WO2014065589A1 - Souche tc1 de lysinibacillus sphaericus, préparation microbienne pour la lutte contre des maladies de plantes comprenant ladite souche, et procédé de lutte contre une maladie de plantes l'utilisant - Google Patents

Souche tc1 de lysinibacillus sphaericus, préparation microbienne pour la lutte contre des maladies de plantes comprenant ladite souche, et procédé de lutte contre une maladie de plantes l'utilisant 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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Korean (ko)
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김진철
최경자
최용호
장경수
윤미영
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한국화학연구원
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Priority claimed from KR1020120118043A external-priority patent/KR102015051B1/ko
Priority claimed from KR1020120117986A external-priority patent/KR101956833B1/ko
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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

La présente invention concerne : une nouvelle souche TC1 de Lysinibacillus sphaericus présentant une activité d'inhibition de la croissance de divers bactéries et champignons pathogènes ; une préparation microbienne pour la lutte contre des maladies de plantes comprenant celle-ci ; et un procédé de lutte contre des maladies de plantes à l'aide de la préparation microbienne. La préparation microbienne selon la présente invention est écologique et peut présenter un effet exceptionnel de lutte contre des maladies de plantes, même dans une petite quantité, et peut augmenter le potentiel commercial et la productivité de plantes en culture et prévient les problèmes provoqués par la persistance de produits agrochimiques, et est utile dans la conservation d'écosystème agricole et dans la lutte contre des maladies intégrées.
PCT/KR2013/009480 2012-10-23 2013-10-23 Souche tc1 de lysinibacillus sphaericus, préparation microbienne pour la lutte contre des maladies de plantes comprenant ladite souche, et procédé de lutte contre une maladie de plantes l'utilisant WO2014065589A1 (fr)

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KR10-2012-0117986 2012-10-23
KR1020120118043A KR102015051B1 (ko) 2012-10-23 2012-10-23 바실러스 벨레젠시스 ｇ341 균주 및 리시니바실러스 스패리쿠스 ｔｃ1 균주를 포함하는 식물병 방제용 미생물 제제 및 이를 이용한 식물병 방제 방법
KR10-2012-0118043 2012-10-23
KR1020120117986A KR101956833B1 (ko) 2012-10-23 2012-10-23 리시니바실러스 스패리쿠스 ｔｃ1 균주 및 이를 이용한 식물병 방제 방법

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WO2015114552A1 (fr) * 2014-01-29 2015-08-06 University Of Pretoria Souches rhizobactériennes stimulant la croissance de plantes et leurs utilisations
CN108676741A (zh) * 2018-04-26 2018-10-19 南京农业大学 一种功能性复合微生物育苗基质及其制备方法与应用
CN112795501A (zh) * 2020-12-22 2021-05-14 华中农业大学 分离自水虻肠道的贝莱斯芽胞杆菌d2406及应用
CN113278551A (zh) * 2021-05-20 2021-08-20 福建省农业科学院植物保护研究所 一株伯克霍尔德氏菌、包括伯克霍尔德氏菌的菌剂、菌肥及制备方法和应用
CN113355262A (zh) * 2021-05-20 2021-09-07 福建省农业科学院植物保护研究所 一株伯克霍尔德氏菌、包括伯克霍尔德氏菌的菌剂、菌肥及制备方法和应用

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015114552A1 (fr) * 2014-01-29 2015-08-06 University Of Pretoria Souches rhizobactériennes stimulant la croissance de plantes et leurs utilisations
CN108676741A (zh) * 2018-04-26 2018-10-19 南京农业大学 一种功能性复合微生物育苗基质及其制备方法与应用
CN108676741B (zh) * 2018-04-26 2022-04-22 南京农业大学 一种功能性复合微生物育苗基质及其制备方法与应用
CN112795501A (zh) * 2020-12-22 2021-05-14 华中农业大学 分离自水虻肠道的贝莱斯芽胞杆菌d2406及应用
CN113278551A (zh) * 2021-05-20 2021-08-20 福建省农业科学院植物保护研究所 一株伯克霍尔德氏菌、包括伯克霍尔德氏菌的菌剂、菌肥及制备方法和应用
CN113355262A (zh) * 2021-05-20 2021-09-07 福建省农业科学院植物保护研究所 一株伯克霍尔德氏菌、包括伯克霍尔德氏菌的菌剂、菌肥及制备方法和应用

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