KR101363163B1 - Naramycin of suppression against bacterial blight of rice caused by bacterial pathogen - Google Patents
Naramycin of suppression against bacterial blight of rice caused by bacterial pathogen Download PDFInfo
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Abstract
본 발명은 벼흰잎마름병 억제용 나라마이신에 관한 것이다. 보다 구체적으로 방선균 추출물 유래 나라마이신의 벼흰잎마름병 억제능에 관한 것이다.
본 발명에 의한 나라마이신은 벼에서 벼흰잎마름병을 저해활성을 가지므로 이 성분을 농약으로 또는 이 물질을 포함하는 발효산물 생물농약으로 사용할 수 있다. The present invention relates to naramycin for inhibiting rice blight. More specifically, the actinomycetes-derived Nara mycin relates to the inhibition of rice leaf blight.
Naramycin according to the present invention has an inhibitory activity against rice leaf blight in rice and can be used as a pesticide or as a fermentation product biopesticide containing the substance.
Description
본 발명은 벼흰잎마름병 억제용 나라마이신에 관한 것이다. 보다 구체적으로 방선균 추출물 유래 나라마이신의 벼흰잎마름병 억제능에 관한 것이다.The present invention relates to naramycin for inhibiting rice blight. More specifically, the actinomycetes-derived Nara mycin relates to the inhibition of rice leaf blight.
벼의 흰잎마름병(Bacterial blight)은 벼흰잎마름병원균(Xanthomonas oryzae pv. oryzae)이 월동기간 동안 기주식물로 알려진 겨풀, 나도겨풀 등의 땅속줄기 또는 뿌리주위나 병든 볏짚, 벼 그루터기에 잠복해 있다가 관개수에 의해 논으로 이동한 후, 벼잎의 물구멍과 공기구멍 부위, 절단된 뿌리 등에 침입하여 벼에 감염되고, 감염된 벼에서 증식하여 2차로 전염되는 것에 의해 7월초~수확기, 특히 7월 상순~8월 중순에 발병하는 병해로, 최종적으로 벼가 말라 죽게 되어 수확량의 감소를 초래하므로 농가에 피해가 크다.Bacterial blight of rice is dormant in the stalks, roots, or sick stumps of rice, which are known as host plants during the wintering period, After being moved to the rice field by irrigation water, it penetrates into water holes, air holes, and cut roots of rice leaves and is infected with rice. It propagates in infected rice and is transmitted by the second order, The disease that occurs in the middle of August causes the rice to dry down eventually, which leads to a decrease in the yield, which causes great damage to the farmhouse.
따라서, 종래에는 이러한 벼의 흰잎마름병을 예방 및 방제하기 위하여 저수지나 관계수로에 있는 병원균의 기주식물을 제거하는 방법; 질소질 비료의 사용량이 많으면 질소질 비료에 의해 벼가 감수성으로 되어 흰잎마름병의 발병이 촉진되므로, 질소질 비료의 사용량을 조절하는 방법; 벼가 물에 잠기지 않도록 하고, 만약 잠기게 되면 최대한 빨리 물을 빼주어 침수시간을 짧게 하여 병원균과의 접촉을 차단하여 병의 전염을 방지하는 방법; 약제를 사용한 방법 등을 사용하여 왔다.Therefore, in the related art, a method of removing host plants of pathogens in reservoirs or related waterways in order to prevent and control rice leaf blight of rice; When the amount of nitrogenous fertilizer is used a lot, since the rice becomes susceptible to nitrogenaceous fertilizer to promote the development of white leaf blight, the method of controlling the amount of nitrogenous fertilizer; To prevent rice from submerging, and if soaked, to drain the water as soon as possible to shorten the immersion time to prevent contact with pathogens to prevent transmission of the disease; A method using a medicament has been used.
그러나, 상기한 방법들은 그 효과가 매우 미미한 실정이다. 특히, 약제방제법은 벼흰잎마름병원균을 조기에 발견하여 미연에 벼의 흰잎마름병을 예방 또는 방제하여 농가의 피해를 최소화할 수 있는 새로운 방제방법의 개발이 필요하였지만, 현재는 벼흰잎마름병원균의 검출을 위해서 선택배지를 이용하거나 병원균의 생리, 생화학적 특성의 검정을 수행하는 방법에 의해 검정하거나, 또는 병이 어느 정도 진행된 상태에서 육안으로 병을 인지하여 왔을 뿐이다. However, the above methods have very little effect. In particular, the pharmaceutic control method required the development of a new control method to minimize the damage to farmers by preventing the early detection of rice leaf blight pathogens and preventing or controlling rice leaf blight in rice. For this purpose, it has only been recognized by the use of selective medium, by the method of performing the physiological and biochemical properties of the pathogen, or by the naked eye with some progress.
이에 벼흰잎마름병을 억제하는 효과적인 천연 생리활성물질을 개발하기에 이르렀다.This led to the development of an effective natural physiologically active substance that suppresses rice leaf blight.
본 발명은 나라마이신을 유효성분으로 함유하는 벼흰잎마름병균 억제용 조성물을 제공하는 것이다. The present invention is to provide a composition for inhibiting rice leaf blight bacteria containing nara mycin as an active ingredient.
본 발명은 나라마이신을 유효성분으로 함유하는 벼흰잎마름병균 억제용 조성물에 관한 것이다. The present invention relates to a composition for inhibiting rice leaf blight bacteria containing Nara mycin as an active ingredient.
또한, 본 발명은 방선균 추출물을 유효성분으로 함유하는 벼흰잎마름병균 억제용 조성물에 관한 것이다.The present invention also relates to a composition for inhibiting rice leaf blight bacteria containing actinomycetes extract as an active ingredient.
또한, 본 발명은 방선균(Streptomyces sp .)의 배양액을 유기용매로 추출하는 단계; 및 상기 유기용매 추출물을 알코올에 용해시킨 후 이에 크로마토그래피를 실시하는 단계를 포함하는 방선균으로부터 나라마이신을 추출하는 방법에 관한 것이다.The present invention also relates to the use of Streptomyces sp .) is extracted with an organic solvent; And it relates to a method for extracting naramycin from actinomycetes comprising the step of dissolving the organic solvent extract in alcohol and then performing chromatography thereon.
본 발명에 의한 나라마이신은 벼에서 벼흰잎마름병을 저해활성을 가지므로 이 성분을 농약으로 또는 이 물질을 포함하는 발효산물 생물농약으로 사용할 수 있다. Naramycin according to the present invention has an inhibitory activity against rice leaf blight in rice and can be used as a pesticide or as a fermentation product biopesticide containing the substance.
본 발명의 방법에 의해 목화, 아마, 덩굴 식물(vine), 과실 또는 채소 작물, 묘목, 주요 작물, 예컨대 벼과 종(Graminae sp.)(예를 들어 옥수수, 잔디 또는 밀, 쌀, 보리 및 라이밀과 같은 곡물), 꽃, 원예 및 산림 작물뿐 아니라 이들 작물의 유전자적으로 변형된 상동체가 보호될 수 있다.Cotton, flax, vine, fruit or vegetable crops, seedlings, major crops such as Graminae sp. (E.g. corn, grass or wheat, rice, barley and lymil) by the process of the invention; Same grains), flowers, horticultural and forest crops, as well as genetically modified homologs of these crops.
도 1은 활성 물질 분리 중 HPLC 분획의 활성에 관한 것으로서, (A)는 HPLC chromatogram이고, (B)는 벼생엽절편에 대한 분획별 활성에 관한 것이다.
도 2는 활성 물질의 질량분석 결과에 관한 것이다.
도 3은 활성 물질로 동정된 나라마이신의 구조 및 기보고된 문헌에 관한 것이다.Figure 1 relates to the activity of the HPLC fraction during active material separation, (A) is HPLC chromatogram, (B) relates to fractional activity on the rice leaf slice.
2 relates to the mass spectrometry results of the active substance.
3 relates to the structure of naramycin identified as active substance and to previously reported literature.
본 발명은 나라마이신을 유효성분으로 함유하는 벼흰잎마름병균 억제용 조성물에 관한 것이다. The present invention relates to a composition for inhibiting rice leaf blight bacteria containing Nara mycin as an active ingredient.
상기 나라마이신은 방선균으로부터 유래될 수 있다.
The naramycin may be derived from actinomycetes.
본 발명에서 나라마이신(cycloheximide)은 하기 화학식 1의 구조를 가질 수 있으며, 벼흰잎마름병균에 대한 동등한 작용을 나타내는 한, 이들 화합물뿐만 아니라 이의 염 또는 유도체를 사용할 수 있다.Naramycin (cycloheximide) in the present invention may have a structure of the formula (1), as long as it shows an equivalent action on rice blight bacteria, these compounds as well as salts or derivatives thereof may be used.
[화학식 1][Formula 1]
본 발명의 일실시예에서, 본 발명의 조성물은 이에 제한되지 않으나, 상기 나라마이신을 0.001 내지 90 중량부, 또는 0.001 내지 70 중량부를 함유할 수 있다.In one embodiment of the present invention, the composition of the present invention is not limited thereto, but may contain 0.001 to 90 parts by weight, or 0.001 to 70 parts by weight of naramycin.
본 발명의 일실시예에서, 본 발명의 조성물은 상기 나라마이신 외에 본 발명이 목적으로 하는 주 효과를 손상시키지 않는 범위 내에서, 주 효과에 상승효과를 줄 수 있는 다른 성분 등을 함유할 수 있다.In one embodiment of the present invention, the composition of the present invention may contain other ingredients that can give a synergistic effect to the main effect, in addition to the naramycin within a range that does not impair the main effect of the present invention. .
또한, 본 발명은 방선균 추출물을 유효성분으로 함유하는 벼흰잎마름병균 억제용 조성물에 관한 것이다. 상기 방선균 추출물에는 나라마이신이 포함될 수 있다.The present invention also relates to a composition for inhibiting rice leaf blight bacteria containing actinomycetes extract as an active ingredient. The actinomycetes extract may include naramycin.
상기 방선균(actinomyces)은 토양·식물체·동물체·하천·해수 등에 균사체 및 포자체로 존재하는 미생물로 세균에 가까운 원핵생물 즉, 세균의 방선균목으로 분류된다. 세포의 크기가 세균과 비슷하며 세포가 곰팡이의 균사처럼 실 모양으로 연결되어 발육하며 그 끝에 포자를 형성한다. 토양 중 방선균은 각종 유기물의 분해, 특히 난분해성 유기물 분해에 중요한 역할을 하며 항생물질을 만들기도 한다. Actinomyces is a microorganism that exists in the form of mycelium and sporophyll in soil, plant, animal, river, seawater, and is classified into prokaryote which is close to bacteria, that is, actinomycetes of bacteria. The cell size is similar to that of bacteria, and the cells grow like a mycelium of fungus in thread-like form and form spores at the end. Actinic actinomycetes play an important role in decomposition of various organic matter, especially degradation of organic matter, and also produce antibiotics.
또한, 본 발명은 상기 조성물을 이용하여 벼흰잎마름병원균을 방제하는 방법에 관한 것이다. 상기 방제방법은 통상적인 식물 병원균의 방제방법이라면 이에 제한되지 않지만, 상기 조성물을 벼흰잎마름병이 발생된 병반 또는 발생이 예상되는 부위에 살포하거나 도포하여 방제하는 방법일 수 있다.In addition, the present invention relates to a method for controlling rice white leaf blight pathogen using the composition. The control method is not limited to the conventional method of controlling plant pathogens, but may be a method of controlling the composition by spraying or applying to the diseased spot or the expected site of rice leaf blight.
또한, 본 발명은 방선균(Streptomyces sp .)의 배양액을 유기용매로 추출하는 단계; 및 상기 유기용매 추출물을 알코올에 용해시킨 후 이에 크로마토그래피를 실시하는 단계를 포함하는 방선균으로부터 나라마이신을 추출하는 방법에 관한 것이다.The present invention also relates to the use of Streptomyces sp .) is extracted with an organic solvent; And it relates to a method for extracting naramycin from actinomycetes comprising the step of dissolving the organic solvent extract in alcohol and then performing chromatography thereon.
상기 유기용매로 추출하는 단계시 사용되는 유기용매는 이에 제한되는 것은 아니지만, 아세톤, 에틸아세테이트, 에테르, 클로로포름, 펜탄, 헥산, 헵탄, 노난, 데칸, 벤젠, 톨루엔, 자이렌, 메탄올, 에탄올, 부탄올 및 주정으로로 이루어진 군에서 선택되는 어느 하나 이상일 수 있다. 여기서 '주정'이란 전분질 원료 또는 당질 원료를 발효시킨 후 증류하여 만든 에탄올을 의미한다. The organic solvent used in the step of extracting with the organic solvent includes but is not limited to acetone, ethyl acetate, ether, chloroform, pentane, hexane, heptane, nonane, decane, benzene, toluene, xylene, methanol, ethanol, And alcoholic beverages. Herein, the term " alcohol " means ethanol produced by fermenting a starch raw material or a saccharide raw material and then distilling it.
상기 유기용매 추출물이 용해되는 알코올은 이에 제한되는 것은 아니나, 메탄올, 에탄올, 부탄올 및 주정으로 이루어진 군에서 선택된 어느 하나 이상을 사용할 수 있으며, 특히, 메탄올을 사용할 수 있다.The alcohol in which the organic solvent extract is dissolved is not limited thereto, but any one or more selected from the group consisting of methanol, ethanol, butanol, and alcohol may be used, and methanol may be used.
상기 크로마토그래피는 이에 제한되는 것은 아니지만, 흡착제로서 실리카겔, 역상실리카겔, 세파덱스류, 다이아이온 레진류, 알루미나, 활성탄, 규산마그네슘, 탄산칼슘, 인산칼슘, 인산마그네슘, 마그네시아 또는 수산화칼륨 등을 사용할 수 있으며, 세파덱스류를 사용할 수 있다. 보다 구체적으로는, 세파덱스 겔 컬럼 크로마토그래피를 수행한 후 고성능 액체 크로마토그래피를 한번 더 수행할 수 있다.
Although the chromatography is not limited thereto, silica gel, reversed phase silica gel, Sephadex, Diaion resins, alumina, activated carbon, magnesium silicate, calcium carbonate, calcium phosphate, magnesium phosphate, magnesia or potassium hydroxide can be used as the adsorbent And Sephadexes can be used. More specifically, high performance liquid chromatography can be performed once more after performing Sephadex gel column chromatography.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.
BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.
<< 실시예Example 1> 1>
1. 토양 방선균 (Soil Actinomycetes ( StreptomycesStreptomyces spsp .. )의 배양액을 에틸아세테이트로 추출하는 단계Extracting the culture solution of) with ethyl acetate
방선균 균주를 G.S.S 배지 10L에 진탕배양하고, 배양이 끝난 후 원심분리기를 통하여 상등액을 얻는다. 그 상등액과 에틸아세테이트를 혼합하여 추출액을 채취하여 40~50℃의 수조에서 감압 농축하여 추출물을 얻는다.
Actinomycetes strains were shaken in 10 L of GSS medium, and after the incubation, a supernatant was obtained through a centrifuge. The supernatant and ethyl acetate are mixed to extract an extract, and the extract is concentrated under reduced pressure in a water bath at 40 to 50 ° C.
2. 에틸아세테이트 추출물을 알코올에 용해시킨 후 크로마토그래피를 실시하는 단계2. Performing chromatography after dissolving ethyl acetate extract in alcohol
상기 단계에서 얻은 에틸아세테이트 추출물 564 mg을 메탄올 2 ㎖에 용해시킨 후 80% 메탄올을 용출용매로 이용하는 세파덱스 LH-20 겔 컬럼 크로마토그래피를 수행하여 검출된 복수 개의 분획들 중 벼생엽절편을 이용한 활성시험을 통해 항균 효과가 있는 활성분획을 수득하였다. 564 mg of ethyl acetate extract obtained in the above step was dissolved in 2 ml of methanol, followed by Sephadex LH-20 gel column chromatography using 80% methanol as an eluent. Through the test, an active fraction having an antimicrobial effect was obtained.
상기 활성분획을 분리하고 감압건조기로 용매를 제거한 후 얻은 잔사(residue)를 메탄올에 용해하여 고성능 액체 크로마토그래피(High Perfomance Liquid Chromatography)를 수행하여 최종 활성물질 나라마이신을 분리하였다.
The active fraction was separated and the residue obtained after removing the solvent with a reduced pressure dryer was dissolved in methanol to perform high performance liquid chromatography (High Perfomance Liquid Chromatography) to separate the final active material naramycin.
<< 실시예Example 2> 항균성 시험 2> antimicrobial test
앞서의 실시예에 기술한 방법에 의하여 균을 배양하고 정제함으로써 얻어진 항균성 물질(나라마이신)을 64 ug/ml 농도에서 0.25ug/ml 농도까지 2배 희석 방법을 사용하여 준비하였고, 이를 하기 표 1의 액체배지에 부가하고 여기에 벼흰잎마름병균(KACC10331)을 접종하여 보존하였다. 시험에 사용할 벼흰잎마름병균은 하기 표 1의 배지를 사용하여 28℃에서 1일간 배양하였으며, 최종균농도가 1.0x108 cfu/ml이 되도록 하여 1/20 희석하여 사용하였다. The antimicrobial substance (naramycin) obtained by culturing and purifying the bacteria by the method described in the previous Example was prepared using a 2-fold dilution method from 64 ug / ml concentration to 0.25 ug / ml concentration, which is shown in Table 1 below. It was added to the liquid medium of and inoculated with rice white leaf blight bacterium (KACC10331) was preserved. The rice leaf blight bacterium used for the test was incubated at 28 ° C. for 1 day using the medium of Table 1 below, and the final bacterial concentration was 1.0x10 8 cfu / ml, and was diluted 1/20.
1일간 배양 후 마이크로플레이트 리더(Microplate reader)를 이용하여 570 nm에서 흡광도를 측정하였다. 그 결과 기존에 알려진 항생제인 DAPG (2,4-Diacetylphloroglucinol)에 비해 높은 활성을 보였는데, 이들의 최소저해농도(MIC)는 표 2와 같다.
After incubation for 1 day, the absorbance was measured at 570 nm using a microplate reader. As a result, it showed higher activity than the known antibiotic DAPG (2,4-Diacetylphloroglucinol), their minimum inhibitory concentration (MIC) is shown in Table 2.
상기 표 2를 참조하면 본 발명의 나라마이신은 기존에 알려진 항생제인 DAPG에 비해 적은 양으로도 항균 활성을 나타내는바, 벼흰잎마름병원균의 방제에 유용하게 사용될 수 있다. Referring to Table 2, the naramycin of the present invention shows an antimicrobial activity in a small amount compared to the conventionally known antibiotic DAPG, it can be useful for the control of rice leaf blight pathogen.
Claims (8)
상기 나라마이신은 방선균으로부터 유래된 것을 특징으로 하는 벼흰잎마름병균 억제용 조성물. The method of claim 1,
Nara mycin is a composition for inhibiting rice leaf blight bacteria, characterized in that derived from actinomycetes.
상기 방선균 추출물은 나라마이신을 포함하는 것을 특징으로 하는 벼흰잎마름병균 억제용 조성물.5. The method of claim 4,
The actinomycetes extract is a composition for inhibiting rice leaf blight bacteria, characterized in that it comprises nara mycin.
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