KR100475559B1 - A microbes pesticide active against brevibacillus compost rot boil effect and process for preparation thereof - Google Patents

A microbes pesticide active against brevibacillus compost rot boil effect and process for preparation thereof Download PDF

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KR100475559B1
KR100475559B1 KR10-2003-0045950A KR20030045950A KR100475559B1 KR 100475559 B1 KR100475559 B1 KR 100475559B1 KR 20030045950 A KR20030045950 A KR 20030045950A KR 100475559 B1 KR100475559 B1 KR 100475559B1
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microbial
composting
brevibacillus
compost
organic
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KR20050005955A (en
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성 준 유
김홍기
김영호
황준영
송정영
류재춘
나재범
곽두원
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(주)바이오쉴드
성 준 유
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00Microorganisms, 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
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
    • C12N1/205Bacterial isolates
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION 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
    • A01N63/00Biocides, 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
    • A01N63/10Animals; Substances produced thereby or obtained therefrom
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B09DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09CRECLAMATION OF CONTAMINATED SOIL
    • B09C1/00Reclamation of contaminated soil
    • B09C1/10Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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    • C05FORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00Other organic fertilisers
    • C05F11/08Organic fertilisers containing added bacterial cultures, mycelia or the like
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Abstract

본 발명은 유기물의 퇴비화를 촉진시키는 효과가 있으며 식물병원성 진균에 길항적인 신규한 브레비바실러스속 미생물 균주를 토양으로부터 분리하고, 이를 브레비바실러스 라테로스포러스(기탁번호 KCTC 10478BP)로 명명하고, 퇴비부숙촉진과 식물병을 방제하기에 적합한 미생물 제제로 제조하는 방법에 관한 것이다. 상세하게는 본 발명 균주의 세포 및 배양액을 천연 및 합성 제오라이트를 포함한 다양한 부형제를 혼합하여 처리하면 퇴비화 과정중에 일어나는 부숙 및 유기물의 완숙화와 부숙화 과정중에 발생되는 암모니아 가스, 유기산 및 휘발성 성분 등 악취성분의 감소에 효과가 있고, 식물 병원성 진균인 뿌리썩음병(Fusarium solani), 시들음병(Fusarium oxysporum), 모잘록병(Pythium sp), 잿빛곰팡이병(Botrytis cinerea), 탄저병(Colletotrichum gloeosporioides), 점무늬병(Alternaria panax), 역병(Phytophthora capsici), 인삼뿌리썩음병(Cylindrocarpon destructans)등에 특이적으로 항진균 효과가 있다. 상기 미생물은 항진균 활성에 더하여 유기물 분해능, 농약에 대한 내성 및 축산폐기물 등의 유기물 분해능을 지니고 있으므로 환경보전 및 정화 작용이 있는 다기능적 미생물 제제로 유용하게 사용될 수 있다.The present invention has the effect of promoting the composting of organic matter and isolates a new Brevibacillus microbial strain antagonistic to phytopathogenic fungi from the soil, named this Brevibacillus Laterosporus (Accession No. KCTC 10478BP), compost The present invention relates to a method for preparing a microbial agent suitable for promoting homeostasis and controlling plant diseases. Specifically, when the cells and cultures of the strain of the present invention are mixed with various excipients including natural and synthetic zeolites, odors such as ammonia gas, organic acids, and volatile components generated during maturation and maturation of organic matter and organic matter occur during composting. It is effective in reducing constituents, and is a plant pathogenic fungus, Fusarium solani , Fusarium oxysporum , Pythium sp, Botrytis cinerea , Colletotrichum gloeosporioides , and spotty disease ( Alternaria panax ). , Anti-fungal effect on phytophthora capsici , ginseng root rot ( Cylindrocarpon destructans ). In addition to antifungal activity, the microorganism has an organic decomposition ability, resistance to pesticides and organic matter degradation such as livestock waste, and thus may be usefully used as a multifunctional microbial preparation having environmental conservation and purification.

Description

신규한 브레비바실러스속 미생물 균주를 이용한 퇴비부숙 효과와 항진균 활성이 있는 미생물 제제 및 그 제조방법{A microbes pesticide active against brevibacillus compost rot boil effect and process for preparation thereof}A microbes pesticide active against brevibacillus compost rot boil effect and process for preparation according to compost maturity effect and antifungal activity using novel Brevibacillus microbial strain
본 발명은 브레비바실러스속 미생물 균주를 이용한 퇴비부숙 효과와 항진균 활성이 있는 미생물 제제 및 그 제조방법에 관한 것으로, 특히 식물병원성 진균에 길항적인 신규한 브레비바실러스속 미생물 균주를 토양으로부터 분리하여 이를 브레비바실러스 라테로스포러스(기탁번호 KCTC 10478BP)로 명명하고, 퇴비부숙촉진과 식물병을 방제하기에 적합한 미생물 제제로 제조한 다음, 균주의 세포 및 배양액을 천연 및 합성 제오라이트를 포함한 다양한 부형제를 혼합하여 처리하면 퇴비화 과정 중에 일어나는 부숙 및 유기물의 완숙화와 부숙화 과정 중에 발생되는 암모니아 가스, 유기산 및 휘발성 성분 등 악취성분의 감소에 효과가 있고, 식물 병원성 진균인 후사리움 솔라니(Fusarium solani : 뿌리썩음병), 후사리움 옥시스포름(Fusarium oxysporum : 시들음병), 피시움 속(Pythium sp. : 모잘록병), 보트리티스 시네레아(Botrytis cinerea : 잿빛 곰팡이병), 콜레토트리쿰 글로에오스포리오이데스(Colletotrichum gloeosporioides : 탄저병), 알터나리아 파낙스(Alternari apanax : 점무늬병), 파이토프쏘라 칵토름(Phytophthora capsici : 역병), 실린드로카폰 데스트럭탄스(Cylindrocarpon destructans : 인삼뿌리썩음병)등에 특이적으로 항진균 효과가 있는 퇴비부숙 효과와 항진균 활성이 있는 미생물 제제 및 그 제조방법에 관한 것이다.The present invention relates to a microbial agent having a compost maturity effect and antifungal activity using the Brevibacillus microbial strains and a method for producing the same, in particular, a novel Brevibacillus microbial strain antagonistic to phytopathogenic fungi from the soil and Named Brevibacillus Laterosporus (Accession No. KCTC 10478BP), prepared as a microbial preparation suitable for promoting compost maturation and controlling plant diseases, and then mixing the cells and cultures of the strain with various excipients, including natural and synthetic zeolites. When used in the treatment process, it is effective for the maturation of organic matters and organic matters during composting and the reduction of odors such as ammonia gas, organic acids and volatile components during the maturation process, and the Fusarium solani root, a plant pathogenic fungus. Rot), Hussain Solarium oxy spokes name (Fusarium oxysporum: wilt), Pythium sp. ( Mozalok disease), Botrytis cinerea (ash fungus), Colletotrichum gloeosporioides (anthrax), Alternari apanax ), Microbial agent with antifungal effect and antifungal activity that have specific antifungal effect on Phytophthora capsici (Pneumonic plague), Cylindrocarpon destructans (Ginseng root rot) It is about.
일반적으로 산업화의 계속적인 진행으로 생활 여건 및 환경이 좋아지면서 산업체에서 나오는 난분해성 폐수뿐만 아니라 고농도의 유기물을 함유한 축산업 폐기물이 환경문제로 크게 대두되고 있으나, 이러한 축산폐기물은 지속적인 양적 증가뿐만 아니라 농축된 유기물 및 화학물질을 이용한 처리시 상당한 어려움을 더하고 있다. 따라서 축산 폐기물의 생물학적 처리를 통한 고기능을 갖는 생물 비료의 개발은 유기질비료자원의 활용으로 자원의 재활용과 환경오염의 경감을 가져온다. 특히 토양의 비옥도가 낮은 현실에서 가축분뇨의 유기질 비료로의 전환은 그 의의가 크다.In general, as industrial conditions continue to improve, living conditions and the environment improve, not only hardly degradable wastewater from industrial fields but also livestock wastes containing high concentrations of organic matter are emerging as environmental issues. Significant difficulties are added to the treatment with organics and chemicals. Therefore, the development of high-performance biological fertilizers through biological treatment of livestock waste brings about the recycling of resources and the reduction of environmental pollution through the use of organic fertilizer resources. In particular, the conversion of livestock manure to organic fertilizer is significant in the low soil fertility.
유기물질의 퇴비화는 미생물 활동결과 안정화된 퇴비로 전환시키는 과정으로서 여러 요인들이 작용하나 궁극적으로는 미생물의 활성을 촉진시키는 것이라 할 수 있다. 따라서 미생물의 활성을 증가시키기 위해 유용한 미생물 제제의 활용에 대한 검토가 많이 이루어져 왔는데, 락토바실러스 플란타름(Lactobacillus plantarum), 바실러스 서브틸리스(Bacillus subtlis), 페니바실러스 마르세란스(Paenibacillus marcerans) 등을 첨가한 퇴비화 효과에 대한 보고가 있다. 또한 작물의 재배에 있어서 방제가 까다로운 토양전염성 식물병원균들에 있어서 현재까지 뚜렷한 방제 대책이 없어 해마다 작물의 연작시 그 피해가 증가하고 있다.Composting of organic material is a process of converting the result of microbial activity into stabilized compost, but many factors work, but ultimately it promotes the activity of microorganisms. Therefore, many studies have been made on the use of useful microbial agents to increase the activity of microorganisms. There are reports of added composting effects. In addition, there is no clear control measures to soil infectious phytopathogens that are difficult to control in the cultivation of crops.
따라서, 이러한 문제점을 해결하기 위해 무공해 방제기술인 길항 미생물을 이용한 농작물의 병피해 방지기술의 개발이 필요한 실정이다. 이를 위한 방안으로 축산폐기물의 비료화와 식물병을 방제할 수 있는 유용미생물의 선발과 적용은 중요한 과제라 할 수 있다.Therefore, in order to solve this problem, it is necessary to develop a disease prevention technology of crops using antagonistic microorganisms, which are pollution control technologies. For this purpose, the selection and application of useful microorganisms that can fertilize livestock waste and control plant diseases are important tasks.
미부숙된 퇴비를 토양에 사용할 경우 병해충의 먹이를 제공하여 병해충의 피해를 받을 우려가 높고 시설하우스에서는 토양중 발효가 일어나 암모니아, 질산등의 가스발생으로 작물이 피해를 입게 된다. If uncomposed compost is used in the soil, it is highly likely to be damaged by providing pest food. In the facility house, the fermentation occurs in the soil, which causes damage to crops due to gas generation such as ammonia and nitric acid.
따라서 유기성 폐자원의 농업적 재활용과 고품질의 퇴비생산을 위하여 악취경감 및 부숙촉진 미생물에 대한 연구가 절실하다고 할 수 있다. 섬유질을 분해하는 바실러스(Bacillus)속 미생물중에는 글루코스(glucose)를 포함한 다양한 당을 이용하여 산을 생성하고, 특히 질소가 있는 경우 산소가 부족하여도 최소 일주일 정도는 생육이 가능한 미생물로 토양, 마른풀, 먼지 등 자연계에 널리 분포하는 그람 양성(Gram positive)의 간균으로 길이는 2-3㎛로서 호기성이며, 내생포자(endospore)를 형성하는 미생물이 많이 존재한다.Therefore, it is urgent to study odor-reducing and home-promoting microorganisms for agricultural recycling of organic waste resources and producing high quality compost. Among microorganisms of the genus Bacillus, which degrades fiber, acid is produced by using various sugars including glucose, and especially in the case of nitrogen, it is a microorganism capable of growing for at least one week even when oxygen is insufficient. Gram positive bacillus widely distributed in nature such as, dust, etc., 2-3㎛ in length, aerobic, there are many microorganisms forming endospores.
퇴비의 탄소원인 톱밥 등은 셀루로스(cellulose), 헤미셀루로스(hemicellulose)를 비롯하여 리그니(lignin) 및 펙틴(pectin)과 같은 분해되기 어려운 물질로 되어 있으나, 바실러스속 미생물중 일부는 체외분비효소인 씨엠씨아제(CMCase), 자일라나제(xylanase)와 락타제(lactase)등을 분비하여 난분해성 물질을 분해하고 이를 탄소원으로 이용한다. 이처럼 바실러스속 균주는 생육이 양호하고, 다양한 특수성을 가진다는 점에서 보다 강력하고 안정성이 우수한 효소를 생산하는 균주로 유기성 폐자원의 재활용등 농업환경개선 측면에서 활용될 수 있도록 다양한 측면의 연구가 필요하다고 판단된다.Sawdust, a carbon source of compost, is composed of cellulose, hemicellulose, and other difficult-to-decompose substances such as lignin and pectin, but some of the Bacillus microorganisms are secreted in vitro. Phosphorus CMCase, xylanase and lactase are secreted to decompose refractory materials and use them as carbon sources. As such, Bacillus strain is a strain that produces more powerful and stable enzyme in terms of good growth and various specificities, and requires various aspects of research so that it can be utilized in terms of improving the agricultural environment such as recycling of organic waste resources. It is judged that
특히 유기성 폐자원인 축산 폐기물의 안정적인 부숙 퇴비화와 길항효과를 갖게 하는 데는 안정화된 미생물의 적용에 있어서 적당한 담체를 필요로 한다. 미생물이 쉽게 부착 성장될 수 있는 담체의 조건으로 미생물이 충분히 부착될 수 있도록 다공성과 넓은 비표면적을 가져야 한다. 또한 미생물의 안정적인 활성을 위해서는 무엇보다도 미생물에 적합한 영양원이 있어야 한다.In particular, in order to have stable home composting and antagonistic effects of livestock waste, which is an organic waste resource, a suitable carrier is required for the application of stabilized microorganisms. The microorganisms should have a porosity and a large specific surface area so that the microorganisms can be sufficiently attached under conditions of a carrier which can be easily attached and grown. In addition, for the stable activity of microorganisms, a nutrient source suitable for microorganisms must be above all.
본 발명은 식물 병원성 진균에 대한 항진균 활성을 가지며 축분을 포함하는 난분해성 유기물의 분해 촉진 기능이 있는 브레비바실러스속 미생물 균주를 이용하여 환경친화적인 식물병 방제제 및 항진균 활성에 더하여 축산폐기물 등의 유기물 분해촉진제 등의 다기능적 미생물 제제를 제공함에 그 목적이 있다.The present invention uses the Brevibacillus microorganism strain having antifungal activity against plant pathogenic fungi and has a function of promoting degradation of hardly decomposable organic substances including sperm, in addition to environmentally friendly plant disease control agents and antifungal activity, such as livestock waste. An object of the present invention is to provide a multifunctional microbial agent such as an organic decomposition promoter.
실시예 1 : 본 발명 미생물 BS-J1의 분리 및 동정Example 1 Isolation and Identification of Inventive Microorganism BS-J1
농업환경에 유용하다고 판단되는 1종의 미생물을 분리하여 식물병원균에 대한 길항력 검사를 실시한 결과 2종(BS-J1, BS-U1)의 미생물이 우수한 것으로 밝혀졌다. 또한 부숙촉진능을 확인하기 위하여 셀루라제(Cellulase), 아밀라제(Amylase), 프레테아제(Protease), 리파아제(Lipase) 등 4종의 효소 활성능을 확인한 결과 4조의 효소에 대하여 2종(BS-J1, BS-U1) 미생물이 세가지 이상 분해능을 보였다. 그 중 유용미생물 1종(BS-J1)을 선발, 분리하여 이화학적 특성 및 식물 병원균 5종 및 배추 육묘를 대상으로 한 길항력 검정 시험결과 85%이상의 높은 병발생 억제율을 보였다. 선발된 유용미생물에 대한 생리 생화학적인 특성과 형태학적 특성을 조사한 결과 그람양성균으로 확인되었다.As a result of antagonistic testing of phytopathogens by separating one microorganism which is considered useful for agricultural environment, two microorganisms (BS-J1 and BS-U1) were found to be excellent. In addition, four kinds of enzyme activities such as cellulase, amylase, pretease, and lipase were identified to confirm the maturation promoting ability. J1, BS-U1) microorganisms showed more than three resolutions. Among them, one useful microorganism (BS-J1) was selected and isolated, and the physicochemical characteristics and antagonistic assay tests of five plant pathogens and Chinese cabbage seedlings showed a high inhibition rate of more than 85%. Physiological biochemical and morphological characteristics of selected microorganisms were identified as Gram-positive bacteria.
생리학적 분석과 16S rDNA 염기서열 분석을 통하여 최종적으로 선발된 미생물 BS-J1은 브레비바실러스 레테로스포러스(Brevibacillus laterosporus)와 97% 이상의 상동성을 나타내어 동종으로 분류하였다.The finally selected microorganism BS-J1 through physiological analysis and 16S rDNA sequencing was homologous with more than 97% homology with Brevibacillus laterosporus .
시험예 1 : 유용미생물의 분리Test Example 1 Isolation of Useful Microorganisms
병원균에 대한 길항효과와 퇴비부숙 효과특성을 나타내는 유용미생물을 분리하기 위해 작물 재배지 포장과 축산농가 주변의 자연발효퇴비와 토양으로부터 시료를 수집하였다. 시료채취는 지상부 15cm 이하 되는 곳에 근권에서 했으며, 포장별 수집은 포장의 중심으로 일정간격으로 수집하였고, 수집된 토양은 5mm×5mm 체로 걸러 10℃에 보관하면서 실험에 이용하였다.In order to isolate useful microorganisms showing antagonistic and compost maturity effects on pathogens, samples were collected from the fields of crop fields and natural fermentation compost and livestock farms. Sampling was carried out at the root zone of less than 15cm above the ground. Collected by pavement was collected at regular intervals as the center of the pavement, and collected soil was collected at 5 ℃ × 5mm sieve and stored at 10 ℃ for the experiment.
이러한 유용미생물의 분리방법은 Burr 등의 방법을 이용하였다. 유용미생물을 분리하기 위해 채집한 토양을 KBA배지에 희석평판법에 따라 형성되는 균체 콜로니수를 40~80 콜로니 형성 수(Colony Forming Unite:CFU)/페트리디쉬(Pertri Dish)로 조정한 후, 25℃에서 24시간동안 배양하였다. 24시간 배양후 퇴비부숙 유용미생물을 선발하기 위해 효소활성용 검정배지(Table 4-1)에서 셀루로즈(Cellulose), 스타치(Starch), 프로테인(Protein), 리피더(Lipid)에 대한 효소활성을 조사하였으며, 아밀라제(Amylase) 및 셀루라제(Cellulase)의 활성 측정은 Ito등의 방법으로 실시하였고, 프레테아제(Protease) 활성측정은 Yanagida등의 방법, 리파아제(lipase)의 활성측정은 Lesuisse 등의 방법을 사용하여 실시하였다. 또한 식물 병원균에 길항효과가 있는 유용미생물을 선발하기 위해 후사리움 솔라니(Fusarium solani), 피시움 속(Pythium sp.), 보트리티스 시네레아(Botrytis cinerea), 라이족토니아 솔라니(Rhizoctonia solani), 파이토프쏘라 칵토름(Phytophthora cactorum)등 식물병원균들의 포자현탁액(108 spore/ml)을 만들어 각각의 KBA 표면에 분무 접종하였다. 분무후 24기간 동안 병원균 포자가 발아하여 생육하면서 KBA에 형성되어 있는 세균 콜로니에 의해 저지원 형성여부를 조사하였다. 퇴비부숙 효과 및 항진균활성을 나타내는 균주들의 콜로니는 순수분리하여 동결건조한 후 -65℃로 보관하였다.As a method for separating useful microorganisms, Burr et al. The soil collected to separate the useful microorganisms was diluted to KBA medium by adjusting the bacterial colony number to 40-80 colony forming water (CFU) / Pertri Dish, and then 25 ° C. Incubated for 24 hours at. Enzyme activity against cellulose, starch, protein, and lipid in an enzyme activity assay medium (Table 4-1) to select useful microorganisms The activity of amylase and cellulase was measured by Ito method, and the protease activity was measured by Yanagida et al., And the lipase activity was measured by Lesuisse et al. It was carried out using the method of. Fusarium solani , Pythium sp., Botrytis cinerea , Rhizoctonia solani Spore suspensions (10 8 spores / ml) of phytopathogens, such as Phytophthora cactorum , were spray-inoculated onto each KBA surface. Pathogen spores germinated and grown for 24 hours after spraying, and the formation of low support by bacterial colonies formed in KBA was examined. Colonies of strains showing compost maturation and antifungal activity were purely isolated, lyophilized and stored at -65 ° C.
(1-1) 유용미생물의 부숙촉진 활성을 조사하기 위한 효소활성 검정(1-1) Enzyme Activity Assay for Investigating the Maturation Activity of Useful Microorganisms
균주번호Strain number 효소 활성Enzyme activity 10분 동안 80℃에서 온도에 대한 내성Resistance to temperature at 80 ° C. for 10 minutes
셀루라제Cellulase 아밀라제Amylase 프로테아제Protease 리파아제Lipase
BS-J1BS-J1 ++++ ++++++ ++++ ++++ 있음has exist
BS-J2BS-J2 ++ -- ++ ++++ --
BS-J3BS-J3 ++++ ++ ++ -- 있음has exist
BS-J4BS-J4 ++ ++++ ++ ++ --
BS-J5BS-J5 -- ++++ ++++ ++ --
BS-U1BS-U1 ++++ ++++ ++++++ ++++ 있음has exist
BS-U2BS-U2 -- ++ ++++ ++ --
BS-U3BS-U3 ++ ++++ ++ ++++ --
BS-U4BS-U4 ++++ ++ -- ++ --
BS-U5BS-U5 ++++ ++ ++++ -- 있음has exist
※ + : 약간, ++ : 우수, +++ : 매우 우수, - : 활성 없음※ +: slightly, ++: excellent, +++: very good,-: no activity
(1-2) 유용미생물의 식물 병원균에 대한 길성 활성 검정(1-2) Prolonged Activity Assay for Plant Pathogens of Useful Microorganisms
균주 번호Strain number 식물병원균Plant pathogen
파이토프쏘라Phytovsora 보트리티스시네레아Botrytis Cinerea 라이족토니아솔라니Raiatonia Solani 피시움 속 Pisces 후사리움솔라니Husarium Solani
BS-J1BS-J1 ++++++ ++++ ++++++ ++++ ++++++
BS-J2BS-J2 ++++ -- ++ ++ ++++
BS-J3BS-J3 ++ ++ ++ ++++ ++
BS-J4BS-J4 ++++++ ++ ++ ++ ++
BS-J5BS-J5 ++++ -- ++ ++ ++
BS-U1BS-U1 ++++ ++++++ ++++++ ++++ ++++
BS-U2BS-U2 ++++ ++ -- ++++ ++
BS-U3BS-U3 ++ ++++ -- ++ ++++
BS-U4BS-U4 ++++ ++++++ ++ -- ++
BS-U5BS-U5 ++ ++ ++ -- ++
※ 길항 활성※ antagonist activity
- : 활성 없음, + : 약간, ++ : 우수, +++ : 매우 우수-: No activity, +: slightly, ++: excellent, +++: very good
실험예 2 : 본 발명 미생물 BS-J1의 동정Experimental Example 2 Identification of Inventive Microorganism BS-J1
Bergey의 시스테믹 박테리올로지 메뉴얼을 이용해 생리화학적 분석과 광학현미경을 이용한 형태적 관찰로 동정하였고, 또한 이들에 대한 정확한 동정을 위해 16S rDNA 영역에 대한 부분 염기서열(700~800bp) 분석을 실시하여 염기서열을 결정하였다. DDBJ/EMBL 진벵크 데이터베이스(GenBank database)에서 이들의 염기서열과 상동성을 검색한 결과 선발된 본 발명 유용미생물(BS-J1)은 브레비바실러스 라테로스포러스와 97% 이상의 높은 상동성이 나타나 동일 종으로 동정하였다.Bergey's Cystemic Bacterology Manual was used to identify physicochemical and morphological observations using optical microscopy, and partial sequencing (700-800bp) analysis of the 16S rDNA region was carried out for accurate identification. The base sequence was determined. As a result of searching for the nucleotide sequence and homology of the DDBJ / EMBL GenBank database, the useful microorganism (BS-J1) of the present invention was selected and showed high homology of more than 97% with Brevibacillus latererosporus. Identified as species.
(1-3) 본 발명 미생물 브레비바실러스 라테로스포러스(Brevibacillus laterosporus)의 생리·생화학적 특성과 형태학적 특성(1-3) Physiological and biochemical and morphological characteristics of the microorganism Brevibacillus laterosporus of the present invention
특성characteristic 유용미생물BS-J1Useful MicroorganismsBS-J1
그람반응(Gram reaction)Gram reaction ++
액체배지에서 운동성Mobility in Liquid Medium ++
NA, YDC or NBY에서 노란 균총 형성Yellow mycelial formation at NA, YDC or NBY --
형광 색소Fluorescent pigment --
젤라틴 분해Gelatin breakdown ++
혐기 배양Anaerobic culture --
가지(rod) 형성Rod formation --
60℃ 또는 고열에서 생장Grow at 60 ℃ or high temperature ++
배양 초기 Rod 형태Initial rod form of culture ++
내생포자 형성Endogenous spore formation ++
(1-4) 본 발명 미생물 브레비바실러스 라테로스포러스(Brevibacillus laterosporus)의 16s rDNA염기서열 일부(1-4) Part of the 16s rDNA base sequence of the microorganism Brevibacillus laterosporus of the present invention
5'-5'-
GACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGGGTCTTCGGACCCTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTGTGAGACTGGATAACATAGGGAAACTTATGCTAATACOGGATAGGGTTTTGCTTCGCCTGAAGCGGAAAGATGGCGCAAGCTAFCACTTACAGATGGGCOTCGGGCGCATTAGCTAGTTGGYGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCACTAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGCTTTCGGGTCGTAAAGTTCTGTTGTTAGGGAAGAAACAGTGCTATTTAAATAAGGTAGCACCTTGACGGTACCTAACGAGAAAGCCACGGCTAACTACGTCGCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGCTATGTAAGTCTGATGTTAAAGCCCGAGGCTCAACCTCGGTTCGCATTGGAAACTGTGTANCTTGAGTGCAGGAGAGGAAAGTGGTATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGGGAAACACCAGTGGCAAAGGCGACTTTCTGGCCTGTAACTGACC-3'GACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGGGTCTTCGGACCCTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTGTGAGACTGGATAACATAGGGAAACTTATGCTAATACOGGATAGGGTTTTGCTTCGCCTGAAGCGGAAAGATGGCGCAAGCTAFCACTTACAGATGGGCOTCGGGCGCATTAGCTAGTTGGYGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCACTAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGCTTTCGGGTCGTAAAGTTCTGTTGTTAGGGAAGAAACAGTGCTATTTAAATAAGGTAGCACCTTGACGGTACCTAACGAGAAAGCCACGGCTAACTACGTCGCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGCTATGTAAGTCTGATGTTAAAGCCCGAGGCTCAACCTCGGTTCGCATTGGAAACTGTGTANCTTGAGTGCAGGAGAGGAAAGTGGTATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGGGAAACACCAGTGGCAAAGGCGACTTTCTGGCCTGTAACTGACC-3 '
(1-5) 16s rDNA 염기서열 분석을 통한 본 발명 미생물 브레비바실러스 라테로스포러스(Brevibacillus laterosporus)의 계통도(1-5) Schematic diagram of the microorganism Brevibacillus laterosporus of the present invention through 16s rDNA sequencing
실시예 2 : 본 발명 미생물 BS-J1의 대량배양 및 제제화(C 타입, G 타입)Example 2 Mass Culture and Formulation of Inventive Microorganism BS-J1 (Type C, Type G)
유용미생물이 자연 환경생태계 또는 퇴비화 과정중에서 생존을 유지하면서 왕성한 유기물 분해 촉진력 및 식물병원균에 대한 길항활성을 유지하기 위해서는 영양분공급(단기적, 중장기간 이용가능한 영양분), 길항세균의 보호 등이 필요하다. 이러한 제반요인을 만족시키기 위해 여러가지 부형제들을 포함하여 다음과 같은 타입의 제형을 개발하였다.In order for the useful microorganisms to survive in the natural environmental ecosystem or in the composting process, it is necessary to supply nutrients (short-term, medium- and long-term available nutrients), and to protect antagonistic bacteria in order to maintain active organic decomposition promotion and antagonistic activity against plant pathogens. . To satisfy these factors, the following types of formulations were developed, including various excipients.
C 타입의 성분 : 톱밥, 쌀겨, 어분, 제올라이트, 마슘, 인슘, 분당, 미생물균체 및 그 배양액.Ingredients of type C: sawdust, rice bran, fishmeal, zeolite, calcium, phosphate, bun sugar, microbial cells and their cultures.
G 타입의 성분 : 톱밥, 쌀겨, 어분, 제올라이트, 마슘, 인슘, 쑈디카본, 분당, 미물균체 및 그 배양액.Ingredients of type G: sawdust, rice bran, fish meal, zeolite, calcium, phosphorus, chandicarbon, bunsang, microbial cells and their cultures.
위에서 나열한 각각의 배양합성 타입 제형들은 일정 성분의 부형제를 일정비율로 조합한 후에 유용미생물에 적합한[BS-J1: PYG 액체배지(펩톤(Peptone) 5g , 이스트 엑스트라(Yeast extract) 5g, 글루코스(Glucose) 10g, K2HP04 1g, 비프 엑스트렉트(Beef extract) 1g, D.W 1ℓ, pH 7.2]에서 108~109농도로 배양된 유용미생물을 섞어준 후에 1주일간 교반기 내에서 교반시켜주면서 충분히 배양한다. 1주일간 배양된 제형에 어분 등의 영양원을 넣어준 다음, 교반기 내에서 교반시켜주면서 2주일간 배양하여 제형을 완성하였다. 최종 산물을 107 CFU/g(유용미생물농도)의 제형이 완성된다.Each of the above-mentioned culture synthesis type formulations was combined with a certain proportion of excipients in a certain ratio, and then used for a useful microorganism [BS-J1: PYG liquid medium (Peptone 5g, Yeast extract) 5g, Glucose 10g, 1g K2HP04, 1g Beef Extract, 1g DW, 1l, pH 7.2], mixed with useful microorganisms incubated at a concentration of 10 8 ~ 10 9, and incubated with stirring in a stirrer for 1 week. Nutrient such as fishmeal was added to the formulation cultured for 1 week, followed by incubation for 2 weeks with stirring in a stirrer to complete the formulation of 10 7 CFU / g (utility microorganism concentration).
실시예 3 : 본 발명 미생물 제제(C 타입, G 타입)의 퇴비부숙 효과Example 3: composting effect of the present microbial preparations (type C, G type)
본 발명은 퇴비화를 촉진시키는 미생물을 제제화하여 퇴비화 과정중에 일어나는 부숙 유기물의 완숙한 과정에 대한 적합성을 규명하고, 각종 유기 및 무기성분의 구성비율을 조사하여 물리화학적 변화를 살펴보았고, 이와 동시에 부숙화 과정중에 발생되는 암모니아가스, 휘발성 유기산성분 등 악취성분의 발생특성에 대하여 상대 비교 분석하여 평가함으로써 악취발생량이 적은 고품질의 퇴비를 생산할 수 있는 미생물 제제를 완성하였다.The present invention is to formulate the microorganisms to promote the composting to determine the suitability for the mature process of the maturation organic matter occurring during the composting process, and to investigate the physicochemical changes by examining the composition ratio of various organic and inorganic components, and at the same time Relative comparative analysis of the development characteristics of malodorous components such as ammonia gas and volatile organic acid components generated during the process was carried out to complete a microbial preparation capable of producing high-quality compost with a low amount of malodors.
처리구별 미생물 및 담체처리양은 처리구별 무게중량에 대비하여 처리하였으며, 미생물은 1%, 미생물 제제는 무게중량대비 4.5%로 처리하였다. 각각의 처리내용은 대조구(C), 미생물 단독 처리구(M), C처리구(C+M), G처리구(G+M)등 4개의 처리구를 두고 실험을 실시하였다.The amount of microorganisms and carriers by treatment group was treated relative to the weight weight of each treatment group, and 1% of microorganisms and 4.5% of microbial formulations were treated. Each treatment was experimented with four treatments, control (C), microbial alone treatment (M), C treatment (C + M), G treatment (G + M).
퇴비화 과정중 각종 유기 및 무기성분 등의 물리화학적 변화를 분석한 결과는 다음과 같다.The results of analyzing the physicochemical changes of various organic and inorganic components during the composting process are as follows.
1. 퇴비화의 반응기간 동안 pH 변화는 발효초기엔 별 영향이 없었으며, 기질의 분해로 인한 암모니아 등의 영향에 의해 상승 후 기간이 지남에 따라 감소하였으며 pH가 8.5~9.0 정도를 유지하였다.1. During the composting period, the pH change was not affected in the early stage of fermentation. The pH was decreased over the period after the rise due to the ammonia effect of substrate decomposition.
2. 퇴비화 과정중 온도의 변화는 모든 처리구에서 실험 개시 후 2~3일까지 온도의 급격한 상승으로 60℃정도까지 도달하였으며, 그 이후에는 감소하여 25일에는 37~39℃까지 감소하였다.2. The temperature change during the composting process reached up to 60 ℃ due to the rapid increase in temperature from 2 to 3 days after the start of the experiment, and then decreased to 37 ~ 39 ℃ on the 25th.
3. 총탄소의 함량은 다소 감소하는 현상을 보였으나, 각 처리구마다 탄소의 차이는 온도변화와 유사한 양상을 보였다.3. The total carbon content showed a slight decrease, but the difference of carbon in each treatment was similar to the temperature change.
4. 전질소는 천연 C처리구와 G처리구는 퇴비화 25일 이후에 1.71~1.77%로 타 처리구보다 높게 나타났다.4. The total nitrogen of natural C and G treatments was 1.71 ~ 1.77% higher than other treatments after 25 days of composting.
5. C/N율은 부숙이 진행됨에 따라 퇴비화 초기에 약간 증가하다가 감소하여 25일쯤 C/N율이 21.4~30.0으로 감소하였다. C처리구와 G처리구의 최종 C/N율이 대조구와 미생물 단독 처리구의 최종 C/N율보다 낮게 나타났다. 이는 탄소함량이 감소와 질소함량의 증가가 다른 대조구와 미생물 단독 처리구보다 크게 나타났기 때문인 것으로 보인다.5. The C / N rate increased slightly during the composting period and then decreased as the maturity progressed. Then, the C / N rate decreased from 21.4 to 30.0 at 25 days. Final C / N rates of C and G treatments were lower than those of control and microbial treatments. This may be because the decrease in carbon content and the increase in nitrogen content were greater than those of other control and microbial treatment alone.
6. 퇴비화 과정중 모든 처리구의 인산함량 변화추이가 전반적으로 증가하는 경향을 나타냈다.6. In the composting process, there was a general trend of increasing phosphate content of all treatments.
7. 가리의 성분함량 변화는 퇴비화가 진행될수록 약간 증가하는 경향을 보였다.7. Changes in the composition of the girly tended to increase slightly as composting progressed.
8. N03--N은 96~106mg/kg정도이었으나 퇴비화가 진행될수록 계속적으로 증가하였고 C처리구 G처리구가 다른 처리구보다 질산태질소의 함량이 높았으며, NH4+ -N 함량은 퇴비화가 진행될수록 계속적으로 감소하는 경향을 보였다.8. N03--N was 96 ~ 106mg / kg, but it increased continuously as composting progressed. The treatment of G treatment C had higher nitrogen nitrogen than other treatments, and NH4 + -N content continued to increase as composting progressed. It tended to decrease.
9. 퇴비화 주발효 기간 후 완료단계에서 퇴비의 분석결과 비료공정 규격의 중금속의 함량 모두 농림부 고시 제 2002-7호의 부산물비료 규격이하로 검출되었다.9. Composting As a result of the compost analysis at the completion stage after the main fermentation period, the contents of heavy metals in the fertilizer process standard were found to be less than the by-product fertilizer standard of Notice No. 2002-7.
돈분의 퇴비화 과정중 악취물질로서 중요한 공정제어 인자로 이용되는 염기성가스인 암모니아 가스(Ammonia gas)와 강산성가스인 유기산(Volitie organicacid)의 발생 특성에 대하여 상대비교 분석결과는 다음과 같다.The comparative results of the characteristics of ammonia gas, a basic gas used as an odorous substance during the composting process of swine manure, and organic acid, a strong acid gas, are as follows.
1. 퇴비화 2일후부터 암모니아가스의 발생이 급격히 증가후 이후 감소하는 경향을 보였으며, C와 G처리구에서 암모니아 가스의 발생 농도가 대조구 및 미생물 단독처리구와 비교하여 최고 3배이상 크게 감소한 결과를 보였다.1. After 2 days of composting, ammonia gas was rapidly increased and then decreased, and ammonia gas concentration was decreased up to 3 times in C and G treatments compared to control and microbial treatments. .
2. 퇴비화 과정중 휘발성 유기산 성분인 부틸릭 산(butyric acid), 이소-부틸릭 발레릭 산(iso-butyric valeric acid), 이소-발레릭 산(iso-valeric acid), 프로피오닉 산(propionic acid), 아세틱 산(acetic acid)의 정량적 분석을 통하여 부숙화 정도를 평가한 결과 프로피오닉산(propionic acid)이 퇴비화 과정중 발생한 휘발성 유기산 중에 서 가장 많은 함량이 검출되었고, 전체적으로 대구조>미생물 단독 처리구>C처리구>G처리구 순으로 퇴비화 과정중 휘발성 유기산의 감소 효율이 낮은 것으로 나타났다.2. Butyric acid, iso-butyric valeric acid, iso-valeric acid, propionic acid ), Quantitative analysis of acetic acid showed that the highest content of propionic acid was found in volatile organic acids generated during composting process. In the order of treatment> C treatment> G treatment, the reduction efficiency of volatile organic acids during composting was low.
실시예 4 : 본 발명 미생물 BS-J1의 배출 모잘록병에 대한 병방제 효과Example 4 Effect of the Disease Control of the Invented Microorganism BS-J1 on Exhaust Mozal Disease
본 발명 유용미생물들의 식물병방제 효과를 알아보기 PDB에서 3일간 25℃에서 배양한 다음, 이들을 8000rpm에서 10분간 원심분리하여 균체를 회수하였다. 이어서 K2HP04 7H20 1% 용액으로 다시 세척 후 균체를 회수하여 1.5% 메칠 셀루로스(Methyl Cellulouse) 용액과 섞고 배추유모에 처리하였다. 배추유묘에 병을 일으키는 라이족토니아 솔라니(Rhizoctonia solani)를 이용한 방제 효과를 실내에서 포트 검정 후 이 병율 조사는 다음과 같은 방법으로 실시하였다.To find out the phytopathogenic effect of the useful microorganisms of the present invention was incubated in PDB for 3 days at 25 ℃, these were centrifuged at 8000rpm for 10 minutes to recover the cells. After washing again with K 2 HP0 4 7H 2 0 1% solution, the cells were recovered, mixed with 1.5% methyl cellulose (Methyl Cellulouse) solution and treated with cabbage mother. After the pot test was conducted indoors to control the effect of Rhizoctonia solani, which causes cabbage seedlings, the prevalence was investigated by the following method.
(a×1)+(b×2)+(c×3)+(d×4)                 (a × 1) + (b × 2) + (c × 3) + (d × 4)
* 이병지수(%)= -------------------------- x 100* Bite Index (%) = -------------------------- x 100
개체수 × 4                        Population × 4
뿌리에 나타난 병반의 크기에 따라 이병지수를 계산하였으며 0.1%~20%를 a, 10.1%~40%를 b, 30.1%~50%를 c 그리고 50% 이상을 d로 이병지수로 하여 발병율을 계산하였다.The morbidity index was calculated according to the size of the lesion on the root, and the incidence rate was calculated from 0.1% to 20% as a, 10.1% to 40% as b, 30.1% to 50% as c and at least 50% as d. It was.
유용미생물의 배추 모잘록병에 대한 병방제 효과Effectiveness of Microorganisms against Cabbage Mozolox Disease
유용미생물 처리* (BS-J1)Useful Microbial Treatment * (BS-J1) 무처리구 No treatment
유묘수Seedling 2020 2020
모잘록병Mozalok disease 99 1919
건강한 유모 비율(%)Healthy Nanny Ratio (%) 5555 55
방제가(%)**Control Price (%) ** 90.990.9 --
* 유묘를 5주씩 이식하고, 실험은 4반복실시. 포트당 50㎖씩 미생물 현탁액(BS-J1 : 2.4 x 106cell/㎖)을 처리.* Seedlings were transplanted every 5 weeks and the experiment repeated 4 times. Treat microbial suspension (BS-J1: 2.4 x 10 6 cells / ml) at 50 ml per pot.
** 방제가(%)= [1- (처리구/무처리구] x 100.** Controlled value (%) = [1- (treated / untreated) x 100.
이상 실시예와 실험예를 통하여 설명한 바와 같이, 토양에서 선발 분리한 본 발명 미생물 브레비바실러스 라테로스포러스(Brevibacillus laterosporus)는 퇴비부숙 촉진 및 축분을 비롯한 폐기물성 자원의 재활용이라는 측면에서 유용할 뿐만아니라, 식물병에 대하여 방제 효과가 있다는 측면에서 농업에도 매우 유용한 발명이라 할 수 있다.As described through the above examples and experimental examples, the microorganism Brevibacillus laterosporus of the present invention selected and separated from the soil is not only useful in terms of promoting compost maturation and recycling of waste materials including animal stock. In addition, it can be said to be a very useful invention in agriculture in that it is effective in controlling plant diseases.

Claims (3)

  1. 퇴비부숙 촉진 및 식물병원균에 길항력이 있는 미생물 브레비바실러스 라테로포러스(Brevibacillus laterosporus)(기탁번호 KCTC 10478BP).Microbial Brevibacillus laterosporus (Accession No. KCTC 10478BP) which promotes compost maturation and antagonizes phytopathogens.
  2. 제 1항의 상기 미생물 또는 이들 혼합물을 유효성분으로 함유하는 것을 특징으로 하는 토양개량, 퇴비부숙촉진 및 식물병원성 진균 방제용 미생물 제제.A microorganism preparation for soil improvement, compost maturation and phytopathogenic fungi, comprising the microorganism of claim 1 or a mixture thereof as an active ingredient.
  3. 제 1항의 상기 미생물을 배양하여 수득한 세균 세포, 또는 이들 세포가 생산한 유용물질들을 통상의 부형제에 혼합하여 제조하는 것을 특징으로 하는 퇴비부숙 및 식물 병원성 진균 방제용 미생물 제제의 제조 방법.A method for producing a microbial preparation for composting and controlling plant pathogenic fungi, wherein the bacterial cells obtained by culturing the microorganisms of claim 1 or useful materials produced by these cells are mixed with conventional excipients.
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KR102080866B1 (en) 2018-11-26 2020-02-24 대한민국 Primer set for detection Ilyonectria mors-panacis, including kits and identification methods
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KR20160050716A (en) * 2014-10-30 2016-05-11 대한민국(농촌진흥청장) Brevibacillus R2a23 and its use
KR101656228B1 (en) 2014-10-30 2016-09-09 대한민국 Brevibacillus R2a23 and its use
KR101656230B1 (en) 2014-10-30 2016-09-09 대한민국 Brevibacillus R1a13 and its use

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