KR20050040154A - A novel streptomyces sp. lim6 having protecting ability for rice blast disease - Google Patents

A novel streptomyces sp. lim6 having protecting ability for rice blast disease Download PDF

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KR20050040154A
KR20050040154A KR1020030075204A KR20030075204A KR20050040154A KR 20050040154 A KR20050040154 A KR 20050040154A KR 1020030075204 A KR1020030075204 A KR 1020030075204A KR 20030075204 A KR20030075204 A KR 20030075204A KR 20050040154 A KR20050040154 A KR 20050040154A
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rice
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rice blast
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임융호
송재경
조광연
최경자
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학교법인 건국대학교
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/04—Actinomyces

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Abstract

본 발명은 벼 도열병에 대한 방제효과를 가지는 신규한 방선균속 lim6 균주(KACC91027), 상기 균주를 이용한 벼 도열병의 방제방법 및 상기 균주를 함유하는 생화학농약제제에 관한 것이다. The present invention relates to a novel actinomycetes lim6 strain (KACC91027) having a control effect against rice blasts, a method for controlling rice blasts using the strains, and a biochemical pesticide preparation containing the strains.

본 발명에 따른 균주는, 기존의 화학농약에서 나타날 수 있는 과다사용으로 인한 생태계의 파괴 없이도, 기존 화학농약보다 우수한 벼 도열병 방제효과를 가지므로 생물농약으로서 유용하다.The strain according to the present invention is useful as a biopesticide because it has a superior rice pest control effect than conventional chemical pesticides without destroying the ecosystem due to overuse that may occur in conventional chemical pesticides.

Description

벼 도열병에 대한 방제효과를 가지는 신규한 방선균 속 ｌｉｍ6 균주{A novel Streptomyces sp. lim6 having protecting ability for rice blast disease} A novel Streptomyces sp. Genus A. Streptomyces sp. lim6 having protecting ability for rice blast disease}

본 발명은 벼 도열병에 대한 방제효과를 가지는 신규한 방선균속 lim6 균주(KACC91027), 상기 균주를 이용한 벼 도열병의 방제방법 및 상기 균주를 함유하는 생화학농약제제에 관한 것이다. The present invention relates to a novel actinomycetes lim6 strain (KACC91027) having a control effect against rice blasts, a method for controlling rice blasts using the strains, and a biochemical pesticide preparation containing the strains.

국내에 재배되고 있는 농작물에 피해를 주는 병해충은 4,157종이 기록되어 있으나, 현재 발생이 확인된 것으로는 1,199종(병 574, 해충 625)으로 농작물 재배기간 및 저장중 농산물에 기생, 섭취함으로서 막대한 손실을 초래하고 있는 현실이다.There are 4,157 pests that cause damage to crops grown in Korea, but 1,199 species (bottle 574 and 625 pests) have been confirmed to occur at present. It is bringing reality.

이와 같은 병해충이 농작물을 가해하여 피해를 주므로 이들을 농작물로부터 배척하거나 방제하기 위한 가장 손쉬운 수단의 하나가 방제제인 농약이라 할 수 있으며, 으레 농작물에 병해충 및 잡초가 발생하면 농약을 사용하는 것이 당연한 것으로 인식되어있다. 그러나 편리한 방제수단인 농약이 최근에 안전성 문제, 잔류문제(농작물, 토양, 환경), 자연생태계에 미치는 여러 문제, 공해유발 등 유해론이 제기되면서 부정적 시각이 점차 증대되는 경향이 높다하겠다. As these pests cause damage to crops, one of the easiest means to exclude or control them from crops is called pesticides, which are recognized as natural when pests and weeds occur on crops. It is. However, the negative view is likely to increase gradually as pesticides, which are convenient control measures, have recently been presented with harmful issues such as safety issues, residual problems (crops, soil, environment), problems on the natural ecosystem, and pollution.

이러한 여러 가지 문제점이 있다하여도 현재 사용 중 인 모든 농약의 사용을 저지할 수 없는 현실로 볼 때 현재의 농약보다 안전하고 부작용이 적은 저독성의 안전한 합성농약 또는 미생물을 활용한 생물학적 방제방법이 각국에서 연구되어 왔다.Even though there are various problems, it is impossible to prevent the use of all pesticides currently in use. Has been studied.

미생물을 이용하여 병해충을 방제한 연구역사는 1880년에 독일에서 곰팡이로 딱정벌레목해충을 방제하기 위한 시도가 최초이며 이후 많은 연구가 이루어져 세계적으로 약 70여종이 미생물 농약으로 실용화되어 있다.The research history of controlling pests using microorganisms was the first attempt to control beetle pests with a fungus in Germany in 1880. Since then, many studies have been carried out and about 70 species have been put to practical use as microbial pesticides worldwide.

외국에서의 개발 현황을 살펴보면, 미생물 자체를 이용한 생물 농약으로서 많은 연구가 시도되고 있는 바, 이는 미생물 자체를 이용한 병해방제 작용으로서 각종 식물병원균에 대해 증식억제작용, 기생작용, 항생작용 및 포식작용을 나타내는 길항미생물을 이용하는 연구가 대표적이다. Looking at the current state of development in foreign countries, many researches have been attempted as biopesticides using microorganisms themselves. This is a pest control action using microorganisms itself. Representative studies using antagonistic microorganisms are representative.

생물농약은 1927년 미국에서 감자 더뎅이병 방제에 방선균을 이용한 것이 최초이며, 일종의 면역작용을 나타내게 하는 교차보호를 이용한 바이러스 방제, 곰팡이를 이용한 고구마줄기 썩음병에 대한 연구가 1951년 시도되었다.Bio pesticides were the first to use actinomycetes to control potato beetle disease in the United States in 1927. In 1951, a study was carried out on virus control using cross-protection and sweet potato stem rot using mold.

또한, 국내에서는 벼 도열병균, 회색세균성 배추 무름병병균 및 캔디다균 등에 효과가 있는, 특히 벼 도열병에 대하여 활성을 나타내는 항균성 항생물질이 분리된바 있다 (김시관, 김창환, 1989, 아세톡시시클로헥시미드의 생리활성 및 그 생산 균주, 한국산업미생물학회지, 17:307-312).In addition, in Korea, antimicrobial antibiotics, which are effective against rice blast, gray bacterium cabbage, and Candida, have been isolated (Kim, Kwan, Kim, Chang Hwan, 1989, Acetoxycycloheximide). Biological Activity and Its Production Strains, Journal of the Korean Society for Industrial Microbiology, 17: 307-312).

벼에 발생하는 병에는 벼 흰잎마름병, 벼 잎집무늬마름병, 벼모 썩음병, 키다리병 등이 있으며, 그 중에서도 벼 도열병은 Magnaporthe grisea에 의해 발병하는데 우리나라 벼 재배에서 가장 무서운 병이다. 벼 도열병은 벼의 전생육기를 통하여 잎, 마디, 이삭목, 이삭가지, 볍씨 등에 발생하여 경제적 피해를 입힌다. 잎에서는 처음 암녹갈색의 작은 무늬가 생겨 나중에 긴 방추형이 되며, 발병이 심할 때는 온 포기가 자라지 않고 주저 않는다. 온도가 25~28℃ 정도의 적온이고 습도가 높으면 병반에 분생포자가 형성된다. 이 포자는 한 병반에서 많은 수가 형성되고 바람에 의해 운반되므로 환경이 발병에 적당하여 병이 일어나면 급속하게 확산되는 식물병이다. 벼 도열병 방제를 위한 저항성 품종의 사용은 신규 레이스의 출현으로 저항성 벼의 병 저항성이 쉽게 무너지는 단점이 있다.Diseases that occur in rice include rice leaf blight, rice leaf blight, rice rot and kidney disease. Among them, rice blast is caused by Magnaporthe grisea . Rice blasts cause economic damage from leaves, nodes, ear trees, ear branches, rice seeds, etc. through the entire growing season of rice. In the leaves, first, dark green brown small patterns are formed, and later long fusiform. When the onset is severe, whole abandonment does not grow and does not hesitate. If the temperature is about 25 ~ 28 ℃ and the humidity is high, conidia are formed on the lesion. This spore is a plant disease that forms a large number in one bottle and is carried by the wind, so that the environment is suitable for the onset and rapidly spreads when the disease occurs. The use of resistant varieties for rice blast control has the disadvantage that the resistance of resistant rice to rice is easily broken by the emergence of a new race.

따라서 방제를 위해서는 주로 살균제를 이용한 화학적 방제에 의존하고 있으며 현재 사용되고 있는 약제는 tricyclazole과 blasticidin-S 등이 있다. 그러나 최근에는 농약에 대한 일반인의 인식이 나쁘며 또한 단위면적 당 농가의 소득을 증가하기 위하여 작물 재배 시에 농약을 사용하지 않는 무농약 재배 및 유기농 재배 등의 농법을 사용하는 농가가 점차 증가하고 있다. 따라서 미생물 및 천연물을 이용한 생물농약에 대한 요구는 점차 증가하고 있어 이를 위한 새로운 방제 방법의 개발이 필요한 실정이다.Therefore, for the control, we mainly rely on chemical control using fungicides, and currently used drugs include tricyclazole and blasticidin-S. However, in recent years, farmers are increasingly using farming methods such as non-pesticide cultivation and organic cultivation, which do not use pesticides to grow crops, in order to increase the income of farmers per unit area. Therefore, the demand for biopesticides using microorganisms and natural products is gradually increasing, and the development of new control methods is required.

이에 본 발명자들은 벼 도열병 방제에 뛰어난 효과를 나타내면서, 환경친화적인 생화학농약제제를 개발하고자 예의 노력한 결과, 토양에서 분리한 방선균(Streptomyce sp.)이 벼 도열병에 탁월한 방제효과를 나타내는 것을 확인하고 본 발명을 완성하였다.Therefore, the present inventors have shown an excellent effect on the control of rice blast, and as a result of intensive efforts to develop an eco-friendly biochemical pesticide formulation, it was confirmed that the Streptomyce sp. Isolated from the soil shows excellent control effect on rice blast Was completed.

본 발명의 목적은 벼 도열병에 대한 방제효과를 가지는 신규한 방선균속 (Streptomyces sp.) lim6(KACC91027) 균주를 제공하는데 있다.An object of the present invention is to provide a novel Streptomyces sp. Lim6 (KACC91027) strain having a control effect against rice blast.

본 발명의 다른 목적은 상기 균주를 이용한 벼 도열병의 방제방법을 제공하는데 있다.Another object of the present invention is to provide a method for controlling rice blast using the strain.

본 발명의 또 다른 목적은 상기균주를 함유하는 것을 특징으로 하는 생화학농약제제 및 그 제조방법을 제공하는데 있다. Still another object of the present invention is to provide a biochemical pesticide preparation and a method for producing the same, comprising the strain.

상기의 목적을 달성하기 위하여, 본 발명은 벼 도열병에 대하여 방제효과를 가지는 방선균 속 (Streptomyces sp.) lim6 (KACC91027) 균주를 제공한다.In order to achieve the above object, the present invention provides a Streptomyces sp . Lim6 (KACC91027) strain having a control effect against rice blast.

본 발명은 또한, 벼 도열병에 대하여 방제효과를 가지고, 서열 1의 16S rRNA 염기서열을 가지는 방선균 속 (Streptomyces sp.) 균주를 제공한다.The present invention also provides a Streptomyces sp . Strain having a control effect against rice blasts and having a 16S rRNA sequence of SEQ ID NO: 1.

본 발명은 또한, 상기의 균주를 배양하는 것을 특징으로 하는 생화학농약제제의 제조방법을 제공하며, 상기 배양은 베넷(Bennet)배지에서 25~30℃에서 7~14일간 수행하는 것을 특징으로 할 수 있다.The present invention also provides a method for producing a biochemical pesticide preparation, characterized in that the culturing of the strain, the culturing may be characterized in that carried out for 7-14 days at 25 ~ 30 ℃ in Bennett (Bennet) medium. have.

본 발명은 또한, 상기 균주를 이용하는 것을 특징으로 하는 벼 도열병의 방제방법을 제공한다.The present invention also provides a method for controlling rice blast, characterized in that using the strain.

본 발명은 또한, 상기 균주 또는 그 배양액을 유효성분으로 함유하는 것을 특징으로 하는 생화학농약제제를 제공한다. The present invention also provides a biochemical pesticide preparation comprising the strain or its culture as an active ingredient.

상기 배양액은 베넷(Bennet)배지에서 25~30℃에서 7~14일간 배양한 것임을 특징으로 할 수 있고, 상기 생화학농약제제는 벼 도열병 방제용인 것을 특징으로 할 수 있다.The culture solution may be characterized in that it is incubated for 7-14 days at 25 ~ 30 ℃ in Bennett (Bennet) medium, the biochemical pesticides may be characterized in that for the control of rice blast.

본 발명의 방선균 속(Streptomyces sp.)lim6 균주는, 2003년 2월 14일 대한민국 농업생명공학원구원(경기도 수원시 권선구 서둔동 225 (우)441-707)에 방선균 속(Streptomyces sp.) lim6이라는 균주명과 기탁번호 KACC91027로 기탁하였다.Streptomyces genus of the present invention (Streptomyces sp.) Lim6 strains, in February 2003 the 14th Republic of Korea Agricultural Biotechnology nine won the strain of (Gwonseon-gu Suwon seodundong 225 (R) 441-707) in actinomycetes (Streptomyces sp.) Lim6 the name Deposited under accession number KACC91027.

이하 본 발명을 간단히 설명한다.The invention is briefly described below.

본 발명의 신규한 방선균속 (Streptomyces sp.) lim6(KACC91027) 균은 항균성을 가진 세균으로서, 16S rRNA의 유전자 염기배열 구조가 하기와 같은 특이한 구조를 가지는 것을 특징으로 하며, 16S rRNA의 전체염기서열에 있어서 방선균 스트렙토마이세스 아우레오파시엔스 (Streptomyces aureofaciens)와 98%의 상동성을 가지고, 벼 도열병에 있어서 탁월한 방제효과를 가진다.The novel Streptomyces sp. Lim6 (KACC91027) of the present invention is a bacterium having antimicrobial properties, characterized in that the gene base sequence structure of 16S rRNA has the following specific structure, and the entire nucleotide sequence of 16S rRNA. It has 98% homology with Streptomyces aureofaciens and has excellent control effect in rice blast.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

실시예 1. 신규한 방선균 속 (Example 1 Novel Actinomycetes Streptomyces Streptomyces sp.) KACC91027 균의 분리 및 동정sp.) Isolation and Identification of KACC91027

배양배지Culture medium

선발된 균주 KACC91027의 현탁배양을 위하여 방선균 배양에 이용되는 Bennet 배지를 이용하였다. 배지의 조성은 Agar 15 g/L, Beef extract 1 g/L, Yeast extract 1 g/L, Tryptone 2 g/L, Glycerol 10 g/L로 조성되며, 배양온도는 28℃, pH 7.2에서 진탕시키며 10일간 배양하였다.Bennet medium used for actinomycetes culture was used for suspension culture of the selected strain KACC91027. The medium was composed of Agar 15 g / L, Beef extract 1 g / L, Yeast extract 1 g / L, Tryptone 2 g / L, Glycerol 10 g / L. Incubated for 10 days.

형태학적 동정Morphological Identification

순수 분리한 신규한 방선균 속 (Streptomyces sp.) lim6(KACC91027) 균주를 Bennet 배지에서 배양하여 주사현미경(Scanning Electron Microscope)으로 형태를 관찰하였다 (도 1).The pure Streptomyces sp. Lim6 (KACC91027) strain was isolated from the cultures in Bennet's medium and observed by scanning electron microscope (FIG. 1).

16S rRNA 유전자염기서열 분석16S rRNA gene base sequence analysis

신규균 방선균속 (Streptomyces sp.) lim6(KACC91027) 균의 유전적 분석을 실시하고, 이를 동정하기 위하여 16S rRNA 유전자의 염기서열분석을 다음과 같이 수행하였다. DNA 추출을 위하여 Bennet 배지에 7일간 현탁배양한 후 배양액을 원심분리 하여 cell을 분리하였다. DNA 추출은 Genomic DNA Extraction Kit (Intron)를 이용하여 추출하였다.Genetic analysis of the novel Streptomyces sp. Lim6 (KACC91027) strain was performed, and sequencing of the 16S rRNA gene was performed as follows. The suspension was cultured in Bennet medium for 7 days for DNA extraction, and then the cells were separated by centrifugation. DNA extraction was performed using Genomic DNA Extraction Kit (Intron).

PCR은 반응용액(10 mM Tris, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM dNTPs; 1 unit Taq DNA polymerase)에 0.2㎍ genomic DNA , 0.25 μM 16S rRNA 유전자 프라이머 4 쌍이 표 1과 같이 조합하여 증폭하였다. 증폭된 DNA 절편의 크기가 A조합은 900 base-pair (bp), B조합은 480 bp, C조합은 1200 bp, D조합은 800 bp가 되도록 하였다. PCR 반응 (초기 94℃ 5분 1회; 94℃ 1분, 55℃ 1분, 72℃ 1분, 35cycle; 72℃ 5분 1회)한 후 증폭된 16S rRNA 유전자 일부분을 pGEM-T Easy vector (Promega co.)에 ligation한 후, 대장균 DH5α에 형질전환 하였다.PCR was carried out in a reaction solution (10 mM Tris, pH 8.3; 50 mM KCl; 1.5 mM MgCl 2 ; 0.2 mM dNTPs; 1 unit Taq DNA polymerase), and 4 pairs of 0.2 μg genomic DNA and 0.25 μM 16S rRNA gene primers were prepared as shown in Table 1. By amplification. The size of the amplified DNA fragments was 900 base-pair (bp) for A combination, 480 bp for B combination, 1200 bp for C combination, and 800 bp for D combination. After PCR reaction (initial 94 ℃ 5 minutes once; 94 ℃ 1 minutes, 55 ℃ 1 minutes, 72 ℃ 1 minutes, 35 cycles; 72 ℃ 5 minutes once) and then amplified 16S rRNA gene portion of pGEM-T Easy vector ( After ligation to Promega co.), E. coli DH5α was transformed.

형질전환된 대장균을 선발배지(1.5% agar, 50mg/L ampicillin, 100uM IPTG, 50mg/L X-gal)에 도발하여 37℃에서 12 시간 배양하였다. 증폭된 16S rRNA 유전자조각의 vector로의 삽입은 blue/white colony로 1차 선발하고, 이중 white colony만을 LB배지에 접종하여 배양하였다. 배양액은 플라스미드분리를 위하여 원심분리한 후 Alkaline lysis방법을 이용하였다. 분리된 플라스미드는 제한효소 EcoRI으로 절단 후 전기영동을 통하여 증폭된 16S rRNA 유전자조각의 삽입을 확인하였다.Transformed Escherichia coli was provoked in selection medium (1.5% agar, 50mg / L ampicillin, 100uM IPTG, 50mg / L X-gal) and incubated at 37 ° C for 12 hours. Insertion of the amplified 16S rRNA fragment into the vector was first selected as a blue / white colony, and only white colony was inoculated into LB medium and cultured. The culture was centrifuged for plasmid separation and Alkaline lysis was used. The isolated plasmid was digested with restriction enzyme Eco RI and confirmed insertion of amplified 16S rRNA fragment through electrophoresis.

증폭된 16S rRNA 유전자를 염기서열분석장치(ABI3100)를 통하여 분석한 결과, 서열 1의 염기서열을 가진다. 이를 NCBI(National Center for Biotechnology Information)의 BLAST를 이용하여 homology를 확인하였고 그 결과 방선균 스트렙토마이세스 아우레오파시엔스 (Streptomyces aureofaciens)와 98%의 상동성을 보였다 (도 2).The amplified 16S rRNA gene was analyzed by a sequencing device (ABI3100) and found to have the nucleotide sequence of SEQ ID NO: 1. The homology was confirmed using BLAST of the National Center for Biotechnology Information (NCBI), and the result showed homology of 98% with Streptomyces aureofaciens (FIG. 2).

16S rRNA 유전자의 PCR 및 염기서열분석에 사용된 프라이머Primers used for PCR and sequencing of 16S rRNA genes 프라이머 쌍Primer pair 염 기 서 열Base standing AA 5'-CAAGCGTTGTCCGGAATTAT-3' (서열 2)5'-CAAGCGTTGTCCGGAATTAT-3 '(SEQ ID NO: 2) 5'-TTCGGGTGTTACCGACTTTC-3' (서열 3)5'-TTCGGGTGTTACCGACTTTC-3 '(SEQ ID NO: 3) BB 5'-CAAGCGTTGTCCGGAATTAT-3' (서열 4)5'-CAAGCGTTGTCCGGAATTAT-3 '(SEQ ID NO: 4) 5'-ATCTCTGGATGTTTCCGGT-3' (서열 5)5'-ATCTCTGGATGTTTCCGGT-3 '(SEQ ID NO: 5) CC 5'-GATACGACTCAGGACCGCAT-3' (서열 6)5'-GATACGACTCAGGACCGCAT-3 '(SEQ ID NO: 6) 5'-TTCGGGTGTTACCGACTTTC-3' (서열 7)5'-TTCGGGTGTTACCGACTTTC-3 '(SEQ ID NO: 7) DD 5'-GATACGACTCAGGACCGCAT-3' (서열 8)5'-GATACGACTCAGGACCGCAT-3 '(SEQ ID NO: 8) 5'-ATCTCTGGATGTTTCCGGT-3' (서열 9)5'-ATCTCTGGATGTTTCCGGT-3 '(SEQ ID NO: 9)

실시예 2.방선균속 (Example 2 Radiation Strain ( Streptomyces Streptomyces sp.) lim6(KACC91027) 균 배양액의 벼도열병 방제 효과sp.) Effect of lim6 (KACC91027) culture on control of rice fever

시험 대상 벼 품종은 Oryza sativa cv Nakdong이며, 벼 도열병균은 Magnaporthe grisea이며 벼풍종 낙동에 대하여 감수성인 KJ201 레이스를 사용하여 실험하였다. 벼 종자를 최아시켜 수도용상토(부농사)에 파종하고 25±5℃의 온실에서 약 4주 동안 재배하여 준비한 약 3~4엽기의 벼를 사용하여 방제효과를 조사하였다.The rice varieties tested were Oryza sativa cv Nakdong, and the rice blast bacteria were Magnaporthe grisea and the KJ201 race was susceptible to rice cultivar Nakdong. Rice seeds were seeded and sown on aquatic soils (sub-farming), and the control effect was investigated using about 3-4 leaf rice prepared and grown for about 4 weeks in a greenhouse of 25 ± 5 ℃.

벼 도열병균을 감자즙액배지(potato dextrose broth, PDB)에 접종하고 25℃에서 7일 동안 진탕배양 하였다. 액체배지에서 자란 균사체를 Waring blender를 사용하여 자른 후에 멸균하여 식힌 쌀겨배지(쌀겨 20g, dextrose 10g, agar 2g, 증류수 1L)에 넣고 잘 흔들어주고 Petri-dish에 부어 굳힌 후에 25℃ 항온기에서 7일 동안 배양하였다. 배양한 균의 공중균사를 제거하고 형광등 하에 두어 형성된 포자를 수확하고 포자농도를 ml 당 5 x 105 개로 조정하고 이를 접종원으로 사용하였다.Rice blast bacteria were inoculated in potato juice medium (potato dextrose broth, PDB) and shaken at 25 ° C. for 7 days. Mycelium grown in liquid medium was cut using a Waring blender and sterilized in cooled rice bran medium (rice bran 20g, dextrose 10g, agar 2g, distilled water 1L), shaken well, poured into Petri-dish and hardened for 7 days in a thermostat. Incubated. Spores formed by removing the aerial hyphae of the cultured bacteria and placed under a fluorescent lamp were harvested, and the concentration of spores was adjusted to 5 x 10 5 per ml and used as an inoculum.

미생물 배양액의 방제효과를 조사하기 위하여 미생물 배양액에 250 mg/L 농도가 되도록 Tween 20을 첨가하고 벼 폿트에 경엽처리 한 후에 24시간 동안 풍건하였다. 미생물 배양액을 처리한 벼 잎에 위에서 준비한 벼 도열병균 포자현탁액을 spray하여 접종하였다. 접종한 벼는 25℃ 습실상에 넣고 1일 동안 습실처리한 후에 25℃, 상대습도 80%의 항온항습실에서 4일 동안 발병을 유도하며 재배하고 병반면적율을 조사하였다. To investigate the control effect of the microbial culture, Tween 20 was added to the microbial culture to have a concentration of 250 mg / L, and the leaves were air dried for 24 hours. The rice leaves treated with the microbial culture solution were inoculated by spraying the spore suspension of rice blast fungus prepared above. Inoculated rice was incubated for 1 day after incubation at 25 ° C. incubation for 4 days in a constant temperature and humidity room at 25 ° C. and a relative humidity of 80%.

실시예 1의 방법으로 배양한 방선균속 (Streptomyces sp.) lim6(KACC91027) 균의 배양액(미생물 세포내 물질이 포함된)을 처리한 결과와 무처리구를 비교하였다 (도 3). 일반적으로 Magnaporthe grisea에 의해 벼 도열병이 심하게 발생하면 잎이 죽게 된다. 도 3을 살펴보면 방선균속 (Streptomyces sp.) lim6(KACC91027) 균의 배양액 무처리구(오른쪽)와 처리구(왼쪽) 모두 Magnaporthe grisea를 접종하여 발병시켰으나, 무처리구는 잎이 모두 죽은 것으로 관찰되었고, 처리구는 방제효과로 인해 살아남은 것으로 관찰되었다.The results of treatment of the culture solution (containing microbial intracellular material) of Streptomyces sp. In general, a severe rice infestation caused by Magnaporthe grisea causes the leaves to die. Referring to FIG. 3, both untreated (right) and treated (left) cultures of Streptomyces sp. Lim6 (KACC91027) were inoculated with Magnaporthe grisea . Has been observed to survive.

병의 발생을 억제하는 방제가는 다음의 식으로 계산하였다.The control value which suppresses disease occurrence was calculated by the following formula.

처리구의 병반면적율Lesion area ratio of treatment area

방제가(%) = ( 1 - ──────────────── ) X 100    Control Value (%) = (1-────────────────) X 100

무처리구의 병반면적율Lesion area ratio of untreated area

결과로서, 방선균속 (Streptomyces sp.) KACC91027 균의 배양액을 처리한 경우 벼 도열병 방제가가 80%로 나타났다. 이로 부터, 본 발명의 KACC91027 균은 우수한 벼 도열병 방제효과를 가지는 것을 확인할 수 있었다.As a result, the rice blast control value was 80% when the culture solution of Streptomyces sp. From this, it was confirmed that the KACC91027 bacteria of the present invention has excellent rice blast control effect.

본 발명의 생화학제제는 본 발명의 균주의 배양액 또는 배양물을 포함하는 다양한 제제의 형태 즉 액제, 수화제, 분제, 입제, 유제 등으로 제조하여 사용할 수 있다. The biochemical preparations of the present invention can be prepared and used in the form of various preparations including the culture or culture of the strain of the present invention, that is, liquids, hydrates, powders, granules, emulsions and the like.

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

본 발명은 벼 도열병 방제효과를 가진 신규한 방선균 속 (Streptomyces sp.) lim6(KACC91027) 균, 이를 이용한 벼 도열병 방제방법 및 상기 균주를 함유하는 생화학농약제제를 제공하는 효과가 있다.The present invention has the effect of providing a novel Streptomyces sp. Lim6 (KACC91027) bacteria having a rice blast control effect, a rice blast control method using the same and a biochemical pesticide preparation containing the strain.

본 발명에 따른 균주는, 기존의 화학농약에서 나타날 수 있는 과다사용으로 인한 생태계의 파괴 없이도, 기존 화학농약보다 높은 벼 도열병 방제효과를 나타내므로 생물농약으로 유용하다. The strain according to the present invention is useful as a biopesticide because it exhibits a higher rice blast control effect than conventional chemical pesticides without destroying the ecosystem due to overuse that may occur in conventional chemical pesticides.

도 1은 신규한 방선균속 (Streptomyces sp.) lim6(KACC91027) 균주의 주사현미경(Scanning Electron Microscope) 사진이다.1 is a scanning electron micrograph of a novel Streptomyces sp. Lim6 (KACC91027) strain.

도 2는 신규한 방선균속 (Streptomyces sp.) lim6(KACC91027) 균의 16S rRNA 유전자분석 결과이다.Figure 2 is a 16S rRNA gene analysis of the novel Streptomyces sp. Lim6 (KACC91027).

도 3은 방선균속 (Streptomyces sp.) lim6(KACC91027) 균의 배양액을 처리하여 도열병이 방제된 벼(오른쪽)와 도열병이 심하게 발병된 무처리벼(왼쪽)를 나타낸 것이다.Figure 3 shows the rice (right) and the untreated rice (left) in which the blast was severely treated by treating the culture medium of Streptomyces sp. Lim6 (KACC91027).

<110> KonKuk University <120> A novel Streptomyces sp. lim6 having protecting ability for rice blast disease <130> KUP03-037 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 1590 <212> DNA <213> Streptomyces sp. lim6 <400> 1 gacgtcgcat gctcccggcc gccatggcgg ccgcgggaat tcgatagagt ttgatcctgg 60 ctcaggacga acgctggcgg cgtgcttagc acatgcaagt cgaacggtga agcccttcgg 120 ggtggatcag tggcgaacgg gtgagtaaca cgtgggcaat ctgccctgca ctctgggaca 180 agccctggaa acggggtcta ataccggata tgaccttcct ccgcatgggg gttggtggaa 240 agctccggcg gtgcaggatg agcccgcggc ctatcagctt gttggtgggg taatggccta 300 ccaaggcgac gacgggtagc cggcctgaga gggcgaccgg ccacactggg actgagacac 360 ggcccagact cctacgggag gcagcagtgg ggaatattgc acaatgggcg caagcctgat 420 gcagcgacgc cgcgtgaggg atgacggcct tcgggttgta aacctctttc agcaggggag 480 aagcgcaagt gacggtacct gcagaagaag caccggctaa ctacgtgcca gcagccgcgg 540 taatacgtag ggtgcgagcg ttgtccggaa ttattgggcg taaagagctc gtaggcggcc 600 tgtcgcgtcg gatgtgaaag cccggggctt aaccccgggt ctgcattcga tacgggcagg 660 ctagagtgtg gtaggggaga tcggaattcc tggtgtagcg gtgaaatgcg cagatatcag 720 gaggaacacc ggtggcgaag gcggatctct gggccattac tgacgctgag gagcgaaagc 780 gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacgtt gggaactagg 840 tgttggcgac attccacctc gtcgtttccg cacctaacgc attaagttcc ccgcctgggg 900 agtacgcccg caaggctaaa actctaagaa ttgacggggg cccgcacaag cagcggagca 960 tgtggcttaa ttcgacgcaa cgcgaagaac cttaccaagg cttgacatat gccggaaaca 1020 tccagagatg ggtgccccct tgtggtcggt atacaggtgg tgcatggttg tcgtcagctc 1080 gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgttct gtgttgccag 1140 cgagtaatgt cggggactca caggagactg ccggggtcaa ctcggaggaa ggtggggacg 1200 acgtcaaatc atcatgcccc ttatgtcttg ggctgcacac gtgctacaat ggtcggtaca 1260 aagggctgcg atgccgtgag gcggagcgaa tcccaaaaag ccggcctcag ttcggattgg 1320 ggtctgcaac tcgaccccat gaagttggag ttgctagtaa tcgcagatca gcatgctgcg 1380 gtgaatacgt tcccgggcct tgtacacacc gcccgtcacg tcacgaaagt cggtaacacc 1440 cgaagccggt ggcctaaccc tctgggatgg agccgtcgaa ggtgggacca gcgattggga 1500 cgaagtcgta acaaggtagc cgtaatcact agtgaattcg cggccgcctg caggtcgacc 1560 atatgggaga gctcccaacg cgttggatgc 1590 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 caagcgttgt ccggaattat 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 ttcgggtgtt accgactttc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 caagcgttgt ccggaattat 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 atctctggat gtttccggt 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 gatacgactc aggaccgcat 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 ttcgggtgtt accgactttc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 gatacgactc aggaccgcat 20 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 atctctggat gtttccggt 19<110> KonKuk University <120> A novel Streptomyces sp. lim6 having protecting ability for rice blast disease <130> KUP03-037 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 1590 <212> DNA <213> Streptomyces sp. lim6 <400> 1 gacgtcgcat gctcccggcc gccatggcgg ccgcgggaat tcgatagagt ttgatcctgg 60 ctcaggacga acgctggcgg cgtgcttagc acatgcaagt cgaacggtga agcccttcgg 120 ggtggatcag tggcgaacgg gtgagtaaca cgtgggcaat ctgccctgca ctctgggaca 180 agccctggaa acggggtcta ataccggata tgaccttcct ccgcatgggg gttggtggaa 240 agctccggcg gtgcaggatg agcccgcggc ctatcagctt gttggtgggg taatggccta 300 ccaaggcgac gacgggtagc cggcctgaga gggcgaccgg ccacactggg actgagacac 360 ggcccagact cctacgggag gcagcagtgg ggaatattgc acaatgggcg caagcctgat 420 gcagcgacgc cgcgtgaggg atgacggcct tcgggttgta aacctctttc agcaggggag 480 aagcgcaagt gacggtacct gcagaagaag caccggctaa ctacgtgcca gcagccgcgg 540 taatacgtag ggtgcgagcg ttgtccggaa ttattgggcg taaagagctc gtaggcggcc 600 tgtcgcgtcg gatgtgaaag cccggggctt aaccccgggt ctgcattcga tacgggcagg 660 ctagagtgtg gtaggggaga tcggaattcc tggtgtagcg gtgaaatgcg cagatatcag 720 gaggaacacc ggtggcgaag gcggatctct gggccattac tgacgctgag gagcgaaagc 780 gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacgtt gggaactagg 840 tgttggcgac attccacctc gtcgtttccg cacctaacgc attaagttcc ccgcctgggg 900 agtacgcccg caaggctaaa actctaagaa ttgacggggg cccgcacaag cagcggagca 960 tgtggcttaa ttcgacgcaa cgcgaagaac cttaccaagg cttgacatat gccggaaaca 1020 tccagagatg ggtgccccct tgtggtcggt atacaggtgg tgcatggttg tcgtcagctc 1080 gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgttct gtgttgccag 1140 cgagtaatgt cggggactca caggagactg ccggggtcaa ctcggaggaa ggtggggacg 1200 acgtcaaatc atcatgcccc ttatgtcttg ggctgcacac gtgctacaat ggtcggtaca 1260 aagggctgcg atgccgtgag gcggagcgaa tcccaaaaag ccggcctcag ttcggattgg 1320 ggtctgcaac tcgaccccat gaagttggag ttgctagtaa tcgcagatca gcatgctgcg 1380 gtgaatacgt tcccgggcct tgtacacacc gcccgtcacg tcacgaaagt cggtaacacc 1440 cgaagccggt ggcctaaccc tctgggatgg agccgtcgaa ggtgggacca gcgattggga 1500 cgaagtcgta acaaggtagc cgtaatcact agtgaattcg cggccgcctg caggtcgacc 1560 atatgggaga gctcccaacg cgttggatgc 1590 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 caagcgttgt ccggaattat 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 ttcgggtgtt accgactttc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 caagcgttgt ccggaattat 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 atctctggat gtttccggt 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 gatacgactc aggaccgcat 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 ttcgggtgtt accgactttc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 gatacgactc aggaccgcat 20 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 atctctggat gtttccggt 19

Claims (8)

벼 도열병에 대하여 방제효과를 가지는 방선균 속 (Streptomyces sp.) lim6 (KACC91027) 균주. Streptomyces sp . Lim6 (KACC91027) strain having a control effect against rice blasts. 벼 도열병에 대하여 방제효과를 가지고, 서열 1의 16S rRNA 염기서열을 가지는 방선균 속 (Streptomyces sp.) 균주. Streptomyces sp . Strain having a control effect against rice blast, and having the 16S rRNA sequence of SEQ ID NO: 1. 제1항 또는 제2항의 균주를 배양하는 것을 특징으로 하는 생화학농약제제의 제조방법.A method for producing a biochemical pesticide preparation, comprising culturing the strain of claim 1 or 2. 제3항에 있어서, 상기 배양은 베넷(Bennet)배지에서 25~30℃에서 7~14일간 수행하는 것을 특징으로 하는 생화학농약제제의 제조방법.The method of claim 3, wherein the culturing is carried out for 7-14 days at 25 ~ 30 ℃ in Bennett medium (Bennet). 제1항 또는 제2항의 균주를 이용하는 것을 특징으로 하는 벼 도열병의 방제방법.A method for controlling rice blast, characterized by using the strain of claim 1 or 2. 제1항 또는 제2항의 균주 또는 그 배양액을 유효성분으로 함유하는 것을 특징으로 하는 생화학농약제제.A biochemical pesticide preparation comprising the strain of claim 1 or 2 or a culture thereof as an active ingredient. 제6항에 있어서, 배양액은 베넷(Bennet)배지에서 25~30℃에서 7~14일간 배양한 것임을 특징으로 하는 생화학농약제제.The biochemical pesticide preparation according to claim 6, wherein the culture solution is incubated at Bennett for 7 to 14 days at 25 to 30 ° C. 제6항에 있어서, 벼 도열병 방제용인 것을 특징으로 하는 생화학농약제제.The biochemical pesticide preparation according to claim 6, which is used for rice blast control.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11466247B2 (en) 2017-09-19 2022-10-11 Evogene Ltd. Bacterial genes and isolates for conferring insect resistance

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11466247B2 (en) 2017-09-19 2022-10-11 Evogene Ltd. Bacterial genes and isolates for conferring insect resistance

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