KR102612090B1 - A microbial agent comprising Bacillus wiedmannii having antagonistic activity against Fusarium oxysporum and a composition for preventing Gastrodia elata rot comprising it - Google Patents
A microbial agent comprising Bacillus wiedmannii having antagonistic activity against Fusarium oxysporum and a composition for preventing Gastrodia elata rot comprising it Download PDFInfo
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- fusarium oxysporum
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
Abstract
본 발명에서는 푸사리움 옥시스포럼 (Fusarium oxysporum)에 길항활성을 갖는 바실러스 위드만니를 포함하는 미생물 제제 및 이를 포함하는 천마썩음병 방제용 조성물이 개시된다.
본 발명에 따른 미생물 제제 및 조성물은 천마썩음병의 원인 병원균인 푸사리움 옥시스포럼에 대한 길항 작용이 우수하여 천마썩음병을 효과적으로 방제할 수 있다. 또한 본 발명에 따른 미생물 제제 및 조성물은 천마 재배에 환경공해를 주지 않고 인체에 독성이 없는 친환경 천마썩음병 방제제의 안정적 공급으로 국내의 천마 재배 농가의 경영비용 절감, 환경오염 감소, 경쟁력 높은 고품질 천마 제품의 안정적 공급에 기여할 수 있다.The present invention discloses a microbial preparation containing Bacillus withmani, which has antagonistic activity against Fusarium oxysporum , and a composition for controlling horse rot disease containing the same.
The microbial preparation and composition according to the present invention have excellent antagonistic action against Fusarium oxysporum, the causative pathogen of horse rot disease, and can effectively control horse rot disease. In addition, the microbial preparation and composition according to the present invention do not cause environmental pollution to the cultivation of Thousand Horses and provide a stable supply of eco-friendly Thousand Horse Rot disease control agents that are non-toxic to the human body, thereby reducing management costs for domestic Thousand Horse cultivation farms, reducing environmental pollution, and providing high quality, highly competitive Thousand Horses. It can contribute to the stable supply of products.
Description
본 발명은 푸사리움 옥시스포럼 (Fusarium oxysporum)에 길항활성을 갖는 바실러스 위드만니를 포함하는 미생물 제제 및 이를 포함하는 천마썩음병 방제용 조성물에 관한 것으로서, 더 상세하게는 바실러스 위드만니(Bacillus wiedmannii) 균주 또는 그의 배양액을 유효성분으로 포함하는 푸사리움 옥시스포럼 방제용 미생물 제제, 천마썩음병 방제용 조성물 및 이를 이용한 천마썩음병을 방제하는 방법에 관한 것이다. The present invention relates to a microbial agent containing Bacillus wiedmanni, which has antagonistic activity against Fusarium oxysporum , and a composition for controlling horse rot disease containing the same. More specifically, it relates to Bacillus wiedmannii . ) It relates to a microbial agent for controlling Fusarium oxysporum containing the strain or its culture as an active ingredient, a composition for controlling horse rot, and a method for controlling horse rot using the same.
천마(Gastrodia elata)는 난초과의 다년생 기생식물로 한국, 중국, 일본, 타이완 등지에 분포한다. 최적의 생육조건은 해발 400~800m의 연평균온도 10~15℃, 평균습도 70%인 활엽수림이 있는 반음지에서 잘 자라는 것으로 알려져 있다. 천마는 덩이줄기 형태로 가로로 뻗으며 길이 10~18cm, 지름 3~4cm 정도로서 옆으로 뚜렷하지 않은 테가 있다. 약재로 이용되는 천마는 중풍, 고혈압, 현기증, 신경성 질환 등에 효능이 좋아 두부질환의 예방과 치료제로 이용되어 왔다 (특허등록 10-1771788호). Gastrodia elata is a perennial parasitic plant of the orchid family and is distributed in Korea, China, Japan, and Taiwan. It is known that the optimal growth conditions are a semi-shady area with a broad-leaved forest at an altitude of 400 to 800 m, an average annual temperature of 10 to 15 degrees Celsius, and an average humidity of 70%. Cheonma extends horizontally in the form of a tuber, is about 10 to 18 cm long and 3 to 4 cm in diameter, and has indistinct edges on the sides. Cheonma, which is used as a medicinal herb, is effective in treating stroke, high blood pressure, dizziness, and nervous diseases, and has been used as a preventative and therapeutic agent for head diseases (Patent Registration No. 10-1771788).
중요 산림약용 식물인 천마는 각종 신경계통의 질병 특히 뇌와 관련된 질환에 우수한 약효를 나타내는 것으로 알려지면서 빠르게 소비가 늘고 있다. 우리나라 천마의 주 생산단지는 무주를 중심으로, 상주, 김천, 산청, 진안 등 여러 지역에서 재배되어 왔다.Consumption of Cheonma, an important forest medicinal plant, is rapidly increasing as it is known to have excellent medicinal properties for various diseases of the nervous system, especially brain-related diseases. The main production complex of Cheonma in Korea is Muju, and it has been cultivated in various regions such as Sangju, Gimcheon, Sancheong, and Jinan.
그런데 천마는 재배시 썩음병에 취약하며 수확 후에도 병원균 등으로 인해 저장이 어려운 것으로 알려져 있다. 천마는 뽕나무버섯에 기생하는 기생식물로 버섯의 생장을 위해 농약이나 화학적 비료를 사용하지 않은 100% 친환경 농법으로 재배되어야 하기 때문에 썩음병 원인 병원균의 방제를 위해서는 재배환경을 개선해 주는 물리적 방법이나 미생물 제제를 이용한 미생물학적 방법으로 방제를 사용할 수 밖에 없다. 종래에는 물리적 방제에 주로 의존해 왔으나, 최근에는 미생물학적 방제에 관한 연구도 시도되고 있으며 천마썩음병 방제용 미생물 제제의 개발이 요구되고 있는 실정이다.However, it is known that Cheonma is vulnerable to rot when cultivated and that it is difficult to store it after harvest due to pathogens. Cheonma is a parasitic plant that lives on mulberry mushrooms and must be cultivated using 100% eco-friendly farming methods without the use of pesticides or chemical fertilizers for the growth of mushrooms. Therefore, physical methods or microbial agents that improve the cultivation environment are needed to control pathogens that cause rot disease. There is no choice but to use microbiological methods for control. In the past, it has mainly relied on physical control, but recently, research on microbiological control has been attempted, and the development of microbial agents for controlling horse rot disease is in demand.
이에 본 발명자들은 종래 기술에서의 요구에 부응하기 위해 천마썩음병을 연구하던 도중, 천마썩음병 원인 병원균인 푸사리움 옥시스포럼에 대해 길항활성이 우수한 바실러스 균주를 동정하게 되어 본 발명을 완성하게 되었다.Accordingly, while researching horse rot disease in order to meet the needs of the prior art, the present inventors identified a Bacillus strain with excellent antagonistic activity against Fusarium oxysporum, the causative pathogen of horse rot disease, and completed the present invention.
따라서 본 발명의 목적은 바실러스 위드만니(Bacillus wiedmannii) 균주 또는 그의 배양액을 유효성분으로 포함하는 푸사리움 옥시스포럼 방제용 미생물 제제를 제공하는 것이다. Therefore, the purpose of the present invention is to provide a microbial agent for controlling Fusarium oxysporum containing Bacillus wiedmannii strain or its culture medium as an active ingredient.
본 발명의 다른 목적은 상기 미생물 제제를 포함하는 천마썩음병 방제용 조성물을 제공하는 것이다. Another object of the present invention is to provide a composition for controlling horse rot disease containing the above microbial agent.
본 발명의 다른 목적은 상기 미생물 제제를 이용하여 푸사리움 옥시스포럼의 생육을 억제하는 방법을 제공하는 것이다. Another object of the present invention is to provide a method of inhibiting the growth of Fusarium oxysporum using the microbial agent.
본 발명의 다른 목적은 상기 미생물 제제 또는 상기 조성물을 이용하여 천마썩음병을 방제하는 방법을 제공하는 것이다. Another object of the present invention is to provide a method for controlling horse rot using the microbial agent or the composition.
상기 본 발명의 목적을 달성하기 위하여, 본 발명은 바실러스 위드만니 균주 또는 그의 배양액을 유효성분으로 포함하는 푸사리움 옥시스포럼 방제용 미생물 제제를 제공한다.In order to achieve the object of the present invention, the present invention provides a microbial preparation for controlling Fusarium oxysporum containing a Bacillus withmani strain or a culture medium thereof as an active ingredient.
상기 바실러스 위드만니 균주는 천마썩음병의 병원균인 푸사리움 옥시스포럼의 생육을 억제하는 특성을 가지며, 이와 같은 특성으로 인하여 천마 재배에서 큰 문제가 되는 천마썩음병을 방제할 수 있다. The Bacillus withmanni strain has the property of suppressing the growth of Fusarium oxysporum, the pathogen of horse rot, and due to this characteristic, it can control horse rot, which is a major problem in horse rot.
본 발명에서 '썩음병'은 식물의 뿌리나 땅속줄기가 분해되거나 썩는 증상이다. 감염되면 뿌리는 내부 조직이 파괴되어 갈색으로 변하고 이후 검게 썩는다. 잔뿌리에서 피해가 더욱 심해 뿌리가 갈라지고 쉽게 뽑힌다. 썩음병에 감염된 천마는 둥글고 회색빛의 갈색 반점을 보였으며, 시간이 지나면서 덩이줄기에 있는 반점은 더 큰 검은 병변으로 합쳐진다. 천마썩음병의 주요 병원균 중 하나로는 푸사리움 옥시스포럼이 알려져 있고, 이 병원균의 생육을 억제할 경우 천마썩음병을 발생을 감소시킬 수 있다.In the present invention, 'rot' is a condition in which the roots or underground stems of plants decompose or rot. When infected, the internal tissues of the roots are destroyed, turning brown and then rotting black. Damage is more severe in the fine roots, causing the roots to split and be easily pulled out. Chonma infected with rot disease shows round, grayish-brown spots, and over time, the spots on the tubers merge into larger black lesions. Fusarium oxysporum is known to be one of the main pathogens of horse rot disease, and suppressing the growth of this pathogen can reduce the occurrence of horse rot disease.
본 발명자들은 천마썩음병 원인 병원균인 푸사리움 옥시스포럼 균주의 생육을 저해하는 길항활성을 나타내는 균주를 탐색하던 중 천마에서 길항 균주 NIFoS_BW01를 분리하였다 (도 1 및 도 2).The present inventors isolated the antagonistic strain NIFoS_BW01 from Cheonma while searching for strains that exhibit antagonistic activity that inhibits the growth of Fusarium oxysporum strains, the causative pathogen of Cheonma rot disease (FIGS. 1 and 2).
본 발명에서 분리된 길항 균주인 NIFoS_BW01를 동정하여 균주의 분류상의 위치를 비교한 결과 바실러스 위드만니와 99% 일치성을 나타냈다. 이를 근거로 본 발명의 균주를 '바실러스 위드만니 NIFoS_BW01'로 명명하였다 (도 4 및 도 5).NIFoS_BW01, an antagonistic strain isolated in the present invention, was identified and its taxonomic position was compared, showing 99% identity with Bacillus withmanni. Based on this, the strain of the present invention was named 'Bacillus withmanni NIFoS_BW01' (Figures 4 and 5).
본 발명의 일 실시예에 따르면, 본 발명의 상기 균주는 푸사리움 옥시포럼의 생육을 억제하는 것을 확인하였다 (도 3). 이와 같은 결과는 본 발명에서 개발한 균주가 천마썩음병을 효과적으로 방제할 수 있음을 시사하는 것이다. 따라서, 본 발명의 바실러스 위드만니 균주는 천마썩음병 원인 병원균에 대하여 우수한 길항 활성을 갖고 있으며, 환경공해와 인체독성이 없어 환경 친화적인 천마썩음병 병원균 방제용 미생물로 이용될 수 있다.According to one embodiment of the present invention, the strain of the present invention was confirmed to inhibit the growth of Fusarium oxyphorum (FIG. 3). These results suggest that the strain developed in the present invention can effectively control horse rot disease. Therefore, the Bacillus withmani strain of the present invention has excellent antagonistic activity against the pathogen that causes horse rot disease, and can be used as an environmentally friendly microorganism for controlling horse rot disease pathogens as it is free from environmental pollution and is not toxic to humans.
본 발명에서 미생물 제제는 미생물의 안정적인 제제화를 목적으로, 수화제, 입제 또는 캡슐화의 형태로 제제될 수 있다.In the present invention, the microbial preparation may be formulated in the form of a wettable powder, granule or encapsulation for the purpose of stable preparation of microorganisms.
본 발명의 미생물 제제에서, 상기 균주 또는 배양액을 미생물 제제에 포함된 채로 공급될 수도 있고, 장기간 보존을 위해 별도로 보관하였다가 사용 직전에 혼합하여 사용할 수도 있다. 상기 균주를 장기간 보존하기 위해 별도로 공급되는 경우에는 글리세롤 저장용액에 -70℃ 이하로 보존하거나 -20 내지 -80℃에서 동결건조 보존하여 사용할 수 있다.In the microbial preparation of the present invention, the strain or culture medium may be supplied included in the microbial preparation, or may be stored separately for long-term preservation and mixed immediately before use. If the strain is supplied separately for long-term storage, it can be stored in a glycerol storage solution at -70°C or lower or freeze-dried and preserved at -20 to -80°C.
본 발명의 수화제는 미생물을 접종한 고체배지를 건조시켜 분쇄한 후 계면활성제 및 증량제/영양제를 첨가하여 혼합하여 제조할 수 있다. 상기에서 계면활성제로는 폴리카복실레이트, 소듐 리그노설포네이트, 칼슘 리그노설포네이트, 소듐 다이알킬 설포석시네이트, 소듐 알킬 아릴 설포네이트, 폴리옥시에틸렌 알킬 페닐 에테르, 소듐트리폴리포스페이트, 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 하나 또는 둘 이상을 사용하며, 증량제 및 영양제로는 콩가루, 쌀, 밀, 황토, 규조토, 덱스트린 (dextrin), 포도당 및 전분으로 이루어진 군으로부터 선택되는 하나 또는 둘 이상을 사용할 수 있다.The wettable powder of the present invention can be prepared by drying and pulverizing a solid medium inoculated with microorganisms, then adding a surfactant and an extender/nutrient and mixing them. In the above, the surfactants include polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, and polyoxyethylene. The group consisting of alkyl aryl 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. One or two or more selected from the group consisting of soybean flour, rice, wheat, red clay, diatomaceous earth, dextrin, glucose and starch may be used as the extender and nutrient.
또한, 본 발명의 입제는 미생물을 접종한 고체배지를 건조시켜 분쇄한 후 계면활성제, 증량제/영양제 및 붕해제를 첨가하여 제조할 수 있다. 상기에서 계면활성제로는 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 하나 또는 둘 이상을 사용하며, 증량제 및 영양제로는 콩가루, 쌀, 밀, 황토, 규조토, 덱스트린, 포도당 및 전분으로 이루어진 군으로부터 선택되는 하나 또는 둘 이상을 사용하며, 붕해제로는 벤토나이트 (bentonite), 탈크 (talc), 다이아라이트 (dialite), 카올린 (kaolin) 및 칼슘 카보네이트 (calcium carbonate)로 이루어진 군으로부터 선택되는 하나 또는 둘 이상을 사용할 수 있다.Additionally, the granules of the present invention can be prepared by drying and pulverizing a solid medium inoculated with microorganisms and then adding surfactants, extenders/nutrients, and disintegrants. In the above, the surfactants include polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene. The group consisting of alkyl aryl 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. One or two or more selected from the group consisting of soybean flour, rice, wheat, red clay, diatomaceous earth, dextrin, glucose and starch are used as extenders and nutrients, and bentonite is used as a disintegrant. One or two or more selected from the group consisting of bentonite, talc, dialite, kaolin, and calcium carbonate may be used.
또한, 본 발명의 입제는 미생물에 표면 활성제, 비활성 담체, 보존제, 습윤제, 공급촉진제, 유인제, 캡슐화제, 결합제, 유화제, 염료, UV 보호제, 완충제 및 흐름제로 이루어진 군으로부터 선택되는 하나 또는 둘 이상의 것을 추가로 첨가하여 제조할 수 있다.In addition, the granule of the present invention is one or more selected from the group consisting of surface active agents, inert carriers, preservatives, wetting agents, feeding accelerators, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protective agents, buffers and flow agents to microorganisms. It can be manufactured by additionally adding.
본 발명의 다른 목적에 따라서, 본 발명은 상기 미생물 제제를 포함하는 천마썩음병 방제용 조성물을 제공한다. According to another object of the present invention, the present invention provides a composition for controlling horse rot disease comprising the above microbial agent.
본 발명에서 조성물은 농약 조성물, 식물 영양제 조성물, 토양개량제 조성물 등으로 제품화될 수 있다. In the present invention, the composition can be commercialized as a pesticide composition, plant nutrient composition, soil conditioner composition, etc.
상기 미생물 제제 또는 조성물은 천마 또는 그 주위의 토양에 적용하여 썩음병 원인 병원균을 방제할 수 있고 썩음병의 발생을 저해시킬 수 있다. 미생물 제제 또는 조성물을 적용하는 방법으로는, 상기 미생물 제제 또는 조성물을 직접 도말 (spreading), 살포 (spraying), 침지(soaking)하거나, 상기 미생물 제제 또는 조성물을 질석, 규조토, 퍼라이트에 흡착시켜 산포하거나, 경작 예정지관리 시에는 토양을 갈아엎는 중에 균주 배양액 또는 흡착시킨 균주를 섞는 방법을 이용할 수 있다. 이 밖에도 알려진 농약 투여법, 미생물 적용법 등이 이용될 수 있다.The microbial preparation or composition can be applied to the ground rot or the soil around it to control pathogens that cause rot and can inhibit the development of rot. Methods of applying the microbial agent or composition include directly spreading, spraying, or soaking the microbial agent or composition, or adsorbing the microbial agent or composition onto vermiculite, diatomaceous earth, or perlite and spreading it. , When managing land scheduled for cultivation, a method of mixing strain culture medium or adsorbed strains while plowing the soil can be used. In addition, known pesticide administration methods and microorganism application methods can be used.
상기 미생물 제제 또는 조성물의 적용은 천마나 이에 근접한 토양에 바실러스 위드만니 균주를 집중하여 살포하고, 미생물의 유효량은 경작지 면적 (㎡) 당 미생물의 수가 1 X 107 내지 5 X 107 마리가 살포되는 것일 수 있으며, 바람직하게는 4 X 107 마리일 수 있다. 또한, 미생물의 적용은 미생물 제제 또는 조성물에 포함되는 미생물의 유효량이 ㎖ 당 1 X 106 내지 1 X 109 마리의 미생물 농도로 희석한 용액을 천마에 직접 살포하거나, ㎖ 당 1 X 106 내지 1 X 109마리의 미생물 농도로 희석한 용액에 천마를 1 내지 2시간 동안 침지시킬 수 있다. The application of the microbial preparation or composition is carried out by concentrating and spraying the Bacillus Weedmanni strain on Cheonma or soil close to it, and the effective amount of microorganisms is 1 It may be, preferably 4 In addition, the application of microorganisms is carried out by directly spraying a solution diluted to a concentration of 1 Thousand horses can be immersed in a solution diluted to a concentration of 1 x 10 9 microorganisms for 1 to 2 hours.
본 발명의 또 다른 목적은 상기 미생물 제제를 이용하여 푸사리움 옥시스포럼의 생육을 억제하는 방법을 제공한다. Another object of the present invention is to provide a method for inhibiting the growth of Fusarium oxysporum using the microbial agent.
본 발명의 또 다른 목적에 따라서 상기 미생물 제제 또는 상기 조성물을 이용하여 천마썩음병을 방제하는 방법을 제공한다. According to another object of the present invention, a method for controlling horse rot using the microbial agent or the composition is provided.
억제 또는 방제하는 방법은 상기 조성물을 천마 또는 이에 근접한 토양에 집중하여 살포하거나, 침지시켜서 병원균인 푸사리움 옥시스포럼의 생육을 억제하여 천마썩음병을 방제할 수 있으며, 구체적인 내용은 상기에서 설명한 바와 같다.The method of inhibition or control is to spray or immerse the composition concentrated in the soil close to the groundhog or the soil to suppress the growth of the pathogen Fusarium oxysporum, thereby controlling the sickle rot. The details are as described above. .
본 발명에 따른 미생물 제제 및 조성물은 천마썩음병의 원인 병원균인 푸사리움 옥시스포럼에 대한 길항 작용이 우수하여 천마썩음병을 효과적으로 방제할 수 있다. The microbial preparation and composition according to the present invention have excellent antagonistic action against Fusarium oxysporum, the causative pathogen of horse rot disease, and can effectively control horse rot disease.
또한 본 발명에 따른 미생물 제제 및 조성물은 천마 재배에 환경공해를 주지 않고 인체에 독성이 없는 친환경 천마썩음병 방제제 또는 농약의 안정적 공급으로 국내의 천마 재배 농가의 경영비용 절감, 환경오염 감소, 경쟁력 높은 고품질 천마 제품의 안정적 공급에 기여할 수 있다. In addition, the microbial preparation and composition according to the present invention do not cause environmental pollution to the cultivation of the wild horse, and provide a stable supply of eco-friendly green horse rot control agents or pesticides that are non-toxic to the human body, thereby reducing management costs for domestic horseradish cultivation farms, reducing environmental pollution, and increasing competitiveness. It can contribute to the stable supply of high-quality Cheonma products.
도 1은 본 발명에 따라 천마에서 분리된 푸사리움 옥시스포럼에 길항 균주인 바실러스 위드만니 NIFoS_BW01의 사진이다 (B5).
도 2는 후보 균주들의 푸사리움 옥시스포럼에 대한 길항활성 시험 결과를 보여주는 사진이다. 1 ~ 20 : 후보균주 번호, C : 대조구
도 3은 본 발명에 따른 바실러스 위드만니 NIFoS_BW01를 포함하는 미생물 제제의 푸사리움 옥시스포럼에 대한 방제 효과를 보여주는 사진이다.
도 4는 본 발명에 따른 바실러스 위드만니 NIFoS_BW01의 16s rRNA 유전자 염기서열을 나타낸다.
도 5는 본 발명의 바실러스 위드만니 NIFoS_BW01의 분류학상 위치를 나타내는 계통도이다. Figure 1 is a photograph of Bacillus withmanni NIFoS_BW01, an antagonistic strain against Fusarium oxysporum isolated from Cheonma according to the present invention (B5).
Figure 2 is a photograph showing the results of an antagonistic activity test of candidate strains against Fusarium oxysporum. 1 to 20: candidate strain number, C: control
Figure 3 is a photograph showing the control effect of a microbial preparation containing Bacillus withmani NIFoS_BW01 according to the present invention on Fusarium oxysporum.
Figure 4 shows the 16s rRNA gene base sequence of Bacillus withmani NIFoS_BW01 according to the present invention.
Figure 5 is a systematic diagram showing the taxonomic position of Bacillus withmanni NIFoS_BW01 of the present invention.
이하, 본 발명의 이해를 돕기 위하여 구체적인 실시예를 통하여 본 발명의 구성 및 효과를 보다 상세히 설명하기로 한다. 그러나 하기 실시예는 본 발명을 보다 명확하게 이해시키기 위하여 예시한 것일 뿐이며, 본 발명의 권리범위가 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, in order to facilitate understanding of the present invention, the configuration and effects of the present invention will be described in more detail through specific examples. However, the following examples are merely illustrative for a clearer understanding of the present invention, and the scope of the present invention is not limited by the following examples.
실시예 1: 길항 균주 후보 탐색Example 1: Search for antagonistic strain candidates
천마썩음병 병원균인 푸사리움 옥시스포럼의 생육을 억제하는 천마 내생균인 길항 미생물을 탐색하기 위하여, 천마썩음병변을 가진 총 50개의 천마를 수집하여 썩음병 병변에서도 생육할 수 있는 길항 미생물을 탐색하였다. In order to search for antagonistic microorganisms, which are endophytic bacteria that inhibit the growth of Fusarium oxysporum, the pathogen of Fusarium oxysporum, a total of 50 horse rot lesions were collected and antagonistic microorganisms that could grow in rot disease lesions were searched.
구체적으로는 천마 표면의 갈색의 병반과 표면이 변형된 부분을 선발하여 0.1% NaOCl에 3분 동안 침지 후 70% 에탄올에 2분 침지 후 멸균수로 2~3회 헹구고 충분히 건조시켜 수분을 제거하여 표면소독을 해 주었다. 병반 조직을 절단하여 조각을 얻고 이것을 각각 Tryptic Soy Agar(TSA) 배지에 치상하여 자라나는 균을 루프와 니들을 이용하여 분리하였다. 1차로 세균 63종이 분리되었다. 분리된 균주들은 20% 글리세롤에 현탁하고 -80℃ 초저온냉동고(deep freezer)에 보관하면서 추가 시험을 하였다.Specifically, select brown lesions and surface deformation areas on the surface of Cheonma, immerse them in 0.1% NaOCl for 3 minutes, then immerse them in 70% ethanol for 2 minutes, rinse 2-3 times with sterilized water, and dry sufficiently to remove moisture. The surface was disinfected. The lesion tissue was cut to obtain pieces, each piece was placed on Tryptic Soy Agar (TSA) medium, and the growing bacteria were separated using a loop and needle. Initially, 63 species of bacteria were isolated. The isolated strains were suspended in 20% glycerol and stored in a deep freezer at -80°C for further testing.
분리된 63종의 균주를 콜로니의 모양, 색, 중심부의 형태, 주변부의 형태 등 형태학적으로 다른 특성을 가진 세균 20개의 후보 균주들를 2차로 분리하였다. 그 결과를 도 1에 도시하였다. From the 63 isolated strains, 20 candidate strains of bacteria with morphologically different characteristics, such as colony shape, color, center shape, and peripheral shape, were separated secondarily. The results are shown in Figure 1.
실시예 2: 푸사리움 옥시스포럼 생육 억제 시험 및 길항 균주 최종 선발Example 2: Fusarium oxysporum growth inhibition test and final selection of antagonistic strains
천마썩음병 병원균인 푸사리움 옥시스포럼은 국립산림과학원 산림생명공학연구과에서 보관중이였던 균주를 사용하였다. For Fusarium oxysporum, the pathogen of horse rot disease, a strain stored in the Forest Biotechnology Research Department of the National Institute of Forest Science was used.
천마썩음병 병원균인 푸사리움 옥시스포럼에 대하여 실시예 1에서 분리된 후보 균주들의 항균활성을 갖는지 알아보기 위하여 대치배양을 실시하였다. 후보 균주들은 Tryptic Soy Broth(TSB) 배지에 28℃, 160rpm으로 24시간 진탕 배양하였다. Replacement cultures were performed to determine whether the candidate strains isolated in Example 1 had antibacterial activity against Fusarium oxysporum, a horse rot disease pathogen. Candidate strains were cultured in Tryptic Soy Broth (TSB) medium at 28°C with shaking at 160 rpm for 24 hours.
푸사리움 옥시스포럼은 Potato Dextrose Agar(PDA)배지에 25℃, 7일간 암조건으로 배양하였고, 배양된 병원균의 균사 끝 부분을 5 mm 직경의 코크 보러(cork borer)로 아가 플러그(agar plug)를 만들어 실험에 이용하였다. 대치배양을 위하여 Tryptic Soy Agar(TSA)와 Potato Dextrose Agar(PDA)를 1:1 혼합한 배지를 만들고 5 mm 직경의 병원균 아가 플러그를 배지 한 가운데에 접종하였다. 그리고 주변 3곳에 5 mm 직경의 코크 보러로 구멍을 내었다. 이후에 배양한 후보 균주는 O.D값을 1.0으로 조정한 후 20 ㎕씩 구멍에 접종하였다. 병원균과 후보 균주가 접종된 TSA와 PDA의 1:1 혼합 배지는 25℃ 배양기에서 7일 동안 암조건으로 배양하였고, 그 결과를 표 1 (-: 길항성 없음, +:길항성 약, ++: 길항성 중, +++: 길항성 강) 및 도 2에 나타냈다: Fusarium oxysporum was cultured on Potato Dextrose Agar (PDA) medium at 25°C in the dark for 7 days, and the end of the hyphae of the cultured pathogen was plugged with an agar plug using a cork borer with a diameter of 5 mm. was created and used in the experiment. For replacement culture, a 1:1 mixture of Tryptic Soy Agar (TSA) and Potato Dextrose Agar (PDA) was prepared, and a 5 mm diameter pathogen agar plug was inoculated in the center of the medium. Then, holes were made with 5 mm diameter cork bores at three surrounding locations. Afterwards, the cultured candidate strains were inoculated into each hole at 20 ㎕ after adjusting the O.D. value to 1.0. A 1:1 mixed medium of TSA and PDA inoculated with pathogens and candidate strains was cultured under dark conditions in an incubator at 25°C for 7 days, and the results are shown in Table 1 (-: no antagonist, +: antagonist drug, ++ : antagonistic, +++: antagonistic) and shown in Figure 2:
표 1 및 도 2에 나타낸 바와 같이, 후보 균주들 중 푸사리움 옥시스포럼에 강한 길항활성을 보이는 균주 NIFoS_BW01 (B5, 도 2에서 5번)을 확인하였다. As shown in Table 1 and Figure 2, among the candidate strains, strain NIFoS_BW01 (B5, number 5 in Figure 2) showing strong antagonistic activity against Fusarium oxysporum was identified.
선발된 균주 NIFoS_BW01를, 상기와 동일한 조건에서 배양된 푸사리움 옥시스포럼과 선발된 균주를 PDA 배지에서 대치배양을 실시하였다. 25℃ 배양기에서 7일 동안 암조건으로 배양하였고 이 후 억제된 균사의 길이를 측정하였다. 억제율(inhibition rate)은 다음 식에 따라 계산하였으며, 균사생장 억제율은 40.9%로 확인되었다. 배양 결과 사진은 도 3에 나타냈다: The selected strain NIFoS_BW01 was replaced with Fusarium oxysporum cultured under the same conditions as above and the selected strain was cultured in PDA medium. The cells were cultured in a 25°C incubator under dark conditions for 7 days, and then the length of the inhibited hyphae was measured. The inhibition rate was calculated according to the following equation, and the mycelial growth inhibition rate was confirmed to be 40.9%. A picture of the culture results is shown in Figure 3:
(상기 식에서, R은 박테리아 콜로니로부터 떨어진 곰팡이 콜로니의 최대 반경이고, r은 박테리아 콜로니 반대편에 있는 곰팡이 콜로니의 반경임)(In the above equation, R is the maximum radius of the fungal colony away from the bacterial colony, and r is the radius of the fungal colony on the other side of the bacterial colony)
도 3에 도시된 바와 같이, 선발된 NIFoS_BW01 균주에 의해 푸사리움 옥시스포럼의 균사 생장이 현저하게 억제됨을 확인할 수 있다. As shown in Figure 3, it can be confirmed that the mycelial growth of Fusarium oxysporum is significantly inhibited by the selected NIFoS_BW01 strain.
실시예 3: 선발된 길항 균주의 동정Example 3: Identification of selected antagonistic strains
실시예 2에서 선발된 균주 NIFoS_BW01를 동정하기 위하여 Macrogen(서울, 한국)에 의뢰하여 NIFoS_BW01에 16s rRNA 유전자 염기서열을 분석하였다. 분석된 16s rRNA의 유전자 염기서열은 도 4 및 서열번호 1에 나타냈다.To identify the strain NIFoS_BW01 selected in Example 2, Macrogen (Seoul, Korea) was contacted and the 16s rRNA gene base sequence of NIFoS_BW01 was analyzed. The gene sequence of the analyzed 16s rRNA is shown in Figure 4 and SEQ ID NO: 1.
상기 균주의 분류상의 위치를 확인하기 위하여 가장 많이 이용되는 미생물의 16s rRNA 유전자 염기서열 비교 데이터를 근거로 NIFoS_BW01 균주의 16s rRNA 유전자 염기서열은 바실러스 위드만니 16s rRNA 유전자 염기서열과 99% 일치성을 나타낸다는 것을 확인할 수 있었다 (도 5, 표 2). Based on the comparative data of the 16s rRNA gene base sequence of the most commonly used microorganisms to confirm the taxonomic position of the above strain, the 16s rRNA gene base sequence of strain NIFoS_BW01 has 99% identity with the 16s rRNA gene base sequence of Bacillus Widmanni. It was confirmed that (Figure 5, Table 2).
상기와 같은 균주 동정 결과를 기초하여, 상기 NIFoS_BW01 균주를 바실러스 위드만니로 분류하였고, '바실러스 위드만니 NIFoS_BW01'로 명명하였다. Based on the above strain identification results, the NIFoS_BW01 strain was classified as Bacillus withmanni and named 'Bacillus withmanni NIFoS_BW01'.
<110> National Institute of Forest Science <120> A microbial agent comprising Bacillus wiedmannii having antagonistic activity against Fusarium oxysporum and a composition for preventing Gastrodia elata rot comprising it <130> P-10445 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1497 <212> DNA <213> Bacillus wiedmannii <220> <221> rRNA <222> (1)..(1497) <223> 16s rRNA gene <400> 1 ttcgtcagga tgaacgctgg cggcgtgcct aatacatgca agtcgagcga atggattaag 60 agcttgctct tatgaagtta gcggcggacg ggtgagtaac acgtgggtaa cctgcccata 120 agactgggat aactccggga aaccggggct aataccggat aacattttga actgcatggt 180 tcgaaattga aaggcggctt cggctgtcac ttatggatgg acccgcgtcg cattagctag 240 ttggtgaggt aacggctcac caaggcaacg atgcgtagcc gacctgagag ggtgatcggc 300 cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg 360 caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggcttt cgggtcgtaa 420 aactctgttg ttagggaaga acaagtgcta gttgaataag ctggcacctt gacggtacct 480 aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg 540 ttatccggaa ttattgggcg taaagcgcgc gcaggtggtt tcttaagtct gatgtgaaag 600 cccacggctc aaccgtggag ggtcattgga aactgggaga cttgagtgca gaagaggaaa 660 gtggaattcc atgtgtagcg gtgaaatgcg tagagatatg gaggaacacc agtggcgaag 720 gcgactttct ggtctgtaac tgacactgag gcgcgaaagc gtggggagca aacaggatta 780 gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttagaggg tttccgccct 840 ttagtgctga agttaacgca ttaagcactc cgcctgggga gtacggccgc aaggctgaaa 900 ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa 960 cgcgaagaac cttaccaggt cttgacatcc tctgaaaacc ctagagatag ggcttctcct 1020 tcgggagcag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg 1080 ttaagtcccg caacgagcgc aacccttgat cttagttgcc atcattaagt tgggcactct 1140 aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc 1200 ttatgacctg ggctacacac gtgctacaat ggacggtaca aagagctgca agaccgcgag 1260 gtggagctaa tctcataaaa ccgttctcag ttcggattgt aggctgcaac tcgcctacat 1320 gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct 1380 tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt ggggtaacct 1440 ttttggagcc agccgcctaa ggtgggacag atgattgggg tgaagtctaa aaggggg 1497 <110> National Institute of Forest Science <120> A microbial agent comprising Bacillus wiedmannii having antagonistic activity against Fusarium oxysporum and a composition for preventing Gastrodia elata rot comprising it <130> P-10445 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1497 <212> DNA <213> Bacillus wiedmannii <220> <221>rRNA <222> (1)..(1497) <223> 16s rRNA gene <400> 1 ttcgtcagga tgaacgctgg cggcgtgcct aatacatgca agtcgagcga atggattaag 60 agcttgctct tatgaagtta gcggcggacg ggtgagtaac acgtgggtaa cctgcccata 120 agactgggat aactccggga aaccggggct aataccggat aacattttga actgcatggt 180 tcgaaattga aaggcggctt cggctgtcac ttatggatgg acccgcgtcg cattagctag 240 ttggtgaggt aacggctcac caaggcaacg atgcgtagcc gacctgagag ggtgatcggc 300 cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg 360 caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggcttt cgggtcgtaa 420 aactctgttg ttagggaaga acaagtgcta gttgaataag ctggcacctt gacggtacct 480 aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg 540 ttatccggaa ttaattgggcg taaagcgcgc gcaggtggtt tcttaagtct gatgtgaaag 600 cccacggctc aaccgtggag ggtcattgga aactgggaga cttgagtgca gaagaggaaa 660 gtggaattcc atgtgtagcg gtgaaatgcg tagagatatg gaggaacacc agtggcgaag 720 gcgactttct ggtctgtaac tgacactgag gcgcgaaagc gtggggagca aacaggatta 780 gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttagaggg tttccgccct 840 ttagtgctga agttaacgca ttaagcactc cgcctgggga gtacggccgc aaggctgaaa 900 ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa 960 cgcgaagaac cttaccaggt cttgacatcc tctgaaaacc ctagagatag ggcttctcct 1020 tcgggagcag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg 1080 ttaagtcccg caacgagcgc aacccttgat cttagttgcc atcattaagt tgggcactct 1140 aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc 1200 ttatgacctg ggctacacac gtgctacaat ggacggtaca aagagctgca agaccgcgag 1260 gtggagctaa tctcataaaa ccgttctcag ttcggattgt aggctgcaac tcgcctacat 1320 gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct 1380 tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt ggggtaacct 1440 ttttggagcc agccgcctaa ggtgggacag atgattgggg tgaagtctaa aaggggg 1497
Claims (9)
상기 균주는 천마 내생균이고,
상기 제제는 푸사리움 옥시스포럼의 균사생장 억제율이 40.9%인 것인, 미생물 제제.
A microbial preparation for controlling Fusarium oxysporum containing Bacillus wiedmannii strain or its culture medium as an active ingredient,
The strain is an endophyte of Cheonma,
The preparation is a microbial preparation having a mycelial growth inhibition rate of Fusarium oxysporum of 40.9%.
The microbial preparation for controlling Fusarium oxysporum according to claim 1, wherein the microbial preparation is in any form selected from the group consisting of wettable powders, granules, and capsules.
The microbial preparation for controlling Fusarium oxysporum according to claim 1, wherein the microbial preparation is prepared by additionally adding at least one selected from the group consisting of surfactants, weighting agents, and nutrients.
The microbial agent for controlling Fusarium oxysporum according to claim 1, wherein the microbial agent is applied by any method selected from the group consisting of direct spreading, spraying, and soaking. .
A method of inhibiting the growth of Fusarium oxysporum using the microbial agent according to claim 1.
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