WO2018093199A2 - Novel microorganism bacillus oryzicola yc7011 producing bacillopeptin-series cyclic lipopeptide, and microbial formulation including same - Google Patents

Abstract

When a plant endophytic bacteria Bacillus oryzicola YC7011 culture medium and a material separated therefrom is used to treat the rhizosphere and seeds of a plant, resistance is induced in the plant such that a control effect against plant disease and pests is exhibited, and a plant growth accelerating effect is exhibited. Moreover, a growth inhibiting effect against plant and human pathogenic fungi is exhibited. Therefore, the present invention provides six resistance-inducing novel cyclic lipopeptides and three known materials (Bacillopeptin A, B, and C) which, as metabolites of Bacillus oryzicola YC7011, have plant disease and pest control effects, which are due to inducing resistance, as well as growth accelerating and antifungal effects. Provided is a novel material which can be used in not only an environmentally friendly multifunctional natural plant protecting agent, but also in various industrial fields, by mass culturing Bacillus oryzicola YC7011, and separating and purifying a culture medium.

Description

Novel microbial Bacillus ORIGICOLA YC7011 producing a cyclic lipopeptides of the Bacillopeptin family and microbial preparations comprising the same

This application claims the priority of Korean Patent Application No. 10-2016-0154297 filed on November 18, 2016, the entirety of which is a reference of the present application.

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).

As the world's problems such as environmental pollution, destruction of ecosystems caused by the misuse of chemical pesticides, causing human poisoning, resistance to pests and weeds are raised, many efforts have been made to establish an eco-friendly and sustainable agricultural production system that minimizes the production of safe food and environmental pollution. Doing In Korea, the government has established a five-year plan to promote eco-friendly agriculture in four phases since 2001, reducing the use of chemical pesticides and fertilizers to less than half, increasing eco-certified production farms, and expanding the eco-friendly agricultural market. I have been pushing. The eco-friendly agricultural market is steadily rising as the scale of eco-friendly agriculture increases and consumer demand for agricultural foods that do not use chemical pesticides or synthetic fertilizers increases. Accordingly, as the use of organic synthetic pesticides and chemical fertilizers is reduced, the use of natural plant protection agents for controlling pests, which are excellent in environmental protection and human safety, can replace their role.

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. Among them, the method using microorganisms is widely used, and antagonistic microorganisms capable of directly inhibiting plant pathogens and plant growth promoting Rhizobacteria (PGPR), which promote plant growth, are the most developed and used products at home and abroad. have. 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. These microorganisms promote plant growth through antagonism such as nutrient supply to plants, secretion of various hormones that promote root growth, and antibacterial substance release, or induce plant defense mechanisms before infecting pathogens. It is known to suppress (Non-Patent Paper 1-4)

Various useful microorganisms are used as natural plant protection agents for controlling pests that cause problems in many crops, but the results of research on controlling pests of rice are very rare (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).

Recently, 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).

Until now, 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. However, when 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. The first three amino acids of the iturin family ( _L- Asx (x = p, n), _D- Tyr, _D- Asn) are well conserved and are known to play an important role in antimicrobial activity. 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. In addition, 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).

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. In particular, as the research on these effects is active, 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). However, it has never been reported that these substances induce host plants resistance to pests.

Recently, 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. In the present patent, 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.

[references]

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Therefore, 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 ).

It also provides a 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.

It also provides a microbial agent comprising the Bacillus oryzicola ( Bacillus oryzicola ) YC7011 culture.

In addition, 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.

 (Formula 1)

In addition, the active ingredient provides a microbial agent, characterized in that it has a structure of any one of the following formulas 2-10.

 (Formula 2)

(Formula 3)

(Formula 4)

(Formula 5)

(Formula 6)

(Formula 7)

(Formula 8)

Formula 9

Formula 10

In addition, 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.

In addition, the pathogenic fungus provides a microbial agent, characterized in that Fusarium fujikuroi causing rice stingray disease or Candida albicans causing human inflammation.

According to the present invention, 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. By using 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,

6 is a schematic diagram of the separation and purification process of G4B and G4C from fraction G of the primary separated material,

7 is a schematic diagram illustrating the separation and purification process of Bacillopeptin A (H4E) from the primary separation substance H,

8 is a separation and purification method of I4B, Bacillopeptin C (I4D), Bacillopeptin B (I4C), I4E and I4F from fraction I of the primary isolated material,

9 is a structural formula of F2D2,

10 is a graph of 1H-NMR analysis in solvent CD3OD for spectroscopic structural characterization of F2D2;

FIG. 11 is a graph of 1H-NMR analysis in solvent DMSO-d6 for spectral structural characterization of F2D2. FIG.

12 is a graph of 13 C-NMR analysis in solvent DMSO-d6 for the spectroscopic characterization of F2D2;

13 is a graph obtained using HR-ESI-TOF-MS for mass spectrometry of F2D2;

14 is a structural formula of G4B,

15 is a graph of 1H-NMR analysis in solvent CD3OD for spectroscopic structural characterization of G4B,

16 is a graph of 1H-NMR analysis in solvent DMSO-d6 for spectroscopic structural characterization of G4B,

17 is a graph of 13 C-NMR analysis in solvent DMSO-d6 for spectroscopic structural characterization of G4B,

18 is a graph of COSY-NMR analysis for spectroscopic structural characterization of G4B,

19 is a graph of HSQC-NMR analysis for spectroscopic structural characterization of G4B,

20 is a graph of HMBC-NMR analysis for spectroscopic structural characterization of G4B,

21 is a graph of TOCSY-NMR analysis for spectroscopic structural characterization of G4B,

22 is a graph of ROESY-NMR analysis for spectroscopic structural characterization of G4B,

23 is a graph obtained using HR-ESI-TOF-MS for mass spectrometry of G4B,

24 is a structural formula of Bacillopeptin A (H4E),

25 is a graph of 1H-NMR analysis in solvent CD3OD for spectroscopic structural characterization of Bacillopeptin A (H4E),

FIG. 26 is a graph of 1H-NMR analysis in solvent DMSO-d6 for spectroscopic structural characterization of Bacillopeptin A (H4E),

27 is a graph of 13C-NMR analysis in solvent DMSO-d6 for spectroscopic structural characterization of Bacillopeptin A (H4E),

28 is a graph obtained using HR-ESI-TOF-MS for mass spectrometry of Bacillopeptin A (H4E),

29 is a structural formula of I4E,

30 is a graph of 1 H-NMR analysis in solvent CD3OD for spectroscopic characterization of I4E,

FIG. 31 is a graph of 1 H-NMR analysis in solvent DMSO-d6 for spectral spectrometry of I4E. FIG.

32 is a graph of 13 C-NMR analysis in solvent DMSO-d6 for the spectroscopic characterization of I4E,

33 is a graph obtained using HR-ESI-TOF-MS for mass spectrometry of I4E,

34 is a structural formula of Bacillopeptin B (I4C),

35 is a graph of 1H-NMR analysis in solvent CD3OD for spectroscopic structural characterization of Bacillopeptin B (I4C),

36 is a graph of 13C-NMR analysis in solvent CD3OD for spectroscopic structural characterization of Bacillopeptin B (I4C),

37 is a graph of 13C-NMR analysis in solvent DMSO-d6 for the spectroscopic characterization of Bacillopeptin B (I4C),

38 is a graph of COSY-NMR analysis for spectroscopic structural characterization of Bacillopeptin B (I4C),

39 is a graph of HSQC-NMR analysis for spectroscopic structural characterization of Bacillopeptin B (I4C),

40 is a graph of ROSEY-NMR analysis for spectroscopic structural characterization of Bacillopeptin B (I4C),

41 is a graph of TOCSY-NMR analysis for the spectroscopic structure of Bacillopeptin B (I4C),

42 is a graph obtained using HR-ESI-TOF-MS for mass spectrometry of Bacillopeptin B (I4C),

43 is a structural formula of Bacillopeptin C (I4D),

44 is a graph of 1H-NMR analysis in solvent CD3OD for spectroscopic structural characterization of Bacillopeptin C (I4D),

45 is a graph of 1H-NMR analysis in solvent DMSO-d6 for spectroscopic structural characterization of Bacillopeptin C (I4D),

46 is a graph of 13 C-NMR analysis in solvent DMSO-d6 for the spectroscopic characterization of Bacillopeptin C (I4D),

47 is a graph obtained using HR-ESI-TOF-MS for mass spectrometry of Bacillopeptin C (I4D),

48 is a structural formula of G4C,

FIG. 49 is a graph of 1 H-NMR analysis in solvent DMSO-d6 for spectroscopic structural characterization of G4C. FIG.

FIG. 50 is a graph of 13 C-NMR analysis in solvent DMSO-d6 for spectroscopic structural characterization of G4C. FIG.

51 is a graph obtained using HR-ESI-TOF-MS for mass spectrometry of G4C,

52 is a structural formula of I4B,

FIG. 53 is a graph of 1 H-NMR analysis in solvent CD3OD for spectroscopic characterization of I4B. FIG.

FIG. 54 is a graph of 1 H-NMR analysis in solvent DMSO-d6 for spectroscopic characterization of I4B. FIG.

FIG. 55 is a 13C-NMR analysis graph in solvent DMSO-d6 for spectroscopic structure characterization of I4B. FIG.

56 is a graph of COSY-NMR analysis for spectroscopic 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,

59 is a graph obtained using HR-ESI-TOF-MS for mass spectrometry of I4B;

60 is a structural formula of I4F,

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.

63 is a graph of 13 C-NMR analysis in solvent DMSO-d6 for the spectroscopic characterization of I4F.

64 is a graph obtained using HR-ESI-TOF-MS for mass spectrometry of I4F;

65 is a photograph of the control effect of rice blight control by inducing resistance of the substances Bacillopeptin A, B and the novel substance I4E,

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.

Hereinafter, the present invention will be described in detail through examples. Prior to this, terms or words used in the present specification and claims should not be construed as being limited to the common or dictionary meanings, and the inventors should properly explain the concept of terms in order to best explain their own invention. Based on the principle that it can be defined, it should be interpreted as meaning and concept corresponding to the technical idea of the present invention. Therefore, the configuration of the embodiments described herein is only one of the most preferred embodiments of the present invention and does not represent all of the technical idea of the present invention, various equivalents and modifications that can replace them at the time of the present application It should be understood that there may be

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.

In other words, as a result of searching for bacteria isolated from the pot experiment in Cheil Green Industry Co., Ltd., it was confirmed that the isolated strains had all the above-mentioned effects, and the sequencing and analysis of the nucleotide sequence of 16S rRNA gene and gene analysis (BOX-PCR) were performed. As a result of the experiment, it was identified as a microorganism belonging to the genus Bacillus sp. ( Bacillus oryzicola YC7011 ) was named.

The microorganism 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). Morphological and physiological biochemical classification and DNA-DNA hybridization, which are common methods for classifying microorganisms, are limited to clearly classify similar species. Marques et al. (Marques AS A., Marchaison A., Garden L., and Samson R. (2008) .BOX-PCR-based indentification of bacterial species belonging to Pseudomonas syringae-P. viridiflava group.Genetics and Molecular Biology 31 (1): 106-115) or Cherif et al. (Cherif A., Brusetti L., Borin S., Rizzi A., Boudabous A., Khyami-Horani H., and Daffonchio D. (2003) Genetic relationship in the Bacillus cereus group by rep-PCR fingerprinting and sequencing of Bacillus anthracis -specific rep- Genetic analysis (BOX-PCR) methods, such as PCR fragment J Micro Biol 94: 1108-1119), may be used to clarify similar species. Therefore, the same pattern of microorganisms as a result of genetic analysis (BOX-PCR) can be treated as the same microorganism as the Bacillus original cola of the present invention.

According to a preferred embodiment of the present invention, the Bacillus origola ( Bacillus oryzicola) may comprise a 16S rRNA having a nucleotide sequence set forth in SEQ ID NO: 1.

In addition, 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.

In the present invention, 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. Among the isolated Bacillopeptins, a new substance induces resistance from host plants, and shows control effects against rice bleeding and rice planting. In addition, 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.

Accordingly, 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.

Therefore, 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.

Hereinafter, the present invention will be described in detail with reference to Examples.

Isolation and Identification of YC7011 Strains

(1) Isolation of YC7011 Strain

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. Was taken for surface sterilization is a root piece 1.0g confirmation piece 10-fold diluted with sterile distilled water 9.0㎖ (10 ^-3) and further diluted ^(10-4 and ^10-5) are sequentially tenfold, the 1/10 dilution Dispense into TSA medium and spread evenly. After culturing the plated medium at 28 ° C. for 2-3 days, a single colony was isolated purely and named YC7011 strain.

(2) Identification of YC7011 strain

The isolated YC7011 strain was a gram-positive, rod-shaped aerobic bacterium, and had no motility. YC7011 strains are grown at 13 ℃ to 60 ℃, 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.

Meanwhile, the nucleotide sequence of the 16S rRNA of the YC7011 strain was determined (1513bp, see FIG. 1), and a homology search was performed using Genbank and EzTaxon-e database servers. As a result, 100% with Bacillus oryzicola YC7010 and Bacillus methylotrophicus KACC13105 ^T And 99.87% were found to be similar. Figure 2 shows the phylogenetic tree made by 16S rRNA gene sequence analysis of the YC7011 strain. The intersection number in FIG. 2 represents the bootstrap value resulting from 1000 iterations.

In addition, Versalovic et al. (Versalovic J., Schneider M., Bruijn FJ and Lupski JR (1994) Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol Cell Biol 5: 25-40) was performed for genetic analysis (BOX-PCR). GoTaq Green Master Mix (Promega) and primers (BOXAR1, 5'-CATCGGCAAGGCGACGCTGACG-3 ') were used for gene analysis from DNA extracted from each strain. 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. As shown in FIG. 2, 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.

Therefore, 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.

Example 1 Material Separation and Purification from Cultures of Bacillus O. Coli YC7011 Strain

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).

In order to know the molecular weight and molecular formula of the isolated active material, the purified sample was dissolved in methanol and measured by HR-ESI-TOF-MS mass spectrometry. In addition, 1D-NMR or 2D-NMR (1H, 13C, COZY, HSQC, HMBC, TOCSY, ROESY) to analyze the structure of the material was analyzed. The nine isolated substances were confirmed to have a cyclic Bacillopeptin-based structure including Bacillopeptin A, B, and C (Table 1). All isolated substances contained the ring structure of Bacillopeptin ( _L -Asn1- _D -Tyr- _D -Asn2- _L -Ser1- _L -Glu- _D -Ser2- _L -Thr-β-AA) and different beta amino acids for each substance. It consists of a chain structure (C _10- C ₁₇ ) and the NMR results for the structure and structural analysis of the identified material are shown in the figure (Figs. 4 to 64). Of these substances, the novel substances identified in the present invention were named Bacillopeptin D, F, G, H, I, X, respectively (Table 1).

Example 2: Inhibition effect of rice hopper by the host resistance induction of Bacillus O. coli YC7011 culture separation material

In order to test the efficacy of the control of the rice plant by the induction of the host resistance of the Bacillus O. Coli YC7011 culture medium was investigated as follows.

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 ℃, 80% relative humidity) for 12 days. 100 µg / ml of 9 pure substances separated from seedlings grown for 12 days were treated with 300 µl / week. After 5 days of treatment, they were placed on plant growth and then transferred to a pot containing sterile topsoil (100g). Covered with After 7 days of irradiating the rice plant, the damage of rice leaves was checked.

Table 2 below shows the results of investigation of the control effect of the rice plant by the induction of resistance of each material.

Nine isolates were treated with rice seedlings, respectively, and after radiating rice planting, all of the seven material treatment groups except for F2D2 and G4C material treatments showed the effect of controlling rice extinction by inducing rice resistance. Among them, the treatment groups that showed more than 70% of control value were H4E (Bacillopeptin A), I4C (Bacillopeptin B) and I4E new material treatment groups, and 70.59%, 85.29%, and 88.24%, respectively.

As a result, when the material isolated from the YC7011 strain culture solution was treated, plant rice resistance was induced, resulting in a control effect on rice hopper. In particular, it was confirmed that the new drug I4E had the highest resistance inducing effect.

Example 3: Control of Rice Blight Control Effect by Host Resistance Induction of Bacillus O. Coli YC7011 Culture Separation Material

In order to assay the rice blight control effect by inducing host resistance of the Bacillus O. coli YC7011 culture isolate, the following investigation was performed.

After soaking rice seeds (Ipumbap) in a constant water, picking the soaked seeds, soaked in 2% sodium hypochlorite for 5 minutes and sterilized the first surface, and then again put in 70% alcohol for 5 minutes to sterilize the second surface. Surface sterilized seeds were 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 MS medium (MS 2.2g / L, agar 8g / L, pH 6.0) and grown on plant growth (28-30 ℃, 80% relative humidity) for 5 days. 100 µg / ml of 9 pure substances separated from seedlings grown for 5 days were treated with 300 µl / week and grown for 5 days on plant growth.

Five days after treatment with nine substances, a Bukholderia glumae suspension (5 × 10 ⁷ cfu / ml, OD ₆₀₀ = 0.1) was inoculated into the inoculation needles and inoculated onto three leaves in three replicates on each leaf. 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. A comparison photograph of the substances Bacillopeptin A, B, and the novel substance I4E, which showed high control value against rice bran, is shown in FIG. 65.

Referring to Table 3, five days after treatment of seedlings with 9 kinds of seedlings, pathogens were inoculated to control rice blight control. H4E (Bacillopeptin A) treatment showed the highest 93.26% control among other treatments. Next, new substance I4E was 82.90%, and I4C (Bacillopeptin B) was 72.54%. Although the control value differed slightly according to the treatment material of the separated material, rice resistance was induced by the treatment of the separated material, and it was confirmed that the control effect on the rice blight was shown.

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 soaked 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 ℃, 80% relative humidity) for 5 days. 100 μg / ml of 9 pure substances isolated from seedlings grown for 5 days were treated with 200 μl / week and grown for 5 days on plant growth, and then stem length and fresh weight were measured.

Table 4 below shows the results of promoting rice growth after treatment with nine substances.

Referring to Table 4, after measuring the length and fresh weight of rice stems after 5 days of treating 9 kinds of substances separated from YC7011, there was a little difference depending on the substance, but compared with the untreated and BTH treated groups. It was confirmed that the growth promoting effect by, and in the case of the new material I4E, excellent growth promoting effect of 76.3% compared to other materials was confirmed.

Example 5 Investigation of Inhibitory Effect of Bacillus O. Coli YC7011 Culture Isolates on Plant and Human Pathogens

In order to investigate the inhibitory effect of the nine pure substances isolated from Bacillus O. coli YC7011 against plant and human pathogenic fungi, the antimicrobial activity was examined as follows.

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 μg / ml of 9 substances were treated with 200 μl 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.

Human pathogen suppression investigation was carried out by suspending Candida albicans incubated overnight at 37 ° C. in sterile distilled water (OD ₆₀₀ = 0.1) and smearing 0.1 ml of the suspension in the medium, followed by 9 substances (1,000 µg / ml, 200 µl). The soaked paper disk was placed and incubated at 37 ° C. for 24 hours to determine the extent of bacterial growth inhibition.

Table 5 below shows the antimicrobial activity of the nine plant materials for the main plant and human pathogen fungi.

Referring to Table 5, 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

* In order to test the efficacy of aphid control by inducing host resistance of Bacillus O. coli YC7011 culture, Bacillus origination coke YC7007, Bacillus Belle Zen sheath (Bacillus velezensis KCTC13012), Bacillus methylation in trophy kusu (Bacillus methylotrophicus KACC13105), Bacillus amyl Lowry quinolyl Pacific Enschede Francisco tarum (Bacillus amyloliquefaciens subsp. Plantarum KACC13105) strains also control as follows with The effect was investigated.

Arabidopsis seedlings grown for 4 weeks on plant growth (28-30 ° C, 80% relative humidity) 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.

Referring to FIG. 66, 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

In order to test the efficacy of the control of C. erysipelas by inducing host resistance of Bacillus O. coli YC7011 culture, Bacillus origination coke YC7007, Bacillus Belle Zen sheath (Bacillus velezensis KCTC13012), Bacillus methylation in trophy kusu (Bacillus methylotrophicus KACC13105), Bacillus amyl Lowry quinolyl Pacific Enschede Francisco tarum (Bacillus amyloliquefaciens subsp. Plantarum KACC13105) strains also control as follows with The effect was investigated.

Rice seedlings grown for 10 days on plant growth (28-30 ° C, 80% relative humidity) 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. After 5 days of treatment, 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.

Referring to Figure 67, 5 days after the treatment of each strain culture, as a result of spinning the rice hopper, 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.

Preferred embodiments of the present invention described above are disclosed to solve technical problems, and those skilled in the art to which the present invention pertains will be capable of various modifications, changes, additions, etc. within the spirit and scope of the present invention. Such changes, modifications and the like should be considered to be within the scope of the following claims.

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13085BP

Deposit date: 20160830

According to the present invention, 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. By using 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.

[Deposit]

[Deposited translation]

Claims (7)

Novel microorganism Bacillus oryzicola YC7011 (Accession No .: KCTC13085BP), which produces cyclic lipopeptides of the bacillopeptin family. The method of claim 1, The Bacillus oryzicola ( Bacillus oryzicola ) YC7011 is characterized in that it comprises a 16S rRNA having a nucleotide sequence of SEQ ID NO: 1 Bacillus oryzicola ( Bacillus oryzicola ) YC7011. Microbial agent comprising the Bacillus oryzicola YC7011 culture of claim 1. The method of claim 3, The microbial agent is a microbial agent, characterized in that it comprises an active ingredient having a structure in which the beta amino acid chain of (C10 ~ C17) is bonded to the cyclic structure of the bacillopeptin series represented by the following formula (1). (Formula 1)

The method of claim 4, wherein The active ingredient is a microbial agent, characterized in that any one or more selected from the group consisting of Formula 2 to 10 below. (Formula 2)

(Formula 3)

(Formula 4)

(Formula 5)

(Formula 6)

(Formula 7)

(Formula 8)

(Formula 9)

Formula 10

The method of claim 3, The microbial agent is a microbial agent, characterized in that the aphid control, control of rice planting, inhibiting the growth of pathogenic fungi, inducing host resistance of the plant root zone or promoting the growth of plants. The method of claim 6, The pathogen is a microbial agent, characterized in that Fusarium fujikuroi causing rice stingray disease or Candida albicans causing human inflammation.

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