KR102641016B1 - Novel Bacillus velezensis strain and uses thereof - Google Patents
Novel Bacillus velezensis strain and uses thereof Download PDFInfo
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- KR102641016B1 KR102641016B1 KR1020210177917A KR20210177917A KR102641016B1 KR 102641016 B1 KR102641016 B1 KR 102641016B1 KR 1020210177917 A KR1020210177917 A KR 1020210177917A KR 20210177917 A KR20210177917 A KR 20210177917A KR 102641016 B1 KR102641016 B1 KR 102641016B1
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- phytophthora
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
Abstract
본 발명은 신규 바실러스 벨레젠시스(Bacillus velezensis) 균주 및 이의 식물 역병균 방제 용도에 관한 것이다.
본 발명의 신규 바실러스 벨레젠시스(Bacillus velezensis) 균주는 역병균에 대해 우수한 방제 효과를 나타내는바, 항진균, 항균, 살균, 소독 등의 목적하는 분야에서 다양하게 활용될 수 있다.The present invention relates to a new Bacillus velezensis strain and its use for controlling plant blight.
The new Bacillus velezensis strain of the present invention exhibits an excellent control effect against late blight bacteria and can be used in various fields such as antifungal, antibacterial, sterilizing, and disinfecting purposes.
Description
본 발명은 신규 바실러스 벨레젠시스(Bacillus velezensis) 균주 및 이의 식물 역병균 방제 용도에 관한 것이다.The present invention relates to a new Bacillus velezensis strain and its use for controlling plant blight.
최근 논 작물 전환, 이상기후, 원예작물 집약재배와 연작 등으로 인해 역병 등과 같은 토양 전염성 병해가 증가하고 있다. 식물에 역병을 일으키는 원인균은 파이토프토라(Phytophthora) 속에 속하는 진균으로, 분류학상 난균류(Oomycetes)에 속하고, 난균류(Oomycetes)는 상위 분류학상 갈색 조류(brown algae)에 속한다. 파이토프토라(Phytophthora) 속 진균 중에서도, 특히 파이토프토라 소제(Phytophthora sojae)는 대두의 줄기와 뿌리 부패를 일으키는 토양 매개 식물 병원체로서 대부분의 콩 재배 지역에서 만연한 질병이며 작물 손실의 주요 원인이다.Recently, soil infectious diseases such as late blight are increasing due to the conversion of rice paddy crops, abnormal climate, intensive cultivation of horticultural crops, and continuous cropping. The causative bacterium that causes late blight in plants is a fungus belonging to the genus Phytophthora . Taxonomically, it belongs to Oomycetes, and Oomycetes belongs to brown algae in higher taxonomy. Among the fungi of the genus Phytophthora , Phytophthora sojae in particular is a soil-borne plant pathogen that causes stem and root rot of soybeans, is a prevalent disease in most soybean growing areas, and is a major cause of crop loss.
현재 난균류(Oomycetes)에 대한 연구가 많이 진행되지 않아 생물학적 지식이 제한되어 있으며, 이미 개발된 다수의 항곰팡이 제제가 난균류(Oomycetes) 계열 역병에는 효과적이지 못하다는 것은 주지의 사실이다. 예를 들어, Phytophthora 속의 방제에 상업적으로 사용되는 유기합성농약들(메타라실(metalaxyl), 디메토몰프(dimethomorph), 플루아지남(fluazinam), 파목사돈(famoxadone), 아즈옥시스트로빈(azoxystrobin), 시목사닐(cymoxanil), 프로파모카브(propamocarb), 포세트일(fosetyl), 만코제브(mancozeb), 족스아미드(zoxamide), 에타보삼클로타노일(ethaboxam chlrothalonoil))중에서 메타라실이 가장 널리 사용되는데, 최근 10년간 이것에 저항성을 보이는 균주가 출현되었고 점점 증가되는 추세이다. 따라서, 이미 개발된 합성농약들의 방제 효과는 감소되고 있으며, 농약의 대량 살포가 효과를 발휘할 수는 있으나, 이 경우 토양과 작물에 대한 잔류독성 문제가 심각해지는 문제점이 있다.Currently, not much research has been conducted on Oomycetes, so biological knowledge is limited, and it is a well-known fact that many antifungal agents that have already been developed are not effective against Oomycetes-type plague. For example, organic synthetic pesticides commercially used to control the genus Phytophthora (metalaxyl, dimethomorph, fluazinam, famoxadone, azoxystrobin) ), cymoxanil, propamocarb, fosetyl, mancozeb, zoxamide, and ethaboxam chlrothalonoil), metaracil is the best. It is widely used, and in the past 10 years, strains showing resistance to it have emerged and are increasing. Therefore, the control effect of synthetic pesticides that have already been developed is decreasing, and although mass spraying of pesticides can be effective, in this case, the problem of residual toxicity to soil and crops becomes serious.
이에, 상기 합성농약을 대체하기 위해 미생물 등으로부터 유래하는 천연 항진균제를 개발하지 위한 노력이 있어왔으나, 특별한 수단을 개발하지 않는다면 생산비가 높아서 시장 경쟁성이 떨어지므로 이들의 생합성 유전자를 분리하여 발현을 높이려는 시도가 있었다(비특허문헌 1, 2).Accordingly, efforts have been made to develop natural antifungal agents derived from microorganisms to replace the above-mentioned synthetic pesticides, but unless special means are developed, production costs are high and market competitiveness is low. Therefore, their biosynthetic genes are isolated to increase expression. There was an attempt (Non-patent Documents 1 and 2).
그러나, 파이토프토라(Phytophthora) 속 진균에 대해 현저한 효과를 나타내는 방제제는 아직까지 없는 실정이다.However, there is still no control agent that shows significant effectiveness against fungi of the genus Phytophthora .
이러한 배경하에, 본 발명자들은 신규 바실러스 벨레젠시스(Bacillus velezensis) 균주를 동정하고, 상기 균주가 파이토프토라 속 진균, 특히 파이토프토라 소제에 대해 항진균 활성을 가지는 것을 확인함으로써, 본 발명을 완성하였다.Against this background, the present inventors identified a new Bacillus velezensis strain and confirmed that the strain has antifungal activity against Phytophthora fungi, especially Phytophthora microorganisms, thereby completing the present invention. .
본 발명의 하나의 목적은 기탁번호 KACC 92360P로 기탁된 바실러스 벨레젠시스 KJB-1(Bacillus velezensis KJB-1) 균주를 제공하는 것이다.One object of the present invention is to provide a strain of Bacillus velezensis KJB-1 deposited with accession number KACC 92360P.
본 발명의 다른 하나의 목적은 상기 균주, 이의 포자, 이의 배양물, 상기 배양물의 농축물, 추출물 및 상기 배양물의 건조물을 포함하는, 항진균 조성물을 제공하는 것이다.Another object of the present invention is to provide an antifungal composition comprising the strain, its spores, its culture, the concentrate of the culture, the extract, and the dried material of the culture.
본 발명의 또 하나의 목적은 상기 항진균 조성물을 포함하는 항진균 조성물을 포함하는, 역병균 방제용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for controlling late blight, comprising an antifungal composition containing the antifungal composition.
본 발명의 또 하나의 목적은 상기 항진균 조성물을 식물체에 처리하는 단계를 포함하는, 역병균 방제 방법을 제공하는 것이다.Another object of the present invention is to provide a method for controlling late blight bacteria, including the step of treating plants with the antifungal composition.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description described below.
상기 목적을 달성하기 위한 본 발명의 하나의 양태는, 기탁번호 KACC 92360P로 기탁된 바실러스 벨레젠시스 KJB-1(Bacillus velezensis KJB-1) 균주를 제공한다.One aspect of the present invention for achieving the above object provides a Bacillus velezensis KJB-1 strain deposited under the accession number KACC 92360P.
본 발명에 있어서, 상기 바실러스 벨레젠시스 KJB-1(Bacillus velezensis KJB-1) 균주는 20 여개의 바실러스 속 균주 중 파이토프토라(Phytophthora) 속 진균에 대한 항진균 활성을 가지는 균을 선발한 결과 선별된 균주로서, 이를 바실러스 벨레젠시스 KJB-1라 명명하고, 부다페스트 조약 하의 국제기탁기관인 씨앗은행(국립농업과학원 미생물은행, Korean Agricultural Culture Collection, KACC)에 2021년 8월 3일자로 기탁하여 수탁번호 KACC 92360P를 부여 받았다.In the present invention, the Bacillus velezensis KJB-1 strain was selected as a result of selecting bacteria with antifungal activity against fungi of the genus Phytophthora among about 20 strains of the genus Bacillus. As a strain, it was named Bacillus belegensis KJB-1, and it was deposited in a seed bank (Korean Agricultural Culture Collection, KACC), an international depository institution under the Budapest Treaty, on August 3, 2021, with accession number KACC. 92360P was granted.
본 발명에 따른 바실러스 벨레젠시스 KJB-1는 항진균 활성을 갖는 것일 수 있다.Bacillus belegensis KJB-1 according to the present invention may have antifungal activity.
본 발명에 따른 바실러스 벨레젠시스 KJB-1는 역병균 억제 활성을 갖는 것일 수 있다.Bacillus belegensis KJB-1 according to the present invention may have antibacterial activity.
본 발명의 용어, "역병균"은 식물에서 줄기 및/또는 뿌리에 부패를 일으키는 식물 병원균을 의미한다. 본 발명에 있어서, 상기 역병균은 파이토프토라 속 진균일 수 있고, 구체적으로 대두의 줄기와 뿌리에 부패를 일으키는 콩 역병균인 파이토프토라 소제(Phytophthora sojae)일 수 있다.As used herein, the term "blight bacteria" refers to plant pathogens that cause rot in stems and/or roots in plants. In the present invention, the blight bacteria may be a fungus of the genus Phytophthora, and specifically may be Phytophthora sojae, a soybean blight fungus that causes rot in the stems and roots of soybeans.
본 발명의 일 구현예에서는, 본 발명의 신규 균주의 파이토프토라 소제(Phytophthora sojae)에 대한 억제 활성을 확인한 결과 표 1의 바실러스 속의 타 균주 대비 바실러스 벨레젠시스 KJB-1의 역병균 억제능이 가장 우수한 것을 확인하였다(도 2).In one embodiment of the present invention, as a result of confirming the inhibitory activity of the new strain of the present invention against Phytophthora sojae, the antibacterial activity of Bacillus belegensis KJB-1 was the highest compared to other strains of the Bacillus genus in Table 1. It was confirmed that it was excellent (Figure 2).
본 발명에 따른 바실러스 벨레젠시스 KJB-1는 역병균의 균사 생장 억제 활성을 갖는 것일 수 있다.Bacillus belegensis KJB-1 according to the present invention may have the activity of inhibiting mycelial growth of late blight bacteria.
본 발명의 일 구현예에서는, 본 발명의 신규 균주의 파이토프토라 소제(Phytophthora sojae)에 대한 균사 생장 억제 활성을 확인한 결과 역병균 균사 생장 억제능을 나타내는 것을 확인하였으며(표 2 및 도 3), 기등록된 농약인 Pesticide C와 비슷한 정도 혹은 그 이상의 수준으로 역병균 균사 생장 억제능을 나타내는 것을 확인하였다(도 4).In one embodiment of the present invention, as a result of confirming the mycelial growth inhibitory activity of the new strain of the present invention against Phytophthora sojae, it was confirmed that it exhibits the ability to inhibit the mycelial growth of late blight bacteria (Table 2 and Figure 3), It was confirmed that it exhibits the ability to inhibit the growth of late blight mycelium at a level similar to or higher than that of Pesticide C, a registered pesticide (Figure 4).
즉, 본 발명에 따른 바실러스 벨레젠시스 KJB-1는 역병균인 파이토프토라 속 진균에 대해 우수한 항진균 활성을 가지며, 구체적으로, 콩 역병균인 파이토프토라 소제에 대해 우수한 항진균 활성을 갖는 것일 수 있다.In other words, Bacillus belegensis KJB-1 according to the present invention may have excellent antifungal activity against fungi of the genus Phytophthora, a latent pathogen, and specifically, may have excellent antifungal activity against Phytophthora microorganisms, a soybean latent pathogen. there is.
본 발명의 다른 하나의 양태는, 본 발명의 균주, 균주의 포자, 균주의 배양물, 상기 배양물의 농축물, 추출물 및 상기 배양물의 건조물을 포함하는 항진균 조성물을 제공한다.Another aspect of the present invention provides an antifungal composition comprising the strain of the present invention, spores of the strain, culture of the strain, concentrate of the culture, extract, and dried material of the culture.
이때, 균주의 상태는 액상 상태 또는 건조 상태일 수 있으나, 이에 제한되는 것은 아니다.At this time, the state of the strain may be liquid or dry, but is not limited thereto.
상기 항진균 조성물은 파이토프토라 속 진균, 구체적으로 파이토프토라 소제에 대해 항진균 활성을 갖는 것일 수 있다.The antifungal composition may have antifungal activity against fungi of the genus Phytophthora, specifically against Phytophthora microorganisms.
본 발명의 용어, "포자"는 세균 등의 번식 세포를 말하며, 본 출원의 목적상 상기 포자는 본 발명의 신규 균주의 번식 세포를 의미할 수 있다.As used herein, the term “spore” refers to a reproductive cell such as a bacterium, and for the purposes of this application, the spore may refer to a reproductive cell of the new strain of the present invention.
본 발명의 용어, "배양물"은 상기 균주를 배양한 다음 수득한 산물을 의미하며, 균체를 포함한 배양 원액일 수 있으며, 또한 배양 균주, 배양 상등액을 제거하거나 농축한 균체일 수 있다. 상기 배양물의 조성은 통상의 바실러스 속 균주의 배양에 필요한 성분뿐 아니라, 바실러스 속 균주의 생장에 상승적으로 작용하는 성분을 추가적으로 포함할 수 있으며, 이에 따른 조성은 당업계의 통상의 기술을 가진 자에 의하여 용이하게 선택될 수 있다.As used herein, the term “culture” refers to a product obtained after culturing the above-mentioned strain, and may be a culture stock solution containing bacteria, or may be a culture strain or a culture supernatant obtained by removing or concentrating the bacteria. The composition of the culture may include not only components necessary for cultivating conventional Bacillus strains, but also additional components that act synergistically on the growth of Bacillus strains, and the resulting composition can be determined by those skilled in the art. can be easily selected.
본 발명의 용어, "농축물"은 상기 배양물을 농축한 것을 의미한다.As used herein, the term “concentrate” means concentrating the culture.
본 발명의 용어, "추출물"은 상기 배양물 또는 이의 농축물로부터 추출한 것을 의미하며, 본 발명의 파이토프토라 속 진균, 구체적으로 파이토프토라 소제에 특이적 항진균 효과를 나타낼 수 있는 추출물인 한, 이에 제한되지 않고, 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 또는 이들 조정제물 또는 정제물, 이를 분획한 분획물을 모두 포함할 수 있다.The term "extract" of the present invention refers to an extract from the culture or a concentrate thereof, and is an extract that can exhibit a specific antifungal effect on the Phytophthora genus fungi of the present invention, specifically Phytophthora microorganisms, It is not limited thereto, and may include all extracts, diluted or concentrated extracts, dried products obtained by drying the extracts, crude or purified products thereof, and fractions thereof.
본 발명의 용어, "건조물"은 상기 배양물, 이의 농축물, 이의 추출물, 및 이의 분획물을 건조한 것을 의미할 수 있다. 건조방법은 통풍건조, 자연건조, 분무건조 및 동결건조가 가능하지만, 이에 제한되는 것은 아니다.As used herein, the term “dried product” may refer to dried culture, its concentrate, its extract, and its fraction. Drying methods include, but are not limited to, ventilation drying, natural drying, spray drying, and freeze drying.
본 발명의 신규 균주는 통상적인 바실러스 속 균주의 배양방법을 통해 배양할 수 있으나, 이에 제한되는 것은 아니다. 배지로는 천연배지 또는 합성배지를 사용할 수 있다. 배지의 탄소원으로는 예를 들어, 글루코오스, 수크로오스, 덱스트린, 글리세롤, 녹말 등이 사용될 수 있고, 질소원으로는 펩톤, 육류 추출물, 효모 추출물, 건조된 효모, 대두, 암모늄염, 나이트레이트 및 기타 유기 또는 무기 질소 함유 화합물이 사용될 수 있으나, 이러한 성분에 한정되는 것은 아니다. 배지에 포함되는 무기염으로는 마그네슘, 망간, 칼슘, 철, 인 등이 사용될 수 있으나, 이들에 한정되는 것은 아니다. 상기 탄소원, 질소원 및 무기염의 성분 이외에 아미노산, 비타민, 핵산 및 그와 관련된 화합물들이 배지에 첨가될 수 있다.The new strain of the present invention can be cultured through a conventional culture method of a Bacillus genus strain, but is not limited thereto. As a medium, natural medium or synthetic medium can be used. For example, glucose, sucrose, dextrin, glycerol, starch, etc. can be used as carbon sources of the medium, and peptone, meat extract, yeast extract, dried yeast, soybeans, ammonium salts, nitrates, and other organic or inorganic sources can be used as nitrogen sources. Nitrogen-containing compounds may be used, but are not limited to these components. Inorganic salts included in the medium may include magnesium, manganese, calcium, iron, and phosphorus, but are not limited to these. In addition to the carbon source, nitrogen source, and inorganic salt components, amino acids, vitamins, nucleic acids, and compounds related thereto may be added to the medium.
상기 항진균 조성물은 본 발명의 신규 균주를 배양하는 과정에서 생성되는 기타 부산물을 함께 포함할 수 있다.The antifungal composition may also include other by-products produced during the process of cultivating the new strain of the present invention.
본 발명에 있어서, 상기 항진균 조성물은, 농약, 의약, 생활용품, 식품 등에서 항진균, 항균, 살균, 소독, 방부 등의 목적을 달성하기 위해 광범위하게 사용될 수 있다. 구체적으로, 농업에 있어서는 항진균, 항균, 살균, 소독의 목적으로, 생활용품에 있어서는 미생물과 직접 연관된 제품에 사용되거나 청소용 세정제, 식기세척용 세정제 등에 방부나 항진균 또는 살균 목적으로 사용될 수 있으나, 이에 한정되는 것은 아니다. 또한, 본 발명의 항진균 조성물은 항생제 대체제 또는 항진균 포장재 등으로 사용될 수 있다. In the present invention, the antifungal composition can be widely used to achieve purposes such as antifungal, antibacterial, sterilizing, disinfecting, and preservative purposes in pesticides, medicines, household goods, foods, etc. Specifically, in agriculture, it can be used for antifungal, antibacterial, sterilizing, and disinfecting purposes, and in household goods, it can be used in products directly related to microorganisms, or in cleaning detergents and dishwashing detergents, etc., for preservative, antifungal, or sterilizing purposes, but is limited to this. It doesn't work. Additionally, the antifungal composition of the present invention can be used as an antibiotic substitute or antifungal packaging material.
본 발명의 또 하나의 양태는, 본 발명의 항진균 조성물을 포함하는 역병균 방제용 조성물을 제공한다.Another aspect of the present invention provides a composition for controlling late blight bacteria comprising the antifungal composition of the present invention.
상기 항진균 조성물은 상술한 바와 같다.The antifungal composition is as described above.
본 발명의 조성물은 액제, 입제, 분제, 유제, 오일제, 수화제, 및 도포제로 이루어지는 군 중에서 선택되는 어느 하나 이상의 형태일 수 있고, 제형화를 위해 다양한 성분을 포함할 수 있으며, 예를 들어, 액체 담체, 고체 담체, 계면활성제 또는 보조제 등이 포함될 수 있다. The composition of the present invention may be in the form of one or more selected from the group consisting of liquids, granules, powders, emulsions, oils, wetters, and coating agents, and may contain various ingredients for formulation, for example, Liquid carriers, solid carriers, surfactants, or auxiliaries may be included.
상기 액체 담체로는 물(water), 식물유(vegetable oil), 에탄올(ethanol) 등이 사용될 수 있으며, 상기 식물유에는 대두유, 유채씨기름, 야자유, 팜핵유, 미강유, 옥수수유, 팜유, 올리브유 등이 포함되고, 이에 제한되지 않는다. 상기 고체 담체로는 광물 분말, 젤라틴, 알긴산 등이 사용될 수 있으며, 이에 제한되지 않는다. 상기 광물 분말로는 양이온 점토(cation clay), 벤토나이트(bentonite), 카올린(kaolin), 탈크(Talc), 규조토(diatomaceous earth) 등이 사용될 수 있으며, 이에 제한되지 않는다. 상기 계면활성제로는 에틸렌옥사이드(Ethyleneoxide)계, 다이에탄올아민(Diethanolamine)계, 솔비톨(Sorbitol)계, 글리세린(Glycerine)계 등이 사용될 수 있으며, 이에 제한되지 않는다. 상기 보조제로는 증량제, 부동액, 용제, 증점제, 및 전착제 등이 1종 이상 사용될 수 있으며, 이에 제한되지 않는다. The liquid carrier may be water, vegetable oil, ethanol, etc., and the vegetable oil may include soybean oil, rapeseed oil, palm oil, palm kernel oil, rice bran oil, corn oil, palm oil, olive oil, etc. Included, but not limited to. The solid carrier may include mineral powder, gelatin, alginic acid, etc., but is not limited thereto. The mineral powder may include, but is not limited to, cation clay, bentonite, kaolin, talc, and diatomaceous earth. The surfactant may include, but is not limited to, ethylene oxide, diethanolamine, sorbitol, and glycerine. The auxiliary agent may include one or more of an extender, antifreeze, solvent, thickener, and electrodeposition agent, but is not limited thereto.
본 발명의 조성물은 식물 방제용 조성물에 통상 사용되는 임의의 적합한 부형제를 추가로 포함할 수 있으며, 이러한 부형제는, 예를 들어 보존제, 습윤제, 분산제, 현탁화제, 완충제, 안정화제 또는 등장화제 등일 수 있으나, 이에 한정되는 것은 아니다.The composition of the present invention may further comprise any suitable excipients commonly used in compositions for plant control, and such excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents, or tonicity agents. However, it is not limited to this.
본 발명에 있어서, 상기 역병균은 콩 역병균일 수 있으며, 구체적으로 파이토프토라 속 균주일 수 있고, 보다 구체적으로 파이토프토라 소제일 수 있다.In the present invention, the late blight bacteria may be soybean blight bacteria, specifically, may be a strain of the genus Phytophthora, and more specifically may be Phytophthora microorganisms.
본 발명의 또 하나의 양태는, 본 발명의 항진균 조성물을 식물체에 처리하는 단계를 포함하는 역병균 방제 방법을 제공한다.Another aspect of the present invention provides a method for controlling late blight bacteria, including the step of treating plants with the antifungal composition of the present invention.
상기 항진균 조성물은 상술한 바와 같다.The antifungal composition is as described above.
본 발명의 방제 방법은 특별이 제한되지 않으며 상기 항진균 조성물의 형태에 따라 적절하게 선택되어 수행될 수 있다. 구체적으로, 상기 방제 방법은 경엽처리, 토양처리, 침지처리, 가지처리, 및 농기구 처리로 이루어지는 군 중에서 선택되는 어느 하나 이상일 수 있으나, 이에 제한되지 않는다.The control method of the present invention is not particularly limited and may be appropriately selected and performed depending on the form of the antifungal composition. Specifically, the control method may be any one or more selected from the group consisting of foliage treatment, soil treatment, soaking treatment, branch treatment, and farming equipment treatment, but is not limited thereto.
상기 방제 방법에 있어서, 본 발명의 조성물은 단독으로 처리하거나 다른 방제제와 병용하여 투여될 수 있고 종래의 방제제와는 순차적 또는 동시에 투여될 수 있으며 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 처리(살포)량 또는 처리 시간 간격은 식물체에 따라 다를 수 있고, 당업자에 의해 용이하게 결정될 수 있다. 본 발명의 조성물의 바람직한 처리량은 식물체의 생육정도, 경작지 환경, 식물바이러스병의 발병 정도 등을 고려하여 당업자에 의해 적절하게 조절할 수 있으며, 이에 제한되지 않는다.In the above control method, the composition of the present invention can be treated alone or administered in combination with other control agents, can be administered sequentially or simultaneously with conventional control agents, and can be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects. The treatment (spraying) amount or treatment time interval may vary depending on the plant and can be easily determined by a person skilled in the art. there is. The preferred treatment amount of the composition of the present invention can be appropriately adjusted by a person skilled in the art in consideration of the growth degree of the plant, the cultivation environment, the degree of occurrence of plant virus diseases, etc., but is not limited thereto.
본 발명의 조성물은 일반적으로 0.1 내지 50 ㎍/mL의 농도, 구체적으로는 0.5 내지 50 ㎍/mL의 농도, 보다 구체적으로는 1 내지 50 ㎍/mL의 농도로 식물체에 처리되는 것일 수 있으나, 이에 제한되지 않는다. 본 발명의 조성물을 제조, 보관, 및 운반할 경우 일반적으로 식물체에 처리시 농도보다 1 내지 100,000배의 농축 농도, 구체적으로는 100 내지 50,000배의 농축 농도, 보다 구체적으로는 500 내지 10,000배의 농축 농도로 제조, 보관, 및 운반할 수 있으나, 이에 제한되지 않는다.The composition of the present invention may be treated on plants at a concentration of generally 0.1 to 50 μg/mL, specifically 0.5 to 50 μg/mL, and more specifically 1 to 50 μg/mL. Not limited. When manufacturing, storing, and transporting the composition of the present invention, the concentration is generally 1 to 100,000 times the concentration when treated with plants, specifically 100 to 50,000 times the concentration, and more specifically 500 to 10,000 times the concentration. It can be manufactured, stored, and transported in any concentration, but is not limited to this.
본 발명에 있어서, 상기 역병균은 콩 역병균일 수 있으며, 구체적으로 파이토프토라 속 균주일 수 있고, 보다 구체적으로 파이토프토라 소제일 수 있다.In the present invention, the late blight bacteria may be soybean blight bacteria, specifically, may be a strain of the genus Phytophthora, and more specifically may be Phytophthora microorganisms.
본 발명의 신규 바실러스 벨레젠시스(Bacillus velezensis) 균주는 역병균에 대해 우수한 방제 효과를 나타내는바, 항진균, 항균, 살균, 소독 등의 목적하는 분야에서 다양하게 활용될 수 있다.The new Bacillus velezensis strain of the present invention exhibits an excellent control effect against late blight bacteria and can be used in various fields such as antifungal, antibacterial, sterilizing, and disinfecting purposes.
도 1은 5개의 유전자(16S rRNA, gyrA, rpoB, purH, and groEL) 서열을 이용한 신규 바실러스 벨레젠시스 KJB-1(Bacillus velezensis KJB-1) 균주 동정 결과이다.
도 2는 바실러스 벨레젠시스 KJB-1와 다양한 바실러스 속 균주의 역병균 억제능을 비교한 결과이다.
도 3은 바실러스 벨레젠시스 KJB-1에 의한 역병균 균사 생장 억제 결과이다.
도 4는 바실러스 벨레젠시스 KJB-1와 기등록된 농약에 의한 역병균 균사 생장 억제능을 비교한 결과이다.
도 5는 바실러스 벨레젠시스 KJB-1의 PGP(Plant Growth Promoting) 특성을 확인한 결과이다.
도 6은 PCR 증폭에 의한 바실러스 벨레젠시스 KJB-1의 생합성 유전자 검출 결과를 나타낸 도이다. M; 마커(100bp 사다리), 1; 바실리신(bacD, 749bp), 2; 바실로마이신(bmyA, 875bp), 3; 이투린(ituA, 647bp), 4; 서팩틴(srfA, 441bp), 5; 펜기신(fenD, 964bp), 6; 쯔비테르미신(zwiA, 779bp). PCR 산물은 1.0% 아가로스 겔에서 검출되었다.Figure 1 shows the results of identification of a new Bacillus velezensis KJB-1 strain using the sequences of five genes (16S rRNA, gyrA, rpoB, purH, and groEL).
Figure 2 shows the results of comparing the anti-bacterial ability of Bacillus belegensis KJB-1 and various strains of the genus Bacillus.
Figure 3 shows the results of inhibition of mycelial growth of late blight bacteria by Bacillus belegensis KJB-1.
Figure 4 shows the results of comparing the mycelial growth inhibition ability of Bacillus belegensis KJB-1 and a previously registered pesticide.
Figure 5 shows the results of confirming the PGP (Plant Growth Promoting) characteristics of Bacillus belegensis KJB-1.
Figure 6 shows Bacillus belegensis by PCR amplification. This diagram shows the results of detection of the biosynthesis gene of KJB-1. M; marker (100bp ladder), 1; bacillicin ( bacD , 749 bp), 2; bacillomycin ( bmyA , 875 bp), 3; iturin ( ituA , 647bp), 4; surfactin ( srfA , 441 bp), 5; Fengyxin ( fenD , 964bp), 6; Zwithermicin ( zwiA , 779bp). PCR products were detected on a 1.0% agarose gel.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples.
실시예 1: 신규 바실러스 벨레젠시스 균주 동정Example 1: Identification of new Bacillus belegensis strains
5개의 유전자(16S rRNA, gyrA, rpoB, purH, and groEL) 서열을 이용하여, 바실러스 벨레젠시스 KJB-1(Bacillus velezensis KJB-1)를 동정하였다. Bacillus velezensis KJB-1 was identified using the sequences of five genes (16S rRNA, gyrA, rpoB, purH, and groEL).
구체적으로, 2일 동안 28℃의 TSA 배지에서 배양한 KJB-1의 단일 콜로니를 TSB 배지 3mL가 들어 있는 시험관으로 옮겼다. 시험관을 28℃, 200rpm 조건의 진탕배양기에서 배양한 후, 배양액을 수득하여 Wizard® Genomic DNA Purification Kit(Pomega, USA)를 이용하여 게놈 DNA를 추출하였다. 추출한 게놈 DNA를 주형으로 하여 PCR을 이용해 16S rRNA를 증폭하였다. Specifically, a single colony of KJB-1 cultured in TSA medium at 28°C for 2 days was transferred to a test tube containing 3 mL of TSB medium. After culturing the test tube in a shaking incubator at 28°C and 200 rpm, the culture medium was obtained and genomic DNA was extracted using the Wizard ® Genomic DNA Purification Kit (Pomega, USA). 16S rRNA was amplified using PCR using the extracted genomic DNA as a template.
PCR 증폭에 사용된 프라이머 세트는 하기 표 1과 같다.The primer sets used for PCR amplification are listed in Table 1 below.
16S rRNA 유전자의 PCR은 초기 변성반응(initial denaturation; 95℃ 3분) 후, 증폭반응(변성; 95℃ 30초, 결합; 55℃ 45초, 신장; 72℃ 1분)을 30회 반복하였으며, 최종 신장반응은 72℃에서 5분간 실시하였다. gyrA와 purH 유전자의 PCR은 초기 변성반응(95℃ 5분) 후, 증폭반응(변성; 95℃ 40초, 결합; 55℃ 1분, 신장; 68℃ 2분)을 35회 반복하였으며, 최종 신장반응은 68℃에서 4분간 실시하였다. rpoB와 groEL 유전자의 PCR은 초기 변성반응(94℃ 5분) 후, 증폭반응(변성; 94℃ 30초, 결합; 55℃ 30초, 신장; 72℃ 1분)을 35회 반복하였으며, 최종 신장반응은 72℃에서 4분간 실시하였다. 증폭한 PCR 산물은 전기영동하여 산물 존재 여부 및 크기를 확인하였다. PCR 산물은 1.0% 아가로스 겔에서 검출되었다.PCR of the 16S rRNA gene involved initial denaturation (95°C for 3 minutes), followed by amplification (denaturation; 95°C for 30 seconds; binding; 55°C for 45 seconds; extension; 72°C for 1 minute) repeated 30 times. The final elongation reaction was performed at 72°C for 5 minutes. PCR of the gyrA and purH genes was performed 35 times with an initial denaturation reaction (95°C for 5 minutes) followed by amplification (denaturation; 95°C for 40 seconds; binding; 55°C for 1 minute; elongation; 68°C for 2 minutes). The final elongation was performed 35 times. The reaction was carried out at 68°C for 4 minutes. PCR of the rpoB and groEL genes was performed 35 times with an initial denaturation reaction (94°C for 5 minutes) followed by amplification (denaturation; 94°C for 30 seconds; binding; 55°C for 30 seconds; elongation; 72°C for 1 minute). The final elongation was performed 35 times. The reaction was carried out at 72°C for 4 minutes. The amplified PCR product was subjected to electrophoresis to confirm the presence and size of the product. PCR products were detected on a 1.0% agarose gel.
그 결과, 최종 분리된 신규한 바실러스 벨레젠시스 균주는 바실러스 벨레젠시스 KJB-1(Bacillus velezensis KJB-1)로 명명하였다(도 1).As a result, the new finally isolated Bacillus velezensis strain was named Bacillus velezensis KJB-1 (Figure 1).
실시예 2: 신규 바실러스 벨레젠시스 균주의 역병균 억제능 확인Example 2: Confirmation of anti-bacterial ability of new Bacillus belegensis strain
다양한 바실러스 속 균주에 대해 콩에서 역병을 일으키는 역병균인 파이토프토라 소제(Phytophthora sojae) KACC40412에 대한 억제능을 확인하였다.The inhibitory ability of various Bacillus strains against Phytophthora sojae KACC40412, a late blight fungus that causes late blight in soybeans, was confirmed.
구체적으로, 7일 동안 26℃의 V8 아가 배지에서 배양한 P. sojae의 직경 7mm 아가 플러그를 V8 아가 플레이트의 중앙에 접종하였다. 아가 플러그 접종 후 24시간 뒤, 2일 동안 28℃의 TSA 배지에서 배양한 각 바실러스 균주의 단일 콜로니를 TSB 배지 5mL가 들어 있는 시험관으로 옮긴 후 시험관을 28℃, 200rpm 조건의 진탕배양기에 넣어 1일간 배양한 각 바실러스 균주 현탁액 50㎕(O.D.600에서 0.3)을 접종된 아가 플러그 양쪽 옆에 떨어뜨렸다. P. sojae와 각 바실러스 균주의 접종이 완료된 V8 아가 배지를 26℃에서 7일 동안 배양 후 직경을 측정하였다. 가로 길이와 세로 길이의 평균을 그래프에 나타내었으며, 4회 반복 실험하였다.Specifically, agar plugs with a diameter of 7 mm of P. sojae cultured in V8 agar medium at 26°C for 7 days were inoculated into the center of the V8 agar plate. 24 hours after agar plug inoculation, a single colony of each Bacillus strain cultured in TSA medium at 28°C for 2 days was transferred to a test tube containing 5 mL of TSB medium, and the test tube was placed in a shaking incubator at 28°C and 200 rpm for 1 day. 50㎕ of each cultured Bacillus strain suspension (0.3 at OD600) was dropped on both sides of the inoculated agar plug. V8 agar medium inoculated with P. sojae and each Bacillus strain was cultured at 26°C for 7 days and then the diameter was measured. The average of the horizontal and vertical lengths is shown in the graph, and the experiment was repeated four times.
여기에서 사용된 바실러스 속 균주는 하기 표 2와 같다.The Bacillus genus strains used here are shown in Table 2 below.
(KACC 92360P)Deposit of patented microorganisms
(KACC 92360P)
보유 균주itself
strains possessed
그 결과, 도 2와 같이 바실러스 속의 타 균주 대비 바실러스 벨레젠시스 KJB-1의 역병균 억제능이 가장 우수한 것을 확인하였다.As a result, as shown in Figure 2, it was confirmed that Bacillus belegensis KJB-1 had the best antibacterial activity compared to other strains of the Bacillus genus.
실시예 3: 신규 바실러스 벨레젠시스 균주의 역병균 균사 생장 억제능 확인Example 3: Confirmation of the ability of the new Bacillus belegensis strain to inhibit the growth of mycelial blight bacteria
상기 실시예 2에서 바실러스 벨레젠시스 KJB-1의 우수한 콩 역병균 억제능을 확인하였는 바, 상기 역병균의 균사 생장 억제능을 확인하였다.In Example 2, the excellent ability of Bacillus belegensis KJB-1 to inhibit soybean late blight bacteria was confirmed, and the mycelial growth inhibitory ability of the late blight bacteria was confirmed.
구체적으로, 7일 동안 26℃의 V8 아가 배지에서 배양한 P. sojae의 직경 7mm 아가 플러그를 V8 아가 플레이트의 중앙에 접종하였다. 2일 동안 28℃의 TSA 배지에서 배양한 KJB-1을 접종된 아가 플러그 양 옆에 일직선 모양으로 접종하였다. 26℃에서 7일 동안 배양 후 직경을 측정하였다. 측정된 직경의 값을 근거하여 억제율을 계산하였으며, 3회 반복 실험하였다. Specifically, agar plugs with a diameter of 7 mm of P. sojae cultured in V8 agar medium at 26°C for 7 days were inoculated into the center of the V8 agar plate. KJB-1 cultured in TSA medium at 28°C for 2 days was inoculated in a straight line on both sides of the inoculated agar plug. After culturing at 26°C for 7 days, the diameter was measured. The inhibition rate was calculated based on the measured diameter value, and the experiment was repeated three times.
그 결과, 하기 표 3 및 도 3에 나타낸 바와 같이 바실러스 벨레젠시스 KJB-1은 역병균 균사 생장 억제능을 나타내는 것을 확인하였다.As a result, as shown in Table 3 and Figure 3 below, it was confirmed that Bacillus belegensis KJB-1 exhibits the ability to inhibit the growth of late blight mycelium.
1대조구의 조사값-처리구의 조사값/대조구의 조사값*100 1 Survey value of control group - Survey value of treatment group / Survey value of control group * 100
또한, 기등록된 농약인 Pesticide C를 양성대조군으로 하여 상기와 동일한 방법으로 콩 역병균에 대한 균사 생장 억제능을 비교한 결과, 도 4에 도시한 바와 같이, 바실러스 벨레젠시스 KJB-1는 Pesticide C와 비슷한 정도 혹은 그 이상의 역병균 균사 생장 억제능을 나타내는 것을 확인하였다.In addition, as a result of comparing the mycelial growth inhibition ability against soybean late blight bacteria in the same manner as above using Pesticide C, a previously registered pesticide, as a positive control, as shown in Figure 4, Bacillus belegensis KJB-1 is Pesticide C It was confirmed that it exhibits a similar or greater ability to inhibit the growth of mycelial blight bacteria.
실시예 4: 신규 바실러스 벨레젠시스 균주의 PGP(Plant Growth Promoting) 특성 확인Example 4: Confirmation of PGP (Plant Growth Promoting) characteristics of new Bacillus belegensis strains
바실러스 벨레젠시스 KJB-1의 PGP 특성을 다음과 같이 분석하였다.The PGP characteristics of Bacillus belegensis KJB-1 were analyzed as follows.
4-1. IAA(Indole-3-acetic acid) 생산량4-1. IAA (Indole-3-acetic acid) production
28℃의 TSA 배지에서 2일 동안 배양한 KJB-1의 단일 콜로니를 5mM L-트립토판(Trp)이 함유된 King's B 배지 5mL가 들어 있는 시험관으로 옮긴 후 28℃, 200rpm 조건의 진탕배양기에서 1일간 배양하였다. 1mL의 배양액을 100mL의 5mM Trp가 함유된 King's B 배지가 들어있는 플라스크로 옮긴 후, 2일 동안 28℃, 200rpm 조건의 진탕배양기에서 배양하였다. 배양액을 15분 동안 4℃에서 8,000 x g 조건에서 원심분리 한 후, 상등액 1ml와 Salkowski 시약 1ml를 30분 동안 암실에서 반응시켰다. 5mM Trp이 함유된 King's B 배지 1ml와 Salkowski 시약 1ml를 30분동안 암실에서 반응시킨 용액을 standard로 하여 530 nm에서 흡광도를 측정하였다.A single colony of KJB-1 cultured for 2 days in TSA medium at 28°C was transferred to a test tube containing 5 mL of King's B medium containing 5mM L-tryptophan (Trp) and cultured in a shaking incubator at 28°C and 200 rpm for 1 day. Cultured. 1 mL of the culture medium was transferred to a flask containing 100 mL of King's B medium containing 5mM Trp, and then cultured in a shaking incubator at 28°C and 200 rpm for 2 days. The culture was centrifuged at 8,000 Absorbance was measured at 530 nm using a solution in which 1 ml of King's B medium containing 5mM Trp and 1 ml of Salkowski reagent were reacted in the dark for 30 minutes as a standard.
4-2. 사이드로포어(Siderophore) 함량4-2. Siderophore content
28℃의 TSA 배지에서 2일 동안 배양한 KJB-1의 단일 콜로니를 CDLIM(Chemically Defined Low Iron Medium) 배지(리터당: 2 g K2SO4, 3 g K2HPO4, 1 g NaCl 및 5 g NH4Cl with trace elements: 80 mg MgSO4·7H2O, 2 mg ZnSO4·7H2O, 100 mg CaCl2·2H2O, 5 μg CuSO4, 35 μg MnSO4·4H2O, 200 μg thiamine·HCl 및 25 ml glycerol; pH 7.0 ± 0.1) 5mL가 함유된 시험관으로 옮긴 후 28℃, 200rpm 조건의 진탕배양기에서 1일간 배양하였다. 1mL의 배양액을 100mL의 CDLIM 배지가 들어있는 플라스크로 옮긴 후 3일 동안 28℃, 200rpm 조건의 진탕배양기에서 배양하였다. 배양액을 10분 동안 4℃에서 8,000 x g 조건으로 원심분리 한 후, 상등액을 동일한 부피의 에틸 아세테이트로 추출하였다. 추출물을 증발기(evaporator)로 농축하고 1ml의 DMSO(Dimethyl Sulfoxide)로 회수하였다. 농축액 1ml와 CAS(Chrome azurol S) 시약 1ml를 반응시켰다. DMSO 1ml와 CAS 시약 1ml를 반응시킨 용액을 standard로 하고 630 nm에서 흡광도를 측정하여 사이드로포어 단위 백분율을 다음 식으로 계산하였다. A single colony of KJB-1 cultured for 2 days in TSA medium at 28°C was cultured in CDLIM (Chemically Defined Low Iron Medium) medium (per liter: 2 g K 2 SO 4 , 3 g K 2 HPO 4 , 1 g NaCl, and 5 g NH 4 Cl with trace elements: 80 mg MgSO 4 ·7H 2 O, 2 mg ZnSO 4 ·7H 2 O, 100 mg CaCl 2 ·2H 2 O, 5 μg CuSO 4 , 35 μg MnSO 4 ·4H 2 O, 200 μg It was transferred to a test tube containing 5 mL of thiamine·HCl and 25 ml of glycerol (pH 7.0 ± 0.1) and cultured for 1 day in a shaking incubator at 28°C and 200 rpm. 1 mL of culture was transferred to a flask containing 100 mL of CDLIM medium and cultured in a shaking incubator at 28°C and 200 rpm for 3 days. The culture was centrifuged at 8,000 xg at 4°C for 10 minutes, and the supernatant was extracted with an equal volume of ethyl acetate. The extract was concentrated using an evaporator and recovered with 1 ml of DMSO (Dimethyl Sulfoxide). 1ml of the concentrate was reacted with 1ml of CAS (Chrome azurol S) reagent. A solution of 1 ml of DMSO and 1 ml of CAS reagent was used as a standard, the absorbance was measured at 630 nm, and the percentage of siderophore units was calculated using the following formula.
siderophore 단위 백분율 = [(Ar - As)/Ar] Х 100Percentage of siderophore units = [(Ar - As)/Ar] Х 100
상기 Ar은 CDLIM/CAS 용액의 흡광도, As는 샘플 흡광도.Ar is the absorbance of the CDLIM/CAS solution, and As is the sample absorbance.
4-3. ACC(1-aminocyclopropane-1-carboxylic acid)-데미나아제(deaminse) 활성 여부4-3. ACC (1-aminocyclopropane-1-carboxylic acid)-deaminase activity
28℃의 TSA 배지에서 2일 동안 배양한 KJB-1의 단일 콜로니를 TSB 배지 5mL가 들어 있는 시험관으로 옮긴 후 28℃, 200rpm 조건의 진탕배양기에서 1일간 배양하였다. 50μL 의 배양액을 5mL의 DF salts minimal 배지(리터당: 4 g KH2PO4, 6 g Na2HPO4, 0.2 g MgSO4·7H2O, 2 g 글루코스(glucose), 2 g 글루콘산(gluconic acid) 및 2 g 시트르산(citric acid) with trace elements: 1 mg FeSO4·7H2O, 10 μg H3BO3, 11.1 μg MnSO4·H2O, 124.6 17 μg ZnSO4·7H2O, 78.22 μg CuSO4·5H2O, 10 μg MoO3, pH 7.2 및 2 g (NH4)2SO4) 가 들어 있는 시험관으로 옮겼다. 시험관을 28℃, 200rpm 조건의 진탕배양기에서 2일간 배양하였다. 50 μL의 배양액을 5mL의 DF salts minimal 배지((NH4)2SO4 대신 3mM의 ACC를 포함)가 들어 있는 시험관으로 옮긴 후 28℃, 200rpm 조건의 진탕배양기에서 3일간 배양하였다. ACC 데미나아제 활성은 3mM ACC를 함유하는 DF salts minimal 배지에서 균주의 성장률에 의해 측정하였다.A single colony of KJB-1 cultured in TSA medium at 28°C for 2 days was transferred to a test tube containing 5 mL of TSB medium and cultured in a shaking incubator at 28°C and 200 rpm for 1 day. 50 μL of culture medium was added to 5 mL of DF salts minimal medium (per liter: 4 g KH 2 PO 4 , 6 g Na 2 HPO 4 , 0.2 g MgSO 4 ·7H 2 O, 2 g glucose, 2 g gluconic acid) ) and 2 g citric acid with trace elements: 1 mg FeSO 4 ·7H 2 O, 10 μg H 3 BO 3 , 11.1 μg MnSO 4 ·H 2 O, 124.6 17 μg ZnSO 4 ·7H 2 O, 78.22 μg It was transferred to a test tube containing CuSO 4 ·5H 2 O, 10 μg MoO 3 , pH 7.2, and 2 g (NH 4 )2SO 4 ). The test tube was cultured in a shaking incubator at 28°C and 200 rpm for 2 days. 50 μL of the culture medium was transferred to a test tube containing 5 mL of DF salts minimal medium (containing 3mM ACC instead of (NH 4 )2SO 4 ) and cultured for 3 days in a shaking incubator at 28°C and 200 rpm. ACC deminase activity was measured by the growth rate of the strain in DF salts minimal medium containing 3mM ACC.
4-4. 인산염 가용화(Phosphate solubilization) 지수4-4. Phosphate solubilization index
28℃의 TSA 배지에서 2일 동안 배양한 KJB-1의 단일 콜로니를 TSB 배지 5mL가 들어 있는 시험관으로 옮긴 후 28℃, 200rpm 조건의 진탕배양기에 배양하였다. PVK(Pikovskaya) 아가 배지(리터당: 10 g 글루코스, 5 g Ca3(PO4), 0.5 g (NH4)2SO4, 0.2 g NaCl, 0.1 g MgSO4·7H2O, 0.2 g KCl 및 0.5 g 효모 추출물(yeast extract) with trace elements: 2 mg FeSO4·7H2O 및 2 mg MnSO4·H2O) 가운데에 놓인 페이퍼 디스크에 20 μl의 배양액(OD600 0.3)을 접종하여 10일간 배양한 후, halo 생성을 관찰하고, 인산염 가용화 지수(Phosphate solubilization index, PSI)를 다음 식으로 계산하였다.A single colony of KJB-1 cultured for 2 days in TSA medium at 28°C was transferred to a test tube containing 5 mL of TSB medium and cultured in a shaking incubator at 28°C and 200 rpm. PVK (Pikovskaya) agar medium (per liter: 10 g glucose, 5 g Ca 3 (PO 4 ), 0.5 g (NH 4 )2SO 4 , 0.2 g NaCl, 0.1 g MgSO 4 ·7H 2 O, 0.2 g KCl and 0.5 g Yeast extract with trace elements: 2 mg FeSO 4 ·7H 2 O and 2 mg MnSO 4 ·H 2 O) 20 μl of culture medium (OD600 0.3) was inoculated on a paper disk placed in the center and cultured for 10 days. , halo production was observed, and the phosphate solubilization index (PSI) was calculated using the following equation.
Phosphate solubilization index (PSI) = [(콜로니 직경 + halo zone 직경)/ 콜로니 직경]Phosphate solubilization index (PSI) = [(colony diameter + halo zone diameter)/colony diameter]
그 결과, 하기 표 4에 나타낸 바와 같이, 바실러스 벨레젠시스 KJB-1는 IAA을 79.5±6.1 μg ml-1로 생산하고, 사이드로포어를 89.45±2.62% 함유하고, 인산염 가용화 지수는 0이며, ACC-데미나아제 활성은 없음을 확인하였다.As a result, as shown in Table 4 below, Bacillus velegensis KJB-1 produces IAA at 79.5 ± 6.1 μg ml-1, contains 89.45 ± 2.62% of siderophores, and has a phosphate solubilization index of 0; It was confirmed that there was no ACC-deminase activity.
상기 균주의 IAA 생산량, 사이드로포어 함량 및 인산염 가용화 지수에 대한 결과는 도 5에 도시하였다.The results of the IAA production, siderophore content, and phosphate solubilization index of the strain are shown in Figure 5.
strain
strain
(μg ml-1)IAA production
(μg ml-1)
실시예 5: 신규 바실러스 벨레젠시스 균주의 길항활성 관련 유전자 검출Example 5: Detection of genes related to antagonistic activity of new Bacillus belegensis strains
바실러스 벨레젠시스 KJB-1의 길항활성 관련 생합성 유전자인 바실리신(bacD, 749bp), 바실로마이신(bmyA, 875bp), 이투린(ituA, 647bp), 서팩틴(srfA, 441bp), 펜기신(fenD, 964bp) 및 쯔비테르미신(zwiA, 779bp) 유전자를 PCR을 통해 검출하였다.The antagonistic activity-related biosynthetic genes of Bacillus belegensis KJB-1 are bacillicin ( bacD , 749 bp), bacillomycin ( bmyA , 875 bp), iturin ( ituA , 647 bp), surfactin ( srfA , 441 bp), and penicin ( fenD , 964 bp) and zwittermicin ( zwiA , 779 bp) genes were detected by PCR.
구체적으로, 2일 동안 28℃의 TSA 배지에서 배양한 KJB-1의 단일 콜로니를 TSB 배지 3mL 가 들어 있는 시험관으로 옮긴 후 28℃, 200rpm 조건의 진탕배양기에서 배양하였다. 배양액을 Wizard® Genomic DNA Purification Kit를 이용하여 게놈 DNA를 추출하였다. 추출한 게놈 DNA를 주형으로 하여 PCR을 수행하였다. Specifically, a single colony of KJB-1 cultured in TSA medium at 28°C for 2 days was transferred to a test tube containing 3 mL of TSB medium and then cultured in a shaking incubator at 28°C and 200 rpm. Genomic DNA was extracted from the culture medium using the Wizard ® Genomic DNA Purification Kit. PCR was performed using the extracted genomic DNA as a template.
PCR 증폭에 사용된 프라이머 세트는 하기 표 5와 같다.The primer sets used for PCR amplification are listed in Table 5 below.
(Bacilysin) bacD
(Bacilysin)
(2008)Chung et al.
(2008)
(Bacillomycin D) bmyA
(Bacillomycin D)
(Fengycin) fenD
(Fengycin)
(Iturin A) ituA
(Iturin A)
(Surfactin) srfA
(Surfactin)
(Zwittermicin A) zwiA
(Zwittermicin A)
PCR은 초기 변성반응(95℃ 3분) 후, 증폭반응(변성; 95℃ 30초, 결합; 55℃ 45초, 신장; 72℃ 1분)을 30회 반복하였으며, 최종 신장반응은 72℃에서 5분간 실시하였다. 증폭한 PCR 산물은 전기영동 하여 산물 존재 여부 및 크기를 확인하였다. PCR 산물은 1.0% 아가로스 겔에서 검출되었다.PCR was performed 30 times with an initial denaturation reaction (95°C for 3 minutes) followed by an amplification reaction (denaturation; 95°C for 30 seconds; binding; 55°C for 45 seconds; extension; 72°C for 1 minute). The final extension reaction was performed at 72°C. This was carried out for 5 minutes. The amplified PCR product was subjected to electrophoresis to confirm the presence and size of the product. PCR products were detected on a 1.0% agarose gel.
그 결과, 도 6에 도시한 바와 같이, 바실리신, 바실로마이신, 서팩틴 및 펜기신이 검출되어, 바실러스 벨레젠시스 KJB-1이 상기 4종의 유전자를 보유하고 있음을 확인하였다.As a result, as shown in Figure 6, bacillicin, bacillomycin, surfactin, and penicin were detected, confirming that Bacillus velegensis KJB-1 possesses the above four genes.
상기 바실러스 벨레젠시스 KJB-1 균주는 부다페스트 조약 하의 국제기탁기관인 국립농업과학원 미생물은행(Korean Agricultural Culture Collection, KACC)에 2021년 8월 3일자로 기탁하여 수탁번호 KACC 92360P를 부여 받았다.The Bacillus belegensis KJB-1 strain was deposited on August 3, 2021 at the Korean Agricultural Culture Collection (KACC) of the National Academy of Agricultural Sciences, an international depositary institution under the Budapest Treaty, and was given accession number KACC 92360P.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.
<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Novel Bacillus velezensis strain and uses thereof <130> KPA211673-KR <160> 22 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R <400> 2 ggttaccttg ttacgactt 19 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> gyrA_F <400> 3 cagtcaggaa atgcgtacgt ctt 23 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> gyrA_R <400> 4 caaggtaatg ctccaggcat tgct 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 2292f <400> 5 gacgtgggat ggctacaact 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 3354r <400> 6 attgtcgcct ttaacgatgg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 70f <400> 7 acagagcttg gcgttgaagt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1013r <400> 8 gcttcttggc tgaatgaagg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 550f <400> 9 gagcttgaag tkgttgaagg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1497r <400> 10 tgagcgtgtw acttttgtwg 20 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BACD-F1 <400> 11 aaaaacagta ttggtyatcg ctga 24 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> BACD-R1 <400> 12 ccatgatgcc ttckatrctg at 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BACC1F <400> 13 gaaggacacg gcagagagtc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BACC1R <400> 14 cgctgatgac tgttcatgct 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FEND1F <400> 15 tttggcagca ggagaagttt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FEND1R <400> 16 gctgtccgtt ctgctttttc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITUD1F <400> 17 gatgcgatct ccttggatgt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITUD1R <400> 18 atcgtcatgt gctgcttgag 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SUR3F <400> 19 acagtatgga ggcatggtc 19 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SUR3R <400> 20 ttccgccact ttttcagttt 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ZWITF2 <400> 21 ttgggagaat atacagctct 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ZWITR1 <400> 22 gaccttttga aatgggcgta 20 <110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Novel Bacillus velezensis strain and uses thereof <130> KPA211673-KR <160> 22 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R <400> 2 ggttaccttg ttacgactt 19 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> gyrA_F <400> 3 cagtcaggaa atgcgtacgt ctt 23 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> gyrA_R <400> 4 caaggtaatg ctccaggcat tgct 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 2292f <400> 5 gacgtgggat ggctacaact 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223>3354r <400> 6 attgtcgcct ttaacgatgg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223>70f <400> 7 acagagcttg gcgttgaagt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1013r <400> 8 gcttcttggc tgaatgaagg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223>550f <400> 9 gagcttgaag tkgttgaagg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1497r <400> 10 tgagcgtgtw acttttgtwg 20 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223>BACD-F1 <400> 11 aaaaacagta ttggtyatcg ctga 24 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> BACD-R1 <400> 12 ccatgatgcc ttckatrctg at 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BACC1F <400> 13 gaaggaacacg gcagagagtc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BACC1R <400> 14 cgctgatgac tgttcatgct 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FEND1F <400> 15 tttggcagca ggagaagttt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FEND1R <400> 16 gctgtccgtt ctgctttttc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITUD1F <400> 17 gatgcgatct ccttggatgt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITUD1R <400> 18 atcgtcatgt gctgcttgag 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SUR3F <400> 19 acagtatgga ggcatggtc 19 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SUR3R <400> 20 ttccgccact ttttcagttt 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223>ZWITF2 <400> 21 ttggggagaat atacagctct 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223>ZWITR1 <400> 22 gaccttttga aatgggcgta 20
Claims (10)
A Bacillus velezensis KJB-1 strain deposited under the accession number KACC 92360P, which has antifungal activity against the pestilential pathogen Phytophthora sojae .
The strain according to claim 1, wherein the strain has Phytophthora small blight pathogen inhibitory activity.
The strain of claim 1, wherein the strain has mycelial growth inhibitory activity of Phytophthora small blight bacteria.
Phytophthora preparations comprising Bacillus velezensis KJB-1 ( Bacillus velezensis KJB-1) strain deposited with accession number KACC 92360P, its spores, its culture, concentrate of the culture, extract, and dried material of the culture Antifungal composition for.
A composition for controlling Phytophthora late blight bacteria, comprising the antifungal composition of claim 7.
The composition of claim 8, wherein the late blight bacteria are soybean late blight bacteria.
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