WO2018093199A9 - Nouveau micro-organisme bacillus oryzicola yc7011 produisant un lipopeptide cyclique en série de bacillopeptine, et formulation microbienne le comprenant - Google Patents

Nouveau micro-organisme bacillus oryzicola yc7011 produisant un lipopeptide cyclique en série de bacillopeptine, et formulation microbienne le comprenant Download PDF

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WO2018093199A9
WO2018093199A9 PCT/KR2017/013111 KR2017013111W WO2018093199A9 WO 2018093199 A9 WO2018093199 A9 WO 2018093199A9 KR 2017013111 W KR2017013111 W KR 2017013111W WO 2018093199 A9 WO2018093199 A9 WO 2018093199A9
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bacillus
plant
formula
bacillopeptin
rice
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WO2018093199A3 (fr
WO2018093199A2 (fr
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정영륜
엠디하룬오라시드
김근곤
이정은
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주식회사 제일그린산업
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    • 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
    • A01N63/50—Isolated enzymes; Isolated proteins
    • 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
    • A01N63/22—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • the present invention relates to a novel microorganism Bacillus ORIGINALA YC7011 and a microbial agent comprising the same to produce a cyclic lipopeptides of the Bacillopeptin family.
  • the present invention supports the 'Industrial Convergence Source Technology Development Project' supported by the Ministry of Trade, Industry and Industry in 2014 (Project No .: 10044909, Project name: Development of the effect-sustaining broad-scale probiotic crop protection agent) and the 'Next-generation biotechnology' supported by the Rural Development Administration in 2016.
  • This study was derived from the research conducted as part of Green 21 Genome Utilization Research Project (Task No .: PJ011049, Assignment Name: Whole Genome Function Analysis and Mechanism of Action of New Rice Probiotic Bacteria).
  • Natural plant protection agents are largely divided into extracts derived from natural products and agricultural microorganisms. In the case of Korea, the former is mainly developed for insecticides and the latter is used for fungicides.
  • Microorganisms useful for agriculture are widely distributed in the natural environment, such as symbiosis in the soil, plant roots, and plant tissues. Since they are diverse and secrete various metabolites, they are developed as natural plant protection agents and effectively used for eco-friendly agriculture. have.
  • Natural plant protection agents using microorganisms can be used in three ways, depending on the main ingredient used: microorganisms themselves, metabolic antibiotics produced by microbial fermentation, and microbial metabolism as a leading compound for new pesticide synthesis. And a method of using the substance.
  • microorganisms used include fungi Trichoderma harzianum , bacteria Bacillus subtilis , Bacillus amyloliquefaciens , Bacillus cereus , Bacillus pasteurii , Bacillus methylotrophicus , Pseudomonas fluorescens , Paenibacillus spp., Streptomyces spp. Lysobacter spp. And the like are well known as main ingredients.
  • Non-Patent Paper 1-4 Non-Patent Paper 1-4
  • Non-Patent Document 2 Some studies on the important pathogens of rice have shown that antagonism of Streptomyces sp. And Bacillus sp., Or Bacillus vallismortis EXTN-1 and two antagonistic microorganisms Pseudomonas fluorescens mc75 and pc78 for the control of rice leaf blight. There have been reports of effective control of rice plant leaf blight. There have been studies using P. fluorescens and Bacillus cereus to suppress Fusarium moniliforme and Fusarium fujikuroi , which cause dyskinesia and rice blight (Non-Patent Paper 4-8).
  • Non-Patent Document 9-12 Treatment of rice seed or root with a new bacterium Bacillus oryzicola YC7007 isolated from the rhizosphere has been reported to induce host resistance to bacterial blight, leaf blight and long leg disease. It was confirmed and announced for the first time in the world that the resistance to rice is also induced (Non-Patent Document 9-12).
  • Non-Patent Document 11-12 discloses a method for treating plant diseases.
  • the development or control of natural plant protection agents using microorganisms has been mostly based on the direct inhibition of plant pathogens or the study of growth promoting effects.
  • the microorganisms are treated with seeds or rhizospheres, there are few studies around the world on crop pest control effects and suppression mechanisms (Non-Patent Document 11-12).
  • Bacillus sp. Strain which is widely used in natural plant protection agents, is the most characteristic of antifungal, antibacterial, and promoting the growth and resistance of host plants, forms endospores, and is stable to heat and survives in harsh environments for a long time. It is a microorganism that can be used and commercialized so that it is most efficiently used commercially. Some strains are known to promote plant growth and induce plant defense mechanisms before being infected by pathogens through various hormonal secretions and various antagonisms that directly or indirectly nourish the plant, promote root growth (Non-Patent Document 2) -3, 12). Bacillus thuringiensis, among many Bacillus, has been known for a long time since it produces toxins and directly kills pests. On the other hand, several kinds of Bacillus strains that exhibit plant disease control and plant growth promoting effects have been reported to produce various metabolites.
  • Antimicrobial agents isolated from various Bacillus strains are the most well known cyclic lipopeptides such as Iturins, Surfectins and Fengycins. And various lipopeptide structures according to fatty acid sequences, lengths, and properties linked to the cyclic amino acids (Non Patent Literature 14-17). Iturin cyclic lipopeptide antimicrobials are produced in B. subtilis strains, including Iturins AE, Bacillomycines D, F, L, Lc, Mycosubtilin, Bacillopeptins A, B, and C.
  • Iturin C is a structure in which L -Asn-1 of Iturin A is substituted with L -Asp-1. Iturin C does not have the antibiotic activity of Iturin A. In addition, methylation of the phenyl group of D -Tyr-2 of Iturin A was confirmed to decrease the activity.
  • Bacillopeptins is connected to L and L -Glu -Asn instead of L and L -Gln -Asp similar in structure and Bacillomycin L, but Bacillomycin L.
  • Bacillopeptin C is only slightly structurally different from Bacillopeptin A or B, but the inhibitory effect on mold and yeast is found only in Bacillopeptin C.
  • Mycosubtilin has a structure in which Iturin A and two amino acids ( D- Ser 6. L -Asn 7) are changed, but its antibacterial activity is stronger than that of Iturin A (Non Patent Literature 14, 17, 19).
  • Recently, studies have been conducted to identify the substances by separating the lipopeptides from microbial strains that exhibit antimicrobial activity against plant diseases, to identify substances, or to identify structural genes and to identify the effects of these substances on plants.
  • Non Patent Literature 15, 18-22 are studies have been conducted to identify the substances by separating the lipopeptides from microbial strains that exhibit antimicrobial activity against plant diseases, to identify substances, or to identify structural genes and to identify the effects of these substances on plants.
  • the various lipopeptides produced by Bacillus are known to be environmentally friendly and harmless to humans, which have shown antagonism, antibacterial and antifungal action, and biosurfactants, which help root microorganisms settle, as well as inducing resistance of host plants. have.
  • these substances are known to be applicable to various industrial fields such as biotechnology, food and pharmaceutical fields as well as natural plant protection agents (Non-Patent Document 15).
  • Non-Patent Document 15 Non-Patent Document 15
  • the inventors of the bacillus O. coli YC7011 isolated from the rice root area during the pot experiment in Cheil Green Industrial Co., Ltd. is an endogenous bacterium that not only inhibits the growth of important path bacteria and fungi of rice, but also induces rice disease resistance and promotes plant growth. Also shown.
  • the multifunctional microorganism YC7011 strain has been identified for the first time in the world that the resistance to insect pests by the root zone or seed treatment to isolate and purify the active substance to reveal the structure. It also reveals the function of these new materials and can be used to develop natural plant protection agents and methods of use for the biological industry in various fields.
  • Non-Patent Document 1 Ryu, C.M. (2013). Promoting plant protection by root-associated microbes. Plant Pathol J 29: 123-124.
  • Non-Patent Document 2 McSpadden Gardener B. (2010). Biocontrol of plant pathogens and plant growth promotion by Bacillus. In: Recent Developments in Management of Plant Disease, Plant Pathology in the 21st Century. Eds. By U.Gisi, I. Chet and M.L. Gullino, Chapt. 6, pp. 71-79. Springer-Amsterdam.
  • Non-Patent Document 3 Lee, S.K., Sohn, H.B., Kim, G.G., and Chung, Y.R. (2006). Enhancement of biological control of Botrytis cinerea on cucumber by foliar sprays and bed potting mixes of Trichoderma harzianum YC459 and its application on tomato in the greenhouse. Plant Pathol J 22 (3): 283-288.
  • Non-Patent Document 4 Sung, K.C. and Chung, Y. R. (1997). Enhanced suppression of rice sheath blight using combination of bacteria which produce chitinases or antibiotics. In: Proceedings of the 4th International Workshop on Plant Growth Promoting Rhizobacteria Present Status and Future Prospects, eds. By A. Ogoshi, K. Kobayashi, Y. Homma, F. Kodama, N. Konodo and S. Akino. pp.370-373. OECD, Paris.
  • Non-Patent Document 5 Choi, G.J., Kim, J.C., Park, E.J., Choi, Y.H., Jang, K.S., Lim, H.K., Cho, K.Y. and Lee, S.W. (2006) Biological control activity of two isolates of Pseudomonas fluorescens against rice sheath blight. Plant Pathol J 22: 289-294.
  • Non-Patent Document 6 Park, K.S., Paul, D. and Yeh, W.H. (2006). Bacillus vallismortis EXTN-1 mediated growth promotion and disease suppression in rice. Plant Pathol J 22: 278-282.
  • Non-Patent Document 7 Kazempour, M.N. and Elahinia, S.A. (2007). Biological control of Fusarium fujikuroi, the causal agent of bakanae disease by rice associated antagonistic bacteria. Bulg J Agric Sci 13: 393-408.
  • Non-Patent Document 8 Rosales, A.M. and Mew, T. W. (1997). Suppression of Fusarium moniliforme in rice by rice-associated antagonistic bacteria. Plant Dis. 81: 49-52.
  • Non-Patent Document 9 Chung, E.J., Hossain, M.T., Khan, A., Kim, K.H., Jeon, C.O., and Chung, Y.R. (2015). Bacillus oryzicola sp. nov., and endophytic bacterium isolated from the roots of rice with antimicrobial, plant growth promoting, and systemic resistance inducing activities in rice. Plant Pathol J 31 (2): 152-164.
  • Non-Patent Document 10 Hossain, M.T., Khan, A., Rashid, M. and Chung, E.J., Chung, Y.R. (2015). Development of a novel endophytic Bacillus species as a microbial inoculant to control seed-borne rice diseases and brown plant hopper. In: 10th International Plant Growth Promoting Rhizobacteria Workshop. Program and Abstract book. p.47.
  • Non-Patent Document 11 Zebelo, S., Song, Y., Kloepper, J.W. and Fadamiro, H. (2016). Rhizobacteria activates (+)-(-) cadinene synthase genes and induces systemic resistance in cotton against beet armyworm (Spodoptera exigua). Plant Cell and Environment 39: 935-943.
  • Non-Patent Document 12 Hossain, M.T., Khan, A., Chung, E.J., Rashid, M.H., and Chung, Y.R. (2016). Biological control of rice bakanae by an endophytic Bacillus oryzicola YC7007. Plant Pathol J 32 (3): 228-241.
  • Non-Patent Document 13 Kwon, YS, Lee, DY, Rakwal, R., Baek, SB, Lee, JH, Kwak, YS, Seo, JS, Chung, WS, Bae, DW, and Kim , SG (2016). Proteomic analyses of the interaction between the plant-growth promoting rhizobacterium Paenibacillus polymyxa E681 and Arabidopsis thaliana. Proteomics 16: 122-135.
  • Non-Patent Document 14 (Non-Patent Document 14)
  • Non-Patent Document 14 Govindasamy V., Senthilkumar M., Magheshwaran V., Kumar U., Bose P., Sharma V., and Annapurna K. (2010). Bacillus and Paenibacillus spp .: Potential PGPR for Sustainable Agriculture. Plant Growth and Health Promoting Bacteria. Microbiology Monographs V18: 333-364.
  • Non-Patent Document 15 Ongena M., and Jacques P. (2008). Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol 16 (3): 115-125.
  • Non-Patent Document 16 Luo C., Liu X., Zhou X., Guo J., Truong J., Wang X., Zhou H., Li X., and Chen Z. (2015) . Unusual biosynthesis and structure of Locillomycins from Bacillus subtilis 916. Appl Environ Microbiol 81 (19): 6601-9.
  • Non-Patent Document 17 Kajimura Y., Sugiyama M., and Kaneda M. (1995). Bacillopeptins, New cyclic lipopeptide antibiotics from Bacillus subtilis FR-2. J Antibiot (Tokyo) 48 (10): 1095-103.
  • Non-Patent Document 18 Chen XH, Koumoutsi A., Scholz R., Eisenreich A., Schneider K., Heinemeyer I., Morgenstern B., Voss B., Hess WR, Reva O., Junge H., Voigt B., Jungblut PR, Vater J., Sussmuth R., Liesegang H., Strittmatter A., Gottschalk G., and Borriss R. (2007). Comparative analysis of the complete genome sequence of the plant growth-promoting bacteriaum Bacillus amyloloquefaciens FZB42. Nat Biotechnol. 25 (9): 1007-14.
  • Non-Patent Document 19 Thasana N., Prapagdee B., Rangkadilok N., Sallabhan R., Aye S. L., Ruchirawat S., and Loprasert S. (2010). Bacillus subtilis SSE4 produces subtulene A, a new lipopeptide antibiotic possessing an unusual C15 unsaturated b-amino acid. FEBS Lett 584 (14): 3209-14.
  • Non-Patent Document 20 Ma Z., Hu J., Wang X., and Wang S. (2013). NMR spectroscopic and MS / MS spectrometric characterization of a new lipopeptide antibiotic bacillopeptin B1 produced by a marine sediment-derived Bacillus amyloliquefaciens SH-B74. J Antibiot (Tokyo) 67 (2): 175-178.
  • Non-Patent Document 21 Yamamota S., Shiraishi S., and Suzuki S. (2015). Are cyclic lipopeptides produced by Bacillus amyloliquefaciens S13-3 responsible for the plant defense response in strawberry against Colletotrichum gloeosporidides Lett Appl Microbiol 60 (4): 379-86.
  • Non-Patent Document 22 Mukherjee, A.K., and Das, K. (2005). Correlation between diverse cyclic lipopeptides production and regulation of growth and substrate utilization by Bacillus subtilis strains in a particular habitat. FEMS Microbiol Ecol 54 (3): 479-89.
  • the present invention was confirmed that the treatment of plant endogenous bacillus Bacillus O. coli YC7011 to the root zone or seed of the host plant has a resistance induction effect and plant growth promoting effect, by separating and purifying the substances produced by this strain to antibacterial action and major
  • the object is to provide a structure of a novel substance that can induce resistance to plant diseases and pests and can promote the growth of plants.
  • the present invention to solve the above-mentioned problem is novel microorganism Bacillus origination coke (Bacillus oryzicola) YC7011 or the Bacillus origination coke (Bacillus oryzicola) Bacillus origination coke (Bacillus having the same pattern YC7011 and genetic analysis (BOX-PCR) Results oryzicola ).
  • Bacillus origination coke (Bacillus oryzicola), it characterized in that the Bacillus origination coke (Bacillus oryzicola) YC7011 comprises a 16S rRNA having the nucleotide sequence represented by SEQ ID NO: 1.
  • Bacillus oryzicola Bacillus oryzicola ) YC7011 culture.
  • the microbial preparation provides a microbial preparation comprising an active ingredient having a structure in which a beta amino acid chain of C8 to C20 is bonded to a cyclic structure of the bacillopeptin family represented by the following Formula 1.
  • the active ingredient provides a microbial agent, characterized in that it has a structure of any one of the following formulas 2-10.
  • the microbial agent provides a microbial agent, characterized in that it has aphid control, rice pest control, inhibiting the growth of pathogenic fungi, inducing host resistance of plant root zone or promoting plant growth.
  • the pathogenic fungus provides a microbial agent, characterized in that Fusarium fujikuroi causing rice stingray disease or Candida albicans causing human inflammation.
  • Bacillus oryzicola YC7011 and the substances isolated from the culture medium of this strain was confirmed to show resistance to host plants to control plant pests and to promote growth.
  • these multifunctional microorganisms and substances they have the ability to promote plant growth, inhibit phytopathogens, and induce host pest resistance. Can provide.
  • 1 is a 16S rRNA sequence of the Bacillus O. coli YC7011 strain
  • Figure 2 is a phylogenetic tree made by 16S rRNA gene sequencing of Bacillus O. coli YC7011 strain
  • Figure 3 shows the result of gene analysis (BOX-PCR) of Bacillus O. coli YC7011 strain and similar strains
  • Figure 4 is a schematic diagram of the material separation and purification from the culture medium of Bacillus O. Coli YC7011 strain
  • Figure 5 is a schematic diagram of the separation and purification process of F2D2 from fraction F of the primary separated material
  • FIG. 6 is a schematic diagram of the separation and purification process of G4B and G4C from fraction G of the primary separated material
  • FIG. 7 is a schematic diagram illustrating the separation and purification process of Bacillopeptin A (H4E) from the primary separation substance H,
  • I4B Bacillopeptin C
  • I4C Bacillopeptin B
  • I4E I4F from fraction I of the primary isolated material
  • FIG. 11 is a graph of 1H-NMR analysis in solvent DMSO-d6 for spectral structural characterization of F2D2.
  • FIG. 11 is a graph of 1H-NMR analysis in solvent DMSO-d6 for spectral structural characterization of F2D2.
  • 17 is a graph of 13 C-NMR analysis in solvent DMSO-d6 for spectroscopic structural characterization of G4B,
  • FIG. 26 is a graph of 1H-NMR analysis in solvent DMSO-d6 for spectroscopic structural characterization of Bacillopeptin A (H4E),
  • FIG. 31 is a graph of 1 H-NMR analysis in solvent DMSO-d6 for spectral spectrometry of I4E.
  • FIG. 31 is a graph of 1 H-NMR analysis in solvent DMSO-d6 for spectral spectrometry of I4E.
  • FIG. 49 is a graph of 1 H-NMR analysis in solvent DMSO-d6 for spectroscopic structural characterization of G4C.
  • FIG. 50 is a graph of 13 C-NMR analysis in solvent DMSO-d6 for spectroscopic structural characterization of G4C.
  • FIG. 53 is a graph of 1 H-NMR analysis in solvent CD3OD for spectroscopic characterization of I4B.
  • FIG. 54 is a graph of 1 H-NMR analysis in solvent DMSO-d6 for spectroscopic characterization of I4B.
  • FIG. 54 is a graph of 1 H-NMR analysis in solvent DMSO-d6 for spectroscopic characterization of I4B.
  • FIG. 55 is a 13C-NMR analysis graph in solvent DMSO-d6 for spectroscopic structure characterization of I4B.
  • FIG. 55 is a 13C-NMR analysis graph in solvent DMSO-d6 for spectroscopic structure characterization of I4B.
  • 57 is a graph of HSQC-NMR analysis for the spectroscopic structure of I4B;
  • 58 is a graph of HMBC-NMR analysis for spectroscopic characterization of I4B
  • 61 is a graph of 1 H-NMR analysis in solvent CD3OD for spectroscopic structural characterization of I4F
  • FIG. 62 is a 1H-NMR analysis graph in solvent DMSO-d6 for spectroscopic structural characterization of I4F.
  • FIG. 62 is a 1H-NMR analysis graph in solvent DMSO-d6 for spectroscopic structural characterization of I4F.
  • 63 is a graph of 13 C-NMR analysis in solvent DMSO-d6 for the spectroscopic characterization of I4F.
  • 66 is a graph showing the effect of controlling the aphid on the culture medium of YC7011
  • 67 is a graph showing the results of the control effect on the rice hopper of YC7011 culture.
  • the present inventors can suppress the growth of plant pathogens and bacteria in a wide range, and at the same time, isolate and mass-cultivate a multifunctional plant endogenous bacterium that has a specific effect on rice, which is a host plant, to induce disease resistance and promote plant growth. This has led to the discovery of new strains that can be used as new forms of biopesticides with microbial fertilizer efficacy.
  • Bacillus origola isolated from the present invention is proposed by Dunlap et al. (Dunlap CA, Kim SJ, Kwon SW, and Rooney AP (2015) Bacillus velezensis is not a later heteotypic synonym of Bacillus amyloliquefaciens ; Bacillus methylotrophicus , Bacillus amyloliquefaciens subsp. plantarum and Bacillus oryzicola are later heterotypic synonyms of Bacillus velezensis based on phylogenomics.Int J Syst Evol Microbiol 66: 1212-1217).
  • the Bacillus origola ( Bacillus oryzicola) may comprise a 16S rRNA having a nucleotide sequence set forth in SEQ ID NO: 1.
  • the present inventors have identified a structure by separating substances having a control effect and growth promoting effect against plant pests due to resistance induction from the culture medium of the novel Bacillus O. Co., Ltd. YC7011. Confirmed.
  • Bacillus O. coli YC7011 materials obtained from the culture solution of Bacillus O. coli YC7011 are Bacillopeptin A published in Kajimura et al. (Non-Patent Document 17: Kajimura Y., et al, 1995 Bacillopeptins, New cyclic lipopetide Antibiotics from Bacillus subtilis FR-2). Bacillopeptin-type cyclic lipopeptides, including B and C.
  • Bacillopeptin-type cyclic lipopeptides including B and C.
  • a new substance induces resistance from host plants, and shows control effects against rice bleeding and rice planting.
  • the plant pathogen Fusarium fujikuroi which causes dyskinesia , as well as the growth inhibitory effect on Candida albicans , a pathogen causing inflammation in the human body, was confirmed.
  • the present invention provides a microbial preparation comprising Bacillus origola YC7011 strain or a culture thereof.
  • the microbial agent may exhibit aphid control, rice pest control, inhibiting the growth of pathogenic fungi, inducing host resistance of the plant root zone or promoting plant growth.
  • the microbial preparation may be used as a preparation for controlling aphids, for controlling rice planthoppers, for inhibiting the growth of pathogenic fungi, for inducing host resistance of plant root zones or for promoting plant growth.
  • the microbial preparation containing Bacillus origola YC7011 strain according to the present invention or a culture solution thereof as an active ingredient is a component other than the active ingredient, for example, a predetermined carrier, a caking additive, a thickener, a fixing agent, an antiseptic agent, a preservative, a solvent. , Stabilizers, antioxidants, ultraviolet rays, crystal precipitation inhibitors, antifoams, physical properties enhancers, colorants and the like.
  • the YC7011 strain was isolated from the root tissue of rice root during rice pot experiment in Cheil Green Industrial Co., Ltd. First, in order to separate plant endogenous bacteria, the surface of the root cut pieces was immersed in 1% sodium hypochlorite (NaOCl) solution for 10 minutes to sterilize the surface. These pieces were placed on 1/10 TSA medium (Tryptic Soy Broth 3g, agar 16g / 1L of distilled water), and the surface was examined for bacterial sterilization while culturing for 2-3 days.
  • NaOCl sodium hypochlorite
  • the isolated YC7011 strain was a gram-positive, rod-shaped aerobic bacterium, and had no motility.
  • YC7011 strains are grown at 13 °C to 60 °C, pH 4 ⁇ 12.0, gelatin degradation, carboxymethyl cellulose undegraded, arbutin, starch, dilactose, diglucose, glycogen, en-machyl-glucosamine, genthiobiose degradation, Phenotypic characteristics of naphthol-ACE-biay-phosphohydrolase and N-acecyl-beta-glucosaminidase enzyme were confirmed.
  • FIG. 2 shows the nucleotide sequence of the 16S rRNA of the YC7011 strain. The intersection number in FIG. 2 represents the bootstrap value resulting from 1000 iterations.
  • PCR conditions include initial denaturation at 95 ° C for 7 minutes, 35 times denaturation, annealing, and extension at 90 ° C for 30 seconds, 40 ° C for 1 minute, The final extension was amplified by reacting at 72 ° C for 10 minutes for 3 minutes at 72 ° C.
  • the PCR product obtained through this process was electrophoresed on 1% LE agarose gel (Seakem) to obtain a result.
  • the YC7011 strain showed the same pattern as the YC7007 strain and the YC7010 strain, and different patterns from Bacillus velezensis KCTC13102 and Bacillus methylotrophicus KACC13105 T.
  • the microorganism of the present invention was named Bacillus oryzicola YC7011 strain based on the genetic analysis result obtained above, and received the accession number KCTC13085BP from KRCC (Korean Collection for Type Cultures) on August 30, 2016.
  • Substance separation and purification are divided into the culturing step of Bacillus O. coli YC7011 and the step of purifying the material from the culture medium after the cultivation.
  • the cultivation conditions of Bacillus O. coli YC7011 are optimal temperature in 1/10 TSB medium (Tryptic Soy Broth 3g / Casein digest 1.7g, soybean digest 0.3g, glucose 0.25g, sodium chloride 0.5g potassium diphosphate 0.25g / distilled water 1L) Shake culture was carried out at 120 rpm for 28 hours, optimal pH 7.0, incubation time 48 hours. 20 liters of the culture solution obtained after the culture was concentrated with methanol, and the brown solid obtained was dissolved in water and chromatographed with a C18 flash column. Each fraction was developed by mixing water and methanol in a predetermined ratio, and the active fractions were collected, and nine substances were separated and purified using HPLC and Silica MPLC (FIGS. 4-8).
  • the purified sample was dissolved in methanol and measured by HR-ESI-TOF-MS mass spectrometry.
  • 1D-NMR or 2D-NMR (1H, 13C, COZY, HSQC, HMBC, TOCSY, ROESY) was analyzed.
  • the nine isolated substances were confirmed to have a cyclic Bacillopeptin-based structure including Bacillopeptin A, B, and C (Table 1).
  • Example 2 Inhibition effect of rice hopper by the host resistance induction of Bacillus O. coli YC7011 culture separation material
  • Rice seeds (Shin Dong-jin) were soaked in tap water, and only the seed that sank was used. The soaked seeds were immersed in 2% sodium hypochlorite (NaOCl) for 5 minutes, then sterilized by primary surface sterilization for 5 minutes in 70% alcohol and rinsed several times with sterile distilled water. The surface sterilized seeds were immersed in sterile distilled water and placed in a dark condition at 30 ° C. for 3 days, and sprouted while changing the water every day. The sprouted seeds were planted on Murashige & Skoog medium (MS 2.2g / L, agar 8g / L, pH 6.0) and grown in plant growth (28-30 °C, 80% relative humidity) for 12 days.
  • Murashige & Skoog medium MS 2.2g / L, agar 8g / L, pH 6.0
  • Table 2 below shows the results of investigation of the control effect of the rice plant by the induction of resistance of each material.
  • Example 3 Control of Rice Blight Control Effect by Host Resistance Induction of Bacillus O. Coli YC7011 Culture Separation Material
  • the pathogen suspension was incubated in R2A medium for 24 hours and then centrifuged to suspend bacterial cells in a 10 mM MgSO 4 solution and to adjust the concentration appropriately.
  • Lesion necrosis was examined after placing on growth for 10 days after inoculation of pathogens. The incidence was divided into 0 ⁇ 3 (0: no symptom, 1: small necrotic speckle, 2: several necrotic specks combined, large gallbladder, 3: total necrosis). It was calculated by the formula of.
  • Table 3 below shows the results of the rice blight control effect by the induction of rice resistance of the separation material.
  • Example 4 Growth Growth Efficacy Assay of Rice by Bacillus O. Coli YC7011 Culture Separation Material Treatment
  • Bacillus ORigola Cola YC7011 Culture In order to assay the growth promoting effect of rice by the treatment of separated substances, the plant growth promoting effect was examined as follows.
  • Rice seeds (a la carte rice) was soaked in a constant water, and only the sinking seeds were used. The soaked seeds were soaked in 2% sodium hypochlorite for 5 minutes and then sterilized by primary surface sterilization, and then immersed in 70% alcohol for 5 minutes and then rinsed with sterile distilled water several times. The surface sterilized seeds were immersed in sterile distilled water and placed in a dark condition at 30 ° C. for 3 days, and sprouted while changing the water every day. The sprouted seeds were planted on MS medium (MS 2.2g / L, agar 8g / L, pH 6.0) and grown on plant growth (28-30 °C, 80% relative humidity) for 5 days. 100 ⁇ g / ml of 9 pure substances isolated from seedlings grown for 5 days were treated with 200 ⁇ l / week and grown for 5 days on plant growth, and then stem length and fresh weight were measured.
  • MS medium MS 2.2g / L, agar 8g / L, pH 6.0
  • Table 4 below shows the results of promoting rice growth after treatment with nine substances.
  • the antimicrobial activity of the nine substances was investigated by confrontation bioassay against one of the important plant pathogens, Fusarium fujikuroi , and one species of Candida albicans that cause inflammation in the human body. Confirmed.
  • the phytopathogenic fungi and nine substances were inhibited by using a paper disc method in a growth medium (16g agar / 1 liter of distilled water) 1 / 5PDA and R2A containing 5g potato agar sugar medium (PDB, Difco).
  • the effect was investigated.
  • the paper discs (5 mm in diameter) were placed 1 cm from the edge of the incubator, and each 100 ⁇ g / ml of 9 substances were treated with 200 ⁇ l of the paper discs, soaked enough, and the pathogens grown in PDA medium for 4 days.
  • the mycelial disc (6 mm) was placed and incubated at 28 ° C. for 3-5 days.
  • Table 5 below shows the antimicrobial activity of the nine plant materials for the main plant and human pathogen fungi.
  • antimicrobial activity against plant pathogens of 9 substances isolated from YC7011 strain culture was shown in H4E, I4E, I4C, I4B and I4F, and antimicrobial activity against human pathogens was grown in I4E and I4F substances. It was confirmed that the inhibitory effect.
  • I4E a novel substance, exhibited both inhibitory activity against plant and human pathogenic fungi.
  • Example 6 Aphid Control Efficacy Assay by Induction of Host Resistance of Bacillus O. Coli YC7011 Culture
  • Arabidopsis seedlings grown for 4 weeks on plant growth were transferred to a pot containing sterile topsoil (100 g), and 7 days after planting, a suspension of the above strains (2x10 7 ) was added to each treatment. 10 ml of cfu / L) were aliquoted into the soil. Strain suspension preparation was performed by shaking culture for one night at 28 ° C. in 1 / 10TSB medium and then centrifuging to suspend bacterial cells in 10 mM MgSO 4 solution and adjust the concentration appropriately. After 7 days of treatment, aphids (5 mice / week) were spun into each treatment group for 8 weeks per treatment, and the number of aphids per port was confirmed 7 days after aphid spinning.
  • 66 shows the aphid control effect results by induction of host resistance after treatment of the culture medium of each strain.
  • aphids were radiated after 7 days of treatment of each strain culture, and the numbers of aphids of B. oryzicola YC7007 and YC7011 treatment were untreated and B. velezensis KCTC13012, B. methylotrophicus KACC13105, B. amyloliquefaciens subsp. much less than the number of aphids plantarum KACC13105 treatments could see a statistically significant difference. Therefore, B. oryzicola YC7007 and YC7011 cultures were treated on Arabidopsis to induce host resistance and showed aphid control effects.
  • Example 7 Insecticidal Control Efficacy Assay by Inducing Host Resistance of Bacillus O. Coli YC7011 Culture
  • Rice seedlings grown for 10 days on plant growth were transferred to a pot containing sterile topsoil (100 g), and 2 days after planting, a suspension of the above strains (2x10 7 cfu) was added to each treatment. / L) 10 ml were aliquoted into the topsoil.
  • Strain suspension preparation was performed by shaking culture for one night at 28 ° C. in 1 / 10TSB medium and then centrifuging to suspend bacterial cells in 10 mM MgSO 4 solution and adjust the concentration appropriately.
  • rice bran (15 rats / week) was irradiated to each treatment for 5 weeks per treatment, and after 7 days of irradiation, rice leaf damage per pot was confirmed.
  • Figure 67 shows the results of the control of the rice hopper by the host resistance induction after treatment of the culture medium of each strain.
  • B. oryzicola YC7007 and YC7011 treatment of the damage caused by the rice hopper was untreated and B. velezensis KCTC13012, B. methylotrophicus KACC13105, B. amyloliquefaciens subsp. plantarum KACC13105 treatment was significantly lower than the damage degree. Therefore, B. oryzicola YC7007 and YC7011 cultures were treated in the rice root area to induce host resistance, and it was confirmed that the effect of controlling rice planthopper.
  • Bacillus oryzicola YC7011 and the substances isolated from the culture medium of this strain was confirmed to show resistance to host plants to control plant pests and to promote growth.
  • these multi-functional microorganisms and substances they have the ability to promote plant growth, inhibit phytopathogens, and induce host pest resistance, thereby providing excellent microbial agents that can simultaneously function as multifunctional natural plant protection agents, plant fortifying agents and microbial fertilizers. It can provide excellent industrial applicability.

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Abstract

Lorsqu'un milieu de culture de bactéries endophytes des plantes Bacillus oryzicola YC7011 et un matériel séparé à partir de celui-ci est utilisé pour traiter la rhizosphère et les graines d'une plante, une résistance est induite dans la plante de sorte à présenter un effet de lutte contre les maladies et les parasites des plantes et à présenter un effet d'accélération de croissance de la plante. De plus, un effet d'inhibition de la croissance contre les champignons pathogènes des plantes et chez les humains est obtenu. Par conséquent, la présente invention concerne six nouveaux lipopeptides cycliques induisant une résistance ainsi que trois matériels connus (Bacillopeptine A, B et C) qui, en tant que métabolites de Bacillus oryzicola YC7011, présentent des effets de lutte contre les maladies et les parasites des plantes, qui sont dus à des effets d'induction de résistance, ainsi que d'accélération de croissance et antifongiques. L'invention concerne un nouveau matériel pouvant être utilisé non seulement dans un agent de protection des plantes naturel multifonctionnel respectueux de l'environnement, mais également dans divers domaines industriels, par culture en masse de Bacillus oryzicola YC7011, et séparation et purification d'un milieu de culture.
PCT/KR2017/013111 2016-11-18 2017-11-17 Nouveau micro-organisme bacillus oryzicola yc7011 produisant un lipopeptide cyclique en série de bacillopeptine, et formulation microbienne le comprenant WO2018093199A2 (fr)

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