml in 500 ml Erlenmeyer flask and the strain 1% inoculation. Using a two-way rubber cork, one line was set at lcm height from the culture and connected to a glass tube 1cm in diameter and 15cm in height to capture volatiles. Was set to a height of 10cm from the Erlenmeyer flask culture and set so that air can be injected from the outside. The glass tube is filled with 150 mg of charcoal and then volatile The material was trapped. After incubation at 30 and 150 rpm for 3 days, the charcoal was harvested, dissolved in methanol and filtered to obtain pure volatiles only.
수득한 휘발성 물질의 성분 분석은 GC/MS를 이용하여 다음과 같이 측정하였다. GC/MS기기는 HP 7890A질량분석 검출기 (Mass selective deterctor, Agilent Technologies Inc., Chandler, AZ, USA)가 장착된 GC/MS 기기를 사용하였으며， 분리용 칼럼은 DB-WAX(30 mX 0.25 mm, I.d., 0.25 mm필름 두께， J&W, Folsom, CA, USA)를 사용하였다. 이때 오본의 온도는 30°C에서 5분간 유지한 다음 10°C/분의 속도로 250°C까지 상승시켰으며, 캐리어 기체 (carrier gas)의 유속은 1ml/분 (He)로 유지하였다.분리된 화합물의 이은화는 전자 충격 이은화 (electron impact ionization, EI -mode) 방법으로 수행하였으며 이온화 전압 (ionization voltage)과 이온 소스 (ion source)의 온도는 각각 70 eV와 230°C로 설정하였고， 분석할 분자량의 범위는 40-350 (m/z)로 설정하였으며， GC 투입기 (injector)의 온도는 250°C로 설정하였다. Component analysis of the obtained volatiles was measured as follows using GC / MS. The GC / MS instrument was a GC / MS instrument equipped with an HP 7890A mass spectrometer (Mass selective deterctor, Agilent Technologies Inc., Chandler, AZ, USA). The separation column was DB-WAX (30 mX 0.25 mm, Id, 0.25 mm film thickness, J & W, Folsom, CA, USA). At this time, the temperature of Aubon was maintained at 30 ° C. for 5 minutes and then increased to 250 ° C. at a rate of 10 ° C./min, and the flow rate of carrier gas was maintained at 1 ml / min (He). The silverization of the compounds was carried out by electron impact ionization (EI-mode) method, and the ionization voltage and ion source temperature were set at 70 eV and 230 ° C, respectively. The molecular weight range was set to 40-350 (m / z) and the temperature of the GC injector was set to 250 ° C.
GC/MS에 의해 총 이은화 크로마토그램 (total ionization chromatogram, Total ionization chromatogram, by GC / MS
TIC)에 분리된 각 피크의 휘발성 성분 분석은 질량 스펙트럼 라이브러리 (mass spectrum library, Wiley 275&N, NBS 75K)와 질량 스펙트럼 데이터 북 (mass spectral data book)의 스펙트럼， 및 문헌상의 보류 지수 (retention index)등을 근거로 하였으며, 머무름 시간 및 면적율에 따른 물질 동정 결과를 하기 표 4에 나타내었다. Volatile component analysis of each peak separated in the TIC) includes the mass spectrum library (Wieley 275 & N, NBS 75K), the spectra of the mass spectral data book, and the retention index in the literature. Based on the results, the material identification results according to the retention time and area ratio are shown in Table 4 below.
【표 4] [Table 4]
Figure imgf000022_0001
표 4에서 나타낸 바와 같이， 본 발명의 리시니바실러스 스패리쿠스 TC1 균주로부터 생산되는 휘발성 물질은 테트라클로로에틸렌， 3-메틸 -1-부탄올， 메틴피라진, 사이클로핵사논, 2 ,5-디메틸피라진， Ν,Ν-디메틸포름아마이드， 및 2-(2-에특시에특시) -에탄올임을 알 수 있었다. 실시예 11: 바실러스 벨레젠시스 G341 균주와 리시니바실러스 스패리쿠스 TC1균주의 흔합 액체 배양액 제조 ¾ 두 균주의 액체 배양액을 제조하기 위하여, -80°C에서 보관하던 바실러스 벨레젠시스 G341 균주와 리시니바실러스 스패리쿠스 TC1 균주를 트립틱소이한천배지 (TSA)에 접종한 후 30°C에서 1일간 배양하였다. 각각의 균주로부터 단일 콜로니를 떼어 트립틱소이액체배지 (TSB)에 접종한 후 30°C에서 150 rpm으로 24시간 배양한 배양액을 접종원으로 사용하여 500 ml 삼각플라스크에 200 ml의 TSB가 들어있는 배지에 최종 1%가 되도톡 동시에 접종하였다. 30°C에서 150 rpm으로 3일간 배양한 후 획득한 두 균주의 흔합 배양액을 이후 실험에 사용하였다. 실시예 12: 흔합배양액의 토마토풋마름병 방제 효과 상기 실시예 11에서 제조한 두 균주의 흔합 배양액을 이용하여 랄스토니아 솔라나세아럼
Figure imgf000023_0001
solanacearum)^] 의한 토마토 풋마름병 발병 억제효과를 토양관주 방법으로 검정하였다.
Figure imgf000022_0001
As shown in Table 4, the volatiles produced from the Ricinibacillus sparycus TC1 strain of the present invention are tetrachloroethylene, 3-methyl-1-butanol, methinepyrazine, cyclonuxanone, 2,5-dimethylpyrazine, It was found that Ν, Ν-dimethylformamide and 2- (2-especially) -ethanol. Example 11 Preparation of a Mixed Liquid Culture of Bacillus Velensis G341 Strains and Ricinibacillus Sparicus TC1 Strains ¾ To prepare liquid cultures of two strains, Bacillus Velensis G341 strains and lis stored at −80 ° C. Cinibacillus spalicus TC1 strain was inoculated in tryptic agar medium (TSA) and then incubated at 30 ° C for 1 day. Single colony from each strain was inoculated in tryptic soy liquid medium (TSB) and cultured for 24 hours at 150 rpm at 30 ° C as a source of inoculum using 200 ml of TSB in a 500 ml Erlenmeyer flask Was inoculated simultaneously at the final 1%. Mixed cultures of the two strains obtained after incubating at 150 rpm at 30 ° C for 3 days were used in the experiments later. Example 12 Effect of Controlling Tomato Foot Blight on Mixed Culture Solution Ralstonia solanacerum using the mixed culture medium of two strains prepared in Example 11
Figure imgf000023_0001
Solanacearum) ^] inhibited the effects of tomato foot blight on the soil by irrigation method.
토마토 풋마름병 발병 유도 시， 병원균을 처리하기 3일 전에 G341 및 TC1 균주를 각각 1/2배로 회석한 후， 각각의 균주 단독 배양액 (G341군 및 TC1군)， 및 G341 및 TC1 균주를 2:1, 1:1 및 1:2로 달리 흔합한 흔합 배양액을 각각 포트당 20 ml씩 토양에 관주처리한 것을 제외하고는， 상기 실시예 4에 기재된 것과 동일한 방식으로 풋마름병 발병률을 측정하였다. 그 결과는 도 6에 나타내었다. W At the induction of tomato foot blight, G341 and TC1 strains were halved three times each before treatment of the pathogen, and each strain alone (G341 and TC1 groups), and G341 and TC1 strains were 2: 1. The incidence of foot blight was measured in the same manner as described in Example 4, except that the mixed cultures were mixed in different amounts of 1: 1 and 1: 2 in the soil by 20 ml per pot, respectively. The results are shown in FIG. W
도 6에서와 같이， 바실러스 벨레젠시스 G341균주 배양액 (1/2배 희석액) 단독 처리군의 경우 47%의 방제효과를 보였고, 리시니바실러스 스패리쿠스 TC1 균주 배양액 (1/2배 희석액)단독 처리군의 경우 80%의 방제효과를 보였다. 반면 두 균주의 배양액 (1/2배 희석액)을 2:1， 1:1 및 1:2의 비율로 흔합 처리한 흔합군으로부터는 모두 80% 이상의 높은 방제효과를 보였으며， 특히 바실러스 벨레젠시스 G341 균주와 리시니바실러스 스패리쿠스 TC1 균주의 배양액을 1:1 비율로 흔합하여 처리한 경우 가장 높은 방제가， 즉 100%의 방제효과를 나타냄을 알 수 있었다. As shown in Fig. 6, the Bacillus velensis G341 strain culture (1 / 2-fold dilution) alone treatment group showed a 47% control effect, Ricinibacillus spalicus TC1 strain culture (1 / 2-fold dilution) alone The treatment group showed 80% control effect. On the other hand, the mixed group of two strains (1 / 2-fold dilution) at the ratios of 2: 1, 1: 1 and 1: 2 showed a high control effect of more than 80%, especially Bacillus Velensis When the mixture of the G341 strain and the Lysinibacillus sp. Liqueur TC1 strain was treated in a 1: 1 ratio, it was found that the highest control, that is, 100% control effect was obtained.
이 실험을 통하여, 두 균주의 흔합 배양액을 특히 1:1 비율로 흔합하여 처리할 경우 가장 높은 효과를 보이는 것을 확인할 수 있었으며， 이후의 모든 식물병에 대한 방제효과는 바실러스 벨레젠시스 G341 균주와 리시니바실러스 스패리쿠스 TC1 균주를 1:1의 비율로 흔합한 흔합 배양액을 이용하여 실험을 실시하였다. 실시예 13: 흔합 배양액의 고추 역병 방제 효과 상기 실시예 11에서 제조한 흔합 배양액을 이용하여 파이토프토라 캡사이시 (/¾Κ θρΑ ΛθΓ3 에 의한 고추 역병 발병 억제효과를 토양관주 방법으로 검정하였다.  Through this experiment, it was confirmed that the mixed cultures of the two strains showed the highest effect especially when mixed and treated in a 1: 1 ratio, and the control effect on all subsequent plant diseases was the Bacillus Velensis G341 strain and Lee. The experiment was carried out using a mixed culture solution of the Synibacillus spalicus TC1 strain in a ratio of 1: 1. Example 13: Effect of controlling pepper late blight of mixed culture medium The inhibitory effect of pepper late blight onset by phytophthora capsaici (/ ¾Κ θρΑ ΛθΓ3) was assayed using the mixed culture medium prepared in Example 11 by soil irrigation method.
구체적으로, 부강 고추 유묘를 사용하여 지름 7.0 cm인 플라스틱 포트 내의 원예용 상토에 이식하여 6엽기까지 키운 후， G341 균주 및 TC1 균주 배양액을 단독으로 또는 1:1 비율로 흔합한 흔합 배양액 20 ml를 포트당 20 ml씩 토양에 관주처리하였다. 배양액 처리 3일 후에 고추 ^^^ Phytophthora capsici)^ 유주자낭 현탁액 (lx 104 유주자낭 /ιι )을 각각의 포트에 20 1 씩 관주 처리함으로써 식물 병원균을 접종하였다. 고추 역병의 발병 억제 효과는 10일간 관찰하였다. 이때, 비교를 위해 대조구 (무처리구)는 배양액 대신 증류수만을 처리하여 상기와 동일한 방식으로 수행하였으며, 각 처리당 10본씩 2회 반복 실험하였다. 고추 역병 발병률은 식물이 아주 싱싱한 경우는 0， 잎이 약간 시든 경우 (25¾» 미만)는 1, 중간 정도 시든 경우 (50% 미만)는 2， 심하게 시든 경우 (75% 정도)는 3， 완전히 시들어 고사한 경우를 4로 등급을 정하였으며, 이를 근거로 발병 억제율은 { (대조구의 발병를-처리구의 발병를 )/대조구의 발병률 }x 100으로 계산하였다. Specifically, after transplanting to gardening topsoil in plastic pots with a diameter of 7.0 cm using Bujin pepper seedlings to grow up to six leaves, 20 ml of mixed culture medium of G341 and TC1 strains alone or in a 1: 1 ratio The soil was irrigated 20 ml per pot. Three days after the culture treatment, red pepper ^^^ Phytophthora capsici) ^ scapula sac suspension (lx 10 4 saponica sac / ιι) was inoculated with each plant by 20 1 irrigation to inoculate plant pathogens. Inhibitory effect of pepper blight was observed for 10 days. At this time, for comparison, the control (no treatment) was performed in the same manner as above by treating only distilled water instead of the culture, and repeated 10 experiments for each treatment. The incidence of pepper blight is 0 when the plant is very fresh, 1 when the leaf is slightly soaked (less than 25¾ »), 2 when it is moderately soaked (less than 50%) and 3 when it is severely soaked (about 75%). The death rate was rated as 4, and on this basis, the inhibition rate was calculated as {(onset control-onset of treatment) / onset of control} x 100.
도 7에서 보는 바와 같이 바실러스 벨레젠시스 G341 균주 및 리시니바실러스 스패리쿠스 TC1 균주의 배양액의 단독 처리구는 각각 38% 및 58%의 방제효과를 보인 반면， 1:1흔합 배양액 처리구는 79)의 높은 방제효과를 보였다. 이를 통하여 두 균주의 흔합 배양액이 단독 배양액보다 고추 역병에도 높은 효과를 보인다는 사실을 발견하였다. 실시예 14: 흔합배양액 분무 건조물의 토마토풋마름병 방제 효과 상기 실시예 11에서와 같이， 두 균주의 각각의 배양액 1 L에 10 g의 화이트카본 및 10 g의 덱스트린을 첨가하여 섞은 후 주입구 (inlet) 온도 165°C와 배출구 (outlet) 온도 94°C로 분무 건조하여 분무 건조 시료를 제조하였다. As shown in FIG. 7, the treatment alone of the culture medium of the Bacillus bellensis G341 strain and the Lysinibacilli sp. Liqueur TC1 strain showed 38% and 58% control effects, respectively, whereas the 1: 1 mixed culture treatment group was 79). High control effect was shown. Through this, it was found that the mixed culture of the two strains showed a higher effect on the pepper blight than the single culture. Example 14 Effect of Controlling Tomato Foot Blight on Mixed Culture Spray Spray Drying As in Example 11, 10 g of white carbon and 10 g of dextrin were added to 1 L of each culture of the two strains, followed by inlet. Spray dried samples were prepared by spray drying at a temperature of 165 ° C. and an outlet temperature of 94 ° C.
이렇게 제조한 분무 건조 시료를 50배 희석한 회석액을 상기 실시예 12에 기재된 것과 동일한 방식으로 토마토 풋마름병에 대한 단독 처리 및 흔합 처리에 의한 방제효과를 조사하여 그 결과를 도 8에 나타내었다.  The dilution solution 50 times diluted with the spray dried sample thus prepared was examined in the same manner as described in Example 12 to investigate the control effect by the single treatment and the mixed treatment on tomato foot blight, and the results are shown in FIG. 8.
도 8에서 나타난 바와 같이， 리시니바실러스 스패리쿠스 TC1 균주의 단독 배양액도 방제효과가 우수하지만， 벨레젠시스 G341 균주 배양액과의 흔합물이 더 강한 방제효과를 보임을 알 수 있었다. 실시예 15: 흔합배양액의 항세균활성 효과 두 균주의 단독 배양액 및 흔합 배양액에서의 항세균활성 물질의 생산량을 확인하기 위하여， in vitro 항세균 활성을 랄스토니아 솔라나세아럼 균주를 이용하여 조사하였다. As shown in FIG. 8, the single culture medium of the Lysinibacillus spalicus TC1 strain was also excellent in the control effect, but the mixture with the Belizesis G341 strain culture medium showed a stronger control effect. Example 15 Antibacterial Activity Effect of the Mixed Culture Solution In order to confirm the production of the antibacterial active material in the single culture and the mixed culture of the two strains, the in vitro antibacterial activity was measured by Ralstonia. Investigations were carried out using Solanaserum strains.
상기 실시예 11에서 기재된 것과 동일한 방식으로 두 균주를 전배양하였다. 이어, 단독 배양액의 경우 각각의 전배양액 1%를 트립틱소이액체배지에 접종하고， 흔합 배양액의 경우 각각의 전배양액에서 0.5%씩 취하여 하나의 삼각 플라스크 내의 TSB 배지에 접종하였다. 이렇게 접종한 배지는 모두 30°C에서 150rpm으로 3일간 배양하였다.3일간 배양하면서 매 12시간마다 1 ml씩 수확하여 막으로 여과하여 제균한 다음 항세균 활성을 조사하였다. Both strains were precultured in the same manner as described in Example 11 above. Subsequently, 1% of each preculture was inoculated in tryptic soy medium for single culture, and 0.5% of each preculture was inoculated in TSB medium in one Erlenmeyer flask for mixed culture. All the inoculated medium was incubated at 30 ° C. at 150 rpm for 3 days. During 3 days, 1 ml of every 12 hours was harvested, filtered through a membrane, and sterilized, and then examined for antibacterial activity.
조사 방법은， 랄스토니아솔라나세럼 균주가 0.5X 108 CFU/ml 수준으로 제조된 현탁액을 1% 접종한 트립틱소이한천배지가 담긴 페트리 디쉬 상에 지름 8 隱의 페이퍼 디스크 (paper disc)를 치상한 후 각각의 디스크에 30 ^씩 배양여액을 점적하고， 30°C에서 2일간 배양한 후 저지원의 지름을 측정하는 방식으로 수행되었다. 그 결과를 도 9에 나타내었다. Investigation method, paper disc diameter of 8 mm on a Petri dish containing tryptic soy agar medium inoculated with a 1% inoculation of the Ralstonia solana serum strain prepared at a level of 0.5 × 10 8 CFU / ml. After quenching was added to the culture filtrate 30 ^ each disc, incubated for 2 days at 30 ° C was carried out by measuring the diameter of the low support. The results are shown in FIG.
도 9에 나타난 바와 같이 바실러스 벨레젠시스 G341균주 (최대 약 1.05 cm), 및 두 균주의 흔합 배양액 (G&T)에서는 강한 항세균활성을 보였다.특히 두 균주를 흔합한 흔합 배양액에서 더 강한 항세균활성 (최대 약 1.55 cm)을 보였다.  As shown in Fig. 9, the bacterium B. regensis G341 strain (up to about 1.05 cm), and the mixed cultures (G & T) of the two strains showed strong antibacterial activity. In particular, the stronger antibacterial activity in the mixed cultures of the two strains (Up to about 1.55 cm).
이 실험은 G341균주는 비휘발성 항세균활성 물질을 생산하며， 또한 바실러스 벨레젠시스 G341 균주는 리시니바실러스 스패리쿠스 TC1 균주와 함께 존재할 때 더 많은 양의 비휘발성 항세균활성 물질을 생산한다는 것을 나타낸다. 실시예 16: 흔합배양액, 균체 및 상층액의 토마토풋마름병 방제 효과 상기 실시예 15에 기재된 것과 동일한 방식으로 배양한' 두 균주의 흔합 배양액의 토마토 풋마름병에 대한 방제효과를 조사하였다.  This experiment shows that strain G341 produces nonvolatile antibacterial actives, and that the Bacillus Velensis G341 strain produces higher amounts of nonvolatile antibacterial actives when present with the Lysinibacilli sp. Indicates. Example 16: Control effect of tomato foot blight on mixed culture medium, cells and supernatant The control effect of the mixed culture of two strains cultured in the same manner as described in Example 15 on the tomato foot blight was investigated.
흔합 배양액， 흔합 배양액을 제거한 균체 및 균체를 제거한 상층액을 비교하는 방식으로 실험을 수행하였으며， 토마토 풋마름병 발병방법 및 방제효과를 확인하기 위한 시료처리 방법은 상기 실시예 12에 기재된 방식과 동일하게 수행하였다. The experiment was carried out by comparing the mixed culture medium, the cells from which the mixed culture medium was removed, and the supernatant from which the cells were removed. The sample treatment method for confirming the control effect was performed in the same manner as described in Example 12.
그 결과， 도 10에서 나타난 바와 같이， 두 균주의 흔합 배양액에서도 방제효과가 나타났지만 특히 균체를 제거한 상층액에서 가장 우수한 방제효과를 확인할 수 있었으며， 균체만 처리했을 경우에는 방제 효과가 전혀 없음을 알 수 있었다.  As a result, as shown in Figure 10, the control effect appeared in the mixed culture of the two strains, in particular, the best control effect was confirmed in the supernatant from which the cells were removed, it was found that there is no control effect when only the cells treated Could.
하지만 본 실험의 결과가 농가 포장에서도 균주의 역할이 미미하다는 것을 시사하는 것은 아니다. 왜냐하면 바실러스 벨레젠시스 G341 균주의 경우 식물내생미생물이기 때문에 작물의 뿌리에서 서식하면서 꾸준히 항균물질을 생산하여 약효를 나타낼 수 있으며, 특히 바실러스 벨레젠시스 G341 균주는 리시니바실러스 스패리쿠스 TC1 균주와 함께 있을 때 항세균활성 물질의 생산량이 증가하므 S 균체도 포장에서 방제효과를 보이는데 큰 역할을 할 것이라 판단된다. 실시예 17: 흔합배양액 분무건조 시료의 토마토풋마름병 방제에 대한 치료또는 예방효과 상기 실시예 15에 기재된 것과 동일한 방식으로 제조한 두 균주의 흔합 배양액을 사용하여 상기 실시예 14에 기재된 방식에 따라 분무 건조 시료를 제조하였으며， 이를 이용하여 토마토 풋마름병에 대한 방제 효과를 조사하였다.  However, the results of this experiment do not suggest that the role of the strain in farm packaging. Because the Bacillus Velensis G341 strain is an endogenous microorganism, it can show its efficacy by steadily producing antimicrobial substances while inhabiting the roots of crops. When present, the production of antimicrobial active substances increases, so it is expected that S cells also play a significant role in controlling the packaging. Example 17 Treatment or Preventive Effect of Mixed Culture Spray Spray Drying Sample on Tomato Foot Blight Control Sprayed according to the method described in Example 14 above using a mixed culture of two strains prepared in the same manner as described in Example 15 above. A dry sample was prepared and used to investigate the control effect on tomato green blight.
분무 건조 시료는 100배 또는 50배로 증류수로 희석한 후 토마토 풋마름병균 (랄스토니아 솔라나세아럼)을 접종하기 7일 전， 3일 전， 1일 전 및 1일 후에 관주 처리하였다. 병원균을 접종한 지 10일 후에 발병도를 조사하였고， 처리당 5반복에 2회 반복 실험하여 그 결과를 도 11에 나타내었다.  Spray dried samples were diluted 100- or 50-fold with distilled water and then irrigated 7 days, 3 days, 1 day and 1 day after inoculation of tomato fusiform bacillus (Ralstonia solanacerum). The incidence was examined 10 days after inoculation of the pathogen, and the results were shown in FIG. 11 by repeating the experiment twice with 5 repetitions per treatment.
도 11에 나타난 바와 같이，흔합 배양액의 분무건조 시료는 병원균 접종 As shown in Figure 11, spray dried sample of the mixed culture solution pathogen inoculation
3일전 처리시 가장 높은 방제효과를 보였다. 이 실험 결과 두 균주의 흔합 배양액으로 제조한 미생물 제제는 치료 효과보다는 예방 효과가 더 크다는 사실을 알게 되었다. 이에 따라 본 발명의 흔합 배양액 제품을 포장에 적용할 경우， 식물병 발병 전이나 초기에 약제를 처리하는 것이 방제효과를 높일 수 있다는 것을 나타낸다. 실시예 18: 바실러스 벨레젠시스 G341 균주 배양액 에틸아세테이트 추출물의 토마토풋마름병 방제효과 상기 실시예 15에서 확인되는 바와 같이， 리시니바실러스 스패리쿠스 TC1 균주는 비휘발성 항세균활성 물질을 생산하지 않은 반면， 바실러스 벨레젠시스 G341 균주는 비휘발성 항세균활성 물질을 생산한다. Three days before treatment, the highest control effect was obtained. The result of this experiment is the combination of two It was found that the microbial preparations prepared from the cultures were more prophylactic than therapeutic. Accordingly, when the mixed culture product of the present invention is applied to packaging, it is indicated that treatment with drugs before or during plant disease can enhance the control effect. Example 18 Tomato Foot Blight Control Effect of Ethyl Acetate Extract from Bacillus Velensis G341 Strain Culture As confirmed in Example 15 above, Ricinibacillus spalicus TC1 strain did not produce nonvolatile antibacterial activity , Bacillus Velensis G341 strain produces a nonvolatile antibacterial active material.
바실러스 벨레젠시스 G341균주가 생산하는 비휘발성 항세균활성 물질을 분리하기 위하여 바실러스 벨레젠시스 G341 균주의 배양 상징액을 동량의 에틸아세테이트를 이용하여 2회 분획 추출하였다. 상기에서 수득한 에틸아세테이트층을 감압농축한 후 메탄올로 용해하고 트원 20용액 (250 ppm)에 희석한 후 토마토 풋마름병균 접종 3일전에 33.3 yg/ml, 1000 yg/ml 및 2000 yg/ml의 양으로 토마토에 관주처리하였다. 병원균 접종 10일 후에 발병도를 조사하였고, 처리당 5반복에 2회 반복 실험하였다.  In order to isolate the nonvolatile antibacterial active material produced by the Bacillus Velensis G341 strain, the culture supernatant of the Bacillus Velensis G341 strain was extracted twice using the same amount of ethyl acetate. The ethyl acetate layer obtained above was concentrated under reduced pressure, dissolved in methanol, diluted in 20 solution (250 ppm) of 3, and 33.3 yg / ml, 1000 yg / ml and 2000 yg / ml three days before inoculation The tomatoes were irrigated with sheep. The incidence was examined 10 days after inoculation of the pathogen and repeated twice with 5 replicates per treatment.
도 12에 나타난 바와 같이， 바실러스 벨레젠시스 G341 균주 배양액의 에틸아세테이트 추출물은 토마토 풋마름병에 대하여 농도 의존적으로 방제효과를 보였다. 실시예 19: 바실러스 벨레젠시스 G341 균주로부터 비휘발성 항세균활성 성분의 분리 및 동정 바실러스 벨레젠시스 G341균주가 생산하는 비휘발성 항세균활성 물질을 분리하기 위하여， 상기 균주를 200 ml의 트립틱소이액체배지에 접종한 다음， As shown in FIG. 12, the ethyl acetate extract of Bacillus bellensis G341 strain culture showed a concentration-dependent control effect against tomato foot blight. Example 19 Isolation and Identification of Nonvolatile Antibacterial Active Ingredients from Bacillus Velensis G341 Strain In order to isolate the nonvolatile antibacterial active material produced by the Bacillus Velensis G341 strain, the strain was 200 ml of tryptic soy. After inoculating liquid medium,
30°C에서 3일간 150rpm으로 진탕 배양하였다.상기에서 수득한 배양액을 8,000 g에서 10분 원심분리하여 배양 상징액 (3 L)를 확득한 다음, 이를 동량의 에틸아세테이트로 순차적으로 2회씩 분획하였다. 얻어진 에틸아세테이트층을 감압농축하여 550 mg의 분획물을 얻었다. 이를 이용하여 역상 컬럼인 sep-pak에 가한 다음 100% 물에서 100% 메탄을로 농도를 조절하여 용출하였다. Shake culture was carried out at 150 rpm for 3 days at 30 ° C. The culture solution obtained above was 8,000 After centrifugation at g for 10 minutes to obtain the culture supernatant (3 L), it was fractionated twice in sequence with the same amount of ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure to obtain 550 mg of a fraction. Using this was added to the reverse phase sep-pak eluted by adjusting the concentration to 100% methane in 100% water.
랄스토니아 솔라나세아럼에 대해 항균활성을 나타내는 분획물을 모아 감압농축하여 240 mg의 용출물을 획득하였다. 세파덱스 컬럼을 통하여 획득한 분획물을 10 mg/ml 수준으로 메탄올로 용해한 다음 페이퍼 디스크에 30 μ 1씩 점적 후 건조하고, 랄스토니아 솔라나세아럼이 접종된 트립틱소이한천배지에 치사하였다. 30°C에서 배양하면서 항균활성을 나타내는 부분의 지름을 측정하였다. 무처리구의 경우 메탄올만 30 μ ΐ 처리하였다. Fractions showing antimicrobial activity against Ralstonia solanacerum were collected and concentrated under reduced pressure to obtain 240 mg of eluate. The fraction obtained through the Sephadex column was dissolved in methanol at the level of 10 mg / ml, then dipped in a 30 μ 1 drop on a paper disk, and dried, and then killed in a tryptic soy agar medium inoculated with Ralstonia solanacerum. The diameter of the part showing antimicrobial activity was measured while incubating at 30 ° C. In the untreated section, only 30 μl of methanol was treated.
얻어진 활성 분획물 6.4 mg으로부터 활성물질을 분리하기 위해 클로로포름: 메탄올: 물 용매 (65:25:4)를 이용하여 분취용 TLC에 전개하였다. 최종적으로 활성물질 2개 (화합물 1과 2)를 분리하였다. 상기에서 분리된 활성물질 중 화합물 1의 구조를 분석하기 위하여， Development of preparative TLC was carried out using chloroform: methanol: water solvent (65: 25: 4) to separate the actives from 6.4 mg of the active fraction obtained. Finally, two active substances (compounds 1 and 2) were separated. In order to analyze the structure of compound 1 in the active substance separated from the above,
UV-Vis 스펙트럼과 EI/MS를 이용하였다. 화합물 1을 메탄올에 용해한 후 흡수 스펙트럼을 측정한 결과， 233 nmCband I)와 270 nm(band II)에서 최대 흡광도를 보였다 (도 13). 화합물 1의 분자량은 EI/MS를 이용하여 측정하였으며， 질량 분석결과 z 559 [Μ-ΗΓ의 분자이온 피크가 관측되어 분자량올 560으로 결정하였다. 또한, 화합물 1의 NMR 결과는 바실러스 서브틸리스 Siyb /7/s)로부터 생산되는 물질인 옥시디피시딘 (oxydifficidin)의 결과와 일치함을 확인함으로써 화합물 1을 옥시디피시딘으로 동정하였다. UV-Vis spectra and EI / MS were used. After dissolving Compound 1 in methanol, absorption spectra were measured to show maximum absorbance at 233 nmCband I) and 270 nm (band II) (FIG. 13). The molecular weight of Compound 1 was measured using EI / MS, and the molecular ion peak of z 559 [Μ-ΗΓ was observed as a result of mass spectrometry to determine the molecular weight of 560. In addition, NMR results of compound 1 was identified as oxydipicidin by confirming that the results are consistent with the results of oxydifficidin, a material produced from Bacillus subtilis Siyb / 7 / s).
한편, 화합물 2의 구조를 분석하기 위하여， UV-Vis 스펙트럼과 EI/MS를 이용하였다.화합물 2를 메탄을에 용해한 후 흡수 스펙트럼을 측정한 결과, 233 nmCband I)와 270 nmCband Π)에서 최대 흡광도를 보였다 (도 13). 화합물 2의 분자량은 EI/MS를 이용하여 측정하였으며, 질량 분석결과 m/z 544 [M-H]ᅳ의 분자이은 피크가 관측되어 분자량을 544로 결정하였다. 또한, 화합물 2의 NMR 결과는 바실러스 서브틸리스로부터 생산되는 물질인 디피시딘 (difficidin)의 결과와 일치함을 확인함으로써 화합물 2를 디피시딘으로 동정하였다. In order to analyze the structure of Compound 2, UV-Vis spectrum and EI / MS were used. As a result of measuring absorption spectrum after dissolving Compound 2 in methane, the maximum absorbance at 233 nmCband I) and 270 nmCband Π) was obtained. (FIG. 13). The molecular weight of Compound 2 was measured using EI / MS. As a result of mass spectrometry, a molecular peak of m / z 544 [MH] was observed and the molecular weight was determined to be 544. In addition, NMR of compound 2 Compound 2 was identified as dipidine by confirming that the results were consistent with the results of dipicidin, a substance produced from Bacillus subtilis.
두 화합물의 구조는 도 14에 기재된 바와 같으며, 도 14에서 R이 H인 경우 디피시딘을 나타내고 R이 0H인 경우 옥시디피시딘을 나타낸다. 실시예 20: 바실러스 벨레젠시스 G341 균주 배양액으로부터 분리한 화합물의 항균활성 상기 실시예 19에서 분리한 항균물질이 식물병 세균에 항균 활성을 나타내는지 확인하기 위하여， 대표적인 그람음성세균 및 그람양성세균을 대상으로 96-웰 플레이트를 이용하여 항세균활성을 조사하였다.  The structures of the two compounds are as described in FIG. 14, and in FIG. 14 represent dipicidine when R is H and oxydipicidine when R is 0H. Example 20 Antimicrobial Activity of Compounds Isolated from Bacillus Velensis G341 Strain Culture Medium To determine whether the antimicrobial material isolated in Example 19 exhibited antimicrobial activity against plant disease bacteria, representative Gram-negative bacteria and Gram-positive bacteria were Antibacterial activity was examined using 96-well plates.
구체적으로， 하기 표 5에 기재된 바와 같은 각각의 식물병 세균의 단일 콜로니를 트립틱 소이 액체 배지에 접종한 후， 30°C 및 150 rpm으로 1일 내지 2일 진탕 배양하여 준비하였다. 준비된 식물병 세균은 IX 108 CFU/ml로 조절하여 현탁액을 제조한 후 멸균한 TSB 배지로 1,000배 희석하였다. 세균이 접종된 배지를 100 μΐ씩 96-웰 플레이트에 분주한 후， 상기 실시예 19에서 분리한 두 화합물 (옥시디피시딘 및 디피시딘)을 메탄을과 물의 흔합액으로 용해한 후 최종 농도가 200， 100， 50， 25, 12.5, 6.25， 3.12， 1.56 및 0.78 yg/ml이 되도록 처리하였다. 이때, 무처리구는 메탄올만 최종농도가 0.5¾가 되도록 처리하였다. 처리당 3반복으로 실험을 실시하였으며， 30°C에서 1일 내지 2일 배양하면서 MIC 및 IC50을 측정하여 그 결과를 표 5에 나타내었다. Specifically, a single colony of each plant disease bacterium as described in Table 5 below was inoculated in tryptic soy liquid medium, and then prepared by shaking culture for 1 to 2 days at 30 ° C and 150 rpm. Prepared plant disease bacteria were adjusted to IX 10 8 CFU / ml to prepare a suspension and diluted 1,000-fold with sterile TSB medium. After dispensing the bacteria-inoculated medium into a 96-well plate by 100 μΐ, the two compounds (oxydipicidine and dipidine) isolated in Example 19 were dissolved in a mixture of methane and water, and the final concentration was 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 yg / ml. At this time, the treatment was treated so that only the final concentration of 0.5 ¾ methanol. The experiment was carried out in three repetitions per treatment, and MIC and IC 50 were measured while incubating at 30 ° C. for 1 to 2 days, and the results are shown in Table 5.
【표 5】 Table 5
Figure imgf000031_0001
그 결과 표 5에서 나타낸 바와 같이， 두 화합물은 그람 양성 세균과 그람 음성 세균 둥 다양한 식물병 세균에 대해 항균활성을 나타내는 것을 확인할 수 있었으며， 특히 랄스토니아 솔라나세아럼에 대해 우수한 항균활성을 보이는 것을 알 수 있었다. 또한， 옥시디피시딘에 비해 디피시딘이 좀 더 넓은 항균활성 스펙트럼을 갖는 것으로 나타났다. 실시예 21: 바실러스 벨레젠시스 G341 균주가 생산하는 휘발성 물질의 항세균 활성 바실러스 벨레젠시스 G341 균주의 휘발성 물질의 항세균 활성을 페트리디쉬를 이용하여 조사하였다.
Figure imgf000031_0001
As a result, as shown in Table 5, it was confirmed that the two compounds exhibit antimicrobial activity against gram-positive bacteria and Gram-negative bacteria, and various plant disease bacteria, and particularly shows excellent antimicrobial activity against Ralstonia solanacerum. I could see that. In addition, difficidine was found to have a broader antimicrobial activity spectrum than oxydipicidine. Example 21 Antibacterial Activity of Volatile Substances Produced by Bacillus Velensis G341 Strain The antibacterial activity of the volatiles of Bacillus veregensis G341 strain was investigated using Petri dishes.
구체적으로，트립틱 소이 한천 배지를 페트리디쉬에 분주한 다음 배지의 중앙으로부터 1 cm의 배지를 완전히 제거하여 확산에 의하여 병원 세균의 생장이 억제되지 않도록 하였다. 실험에 사용하기 위하여， 랄스토니아 솔라나세아럼， 아시도보락스 아베내 아종 카를리애， 아그로박테리움 투메파시엔스， 버크홀더리아 글루매， 펙토박테리움 카로토보라 아종 카로토보라 펙토박테리움 크리산테미， 슈도모나스 시린개 변종 액티니데， 슈도모나스 시린개 변종 라크리만스， 산토모나스 악소노포디스 및 클라비박터 미시가넨시스 아종 균주는 트립틱소이액체배지에서 3(rc조건에서 1일간 배양되었다. Specifically, tryptic soy agar medium is dispensed into Petri dishes, and then 1 cm of medium is completely removed from the center of the medium to spread the pathogen. Growth was not inhibited. For use in experiments, Ralstonia Solanace Arum, Ashidovorax Avena subspecies Carlia, Agrobacterium tumefaciens, Berkholderia glutama, Pectobacterium cartobora subspecies Cartobora pectobacte Leeum chrysanthemi, Pseudomonas syringe strain Actinide, Pseudomonas syringe strain Lacrimans, Santomonas axonopodis and Clavibacter microgigansis subspecies strains were cultured on tryptic soy liquid medium (3 days under rc conditions). Incubated.
배지의 한쪽 면에， 상기 병원균의 현탁액 3 μ ΐ을 각각 취하여 약 1 cm 간격으로 점적 접종하였다. 반대편 배지에는 바실러스 벨레젠시스 G341 균주와 리시니바실러스 스패리쿠스 TC1 균주 배양액을 50 μ ΐ씩 플레이트의 표면에 균질하게 도말하였다. 휘발성 물질이 플레이트 밖으로 나가지 않도록 플레이트를 파라필름으로 밀봉한 다음 30°C에서 1일간 배양하였다. 모든 실험은 3회 실시하였으며 병원성 세균의 생장정도를 무처리구와 비교하여 저해 활성을 측정하였다. 실험결과는 하기 표 6에 나타내었다. On one side of the medium, 3 μl of the suspension of the pathogen was respectively inoculated and inoculated at intervals of about 1 cm. On the other side of the medium, 50 μl of Bacillus velogenis G341 strain and Ricinibacillus spalicus TC1 strain culture medium were plated homogeneously on the surface of the plate. The plate was sealed with parafilm to prevent volatiles from leaving the plate and then incubated at 30 ° C. for 1 day. All experiments were carried out three times and the inhibitory activity was measured by comparing the growth of pathogenic bacteria with no treatment. The experimental results are shown in Table 6 below.
【표 6] [Table 6]
Figure imgf000032_0001
표 6에 나타낸 바와 같이 바실러스 벨레젠시스 G341 균주는 다양한 식물병 세균에 대해 항균활성을 나타내는 휘발성 물질을 생산한다는 것을 확인할 수 있었으며，특히 아그로박테리움 투메파시엔스， 버크홀더리아 글루매， 랄스토니아 솔라나세아럼 및 클라비박터 미시가넨시스 아종 미시가넨시스에 대하여 높은 활성을 보였다. 실시예 22: 바실러스 벨레젠시스 G341 균주가 생산하는 휘발성 물질의 항진균활성 바실러스 벨레젠시스 G341 균주가 생산하는 휘발성 물질이 식물병 곰광이의 균사 생육에 미치는 영향을 살펴보기 위하여， 대표적인 식물병 곰광이균을 대상으로 곰광이 훈증 플레이트 분석 (mycofumigation plate assay) (Strobel et al. , 2001， Microbiology 147:2943— 2950)을 통해 확인하였다.
Figure imgf000032_0001
As shown in Table 6, it was confirmed that the Bacillus bellensis G341 strain produces a volatile substance exhibiting antimicrobial activity against various phytopathogenic bacteria. It showed high activity against Solanasearum and Clavibacter microgigansis subspecies microgigansis. Example 22 Antifungal Activity of Volatile Substances Produced by the Bacillus Velensis G341 Strain To examine the effect of volatiles produced by the Bacillus vegetans G341 strain on the mycelial growth of plant diseases, This was confirmed by mycofumigation plate assay (Strobel et al., 2001, Microbiology 147: 2943-2950).
실험 방법은 한 플레이트가 반으로 나누어진 I플레이트를 이용하였으며， 상기 플레이트의 한쪽 면에는 감자한천배지 (Potato Dextrose Agar, Bee ton and Dickinson Co. , Farnklins Lakes , NJ, USA)를 분주하였고， 다른 한쪽 면에는 균주는 트립틱소이한천배지를 분주하였다. 실험에 사용된 식물병 곰광이균은 리족토니아 솔라니 (RS), 마그나포르테 오리재 (M0), 보트리티스 씨네레아 (BC), 콜레토트리쿰 코코데스 (CC), 푸자리움 옥시스포룸 f. sp. 라파니 (F0), 스클레로티니아 스클레로티오룸 (SS), 파이토프토라 캡사이시 (PC) 및 파이토프토라 인페스탄스 (PI)를， 각각 감자한천배지 배지에서 25t에서 3일내지 7일간 배양한 후 사용하였다.  Experimental method used an I plate divided into one half plate, one side of the plate was dispensed potato agar medium (Potato Dextrose Agar, Bee ton and Dickinson Co., Farnklins Lakes, NJ, USA), and the other side In cotton, the strain dispensed tryptic agar medium. Phytopathogenic fungi used in the experiments were Lyactonia solani (RS), Magna Forte duck (M0), Botrytis cinerea (BC), Colletotricum cocodes (CC), Fuzarium oxysporum f . sp. Raphani (F0), Sclerotinia sclerothiorum (SS), phytoptotora capsaish (PC) and phytophthora infestans (PI), respectively, 25 to 3 days in potato agar medium It was used after culturing for 7 days.
구체적으로， 1일 동안 트립틱소이액체배지에서 30°C 및 150 rpm으로 전배양한 바실러스 벨레젠시스 G341 균주 배양액 50μ 1를 트립틱소이한천배지를 분주한 플레이트에 균질하게 도말하였다. 그리고 플레이트의 다른 한쪽 면에는， 감자한천배지 배지에서 왕성하게 자라고 있는 균주의 가장자리에서 지름 8 mm의 코르크 보어러를 이용하여 균체를 포함하고 있는 한천배지를 떼어낸 것을 접종하였다. 상기 플레이트를 파라 필름으로 밀봉한 다음 28°C에서 3-5일간 배양하면서 식물병 곰광이의 균사 생육저해 정도를 측정하였고 그 결과를 하기 표 7에 나타내었다. Specifically, 50 μl of Bacillus Velensis G341 strain culture medium precultured at 30 ° C and 150 rpm in a tryptic soy medium for 1 day was homogeneously plated on a plate dispensed with tryptic soy agar medium. And on the other side of the plate, they grow vigorously on potato agar badges. The agar medium containing the cells was inoculated using a cork bore with a diameter of 8 mm at the edge of the strain. The plate was sealed with a para film and then cultured at 28 ° C. for 3-5 days, and the degree of mycelial growth of the plant disease bear was measured. The results are shown in Table 7 below.
균사 생육 저해율은 (무처리구 병원균의 균사 지름 - 처리구 병원균의 균사 지름 )/무처리구 병원균의 균사 지름 X 100의 식에 따라 계산하였다.  The mycelial growth inhibition rate was calculated according to the formula of (mycelial diameter of untreated pathogen-mycelial diameter of treated pathogen) / mycelial diameter of untreated pathogen X 100.
【표 7]
Figure imgf000034_0001
[Table 7]
Figure imgf000034_0001
RS: 리족토니아 솔라니 ( oc OT/a solani) ,  RS: Lizatonia Solani (oc OT / a solani)
MO: 마그나포르테 \^( iagnapor ie oryzae) ,  MO: Magna Forte \ ^ (iagnapor ie oryzae),
BC: 보트리티스 씨네레아 0¾iryZ s cinerea) ,  BC: Botrytis Cinerea 0¾iryZ s cinerea),
CC: 콜레토트리쿰 3—3췌스 (Sol letotrich n coccodes) ,  CC: Colletricum 3—3 pancreas (Sol letotrich n coccodes),
F0: 푸자리움 옥시스포롬 f . sp. ^ ^A Fusarium oxysporum f. sp. raphani) ,  F0: Fujium oxysporum f. sp. ^ ^ A Fusarium oxysporum f. sp. raphani),
SS: 스클레로티니아 스클레로티오룸 (Sc/e /Tj/a sclerot iorum) ,  SS: sclerotinia sclerotiorum (Sc / e / Tj / a sclerot iorum),
PC: 파이토프토라 캡시사이 (/¾ (¾σΛίΛθΓ3 capsici) ,  PC: phytophthora capcici (/ ¾ (¾σΛίΛθΓ3 capsici),
PI: 파이토프토라 인페스탄스 (/¾yc A ?ora infestans)  PI: Phytophthora infestans (/ ¾yc A? Ora infestans)
표 7에서 나타낸 바와 같이， 푸자리움 옥시스포룸 f.sp. 라파니를 제외한 모든 병원성 곰광이의 균사 생육이 바실러스 벨레젠시스 G341 균주가 생성하는 휘발성 물질에 의해 생육이 저해됨을 알 수 있었다. 실시예 23: 바실러스 벨레젠시스 G341 균주가 생산하는 휘발성 물질의 성분분석 바실러스 벨레젠시스 G341 균주가 생산하는 휘발성 물질의 성분을 분석하기 위하여， DeMilo et al. (1996)의 방법을 변형하여 휘발성 물질을 포집하였다. As shown in Table 7, fujium oxysporum f.sp. The mycelial growth of all pathogenic bears except Raphani was inhibited by the volatiles produced by the Bacillus Velensis G341 strain. Example 23 Component Analysis of Volatile Substances Produced by Bacillus Velensis G341 Strain To analyze the components of volatiles produced by the Bacillus Velensis G341 strain, DeMilo et al. (1996) was modified to capture volatiles.
바실러스 벨레젠시스 G341 균주의 단일 콜로니를 트립틱소이액체배지 (Trypt i c Soy Broth： TSB)에 접종한 후 30 °C에서 150 rpm으로 1일 진탕배양한 후, 500 ml 삼각플라스크에 100 ml의 TSB 배지를 넣고 상기 균주를 1% 접종하였다. 투 -웨이 고무 코르크 (Two-way rubber cork)를 이용하여 하나의 선에는 배양액으로부터 1cm높이의 위치로 설정하였으며 휘발성 물질을 포집할 수 있도록 지름 1cm및 높이 15 cm의 유리관과 연결하였고，다른 하나의 선은 삼각플라스크 배양액으로부터 10 cm의 높이로 설정하였으며 외부로부터 공기가 주입될 수 있도록 설정하였다. 이때 유리관 안에는 150 mg의 차콜로 채운 후 휘발성 물질을 포집하도록 하였다. 30 °C 및 150 rpm으로 3일간 배양한 후 차콜을 수확하였으며， 메탄올에 용해한 후 여과하여 순수한 휘발성 물질만을 획득하였다. A single colony of Bacillus Velensis G341 strain After inoculating Trypt ic Soy Broth (TSB) and shaking culture at 150 rpm at 30 ° C for 1 day, 100 ml of TSB medium was added to a 500 ml Erlenmeyer flask and 1% of the strain was inoculated. . Using a two-way rubber cork, one line was set at a height of 1 cm from the culture medium and connected to a glass tube 1 cm in diameter and 15 cm in height to collect volatiles. The line was set to a height of 10 cm from the Erlenmeyer flask culture and set to allow air to be injected from the outside. At this time, the glass tube was filled with 150 mg of charcoal to collect volatile substances. After incubation at 30 ° C. and 150 rpm for 3 days, the charcoal was harvested, dissolved in methanol, and filtered to obtain only pure volatiles.
수득한 휘발성 물질의 성분 분석은 GC/MS를 이용하여 다음과 같이 측정하였다. GC/MS기기는 HP 7890A질량분석 검출기 (Mass selective deterctor, Agilent Technologies Inc. , Chandler, AZ, USA)가 장착된 GC/MS 기기를 사용하였으며， 분리용 칼럼은 DB— WAX(30 mX 0.25 mm, I.d., 0.25誦필름 두께， 氣 Folsom, CA, USA)를 사용하였다. 이때 오븐의 온도는 30°C에서 5분간 유지한 다음 10°C/분의 속도로 250°C까지 상승시켰으며， 캐리어 기체 (carrier gas)의 유속은 1ml/분 (He)로 유지하였다.분리된 화합물의 이온화는 전자 층격 이온화 (electron impact ionization, EI -mode) 방법으로 수행하였으며 이온화 전압 (ionization voltage)과 이온 소스 (ion source)의 온도는 각각 70 eV와 230°C로 설정하였고， 분석할 분자량의 범위는 40-350 (m/z)로 설정하였으며， GC 투입기 (injector)의 온도는 250 °C로 설정하였다. Component analysis of the obtained volatiles was measured as follows using GC / MS. The GC / MS instrument was a GC / MS instrument equipped with an HP 7890A mass spectrometer (Mass selective deterctor, Agilent Technologies Inc., Chandler, AZ, USA). The column for separation was DB—WAX (30 mX 0.25 mm, Id, 0.25 mm film thickness, Folsom, CA, USA). At this time, the temperature of the oven was maintained at 30 ° C for 5 minutes and then raised to 250 ° C at a rate of 10 ° C / min, and the carrier gas flow rate was maintained at 1 ml / min (He). Ionization of the prepared compounds was carried out by electron impact ionization (EI-mode) method, and the ionization voltage and ion source temperature were set at 70 eV and 230 ° C, respectively. The molecular weight range was set to 40-350 (m / z) and the temperature of the GC injector was set to 250 ° C.
GC/MS에 의해 총 이온화 크로마토그램 (total ionization chromatogram, TIC)에 분리된 각 피크의 휘발성 성분 분석은 질량 스펙트럼 라이브러리 (mass spectrum library, Wiley 275&N, NBS 75K)와 질량 스펙트럼 데이터 북 (mass spectral data book)의 스펙트럼， 및 문헌상의 보류 지수 (retention index)등을 근거로 하였으며， 머무름 시간 및 면적율에 따른 물질 동정 결과를 하기 표 8에 나타내었다. 【표 8】
Figure imgf000036_0001
표 8에서 나타낸 바와 같이， 바실러스 벨레젠시스 G341 균주로부터 생산되는 휘발성 물질은 디메틸디설파이드， 1-부탄올 및 3-히드록시 -2-부타논임을 알 수 있었다. 본 발명을 상기의 구체적인 실시예와 관련하여 기술하였지만, 첨부된 특허청구범위에 의해 정의된 본 발명의 범위 내에서 당 분야의 숙련자는 본 발명을 다양하게 변형 및 변화시킬 수 있다. Volatile component analysis of each peak separated in a total ionization chromatogram (TIC) by GC / MS includes a mass spectrum library (Wieley 275 & N, NBS 75K) and a mass spectral data book. ), And the retention index in the literature, and the results of material identification according to retention time and area ratio are shown in Table 8 below. Table 8
Figure imgf000036_0001
As shown in Table 8, it can be seen that the volatiles produced from the Bacillus bellensis G341 strain are dimethyldisulfide, 1-butanol and 3-hydroxy-2-butanone. Although the present invention has been described in connection with the specific embodiments described above, those skilled in the art can variously modify and change the present invention within the scope of the present invention as defined by the appended claims.
특허 절차상 미생물기탁의 국제적 승인에 관한 부다페스트조약 국제양식 International Convention on the Convention on the International Recognition of Microbial Deposits under Patent Procedures
원기탁에 관한수탁증 하기 국제기탁기관에서 정한 규칙 7.1 에 따라 발행됨 수령인 (기탁자): 김진철  Certificate of Deposition Issued in accordance with Rule 7.1 of the following International Depositary Recipients:
305-606 대한민국 대전광역시 유성구 장동 100 한국화학연구원 (KRICT) 그린화학공정연구본부  Korea Chemical Research Institute (KRICT) Green Chemical Process Research Division, 100, Jang-dong, Yuseong-gu, Daejeon, Korea
Figure imgf000037_0001
특허 절차상 미생물기탁의 국제적 승인에 관한 부다페스트 조약 국제양식
Figure imgf000037_0001
International Convention on the Budapest Convention on the International Approval of Microbial Deposits under Patent Procedures
원기탁에 관한수탁증  Trust certificate about original deposit
하기 국제기탁기관에서 정한 규칙 7.1 에 따라 발행됨 수령인 (기탁자): 김진철  Issued in accordance with Rule 7.1 of the following International Depositary Recipients:
305-606 대한민국 대전광역시 유성구 장동 100 한국화학연구원 (KRICT) 그린화학공정연구본부  Korea Chemical Research Institute (KRICT) Green Chemical Process Research Division, 100, Jang-dong, Yuseong-gu, Daejeon, Korea
Figure imgf000038_0001
Figure imgf000038_0001

Claims

허청구의 범위 Scope of claim
1. 항균 활성을 나타내는 휘발성 물질을 생산하는 리시니바실러스 스패리쿠스 ( s/j/bac/J/ s sphaericus) TCI 균주 (KCTC 12287BP) . 1. Ricinibacillus sp. Liqueur (s / j / bac / J / s sphaericus) TCI strain (KCTC 12287BP), which produces volatiles showing antimicrobial activity.
2. 제 1 항의 리시니바실러스 스패리쿠스 TC1 균주 (KCTC 12287BP) , 상기 균주의 포자， 상기 균주의 배양액 및 상기 배양액의 추출물로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 함유하는， 식물병 방제용 미생물 제제. 2. Plant disease, containing as an active ingredient any one or more selected from the group consisting of the ricinibacillus spalicus TC1 strain (KCTC 12287BP) of claim 1, the spores of the strain, the culture solution of the strain and the extract of the culture solution Microbial preparations for control.
3. 제 2 항에 있어서， 상기 미생물 제제가 바실러스 벨레젠시스 ( 3c///i/s velezensis) G341 균주 (KCTC 11497BP), 상기 균주의 포자， 상기 균주의 배양액 및 상기 배양액의 추출물로 이루어지는 군으로부터 선택되는 어느 하나 이상을 추가로 함유하는， 식물병 방제용 미생물 제제. 3. The group of claim 2, wherein the microbial agent is composed of Bacillus veletensis (3c /// i / s velezensis) G341 strain (KCTC 11497BP), spores of the strain, culture of the strain and extract of the culture solution. Further containing at least one selected from, Microbial agent for controlling plant diseases.
4. 제 3 항에 있어서, 상기 미생물 제제가 바실러스 벨레젠시스 G341 균주， 상기 균주의 포자， 상기 균주의 배양액 및 상기 배양액의 추출물로 이루어지는 군으로부터 선택되는 어느 하나 이상; 및 4. The method according to claim 3, wherein the microbial agent is at least one selected from the group consisting of Bacillus bellensis G341 strain, spores of the strain, culture of the strain and extract of the culture; And
리시니바실러스 스패리쿠스 TC1 균주, 상기 균주의 포자， 상기 균주의 배양액 및 상기 배양액의 추출물로 이루어지는 군으로부터 선택되는 어느 하나 이상을， Any one or more selected from the group consisting of Lysinibacillus spalicus TC1 strain, spores of the strain, culture of the strain and extract of the culture,
1:9 내지 9:1의 중량비로 포함하는 것을 특징으로 하는, 식물병 방제용 미생물 제제.  A microbial agent for controlling plant diseases, comprising a weight ratio of 1: 9 to 9: 1.
5. 제 2 항에 있어서， 상기 식물병이 토마토 풋마름병， 고추 역병， 배추 ᅳ 뿌리혹병 및 토마토 뿌리혹선층으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는， 식물병 방제용 미생물 제제. 5. The microorganism preparation according to claim 2, wherein the plant disease is any one or more selected from the group consisting of tomato green blight, red pepper blight, Chinese cabbage root root disease and tomato root gall layer.
6. 제 2 항에 있어서, 상기 미생물 제제가 랄스토니아 솔라나세아럼
Figure imgf000040_0001
solanacearum) , 아시도보락스 아베내 아종 카를리애 (^c/cforaraA1 avenae subs . cattlyae) , 아그로박테리움 투메파시엔스 (4§ bac e iOT tumefaciens) , 버크홀더리아 글루 Burkholderia glumae) , 펙토박테리움 크리산테미 (/ C 0Z)ac er/iffl chrysanthemi) , 슈도모나스 시린개 변종 액티니데 0¾ei/i/OTi 2as syringae v. act i nidi ae) , 슈도모나스 시린개 .변종 라크리만스 (Z¾e"ciM7c as syringae pv. lachrymans) , 산토모나스 악소노포디스 ( a/z zOTi as axonopodis) , 클라비박터 미시가넨시스 아종 미시가넨시스 ( CVav ba er michiganensis subs . michiganensis) , 리족토니아 솔라니 Rhizoctonia solani) , 마그나포르테 오리재 0 g77a/ ri/?e oryzae) , 보트리티스 씨네레아 ( o ry s cinerea) , 콜레토트리쿰 코코쪠스 (Colletotrichum coccodes) , 스클레로티니아 스클레로티오룸 (5Wero //?/a sclerot iorum) , 파이토프토라 캡사이시 iPhytophthora capsici) , 파이토프토라 인페스탄스 (/¾ ορΛ ?θΓ3 infestans) 및 플라즈모디오포라 브래시캐 ( Vas/ i/p?ara brass icae)S— 이루어진 군으로부터 선택되는 어느 하나 이상의 병원균의 생장을 억제하는 것을 특징으로 하는， 식물병 방제용 미생물 제제. 6. The method of claim 2, wherein the microbial agent is Ralstonia solanacerum
Figure imgf000040_0001
solanacearum), Ashdoborax avenae subspecies Carlia (^ c / cforaraA 1 avenae subs. cattlyae), Agrobacterium tumefaciens (4§ bac e iOT tumefaciens), Burkholderia glu Burkholderia glumae, Pectobacterium Chrysanthemi (/ C 0 Z) ac e r / iffl chrysanthemi), Pseudomonas syringae strain actinide 0¾ei / i / OTi 2as syringae v. act i nidi ae), Pseudomonas syringe .Variant lacrimans (Z¾e "ciM7c as syringae pv.lachrymans), Santomonas axonopodis (a / z zOTi as axonopodis), Clavibacter misganogensis subspecies miganane CVav ba er michiganensis subs. Michiganensis, Rhizoctonia solani, Magna Forte Orija 0 g77a / ri /? E oryzae, o ry s cinerea, cholesterum coco Colletotrichum coccodes, Sclerotinia sclerotiorum (5Wero //? / A sclerot iorum), phytophthora capsici, phytophthora infestans (/ ¾ ορΛ? ΘΓ3 infestans) And Plasmodiophora brasica (Vas / i / p? Ara brass icae) S—, which inhibits the growth of any one or more pathogens selected from the group consisting of microorganisms for controlling plant diseases.
7. 제 3 항에 있어서， 상기 미생물 제제가 랄스토니아 솔라나세아럼 ¾/Si03/a solanacearum), 아시도보락스 아베내 아종7. The microorganism preparation according to item 3, wherein the microbial agent is Ralstonia solanacearum ¾ / S i 03 / a solanacearum), an Ashdoborax avena subspecies
^l"!"^ °11 {Acidovorax avenae subsp. cattlyae) , 아그로박테리움 투메파시엔 ( Agrobact eriiim tumefaciens)， 버크홀더리아 글루口 fl {Burkholderia glumae) , 펙토박테리움 크리산테미 Pectobacterim chrysanthemi)， 슈도모나스 시린개 변종 액티니데 ( ¾e /o/ y?as syringae v. actinidiae) , 슈도모나스 시린개 변종 라크리만스 ( ¾e"oto»(¾?as syringae pv. lachrymans) , 산토모나스 악소노포디스 Λ¾7?0 35 axonopodis) , 클라비박터 미시가넨시스 아종 미시 7}·넨시스 ( Clavibacter ichiganensis subsp . michiganensis)， 리족토니아 솔라니 iRhizoctonia solani) , 마그나포르테 오리재 O!/a^apor^e oryzae) , 보트리티스 씨네레아 (^o ry /s cinerea) , 콜레토트리쿰 코코쪠 ^ Col letotrichiM coccodes) , 스클레로티니아 스클레로티오룸 ( Wero /;3/a sclerotiorum) , 파이토프토라 캡사이시 iPhytophthora capsici)， 파이토프토라 인페스탄스 (/¾y o/?7 ?ora infestans) , 플라즈모디오포라 브래시캐 ( /as iZ/p ra brassicae) , 아시도보락스 콘자시 (Aciclovorax konjaci), 펙토박테리움 카로토보라 아종 로토 Ά근 Pectobacteri carotovora subsp. carotovora) , 산토모나스 유베 1가토리。1" (Xanthomonas euvesicatoria) 및 산토모나스 프르니 (Xanthomonas pn ~ 이루어진 군으로부터 선택되는 어느 하나 이상의 병원균의 생장을 억제하는 것을 특징으로 하는， 식물병 방제용 미생물 제제. ^ l "! " ^ ° 11 {Acidovorax avenae subsp. cattlyae), Agrobacterium tumefaciens, Burkholderia glumae, Pectobacterim chrysanthemi, Pseudomonas syringe variants Actinide (¾e / o / y? As syringae v. Actinidiae), Pseudomonas syringae strain Lacrimans (¾e "oto» (¾? As syringae pv. Lachrymans), Santomonas axonopodis Λ¾7? 0 35 axonopodis Clavibacter ichiganensis subsp. Michiganensis, Lizatonia soni iRhizoctonia solani, Magnaporte duck O! / A ^ apor ^ e oryzae), Botrytis cine ^ O ry / s cinerea, Col letotrichiM coccodes, Sclerotinia sclerotiorum (Wero /; 3 / a sclerotiorum), Phytophthora capsaisi iPhytophthora capsici ， Phytophthora infestans (/ ¾y o /? 7? Ora infestans), Plasmopodora brasica (/ as iZ / p ra brassicae), Aciclovorax konjaci, Pectobacterium Cartovora subspecies Roto Ά muscle Pectobacteri carotovora subsp. Carotovora), Santomonas Juve 1 Gatori. 1 "(Xanthomonas euv esicatoria) and Santomonas prn (Xanthomonas pn ~ microbial agent for controlling plant diseases, characterized in that the growth of any one or more pathogens selected from the group consisting of.
8. 제 2 항에 있어서， 상기 리시니바실러스 스패리쿠스 TC1 균주의 배양액 또는 배양액의 추출물이 테트라클로로에틸렌 (tetrachloroethylene), 3-메틸 -1-부탄을 (3-methy卜 1— butanol), 메틸피라진 (niethylpyrazine) , 사이클로핵사논 (cyclohexanone), 2，5-디메틸피라진 (2,5-dimethylpyrazine), Ν,Ν-디메틸포름아미드 (Ν,Ν-dimethyl formamide) , 8. The method of claim 2, wherein the culture medium or extract of the culture medium of Ricinibacillus sparycus TC1 strain comprises tetrachloroethylene, 3-methyl-1-butane (3-methy 卜 1—butanol), methyl Pyrazine (niethylpyrazine), cyclohexanone, 2,5-dimethylpyrazine, Ν, Ν-dimethyl formamide,
2-(2-에톡시에톡시) -에탄올 (2-(2-ethoxyethoxy)-ethanol) 및 이의 흔합물로 이루어진 군에서 선택되는 휘발성 물질을 포함하는 것을 특징으로 하는， 식물병 방제용 미생물 제제.  A microbial agent for controlling plant diseases, comprising a volatile substance selected from the group consisting of 2- (2-ethoxyethoxy) -ethanol and combinations thereof.
9. 제 3 항에 있어서， 상기 바실러스 벨레젠시스 G341 균주의 배양액 또는 배양액의 추출물이 디피시딘 (difficidin), 옥시디피시딘 (oxydiff icidin) 및 이의 흔합물로 이루어진 군에서 선택되는 비휘발성 물질; 및 디메틸디설파이드 (dimethyldisulfide), 1-부탄올 (1-butanol )，9. The culture medium of claim 3, wherein the culture medium of said Bacillus vegetans G341 strain or Non-volatile substances selected from the group consisting of diphicidin, oxydiff icidin, and combinations thereof; Dimethyldisulfide, 1-butanol,
3-히드록시 -2-부타논 (3-hydroxy-2-butanone) 및 이의 흔합물로 이루어진 군에서 선택되는 휘발성 물질을 포함하는 것을 특징으로 하는, 식물병 방제용 미생물 제제. 3-hydroxy-2-butanone (3-hydroxy-2-butanone) and a combination thereof, characterized in that it comprises a volatile substance selected from the group consisting of, microbial agent for controlling plant diseases.
10. 제 2 항에 있어서， 상기 배양액의 추출물이 배양액을 에틸아세테이트， 부탄을, 물 또는 이들의 흔합용매로 추출된 것임을 특징으로 하는， 식물병 방제용 미생물 제제. 10. The microorganism preparation according to item 2, wherein the extract of the culture solution is obtained by extracting the culture solution with ethyl acetate, butane, water or a mixed solvent thereof.
11. 제 2 항 내지 제 10 항 중 어느 한 항에 따른 식물병 방제용 미생물 제제를 식물 또는 주변 환경에 처리하는 것을 포함하는， 식물병의 방제방법. 11. A method for controlling plant diseases, comprising treating the plant or the surrounding environment with a microbial agent for controlling plant diseases according to any one of items 2 to 10.
12. 제 11 항에 있어서， 상기 식물병이 토마토 풋마름병, 고추 역병, 배추 뿌리혹병 및 토마토 뿌리혹선층으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는， 식물병의 방제방법. 12. The method for controlling plant diseases according to 11 above, wherein the plant diseases are any one or more selected from the group consisting of tomato green blight, red pepper blight, cabbage root knot disease and tomato root gall layer.
13. 제 11 항에 있어서, Λ기
Figure imgf000042_0001
랄스토니아 솔라나세아럼 {Ralstonia solanacearum)， 아시도보락스 아베내 아종 ^Ί-^ o]] (Acidovorax avenae subs . cattlyae) , 아그로박테리움 투메파시엔스 {Agrobacterium tumefaciens)， 버크흘더리아 글루미 K Burkholderia gl umae) , 펙토박테리움 크리산테미 ( ¾c i)bac e/*/dw chrysanthe i) , 슈도모나스 시린개 변종 액티니데 0¾euctono as syringae v. actinidiae) , 슈도모나스 시린개 변종 라크리만스 ( ¾ei/o /7?o/7as syringae pv. lachrymans) , 산토모나스 악 노또디스 iXanthomonas axonopodis) , 클라비박터 미시가넨시스 아종 미시 7넨시스 ( 7av ?ac er michiganensis subsp. michiganensis) , 리족토니아 솔라니 Rhizoctonia solani) , 마그나포르테 오리재 ( w ¾?r /7e oryzae) , 보트리티스 씨네레아0 /5 cinerea) , 콜레토트리쿰 3—3 ^^ CoHetotrichi coccodes) , 스클레로티니아 스클레로티오룸 (5c/ TOi//3/a sclerotiorim) , 파이토프토라 캡사이시 Phytophthora capsici)， 파이토프토라 인페스탄스 (/¾ i¾9?i/?(?ra infestans) , 플라즈모디오포라 efl ] ?1 {Plasmodiphora brass i cae) , 아시도보락스 콘자시 (Acidovorax konjaci), 펙토박테리움 카로토보라 아종 카로토보라 (/ c o?ac er/½7 carotovora subs . carotovora) , 산토모나스 유베치 토리。 (Xanthomonas euvesicatoria) 및 산토모나스 프르니 (Xanthomonas p ni)로— 이루어진 군에서 선택되는 어느 하나 이상의 생장을 억제하는 것을 특징으로 하는， 식물병의 방제방법. 13. The method according to 11, wherein
Figure imgf000042_0001
Ralstonia solanacearum, Ashidoborax avenae subspecies ^ Ί- ^ o ]] (Acidovorax avenae subs. Cattlyae), Agrobacterium tumefaciens, K Burkholderia gloomy K Burkholderia gl umae), Pectobacterium chrysanthemi (¾c i) bac e / * / dw chrysanthe i), Pseudomonas syringae strain Actinide 0¾euctono as syringae v. actinidiae), Pseudomonas syringae strain Lacrimans (¾ei / o / 7? o / 7as syringae pv.lachrymans), Xanthomonas axonopodis), Clavibacter myganigansis subspecies (7av? Ac er michiganensis subsp. Michiganensis), Rhizoctonia solani, Magna Forte Duck (w ¾? R / 7e oryzae), Botrytis Cinerea 0/5 cinerea, Colleto Tricum 3—3 ^^ CoHetotrichi coccodes), Sclerotinia sclerotiorum (5c / TOi // 3 / a sclerotiorim), Phytophthora capsici, Phytoptotora infestans (/ ¾ i¾9? I /? (? Ra infestans), Plasmodiophora efl]? 1 {Plasmodiphora brass i cae), Acidovorax konjaci, Pectobacterium carotobora subspecies co? ac er / ½7 carotovora subs.carotovora), Xanthomonas euvesicatoria, and Xanthomonas pni—inhibit any one or more growth selected from the group consisting of: ， Method of controlling plant disease.
14. 제 11 항에 있어서， 상기 방법이 미생물 제제를 토양에 처리， 식물 표면에 처리, 종자에 코팅， 또는 이들의 조합에 의해 처리하는 것을 특징으로 하는， 식물병의 방제방법. 14. The method of controlling plant diseases according to 11, wherein the method comprises treating the microbial agent with soil, by treating the surface of the plant, coating the seed, or a combination thereof.
15. 테트라클로로에틸렌， 3-메틸 -1-부탄올, 메틸피라진， 사이클로핵사논， 2，5-디메틸피라진, Ν,Ν-디메틸포름아미드， 및 2-(2-에톡시에톡시) -에탄을로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는， 식물병 방제용 조성물. 미시 7넨시스 ( 7av ?ac er michiganensis subsp. michiganensis) , 리족토니아 솔라니 Rhizoctonia solani) , 마그나포르테 오리재 ( w ¾?r /7e oryzae) , 보트리티스 씨네레아 cinerea) , 콜레토트리쿰 3—3 ^^ CoHetotrichi coccodes) , 스클레로티니아 스클레로티오룸 (5c/ TOi//3/a sclerotiorim) , 파이토프토라 캡사이시 Phytophthora capsici)， 파이토프토라 인페스탄스 (/¾ i¾9?i/?(?ra infestans) , 플라즈모디오포라 efl ] ?1 (PJ as/nod iphora brassicae) , 아시도보락스 콘자시 (Acidovorax konjaci), 펙토박테리움 카로토보라 아종 카로토보라 (/ c o?ac er/½7 carotovora subs . carotovora) , 산토모나스 유베치 토리。 (Xanthomonas euvesicatoria) 및 산토모나스 프르니 (Xanthomonas p ni)로 이루어진 군에서 선택되는 어느 하나 이상의 생장을 억제하는 것을 특징으로 하는， 식물병의 방제방법. 15.Tetrachloroethylene, 3-methyl-1-butanol, methylpyrazine, cyclonuxanone, 2,5-dimethylpyrazine, Ν, Ν-dimethylformamide, and 2- (2-ethoxyethoxy) -ethane Comprising any one or more selected from the group consisting of, plant disease control composition. 7av? Ac er michiganensis subsp. Michiganensis, Ryazotonia solani, Magnaforte duckling (w ¾? R / 7e oryzae), Botrytis cinerea cinerea, Choletotricum 3 —3 ^^ CoHetotrichi coccodes), Sclerotinia sclerotiorum (5c / TOi // 3 / a sclerotiorim), Phytophthora capsaisi Phytophthora capsici, Phytophtor infestans (/ ¾ i¾9? i /? (? ra infestans), plasmophora efl]? 1 (PJ as / nod iphora brassicae), Acidovorax konjaci, Pectobacterium carotobora subspecies carobora (/ co ? ac er / ½7 carotovora subs.carotovora), a plant characterized by inhibiting any one or more growths selected from the group consisting of Xanthomonas euvesicatoria and Xanthomonas pni. How to control the disease.
14. 제 11 항에 있어서， 상기 방법이 미생물 제제를 토양에 처리， 식물 표면에 처리, 종자에 코팅， 또는 이들의 조합에 의해 처리하는 것을 특징으로 하는， 식물병의 방제방법. 14. The method of controlling plant diseases according to 11, wherein the method comprises treating the microbial agent with soil, by treating the surface of the plant, coating the seed, or a combination thereof.
15. 테트라클로로에틸렌， 3-메틸 -1-부탄올, 메틸피라진， 사이클로핵사논， 2，5-디메틸피라진, Ν,Ν-디메틸포름아미드， 및 2-(2-에톡시에톡시) -에탄을로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는， 식물병 방제용 조성물. 15.Tetrachloroethylene, 3-methyl-1-butanol, methylpyrazine, cyclonuxanone, 2,5-dimethylpyrazine, Ν, Ν-dimethylformamide, and 2- (2-ethoxyethoxy) -ethane Comprising any one or more selected from the group consisting of, plant disease control composition.
42 42
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