KR102626565B1 - Composition for controlling plant diseases comprising culture solution of Bacillus velezensis CMML20-16 or extract thereof, methods for manufacturing thereof, and methods for plant disease control by using them - Google Patents
Composition for controlling plant diseases comprising culture solution of Bacillus velezensis CMML20-16 or extract thereof, methods for manufacturing thereof, and methods for plant disease control by using them Download PDFInfo
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- KR102626565B1 KR102626565B1 KR1020210179146A KR20210179146A KR102626565B1 KR 102626565 B1 KR102626565 B1 KR 102626565B1 KR 1020210179146 A KR1020210179146 A KR 1020210179146A KR 20210179146 A KR20210179146 A KR 20210179146A KR 102626565 B1 KR102626565 B1 KR 102626565B1
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- cmml20
- strain
- bacillus
- plant diseases
- causative agent
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Abstract
본 발명은 바실러스 벨레젠시스 CMML20-16 균주의 배양액 또는 균주 배양액의 추출물을 포함하는 식물병 방제용 조성물, 이의 제조방법, 및 식물병 방제 방법에 관한 것으로서, 상기 균주의 배양액 또는 이의 추출물은 방제가 어려운 잔디의 주요 토양 병해인 갈색잎마름병 및 라이족토니아마름병을 유발하는 라이족토니아 솔라니에 대해 우수한 방제 효과를 나타내므로 잔디 병해의 방제에 이용할 수 있다.The present invention relates to a composition for controlling plant diseases comprising a culture medium or extract of the strain culture medium of the Bacillus belegensis CMML20-16 strain, a method for producing the same, and a method for controlling plant diseases, wherein the culture medium of the strain or the extract thereof is effective in controlling plant diseases. It has an excellent control effect against Rhizoctonia solani, which causes brown leaf blight and Rhizoctonia blight, which are major soil diseases of difficult lawns, so it can be used to control lawn diseases.
Description
본 발명은 바실러스 벨레젠시스 CMML20-16 균주의 배양액 또는 균주의 배양액의 추출물을 포함하는 식물병 방제용 조성물, 이의 제조방법 및 식물병 방제 방법에 관한 것으로서, 더욱 상세하게는 항균 활성을 갖는 바실러스 벨레젠시스 CMML20-16 균주의 배양액, 균주의 배양액의 추출물에서 확인된 식물병 원인균에 대한 방제 효과에 관한 것이다.The present invention relates to a composition for controlling plant diseases comprising a culture medium of the strain Bacillus belegensis CMML20-16 or an extract of the culture medium of the strain, a method for producing the same, and a method for controlling plant diseases, and more specifically, to a method for controlling plant diseases, and more specifically, to a composition for controlling plant diseases containing the culture medium of the strain Bacillus belegensis CMML20-16 or an extract of the culture medium of the strain. This relates to the control effect against plant disease-causing bacteria identified in the culture medium of the Gensis CMML20-16 strain and extracts of the strain's culture medium.
잔디는 토양의 침식 제어, 먼지 발생 방지, 냉각 기능, 상해 방지 등의 기능적인 측면과 아름다운 경관을 제공하는 휴양적, 심미적인 측면이 있는 식물이다. 이러한 잔디는 전 세계적으로 널리 이용하는 작물 중 하나이며, 한국에서도 여가시간 증가와 레저 스포츠에 관한 관심 증대로 공원, 도심 녹화, 축구장, 야구장, 골프장 등에서 잔디가 광범위하게 사용됨에 따라 잔디 병해가 다양해지고 피해 규모 또한 증가하는 추세이다.Grass is a plant that has functional aspects such as soil erosion control, dust prevention, cooling function, and injury prevention, as well as recreational and aesthetic aspects that provide beautiful scenery. Such grass is one of the crops widely used around the world, and in Korea as well, with increased leisure time and increased interest in leisure sports, grass is widely used in parks, urban greening, soccer fields, baseball fields, golf courses, etc., resulting in a variety of lawn diseases and damage. The size is also increasing.
다양한 잔디의 병해 중 갈색잎마름병 (Brown patch)과 라이족토니아마름병 (Large patch)은 라이족토니아 솔라니 (Rhizoctonia solani)라는 진균에 의해 발병하며, 주로 토양에 분포하기 때문에 발병 시 방제가 어렵고, 식재 상태를 쉽게 바꿀 수 없는 잔디의 특성상 예방 및 방제를 위해 많은 화학비료와 농약을 사용할 수 밖에 없는 실정이다. 특히, 골프장의 경우, 가장 넓은 면적인 페어웨이에서 해당 병해가 발병하기 때문에 방제를 위해 많은 양의 화학 농약이 사용될 수 밖에 없다. 이는 환경 오염, 사람과 가축 독성, 약제 저항성 미생물의 출현을 유발하게 되어 환경에 대한 사회적 우려를 낳고 있다.Among various lawn diseases, brown patch and large patch are caused by a fungus called Rhizoctonia solani . They are mainly distributed in the soil, so they are difficult to control when they occur. Due to the nature of grass, where the planting condition cannot be easily changed, there is no choice but to use a lot of chemical fertilizers and pesticides for prevention and control. In particular, in the case of golf courses, because the disease occurs in the fairway, which is the largest area, large amounts of chemical pesticides have to be used for control. This is causing environmental pollution, toxicity to humans and livestock, and the emergence of drug-resistant microorganisms, raising social concerns about the environment.
또한, 최근 기후변화 대응 전략 중 하나로 화학 농약과 비료의 사용량을 감축하려는 세계적인 움직임에 따라 다양한 미생물 농약 및 제제의 개발이 전세계적으로 이어지고 있다. 생물학적 제제로서 상용화된 대부분의 유용 미생물은 바실러스 속에 속한다. 바실러스 속의 경우 열과 스트레스에 오래 견딜 수 있는 내생포자를 형성하여, 오랜 기간 보관하더라도 활성을 잃지 않는다. 다양한 기능을 가진 물질을 생산하여 식물의 생육을 촉진하고, 식물의 면역기능을 높여 작물이 식물 병원균에 대한 저항성을 높이는 작용을 활성화한다. 또한, 항진균성 리포펩타이드인 설펙틴 (Surfectin), 이튜린 (Iturin), 펜지신 (Fengycin) 등의 물질을 생산하여 식물 병원성 진균을 사멸하는 활성을 나타낸다.In addition, in accordance with the global movement to reduce the use of chemical pesticides and fertilizers as one of the recent climate change response strategies, the development of various microbial pesticides and preparations is continuing around the world. Most useful microorganisms commercialized as biological agents belong to the genus Bacillus. In the case of the Bacillus genus, endospores are formed that can withstand heat and stress for a long time, and do not lose activity even when stored for a long period of time. By producing substances with various functions, it promotes plant growth and increases the plant's immune function, activating the action of increasing the crop's resistance to plant pathogens. In addition, it produces substances such as antifungal lipopeptides such as Surfectin, Iturin, and Fengycin, which have the activity of killing plant pathogenic fungi.
현재 잔디 갈색잎마름병과 라이족토니아마름병 방제용으로 상용화된 국내 제조 미생물 농약의 주요 균주는 바실러스 서브틸리스(Bacillus subtilis) 종을 이용한 홀인원, 테라스, 탑세이버, 재노탄 등이 있으며, 스트렙토마이세스(Streptomyces) 속을 이용한 마이코싸이드, 암펠로마이세스 (Ampellomyces) 속을 이용한 큐펙트, 페니바실루스 (Paenibacillus) 속을 이용한 탑시드 등이 있다.Currently, the main strains of domestically manufactured microbial pesticides commercialized for controlling lawn brown leaf blight and Rhizoctonia blight include Hole-in-One, Terrace, Topsaver, and Xenotan using Bacillus subtilis species, and Streptomyces There are mycosides using the genus Streptomyces , cufects using the genus Ampellomyces , and top seeds using the genus Paenibacillus .
독일의 ABiTEP 에서 생산 중인 Rhizo-Vital은 바실러스 벨레젠시스 (Bacillus velezensis) FKB24 생균의 포자를 이용한 미생물 제재로서 식물의 생육 촉진 및 다양한 토양의 병해를 방제하는데 널리 사용되고 있으며, 스페인 Agricaldes사의 Botrybel은 바실러스 벨레젠시스 (Bacillus velezensis) AH2 의 항진균활성을 이용하여, 보트리티스 시네리아 (Botrytis cinerea)로 인한 잿빛곰팡이병을 방제하는 미생물농약으로 시판되고 있다.Rhizo-Vital, produced by ABiTEP in Germany, is a microbial agent using spores of the live bacteria Bacillus velezensis FKB24 and is widely used to promote plant growth and control various soil diseases. Botrybel, made by Agricaldes in Spain, is a microbial agent made from spores of the live bacteria Bacillus velezensis FKB24. Using the antifungal activity of Bacillus velezensis AH2, it is commercially available as a microbial pesticide to control gray mold disease caused by Botrytis cinerea .
국내에서도 다양한 균주를 이용한 미생물 제제들이 출시되고 있으나, 전체 농약에 비해 차지하는 비중은 매우 적은 편일 뿐만 아니라 잔디의 병해 방제도 몇몇 균주로만 제한되어 있으며, 바실러스 벨레젠시스 (Bacillus velezensis) 균주를 잔디 병해에 적용하는 생물학적 제제의 발명은 알려져 있지 않다.Microbial preparations using various strains are being released in Korea, but not only do they account for a very small proportion of all pesticides, but the control of lawn diseases is also limited to a few strains, and Bacillus velezensis strains are used for lawn diseases. The invention of the biological agent applied is unknown.
따라서, 이러한 바실러스 벨레젠시스 균주를 이용한 항균 생물 방제제의 개발이 시급한 실정이다.Therefore, there is an urgent need to develop antibacterial biological control agents using these Bacillus belegensis strains.
이에 본 발명자들은 바실러스 벨레젠시스 (Bacillus velezensis) CMML20-16 균주의 배양액 또는 균주의 배양액의 추출물을 식물병 원인균에 처리하였을 때 항균 활성이 월등히 우수한 것을 확인하였다.Accordingly, the present inventors confirmed that the antibacterial activity was significantly superior when the culture medium of the Bacillus velezensis strain CMML20-16 or the extract of the strain's culture medium was treated with plant disease-causing bacteria.
본 발명의 목적은 항균 활성을 갖는 바실러스 벨레젠시스 CMML20-16 균주를 제공하는 것이다.The purpose of the present invention is to provide a Bacillus velegensis CMML20-16 strain having antibacterial activity.
본 발명의 다른 목적은 바실러스 벨레젠시스 CMML20-16 균주의 배양액 또는 이의 추출물을 포함하는 식물병 방제용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for controlling plant diseases containing a culture medium of Bacillus velegensis CMML20-16 strain or an extract thereof.
본 발명의 또 다른 목적은 바실러스 벨레젠시스 CMML20-16 균주를 배양하여 배양액을 제조하는 배양 단계를 포함하는 식물병 방제용 조성물의 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing a composition for controlling plant diseases, including a cultivation step of preparing a culture medium by culturing the Bacillus velegensis CMML20-16 strain.
본 발명의 또 다른 목적은 바실러스 벨레젠시스 CMML20-16 균주의 배양액 또는 이의 추출물을 처리하는 조성물 처리 단계를 포함하는 식물병 방제방법을 제공하는 것이다.Another object of the present invention is to provide a plant disease control method comprising the step of treating a composition for treating the culture medium of Bacillus belegensis CMML20-16 strain or an extract thereof.
이하 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태는 항균 활성을 갖는 바실러스 벨레젠시스 (Bacillus velezensis) CMML20-16 균주에 관한 것이다.One aspect of the present invention relates to Bacillus velezensis CMML20-16 strain having antibacterial activity.
본 발명에 있어서 바실러스 벨레젠시스 CMML20-16 균주는 수탁번호 KCTC 14762BP로 기탁된 바실러스 벨레젠시스 CMML20-16 균주인 것일 수 있다.In the present invention, the Bacillus belegensis CMML20-16 strain may be the Bacillus belegensis CMML20-16 strain deposited under the accession number KCTC 14762BP.
본 발명의 바실러스 벨레젠시스 CMML20-16 균주는 한국생명공학연구원 KCTC (Korean Collection for Type Cultures)에 2021년 11월 10일자 수탁번호 KCTC 14762BP로 기탁되었다.The Bacillus velegensis CMML20-16 strain of the present invention was deposited with the Korea Research Institute of Bioscience and Biotechnology KCTC (Korean Collection for Type Cultures) under accession number KCTC 14762BP on November 10, 2021.
본 발명의 다른 일 양태는 바실러스 벨레젠시스 CMML20-16 균주의 배양액 또는 균주의 배양액의 추출물을 포함하는 식물병 방제용 조성물에 관한 것이다.Another aspect of the present invention relates to a composition for controlling plant diseases comprising a culture medium of the Bacillus velegensis CMML20-16 strain or an extract of the culture medium of the strain.
본 발명에 있어서 바실러스 벨레젠시스 CMML20-16 균주는 수탁번호 KCTC 14762BP로 기탁된 바실러스 벨레젠시스 CMML20-16 균주인 것일 수 있다.In the present invention, the Bacillus belegensis CMML20-16 strain may be the Bacillus belegensis CMML20-16 strain deposited under the accession number KCTC 14762BP.
본 명세서상의 용어 “배양액”은 미생물을 배양한 후, 상기 미생물을 포함한 것을 의미한다.The term “culture medium” in this specification refers to containing microorganisms after culturing them.
본 명세서상의 용어 “추출물”은 균주의 배양액에서 분리한 것을 의미하며, 추출물은 분획물을 포함한다.The term “extract” in this specification refers to separation from the culture medium of the strain, and extract includes fractions.
본 명세서상의 용어 “추출물”은 균주의 배양액에서 분리한 것을 의미하며, 추출물은 배양여액을 포함한다.The term “extract” in this specification refers to something isolated from the culture medium of the strain, and extract includes the culture filtrate.
본 명세서상의 용어 “배양 상층액”은 원심분리 방법을 통해 배양액으로부터 대부분의 미생물을 제거한 후 획득한 상층의 액을 의미한다.The term “culture supernatant” in this specification refers to the upper liquid obtained after removing most microorganisms from the culture medium through centrifugation.
본 명세서상의 용어 “배양여액”은 원심분리 및 여과를 수행함으로써 배양액으로부터 균체를 여과하여 제거한 뒤에 남은 액체를 의미한다. 배양여액은 미생물이 자라는 과정에서 형성하여 배출한 물질들이 포함되어 있어서 그 물질을 정제하거나 추출할 수 있다.The term “culture filtrate” in this specification refers to the liquid remaining after filtering and removing bacterial cells from the culture medium by performing centrifugation and filtration. Culture filtrate contains substances formed and excreted during the growth of microorganisms, so the substances can be purified or extracted.
본 발명에 있어서 식물병은 잔디의 갈색잎마름병 (Brown patch), 라이족토니아마름병 (Large Patch), 검은무늬썩음병, 식물 잎의 검은점무늬병, 잿빛곰팡이병, 잔디의 동전마름병 (Dollar spot), 식물 탄저병, 줄기마름병, 시들음병, 뿌리썩음병, 모잘록병, 잔디의 봄마름병, 균핵병 및 흑반병으로 이루어진 군에서 선택된 1종 이상인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, plant diseases include brown patch of grass, large patch, black spot rot, black spot of plant leaves, gray mold, dollar spot of grass, and plant diseases. It may be one or more species selected from the group consisting of anthracnose, stem blight, wilt, root rot, mottled blight, spring blight of grass, sclerotia and black spot, but is not limited thereto.
본 발명에 있어서 식물병은 원인 균주가 라이족토니아 솔라니 (Rhizocctonia solani), 알타나리아 알타르나타 (Alternaria alternata), 보트리티스 시네레아 (Botrytis cinerea), 클라리레이디아 잭소니 (Clarireedia jacksonii), 콜레토트리쿰 글로에오스포리오데스 (Colletotrichum gloeosporiodes), 콜레토트리쿰 속(Colletotrichum sp.), 포모시스 속 (Phomopsis sp.), 푸사리움 옥시포룸 (Fusarium oxysporum), 푸시리움 속 (Fusarium sp.), 피티움 얼티뭄 (Pythium ultimum), 라이족토니아 세레아리스(Rhizoctonia cerealis), 스켈로티니아 스켈로티오룸 (Sclerotinia sclerotiorum) 및 세라토시스티스 핌브리아타 (Ceratocystis fimbriata)으로 이루어진 군에서 선택된 1종 이상인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the causative strains of plant diseases are Rhizocctonia solani , Alternaria alternata , Botrytis cinerea , and Clarireedia jacksonii . , Colletotrichum gloeosporiodes , Colletotrichum sp., Phomopsis sp., Fusarium oxysporum , Fusarium sp. .), Pythium ultimum , Rhizoctonia cerealis , Sclerotinia sclerotiorum , and Ceratocystis fimbriata . It may be more than one species, but is not limited to this.
본 발명에 있어서 식물병 방제용 조성물은 첨가제, 증량제, 영양제 또는 봉해제 등의 부가제를 추가로 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the composition for controlling plant diseases may further include additives such as additives, extenders, nutrients, or sealing agents, but is not limited thereto.
본 발명에 있어서 폴리카복실레이트, 소듐 리그노설포네이트, 칼슘 리그노설포네이트, 소듐 다이알킬 설포석시네이트, 소듐 알킬 아릴 설포네이트, 폴리옥시에틸렌 알킬 페닐 에테르, 소듐 트리폴리포스페이트, 폴리옥시에틸렌 알킬 아릴 포스포릭 에스테르, 폴리옥시에틸렌 알킬 아릴 에테르, 폴리옥시에틸렌 알킬 아릴 폴리머, 폴리옥시알킬온 알킬 페닐 에테르, 폴리옥시에틸렌 노닐 페닐 에테르, 소듐 설포네이트 나프탈렌 포름알데히드, 트리톤 100 및 트윈 80으로 이루어진 군으로부터 선택되는 1종 이상을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, polycarboxylates, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene alkyl aryl selected from the group consisting of phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkylone alkyl phenyl ethers, polyoxyethylene nonyl phenyl ethers, sodium sulfonate naphthalene formaldehyde, Triton 100 and Tween 80. It may include one or more types, but is not limited thereto.
본 발명에 있어서 증량제는 벤토나이트 (bentonite), 탈크 (talc), 다이아라이트 (dialite), 카올린 (kaolin) 및 칼슘 카보네이트 (calcium carbonate)로 이루어진 군으로부터 선택되는 1종 이상을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the extender may include one or more selected from the group consisting of bentonite, talc, dialite, kaolin, and calcium carbonate. It is not limited.
본 발명에 있어서 영양제는 스킴 밀크 (skim milk, 배지), 콩가루, 쌀, 밀, 황토, 규조토, 벤토나이트 (bentonite), 덱스트린, 포도당 및 전분으로 이루어진 군으로부터 선택되는 1종 이상을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the nutritional agent may include one or more selected from the group consisting of skim milk, soybean flour, rice, wheat, red clay, diatomaceous earth, bentonite, dextrin, glucose, and starch. , but is not limited to this.
본 발명의 다른 일 양태는 바실러스 벨레젠시스 (Bacillus velezensis) CMML20-16 균주를 배양하여 배양액을 제조하는 배양 단계를 포함하는 식물병 방제용 조성물의 제조방법에 관한 것이다.Another aspect of the present invention relates to a method for producing a composition for controlling plant diseases, including a culturing step of culturing the Bacillus velezensis CMML20-16 strain to prepare a culture solution.
본 발명에 있어서 바실러스 벨레젠시스 CMML20-16 균주는 수탁번호 KCTC 14762BP로 기탁된 바실러스 벨레젠시스 CMML20-16 균주인 것일 수 있다.In the present invention, the Bacillus belegensis CMML20-16 strain may be the Bacillus belegensis CMML20-16 strain deposited under the accession number KCTC 14762BP.
본 발명에 있어서 식물병은 잔디의 갈색잎마름병 (Brown patch), 라이족토니아 마름병 (Large Patch), 검은무늬썩음병, 식물 잎의 검은점무늬병, 잿빛곰팡이병, 잔디의 동전마름병 (Dollar spot), 식물 탄저병, 줄기마름병, 시들음병, 뿌리썩음병, 모잘록병, 잔디의 봄마름병, 균핵병 및 흑반병으로 이루어진 군에서 선택된 1종 이상인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, plant diseases include brown patch of grass, large patch of lawn, black spot rot, black spot of plant leaves, gray mold, dollar spot of lawn, and plant diseases. It may be one or more species selected from the group consisting of anthracnose, stem blight, wilt, root rot, mottled blight, spring blight of grass, sclerotia and black spot, but is not limited thereto.
본 발명에 있어서 식물병은 원인 균주가 라이족토니아 솔라니 (Rhizocctonia solani), 알타나리아 알타르나타 (Alternaria alternata), 보트리티스 시네레아 (Botrytis cinerea), 클라리레이디아 잭소니 (Clarireedia jacksonii), 콜레토트리쿰 글로에오스포리오데스 (Colletotrichum gloeosporiodes), 콜레토트리쿰 속(Colletotrichum sp.), 포모시스 속 (Phomopsis sp.), 푸사리움 옥시포룸 (Fusarium oxysporum), 푸시리움 속 (Fusarium sp.), 피티움 얼티뭄 (Pythium ultimum), 라이족토니아 세레아리스(Rhizoctonia cerealis), 스켈로티니아 스켈로티오룸 (Sclerotinia sclerotiorum) 및 세라토시스티스 핌브리아타 (Ceratocystis fimbriata)으로 이루어진 군에서 선택된 1종 이상인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the causative strains of plant diseases are Rhizocctonia solani , Alternaria alternata , Botrytis cinerea , and Clarireedia jacksonii . , Colletotrichum gloeosporiodes , Colletotrichum sp., Phomopsis sp., Fusarium oxysporum , Fusarium sp. .), Pythium ultimum , Rhizoctonia cerealis , Sclerotinia sclerotiorum , and Ceratocystis fimbriata . It may be more than one species, but is not limited to this.
본 발명에 있어서 배양 단계는 배양액을 원심분리 및 여과하여 배양액으로부터 배양여액을 제조하는 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culturing step may include preparing a culture filtrate from the culture solution by centrifuging and filtering the culture solution, but is not limited thereto.
본 발명에 있어서 배양 단계의 배지는 탈지유, 훼이, 카제인 등의 우유 단백질, 당류, 호모 및 엑기스로 이루어진 군에서 선택된 1종 이상을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culture medium may include one or more selected from the group consisting of milk proteins such as skim milk, whey, and casein, sugars, homologs, and extracts, but is not limited thereto.
본 발명에 있어서 배양 단계의 배지는 TSB (Tryptic Soy Broth) 배지, LB (Luria-Bertani broth) 배지, PDB (Potato dextrose broth) 배지, NYDB 배지, KB 배지 및 BSM (Bifidus Selective Medium Broth) 배지로 이루어진 군에서 선택된 1종 이상의 배지인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culture medium is TSB (Tryptic Soy Broth) medium, LB (Luria-Bertani broth) medium, PDB (Potato dextrose broth) medium, NYDB medium, KB medium, and BSM (Bifidus Selective Medium Broth) medium. It may be one or more types of media selected from the group, but is not limited thereto.
본 발명에 있어서 배양 단계의 배지는 TSB (Tryptic Soy Broth) 배지인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culture medium may be TSB (Tryptic Soy Broth) medium, but is not limited thereto.
본 발명에 있어서 식물병 방제용 조성물의 제조방법은 배양액을 농축하는 농축 단계를 추가로 포함하는 것일 수 있다.In the present invention, the method for producing a composition for controlling plant diseases may further include a concentration step of concentrating the culture medium.
본 발명에 있어서 식물병 방제용 조성물의 제조방법은 배양액을 희석하는 희석 단계를 추가로 포함하는 것일 수 있다.In the present invention, the method for producing a composition for controlling plant diseases may further include a dilution step of diluting the culture medium.
본 발명에 있어서 배양 단계는 균주를 pH 5 내지 8, pH 5 내지 7, 예를 들어, pH 6 내지 7에서 배양하는 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culturing step may include culturing the strain at pH 5 to 8, pH 5 to 7, for example, pH 6 to 7, but is not limited thereto.
본 발명에 있어서 배양 단계는 균주를 20 내지 45 ℃, 20 내지 40 ℃, 20 내지 37 ℃, 20 내지 35 ℃, 20 내지 33 ℃, 25 내지 45 ℃, 25 내지 40 ℃, 25 내지 37 ℃, 25 내지 35 ℃, 25 내지 33 ℃, 예를 들어, 30 ℃에서 배양하는 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culturing step is performed by culturing the strain at 20 to 45°C, 20 to 40°C, 20 to 37°C, 20 to 35°C, 20 to 33°C, 25 to 45°C, 25 to 40°C, 25 to 37°C, 25°C. It may include culturing at 35°C to 35°C, 25 to 33°C, for example, 30°C, but is not limited thereto.
본 발명에 있어서 식물병 방제용 조성물의 제조방법은 균체 내의 성분을 추출하는 추출 단계를 추가로 포함하는 것일 수 있다.In the present invention, the method for producing a composition for controlling plant diseases may further include an extraction step of extracting components within the bacterial cells.
본 발명에 있어서 식물병 방제용 조성물의 제조방법은 배양액으로부터 활성 분획을 획득하는 분획 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the method for producing a composition for controlling plant diseases may include a fractionation step of obtaining an active fraction from the culture medium, but is not limited thereto.
본 발명에 있어서 분획 단계는 하기의 단계를 포함하는 것일 수 있다:In the present invention, the fractionation step may include the following steps:
배양액으로부터 상등액을 얻는 상등액 수득 단계;A supernatant obtaining step of obtaining a supernatant from the culture medium;
상등액을 용매로 분획하여 분획물을 얻는 분획 단계.A fractionation step of obtaining fractions by fractionating the supernatant with a solvent.
본 발명에 있어서 분획 단계는 상등액으로부터 침전물을 수득하는 침전물 수득 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the fractionation step may include, but is not limited to, a precipitate obtaining step of obtaining a precipitate from the supernatant.
본 발명에 있어서 침전물 수득 단계는 상등액을 염산에 방치하여 수득하는 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the step of obtaining the precipitate may include, but is not limited to, the step of obtaining the supernatant by leaving it in hydrochloric acid.
본 발명에 있어서 침전물 수득 단계는 상등액을 5000 내지 20000 rpm, 5000 내지 18000 rpm, 5000 내지 16000 rpm, 5000 내지 14000 rpm, 5000 내지 12000 rpm, 10000 내지 20000 rpm, 10000 내지 18000 rpm, 10000 내지 16000 rpm, 10000 내지 14000 rpm, 10000 내지 12000 rpm, 예를 들어, 12000 rpm으로 원심분리하는 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the precipitate obtaining step is performed by rotating the supernatant at 5000 to 20000 rpm, 5000 to 18000 rpm, 5000 to 16000 rpm, 5000 to 14000 rpm, 5000 to 12000 rpm, 10000 to 20000 rpm, 10000 to 18000 rpm, 100 00 to 16000 rpm, It may include centrifuging at 10000 to 14000 rpm, 10000 to 12000 rpm, for example, 12000 rpm, but is not limited thereto.
본 발명에 있어서 분획 단계는 용매로 메탄올, 부탄올, 에틸아세테이트, 에탄올 및 헥산으로 이루어진 군에서 선택된 1종 이상으로 분획하는 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the fractionation step may include fractionating with one or more solvents selected from the group consisting of methanol, butanol, ethyl acetate, ethanol, and hexane, but is not limited thereto.
본 발명에 있어서 분획 단계는 역상 고속액체크로마토그래피 (Reverse phase High performance liquid chromatography, RP-HPLC)로 활성 분획을 분획하는 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the fractionation step may include, but is not limited to, fractionating the active fraction by reverse phase high performance liquid chromatography (RP-HPLC).
본 발명에 있어서 활성 분획은 펜기신 (Fengycin)을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the active fraction may include Fengycin, but is not limited thereto.
본 명세서상 용어 “펜기신”은 농업용 살균제로 사용되는 고리형 리포펩티드로, 고초균 (Bacillus subtilis)에 의해 합성된다. 진균 감염에 대한 면역 반응으로 표적의 세포막을 손상시키는 기능을 한다.As used herein, the term “penicine” is a cyclic lipopeptide used as an agricultural fungicide and is synthesized by Bacillus subtilis . It functions to damage the target's cell membrane as an immune response to fungal infection.
본 발명에 있어서 활성 분획은 설펙틴 (Surfectin)을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the active fraction may include surfectin, but is not limited thereto.
본 명세서상 용어 “설펙틴”은 항생제로 사용되는 강력한 계면활성제로 사용되는 리포펩티드로, 고초균 (Bacillus subtilis)에 의해 합성된다. 항균, 항바이러스, 항진균, 항마이코플라스마 및 용혈을 활성시키는 기능을 한다.As used herein, the term “sulpectin” is a lipopeptide used as a strong surfactant used as an antibiotic and is synthesized by Bacillus subtilis . It functions to activate antibacterial, antiviral, antifungal, antimycoplasma, and hemolysis.
본 발명의 다른 일 양태는 바실러스 벨레젠시스 (Bacillus velezensis) CMML20-16 균주의 배양액 또는 이의 추출물을 처리하는 조성물 처리 단계를 포함하는 식물병 방제방법에 관한 것이다.Another aspect of the present invention relates to a plant disease control method comprising the step of treating a composition for treating a culture medium of Bacillus velezensis CMML20-16 strain or an extract thereof.
본 발명에 있어서 바실러스 벨레젠시스 CMML20-16 균주는 수탁번호 KCTC 14762BP로 기탁된 바실러스 벨레젠시스 CMML20-16 균주인 것일 수 있다.In the present invention, the Bacillus belegensis CMML20-16 strain may be the Bacillus belegensis CMML20-16 strain deposited under the accession number KCTC 14762BP.
본 발명에 있어서 조성물 처리 단계는 살포, 토양관주, 표면살포, 근권 처리, 종자 처리, 침지, 독이 및 훈연시용으로 이루어진 군으로부터 선택되는 1종 이상의 방식으로 수행되는 것일 수 있으나, 이에 한정되는 것은 아니다In the present invention, the composition treatment step may be performed by one or more methods selected from the group consisting of spraying, soil irrigation, surface spraying, root zone treatment, seed treatment, soaking, poisoning, and smoking, but is not limited thereto.
본 발명에 있어서 “살포”는 분무, 미스팅, 아토마이징, 분말살포, 과립살포, 수명시용 및 상시용으로 이루어진 군으로부터 선택되는 1종 이상의 방식으로 수행되는 것일 수 있다.In the present invention, “spraying” may be performed by one or more methods selected from the group consisting of spraying, misting, atomizing, powder spraying, granule spraying, lifetime use, and permanent use.
본 명세서상 용어 “관주”는 토양이나 나무에 구멍을 파서 약액을 주입하는 약제 살포의 한 방법이다.As used herein, the term “drenchment” is a method of spraying a chemical that involves digging a hole in the soil or tree and injecting a chemical solution.
본 명세서상 용어 “토양 관주”는 작물 재배토양에 약액을 주입 또는 살포하는 방법이다.As used herein, the term “soil irrigation” refers to a method of injecting or spraying a chemical solution into crop cultivation soil.
상기 식물병 방제용 조성물의 제조 방법 및 식물병 방제 방법에 있어서, 상기 식물병 방제용 조성물과 중복되는 내용은 본 명세서의 복잡성을 고려하여 생략한다.In the method for producing the composition for controlling plant diseases and the method for controlling plant diseases, content that overlaps with the composition for controlling plant diseases is omitted in consideration of the complexity of the present specification.
본 발명은 바실러스 벨레젠시스 CMML20-16 균주의 배양액 또는 균주 배양액의 추출물을 포함하는 식물병 방제용 조성물, 이의 제조방법, 및 식물병 방제 방법에 관한 것으로서, 상기 균주의 배양액 또는 이의 추출물은 방제가 어려운 잔디의 주요 토양 병해인 갈색잎마름병 및 라이족토니아마름병을 유발하는 라이족토니아 솔라니에 대해 우수한 방제 효과를 나타내므로 잔디 병해의 방제에 이용할 수 있다.The present invention relates to a composition for controlling plant diseases comprising a culture medium or extract of the strain culture medium of the Bacillus belegensis CMML20-16 strain, a method for producing the same, and a method for controlling plant diseases, wherein the culture medium of the strain or the extract thereof is effective in controlling plant diseases. It has an excellent control effect against Rhizoctonia solani, which causes brown leaf blight and Rhizoctonia blight, which are major soil diseases of difficult lawns, so it can be used to control lawn diseases.
도 1은 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주의 16s rRNA 유전자를 이용한 계통도를 나타낸 도이다.
도 2는 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주의 gyrB 유전자를 이용한 상동성을 통해 분석한 유전학적 계통도를 나타낸 도이다.
도 3은 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주의 유전체 분석을 통한 상동성을 확인한 유전학적 계통도를 나타낸 도이다.
도 4는 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주의 배지별 생장곡선을 나타낸 그래프이다.
도 5는 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주의 최적 생장 pH를 나타낸 그래프이다.
도 6은 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주의 최적 생장 온도를 나타낸 그래프이다.
도 7은 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주를 카제인 평판배지에 배양하여 단백질분해효소 활성을 확인한 결과를 나타낸 도이다.
도 8은 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주를 젤라틴 평판 배지에 배양하여 젤라틴 분해효소의 활성을 확인한 결과를 나타낸 도이다.
도 9는 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주의 식물성장촉진인자 형성능을 확인한 결과를 나타낸 그래프이다.
도 10은 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주의 식물 병원균에 대한 생육저지 효과를 확인한 결과를 나타낸 도이다.
도 11은 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주의 갈색잎마름병(Brown Patch)균인 라이족토니아 솔라니 (Rhizoctonia solani) AG2-2(ⅢB)의 생장 저해 효과를 확인한 결과를 나타낸 도이다.
도 12는 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주의 라이족토니아마름병(Large Patch)균인 라이족토니아 솔라니 (Rhizoctonia solani) AG2-2(Ⅳ)의 생장 저해 효과를 확인한 결과를 나타낸 도이다.
도 13은 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주의 갈색잎마름병(Brown Patch)을 발병시킨 잔디에 대한 항균력을 확인한 결과를 나타낸 도이다.
도 14는 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주의 라이족토니아마름병(Large Patch)을 발병시킨 잔디에 대한 항균력을 확인한 결과를 나타낸 도이다.
도 15a는 본 발명의 일 실시예에 따른 역상 HPLC를 이용한 표준물질 펜기신 (500 μg/ml)의 피크 (Peak)를 확인한 결과를 나타낸 크로마토그램이다.
도 15b는 본 발명의 일 실시예에 따른 역상 HPLC를 이용한 바실러스 벨레젠시스 CMML20-16의 피크 (Peak)를 확인한 결과를 나타낸 크로마토그램이다.
도 15c는 본 발명의 일 실시예에 따른 역상 HPLC를 이용한 표준물질 펜기신 (500 μg/ml)의 10분 내지 15분 사이의 피크 (Peak)를 확인한 크로마토그램을 확대한 그래프이다.
도 15d는 본 발명의 일 실시예에 따른 역상 HPLC를 이용한 바실러스 벨레젠시스 CMML20-16의 10분 내지 15분 사이의 피크 (PeaK)를 확인한 크로마토그램을 확대한 그래프이다.
도 16a는 본 발명의 일 실시예에 따른 역상 HPLC를 이용한 표준물질 설펙틴 (500 μg/ml)의 피크 (Peak)를 확인한 결과를 나타낸 크로마토그램이다.
도 16b는 본 발명의 일 실시예에 따른 역상 HPLC를 이용한 바실러스 벨레젠시스 CMML20-16의 피크 (Peak)를 확인한 결과를 나타낸 크로마토그램이다.
도 16c는 본 발명의 일 실시예에 따른 역상 HPLC를 이용한 표준물질 설펙틴 (500 μg/ml)의 60분 내지 90분 사이의 피크 (Peak)를 확인한 크로마토그램을 확대한 그래프이다.
도 16d는 본 발명의 일 실시예에 따른 역상 HPLC를 이용한 바실러스 벨레젠시스 CMML20-16 균주의 10분 내지 15분 사이의 피크 (Peak)를 확인한 결과를 나타낸 크로마토그램이다.
도 17은 본 발명의 일 실시예에 따른 바실러스 벨레젠시스 CMML20-16 균주의 살진균 농약 저항성을 확인하기 위한 콜로니 수 검정 결과를 나타낸 도이다.Figure 1 is a diagram showing a family diagram using the 16s rRNA gene of the Bacillus velegensis CMML20-16 strain according to an embodiment of the present invention.
Figure 2 is a diagram showing a genetic tree analyzed through homology using the gyrB gene of the Bacillus velegensis CMML20-16 strain according to an embodiment of the present invention.
Figure 3 is a diagram showing a genetic tree confirming homology through genome analysis of the Bacillus velegensis CMML20-16 strain according to an embodiment of the present invention.
Figure 4 is a graph showing the growth curve for each medium of the Bacillus velegensis CMML20-16 strain according to an embodiment of the present invention.
Figure 5 is a graph showing the optimal growth pH of the Bacillus velegensis CMML20-16 strain according to an embodiment of the present invention.
Figure 6 is a graph showing the optimal growth temperature of the Bacillus velegensis CMML20-16 strain according to an embodiment of the present invention.
Figure 7 is a diagram showing the results of confirming protease activity by culturing the Bacillus velegensis CMML20-16 strain on casein plate medium according to an embodiment of the present invention.
Figure 8 is a diagram showing the results of confirming the activity of gelatin degrading enzyme by culturing the Bacillus velegensis CMML20-16 strain on a gelatin plate medium according to an embodiment of the present invention.
Figure 9 is a graph showing the results of confirming the plant growth promoting factor forming ability of the Bacillus velegensis CMML20-16 strain according to an embodiment of the present invention.
Figure 10 is a diagram showing the results of confirming the growth inhibition effect of the Bacillus velegensis CMML20-16 strain against plant pathogens according to an embodiment of the present invention.
Figure 11 shows the results of confirming the growth inhibition effect of Rhizoctonia solani AG2-2(ⅢB), a brown patch fungus of Bacillus velegensis CMML20-16 strain according to an embodiment of the present invention. This is a diagram showing .
Figure 12 shows the growth inhibition effect of Rhizoctonia solani AG2-2(IV), a Large Patch bacterium, of the Bacillus velegensis CMML20-16 strain according to an embodiment of the present invention. This diagram shows the results.
Figure 13 is a diagram showing the results of confirming the antibacterial activity of the Bacillus velegensis CMML20-16 strain on grass that developed brown patch disease according to an embodiment of the present invention.
Figure 14 is a diagram showing the results of confirming the antibacterial activity of the Bacillus velegensis CMML20-16 strain on lawns that developed Rhizoctonia blight (Large Patch) according to an embodiment of the present invention.
Figure 15a is a chromatogram showing the results of confirming the peak of the standard substance penicine (500 μg/ml) using reverse-phase HPLC according to an embodiment of the present invention.
Figure 15b is a chromatogram showing the results of confirming the peak of Bacillus velegensis CMML20-16 using reverse-phase HPLC according to an embodiment of the present invention.
Figure 15c is an enlarged graph of a chromatogram confirming the peak between 10 and 15 minutes of the standard substance penicine (500 μg/ml) using reverse-phase HPLC according to an embodiment of the present invention.
Figure 15d is an enlarged graph of a chromatogram confirming the peak (PeaK) between 10 and 15 minutes of Bacillus velegensis CMML20-16 using reverse-phase HPLC according to an embodiment of the present invention.
Figure 16a is a chromatogram showing the results of confirming the peak of the standard sulpectin (500 μg/ml) using reverse-phase HPLC according to an embodiment of the present invention.
Figure 16b is a chromatogram showing the results of confirming the peak of Bacillus velegensis CMML20-16 using reverse-phase HPLC according to an embodiment of the present invention.
Figure 16c is an enlarged graph of a chromatogram confirming the peak between 60 and 90 minutes of the standard sulpectin (500 μg/ml) using reverse-phase HPLC according to an embodiment of the present invention.
Figure 16d is a chromatogram showing the results of confirming the peak between 10 and 15 minutes of the Bacillus velegensis CMML20-16 strain using reverse-phase HPLC according to an embodiment of the present invention.
Figure 17 is a diagram showing the results of a colony count test to confirm the fungicidal pesticide resistance of the Bacillus velegensis CMML20-16 strain according to an embodiment of the present invention.
이하, 본 발명을 하기의 실시예에 의하여 더욱 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것일 뿐이며, 본 발명의 범위가 이들 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
실시예 1. 바실러스 벨레젠시스 CMML20-16 균주의 분자생물학적 분석 및 계통도 분석Example 1. Molecular biological analysis and phylogenetic analysis of Bacillus belegensis CMML20-16 strain
1-1. 균주의 분리1-1. Isolation of strains
식물 병원성균에 방제 활성이 있는 균주는 잔디가 분포되어 있는 전남 화순의 골프장 페어웨이 중지 근권에서 건전한 잔디 근권 (생장하는 뿌리가 물질을 섭취하고 저장함으로써, 결과적으로 구성 생물의 비율이 달라지는 등 다양한 변화를 일으키는 토양 환경)에 존재하는 토양에서 분리하였다. 토양 100 g을 채취하여 멸균 증류수로 10 배씩 단계 희석한 후, 각 100 μl씩 취하여 트립틱 소이 아가 (Tryptic Soy Agar, TSA) 고체 평판 배지에 멸균된 스프레드로 도말하였다. 25 ℃ 항온기에서 3일간 배양한 후, 형성된 세균의 모양에 따라 획선 도말한 후 단일 콜로니로 분리하였다.Strains with activity against plant pathogenic bacteria are found in the root zone of fairways of golf courses in Hwasun, Jeollanam-do, where grass is distributed, in the root zone of healthy grass (growing roots ingest and store substances, resulting in various changes such as changes in the ratio of constituent organisms). It was isolated from soil that exists in the soil environment that causes it. 100 g of soil was collected and diluted 10 times with sterilized distilled water, and then 100 μl each was taken and spread on Tryptic Soy Agar (TSA) solid plate medium with a sterilized spread. After culturing in a thermostat at 25°C for 3 days, the formed bacteria were streaked according to their shape and separated into single colonies.
1-2. 균주의 분자생물학적 분석 및 계통도 분석1-2. Molecular biological analysis and phylogenetic analysis of strains
토양으로부터 분리한 균주는 16s rRNA, 자이레즈 B (gyr B, gyrase B 유전자)의 염기서열 분석을 통해 분자생물학적으로 동정하였다. 균주는 TSB배지 (Trypsin Soy Broth: Bacto Tryptone 15 g, Bacto Soytone 5 g, NaCl 5 g/ℓ)에 접종한 다음 30 ℃에서 2 일간 150 rpm으로 진탕 배양하였다. 그 다음, 수확한 균주는 블로드 앤드 셀 컬쳐 디엔에이 추출 미니 키트 (Blood & Cell culture DNA Mini Kit, 퀴아젠(QIAGEN), 독일)를 이용하여 프로토콜에 따라서 균주의 게놈 DNA (gDNA)를 추출하였다. 추출된 균주의 gDNA와 TaKaRa Ex Taq®DNA Polymerase (다카라바이오, 일본) 및 하기 표 1의 균주의 16s rRNA를 증폭할 수 있는 서열번호 1 (27F) 및 서열번호 2 (1492R) 또는 gyr B를 증폭할 수 있는 서열번호 3 (UP-1) 및 서열번호 4 (UP-2r)을 혼합한 후 PCR을 통해 유전자를 증폭하였다.The strain isolated from soil was identified molecularly through nucleotide sequence analysis of 16s rRNA and gyrase B (gyr B, gyrase B gene). The strain was inoculated into TSB medium (Trypsin Soy Broth: Bacto Tryptone 15 g, Bacto Soytone 5 g, NaCl 5 g/l) and then cultured at 30°C for 2 days with shaking at 150 rpm. Next, the genomic DNA (gDNA) of the harvested strain was extracted according to the protocol using the Blood & Cell culture DNA Mini Kit (QIAGEN, Germany). Amplify gDNA of the extracted strain and TaKaRa Ex Taq®DNA Polymerase (Takara Bio, Japan) and SEQ ID NO. 1 (27F) and SEQ ID NO. 2 (1492R) or gyr B, which can amplify the 16s rRNA of the strains in Table 1 below. After mixing SEQ ID NO: 3 (UP-1) and SEQ ID NO: 4 (UP-2r), the gene was amplified through PCR.
AGAGTTTGATCMTGGCTCAG
GGTTACCTTGTTACGACTT
16S rRNA 유전자를 증폭하기 위한 PCR cycle 조건은 98 ℃ 30초를 시작으로 98 ℃ 15초, 58 ℃ 30초, 72 ℃ 2분을 cycle마다 annealing temperature를 1도씩 낮추어 8번 반복한 후, 98도 15초, 50도 30초, 72도 2분을 30번 반복한 후 72 ℃에서 5분, 4 ℃에서 증폭을 끝냈다. gyrB 유전자를 증폭하기 위한 PCR cycle 조건은 98 ℃ 30초를 시작으로 98 ℃ 15초, 71 ℃ 30초, 72 ℃ 2분을 cycle마다 annealing temperature를 1도씩 낮추어 8번 반복한 후, 98도 15초, 63도 30초, 72도 2분을 30번 반복한 후 72 ℃에서 5분, 4 ℃에서 증폭을 끝냈다. 증폭된 16S rRNA 유전자와 gyrB 유전자PCR 산물들은 마크로젠 (Macrogen, 대한민국)에 염기서열 분석을 의뢰하였다.The PCR cycle conditions for amplifying the 16S rRNA gene were as follows: starting at 98°C for 30 seconds, repeating 8 times 98°C for 15 seconds, 58°C for 30 seconds, and 72°C for 2 minutes with the annealing temperature lowered by 1 degree for each cycle, then 98°C for 15 seconds. After repeating 30 cycles of 30 seconds at 50 degrees and 2 minutes at 72 degrees, amplification was completed at 72°C for 5 minutes and 4°C. The PCR cycle conditions for amplifying the gyrB gene were repeated 8 times, starting at 98°C for 30 seconds, followed by 98°C for 15 seconds, 71°C for 30 seconds, and 72°C for 2 minutes, lowering the annealing temperature by 1 degree for each cycle, and then 98°C for 15 seconds. , 63 degrees for 30 seconds, and 72 degrees for 2 minutes were repeated 30 times, then amplification was completed at 72°C for 5 minutes and 4°C. The amplified 16S rRNA gene and gyrB gene PCR products were submitted to Macrogen (Republic of Korea) for base sequence analysis.
상기 표 2에 염기서열을 기초로 염기서열의 상동성 검사는 등록된 정보 (Genebank database)를 대상으로 블라스트 프로그램 (Blast program, http://www.ncbi.nlm.nhi.gov)에 의해 실행하였다. 그 결과, CMML20-16 균주는 바실러스 벨레젠시스로 동정 되었으며, 부분적인 16s rRNA의 염기서열 (서열번호 5)은 GenBank에 Accession number OL721677번으로 등록되었다.Based on the base sequence in Table 2 above, the homology test of the base sequence was performed using the Blast program (http://www.ncbi.nlm.nhi.gov) against the registered information (Genebank database). . As a result, the CMML20-16 strain was identified as Bacillus bellegensis, and the partial 16s rRNA base sequence (SEQ ID NO: 5) was registered in GenBank as Accession number OL721677.
하기 표 3 및 도 1에서 확인할 수 있듯이 CMML20-16 균주는 바실러스 벨레젠시스 균주와 유전적 유사성을 나타내었다.As can be seen in Table 3 and Figure 1 below, the CMML20-16 strain showed genetic similarity to the Bacillus belegensis strain.
또한, 보다 정확한 CMML20-16 균주의 동정을 위하여 자이레즈 B (gyr B, gyrase B 유전자)의 상동성을 비교하였다.In addition, for more accurate identification of the CMML20-16 strain, the homology of gyrase B (gyr B, gyrase B gene) was compared.
상기 표 4에 염기서열을 기초로 염기서열의 상동성 검사는 등록된 정보 (Genebank database)를 대상으로 블라스트 프로그램 (Blast program, http://www.ncbi.nlm.nhi.gov)에 의해 실행하였다. 그 결과, CMML20-16 균주는 바실러스 벨레젠시스로 동정 되었으며, 부분적인 gyrB의 염기서열 (서열번호 6)은 GenBank에 Accession number OL764353번으로 등록되었다.Based on the base sequences in Table 4 above, the homology test of base sequences was performed using the Blast program (http://www.ncbi.nlm.nhi.gov) against registered information (Genebank database). . As a result, the CMML20-16 strain was identified as Bacillus belegensis, and the partial base sequence of gyrB (SEQ ID NO: 6) was registered in GenBank as Accession number OL764353.
하기 표 5 및 도 2에서 확인할 수 있듯이, CMML20-16 균주는 바실러스 벨레젠시스 균주와 유전적 유사성을 나타내었다.As can be seen in Table 5 and Figure 2 below, the CMML20-16 strain showed genetic similarity to the Bacillus belegensis strain.
따라서, CMML20-16 균주는 바실러스 벨레젠시스 (Bacillus velezensis)로 동정되었으며, 상기 균주를 바실러스 벨레젠시스 CMML20-16로 명명하였고, 2021년 11월 10일자로 대한민국 KCTC (Korean Collection For Type Cultures)에 기탁하여 수탁번호 KCTC14762BP를 부여 받았다.Therefore, the CMML20-16 strain was identified as Bacillus velezensis , and the strain was named Bacillus velezensis CMML20-16, and was registered in the Korean KCTC (Korean Collection For Type Cultures) on November 10, 2021. It was deposited and given accession number KCTC14762BP.
실시예 3. 바실러스 벨레젠시스 CMML20-16 균주의 유전체 해독 및 분석Example 3. Genome decoding and analysis of Bacillus belegensis CMML20-16 strain
Phyzen사 (대한민국)에 의뢰하여 바실러스 벨레젠시스 CMML20-16 (Bacillus velezensis CMML29016)균주의 유전체 해독 및 분석을 진행하였다.We commissioned Phyzen (Korea) to decode and analyze the genome of Bacillus velezensis CMML20-16 ( Bacillus velezensis CMML29016).
구체적으로, CMML20-16 균주를 Illumina Hiseq X Ten을 이용한 Pared-end 방식 (Illumina sequencing)을 통하여 1,719,705 bp를 읽었다. 그 다음, Fastp Program 및 BBDuk을 이용하여 Adaptor sequence 제거와 quality trimming을 수행한 결과 1,315,744,269bp의 유전체를 얻었다. 또한 gridion을 이용한 나노포어 시퀀싱 (Nanopore sequencing) 방법으로 전체 유전체를 읽은 후, 일정 조건에 맞는 더 높은 수준의 데이터를 추출하고 이중 1Gb (1,000,021,893bp)만을 이용하여 유전체를 조립하였다.Specifically, 1,719,705 bp of strain CMML20-16 was read through pared-end method (Illumina sequencing) using Illumina Hiseq Next, adapter sequence removal and quality trimming were performed using the Fastp Program and BBDuk, resulting in a genome of 1,315,744,269 bp. In addition, after reading the entire genome using nanopore sequencing using gridion, higher-level data that met certain conditions was extracted and the genome was assembled using only 1Gb (1,000,021,893bp).
두 가지 방법으로 읽은 유전체는 Unicycler v0.4.8-beta를 이용하여 Hybrid assembly를 진행하였다. 이러한 신규 어셈블리(De novo assembly) 과정을 수행한 결과 총 4,071,981 bp의 게놈 DNA(genome DNA)의 서열과 71,675bp의 플라스미드 DNA (Plasmid DNA)의 서열을 밝혔다. 바실러스 벨레젠시스 CMML20-16의 게놈 DNA는 GenBank에 Accession number CP088973번로 등록이 되었으며, 플라스미드 DNA은 GenBank에 Accession number CP088974번으로 등록되었다.The genome read by two methods was subjected to hybrid assembly using Unicycler v0.4.8-beta. As a result of performing this de novo assembly process, a total of 4,071,981 bp of genomic DNA sequence and 71,675 bp of plasmid DNA sequence were revealed. The genomic DNA of Bacillus velegensis CMML20-16 was registered in GenBank under Accession number CP088973, and the plasmid DNA was registered in GenBank under Accession number CP088974.
그 다음, 유전체 해독 정보에 따라 알려진 균주와 상동성을 조사하였다.Next, homology with known strains was investigated according to genome sequencing information.
도 3에서 확인할 수 있듯이, 바실러스 벨레젠시스 CMML20-16 균주는 Bacillus velezensis GH1-13과 Bacillus velezensis KMU01과 높은 상동성을 나타내는 것을 확인하였다.As can be seen in Figure 3, the Bacillus velezensis CMML20-16 strain was confirmed to exhibit high homology with Bacillus velezensis GH1-13 and Bacillus velezensis KMU01.
AntiSMASH version 2를 이용하여 유전체 내의 2차 대사산물에 대한 주석화(Annotation)을 진행하여 항생물질 생합성 유전자군을 조사하였다. 그 결과, fengycin, surfactin 유사 유전자 군집, bacillomycin D, mersacidin, bacillibactin, bacilysin, macrolactin, bacillaene, difficidin 등의 생합성에 관여하는 유전체를 가지고 있음을 확인할 수 있었다.Annotation of secondary metabolites in the genome was performed using AntiSMASH version 2 to investigate the antibiotic biosynthetic gene group. As a result, it was confirmed that it had genomes involved in the biosynthesis of fengycin, surfactin-like gene cluster, bacillomycin D, mersacidin, bacillibactin, bacilysin, macrolactin, bacillaene, and difficidin.
실시예 3. 균주의 생장 특성Example 3. Growth characteristics of strains
3-1. 균주의 배지 종류별 생장 확인3-1. Check the growth of strains by type of medium
바실러스 벨레젠시스 CMML20-16을 배양하기에 적합한 배지를 선택하기 위해 균주의 다양한 배지에서의 생장을 확인하였다. TSB (Bacto Tryptone 15 g, Bacto Soytone 5 g, NaCl 5 g/ℓ)에 전배양한 후, 각각의 LB (Tryptone 10 g, Yeast extract 5 g, NaCl 10 g/ℓ), TSB (Bacto Tryptone 15 g, Bacto Soytone 5 g, NaCl 5 g/ℓ), PDB (glucose 20 g, potato extract 4 g/ℓ), NYDB (Peptone 1.2g, Yeast extract 1.2 g, Dextrose 0.3 g/ℓ), KB (Peptone 20 g, K2HPO4 1.5g, MgSO4*7H2O 1.5g, Glycerol 15 ㎖/ℓ) 및 BSM(Soytone 0.5 %, Sucrose 2 %/ℓ) 배지 50 mL에 2 %(v/v)가 되게 접종하여 진탕배양기에서 30 ℃, 150 rpm 조건으로 48시간 동안 배양하였다. 그 다음, 각 배양액을 600 nm의 흡광도로 조사하여 바실러스 벨레젠시스 CMML20-16 균주의 생장 정도를 관찰하여, 그 결과를 도 4에 나타내었다.In order to select a suitable medium for cultivating Bacillus belegensis CMML20-16, the growth of the strain in various media was confirmed. After pre-culturing in TSB (Bacto Tryptone 15 g, Bacto Soytone 5 g, NaCl 5 g/ℓ), each LB (Tryptone 10 g, Yeast extract 5 g, NaCl 10 g/ℓ), TSB (Bacto Tryptone 15 g) , Bacto Soytone 5 g, NaCl 5 g/ℓ), PDB (glucose 20 g, potato extract 4 g/ℓ), NYDB (Peptone 1.2g, Yeast extract 1.2 g, Dextrose 0.3 g/ℓ), KB (Peptone 20 g) , K 2 HPO 4 1.5g, MgSO 4 *7H 2 O 1.5g, Glycerol 15 ㎖/ℓ) and BSM (Soytone 0.5 %, Sucrose 2 %/ℓ) inoculated at 2% (v/v) in 50 mL of medium. It was cultured in a shaking incubator at 30°C and 150 rpm for 48 hours. Next, each culture was irradiated with an absorbance of 600 nm to observe the growth degree of the Bacillus belegensis CMML20-16 strain, and the results are shown in Figure 4.
도 4에서 확인할 수 있듯이, 바실러스 벨레젠시스 CMML20-16 균주는 모든 배지에서 생육이 가능하였으며, 특히, TSB 배지에서 생장이 가장 우수한 것으로 확인되었다.As can be seen in Figure 4, the Bacillus belegensis CMML20-16 strain was able to grow in all media, and in particular, it was confirmed that the growth was the best in TSB media.
3-2. 균주의 pH에 따른 생장 확인3-2. Check growth according to the pH of the strain
바실러스 벨레젠시스 CMML20-16 균주를 TSB (Tryptic Soybean Broth) 배지에 전배양하였다. 그 다음, 1N HCl과 1N NaOH로 pH를 조절한 50 mL TSB 배지에 2 %(v/v)가 되도록 접종하였고, 진탕 배양기에서 30 ℃, 150 rpm의 조건으로 배양하였다. 배양 48시간 후, 600 nm의 흡광도를 조사하여 바실러스 벨레젠시스 CMML20-16 균주의 생장 정도를 비교하였으며, 그 결과를 도 5에 나타내었다.Bacillus belegensis CMML20-16 strain was pre-cultured in TSB (Tryptic Soybean Broth) medium. Next, it was inoculated at 2% (v/v) in 50 mL TSB medium whose pH was adjusted with 1N HCl and 1N NaOH, and cultured in a shaking incubator at 30°C and 150 rpm. After 48 hours of incubation, the absorbance at 600 nm was measured to compare the growth degree of the Bacillus belegensis CMML20-16 strain, and the results are shown in Figure 5.
도 5에서 확인할 수 있듯이, 바실러스 벨레젠시스 CMML20-16 균주는 pH 5 내지 8까지 비슷한 생장을 보였으며, pH 6 내지 7에서 가장 적절한 성장을 나타내었다. 대부분의 식물 병원성 진균이 약산성 토양에서 잘 발생하게 되므로 바실러스 벨레젠시스 CMML20-16이 약산성에서 생육이 잘 되는 특성은 바실러스 벨레젠시스 CMML20-16 균주 또는 이의 배양액이 잔디 병해의 미생물 제제로서 우수한 효과를 나타낼 수 있음을 의미한다.As can be seen in Figure 5, the Bacillus velegensis CMML20-16 strain showed similar growth from pH 5 to 8, and showed the most appropriate growth at pH 6 to 7. Since most plant pathogenic fungi grow well in weakly acidic soil, the characteristic of Bacillus velgensis CMML20-16 that grows well in weakly acidic soil is that Bacillus velgensis CMML20-16 strain or its culture medium has an excellent effect as a microbial agent for lawn diseases. This means that it can be displayed.
3-3. 균주의 온도별 생장의 확인3-3. Confirmation of growth of strains by temperature
바실러스 벨레젠시스 CMML20-16 균주를 TSB 배지에 접종하고 1일 동안 전배양 하였다. 그 다음, 배양액을 멸균한 50 ml의 TSB 배지가 들어있는 삼각 플라스크에 접종한 후, 각각의 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃ 및 40 ℃ 온도에서 48시간 동안 배양하였다. 그 다음, 각각의 배양액을 600 nm에서 흡광도를 조사하고, 그 결과를 도 6에 나타내었다.Bacillus belegensis CMML20-16 strain was inoculated into TSB medium and pre-cultured for 1 day. Next, the culture was inoculated into an Erlenmeyer flask containing 50 ml of sterilized TSB medium and cultured for 48 hours at temperatures of 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C. Next, each culture medium was examined for absorbance at 600 nm, and the results are shown in Figure 6.
도 6에서 확인할 수 있듯이, 바실러스 벨레젠시스 CMML20-16 균주는 20 내지 30 ℃에서 잘 자랐으며, 특히, 30 ℃에서 높은 생장률을 나타내었다. 또한, 40 ℃이상의 고온에서도 생장이 가능하였다. 잔디에 치명적인 병해를 입히는 진균의 균사 생장 온도가 25 내지 30 ℃이고, 최적 발병온도가 23 ℃ 임을 고려하였을 때, 바실러스 벨레젠시스 CMML20-16 균주는 20 내지 30 ℃사이에서 생장이 우수하므로, 효과적으로 식물 병원성 진균의 생장 및 활성을 저해할 수 있어 미생물 제제로 우수하며, 특히, 40 ℃ 이상의 고온에서도 생장이 가능하기 때문에 여름에 발병하는 잔디의 병해에도 유용하게 사용될 수 있음을 확인하였다.As can be seen in Figure 6, the Bacillus velegensis CMML20-16 strain grew well at 20 to 30°C, and in particular, showed a high growth rate at 30°C. In addition, growth was possible even at high temperatures above 40°C. Considering that the mycelial growth temperature of the fungus that causes fatal diseases in grass is 25 to 30 ℃ and the optimal onset temperature is 23 ℃, Bacillus belegensis CMML20-16 strain has excellent growth between 20 and 30 ℃, so it is effective It is excellent as a microbial agent because it can inhibit the growth and activity of plant pathogenic fungi. In particular, it has been confirmed that it can be usefully used for lawn diseases that occur in the summer because it can grow even at high temperatures of 40 ℃ or higher.
실시예 4. 균주의 생리학적 및 생화학적 특성Example 4. Physiological and biochemical characteristics of strains
바실러스 벨레젠시스 CMML20-16 균주의 여러 종류의 효소 활성 및 식물 생육 촉진 인자 활성을 분석하기 위해, 균주를 TSB 5 ml에 18시간 이상 전배양한 후, 10 μl의 배양액을 각각의 배지에 치상하여 배양하였다.To analyze the activities of various enzymes and plant growth promoting factors of the Bacillus belegensis CMML20-16 strain, the strain was pre-cultured in 5 ml of TSB for more than 18 hours, and then 10 μl of the culture solution was placed on each medium. Cultured.
셀룰로즈 분해 효소 활성은 CMC 콩고 레드 배지 (CMC Congo red medium; 0.01 g/L MgSO4, 0.05 g/L KH2PO4, 0.01 g/L CaCl2, 6 g/L NaCl, 2g/L (NH4)2SO4, 2 g/L K2HPO4, 1g/L yeast extract, 5 g/L CMC, 20 g/L agar) 염색법을 사용하였다. 단백질 분해효소 활성 검정은 카제인 평판 배지 (Casein agar; skim mik 100 g/L agar 20 g/L)를 사용하였다. 또한, 젤라틴 분해효소 검정은 BHM 평판배지 (0.2 g/l MgSO4, 0.2 g/l CaCl2, 1 g/l KH2PO4, 1 g/l K2HPO4, 1 g/l NH4NO3, 0.05 g/l FeCl3)에 균주를 배양한 후 Frazier’s reagent ( 2 N HCl에 녹인 15 % HgCl2)를 부어 확인하였다. 키틴 분해 효소 활성은 Toshy Agrawal and Anil S Kotasthane (2012)의 콜로이달 키틴 (colloidal chitin) 배지(0.3 g/L MgSO4.7H2O, 3.0 g/L (NH4)2SO4, 2.0 g/L KH2PO4, 1.0 g/L citric acid monohydrate, 15 g/L agar, 200 μl/L Tween-80, 4.5 g/L colloidal chitin 0.15 g/L Bromocresol purple. 이후 pH 5로 조정)를 이용하여 키틴 분해효소 활성을 확인하였다.Cellulolytic enzyme activity was measured using CMC Congo red medium (CMC Congo red medium; 0.01 g/L MgSO 4 , 0.05 g/L KH 2 PO 4 , 0.01 g/L CaCl 2 , 6 g/L NaCl, 2g/L (NH4) 2 SO 4 , 2 g/LK 2 HPO 4 , 1g/L yeast extract, 5 g/L CMC, 20 g/L agar) staining method was used. For the protease activity assay, casein plate medium (Casein agar; skim mik 100 g/L agar 20 g/L) was used. Additionally, the gelatinase assay was performed using BHM plate medium (0.2 g/l MgSO 4 , 0.2 g/l CaCl 2 , 1 g/l KH 2 PO 4 , 1 g/l K 2 HPO 4 , 1 g/l NH 4 NO 3 , the strain was cultured in 0.05 g/l FeCl 3 ) and confirmed by pouring Frazier's reagent (15% HgCl 2 dissolved in 2 N HCl). Chitinase activity was measured using the colloidal chitin medium (0.3 g/L MgSO 4 .7H 2 O, 3.0 g/L (NH 4 ) 2 SO 4 , 2.0 g/L of Toshy Agrawal and Anil S Kotasthane (2012). L KH 2 PO 4 , 1.0 g/L citric acid monohydrate, 15 g/L agar, 200 μl/L Tween-80, 4.5 g/L colloidal chitin 0.15 g/L Bromocresol purple. Then adjust pH to 5) Chitinase activity was confirmed.
바실러스 벨레젠시스 CMML20-16 의 식물성장촉진인자 형성능 확인을 위하여 IAA 생성능을 확인하였다. IAA 형성능은 Patten and Glick (1996)의 방법을 사용하였다. 스타치 카제인 배지 (Starch Casein Broth; Starch, Soluble 10.0 g/L, Casein 1.0 g/L, K2HPO4 0.5g/L ) L-tryptophan을 넣어준 후 균주를 4일간 배양하고 상등액을 Salkowsky reagent (1 ml /0.5 M FeCl3 과 50 ml/ 35% HClO4)와 반응시켜 나타나는 색의 변화를 흡광도를 측정하여 그 결과를 표 6 및 도 7 내지 9에 나타내었다.To confirm the ability of Bacillus velegensis CMML20-16 to form plant growth promoting factors, the ability to produce IAA was confirmed. IAA formation ability was determined using the method of Patten and Glick (1996). After adding L-tryptophan (Starch Casein Broth; Starch, Soluble 10.0 g/L, Casein 1.0 g/L, K 2 HPO 4 0.5g/L), the strain was cultured for 4 days and the supernatant was incubated with Salkowsky reagent ( The absorbance of the color change resulting from reaction with 1 ml/0.5 M FeCl 3 and 50 ml/35% HClO 4 ) was measured, and the results are shown in Table 6 and Figures 7 to 9.
상기 표 6 및 도 7 및 8에서 확인할 수 있듯이, 바실러스 벨레젠시스 CMML20-16 균주는 카제인 평판배지와 젤라틴 분해효소를 분해하며 생장하여 균주 주변의 배지가 투명해 진 것을 확인할 수 있었다. 바실러스 벨레젠시스 CMML20-16은 라이족토니아 솔라니 (Rhizoctonia solani)와 같은 진균의 주요 세포벽 구성성분 중 하나인 단백질을 분해함으로서 식물 병원성 진균의 생장을 저해할 수 있다.As can be seen in Table 6 and Figures 7 and 8, the Bacillus belegensis CMML20-16 strain grew by decomposing the casein plate medium and gelatin decomposing enzyme, and it was confirmed that the medium around the strain became transparent. Bacillus belegensis CMML20-16 can inhibit the growth of plant pathogenic fungi by decomposing protein, which is one of the main cell wall components of fungi such as Rhizoctonia solani .
도 8에서 확인할 수 있듯이, 바실러스 벨레젠시스 CMML20-16 균주는 근권미생물로 식물의 생장을 촉진하는 인자인 IAA를 약 5 μg/ml가량 생성하여 잔디의 건강한 생육을 촉진할 수 있다.As can be seen in Figure 8, the Bacillus belegensis CMML20-16 strain is a rhizosphere microorganism and can promote the healthy growth of grass by producing about 5 μg/ml of IAA, a factor that promotes plant growth.
실시예 5. 항균활성 검정Example 5. Antibacterial activity assay
바실러스 벨레젠시스 CMML20-16 균주의 식물 병원균에 대한 생육저지 효과를 확인하기 위해 대치배양을 하였다.Replacement culture was performed to confirm the growth inhibition effect of Bacillus belegensis CMML20-16 strain against plant pathogens.
잔디 식물 병원균인 라이족토니아 솔라니 AG-1(ⅠA)(Rhizoctonia solani AG-1(ⅠA)), 라이족토니아 솔라니 AG2-2(Ⅳ)(Rhizoctonia solani AG2-2(Ⅳ)), 라이족토니아 솔라니 AG-1(ⅠB) (Rhizoctonia solani AG-1(ⅠB)), 라이족토니아 솔라니 AG-4, 라이족토니아 솔라니 AG2-2(ⅢB)(Rhizoctonia solani AG2-2(ⅢB)) 및 라이족토니아 세레아리스(Rhizoctonia cerealis) 균주를 25 ℃에서 5일간 PDA 배지에서 배양하였다.Rhizoctonia solani AG-1(ⅠA) ( Rhizoctonia solani AG-1(ⅠA)), Rhizoctonia solani AG2-2(IV) ( Rhizoctonia solani AG2-2(Ⅳ)), a lawn plant pathogen Rhizoctonia solani AG-1(ⅠB) ( Rhizoctonia solani AG-1(ⅠB)), Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(ⅢB) ( Rhizoctonia solani AG2-2(ⅢB)) and Rhizoctonia cerealis strains were cultured in PDA medium at 25°C for 5 days.
또한, 알타나리아 알타르나타 (Alternaria alternata), 보트리티스 시네레아 (Botrytis cinerea), 클라리레이디아 잭소니 (Clarireedia jacksonii), 콜레토트리쿰 글로에오스포리오데스 (Colletotrichum gloeosporiodes), 콜레토트리쿰 속(Colletotrichum sp.), 포모시스 속 (Phomopsis sp.), 푸사리움 옥시포룸 (Fusarium oxysporum), 푸사리움 속 (Fusarium sp.), 피티움 얼티뭄 (Pythium ultimum), 라이족토니아 솔라니 AG-3, 스켈로티니아 스켈로티오룸 (Sclerotinia sclerotiorum) 및 세라토시스티스 핌브리아타 (Ceratocystis fimbriata) 균주를 25 ℃에서 5일간 PDA 배지에서 배양하였다.In addition, Alternaria alternata , Botrytis cinerea , Clarireedia jacksonii, Colletotrichum gloeosporiodes , Colletotrichum Colletotrichum sp., Phomopsis sp., Fusarium oxysporum , Fusarium sp., Pythium ultimum , Rhizoctonia solani AG -3, Sclerotinia sclerotiorum and Ceratocystis fimbriata strains were cultured in PDA medium at 25°C for 5 days.
직경 5 mm인 코르크 보러 (Cork borer)를 이용하여 병원균의 균총을 PDA 배지가 분주된 일회용 페트리디쉬 중앙에 치상하였다. 바실러스 벨레젠시스 CMML20-16 균주는 TSB 액체배지에 진탕배양한 후, 총 균수를 1Χ 108 C.F.U/ml 로 일정하게 희석하였다. 희석한 바실러스 벨레젠시스 CMML20-16 균주를 분주된 일회용 페트리디쉬의 3개 부분에 각각 10 μl씩 치상하여 병원균별로 25 ℃ 항온기에서 2 내지 5일간 배양한 후 병원균이 자라는 정도를 측정하였다. 병원균만 배양한 배지와 비교하였을 때, 바실러스 벨레젠시스 CMML20-16을 같이 배양한 경우 병원균의 생장이 저해되는 정도를 백분율로 나타내었고, 그 결과를 하기 표 7 및 도 10에 나타내었다.Using a cork borer with a diameter of 5 mm, the pathogen colony was placed in the center of a disposable Petri dish in which PDA medium was dispensed. Bacillus belegensis CMML20-16 strain was cultured with shaking in TSB liquid medium, and the total number of bacteria was 1Χ 10 8 CFU/ml. It was diluted consistently. 10 μl of the diluted Bacillus belegensis CMML20-16 strain was placed on each of three portions of a disposable petri dish, and each pathogen was cultured in a thermostat at 25°C for 2 to 5 days, and then the growth of the pathogen was measured. When compared to a medium cultured only with pathogens, the degree to which the growth of pathogens was inhibited when cultured with Bacillus belegensis CMML20-16 was expressed as a percentage, and the results are shown in Table 7 and Figure 10 below.
(Alternaria alternata)Altanaria Altarnata
(Alternaria alternata )
(Botrytis cinerea)Botrytis cinerea
( Botrytis cinerea )
(Colletotrichum gloeosporiodes)Colletotrichum gloeosporiodes
( Colletotrichum gloeosporiodes )
(Rhizoctonia solani AG-1(ⅠA))Rhizoctonia solani AG-1(ⅠA)
( Rhizoctonia solani AG-1(ⅠA))
(Rhizoctonia solani AG-1(ⅠB))Rhizoctonia solani AG-1(ⅠB)
( Rhizoctonia solani AG-1(ⅠB))
(Rhizoctonia solani AG-4)Rhizoctonia solani AG-4
( Rhizoctonia solani AG-4)
(Rhizoctonia solani AG2-2(ⅢB))Rhizoctonia solani AG2-2(ⅢB)
( Rhizoctonia solani AG2-2(ⅢB))
(Rhizoctonia solani AG2-2(Ⅳ))Rhizoctonia solani AG2-2(Ⅳ)
( Rhizoctonia solani AG2-2(IV))
(Rhizoctonia solani AG-3)Rhizoctonia solani AG-3
( Rhizoctonia solani AG-3)
(Sclerotinia sclerotiorum)Skelotinia scelotiorum
( Sclerotinia sclerotiorum )
(Ceratocystis fimbriata)Ceratocystis fimbriata
( Ceratocystis fimbriata )
상기 표 7 및 도 10에서 확인할 수 있듯이, 라이족토니아마름병 (라지패취)을 유발하는 라이족토니아 솔라니 (Rhizoctonia solani) AG2-2(Ⅳ), 갈색잎마름병을 유발하는 라이족토니아 솔라니 (Rhizoctonia solani) AG1-1A, AG1-1B, AG2-2(ⅢB), AG4와 봄마름병, 황색마름병등을 유발하는 라이족토니아 세레아리스 (Rhizoctonia cerealis)에서 모두 탁월한 생육 저지능을 보였다. 또한, 그 외의 식물 병원균에 대해서도 높은 항균 활성을 나타내어, 바실러스 벨레젠시스 CMML20-16 은 다양한 식물병에 방제가를 나타냄을 확인할 수 있었다.As can be seen in Table 7 and Figure 10, Rhizoctonia solan i AG2-2(IV), which causes Rhizoctonia blight (large patch), and Rhizoctonia solani, which causes brown leaf blight. ( Rhizoctonia solani ) AG1-1A, AG1-1B, AG2-2(ⅢB), AG4, and Rhizoctonia cerealis, which causes spring blight and yellow blight, all showed excellent growth inhibition ability. In addition, it showed high antibacterial activity against other plant pathogens, and it was confirmed that Bacillus belegensis CMML20-16 exhibits a control value against various plant diseases.
실시예 6. 병원성 균주의 생육저지 활성 평가Example 6. Evaluation of growth inhibition activity of pathogenic strains
바실러스 벨레젠시스 CMML20-16의 잔디 병원균에 대한 직접적인 생육저지 활성을 평가하였다. 갈색잎마름병 (브라운 패취)을 유발하는 라이족토니아 솔라니 (Rhizoctonia solani) AG2-2(ⅢB) 와 라이족토니아마름병(라지 패취) 을 유발하는 라이족토니아 솔라니 (Rhizoctonia solani) AG2-2(Ⅳ)를 바실러스 벨레젠시스 CMML20-16 과 대치배양하고, 현미경으로 확인한 결과를 도 11 및 12에 나타내었다.The direct growth inhibition activity of Bacillus belegensis CMML20-16 against turfgrass pathogens was evaluated. Rhizoctonia solani AG2-2(ⅢB), which causes brown leaf blight (brown patch) and Rhizoctonia solani AG2-2(, which causes Rhizoctonia blight (large patch) IV) was replaced with Bacillus belegensis CMML20-16, and the results confirmed under a microscope are shown in Figures 11 and 12.
바실러스 벨레젠시스 CMML20-16에 의한 갈색잎마름병균인 라이족토니아 솔라니 AG2-2(ⅢB)의 저지환의 경계부분을 현미경 (Olympus BX51, 40배율)으로 확대 촬영하여 균사의 형태적 변형을 확인하였다. 대치배양한 균주는 저지환 경계에서 균사가 충분히 자라지 못하고 마디가 생기며 자라거나, 균사의 일부가 일그러지거나 납작해지는 등 사멸에 이르러 형태가 아예 망가진 것을 관찰하였다.The border of the low ring of Rhizoctonia solani AG2-2(ⅢB), a brown leaf blight caused by Bacillus belegensis CMML20-16, was photographed under a microscope (Olympus BX51, 40x magnification) to confirm the morphological deformation of the hyphae. did. It was observed that the strain cultured as a replacement had its hyphae unable to grow sufficiently at the border of the low-lying ring, growing with nodes, or parts of the hyphae being distorted or flattened, leading to death and completely ruining their form.
즉, 바실러스 벨레젠시스 CMML20-16은 라이족토니아 솔라니 AG2-2(ⅢB)의 정상적인 균사 생장을 저해함을 확인할 수 있었다 (도 11).In other words, it was confirmed that Bacillus belegensis CMML20-16 inhibits the normal mycelial growth of Rhizoctonia solani AG2-2(IIIB) (FIG. 11).
또한, 바실러스 벨레젠시스 CMML20-16에 의한 라이족토니아마름병균인 라이족토니아 솔라니 AG2-2(IV)의 저지환의 경계부분을 현미경 (Olympus BX51, 100배율)으로 확대 촬영하여 균사의 형태적 변형을 확인하였다. 대치배양한 균주는 저지환 경계에서 균사가 갈색으로 변하고, 정상적으로 격벽을 형성하지 못하였으며, 격벽 사이가 짧고 가지치기 (Branching)이 많으며, 일정하지 않은 길이와 형태로 생장하는 것을 확인할 수 있었다.In addition, the border of the low-lying ring of Rhizoctonia solani AG2-2(IV), a Rhizoctonia blight fungus caused by Bacillus belegensis CMML20-16, was photographed under magnification (Olympus BX51, 100x magnification) to determine the morphological characteristics of the hyphae. Deformation was confirmed. In the replacement cultured strain, it was confirmed that the hyphae turned brown at the border of the low ring, failed to form septum normally, had short septa spaces, had a lot of branching, and grew with irregular length and shape.
즉, 바실러스 벨레젠시스 CMML20-16은 라이족토니아 솔라니 AG2-2(Ⅳ)의 정상적인 균사 생장을 저해함을 확인할 수 있었다 (도 12).In other words, it was confirmed that Bacillus belegensis CMML20-16 inhibits the normal mycelial growth of Rhizoctonia solani AG2-2(IV) (FIG. 12).
이를 통해, 바실러스 벨레젠시스 CMML20-16은 주요 잔디 병해인 갈색잎마름병과 라이족토니아마름병에 작용하는 라이족토니아솔라니 (Rhizoctonia solani)의 여러 균주를 방제할 수 있는 능력이 있음을 확인할 수 있었다.Through this, it was confirmed that Bacillus belegensis CMML20-16 has the ability to control several strains of Rhizoctonia solani, which acts on brown leaf blight and Rhizoctonia blight, which are major lawn diseases. .
실시예 7. 식물병 방제 검정Example 7. Plant disease control assay
7-1. 갈색잎마름병 방제효과7-1. Brown leaf blight control effect
바실러스 벨레젠시스 CMML20-16 균주 (이하, CMML20-16 균주)와 실제 방제용도로 사용되는 농약인 아족시스트로빈 (Azoxystrobin)을 원형 포트에 파종하여 키운 크리핑 벤트 그라스에 처리한 후, 갈색잎마름병 (브라운패치, Brown Patch)의 방제능을 확인하였다.After treating Bacillus belegensis CMML20-16 strain (hereinafter referred to as CMML20-16 strain) and Azoxystrobin, a pesticide used for actual control purposes, on creeping bent grass sown and grown in round pots, brown leaf blight disease ( The control effect of Brown Patch was confirmed.
구체적으로, CMML20-16 균주를 TSB 배지에서 30 ℃, 150 rpm으로 24시간 진탕 배양한 후, 1×107 cfu/ml 농도의 배양희석액을 만들어 사용하였다. 대조구로는 화학 농약인 아족시스트로빈 21.7 μg/ml을 제조하여 사용하였다. CMML20-16 균주 배양희석액과 아족시스트로빈을 크리핑벤트 그라스 원형 포트 네 지점에 1 mL씩 3일 간격으로 2회 처리하였다. 1회 처리 1시간 후에 포트 네 지점에 PDB 배지에서 3일간 배양된 갈색잎마름병 병원균인 라이족토니아 솔라니 (Rhizoctonia solani) AG2-2(ⅢB) 균체를 그라인더 (Grinder)를 이용하여 분쇄한 후 하루 더 배양하여 상기와 같은 방법으로 1 mL씩 접종하여 발병시키고, 그 결과를 하기 표 8 및 도 13에 나타내었다.Specifically, the CMML20-16 strain was cultured in TSB medium at 30°C and 150 rpm with shaking for 24 hours, and then a culture dilution with a concentration of 1×10 7 cfu/ml was prepared and used. As a control, 21.7 μg/ml of azoxystrobin, a chemical pesticide, was prepared and used. CMML20-16 strain culture dilution and azoxystrobin were treated twice at 3-day intervals at 1 mL each at four points in the creeping vent glass circular port. 1 hour after one-time treatment , Rhizoctonia solani AG2-2(ⅢB), a brown leaf blight pathogen, cultured for 3 days in PDB medium at four port locations was pulverized using a grinder and then ground for one day. The cells were further cultured and inoculated with 1 mL each in the same manner as above to cause disease, and the results are shown in Table 8 and Figure 13.
상기 표 8 및 도 13에서 확인할 수 있듯이, 무처리구 대비 CMML20-16 균주 처리구에서는 방제가가 약 81.82±4.69 %로 높은 방제가가 나타났다.As can be seen in Table 8 and Figure 13, the control value was high at about 81.82 ± 4.69% in the treatment group with the CMML20-16 strain compared to the untreated group.
7-2. 라이족토니아마름병 방제효과7-2. Rhizoctonia blight control effect
바실러스 벨레젠시스 CMML20-16 균주 (이하, CMML20-16 균주)와 실제 방제용도로 사용되는 농약인 아족시스트로빈 (Azoxystrobin)을 골프장에 식재하는 잔디를 떼어와 원형 포트에 옮겨 키운 후, 처리하여 라이족토니아마름병 (라지패치, Large patch)에 대한 방제능을 확인하였다.Bacillus belegensis CMML20-16 strain (hereinafter referred to as CMML20-16 strain) and Azoxystrobin, a pesticide used for actual control purposes, are grown in round pots by removing the grass planted on the golf course and then treating and growing them. The control effect against zoctonia blight (large patch) was confirmed.
구체적으로, CMML20-16 균주를 TSB 배지에서 30 ℃, 150 rpm으로 24시간 진탕 배양한 후, 1×107 cfu/ml 농도의 배양희석액을 만들어 사용하였다. 대조구로는 화학 농약인 아족시스트로빈 21.7 μg/ml을 제조하여 사용하였다. CMML20-16 균주 배양희석액과 아족시스트로빈을 잔디 원형 포트 네 지점에 1 mL씩 3일 간격으로 2회 처리하였다. 1회 처리 1시간 후에 포트 네 지점에 PDB 배지에서 5일간 배양 후 그라인더를 이용하여 분쇄한 후 하루 더 배양한 라이족토니아마름병 병원균인 라이족토니아 솔라니 (Rhizoctonia solani) AG2-2(IV) 균체를 1 mL씩 접종하여 발병시키고, 그 결과를 하기 표 9 및 도 13에 나타내었다.Specifically, the CMML20-16 strain was cultured in TSB medium at 30°C and 150 rpm with shaking for 24 hours, and then a culture dilution with a concentration of 1×10 7 cfu/ml was prepared and used. As a control, 21.7 μg/ml of azoxystrobin, a chemical pesticide, was prepared and used. CMML20-16 strain culture dilution and azoxystrobin were treated twice at 3-day intervals, 1 mL each, to four circular pots on the lawn. After one hour of treatment, Rhizoctonia solani ( Rhizoctonia solani ) AG2-2(IV) cells, the pathogen responsible for Rhizoctonia blight, were cultured in PDB medium at four port locations for 5 days, pulverized using a grinder, and cultured for another day. The disease was caused by inoculating 1 mL each, and the results are shown in Table 9 and Figure 13 below.
상기 표 9 및 도 14에서 확인할 수 있듯이, 무처리구 대비 CMML20-16 균주 처리구에서는 방제가가 약 91.67 ±4.17%를 나타났으며, 시중에 농약으로 사용되는 아족시스트로빈과 유사한 방제가를 나타낸 것을 확인할 수 있었다.As can be seen in Table 9 and Figure 14, the control value was about 91.67 ± 4.17% in the treatment group with the CMML20-16 strain compared to the untreated group, and it can be confirmed that the control value was similar to azoxystrobin, which is used as a pesticide on the market. there was.
실시예 8. 균주의 항균활성 물질 분석Example 8. Analysis of antibacterial activity of strains
바실러스 벨레젠시스 CMML20-16에서 항균 물질인 리포펩타이드를 추출하였다. 구체적으로, 바실러스 벨레젠시스 CMML20-16를 10 ml의 TSB에 전배양한 후, 200 ml Landy's medium (Glucose 20g, L-glutamic acid 2g, Yeast extract 1g, (NH4)2SO4 2.3g, MgSO4 0.5 g, KCl 0.5 g, KH2PO4 1g, Fe(SO4)3 0.0012 g, MnSO4 0.0014 g, CuSO4 0.0016 g/l)에 2 %(v/v)가 되게 접종하여 진탕 배양기에서 30 ℃, 150 rpm 조건으로 60시간 동안 배양하였다. 배양액을 12,000 rpm으로 4 ℃에서 15분 동안 원심분리하여 상등액을 취하였다. 상기 획득한 각 상등액은 포화된 염산 (35 % HCl)로 pH 2.0까지 맞추고, 4 ℃에서 24시간 동안 방치한 후, 12,000 rpm으로 4 ℃에서 15분 동안 원심분리하여 침전물을 획득하였다. 상기 획득된 침전물을 10 ml 메탄올로 녹이고, 용액은 회전 증발 건조기 (Rotary evaporator)로 건조하고, 얻어진 펠렛을 메탄올 용매 1 ml에 녹인 후 0.45 um의 비-발열 (Non-pyrogenic) 친수성 필터로 여과하여 분석시료로 사용하였다.Lipopeptide, an antibacterial substance, was extracted from Bacillus belegensis CMML20-16. Specifically, Bacillus belegensis CMML20-16 was pre-cultured in 10 ml of TSB, then 200 ml Landy's medium (Glucose 20g, L-glutamic acid 2g, Yeast extract 1g, (NH 4 ) 2 SO 4 2.3g, MgSO 4 0.5 g, KCl 0.5 g, KH 2 PO 4 1g, Fe(SO 4 ) 3 0.0012 g, MnSO 4 0.0014 g, CuSO 4 0.0016 g/l) at a concentration of 2% (v/v) in a shaking incubator. Cultured for 60 hours at 30°C and 150 rpm. The culture was centrifuged at 12,000 rpm at 4°C for 15 minutes and the supernatant was collected. Each of the obtained supernatants was adjusted to pH 2.0 with saturated hydrochloric acid (35% HCl), left at 4°C for 24 hours, and then centrifuged at 12,000 rpm at 4°C for 15 minutes to obtain a precipitate. The obtained precipitate was dissolved in 10 ml of methanol, the solution was dried using a rotary evaporator, and the obtained pellet was dissolved in 1 ml of methanol solvent and filtered through a 0.45 um non-pyrogenic hydrophilic filter. It was used as an analysis sample.
8-1. 항진균성 리포펩타이드인 펜기신의 분석8-1. Analysis of penicine, an antifungal lipopeptide.
항진균성 리포펩타이드 (Lipopeptide)계 항생제 중 펜기신 (Fengycin)을 분석하기 위하여 역상 고속액체크로마토그래피 (Reverse phase High performance liquid chromatography, RP-HPLC)을 이용하였다. Gradient 용매제는 하기 표 10에 기재한 바와 같이 분석을 수행하였다.Reverse phase high performance liquid chromatography (RP-HPLC) was used to analyze Fengycin, one of the antifungal lipopeptide antibiotics. The gradient solvent was analyzed as described in Table 10 below.
용매 A는 0.1% 트리플로오로아세트산 (Trifluoroacetic acid/TFA)가 첨가된 아세토니트릴 (acetonitrile)을, 용매 B는 0.1% 트리플로오로아세트산(Trifluoroacetic acid/TFA)가 첨가된 물 (Water)을 이용하여 수행하였다. 분석에는 Column은 C18 column을 사용하였으며, 유속은 분당 0.8 ml, 흡광도 205nm에서 시행하였다. 표준물질로는 시그마 알드리치에서 구입한 펜기신을 500μg/ml 사용하였다.Solvent A uses acetonitrile with 0.1% trifluoroacetic acid (TFA) added, and solvent B uses water with 0.1% trifluoroacetic acid (TFA) added. carried out. The analysis was performed using a C18 column, with a flow rate of 0.8 ml per minute and an absorbance of 205 nm. As a standard material, 500 μg/ml of penicine purchased from Sigma Aldrich was used.
도 15a 내지 15d에서 확인할 수 있듯이, HPLC 분석 결과 머무름 시간 (Retention time)은 11분 내지 15분 사이에 다수의 피크 (Peak)가 검출되었고, 이들 피크의 머무름 시간과 표준물질인 펜기신의 머무름 시간을 비교한 결과 거의 같은 시간에 피크가 검출되었다.As can be seen in Figures 15a to 15d, as a result of HPLC analysis, a number of peaks were detected between 11 and 15 minutes, and the retention time of these peaks and the retention time of penicine, a standard material, As a result of comparing, the peak was detected at almost the same time.
이를 통해, 바실러스 벨레젠시스 CMML20-16의 항진균성 성분은 리포펩티드계 항생제인 펜기신 (Fengycin)임을 확인할 수 있었다.Through this, it was confirmed that the antifungal ingredient of Bacillus velegensis CMML20-16 is Fengycin, a lipopeptide antibiotic.
8-2. 항진균성 리포펩타이드인 설펙틴의 분석8-2. Analysis of sulpectin, an antifungal lipopeptide.
항진균성 리포펩타이드 (Lipopeptide)계 항생제 중 설펙틴 (Surfectin)을 분석하기 위하여 역상 고속액체크로마토그래피 (Reverse phase High performance liquid chromatography, RP-HPLC)을 이용하였다. Gradient 용매제는 하기 표 11에 기재한 바와 같이 분석을 수행하였다.Reverse phase high performance liquid chromatography (RP-HPLC) was used to analyze surfectin, one of the antifungal lipopeptide antibiotics. The gradient solvent was analyzed as described in Table 11 below.
용매 A는 0.05 % 트리플로오로아세트산 (Trifluoroacetic acid/TFA)가 첨가된 아세토니트릴 (acetonitrile)을, 용매 B는 물 (Water)를 이용하여 수행하였다. 분석에는 컬럼 (Column)은 C18 Column을 사용하였으며, 유속은 분당 1 ml, 흡광도 210 nm에서 수행하였다. 표준물질로는 시그마 알드리치에서 구입한 설펙틴을 500 μg/ml 사용하였다.Solvent A was performed using acetonitrile with 0.05% trifluoroacetic acid (Trifluoroacetic acid/TFA) added, and solvent B was performed using water. The analysis was performed using a C18 column, with a flow rate of 1 ml per minute, and an absorbance of 210 nm. As a standard material, 500 μg/ml of sulpectin purchased from Sigma Aldrich was used.
도 16a 내지 16d에서 확인할 수 있듯이, HPLC 분석 결과 머무름 시간 (Retention time)은 60 내지 90분 사이에 다수의 피크가 검출되었으며, 이들 피크의 머무름 시간과 표준물질인 서텍틴의 머무름 시간을 비교한 결과 거의 같은 시간에 피크가 검출되었다.As can be seen in Figures 16a to 16d, as a result of HPLC analysis, a number of peaks were detected with retention times between 60 and 90 minutes, and as a result of comparing the retention times of these peaks with the retention time of searchtin, a standard material Peaks were detected at approximately the same time.
이를 통해, 바실러스 벨레젠시스 CMML20-16의 항진균성 성분은 리포펩티드계 항생제인 설펙틴 (Surfectin)임을 확인하였다.Through this, it was confirmed that the antifungal ingredient of Bacillus velegensis CMML20-16 is sulfectin, a lipopeptide antibiotic.
실시예 9. 균주의 주요 잔디 살진균 농약에 대한 내성Example 9. Resistance of strains to major turf fungicide pesticides
잔디 병해 방제 및 관리를 위해 아족시스트로빈 (Azoxystrobin) 및 플룩사피록사드 (Fluxapyroxad)가 주로 사용되고 있다. 바실러스 벨레젠시스 CMML20-16 균주가 상기 농약과 혼용이 가능할 경우, 살균제에 대한 사용 저감 효과를 나타낼 수 있으며, 미생물이 토양 및 잔디에 작용하는 시간 동안의 잔디 병해를 억제할 수 있다. 이에, 바실러스 벨레젠시스 CMML20-16 균주의 배양 시 아족시스트로빈 및 플룩사피록사드를 처리하여 균주의 생장 여부를 확인하고 농약과 함께 사용이 가능한지를 평가하였다.Azoxystrobin and Fluxapyroxad are mainly used for lawn disease control and management. When the Bacillus belegensis CMML20-16 strain can be mixed with the above pesticide, it can have an effect of reducing the use of fungicides and can suppress lawn diseases during the time that microorganisms act on soil and grass. Accordingly, when cultivating the Bacillus belegensis CMML20-16 strain, azoxystrobin and fluxapyroxad were treated to confirm the growth of the strain and to evaluate whether it could be used with pesticides.
구체적으로, 바실러스 벨레젠시스 CMML20-16 균주를 10 ml의 TSB에 전배양한 후, TSB에 아족시스트로빈을 각각 50 μg/ml 및 20 μg/ml 농도로, 플룩사피록사드를 각각 50 μg/ml 및 20 μg/ml 농도로 혼합한 후, 24시간 동안 배양하였다. 그 다음, 200 μl의 배양액을 TSA에 평판 도말한 후, 37 ℃에서 12시간동안 배양하여 콜로니를 계수하여 농약에 대한 내성을 평가하였다. 상기 실험은 총 3회 반복으로 실시하였으며, 농약의 무처리구는 Tryptic Soy Broth (Control), Tryptic Soy Broth와 20μg/ml Azoxystrobin을 섞은 후 바실러스 벨레젠시스 CMML20-16을 배양하여 TSA에 도말한 것을 Tryptic Soy Broth + Azoxystrobin (20 μg/ml)으로, Tryptic Soy Broth와 50μg/ml Azoxystrobin을 섞은 후 바실러스 벨레젠시스 CMML20-16을 배양하여 TSA에 도말한 것을 Tryptic Soy Broth + Azoxystrobin (50 μg/ml)으로, Tryptic Soy Broth와 20 μg/ml Fluxapyroxad 을 섞은 후 바실러스 벨레젠시스 CMML20-16을 배양하여 TSA에 도말한 것을 Tryptic Soy Broth + Fluxapyroxad (20 μg/ml)으로, Tryptic Soy Broth와 50 μg/ml Fluxapyroxad 을 섞은 후 바실러스 벨레젠시스 CMML20-16을 배양하여 TSA에 도말한 것을 Tryptic Soy Broth + Fluxapyroxad (50 μg/ml)으로 하여, 그 결과를 도 17에 나타내었다.Specifically, the Bacillus belegensis CMML20-16 strain was pre-cultured in 10 ml of TSB, and then azoxystrobin was added to the TSB at a concentration of 50 μg/ml and 20 μg/ml, and fluxapyroxad was added at a concentration of 50 μg/ml, respectively. After mixing at a concentration of 20 μg/ml and 20 μg/ml, the mixture was cultured for 24 hours. Next, 200 μl of the culture medium was plated on TSA, incubated at 37°C for 12 hours, and colonies were counted to evaluate resistance to pesticides. The above experiment was repeated a total of three times, and the pesticide-free group was Tryptic Soy Broth (Control). After mixing Tryptic Soy Broth and 20 μg/ml Azoxystrobin, Bacillus belegensis CMML20-16 was cultured and smeared on TSA. Broth + Azoxystrobin (20 μg/ml), Tryptic Soy Broth and 50 μg/ml Azoxystrobin were mixed, then Bacillus belegensis CMML20-16 was cultured and smeared on TSA with Tryptic Soy Broth + Azoxystrobin (50 μg/ml). After mixing Tryptic Soy Broth and 20 μg/ml Fluxapyroxad, Bacillus belegensis CMML20-16 was cultured and smeared on TSA with Tryptic Soy Broth + Fluxapyroxad (20 μg/ml), Tryptic Soy Broth and 50 μg/ml Fluxapyroxad. After mixing, Bacillus belegensis CMML20-16 was cultured and smeared on TSA with Tryptic Soy Broth + Fluxapyroxad (50 μg/ml), and the results are shown in Figure 17.
도 17에서 확인할 수 있듯이, 아족시스트로빈 및 플룩사피록사드를 각각 20 μg/ml 씩 처리한 경우도 균주의 생장이 잘 이루어졌다. 특히, 아족시스트로빈 및 플룩사피록사드를 각각 50 μg/ml 씩 처리하였을 때도 균주의 생장이 가능하였으며, 무처리 대조구와 큰 차이를 보이지 않음을 확인할 수 있었다.As can be seen in Figure 17, the strain grew well even when treated with 20 μg/ml each of azoxystrobin and fluxapyroxad. In particular, growth of the strain was possible even when treated with 50 μg/ml each of azoxystrobin and fluxapyroxad, and it was confirmed that there was no significant difference from the untreated control.
현재, 골프장 잔디에 처리하는 아족시스트로빈의 농도는 1 m2 당 50 μg/ml이고, 플룩사피록사드는 1 m2 당 23 μg/ml의 농도로 처리된다. 이 점을 감안할 때, 바실러스 벨레젠시스 CMML20-16 균주는 잔디에 처리하는 살진균 농약과 혼용 또는 교차 처리가 가능함을 확인할 수 있다.Currently, the concentration of azoxystrobin treated on golf course grass is 50 μg/ml per 1 m 2 , and fluxapyroxade is treated at a concentration of 23 μg/ml per 1 m 2 . Considering this, it can be confirmed that Bacillus belegensis CMML20-16 strain can be mixed or cross-treated with fungicidal pesticides applied to lawns.
<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Composition for controlling plant diseases comprising culture solution of Bacillus velezensis CMML20-16 or extract thereof, methods for manufacturing thereof, and methods for plant disease control by using them <130> PN210447 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA 27F forward primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA 1492R reverse primer <400> 2 ggttaccttg ttacgactt 19 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> gyrB UP1 forward primer <400> 3 gaagtcatca tgaccgttct gcaygcnggn ggnaarttyg a 41 <210> 4 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> gyrB UP-2r reverse primer <400> 4 agcagggtac ggatgtgcga gccrtcnacr tcngcrtcng tcat 44 <210> 5 <211> 1400 <212> DNA <213> Artificial Sequence <220> <223> CMML20-16 16S rRNA <400> 5 tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60 acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120 atggttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc acttacagat 180 ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga cgatgcgtag 240 ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300 ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360 gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc cgttcaaata 420 gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480 ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540 tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600 aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660 tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720 gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780 agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840 gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900 catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960 tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020 gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080 ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140 tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacagaa 1200 caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc 1260 gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320 cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380 cccgaagtcg gtgaggtaac 1400 <210> 6 <211> 1064 <212> DNA <213> Artificial Sequence <220> <223> CMML 20-16 gyr B <400> 6 cgccttgtcg accactcttg acgttacggt tcatcgtgac gggaaaatcc actatcaggc 60 gtacgagcgc ggtgtacctg tggctgatct tgaagtgatc ggcgaaactg ataagaccgg 120 aacgattacg cacttcgttc cggacccgga aattttcaaa gaaacaactg tatatgacta 180 tgatctgctt tcaaaccgtg tccgggaatt ggccttcctg acaaaaggcg taaacatcac 240 gattgaagac aaacgtgaag gacaagaacg gaaaaacgag taccactaca aaggcggaat 300 caaaagctat gttgagtact taaaccgttc caaagaagtc gttcatgaag agccgattta 360 tatcgaaggc gagaaagacg gcataacggt tgaagttgca ttgcaataca acgacagcta 420 tacaagcaat atttattctt tcacaaataa tatcaacaca tacgaaggcg gcacgcacga 480 ggccggattt aaaaccggtc tgacccgtgt cataaacgac tatgcaagaa gaaaggggat 540 tttcaaagaa aatgatccga atttaagcgg ggatgatgtg agagaagggc tgactgccat 600 tatttcaatt aagcaccctg atccgcaatt cgaagggcag acgaaaacga agctcggcaa 660 ctccgaagcg agaacgatca ctgatacgct gttttcttct gcgctggaaa cattccttct 720 tgaaaatccg gactcagccc gcaaaatcgt tgaaaaaggt ttaatggccg caagagcgcg 780 gatggcggcg aaaaaagcgc gggaattgac ccggcgcaaa agtgcgcttg agatttccaa 840 tctgccgggc aaactggcgg actgttcttc taaagatccg agcatttccg agctgtatat 900 cgtagagggt gactctgcgg gcggatcagc gaaacaggga cgggaccgtc atttccaagc 960 cattctgccg ctgcgcggta agattctgaa cgttgagaaa gccagacttg ataagattct 1020 ctcaaacaat gaggtcagat caatgatcac ggccctcgga acag 1064 <110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Composition for controlling plant diseases comprising culture solution of Bacillus velezensis CMML20-16 or extract thereof, methods for manufacturing it, and methods for plant disease control by using them <130> PN210447 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA 27F forward primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA 1492R reverse primer <400> 2 ggttaccttg ttacgactt 19 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> gyrB UP1 forward primer <400> 3 gaagtcatca tgaccgttct gcaygcnggn ggnaarttyg a 41 <210> 4 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> gyrB UP-2r reverse primer <400> 4 agcagggtac ggatgtgcga gccrtcnacr tcngcrtcng tcat 44 <210> 5 <211> 1400 <212> DNA <213> Artificial Sequence <220> <223> CMML20-16 16S rRNA <400> 5 tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60 acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120 atggttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc acttacagat 180 ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga cgatgcgtag 240 ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300 ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360 gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc cgttcaaata 420 gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480 ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540 tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600 aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660 tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720 gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780 agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840 gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900 catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960 tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020 gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080 ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140 tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacagaa 1200 caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc 1260 gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320 cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380 cccgaagtcg gtgaggtaac 1400 <210> 6 <211> 1064 <212> DNA <213> Artificial Sequence <220> <223> CMML 20-16 gyr B <400> 6 cgccttgtcg accactcttg acgttacggt tcatcgtgac gggaaaatcc actatcaggc 60 gtacgagcgc ggtgtacctg tggctgatct tgaagtgatc ggcgaaactg ataagaccgg 120 aacgattacg cacttcgttc cggacccgga aattttcaaa gaaacaactg tatatgacta 180 tgatctgctt tcaaaccgtg tccgggaatt ggccttcctg acaaaaggcg taaacatcac 240 gattgaagac aaacgtgaag gacaagaacg gaaaaacgag taccactaca aaggcggaat 300 caaaagctat gttgagtact taaaccgttc caaagaagtc gttcatgaag agccgattta 360 tatcgaaggc gagaaagacg gcataacggt tgaagttgca ttgcaataca acgacagcta 420 tacaagcaat atttattctt tcacaaataa tatcaacaca tacgaaggcg gcacgcacga 480 ggccggattt aaaaccggtc tgacccgtgt cataaacgac tatgcaagaa gaaaggggat 540 tttcaaagaa aatgatccga atttaagcgg ggatgatgtg agagaagggc tgactgccat 600 tatttcaatt aagcaccctg atccgcaatt cgaagggcag acgaaaacga agctcggcaa 660 ctccgaagcg agaacgatca ctgatacgct gttttcttct gcgctggaaa cattccttct 720 tgaaaatccg gactcagccc gcaaaatcgt tgaaaaaggt ttaatggccg caagagcgcg 780 gatggcggcg aaaaaagcgc gggaattgac ccggcgcaaa agtgcgcttg agatttccaa 840 tctgccgggc aaactggcgg actgttcttc taaagatccg agcatttccg agctgtatat 900 cgtagagggt gactctgcgg gcggatcagc gaaacaggga cgggaccgtc atttccaagc 960 cattctgccg ctgcgcggta agattctgaa cgttgagaaa gccagacttg ataagattct 1020 ctcaaacaat gaggtcagat caatgatcac ggccctcgga acag 1064
Claims (9)
잿빛곰팡이병 원인균 보트리티스 시네레아 (Botrytis cinerea);
동전마름병 원인균 클라리레이디아 잭소니 (Clarireedia jacksonii);
모잘록병 원인균 피티움 얼티뭄 (Pythium ultimum);
봄마름병 원인균 라이족토니아 세레아리스(Rhizoctonia cerealis); 및,
흑반병 원인균 세라토시스티스 핌브리아타 (Ceratocystis fimbriata);으로 이루어진 군에서 선택된 1종 이상의 원인균에 대한 항균 활성을 갖는 수탁번호 KCTC 14762BP로 기탁된 잔디의 식물병 방제용 바실러스 벨레젠시스 (Bacillus velezensis) CMML20-16 균주. Rhizoctonia solani , the causative agent of brown leaf blight;
Botrytis cinerea, the causative agent of gray mold disease;
Clarireedia jacksonii, the causative agent of penny blight;
Pythium ultimum , the causative agent of hairy disease;
Rhizoctonia cerealis, the causative agent of spring blight; and,
Ceratocystis fimbriata , the causative agent of black spot disease; Bacillus velezensis CMML20 for controlling plant diseases in grass deposited under the accession number KCTC 14762BP, which has antibacterial activity against one or more causative bacteria selected from the group consisting of Ceratocystis fimbriata -16 strain.
상기 균주가,
갈색잎마름병 원인균 라이족토니아 솔라니 (Rhizocctonia solani);
잿빛곰팡이병 원인균 보트리티스 시네레아 (Botrytis cinerea);
동전마름병 원인균 클라리레이디아 잭소니 (Clarireedia jacksonii);
모잘록병 원인균 피티움 얼티뭄 (Pythium ultimum);
봄마름병 원인균 라이족토니아 세레아리스(Rhizoctonia cerealis); 및,
흑반병 원인균 세라토시스티스 핌브리아타 (Ceratocystis fimbriata);으로 이루어진 군에서 선택된 1종 이상의 원인균에 대한 항균 활성을 갖는 잔디의 식물병 방제용 조성물.A composition for controlling plant diseases in grass containing the culture medium of Bacillus velezensis CMML20-16 strain or extract of the culture medium of the strain deposited with accession number KCTC 14762BP,
The strain,
Rhizoctonia solani , the causative agent of brown leaf blight;
Botrytis cinerea, the causative agent of gray mold disease;
Clarireedia jacksonii, the causative agent of penny blight;
Pythium ultimum , the causative agent of hairy disease;
Rhizoctonia cerealis, the causative agent of spring blight; and,
A composition for controlling plant diseases in grass that has antibacterial activity against one or more types of causative bacteria selected from the group consisting of Ceratocystis fimbriata, the causative bacteria of black spot disease.
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| KR101845708B1 (en) | 2016-11-10 | 2018-04-09 | 대한민국 | New microorganism Bacillus velezensis YP2 or microbial agent comprising the same |
| KR102078252B1 (en) | 2018-12-18 | 2020-02-17 | 경기도 | Bacillus velezensis ARRI17 strain having antifungal activity against Root Rot Pathogen of ginseng and Use Thereof |
| KR102252856B1 (en) | 2020-11-17 | 2021-05-17 | 고애란 | Natural Plant Protection Agents Comprising Bacillus velezensis KCCM12751P Having Antifungal Activity Against Pathogens And Plant Growth Promoting Effect, And Uses Thereof |
| KR102354494B1 (en) | 2021-04-28 | 2022-01-24 | 부산광역시 | Bacillus velezensis BM2021 STRAIN, COMPOSITION COMPRISING FOR BIOLOGICAL CONTROL OF PLANT OR INSECT PATHOGENS USING SAME AND METHOD FOR THE BIOLOGICAL CONTROL USING SAME |
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| KR102078252B1 (en) | 2018-12-18 | 2020-02-17 | 경기도 | Bacillus velezensis ARRI17 strain having antifungal activity against Root Rot Pathogen of ginseng and Use Thereof |
| KR102252856B1 (en) | 2020-11-17 | 2021-05-17 | 고애란 | Natural Plant Protection Agents Comprising Bacillus velezensis KCCM12751P Having Antifungal Activity Against Pathogens And Plant Growth Promoting Effect, And Uses Thereof |
| KR102354494B1 (en) | 2021-04-28 | 2022-01-24 | 부산광역시 | Bacillus velezensis BM2021 STRAIN, COMPOSITION COMPRISING FOR BIOLOGICAL CONTROL OF PLANT OR INSECT PATHOGENS USING SAME AND METHOD FOR THE BIOLOGICAL CONTROL USING SAME |
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