KR102612547B1 - Novel bacteriophage effective for soft rot bacteria and use thereof - Google Patents
Novel bacteriophage effective for soft rot bacteria and use thereof Download PDFInfo
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- KR102612547B1 KR102612547B1 KR1020220044185A KR20220044185A KR102612547B1 KR 102612547 B1 KR102612547 B1 KR 102612547B1 KR 1020220044185 A KR1020220044185 A KR 1020220044185A KR 20220044185 A KR20220044185 A KR 20220044185A KR 102612547 B1 KR102612547 B1 KR 102612547B1
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- bacteriophage
- pectobacterium
- composition
- cabbage
- odoriferum
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Abstract
본 발명은 신규 박테리오파지 및 이의 용도에 관한 것으로, 구체적으로 펙토박테리움 오도리페룸(Pectobacterium odoriferum)에 특이적인 사멸능을 갖는, 기탁번호 KCTC14860BP인 분리된 박테리오파지, 상기 박테리오파지를 유효성분으로 포함하는 농약 조성물, 상기 박테리오파지를 유효성분으로 포함하는 항균제, 상기 박테리오파지를 유효성분으로 포함하는 소독제 또는 세척제, 상기 박테리오파지를 처리하는 단계를 포함하는 펙토박테리움 카로토보룸 박테리아를 용균하는 방법 및 상기 박테리오파지를 처리하는 단계를 포함하는 배추 무름병을 방제하는 방법에 관한 것이다.
본 발명의 신규 박테리오파지는 펙토박테리움 오도리페룸(Pectobacterium odoriferum)에 특이적인 사멸능을 가지며, 내산성 및 내열성이 우수하므로, 상기 펙토박테리움 카로토보룸에 의하여 야기되는 배추 무름병 예방 또는 치료에 활용할 수 있을 뿐 아니라, 향균제, 소독제, 또는 세척제 등에도 광범위하게 이용될 수 있다.The present invention relates to a new bacteriophage and its use, and specifically to an isolated bacteriophage with accession number KCTC14860BP, which has a specific killing ability against Pectobacterium odoriferum , and a pesticide composition containing the bacteriophage as an active ingredient. , an antibacterial agent containing the bacteriophage as an active ingredient, a disinfectant or detergent containing the bacteriophage as an active ingredient, a method for lysing Pectobacterium carotovorum bacteria comprising the step of treating the bacteriophage, and treating the bacteriophage. It relates to a method for controlling soft rot of cabbage comprising the following steps.
The new bacteriophage of the present invention has a specific killing ability against Pectobacterium odoriferum and has excellent acid resistance and heat resistance, so it can be used to prevent or treat cabbage soft rot caused by Pectobacterium carotovorum. In addition, it can be widely used as an antibacterial agent, disinfectant, or cleaning agent.
Description
본 발명은 신규 박테리오파지 및 이의 용도에 관한 것으로, 구체적으로 펙토박테리움 오도리페룸(Pectobacterium odoriferum)에 특이적인 사멸능을 갖는, 기탁번호 KCTC14860BP인 분리된 박테리오파지, 상기 박테리오파지를 유효성분으로 포함하는 농약 조성물, 상기 박테리오파지를 유효성분으로 포함하는 항균제, 상기 박테리오파지를 유효성분으로 포함하는 소독제 또는 세척제, 상기 박테리오파지를 처리하는 단계를 포함하는 펙토박테리움 오도리페룸 박테리아를 용균하는 방법 및 상기 박테리오파지를 처리하는 단계를 포함하는 배추 무름병을 방제하는 방법에 관한 것이다.The present invention relates to a new bacteriophage and its use, and specifically to an isolated bacteriophage with accession number KCTC14860BP, which has a specific killing ability against Pectobacterium odoriferum , and a pesticide composition containing the bacteriophage as an active ingredient. , an antibacterial agent containing the bacteriophage as an active ingredient, a disinfectant or detergent containing the bacteriophage as an active ingredient, a method of lysing Pectobacterium odoriferum bacteria comprising the step of treating the bacteriophage, and the step of treating the bacteriophage. It relates to a method of controlling cabbage soft rot, including.
무름병은 독특한 냄새가 나면서 흐물흐물해져서 썩는 식물의 병해를 지칭하는 것으로, 흔히 연부병이라고도 한다. 무름병에 걸리면 세포벽 중간층의 펙틴질이 녹아 처음에는 물기가 보이는 것에 그치나, 점차로 물러져 썩고 액체처럼 흐물흐물해진다. 무름병은 채소에 많이 나타나는 병해이나, 그 밖에 토마토, 고구마, 감자에도 많이 나타난다.Soft rot refers to a disease of plants that causes them to rot and become mushy with a unique odor, and is also commonly called soft rot. When soft rot occurs, the pectin in the middle layer of the cell wall dissolves and appears watery at first, but it gradually softens, rots, and becomes runny like liquid. Soft rot is a disease that often appears on vegetables, but it also appears on tomatoes, sweet potatoes, and potatoes.
배추를 포함하는 야채에 무름병을 발생시키는 펙토박테리움 오도리페룸(Pectobacterium odoriferum; Po)은 최근 각종 야채에서 무름병을 일으키는 파괴적인 병원체로 부상되고 있다. 펙토박테리움 오도리페룸(Pectobacterium odoriferum; Po)은 숙주 범위가 좁고 치커리에서 잘 분리되는 것으로 여겨져 왔다(Gallois, et al., 1992, Lan 등, 2013). 그러나 다양한 저장 야채에서 무름병을 유발하고, P. 카로토보룸(Waleron et al., 2014, Li et al., 2020)에 의한 증상보다 더 심한 증상을 나타낸다고 보고된다. 상기 병원체는 생장 적온이 26~28℃이나, 다른 식물 병원 세균과는 달리 상온에서도 잘 자라, 여름철 배추 생산에 가장 큰 장애 요인이다. 무름병균은 배추를 비롯하여 거의 모든 식물에 침입하여 발병할 수 있는데, 한번 침입하면 주변의 다른 채소로 옮겨가 발병을 전파한다. Pectobacterium odoriferum (Po), which causes soft rot in vegetables including Chinese cabbage, has recently emerged as a destructive pathogen that causes soft rot in various vegetables. Pectobacterium odoriferum (Po) has a narrow host range and has been considered to be well isolated from chicory (Gallois, et al., 1992, Lan et al., 2013). However, it is reported that it causes soft rot in various stored vegetables and shows more severe symptoms than those caused by P. carotovorum (Waleron et al., 2014, Li et al., 2020). The optimal growth temperature for this pathogen is 26-28℃, but unlike other plant pathogenic bacteria, it grows well at room temperature, making it the biggest obstacle to cabbage production in the summer. Soft rot bacteria can invade almost any plant, including cabbage, and cause disease. Once invaded, it moves to other nearby vegetables and spreads the disease.
최근 웰빙 문화의 확산으로 신선 채소의 소비가 급증하고 있는데, 신선 채소에서도 무름병 발병이 급증하고 있다. 특히, 진열대에 진열한 신선 채소는 신선도 유지를 위하여 수분을 계속 공급하는데, 수분을 통해서도 무름병균이 전파된다. 무름병은 배추나 신선 채소에 한번 발생하면 방제가 매우 까다로운데, 소비자들이 배추나 채소를 익히지 않고, 대부분 그대로 섭취하기 때문이다. 또한, 병원균인 펙토박테리움 오도리페룸은 토양에서 비교적 오랫동안 생존이 가능하며, 농약에 의한 방제 효과도 일반적으로 낮은 것으로 알려져 있다.Recently, the consumption of fresh vegetables is rapidly increasing due to the spread of wellness culture, and the incidence of soft rot is also rapidly increasing in fresh vegetables. In particular, fresh vegetables displayed on shelves are continuously supplied with moisture to maintain freshness, and soft rot bacteria are also spread through moisture. Once soft rot occurs on cabbage or fresh vegetables, it is very difficult to control because most consumers consume cabbage or vegetables as is rather than cooking them. In addition, the pathogen Pectobacterium odoriferum can survive in the soil for a relatively long time, and the control effect of pesticides is generally known to be low.
따라서, 친환경적이면서도 효과적인 방제기술 개발이 시급한데, 지금까지 각종 작물의 병 방제는 주로 저항성 품종의 개발과 농약 사용에 의한 화학적 약제에 크게 의존하였다. 이 중 저항성 품종의 개발에는 많은 기술적, 경제적 그리고 시간적 제약이 따르며, 새로운 저항성 품종을 선발하여도 번식 속도가 빠른 세균이 바로 적응할 수 있는 능력을 키우게 되어 저항성을 상실하게 되는 문제가 발생한다. 그리고 화학 약제로 알려진 항생제는 동물 세균에도 같이 작용하여 항생제 저항성 균을 조장할 수 있는 문제를 발생한다.Therefore, there is an urgent need to develop eco-friendly and effective control technologies. Until now, disease control of various crops has largely relied on the development of resistant varieties and chemical agents through the use of pesticides. Among these, the development of resistant varieties is subject to many technical, economic and time constraints, and even if new resistant varieties are selected, bacteria with a fast reproduction rate develop the ability to adapt immediately, causing the problem of loss of resistance. And antibiotics, known as chemical agents, also act on animal bacteria, causing the problem of promoting antibiotic-resistant bacteria.
이에 대한 대응책으로 생물학적 방제 기법이 현재 부각되고 있는데, 병원균에 대해 길항 및 경쟁 미생물을 분리하여 배양한 후, 토양, 종자 또는 식물체에 직접 처리하여 병원균의 침입을 막거나, 살균 물질을 분비하여 병원성 세균의 증식을 억제하는 방법이 알려져 있고, 세균만을 특이적으로 공격하여 용균시키는 박테리오파지라는 바이러스를 이용하는 방법이 알려져 있다. 이와 같은 생물학적 방제는 유기 합성 농약이 가지는 환경오염, 약해 및 잔류 독성의 문제가 없다는 점에서 매우 바람직한 것으로 받아들여지고 있다.As a countermeasure to this, biological control techniques are currently emerging. They isolate and culture antagonistic and competitive microorganisms against pathogens and then treat them directly with soil, seeds, or plants to prevent the invasion of pathogens, or secrete bactericidal substances to prevent pathogenic bacteria. There is a known method of inhibiting the proliferation of , and a method using a virus called bacteriophage that specifically attacks and lyses only bacteria is known. Such biological control is accepted as highly desirable because it does not have the problems of environmental pollution, toxicity, and residual toxicity that organic synthetic pesticides have.
파지가 발견된 1910년부터 1930년대에 걸쳐 파지는 숙주 특이성과 숙주 세균의 내독소, 외독소 등 그 당시 과학적으로 풀지 못하는 문제 및 1929년 플래밍에 의해 발견된 페니실린과 1943년 워크스만에 의한 스트렙토마이신으로 대표되는 화학적 치료제(약)의 발견으로 인해 그 활용 및 개발이 잠시 주춤하였다.From 1910, when phages were discovered, to the 1930s, phages were used to solve problems that could not be solved scientifically at the time, such as host specificity and endotoxins and exotoxins of host bacteria, as well as penicillin discovered by Fleming in 1929 and streptocystis discovered by Worksman in 1943. Due to the discovery of chemical therapeutic agents (drugs) represented by mycin, their use and development slowed down for a while.
한편, 그 당시에 파지를 치료제로 사용하기에는 몇 가지 문제가 있었다. 그 중, 특이성이 높아 몇몇 균주에만 특이적으로 작용하는 점과 저항성이 높아 사용 후 바로 저항성 균이 나타나 치료제로서의 효과가 바로 없어진다는 것이 가장 문제였다. 반면에 화학적 치료제는 항균 범위가 넓으며, 최초 적용 시에는 내성균 문제도 없었기 때문에 광범위하게 활용되었고, 그 결과 파지 요법은 거의 사라지게 되었다. 그 후로 1950년대에 WHO에 의해 파지 요법은 무효 선언되었으며, 동구권과 구소련연방에서만 그 명맥이 겨우 유지되어 왔다.Meanwhile, there were several problems with using phages as a treatment at that time. Among them, the biggest problem was that it had high specificity, acting specifically on only a few strains, and high resistance, so resistant bacteria appeared immediately after use and the effect as a treatment was immediately lost. On the other hand, chemical treatments have a wide antibacterial spectrum and there was no problem with resistant bacteria when first applied, so they were widely used, and as a result, phage therapy almost disappeared. Since then, phage therapy was declared invalid by the WHO in the 1950s, and its existence has barely been maintained only in the Eastern Bloc and the former Soviet Union.
그러나 1980년대에 들어 파지 요법에 대한 생각이 바뀌게 되는데, 1982년 영국의 연구자그룹인 스미스와 휴긴스는 마우스를 이용한 뇌막염 치료 모델 실험을 하였다. 마우스의 피하 근육 내에 균을 주사하고 반대쪽의 피하에는 균의 특이적인 용균 파지를 주사한 결과, 파지를 주사하지 않은 대조군은 100% 폐사한 것에 비해 파지를 주사한 경우(실험군) 100% 생존하였으며, 항생제(스트렙토마이신 등)의 효과보다도 우월한 것이 확인되었다. However, in the 1980s, ideas about phage therapy changed. In 1982, Smith and Huggins, a group of British researchers, experimented with a meningitis treatment model using mice. As a result of injecting bacteria into the subcutaneous muscle of a mouse and injecting specific lytic phages of the bacteria into the subcutaneous area on the other side, 100% of mice injected with phages (experimental group) survived, compared to 100% death in the control group that was not injected with phages. It was confirmed that the effect was superior to that of antibiotics (streptomycin, etc.).
본 발명자들은 펙토박테리움 오도리페룸의 방제 방법을 개발하고자 예의 연구 노력한 결과, 펙토박테리움 오도리페룸에 특이적으로 용균 활성을 보이고, 여러 가지 환경적 요인 (온도, pH 및 자외선)에 대하여 저항성을 보여, 배추 무름병에 대한 파지테라피로서 사용될 수 있는, 신규 박테리오파지를 분리하여, 본 발명을 완성하였다.The present inventors have made extensive research efforts to develop a method for controlling Pectobacterium odoriferum, and as a result, it shows lytic activity specifically against Pectobacterium odoriferum and is resistant to various environmental factors (temperature, pH, and ultraviolet rays). By isolating a new bacteriophage that can be used as phage therapy for cabbage soft rot, the present invention was completed.
본 발명의 하나의 목적은 펙토박테리움 오도리페룸(Pectobacterium odoriferum)에 특이적인 사멸능을 갖는, 기탁번호 KCTC14860BP인 분리된 박테리오파지를 제공하는 것이다.One object of the present invention is to provide an isolated bacteriophage, accession number KCTC14860BP, which has a specific killing ability against Pectobacterium odoriferum .
본 발명의 다른 하나의 목적은 상기 박테리오파지를 유효성분으로 포함하는 농약조성물을 제공하는 것이다.Another object of the present invention is to provide a pesticide composition containing the bacteriophage as an active ingredient.
본 발명의 또 다른 하나의 목적은 상기 박테리오파지를 유효성분으로 포함하는 항균제를 제공하는 것이다.Another object of the present invention is to provide an antibacterial agent containing the bacteriophage as an active ingredient.
본 발명의 또 다른 하나의 목적은 박테리오파지를 유효성분으로 포함하는 소독제 또는 세척제를 제공하는 것이다.Another object of the present invention is to provide a disinfectant or cleaner containing bacteriophage as an active ingredient.
본 발명의 또 다른 하나의 목적은 상기 박테리오파지를 처리하는 단계를 포함하는 펙토박테리움 오도리페룸(Pectobacterium odoriferumm)을 용균하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for lysing Pectobacterium odoriferum including the step of treating the bacteriophage.
본 발명의 또 다른 하나의 목적은 상기 박테리오파지를 처리하는 단계를 포함하는 배추 무름병을 방제하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for controlling soft rot of cabbage comprising treating the bacteriophage.
본 발명의 또 다른 하나의 목적은 상기 박테리오파지를 유효성분으로 포함하는 펙토박테리움 오도리페룸(Pectobacterium odoriferum) 용균용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for lysing Pectobacterium odoriferum containing the bacteriophage as an active ingredient.
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, 펙토박테리움 펙토박테리움 오도리페룸(Pectobacterium odoriferum)에 특이적인 사멸능을 갖는, 기탁번호 KCTC14860BP인 분리된 박테리오파지를 제공한다.One aspect of the present invention for achieving the above object provides an isolated bacteriophage, accession number KCTC14860BP, which has a specific killing ability for Pectobacterium odoriferum .
본 발명에서의 용어, "박테리오파지(bacteriophage)"란 특정한 타겟 박테리아를 감염시키는 바이러스이다. 단백질 캡시드(capsid)로 싸여 있는 박테리오파지의 머리는 핵산 게놈으로 이루어져 있으며, 몇몇 박테리오파지는 지질 또는 단백질로 구성된 꼬리를 갖는다. 대부분의 박테리오파지는 유전 물질로서 이중나선 DNA를 포함한다.As used herein, the term “bacteriophage” refers to a virus that infects a specific target bacteria. The head of a bacteriophage, enclosed in a protein capsid, consists of a nucleic acid genome, and some bacteriophages have a tail composed of lipids or proteins. Most bacteriophages contain double-stranded DNA as their genetic material.
알려진 박테리오파지의 95% 이상이 카우도비랄레스(Caudovirales) 목(order)에 속한다(Maniloff J and Ackermann H-W. 1998. Arch. Virology. 143: 2051-2063.). 박테리오파지의 주요 세 가지 과(family)는 형태적인 특징으로 구분되며, 특히 꼬리 모양으로 구분된다. 마이오비리대(Myoviridae) 과에 속하는 파지는 이중층의 수축성 꼬리를 가지며, 시포비리대(Siphoviridae) 과에 속하는 파지는 긴 탄성의 꼬리를 가지며, 포도비리대(Podoviridae) 과에 속하는 파지는 짧고 뭉툭한 꼬리를 갖는다(Deveau H, 2006, Appl. Environ. Microbiol. 72: 4338-4346.).More than 95% of known bacteriophages belong to the Caudovirales order (Maniloff J and Ackermann H-W. 1998. Arch. Virology. 143: 2051-2063.). The three major families of bacteriophages are distinguished by their morphological characteristics, especially their tail shape. Phages belonging to the Myoviridae family have a double-layered contractile tail, phages belonging to the Siphoviridae family have a long elastic tail, and phages belonging to the Podoviridae family have a short, blunt tail. (Deveau H, 2006, Appl. Environ. Microbiol. 72: 4338-4346.).
박테리오파지는 물, 흙, 및 식물의 안 또는 밖의 모든 환경에서 찾아볼 수 있다. 박테리오파지는 타겟 박테리아에 부착하여 숙주 안으로 자신의 DNA를 주입하며, 숙주 내에서 자가복제하여 박테리아의 죽음을 야기한다. 박테리오파지는 동물 또는 식물 세포에 대해서는 상대적으로 안전하며(Kutter E and Sulakvelidze A. 2004. CRC Press.), 다른 유익한 미생물에 대해서도 영향을 미치지 않는다. 또한, 박테리오파지는 숙주 박테리아성 환경에서 비교적 쉽게 분리된다.Bacteriophages can be found in all environments, inside or outside water, soil, and plants. Bacteriophages attach to target bacteria, inject their DNA into the host, and self-replicate within the host, causing the death of the bacteria. Bacteriophages are relatively safe for animal or plant cells (Kutter E and Sulakvelidze A. 2004. CRC Press.) and do not affect other beneficial microorganisms. Additionally, bacteriophages are relatively easily isolated from the host bacterial environment.
최근, 박테리오파지는 식물병원성 박테리아, 예를 들어 Dickeya solani(Czajkowski R,2014, Plant Pathol. 63: 758-772.), Pectobacterium carotovorum(Lim JA, J. Microbiol. Biotechnol. 19 23: 1147-1153.), 또는 Ralstonia solanacearum(Young BJ, J. Microbiol. Biotechnol. 22:16 1613-1620.)에 의하여 야기되는 식물 질병을 방제하기 위한 파지테라피(phage therapy)의 물질로서 많이 연구되고 있다. 그러나 박테리오파지의 유전적 다양성으로 인해, 식물 질병 방제를 위해서는 특정 박테리아 특이적으로 사멸능을 갖는 특정 박테리오 파지를 규명하는 것이 필요하다.Recently, bacteriophages have been used against phytopathogenic bacteria, such as Dickeya solani (Czajkowski R, 2014, Plant Pathol. 63: 758-772.), Pectobacterium carotovorum (Lim JA, J. Microbiol. Biotechnol. 19 23: 1147-1153.) , or Ralstonia solanacearum (Young BJ, J. Microbiol. Biotechnol. 22:16 1613-1620.) is being widely studied as a phage therapy material for controlling plant diseases caused by . However, due to the genetic diversity of bacteriophages, it is necessary to identify specific bacteriophages that have the ability to kill specific bacteria in order to control plant diseases.
한편, 본 발명의 박테리오파지는 기존의 알려진 파지와 상이한 신규한 파지로서, 본 발명자들에 의해 펙토박테리움 오도리페룸에 특이적 사멸능을 갖는 것으로 최초로 규명되었다.Meanwhile, the bacteriophage of the present invention is a novel phage different from existing known phages, and was first identified by the present inventors as having a specific killing ability against Pectobacterium odoriferum.
또한 본 발명의 박테리오파지는 본 발명자들에 의해 펙토박테리움 카로토보룸에 대해서도 추가로 특이적인 사멸능을 갖는 것으로 확인되었다.In addition, the bacteriophage of the present invention was confirmed by the present inventors to have an additional specific killing ability against Pectobacterium carotovorum.
본 발명의 일 실시예에서는, 펙토박테리움 오도리페룸에 용균력을 갖는 박테리오파지를 얻기 위하여 오버레이 어세이를 실시하였으며, 이를 통해, 펙토박테리움 오도리페룸에 용균력을 갖는 박테리오파지를 분리하였다. In one embodiment of the present invention, an overlay assay was performed to obtain a bacteriophage having lytic activity on Pectobacterium odoriferum, and through this, bacteriophages with lytic activity on Pectobacterium odoriferum were isolated.
본 발명의 다른 하나의 양태는 상기 박테리오파지를 유효성분으로 포함하는 농약 조성물을 제공한다.Another aspect of the present invention provides a pesticide composition containing the bacteriophage as an active ingredient.
이때, 상기 박테리오파지의 정의는 전술한 바와 같다.At this time, the definition of the bacteriophage is as described above.
*본 발명에서의 용어, "농약"이란, 농작물의 보호와 생장에 사용되는 약제를 의미하며, 농작물의 재배 또는 저장 중 발생하는 병충, 해충, 잡초 등을 방제하는데 사용하는 화학농약 및 생물농약, 농작물의 생리기능을 증진 또는 억제하는데 사용되는 생장조정제, 약효를 증진시키는 보조제 등을 총칭한다.*The term "pesticide" in the present invention refers to a drug used for the protection and growth of crops, including chemical pesticides and biological pesticides used to control pests, pests, and weeds that occur during cultivation or storage of crops. It is a general term for growth regulators used to enhance or suppress the physiological functions of crops and supplements that enhance medicinal efficacy.
본 발명의 농약 조성물은 펙토박테리움 오도리페룸에 특이적인 사멸능을 갖는 박테리오파지를 유효성분으로 포함하므로, 상기 농약 조성물은 배추 무름병(soft rot)의 예방 또는 치료에 사용될 수 있다.Since the pesticide composition of the present invention contains a bacteriophage having a specific killing ability for Pectobacterium odoriferum as an active ingredient, the pesticide composition can be used for the prevention or treatment of cabbage soft rot.
구체적으로, 상기 농약 조성물은 수간주사 조성물, 생물제제 조성물, 비료 조성물, 영양 조성물, 식물 강화 조성물, 식물 보호 조성물, 토양 개량 조성물(soil conditioner composition) 또는 생산량 증가 조성물(yield enhancer composition)일 수 있으나, 이에 제한되는 것은 아니다.Specifically, the pesticide composition may be an injectable composition, a biologic composition, a fertilizer composition, a nutritional composition, a plant strengthening composition, a plant protection composition, a soil conditioner composition, or a yield enhancer composition. It is not limited to this.
상기 농약 조성물의 제형으로는 수화제(wettable powder), 입상 수화제(water dispersible granule), 분제(dusting powder), 액상 수화제(suspension concentrate), 유제(emulsifiable concentrate), 정제(tablet), 액제(liquid) 또는 오일 현탁제(oil suspension)로 제제화될 수 있으나, 이에 제한되는 것은 아니다.Formulations of the pesticide composition include wettable powder, water dispersible granule, dusting powder, suspension concentrate, emulsifiable concentrate, tablet, liquid, or It may be formulated as an oil suspension, but is not limited thereto.
상기 농약 조성물은 유효량의 물과 함께, 하나 이상의 분산제를 포함할 수 있으며, 상기 분산제는 약 2 내지 약 60%(w/w)의 범위로 포함될 수 있다. Rhone Poulenc, Witco, Westvaco, International Speciality products, Crodachemicals, Borregaard, BASF, Rhodia, 등에서 판매되는 다양한 분산제가 농약 조성물에 사용하기 위해 상업적으로 입수가능하다.The pesticide composition may include one or more dispersants along with an effective amount of water, and the dispersants may be included in the range of about 2 to about 60% (w/w). A variety of dispersants are commercially available for use in pesticide compositions, sold by Rhone Poulenc, Witco, Westvaco, International Specialty products, Crodachemicals, Borregaard, BASF, Rhodia, etc.
상기 농약 조성물에 사용될 수 있는 분산제의 구체적인 예로, 폴리비닐피롤리돈, 폴리비닐알코올, 리그노술포네이트, 페닐 나프탈렌 술포네이트, 에톡시화(ethoxylated) 알킬 페놀, 에톡시화지방산, 알콕시화 선형 알코올, 환방향족(polyaromatic) 술포네이트, 소듐 알킬 아릴 술포네이트, 글리세릴에스테르, 말레산 무수물 공중합체, 포스페이트 에스테르, 아릴 술폰산과 포름알데히드의 축합반응 생성물, 알킬아릴 술폰산과 포름알데히드의 축합반응 생성물, 에틸렌 옥시드와 지방산 에스테르의 첨가 반응 생성물, 에틸렌 옥시드와 지방산 에스테르의 첨가반응 생성물의 염, 축합된(condensed) 나프탈렌의 술포네이트, 에틸렌 옥시드와 지방산 에스테르의 첨가 반응 생성물, 에틸렌 옥시드와 지방산 에스테르의 첨가 반응생성물의 염, 리그닌 유도체, 나프탈렌 포름알데히드 축합물, 이소데실술포숙신산하프 에스테르(half ester)의 소듐 염, 폴리카르복실레이트, 소듐 알킬벤젠술포네이트, 술폰화 나프탈렌의 소듐 염, 술폰화 나프탈렌의 암모늄 염, 폴리아크릴산의 염, 페놀술폰산의 염 또는 나프탈렌 술폰산의 염일 수 있으나, 이에 제한되는 것은 아니다.Specific examples of dispersants that can be used in the pesticide composition include polyvinylpyrrolidone, polyvinyl alcohol, lignosulfonate, phenyl naphthalene sulfonate, ethoxylated alkyl phenol, ethoxylated fatty acid, alkoxylated linear alcohol, and ring. Aromatic (polyaromatic) sulfonates, sodium alkyl aryl sulfonates, glyceryl esters, maleic anhydride copolymers, phosphate esters, condensation products of aryl sulfonic acids and formaldehyde, condensation products of alkylaryl sulfonic acids and formaldehyde, ethylene oxide. and addition reaction products of fatty acid esters, salts of addition reaction products of ethylene oxide and fatty acid esters, sulfonates of condensed naphthalene, addition reaction products of ethylene oxide and fatty acid esters, addition of ethylene oxide and fatty acid esters. Salts of reaction products, lignin derivatives, naphthalene formaldehyde condensate, sodium salt of isodecyl sulfosuccinic acid half ester, polycarboxylates, sodium alkylbenzene sulfonate, sodium salt of sulfonated naphthalene, sulfonated naphthalene It may be an ammonium salt, a salt of polyacrylic acid, a salt of phenolsulfonic acid, or a salt of naphthalene sulfonic acid, but is not limited thereto.
또한, 상기 농약 조성물은 습윤제, 결합제, 충진제(filler) 또는 유기 첨가제를 추가로 포함할 수 있다.Additionally, the pesticide composition may further include a wetting agent, binder, filler, or organic additive.
상기 습윤제는 상기 농약 조성물에 약 0.1 내지 5% 범위로 포함될 수 있으며, 구체적인 예로, 알킬 나프탈렌 술포네이트, 소듐 알킬 나프탈렌 술포네이트, 술폰화 알킬카르복실레이트의 소듐 염, 폴리옥시알킬화 에틸 페놀, 폴리옥시에톡시화(polyoxyethoylated) 지방성 알코올, 폴리옥시에톡시화 지방성 아민, 리그닌 유도체, 알칸 술포네이트, 알킬벤젠 술포네이트, 폴리카르복시산의 염, 술포숙신산의 에스테르의 염, 알킬나프탈렌술포네이트, 알킬벤젠술포네이트, 알킬폴리글리콜 에테르 술포네이트, 알킬 에테르 포스페이트, 알킬 에테르 술페이트 또는 알킬 술포숙신산 모노에스테르일 수 있으나, 이에 제한되는 것은 아니다.The wetting agent may be included in the pesticide composition in an amount of about 0.1 to 5%, and specific examples include alkyl naphthalene sulfonate, sodium alkyl naphthalene sulfonate, sodium salt of sulfonated alkylcarboxylate, polyoxyalkylated ethyl phenol, polyoxyalkyl phenol, Ethoxyethoylated fatty alcohols, polyoxyethoxylated fatty amines, lignin derivatives, alkane sulfonates, alkylbenzene sulfonates, salts of polycarboxylic acids, salts of esters of sulfosuccinic acid, alkylnaphthalenesulfonates, alkylbenzene sulfonates. , alkylpolyglycol ether sulfonate, alkyl ether phosphate, alkyl ether sulfate, or alkyl sulfosuccinic acid monoester, but is not limited thereto.
상기 결합제는 상기 농약 조성물에 약 0.1 내지 7%의 범위로 포함될 수 있으며, 구체적인 예로, 폴리비닐 알코올, 리그닌 유도체, 폴리비닐 피롤리돈, 폴리알킬피롤리돈, 카르복시메틸셀룰로오스, 크산탄 검, 폴리에톡시화 지방산, 폴리에톡시화 지방성 알코올, 에틸렌 옥시드 공중합체, 프로필렌 옥시드 공중합체, 폴리에틸렌 글리콜 또는 폴리에틸렌 옥시드일 수 있으나, 이에 제한되는 것은 아니다.The binder may be included in the pesticide composition in an amount of about 0.1 to 7%, and specific examples include polyvinyl alcohol, lignin derivatives, polyvinyl pyrrolidone, polyalkyl pyrrolidone, carboxymethyl cellulose, xanthan gum, poly It may be, but is not limited to, ethoxylated fatty acid, polyethoxylated fatty alcohol, ethylene oxide copolymer, propylene oxide copolymer, polyethylene glycol, or polyethylene oxide.
상기 충진제는 상기 농약 조성물에 약 0.1 내지 10%의 범위로 포함될 수 있으며, 구체적인 예로, 벤토나이트, 서브-벤토나이트(sub-bentonite), 아타풀자이트(attapulgite), 카올리나이트(kaolinite), 몬모릴로나이트, 보크사이트, 수화된 알루미나(hydrated alumina), 하소된(calcined) 알루미나, 규조토(diatomaceousearth), 백악(chalk), 백토(fuller's earth), 돌로마이트, 규조토(kieselguhr), 황토(loess), 프로필리트(prophyllite), 탈크(talc), 버미큘라이트(vermiculite), 석회암(limestone), 천연 및 합성 실리케이트(silicate), 실리카(silica) 또는 고령토(china clay)일 수 있으나, 이에 제한되는 것은 아니다.The filler may be included in the pesticide composition in an amount of about 0.1 to 10%, and specific examples include bentonite, sub-bentonite, attapulgite, kaolinite, montmorillonite, bauxite, Hydrated alumina, calcined alumina, diatomaceous earth, chalk, fuller's earth, dolomite, kieselguhr, loess, propyllite, talc. It may be (talc), vermiculite, limestone, natural and synthetic silicate, silica, or china clay, but is not limited thereto.
상기 유기 첨가제는 상기 농약 조성물에 약 0.01 내지 50%의 범위로 포함될 수 있으며, 구체적인 예로, 다량 영양소(macronutrient), 미량 영양소 퇴비 비료(micronutrient compost fertilizer), 천연 원소(natural element), 천연 유기체(natural organism), 트리코데르마(trichoderma), 부식산(humic acid) 추출물, 바실러스 투린지엔시스(bacillus thuringiensis), 바이러스, 천연 곰팡이, 식물 추출물, 제충국, 생물학적 제어 산물(biological control product), 천연 오일, 천연 추출물, 광물 또는 요소 군일 수 있으나, 이에 제한되는 것은 아니다. 상기 미량 영양소는 아연, 철, 망간, 구리, 보론, 코발트, 바나듐, 셀레늄, 실리콘 또는 니켈일 수 있으나, 이에 제한되는 것은 아니다. 상기 다량 영양소는 질소, 인, 칼륨, 칼슘 또는 마그네슘일 수 있으나, 이에 제한되는 것은 아니다.The organic additive may be included in the pesticide composition in an amount of about 0.01 to 50%, and specific examples include macronutrients, micronutrient compost fertilizer, natural elements, and natural organisms. organism, trichoderma, humic acid extract, bacillus thuringiensis, virus, natural fungus, plant extract, pyrethrum, biological control product, natural oil, natural It may be an extract, a mineral, or a group of elements, but is not limited thereto. The micronutrients may be zinc, iron, manganese, copper, boron, cobalt, vanadium, selenium, silicon, or nickel, but are not limited thereto. The macronutrients may be nitrogen, phosphorus, potassium, calcium, or magnesium, but are not limited thereto.
본 발명의 또 다른 하나의 양태는 상기 박테리오파지를 유효성분으로 포함하는 항균제를 제공한다.Another aspect of the present invention provides an antibacterial agent containing the bacteriophage as an active ingredient.
본 발명에서의 용어, "항균제"란, 미생물을 사멸시키거나 혹은 발육을 억제하는 물질을 의미하며, 방부제, 살균제 및 항생제를 총칭하는 것이다.The term “antibacterial agent” in the present invention refers to a substance that kills or inhibits the growth of microorganisms and is a general term for preservatives, disinfectants, and antibiotics.
본 발명의 박테리오파지는 펙토박테리움 오도리페룸에 대하여 특이적으로 높은 항균 활성을 나타낼 뿐만 아니라, 우수한 내열성 및 내산성을 갖는다. 따라서 상기 박테리오파지를 유효성분으로 포함하는 상기 항균제는 기존의 항균물질에 비하여 약물 내성 또는 저항성을 유도하지 않아 안전하며, 제품수명(life cycling)이 긴 신규 항균제로서 이용될 수 있다.The bacteriophage of the present invention not only exhibits high antibacterial activity specifically against Pectobacterium odoriferum, but also has excellent heat resistance and acid resistance. Therefore, the antibacterial agent containing the bacteriophage as an active ingredient is safe because it does not induce drug resistance or resistance compared to existing antibacterial substances, and can be used as a new antibacterial agent with a long product life cycle.
본 발명의 또 다른 하나의 양태는 상기 박테리오파지를 유효성분으로 포함하는 소독제 또는 세척제를 제공한다.Another aspect of the present invention provides a disinfectant or cleaner containing the bacteriophage as an active ingredient.
본 발명에서의 용어, "소독제"란, 병원세균의 살균을 목적으로 하는 약제를 의미하며, 본 발명에서의 용어, "세척제"란, 세정의 목적에 사용되는 약제를 의미한다.The term "disinfectant" in the present invention refers to a drug used for the purpose of sterilizing pathogenic bacteria, and the term "cleaning agent" in the present invention refers to a drug used for the purpose of cleaning.
본 발명의 박테리오파지는 펙토박테리움 오도리페룸에 대하여 특이적으로 높은 항균 활성을 나타낼 뿐만 아니라, 우수한 내열성 및 내산성을 갖는다. 따라서 상기 박테리오파지를 유효성분으로 포함하는 상기 소독제 또는 세척제는 기존의 약제에 비하여 우수한 살균 활성을 가지며, 제품수명(life cycling)이 긴 신규 소독제 또는 세척제로서 이용될 수 있다.The bacteriophage of the present invention not only exhibits high antibacterial activity specifically against Pectobacterium odoriferum, but also has excellent heat resistance and acid resistance. Therefore, the disinfectant or cleaner containing the bacteriophage as an active ingredient has superior sterilizing activity compared to existing drugs and can be used as a new disinfectant or cleaner with a long product life cycle.
본 발명의 또 다른 하나의 양태는 상기 박테리오파지를 처리하는 단계를 포함하는 펙토박테리움 오도리페룸을 용균하는 방법을 제공한다.Another aspect of the present invention provides a method for lysing Pectobacterium odoriferum comprising the step of treating the bacteriophage.
본 발명에서의 용어, "용균"이란, 박테리아의 세포벽 또는 세포질막이 파괴되어 박테리아가 용해되는 현상을 의미하며, 박테리아가 사멸되는 현상을 의미한다.The term "lysis" in the present invention refers to a phenomenon in which the cell wall or cytoplasmic membrane of the bacteria is destroyed and the bacteria are dissolved, and it means the phenomenon in which the bacteria are killed.
구체적으로, 상기 박테리아를 용균하는 방법은 본 발명의 박테리오파지를 유효성분으로 포함하는 농약 조성물, 항균제, 소독제 또는 세척제의 유효량을 박테리아에 처리하는 단계를 포함할 수 있으나, 이에 제한되는 것은 아니다.Specifically, the method for lysing bacteria may include treating the bacteria with an effective amount of a pesticide composition, antibacterial agent, disinfectant, or detergent containing the bacteriophage of the present invention as an active ingredient, but is not limited thereto.
본 발명의 또 다른 하나의 양태는 상기 박테리오파지를 처리하는 단계를 포함하는 배추 무름병을 방제하는 방법을 제공한다.Another aspect of the present invention provides a method for controlling soft rot of cabbage comprising treating the bacteriophage.
본 발명에서의 용어, “배추 무름병”은 배추의 잎·줄기·뿌리에 수침상의 반점이 생기기 시작하여 빠른 속도로 확대되어 포기 전체가 썩게 되는 병해를 의미한다. The term “cabbage soft rot” in the present invention refers to a disease in which water-soaked spots begin to appear on the leaves, stems, and roots of cabbage and rapidly expand, causing the entire cabbage to rot.
본 발명에서의 용어, "방제"란, 작물에 피해를 주는 각종 병해충을 예방하고 구제하는 것을 의미한다.The term "control" in the present invention means preventing and exterminating various diseases and pests that damage crops.
구체적으로, 상기 배추 무름병을 방제하는 방법은 본 발명의 박테리오파지를 유효성분으로 포함하는 농약 조성물, 항균제, 소독제 또는 세척제의 유효량을 식물의 기부(basal) 또는 식물의 잎 부분에 처리하는 단계를 포함할 수 있으며, 더욱 구체적으로 식물의 병증 부위에 처리하는 단계를 포함할 수 있으나, 이에 제한되는 것은 아니다.Specifically, the method for controlling cabbage soft rot may include treating the base of the plant or the leaf portion of the plant with an effective amount of a pesticide composition, antibacterial agent, disinfectant, or detergent containing the bacteriophage of the present invention as an active ingredient. It may include, and more specifically, treating the diseased part of the plant, but is not limited thereto.
본 발명의 신규 박테리오파지는 펙토박테리움 오도리페룸(Pectobacterium odoriferum)에 특이적인 사멸능을 가지며, 내산성 및 내열성이 우수하므로, 상기 펙토박테리움 오도리페룸에 의하여 야기되는 배추 무름병 예방 또는 치료에 활용할 수 있을 뿐 아니라, 향균제, 소독제, 또는 세척제 등에도 광범위하게 이용될 수 있다.The new bacteriophage of the present invention has a specific killing ability against Pectobacterium odoriferum and has excellent acid resistance and heat resistance, so it can be used to prevent or treat cabbage soft rot caused by Pectobacterium odoriferum. In addition, it can be widely used as an antibacterial agent, disinfectant, or cleaning agent.
도 1은 박테리오파지의 플라그를 나타내는 사진이다.
도 2는 phiPccP-1의 DNA를 제한효소로 처리하여 전기영동으로 확인한 사진이다.
도 3은 현미경을 통해 확인한 박테리오파지의 형태 사진이다.
도 4는 박테리오파지의 온도 및 pH의 안정성을 나타내는 그래프이다.
도 5는 온실 조건에서의 phiPccP-1 박테리오파지를 이용한 배추 무름병 예방 효능을 나타내는 사진 및 그래프이다.
도 6은 배추 잎에서의 phiPccP-1 박테리오파지를 이용한 배추 무름병 예방 효능을 나타내는 사진 및 그래프이다.Figure 1 is a photograph showing a bacteriophage plaque.
Figure 2 is a photograph of phiPccP-1 DNA treated with restriction enzymes and confirmed by electrophoresis.
Figure 3 is a photograph of the morphology of bacteriophage confirmed through a microscope.
Figure 4 is a graph showing the stability of temperature and pH of bacteriophage.
Figure 5 is a photograph and graph showing the efficacy of preventing soft rot of cabbage using phiPccP-1 bacteriophage in greenhouse conditions.
Figure 6 is a photograph and graph showing the efficacy of preventing soft rot of cabbage using phiPccP-1 bacteriophage in cabbage leaves.
이하, 본 발명을 하기 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1: 세균 균주 및 토양 샘플링Example 1: Bacterial strains and soil sampling
본 발명에서 사용된 31개의 P. odoriferum(Pco 또는 pcc로 표기) 균주는 농촌진흥청 에서 받아서 사용되었다. 모든 박테리아 균주는 26℃에서 18시간동안 LB(Luria-Bertani) 아가 플레이트에서 군집화를 위해 배양하였다. 단일 군집으로부터 얻은 Pc 액체 배양액은 LB 브로스 배양액 3mL를 함유한 15mL 원형 바닥 튜브에서 부드럽게 흔들며 26℃에서 배양하였다. 그 후, 박테리아 배양균은 추가 실험을 위한 숙주 박테리아로 사용하였다.31 P. odoriferum (represented as Pco or pcc) strains used in the present invention were received from the Rural Development Administration. All bacterial strains were cultured for colonization on LB (Luria-Bertani) agar plates for 18 hours at 26°C. Pc liquid cultures from a single colony were cultured at 26°C with gentle shaking in 15 mL round bottom tubes containing 3 mL of LB broth. Afterwards, the bacterial culture was used as host bacteria for further experiments.
실시예 2: 박테리오파지의 분리 및 정제Example 2: Separation and purification of bacteriophage
펙토박테리움 오도리페룸을 억제하는 새로운 박테리오파지 후보균들은 한국 평창의 배추밭에서 전형적인 펙토박테리움 오도리페룸 감염 증상을 보이는 배추에서부터 분리하였다. 배추의 썩은 조직을 모아 증류수에 30분간 담지한 후, 흔들어 균질화하였다. 추출된 용액은 원심분리(8,000 x g, 10분, 4℃)에 의해 분리되었으며, 이는 추정 박테리오파지를 모두 포함할 수 있다. 투명한 상등액을 0.22μm pore 크기의 필터(독일 사토리우스)로 여과한 후, 다양한 Po 균주들을 이용하여 오버레이 분석을 실시해 박테리오파지를 얻었다. 이후, 18 hai에서 배양된 LB 아가 배양액에서 클리어 존(clear zone)을 확인하였다. 단일 박테리오파지를 분리하기 위하여, 오버레이 분석을 5번 반복하였다.New bacteriophage candidates that inhibit Pectobacterium odoriferum were isolated from cabbage showing typical symptoms of Pectobacterium odoriferum infection in a cabbage field in Pyeongchang, Korea. Rotten tissues of cabbage were collected, placed in distilled water for 30 minutes, and then shaken to homogenize. The extracted solution was separated by centrifugation (8,000 x g, 10 min, 4°C), which may contain all putative bacteriophages. The transparent supernatant was filtered through a filter with a 0.22 μm pore size (Satorius, Germany), and then overlay analysis was performed using various Po strains to obtain bacteriophages. Afterwards, a clear zone was confirmed in the LB agar culture medium cultured at 18 hai. To isolate single bacteriophages, the overlay analysis was repeated five times.
보다 구체적으로, 상기 오버레이 어세이 및 스팟팅 어세이는 하기 실험방법을 수정하여 수행하였다(Carey-Smith et al. 2006, Kim and Ryu 2011, Kropinski et al. 2009). 5mL의 녹은 LB 연한 한천 (0.4% 아가로스)을 100μL의 박테리아 상등액과 40℃ 이하의 온도에서 혼합하였다. 파지 샘플은 박테리아 상등액이 함유된 LB 연한 한천 5mL에 추가한 뒤, LB 아가 플레이트에 부어 이중의 박테리아 층을 만들었다. 그 후, 이중으로 제작된 플레이트는 호스트 박테리아를 위한 최적의 온도 조건에서 배양하였다.More specifically, the overlay assay and spotting assay were performed by modifying the following experimental method (Carey-Smith et al. 2006, Kim and Ryu 2011, Kropinski et al. 2009). 5 mL of dissolved LB soft agar (0.4% agarose) was mixed with 100 μL of bacterial supernatant at a temperature below 40°C. The phage sample was added to 5 mL of LB soft agar containing bacterial supernatant and then poured onto an LB agar plate to create a double layer of bacteria. Afterwards, the duplicated plates were cultured under optimal temperature conditions for the host bacteria.
실시예 3: 박테리오파지의 증식Example 3: Proliferation of bacteriophages
박테리오파지 증식과 분리 실험은 파지 용해물을 이용하여 수행하였다. 파지 용해물은 P. doriferum strain 14를 이용하여 수행하였으며, 박테리아 배양 방법은 상기에서 서술한 바와 같다.Bacteriophage growth and isolation experiments were performed using phage lysates. Phage lysate was performed using P. doriferum strain 14, and the bacterial culture method was as described above.
보다 구체적으로, 박테리오파지 증식을 위하여 OD600 = 0.5로 조절한 박테리아 배양액을 단일 플라그를 접종하였다. 혼합액은 6hai까지 충분히 증식할 수 있도록 26℃에서 배양하였다.그 후, 다른 잔여 박테리아 세포를 제거하고, 상등액을 원심분리(8,000 x g, 4℃, 10분)하여 제거한 뒤, 0.22μm pore 크기의 필터로 필터링하였다. 파지 용해물의 농도는 상기에서 서술한 스팟팅 어세이를 통해 확인하였다.More specifically, for bacteriophage growth, a single plaque was inoculated into a bacterial culture medium adjusted to OD 600 = 0.5. The mixed solution was cultured at 26°C to ensure sufficient growth up to 6hai. Afterwards, other remaining bacterial cells were removed, the supernatant was removed by centrifugation (8,000 xg, 4°C, 10 minutes), and then filtered with a 0.22μm pore size. Filtered by . The concentration of the phage lysate was confirmed through the spotting assay described above.
실시예 4: 숙주 균주 범위 결정Example 4: Host Strain Range Determination
본 발명의 신규 박테리오파지의 숙주 균주 범위를 조사하기 위하여, 31개의 Po 균주 및 3개의 다른 박테리아를 사용하였다(표 1).To investigate the host strain range of the novel bacteriophage of the present invention, 31 Po strains and 3 other bacteria were used (Table 1).
본 발명의 신규 박테리오파지의 항균활성을 결정하기 위해 한천판에 대한 spot-on-lawn 검사를 P. odoriferum isolates 8종, P. carotovorum, P. brasiliense, 및 P. parmentieri를 포함하는 다른 종의 펙토박테리움 균주 18종, P. atrosepticum, P, betavasculorum, P. wasabiae 및 P. versatile을 포함하는 39종의 박테리아 균주와 4종의 Dickeya spp. 균주를 대상으로 실시하였다. To determine the antibacterial activity of the novel bacteriophage of the present invention, a spot-on-lawn test on agar plates was performed on eight P. odoriferum isolates, P. carotovorum, P. brasiliense, and P. parmentieri, as well as other species of Pectobac. 39 bacterial strains, including 18 theterium strains, P. atrosepticum, P, betavasculorum, P. wasabiae and P. versatile, and 4 Dickeya spp. This was conducted on strains.
보다 구체적으로, 100μL의 108 CFU/mL의 하루동안 배양된 박테리아 상등액을 용해된 LB 연한 한천(0.4% 아가) 5mL과 혼합하고, LB 아가 플레이트에 부어 30분동안 배양하였다. 그 후, 10μL의 108 CFU/mL의 파지 용해액을 3번 플레이트에 접종하였다. 상기 배양은 26℃에서 하루동안 배양하였다. 그 뒤, 플라그인 클리어존의 형성을 측정하여 본 발명의 신규 박테리오 파지 감염에 의한 펙토박테리움 오도리페룸에 대한 용해력을 측정하였다. 참고로, 플라그가 나타나지 않는 플레이트에서는 본 발명의 박테리오파지의 저항성을 나타낸다.More specifically, 100 μL of 10 8 CFU/mL bacterial supernatant cultured for one day was mixed with 5 mL of dissolved LB soft agar (0.4% agar), poured onto an LB agar plate, and cultured for 30 minutes. Afterwards, 10 μL of 10 8 CFU/mL phage lysate was inoculated into plate No. 3. The culture was incubated at 26°C for one day. Afterwards, the formation of the plaque clear zone was measured to measure the lytic power against Pectobacterium odoriferum caused by infection with the new bacteriophage of the present invention. For reference, the plate on which no plaque appears shows the resistance of the bacteriophage of the present invention.
그 결과, 도 1에서 볼 수 있듯이, 플라그가 나타나는 것을 확인하였다.As a result, as can be seen in Figure 1, it was confirmed that plaque appeared.
또한, 표 1에서 볼 수 있듯이, 본 발명의 신규 균주가 펙토박테리움 오도리페룸 균주 8개 중 6개, P. 브레이슬리언스(P. brasilience) 균주 6개 중 5개, P. 카로토보룸 균주 12개 중 6개 및 P. 와사비애 2개 중 1개 대하여 lytic activity를 가짐을 확인하였으며, 다른 Pectobacterium spp. 및 Dickeya spp.에 대한 억제는 관찰되지 않았다. In addition, as can be seen in Table 1, the new strains of the present invention are 6 out of 8 Pectobacterium odoriferum strains, 5 out of 6 P. brasilience strains, and P. caroto. It was confirmed that 6 out of 12 Borum strains and 1 out of 2 P. wasabiae had lytic activity, and other Pectobacterium spp. and Dickeya spp., no inhibition was observed.
이 결과는 본 발명의 신규 박테리오파지인 piPccP-1가 P. 오도리페룸 뿐만 아니라 P. 카로토보룸과 P. 브레이슬리언스도 제어할 수 있음을 나타낸다.These results indicate that piPccP-1, a novel bacteriophage of the present invention, can control not only P. odoriferum but also P. carotovorum and P. braceliance.
실시예 5: DNA 추출 및 제한효소 절단Example 5: DNA extraction and restriction enzyme digestion
Promega wizard DNA clean-up 키트(Promega catalog #A7280)를 이용하여 DNA를 추출하였다.DNA was extracted using the Promega wizard DNA clean-up kit (Promega catalog #A7280).
보다 구체적으로, 20mL의 파지 용해액을 DNase 및 RNase로 30분간, 37℃에서 처리하여 박테리아의 DNA 및 RNA를 제거하였다. 그 뒤, 3M NaCl (최종 농도 10% 및 1M)이 포함된 10mL의 30%(w/v) 폴리에틸렌 글라이콜 8000을 용액에 넣고, 4℃에서 하루동안 반응시켰다. 원심분리기를 통해 파지 파티클인 펠렛을 얻은 뒤, 5mM MgSO4로 부유시켰다. Promega wizard DNA clean-up 키트의 DNA 스핀 컬럼 및 버퍼를 통해 DNA를 추출하고, 겔 전기영동을 통해 이를 확인하였다.More specifically, 20 mL of phage lysate was treated with DNase and RNase for 30 minutes at 37°C to remove bacterial DNA and RNA. Then, 10 mL of 30% (w/v) polyethylene glycol 8000 containing 3M NaCl (final concentrations 10% and 1M) was added to the solution and reacted at 4°C for one day. After obtaining a pellet of phage particles through a centrifuge, it was suspended in 5mM MgSO 4 . DNA was extracted using the DNA spin column and buffer of the Promega wizard DNA clean-up kit, and confirmed through gel electrophoresis.
그 결과, 도 2에서 볼 수 있듯이, EcoRI, BamHI 및 HindIII를 사용하여 본 발명의 박테리오파지의 DNA를 젤 전기영동으로 확인하였다.As a result, as can be seen in Figure 2, the DNA of the bacteriophage of the present invention was confirmed by gel electrophoresis using EcoR I, BamH I, and Hind III.
실시예 6: 박테리오파지 형태Example 6: Bacteriophage morphology
박테리오파지의 형태학적 특징을 분류하기 위하여, 정제된 박테리오파지 스탁을 탄소-코팅 구리 그리드(carbon-coated copper grids)에 놓고, 2% (w/v) 아세트산 우라늄으로 1분간 염색한 뒤, Korea Basic Science Institute(KBSI)의 Tecnai G2 Spirit Twin microscope(FEI,USA)를 통해 확인하였다.To classify the morphological characteristics of bacteriophages, purified bacteriophage stocks were placed on carbon-coated copper grids, stained with 2% (w/v) uranium acetate for 1 minute, and then cultured at Korea Basic Science Institute. It was confirmed through (KBSI) Tecnai G 2 Spirit Twin microscope (FEI, USA).
그 결과, 도 3에서 볼 수 있듯이, 본 발명의 박테리오파지의 형태를 확인하였다.As a result, as can be seen in Figure 3, the form of the bacteriophage of the present invention was confirmed.
이후, 확인된 박테리오파지 형태는 International Committee on Taxonomy of Viruses (King et al. 2011)를 통해 분류하였다.Afterwards, the identified bacteriophage types were classified through the International Committee on Taxonomy of Viruses (King et al. 2011).
실시예 7: 전체 유전자 시퀀스Example 7: Full gene sequence
신규 분리된 파지의 유전적 정보를 분석하고자, 본 발명의 박테리오파지의 전체 유전자를 Genome sequencer PacBio RSII를 통해 시퀀스를 분석하였다. 이후 전체 유전자는 GS de novo assembler software (Canu)를 통해 조합하였다. 또한, 유전적 정보는 다양한 프로그램을 통해 분석되었다. 구체적으로, ORFs (Open reading frames)는 ORFfinder(https://www.ncbi.nlm.nih.gov/orffinder/)를 통해 예측하고, NCBI Blastp (https://blast.ncbi.nlm.nih.gov/Blast.cgi)를 통해 아미노산 시퀀스의 기능을 예측하였다(표 2).In order to analyze the genetic information of the newly isolated phage, the entire gene of the bacteriophage of the present invention was sequenced using the genome sequencer PacBio RSII. Afterwards, the entire gene was assembled using GS de novo assembler software (Canu). Additionally, genetic information was analyzed through various programs. Specifically, ORFs (open reading frames) were predicted through ORFfinder (https://www.ncbi.nlm.nih.gov/orfinder/) and NCBI Blastp (https://blast.ncbi.nlm.nih.gov). /Blast.cgi), the function of the amino acid sequence was predicted (Table 2).
실시예 8: 박테리오파지의 온도, pH 및 자외선에 대한 안정성Example 8: Stability of bacteriophage to temperature, pH and ultraviolet light
실제 환경과 유사한 두 조건에서의 파지의 안정성을 확인하였다.The stability of the phages was confirmed under two conditions similar to the actual environment.
보다 구체적으로, 1mL의 파지 용해액을 1.5mL 튜브에 넣고, 다양한 온도(0℃, 26℃, 45℃ 및 54℃)에서 3주간 처리하였다. 또한, pH 안정성을 평가하기 위하여, SM 버퍼를 준비하고, HCl 및 NaOH를 통해, pH 값이 2, 3, 4, 5, 7, 10, 11 및 12가 되도록 조정하였다. 1.5mL 튜브에 990μL의 pH 용액을 넣고, 10μL의 파지 용해액을 넣은 뒤, 4℃에서 하루동안 반응시켰다. 이후, 스팟팅 어세를 통해 반응 후 파지의 용해력을 확인하였다.More specifically, 1 mL of phage lysate was placed in a 1.5 mL tube and treated at various temperatures (0°C, 26°C, 45°C, and 54°C) for 3 weeks. Additionally, to evaluate pH stability, SM buffer was prepared and adjusted to pH values of 2, 3, 4, 5, 7, 10, 11, and 12 using HCl and NaOH. Add 990 μL of pH solution to a 1.5 mL tube, add 10 μL of phage lysate, and react at 4°C for one day. Afterwards, the lytic power of the phage after reaction was confirmed through a spotting assay.
그 결과, 도 4에서 볼 수 있듯이, 본 발명의 박테리오파지는 0℃ 내지 26℃ 및 pH 4 내지 pH 10에서 가장 뛰어난 안정성을 보였다.As a result, as can be seen in Figure 4, the bacteriophage of the present invention showed the best stability at 0°C to 26°C and pH 4 to pH 10.
또한, 본 발명의 신규 박테리오파지는 60분까지 자외선을 처리했을 때 매우 안정적으로 생존하는 것도 알 수 있었다.In addition, it was found that the new bacteriophage of the present invention survives very stably when treated with ultraviolet rays for up to 60 minutes.
이를 통해, 본 발명의 박테리오파지는 다양한 온도, pH 및 자외선 범위에 대하여 안정적인 것을 확인하였다.Through this, it was confirmed that the bacteriophage of the present invention is stable over various temperatures, pH, and ultraviolet ray ranges.
실시예 9: 온실 실험Example 9: Greenhouse experiment
식물에서의 파지 적용을 확인하기 위하여, 비닐하우스에서 생물적 방제(biocontrol) 어세이를 진행하였다. 배추는 32개의 흙이 담긴 그릇에 심어 3주간 온실에서 재배되었다. 파지테라피의 최적 조건을 확인하기 위하여, 감염다중도(MOI) 0.1, 1 두 조건의 파지-용액을 준비하였다. 또한, 양성대조군으로 아그리마이신(Agrimycin)을 준비하였다. 파지 용액은 고농도의 파지 용해액(108 PFU/mL)을 10mM MgCl2과 계면활성제(silwet)으로 희석하여, 최종 농도가 0.02%가 되도록 한 후, 3주된 배추에 고르게 분사하였다. 2시간동안 반응시킨 뒤, 동일한 방법으로 박테리아 상등액을 분사하였다. 자연 상태에서는 Pco로 인해 식물과 토양 표면부근에서 배추 무름병 증상이 모든 식물들에서 균일하게 발생하였다. 본 발명에 있어서 사용된 식물들은 플라스틱 용기 덮개를 덮어 높은 상대 습도를 유지하며 재배하였다. 다음날, 플라스틱 덮개를 제거하고, 48시간 동안 질병 점수를 확인하였다.To confirm the application of phages in plants, a biological control (biocontrol) assay was conducted in a greenhouse. Cabbage was planted in 32 soil containers and grown in a greenhouse for 3 weeks. In order to confirm the optimal conditions for phage therapy, phage-solutions with a multiplicity of infection (MOI) of 0.1 and 1 were prepared. Additionally, agrimycin was prepared as a positive control. The phage solution was prepared by diluting a high-concentration phage solution (10 8 PFU/mL) with 10mM MgCl 2 and surfactant (silwet) to reach a final concentration of 0.02%, and then spraying it evenly on 3-week-old cabbage. After reacting for 2 hours, the bacterial supernatant was sprayed in the same manner. In natural conditions, cabbage soft rot symptoms occurred uniformly in all plants near the plant and soil surface due to Pco. The plants used in the present invention were grown under plastic container covers and maintained at high relative humidity. The next day, the plastic cover was removed, and the disease score was checked for 48 hours.
그 결과, 도 5에서 볼 수 있듯이, 본 발명의 박테리오파지를 처리하지 않은 배추군에서는 펙토박테리움 오도리페룸(pcc로 표기)에 의해 배추 무름병이 심각한 것을 확인할 수 있는 반면, 박테리오파지를 처리한 배추군에서는 농도 의존적으로 배추 무름병의 증상이 현저히 완화된 것을 확인하였다.As a result, as can be seen in Figure 5, in the cabbage group that was not treated with the bacteriophage of the present invention, it was confirmed that cabbage soft rot was serious due to Pectobacterium odoriferum (noted as pcc), whereas in the cabbage group that was treated with the bacteriophage, the cabbage soft rot disease was confirmed to be serious. It was confirmed that the symptoms of cabbage soft rot were significantly alleviated in a concentration-dependent manner.
이를 통해, 본 발명의 박테리오파지를 이용한 생물학적제제가 배추 무름병을 농도 의존적으로 예방 및 완화하는 효과가 있음을 확인하였다.Through this, it was confirmed that the biological agent using the bacteriophage of the present invention is effective in preventing and alleviating cabbage soft rot in a concentration-dependent manner.
실시예 10: 배추 잎에서의 파지테라피 어세이Example 10: Phagetherapy assay in cabbage leaves
생물학적제제인 박테리오파지의 펙토박테리움 오도리페룸에 의한 배추 무름병 예방 효능에 대해 조사하였다. 지역 시장에서 배추를 구매한 뒤, 대략 같은 크기의 건강한 배추 잎을 선택하였다. 10mM MgCl2과 0.02%의 계면활성제(silwet)로 희석한 고농도의 파지 용해액 109 PFU/mL를 잎 표면에 충분히 분무하였다. 두 시간 뒤, 박테리아 상등액을 10mM MgCl2에서 균질화하였다. 일반적으로, 배추 무름병은 배추 수확 시 생기는 작은 상처에서부터 발생한다. 하루 뒤, 나노드랍을 사용하여 최종 농도가 109 CFU/mL가 되도록 하였다.The efficacy of bacteriophage, a biological agent, in preventing soft rot of cabbage caused by Pectobacterium odoriferum was investigated. After purchasing cabbage at a local market, healthy cabbage leaves of approximately the same size were selected. A high concentration of 10 9 PFU/mL phage solution diluted with 10mM MgCl 2 and 0.02% surfactant (silwet) was sufficiently sprayed on the leaf surface. Two hours later, the bacterial supernatant was homogenized in 10mM MgCl 2 . Generally, cabbage soft rot occurs from small wounds that occur when cabbage is harvested. One day later, nanodrops were used to bring the final concentration to 10 9 CFU/mL.
면도날을 이용하여 배추에 상처를 만든 뒤, 상기에서 준비한 박테리아 상등액에 1분간 담지하여, 배추 무름병을 감염시켰다. 또한, 음성 대조군으로 박테리아가 포함되지 않은 10mM MgCl2를 처리하였다. 잎 표면에서 고르게 반응시키기 위하여, 모든 잎들은 플라스틱 박스에 넣고, 표면이 건조해지지 않게 습도를 유지시켜 26℃에서 배양하고, 다음 날, 병변의 크기를 측정하였다.After making a wound in the cabbage using a razor blade, it was placed in the bacterial supernatant prepared above for 1 minute to infect the cabbage with soft rot. Additionally, as a negative control, 10mM MgCl 2 containing no bacteria was treated. In order to react evenly on the leaf surface, all leaves were placed in a plastic box and incubated at 26°C to maintain humidity to prevent the surface from drying out. The next day, the size of the lesion was measured.
그 결과, 도 6에서 볼 수 있듯이, 펙토박테리움 오도리페룸만 처리한 배추군에서는 배추 무름병에 의한 병변이 크게 발생한 반면, 본 발명의 박테리오파지를 처리한 배추군에서는 현저하게 배추 무름병을 예방 및 완화한 것을 확인하였다.As a result, as can be seen in Figure 6, lesions caused by soft rot of cabbage occurred significantly in the cabbage group treated only with Pectobacterium odoriferum, whereas soft rot of cabbage was significantly prevented and alleviated in the cabbage group treated with the bacteriophage of the present invention. It was confirmed that it was done.
이를 통해, 본 발명의 박테리오파지를 이용한 생물학적제제가 배추 무름병을 효과적으로 예방 및 완화하는 효과가 있음을 확인하였다.Through this, it was confirmed that the biological agent using the bacteriophage of the present invention is effective in effectively preventing and alleviating cabbage soft rot.
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.
Claims (10)
Specific killing of Pectobacterium odoriferum , Pectobacterium carotovorum , Pectobacterium brasilience and Pectobacterium wasabiae An isolated bacteriophage with accession number KCTC14860BP.
A pesticide composition comprising the bacteriophage of claim 1 as an active ingredient.
The pesticide composition according to claim 3, wherein the bacteriophage is used for the prevention or treatment of cabbage soft rot.
The method of claim 3, wherein the pesticide composition includes an injectable composition, a biologic composition, a fertilizer composition, a nutritional composition, a plant strengthening composition, a plant protection composition, a soil conditioner composition, and a yield enhancer composition. A pesticide composition, which is one or more selected from the group consisting of.
An antibacterial agent comprising the bacteriophage of claim 1 as an active ingredient.
Disinfectant containing the bacteriophage of paragraph 1 as an active ingredient
A detergent containing the bacteriophage of claim 1 as an active ingredient.
Pectobacterium odoriferum, Pectobacterium carotovorum , Pectobacterium brasilience and Pectobacter comprising the step of processing the bacteriophage of paragraph 1 Method for lysing Pectobacterium wasabiae .
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