WO2011005042A2 - Nouvelle souche de streptomyces argenteus possédant une activité antimicrobienne contre des agents pathogènes de végétaux - Google Patents

Nouvelle souche de streptomyces argenteus possédant une activité antimicrobienne contre des agents pathogènes de végétaux Download PDF

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WO2011005042A2
WO2011005042A2 PCT/KR2010/004453 KR2010004453W WO2011005042A2 WO 2011005042 A2 WO2011005042 A2 WO 2011005042A2 KR 2010004453 W KR2010004453 W KR 2010004453W WO 2011005042 A2 WO2011005042 A2 WO 2011005042A2
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streptomyces
plant
strain
disease
antimicrobial activity
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Korean (ko)
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WO2011005042A3 (fr
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노문철
이우송
박찬선
김민수
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한국생명공학연구원
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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
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/28—Streptomyces
    • 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
    • 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
    • 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
    • C12N2523/00—Culture process characterised by temperature
    • 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
    • C12R2001/465—Streptomyces

Definitions

  • the present invention includes a novel Streptomyces argentherus strain (KCTC 11524BP) having antibacterial activity against plant pathogens, a pesticide preparation for plant disease control containing the strain as an active ingredient, and culturing the strain. It relates to a method for producing a pesticide preparation for plant disease control.
  • KCTC 11524BP novel Streptomyces argentherus strain
  • the principle of biological control by antagonistic microorganisms is to reduce the density or pathogenesis of pathogens through antibiotics produced by antibiotics, parasitic or phagocytosis by cell wall degrading enzymes, and nutrient competition or niche competition.
  • the main mechanisms of action include cross-protection and induction of host resistance using non-pathogenic strains or weakly-pathogenic strains.
  • the inhibitory action of fungal diseases caused by antibiotics or volatile substances, toxins, and special inhibitors secreted by the genus Trichoderma and gliocladium is known.
  • antagonism by parasitic or phagocytosis has been actively studied in recent years, and the lytic action of pathogens by cell wall degrading enzymes of fungi secreted by antagonistic bacteria is known as the main mechanism of action.
  • Typical microbial agent developed and commercialized in a foreign country is currently tricot der Do not annuum (Trichoderma harzianum) and tricot der Matthew Polis Forum (T. polysporum) made a BINAB T (1988, ER Butts International ) to inhibit Wood rot fungi, writing GlioGard (1990, WR Grace Co.), a bottle control agent for greenhouse flower seedlings using Gliocladium virens , Fstop (1991, Kodak), Trichodex (1997, Israel) Developed by KODIAK (1990, Gustafson) and Pseudomonas fluorescens using seed antagonist Bacillus subtilis) to control cotton lipotonia and Pythium sp.
  • DAGGER (1990, Ecogen Inc.), Pseudomonas Sefar Asia (P. cepacia) for BLUE CIRCLE the seed treatment of multiple crops using (1990, Stine Microbial Products), and antagonistic actinomycetes host Sat My process and the like so Seo irregularities disk (Streptomyces griseoviridis) the Fusarium (Fusarium) wilt, damping-off control agent MYCOSTOP (1985, Kemira Oy Biocontrol of Finland) using.
  • Bacteria among the microorganisms for fungicides patented as microbial pesticides include Bacillus sp. BS555, KCTC 8821P, Bacillus polymoxa B36, FERM BP-6068, Pseudomonas aureofaciens B5, FERM BP- 6067), Enterobacter cloacae B51, FERM BP-6069, and Bom1 (St.
  • KFCC 10992 Bom2 (KFCC 10993) in Streptomyces, an actinomycetes that secrete antifungal substances.
  • Bom2 KFCC 10993
  • a microorganism having an antagonistic capability for pathogenic fungi Bacillus subtilis bar amyl duck keupa City Enschede (Bacillus subtilis var.
  • the fungus has a antagonistic ability against Actinomycetes Streptomyces genus Pseudomonas fluorescence lessons Biovar 1, the fungus is Trichoderma hazinium, Penicillium sp., Aspergillus genus (Aspergillus sp.), redundancy parasitic fungi in Acre help monitor the streak lithium BCP (Acremonium strictum, KCTC 0639BP) This being studied.
  • Korean Patent Registration No. 10-0757298 relates to lysobacter enzymogenes and strain of chromobacterium having plant disease control activity using chitin decomposition ability, but the production ability of antagonist microorganism and chitinase At the same time, there is a disadvantage in that the production of a medium that can be exerted at the same time is not easy, and the strain is different from the Streptomyces argenteo strain of the present invention.
  • One object of the present invention is to provide Streptomyces argenteolus having antimicrobial activity against plant pathogens that cause cayenne blight, anthrax and root rot.
  • Still another object of the present invention is to provide a pesticide preparation for plant disease control and a method of preparing the same, containing the cells, spores, culture medium or mixtures of the Streptomyces argentherus strain as an active ingredient.
  • Another object of the present invention relates to a method for controlling plant diseases using a pesticide preparation containing a Streptomyces argeneorus strain.
  • the novel Streptomyces argentherus strain of the present invention shows excellent antimicrobial activity and inhibitory effect against plant pathogens that cause pepper blight, anthrax, and root rot disease, and microbial pesticide preparations containing the strain have various plant diseases. It is very useful as an environmentally friendly, pollution-free microbial pesticide that shows excellent control effect against and prevents ecosystem destruction.
  • 1 is a sequence of 16S rDNA of Streptomyces argenteo of the present invention 446bp.
  • Figure 3 shows the antimicrobial activity of Streptomyces argenteorus of the present invention against Pythium ultimum .
  • Figure 4 shows the antimicrobial activity of Streptomyces argentherus of the present invention against Rhizoctonia solani .
  • Figure 5 shows the antimicrobial activity of Streptomyces argentherus of the present invention against Botritis cinerea .
  • Figure 6 shows the antimicrobial activity of Streptomyces argentherus of the present invention against Altenaria panax .
  • Figure 7 shows the antimicrobial activity of Streptomyces argentherus of the present invention against Cylidrocarpon destructans .
  • Figure 8 shows the antimicrobial activity of Streptomyces argentherus of the present invention against Fusarium solani.
  • Figure 9 is a graph showing the growth curve of the Streptomyces Argenteorus of the present invention over the incubation time.
  • Figure 10 is a photograph of the pepper laboratory to perform the present invention.
  • Figure 11 is a photograph of the pepper produced by treating the Streptomyces Argenteorus of the present invention.
  • the present invention is a Streptomyces argenteolus strain having an antimicrobial activity against plant pathogens, a pesticide preparation for plant disease control containing the strain as an active ingredient, and a plant comprising the step of culturing the strain. It relates to a method for producing a pesticide preparation for bottle control.
  • the present invention relates to a strain of Streptomyces sp. That has antimicrobial activity against plant pathogens, and more particularly to plants that cause plant pathogens, in particular, cayenne blight, anthrax and root rot. It relates to a novel Streptomyces argentherus strain having antimicrobial activity against pathogens.
  • streptomyces is a representative genus of soil actinomycetes and produces antibiotics, and is a fungi belonging to the actinomycetes Streptomyces family and has a characteristic of forming a conidia in the mycelia. It's a group of people.
  • Streptomyces argenteorus strain of the present invention is preferably Streptomyces argenteolus subsp.toyonakensis .
  • the novel Streptomyces argentheor toyonakensis subspecies is isolated from the soil of good parts, and has a strong antibacterial activity in common to Phytophthora cactorum and Colletotrichum acutatum .
  • the microorganisms were selected and isolated and identified.
  • pepper blight, anthrax, and root rot are representative diseases that cause enormous damage in the harvest of crops.
  • Cayenne pepper disease is a major disease of pepper that occurs in all parts of the roots, stems, leaves, and fruits throughout its growth period.
  • Phytophthora cactorum its pathogen, is very pathogenic. Invade a lot of damage.
  • anthrax in plants is caused by a fungus called gnus Colletotrichum , the main pathogen of which is Colletotrichum acutatum . This fungus destroys leaves, stems, fruits, and flowers.
  • Root rot is a condition in which part or all of the roots of a plant are broken down or rotted.
  • the pathogens Rhizoctonia solani and Pythium ultimum are infected, the internal tissues of the roots are destroyed and black rots.
  • the root's ability to absorb falls, causing the leaves to fall and causing a significant drop in fruition.
  • the present invention provides a strain of Streptomyces argenteorus, which is a kind of biopesticide capable of exhibiting antimicrobial activity against all of the plant pathogens.
  • the biopesticide may be defined as the product of the microorganisms and natural materials, natural enemies, etc. collected and collected in the natural environment to control pests and pathogenic microorganisms, weeds of crops.
  • the strain of the present invention is preferably resistant to the phytophthora japtorum, which is the causative agent of pepper blight, choletotricum akutat, which is the causative agent of anthrax, and the lysatonia solani, which is the causative agent of root rot, and fishium Ultimum. It has antimicrobial activity (FIG. 4).
  • Streptomyces argentheor toyonakensis subspecies of the present invention in addition to the above-mentioned plant pathogens, Fusarium solani and Cylidrocarpon destructans , gray mold, which cause root rot diseases.
  • Botrytis cinerea the causative agent of disease, Pythium utimum , the causative agent of wear and mozzarella disease, Altenaria panax , the causative agent of spots , and Silerotinia sile , a causative agent of fungal nuclear disease It may have an antagonistic activity such as Sclerotinia sclerotiorum , Streptomyces scabies , which is a causative agent of potato beetle disease, but is not limited thereto.
  • the strains of the present invention exhibit strong antimicrobial activity against Pisium Ultimum, Botrytis Ceria, Altenaria Panax and Fusarium Solani (Figs. 3, 5, 6 and 8).
  • these pathogens are most often caused by complex infections that are expressed in correlation rather than alone.
  • the strain of the present invention exhibits strong antagonism against pathogens causing various plant diseases, including pepper blight, anthrax and root rot, the strain is used as an environmentally friendly, non-polluting biopesticide. It can be useful for solving ecosystem destruction and toxicity problem.
  • the present invention relates to a pesticide preparation for controlling plant diseases containing cells, spores, culture medium or mixtures of novel Streptomyces argentherus toyonakensis subspecies as an active ingredient. . More specifically, when the foliar spray of a culture medium containing the microorganism of the present invention to the foliar crops, the causative agent of pepper blight, Phytophthora cactorum , a causative agent of anthrax, Collettotrichum acutatum, root rot disease The present invention relates to a microbial pesticide preparation having a control effect of phytopathogenic microorganisms containing the effect of inhibiting the growth of Rhizoctonia solani .
  • the culture solution included in the pesticide preparation of the present invention is a supernatant from which the strain is removed from the strain culture solution, which can be obtained by centrifugation or filtration, and can be included in the pesticide preparation for controlling plant diseases in a liquid or solid state after drying.
  • a pesticide preparation may be prepared using a carrier usable in a pesticide preparation in addition to the cells of Streptomyces argentheor toyonakensis subspecies, its culture, or its culture extract.
  • the carrier may be any material having a small grain size so that the antagonist can be adsorbed, but is preferably a granular material having a porosity, and may be preferably selected from zeolite, elvan, vermiculite, sand, and activated carbon. Most preferred.
  • the pesticide preparation of the present invention may be prepared by a known method in the pharmaceutical field, and in addition to the carrier, it may be prepared and used in various formulations, including excipients and diluents, and the antifungal active bacteria may be well adhered to the plant leaves or It may also contain a coating flocculant that will degrade well in the soil.
  • the pesticide preparation of the present invention may be formulated as a liquid or emulsion, typically diluted with water and dispersed in powder, or previously formulated with water, or formulated as a water-soluble hydrate, granule, oil suspension. Alternatively, it may be prepared in the form of a ready mix or tank mix with other agrochemically active compounds such as toxic agents or growth regulators.
  • the active ingredient may be used in 0.001 to 90% of the culture solution extract with respect to the entire pesticide formulation, more preferably may be used by diluting 100 to 500 times the strain culture extract according to the present invention.
  • the present invention provides a method for controlling plant diseases by using the pesticide preparation for plant diseases control.
  • the plant disease may be pepper blight, anthrax, or root rot.
  • Pesticides prepared as described above is preferably used as a foliage treatment method on the plant's ground portion from 10 days after planting the crop or just before the onset.
  • the pesticide formulation may be used as a general pesticide formulation such as seed coating agents, microbial nutrients, soil improving agents, in addition to plant disease control agents, but is not limited thereto.
  • the present invention relates to a method for producing a pesticide preparation for plant disease control, comprising the step of culturing Streptomyces argentherus toyonaksis subspecies strain in a medium.
  • the preparation method of the microbial agent of the present invention is a microorganism Streptomyces that is antagonistic against plant pathogens such as Phytoprosora chitorum, Colletotricum akutatum, Raiztonia sorani, fishium Ultimum and the like.
  • Genteorus strain Separating the Genteorus strain from the soil; Identifying the isolated antagonistic microorganism Streptomyces argentherus strain; Treating the spore suspension of the isolated Streptomyces argentherus strain in agar solid medium, inoculating fungal pathogens and measuring the antibacterial activity to assay the antagonism of the Streptomyces argentherus strain; And mass culturing of the Streptomyces argeneorus strain.
  • Separation of the strain may be carried out by purely separating the colonies produced after 2 to 4 days incubation in YM medium after diluting the soil by mixing the sterile water with dilution.
  • the isolated strain may be subjected to 16S rDNA sequencing to identify morphological characteristics and phylogenetic location to identify. 16S rDNA sequence analysis for the strain of the present invention is shown in FIG.
  • the step of assaying the antagonism of the strain is buried in a culture medium cultured strain of the present invention on a paper disk and put on a petri dish mixed with pathogens and cultured for 3 to 4 days at 25 ⁇ 30 °C well grown various pathogens
  • the effect of the strain of the present invention on various pathogens can be investigated with a low support diameter.
  • the mass culture step may be a liquid medium in which yeast extract 3 ⁇ 6 g / L, juicy extract 3 ⁇ 6 g / L, glucose 8 ⁇ 12 g / L is added, at 25 ⁇ 30 °C 120 ⁇ 180rpm It can be performed for 3 to 7 days under the condition.
  • the growth of the bacteria can be measured by measuring the absorbance of the culture solution.
  • Optical Density (OD) means that when a certain wavelength of light passes through a culture of bacteria, the solution can absorb (or scatter) the light, and the amount of light absorbed is dependent on the cell concentration of the solution. It uses the principle of proportionality, and it means the value measured by using an absorbance meter, and it is used because the unknown number of cells can be determined by this absorbance measurement alone.
  • the growth curve of the strain in the culture solution is shown in Figure 9 by measuring the absorbance.
  • the pesticide preparation of the present invention prepared as described above may be sprayed on the crop plantation to measure the number of crops produced per kilogram to analyze the growth of the crop.
  • the results for red pepper are shown in FIG.
  • soil samples were collected from all over the country in order to isolate the microorganisms having antimicrobial activity, using the pepper strains Phytophthora Cactorum and pepper anthrax colletricum akutatum as test strains.
  • Sampling was carried out by selecting a depth of about 15 centimeters of the topsoil layer, and removing foreign matters such as weeds and fallen leaves. It was sealed in a 200 ml glass bottle and transported to a laboratory at 0 to 4 ° C. 1 g of the collected sample was mixed with 10 mL of sterile water and shaken at 28 ° C. for 30 minutes.
  • 16S rDNA of the selected strains were analyzed for homology using the gene bank blast program (Fig. 1).
  • the selected strain exhibited 99% homology with Streptomyces argentherus NRRL B-3230, which was finally confirmed to be a Streptomyces argentherus strain. Therefore, on July 6, 2009, it was deposited with the Korea Institute of Bioscience and Biotechnology, located at 111, Gwahak-ro, Yuseong-gu, Daejeon, Korea.
  • Phytopathogens include a variety of pathogens that cause significant damage to plants, such as Streptomyces scabies (potato bugs ), Pythium utimum KACC 40705 (root rot), Colletotrichum acutatum KACC 40042 (anthrax), Botrytis cinerea KACC 40573 (gray mold), and Cylindrocarpon destructans KACC 41077 (root rot), Sclerotinia sclerotiorum KACC 41065 (bacterial disease), Phytophthora cactorum KACC 40166 (pepper plague), Rhizoctonia solani AG-2-2 (IV) KACC 40123 (root rot)
  • the antimicrobial range was investigated by testing the antagonism of Streptomyces argentherus from Korea Agricultural Microbial Resources Center.
  • the strain of the present invention is the phytophthora pactrum, which is the causative disease It has excellent inhibitory effects against choletotricum akutat, the cause of anthrax, lysatonia solani, the cause of root rot, Pisium Ultimum, the cause of wear and mozzarella, and syllotini cilerothiorum, the cause of mycotic disease.
  • Antagonist microorganism Streptomyces argenteorus strains were cultured using a 2.5 L fermenter.
  • the production medium used was 5 g of starch, 20 g of glucose, 25 g of soy flour, 1 g of juice extract, 4 g of yeast extract, 2 sodium chloride.
  • g, potassium phosphate 0.25 g, and calcium carbonate 2 g were dissolved in 1 L of distilled water, and then a conventional liquid medium having a pH of 7.2 was prepared and used.
  • Species culture was incubated in a 500 ml Erlenmeyer flask using a 100 ml liquid medium containing 5 g / L yeast extract, 5 g / L juicy extract and 10 g / L glucose.
  • the culture conditions were shaken for 120 hours at 28 °C and 150 rpm, inoculated in a 2.5 L fermentation medium containing 1.5 L production medium 5% (v / v) of the culture medium containing Streptomyces argentherus and 28 °C Incubated for 120 hours at 150 rpm, 1 vvm condition (Fig. 9).
  • Streptomyces argeneorus cultured in the same manner as in Example 3 was mixed with vermiculite so that the number of 10 8 cells / 10 ha and sprayed on the pepper planting was investigated for the occurrence of pepper anthrax and late blight.
  • foliar spraying was carried out at the first stage (before seedling) and at the second stage (before the rainy season), and nothing was treated.
  • the growth of pepper was analyzed by measuring the number of peppers per kilogram of the final pepper produced. As shown in Table 3, the microbial treatments were 53 peppers per kg, showing much better growth than the 64 control.

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Abstract

La présente invention concerne une nouvelle souche de Streptomyces argenteus (KCTC 11524BP) possédant une activité antimicrobienne contre des agents pathogènes de végétaux, une préparation agrochimique destinée à prévenir des maladies végétales et contenant la souche en tant que constituant actif, et une méthode de production de la préparation agrochimique destinée à prévenir des maladies végétales et comprenant des étapes au cours desquelles la souche est cultivée. L'invention concerne aussi une nouvelle souche qui a un effet particulièrement bénéfique sur la prévention de la pourriture causée par phytophthora, de l'anthracnose et du pourridié.
PCT/KR2010/004453 2009-07-08 2010-07-08 Nouvelle souche de streptomyces argenteus possédant une activité antimicrobienne contre des agents pathogènes de végétaux WO2011005042A2 (fr)

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KR10-2009-0062319 2009-07-08
KR1020090062319A KR101107331B1 (ko) 2009-07-08 2009-07-08 식물 병원균에 대하여 항균 활성을 갖는 신규한 스트렙토마이세스 아르젠테오루스 균주

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CN114854631A (zh) * 2022-05-12 2022-08-05 中国热带农业科学院热带生物技术研究所 一株海绵来源的生防链霉菌ITBB-ZK-a5及其应用

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KR101836007B1 (ko) 2016-11-29 2018-04-19 충북대학교 산학협력단 해충방제 및 항균활성을 가지는 곤충병원성 곰팡이 메타리지움 아니소플리애 sd4-2 균주

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CN114854631B (zh) * 2022-05-12 2023-06-23 中国热带农业科学院热带生物技术研究所 一株海绵来源的生防链霉菌ITBB-ZK-a5及其应用

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