KR101996530B1 - Bacillus amyloliquefaciens nbc241 and composition comprising the same for control of insect pathogens - Google Patents
Bacillus amyloliquefaciens nbc241 and composition comprising the same for control of insect pathogens Download PDFInfo
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Abstract
본 발명은 바실러스 아밀로리퀴에파션스 NBC241(Bacillus amyloliquefaciens NBC241, 수탁번호 : KCTC13359BP) 균주에 관한 것이다. 보다 구체적으로, 곤충 사육시 발생하는 진균성 병원균에 대한 방제효능을 보유한 바실러스 아밀로리퀴에파션스 NBC241 균주 및 상기 균주의 배양액을 유효성분으로 포함하는 조성물에 관한 것이다. 특히, 꽃무지류 유충의 녹강병 및 백강병에 대한 방제효능을 보유하고 있어, 꽃무지류의 사육시 발생하는 병해를 방제하는데 유용하게 이용할 수 있다.The present invention relates to a strain of Bacillus amyloliquefaciens NBC241 (accession number: KCTC13359BP). More specifically, the present invention relates to a Bacillus amyloliquefaciens NBC241 strain having an antifungal activity against fungal pathogens generated when an insect is raised, and a composition comprising the culture medium of the strain as an active ingredient. In particular, it has an antiseptic effect against the rusty red mildew and the back sting disease of the flowering larva, and can be effectively used for controlling the disease caused by the breeding of the flowering mildew.
Description
본 발명은 바실러스 아밀로리퀴에파션스 NBC241(Bacillus amyloliquefaciens NBC241, 수탁번호 : KCTC13359BP) 균주에 관한 것이다. The present invention relates to a strain of Bacillus amyloliquefaciens NBC241 (accession number: KCTC13359BP).
보다 구체적으로, 곤충에 병을 일으키는 진균성 병원균에 대한 방제효능을 보유한 바실러스 아밀로리퀴에파션스 NBC241 균주 및 상기 균주를 유효성분으로 포함하는 조성물에 관한 것이다. More specifically, the present invention relates to a strain of Bacillus amyloliquefaciens NBC241 having an antifungal activity against fungal pathogens causing diseases in insects, and a composition comprising the strain as an active ingredient.
최근 유용 곤충에 대한 일반적인 관심이 커지고 있으나 대부분 야생 자원의 인공 채집에 의존되어 그 양과 질이 보장되지 못하고 야생 자원도 점차 고갈되어 가고 있는 추세이다. In recent years, there has been a growing interest in useful insects, but most of them are dependent on artificial harvesting of wild resources and their quantity and quality are not guaranteed and wild resources are gradually getting exhausted.
국내 곤충시장의 규모는 2015년 현재 약 3,000억 규모로 2020년 7,000억 규모로 성장할 전망이며 세계곤충시장은 2007년 이미 11조를 넘어섰으며 2020년 38조원으로 성장할 것으로 예상된다.The size of the domestic insect market is expected to grow to about 700 billion by 2020 by 2015 and the world insect market has already surpassed 11 trillion by 2007 and is expected to grow to 38 trillion won by 2020.
또한, 곤충은 천적을 이용한 생물농약(무당벌레, 풀잠자리, 응애 등), 환경정화(쇠똥구리, 연두금파리 등), 약용(누에, 꽃무지 등), 애완(나비류, 사슴벌레, 수서곤충 등), 환경지표 등으로 이용될 뿐 아니라 전세계적으로 미래 식량난에 대비한 식용으로서의 연구도 활발히 진행중이다. In addition, insects can be used as pesticides (ladybugs, rhododendrons, mites, etc.), environmental cleanups (such as dung beetles, , Environmental indicators, etc., as well as research on edible foods for the future food shortage in the world.
산업곤충 시장이 확대되면서 유용곤충의 가치가 향상되었고, 유용곤충 대량사육장이 증가하게 됨에 따라 곤충병원미생물에 의한 피해 또한 증가 추세이나 뚜렷한 해결책은 미흡한 실정이다. 또한, 곤충을 감염시켜 질병을 일으키는 병원균은 진균, 세균, 바이러스 등 다양하나, 병원균에 대한 연구는 매우 미흡한 실정이다. As the industrial insect market expanded, the value of useful insects was improved and the damage caused by insecticide microorganisms also increased as the use of useful insect mass hatcheries increased. However, there are few clear solutions. In addition, pathogens causing diseases by infecting insects include fungi, bacteria, and viruses, but studies on pathogens are very inadequate.
한편, 꽃무지류 유충은 일반적으로 ‘굼벵이’라 불리며 간기능 보호와 중품 등에 좋은 것으로 알려져 있으며, 최근 대학들을 중심으로 꽃무지류 유충에 대한 약리 작용을 연구하고 있다. 곤충의 식용으로의 연구가 활발해 지고 있는 만큼 식용 곤충 사육에 있어 화학 농약의 사용을 제한하여, 섭취시 고영양원으로서 활용할 수 있도록 하는 연구가 진행중이다. On the other hand, the flowering larvae are generally called 'slugs' and are known to be good for protecting liver function and middle products. Recently, they have been studying the pharmacological effects on flowering larvae mainly in the universities. As research on edible insects is becoming more active, studies are underway to limit the use of chemical pesticides in feeding insects and to utilize them as a nutrient source for ingestion.
특히 흰점박이꽃무지 유충은 영양적으로도 고단백 식품으로, 수분제외 시100g당 단백질 함량이 58g에 이른다. 또한, 지난 2014년 8월 30일 식품의약품안전처로부터 한시적 식품원료로 인정받았다. 식약처로부터 한시적 식품원료로 인정받으면 승인받은 형태와 제품으로 식품 판매가 가능하며, 일정기간 문제가 없을 경우 식품 원료로 공식 등록이 가능하다.In particular, white spotted moth larvae are nutritionally high - protein foods, and have a protein content of 100g to 58g excluding moisture. In addition, on August 30, 2014, it was recognized as a temporary food ingredient by the Food and Drug Administration. If it is recognized as a temporary food ingredient from the food service, food can be sold with the approved form and product. If there is no problem for a certain period, it can be officially registered as food ingredient.
이에 본 출원인은 곤충에 병을 일으키는 병원균의 방제효과가 있는 균주를 동정하여 특허기탁이 완료된 상태(KCTC 13359BP)이며, 흰점박이꽃무지 유충사육시 발생하는 녹강병 및 백강병의 방제효과가 있는 조성물을 완성하였다. Accordingly, the present applicant identifies a strain having an effect of controlling a pathogenic bacterium that causes disease in insects and has a patent deposited state (KCTC 13359BP), and is a composition having a control effect against rust mite disease and back disease caused by white spotted moth larvae Completed.
이하, 선행기술을 살펴보면 대한민국 등록특허공보 제10-1189104호에는 신규한 바실러스 아밀로리퀴에파션스 JBC36 균주 및 이를 유효성분으로 포함하는 식물 병원균 방제용 조성물에 관해 기재되어 있다. 개략적으로 살펴보면, 과일류의 녹색곰팡이병과 푸른곰팡이병에 대해 방제효과가 뛰어나기 때문에 감귤과 배, 사과 등의 저장 및 유통 중에 발생하는 여러 가지 병해를 방제하는데 이용할 수 있는 것이다. Hereinafter, in reference to the prior art, Korean Patent Registration No. 10-1189104 discloses a novel Bacillus amyloliquefaciens strain JBC36 and a composition for controlling a plant pathogenic bacterium containing the same as an effective ingredient. In summary, it can be used to control various diseases occurring during storage and distribution of citrus fruits, pears, apples and the like because it has excellent control effect against green fungus disease and blue mold disease of fruits.
다음으로 대한민국 등록특허공보 제10-0838103호에는 신규한 바실러스 아밀로리퀴페션스 씨씨178 및 이를 이용한 잿빛곰팡이병 방제에 관해 기재되어 있다. 개략적으로 살펴보면, 채소재배 중 잿빛곰팡이병의 발생을 억제 효과가 뛰어나 식물 병원균에 대한 생물농약 기능을 할 수 있되, 잔류농약으로 인한 문제점을 방지할 수 있는 것이다. Korean Patent Publication No. 10-0838103 discloses a novel Bacillus amyloliquefaciens seed 178 and a method for controlling gray mold disease using the same. In summary, the excellent effect of inhibiting the occurrence of gray mold disease in vegetable cultivation can prevent the problems caused by the residual pesticide, while it can function as a pesticide for plant pathogens.
상기 선행기술들은 바실러스 아밀로리퀴에파션스(Bacillus amyloliquefaciens)를 이용한 식물 병원균 방제용 조성물에 관한 것으로, 곤충에 병을 일으키는 병원균 방제에 효과적인 바실러스 아밀로리퀴에파션스 NBC241에 대해서는 기재되어 있지 않다. The above prior art relates to a composition for controlling plant pathogens using Bacillus amyloliquefaciens, and does not disclose Bacillus amyloliquefaciens NBC241 which is effective for controlling pathogens causing diseases in insects.
다음으로 대한민국 등록특허공보 제10-1524557호에는 바실러스 아밀로리퀘페이션스 EML-CAP3를 포함하는 살충 조성물에 대하여 기재되어 있다. 간략히 살펴보면 복숭아혹진딧물의 방제용 조성물로 바실러스 아밀로리퀘페이션스 EML-CAP3를 살포 또는 도포하는 단계를 포함하는 곤충 방제 방법에 관한 것이다. Next, Korean Patent Publication No. 10-1524557 discloses a insecticidal composition comprising Bacillus amyloliquefaciens EML-CAP3. The present invention relates to a method for controlling insects including a step of spraying or applying Bacillus amyloliquefaciens EML-CAP3 as a composition for controlling peach aphid.
그러나 곤충을 감염시켜 굳게 만드는 녹강병 및 허옇게 말라죽게 하는 백강병을 방제할 수 있는 방법에 대해서는 기재되어 있지 않다. However, it does not disclose how to control rusty mildew that infects insects and hardens them, and whitish rotten mildew.
본 발명의 기술적 과제는 곤충 사육시 발생하는 진균성 병원균에 대한 바실러스 아밀로리퀴에파션스 NBC241(Bacillus amyloliquefaciens NBC241)균주의 방제효과를 확인하는 것이다. The technical problem of the present invention is to confirm the control effect of Bacillus amyloliquefaciens NBC241 strain against fungal pathogens generated when insects are raised.
또한, 상기 균주의 녹강병 및 백강병 방제효과를 확인하는 것이다. In addition, the effect of controlling the rust and redness of the strain is confirmed.
또한, 상기 균주의 배양액을 유효성분으로 포함하는 조성물에 대한 백강병균 및 녹강병균의 균사신장 억제 효과를 확인하는 것이다. In addition, the mycelial growth inhibitory effect of white algae and green algae on the composition containing the culture medium of the strain as an active ingredient is confirmed.
본 발명은 곤충에 병을 일으키는 병원균에 방제효능을 보유한 바실러스 아밀로리퀴에파션스 NBC241 (수탁번호 : KCTC13359BP) 균주를 제공하여 기술적 과제를 해결하고자 한다. The present invention provides a strain of Bacillus amyloliquefaciens NBC241 (accession number: KCTC13359BP), which has an antifungal activity against pathogenic bacteria causing insects, thereby solving the technical problem.
본 발명에서 상기 곤충은 꽃무지류, 풍뎅이과 및 딱정벌레목으로 이루어진 군으로부터 선택된 1종인 것을 특징으로 한다. In the present invention, the insect is characterized by being selected from the group consisting of a flowering plant, a chafer and a beetle tree.
본 발명에서 상기 꽃무지류 군의 곤충은 흰점박이꽃무지인 것을 특징으로 한다.In the present invention, the insects of the non-flowering group are characterized by being white spotted flowers.
본 발명에서 상기 병원균은 Metarhizium anisopliae 및 Beauveria bassiana 으로 이루어진 군으로부터 선택되는 1종 이상인 것을 특징으로 한다.In the present invention, the pathogen is Metarhizium anisopliae, and Beauveria bassiana .
본 발명은 바실러스 아밀로리퀴에파션스 NBC241 (수탁번호 : KCTC13359BP) 균주 또는 상기 균주의 배양액을 유효성분으로 포함하는, 곤충에 병을 일으키는 병원균 방제용 조성물을 제공하여 기술적 과제를 해결하고자 한다. The present invention aims to solve the technical problem by providing a composition for inhibiting pathogens causing diseases in insects, comprising a strain of Bacillus amyloliquefaciens NBC241 (Accession No .: KCTC13359BP) or a culture solution of the strain as an active ingredient.
본 발명에서 상기 조성물에서 배양액을 유효성분으로 포함하는 경우, 상기 조성물은 바실러스 아밀로리퀴에파션스 NBC241 균주를 LB배지에서 48시간 동안 배양하여 배양액을 제조하는 단계; 상기 제조된 배양액을 15000 rpm에서 20분간 원심분리를 실시하여 바실러스 아밀로리퀴에파션스 NBC241 균체를 제거하여 배양 상등액을 얻는 단계; 상기 배양 상등액을 0.22㎛ 멤브레인 필터로 필터하여 남아있는 균체를 완전히 제거하는 단계를 포함하여 제조되는 것을 특징으로 한다. In the present invention, when the composition contains the culture medium as an active ingredient, the composition may be prepared by culturing the strain of Bacillus amyloliquefaciens NBC241 in LB medium for 48 hours to prepare a culture solution; Centrifuging the prepared culture solution at 15000 rpm for 20 minutes to remove Bacillus amyloliquefaciens NBC241 cells to obtain a culture supernatant; And filtering the supernatant of the culture with a 0.22 mu m membrane filter to completely remove the remaining microbial cells.
본 발명에서 상기 병원균은 Metarhizium anisopliae 및 Beauveria bassiana 으로 이루어진 군으로부터 선택되는 1종 이상인 것을 특징으로 한다. In the present invention, the pathogen is Metarhizium anisopliae, and Beauveria bassiana .
본 발명의 바실러스 아밀로리퀴에파션스 NBC241 균주는 곤충의 사육시 발생하는 진균성 병원균에 대하여 방제효과가 있다. 보다 구체적으로, 흰점박이 꽃무지의 사육시 발생하는 녹강병과 백강병에 대한 방제효과가 있다. The Bacillus amyloliquefaciens strain NBC241 of the present invention has an antifungal effect against fungal pathogens generated when insects are raised. More specifically, there is a control effect against rust and rash that occurs when white spotted flowers are raised.
또한, 본 발명의 바실러스 아밀로리퀴에파션스 NBC241 균주 또는 상기 균주의 배양액을 유효성분으로 포함하는 조성물은 곤충 사육시 발생하는 진균성 병원균에 대하여 방제효과가 있다. In addition, the composition comprising the Bacillus amyloliquefaciens NBC241 strain of the present invention or a culture solution of the strain as an active ingredient has a controlling effect against fungal pathogens generated during insect rearing.
또한, 본 발명의 바실러스 아밀로리퀴에파션스 NBC241 균주의 배양액을 유효성분으로 포함하는 조성물은 백강병균과 녹강병균의 균사신장 억제 효과가 있다. In addition, the composition comprising the culture medium of the Bacillus amyloliquefaciens NBC241 strain of the present invention as an active ingredient has an effect of inhibiting mycelial growth of white algae and rusty bacterium.
도 1은 본 발명의 바실러스 아밀로리퀴에파션스 NBC241에 대한 16s rDNA 염기서열 분석 및 동정결과를 계통학적으로 나타낸 도면이다.
도 2는 본 발명의 바실러스 아밀로리퀴에파션스 NBC241의 녹강병균(Metarhizium anisopliae)과 백강병균(Beauveria bassiana)에 대한 길항 효과를 보여주는 사진이다.
도 3은 본 발명의 바실러스 아밀로리퀴에파션스 NBC241 배양액의 녹강병균(Metarhizium anisopliae)과 백강병균(Beauveria bassiana)에 대한 항균활성 효과를 보여주는 사진이다. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a phylogenetic diagram of 16s rDNA sequence analysis and identification of Bacillus amyloliquefaciens NBC241 of the present invention.
FIG. 2 is a photograph showing the antagonistic effect of Bacillus amyloliquefaciens NBC241 of the present invention against Metarhizium anisopliae and Beauveria bassiana .
3 is a photograph showing the antimicrobial activity effective for rust gangbyeonggyun of wave syeonseu NBC241 culture (Metarhizium anisopliae) and back gangbyeonggyun (Beauveria bassiana) in Bacillus amyl Lowry quinone of the present invention.
본 명세서 및 청구 범위에 사용된 용어나 단어는 통상적이거나 사전적인 의미로 한정해서 해석되어서는 안되며, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다.The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary meanings and the inventor can properly define the concept of the term to describe its invention in the best possible way And should be construed in accordance with the principles and meanings and concepts consistent with the technical idea of the present invention.
따라서 본 명세서에 기재된 실시예, 참조예 및 도면에 기술된 사항은 본 발명의 가장 바람직한 일 예에 불과할 뿐이고 본 발명의 기술적 사상을 모두 대변하는 것은 아니므로, 본 출원시점에 있어서 이들을 대체할 수 있는 다양한 균등물과 변형예들이 있을 수 있음을 이해하여야 한다.Therefore, the embodiments described in the present specification, the reference examples, and the drawings are merely the most preferred examples of the present invention, and not all of the technical ideas of the present invention are described. Therefore, It should be understood that various equivalents and modifications may be present.
본 발명은 곤충에 병을 일으키는 병원균에 방제효과를 나타내는 바실러스 아밀로리퀴에파션스 NBC241 (Bacillus amyloliquefaciens NBC241L, 수탁번호 :KCTC13359BP)를 제공한다. The present invention provides Bacillus amyloliquefaciens NBC241L (accession number: KCTC13359BP), which has a controlling effect on pathogenic bacteria causing insect diseases.
상기 바실러스 바실러스 아밀로리퀴에파션스 NBC241 (Bacillus amyloliquefaciens NBC241L, 수탁번호 :KCTC13359BP) 균주는 특히 흰점박이꽃무지의 사육시 발생하는 녹강병과 백강병에 대하여 탁월한 길항효과를 보이는 것으로 분리 및 동정된 신규한 균주로서 2017. 10. 11. 자로 KCTC13359BP의 수탁번호를 부여받았다. The strain Bacillus amyloliquefaciens NBC241 (Accession No .: KCTC13359BP) is a novel strain isolated and identified as having excellent antagonistic effect against rust and red stomach diseases occurring during the rearing of white spotted flowers As a strain, we received the accession number of KCTC13359BP on October 10, 2017.
상기 균주는 녹강병균인 Metarhizium anisopliae, 백강병균인 Beauveria bassiana 뿐만 아니라 식물병원균인 Penicillium digitatum, Botrytis cinerea, Fusarium oxysporum 이 있으나 이에 한정되는 것은 아니다. The strain was transformed with Metarhizium anisopliae, Beauveria bassiana as well as the plant pathogen Penicillium digitatum , Botrytis cinerea , and Fusarium oxysporum .
본 발명에 따른 바실러스 아밀로리퀴에파션스 NBC241 (수탁번호 :KCTC13359BP)은 흰점박이꽃무지의 사육시 발생하는 진균성 병원균인 녹강병과 백강병에 탁월한 길항효과 및 방제효과를 가지고 있으며, 이는 생물농약의 미생물 제제로 유용하게 사용될 수 있다. Bacillus amyloliquefaciens NBC241 (accession number: KCTC13359BP) according to the present invention has an excellent antagonistic and antifungal effect on fungicidal pathogens such as rust, As a microorganism preparation.
본 발명은 또한 바실러스 아밀로리퀴에파션스 NBC241 (수탁번호 :KCTC13359BP) 균주의 배양액으로부터 분리하여 얻은 곤충 병원균 길항물질을 제공한다. The present invention also provides an insect pathogenic antagonist obtained by isolating from a culture medium of a strain of Bacillus amyloliquefaciens NBC241 (Accession No .: KCTC13359BP).
상기 길항물질은 바실러스 아밀로리퀴에파션스 NBC241 (수탁번호 :KCTC13359BP) 균주를 배양하여 배양액을 형성하는 단계; 상기 배양액으로부터 본 균주를 제거하는단계; 및 균주가 제거된 배양액에 추출용매를 처리하여 단백질을 추출하는 단계를 통하여 얻어질 수 있다. 상기 추출용매는 부탄올을 사용한다. The antagonistic substance may be obtained by culturing a strain of Bacillus amyloliquefaciens NBC241 (Accession No: KCTC13359BP) to form a culture solution; Removing the strain from the culture solution; And extracting the protein by treating the culture medium from which the strain has been removed with an extraction solvent. Butanol is used as the extraction solvent.
이하, 본 발명의 바실러스 아밀로리퀴에파션스 NBC241 (수탁번호 :KCTC13359BP) 균주가 방제효과를 보이는 녹강병과 백강병에 대해 간략히 살펴본다.Bacillus amyloliquefaciens NBC241 (accession number: KCTC13359BP) according to the present invention will be briefly described as a rust-inhibiting agent and a potent anticancer agent showing a controlling effect.
녹강병은 누에 굳음 병의 하나로, 녹강균에 의하여 감염이 되면, 누에가 뽕을 먹지 않고 움직임이 둔해진다. 감염충에는 갈색, 흑색의 대형 병반이 생기며, 죽은 후에는 경화하여 체 표면에 선명의 녹색의 분생포자가 다수 형성되는데, 이것이 감염원이 된다.It is one of the silkworm disease of the silkworm, and when it becomes infected by the rust bacillus, the silkworms do not eat the mongolian and the movement becomes dull. There are large brown and black lesions in the infected worms, and after death, they are hardened to form a large number of fine green warts on the body surface, which becomes the infectious agent.
백강병은 누에가 허옇게 말라죽는 병이라 하여 그렇게 불리는 것이며, 백강병에 걸리면 누에의 피부가 기름에 밴 것처럼 보이고 얼마 후에는 피부에 얼룩반점이 생기거나 설사를 하며 죽은 다음에는 하얗게 되어 굳어진다.It is so called a disease in which silkworms die, and when it gets caught in a backache, the skin of the silkworm appears to be in the oil, and after a while, the skin becomes stained or diarrhea.
실시예Example 1. 미생물의 분리 및 동정 1. Isolation and Identification of Microorganisms
본 발명의 신규한 바실러스 아밀로리퀴에파션스 NBC241 균주는 경북 상주의 낙동강 유역의 토양에서 채취한 샘플에서 희석평판배양법에 의하여 분리하였다. 이후 생성된 콜로니를 이용하여 순수분리 한 후 20% glycerol에 현탁하여 70℃에 보관하며 사용하였다. 선발한 균주는 KOH test를 통하여 그람음성 및 양성의 여부에 대하여 판단하고, Biolog test 및 API kit를 이용하여 탄소원 이용 정도를 조사하였다. The novel Bacillus amyloliquefaciens NBC241 strain of the present invention was isolated by dilute plate culture in samples collected from soil in the Nakdong River basin of Sangju, Gyeongbuk. The resulting colonies were separated and then suspended in 20% glycerol and stored at 70 ° C. The selected strains were tested for Gram negative and positive by KOH test, and biolog test and API kit were used to investigate the degree of carbon source utilization.
또한, Genomic DNA Prep Kit (Solgent Co. Ltd, Korea)를 이용하여 gDNA를 추출하였으며, 27F(5‘GTT TGA TCC TGG CTC AG-3’와 1492R(5‘TAC CTT GTT ACG ACT T-3’프라이머를 사용하여 선발 미생물의 16S rDNA 염기서열을 분석한 후 BLAST program(http://www.ncbi.nlm.nih.gov/BLAST/)을 이용하여 미생물을 동정하였으며, 주요 결과 및 특성은 하기 표1 및 2와 같다. GDNA was extracted using Genomic DNA Prep Kit (Solgent Co. Ltd, Korea), and 27F (5'GTT TGA TCC TGG CTC AG-3 'and 1492R (5'TAC CTT GTT ACG ACT T-3' primer , The microorganisms were identified using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). The main results and characteristics are shown in the following Table 1 ≪ / RTI >
KOH test
KOH test
동정결과
Identification result
Biolog
Biolog
16S rDNA sequencing
16S rDNA sequencing
+
+
B. B.
amyloliquefaciensamyloliquefaciens
B. B.
amyloliquefaciensamyloliquefaciens
구분
division
특성
characteristic
구분
division
특성
characteristic
형태
shape
간상
Large
혐기적 생육
Anaerobic growth
+
+
호기적 생육
Aerobic growth
7% NaCl 에서의 생육
Growth in 7% NaCl
+
+
40℃에서의 생육
Growth at 40 ° C
실시예Example 2. 2. 신규한New 미생물의 Microbial 곤충병원균에 대한 길항 효과Antagonistic effect on insect pathogen
발명한 바실러스 아밀로리퀴에파션스 NBC241 (수탁번호 :KCTC13359BP) 균주를 곤충 사육중에 발생하는 진균성 병원균과의 대치배양을 통하여 길항 효과를 확인하였다. 그 결과 녹강병균(Metarhizium anisopliae)과 백강병균(Beauveria bassiana)의 균사 신장 억제 효과가 우수하였다. (도 2 참조)The antagonistic effect of the invented Bacillus amyloliquefaciens NBC241 strain (accession number: KCTC13359BP) strain was confirmed by incubation with a fungal pathogen occurring during insect rearing. As a result, Metarhizium The inhibitory effect of the height mycelia anisopliae) and back gangbyeonggyun (Beauveria bassiana) was excellent. (See Fig. 2)
병원균
Pathogen
저지원(mm)
Low support (mm)
Metarhizium anisopliae (꽃무지류, 녹강병균)
Metarhizium anisopliae (flowering, rust bran)
2.0
2.0
eauveria bassiana (꽃무지류, 백강병균)
eauveria bassiana (Flowers and flowers, bacitracin)
2.8
2.8
실시예Example 3. 3. 신규한New 미생물의 Microbial 녹강병Rust 및 And 백강병Backgag 방제효과 Control effect
곤충 사육시 발생하는 녹강병(KACC 40969)균 및 백강병(KACC 40039)균을 농촌진흥청 농업유전자원센터로부터 분양받은 후 감자한천배지(PDA; Potato dextrose agar)에서 25℃, 1주일간 배양 후 포자를 채취한 후 105 포자/㎖ 농도로 조절하여 사용하였다. The KACC 40969 and KACC 40039 bacteria were distributed from the Agricultural Genetic Resources Center of RDA and cultured in potato dextrose agar (PDA) at 25 ℃ for 1 week. After collection, the concentration was adjusted to 10 5 spores / ml.
신규한 바실러스 아밀로리퀴에파션스 NBC241 (수탁번호 :KCTC13359BP) 균주를 25℃에서 48시간 동안 배양 후 균체를 회수하여 5x107 세포/㎖ 농도로 조절하였다. 다음으로 멸균수에 현탁하여 참나무 톱밥의 전체 부피의 1%가 되게 첨가하고, 수분을 60% 정도가 되도록 배합하였다. 배합 후, 2일 1회 뒤집어주기를 반복하여 약 10일간 발효를 실시하였다. Wave syeonseu NBC241 to a novel Bacillus amyl quinone Lowry: to recover the (accession number KCTC13359BP) and then cultured at 25 ℃ for 48 hours, strain cells were adjusted to 5x10 7 cells was / ㎖. Next, the mixture was suspended in sterilized water and added to 1% of the total volume of the oak sawdust, and the moisture was blended to about 60%. After mixing, the fermentation was performed for about 10 days by repeating the inverting cycle twice a day.
이후 흰점박이꽃무지 유충을 사육하였으며, 사육 시작시 녹강병균 및 백강병균의 포자를 흰점박이꽃무지 사육용 톱밥에 각각 접종하여 30일 동안 병 발생 정도를 관찰하였다. 그 결과 바실러스 아밀로리퀴에파션스 NBC241 (수탁번호 :KCTC13359BP) 균주를 첨가하지 않은 대조군에 비해 녹강병은 83.3%, 백강병은 73.3%의 방제효과를 나타내었다. Then, white spotted spider mite larvae were raised. At the beginning of breeding, spores of rusty bacterium and white spore bacterium were inoculated into sawdust for white spotted flowers, respectively, for 30 days. As a result, it was 83.3% in rust resistance and 73.3% in backwash compared with the control without Bacillus amyloliquefaciens NBC241 (accession number: KCTC13359BP).
병원균
Pathogen
처리
process
병발생정도
Degree of disease occurrence
방제율(%)
Control rate (%)
녹강병
Rust
무처리
No treatment
40.0
40.0
83.3
83.3
처리구
Treatment
6.7
6.7
백강병
Backgag
무처리
No treatment
50.0
50.0
73.3
73.3
처리구
Treatment
13.3
13.3
실시예Example 4. 4. 신규한New 미생물이 생산한 Microbial 천연길항물질에To natural antagonists 의한 by 백강병과Backache 녹강병의Rusty 균사신장 억제 효과 Mycelial kidney inhibitory effect
본 발명의 바실러스 아밀로리퀴에파션스 NBC241 균주를 LB배지에서 48시간 동안 배양 후 15000rpm에서 20분간 원심분리를 실시하여 균체를 제거하였다. 균체를 제거한 배양 상등액을 다시 0.22㎛ 멤브레인 필터를 이용하여 필터링 하여 남아있는 균체를 완전히 제거하여 순수한 배양액만을 획득한 후, 녹강병균과 백강병균에 대하여 항균활성 검정을 실시하였다. The Bacillus amyloliquefaciens NBC241 strain of the present invention was cultured in LB medium for 48 hours and centrifuged at 15000 rpm for 20 minutes to remove the cells. After removing the cells, the culture supernatant was filtered using a 0.22 μm membrane filter to completely remove the remaining cells to obtain only a pure culture medium. Then, the antimicrobial activity test was carried out on rusty bacilli and white bacilli.
상기 항균활성 검정 결과 바실러스 아밀로리퀴에파션스 NBC241 균주가 제거된 순수한 배양액만으로도 진균성 병원균의 균사신장을 현저히 억제하여 길항효과가 우수함을 확인하였다. (도 3 참조) As a result of the above-mentioned antibacterial activity test, it was confirmed that the mycelial growth of fungal pathogens was remarkably suppressed by only pure culture medium in which Bacillus amyloliquefaciens NBC241 strain was removed, and the antagonistic effect was excellent. (See Fig. 3)
Claims (7)
상기 병원균은 메타라이지움 아니소플리에(Metarhizium anisopliae) 및 보베리아 바시아나(Beauveria bassiana)이고,
상기 바실러스 아밀로리퀴에파션스 NBC241의 세포수는 5 X 107 세포/mL인 것을 특징으로 하는 바실러스 아밀로리퀴에파션스 NBC241 균주.
Bacillus amyloliquefaciens NBC241 (accession number: KCTC13359BP), which has an antifungal activity against pathogenic bacteria causing insect diseases,
The pathogen is Metarhizium anisopliae and Beauveria bassiana ,
The Bacillus amyloliquefaciens Wherein the cell number of NBC241 is 5 x 10 < 7 > cells / mL.
상기 곤충은,
꽃무지류, 풍뎅이과 및 딱정벌레목으로 이루어진 군으로부터 선택된 1종인 것을 특징으로 하는, 바실러스 아밀로리퀴에파션스 NBC241 균주.
The method according to claim 1,
The insect,
A bacterium amyloliquefaciens strain NBC241, characterized in that it is one selected from the group consisting of a flowering, a chafer and a beetle.
상기 병원균은 메타라이지움 아니소플리에(Metarhizium anisopliae) 및 보베리아 바시아나(Beauveria bassiana)이고,
상기 바실러스 아밀로리퀴에파션스 NBC241의 세포수는 5 X 107 세포/mL인 것을 특징으로 하는 곤충에 병을 일으키는 병원균 방제용 조성물.
Bacillus amyloliquefaciens NBC241 (accession number: KCTC13359BP) or a composition for controlling pathogens causing diseases in an insect comprising the culture medium of the strain as an active ingredient,
The pathogen is Metarhizium anisopliae and Beauveria bassiana ,
The Bacillus amyloliquefaciens Wherein the cell number of NBC241 is 5 X 10 7 cells / mL.
상기 조성물에서 배양액을 유효성분으로 포함하는 경우,
상기 조성물은,
바실러스 아밀로리퀴에파션스 NBC241 균주를 LB배지에서 48시간 동안 배양하여 배양액을 제조하는 단계;
상기 제조된 배양액을 15000 rpm에서 20분간 원심분리를 실시하여 바실러스 아밀로리퀴에파션스 NBC241 균체를 제거하여 배양 상등액을 얻는 단계;
상기 배양 상등액을 0.22㎛ 멤브레인 필터로 필터하여 남아있는 균체를 완전히 제거하는 단계를 포함하여 제조되는 것을 특징으로 하는, 곤충에 병을 일으키는 병원균 방제용 조성물.
The method of claim 5,
When the composition contains the culture medium as an active ingredient,
The composition may comprise,
Culturing the Bacillus amyloliquefaciens NBC241 strain in an LB medium for 48 hours to prepare a culture solution;
Centrifuging the prepared culture solution at 15000 rpm for 20 minutes to remove Bacillus amyloliquefaciens NBC241 cells to obtain a culture supernatant;
Filtering the supernatant of the culture with a 0.22 탆 membrane filter to completely remove the remaining cells; and administering the composition for inhibiting pathogens in an insect.
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