KR20030015010A - New biopesticide using WT#3-1 gene from Erwinia pyrifoliae WT#3, novel pathogen that affects Asian pear trees - Google Patents

New biopesticide using WT#3-1 gene from Erwinia pyrifoliae WT#3, novel pathogen that affects Asian pear trees Download PDF

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KR20030015010A
KR20030015010A KR1020010049047A KR20010049047A KR20030015010A KR 20030015010 A KR20030015010 A KR 20030015010A KR 1020010049047 A KR1020010049047 A KR 1020010049047A KR 20010049047 A KR20010049047 A KR 20010049047A KR 20030015010 A KR20030015010 A KR 20030015010A
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hypersensitivity
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임춘근
허장현
박덕환
배후남
백수진
최신건
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주식회사 파이오니아
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Priority to KR10-2002-0048074A priority patent/KR100389143B1/en
Priority to HU0402172A priority patent/HU225133B1/en
Priority to PCT/KR2002/001553 priority patent/WO2003016510A1/en
Priority to CNB028144066A priority patent/CN100494344C/en
Priority to BR0210787A priority patent/BR0210787A/en
Priority to JP2003521819A priority patent/JP3976731B2/en
Priority to CA 2457060 priority patent/CA2457060A1/en
Priority to PL36898102A priority patent/PL368981A1/en
Priority to EP02758919A priority patent/EP1417299A4/en
Priority to AU2002324348A priority patent/AU2002324348B2/en
Publication of KR20030015010A publication Critical patent/KR20030015010A/en
Priority to ZA200400008A priority patent/ZA200400008B/en

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Abstract

PURPOSE: A biopesticide using WT#3-1 gene derived from Erwinia pyrifoliae, a pathogen that affects Asian pear trees is provided. The biopesticide has more improved plant disease resistance and plant growth facilitating effect than the prior biopesticide. CONSTITUTION: A gene encoding a plant hypersensitivity reaction(HR) inducing protein derived of Erwinia pyrifoliae WT#3(KCCM 10283) has the nucleotide sequence of SEQ ID NO: 1. The plant hypersensitivity reaction(HR) inducing protein has the amino acid sequence of SEQ ID NO: 2. A recombinant plasmid pKEP3 contains the gene of SEQ ID NO: 1. A transformant E. coli/pKEP3 (KCCM 10282) is produced by transformation with the recombinant plasmid pKEP3. The plant hypersensitivity reaction(HR) inducing protein is mass-produced by culturing the transformant E. coli/pKEP3 (KCCM 10282); and extracting and purifying the plant hypersensitivity reaction(HR) inducing protein.

Description

신규한 가지검은마름병원균 ＷＴ＃３（ＫＣＣＭ １０２８３）유래 ＷＴ＃３－１유전자를 이용한 새로운 생물농약{New biopesticide using WT＃3-1 gene from Erwinia pyrifoliae WT＃3, novel pathogen that affects Asian pear trees}New biopesticide using WT ＃ 3-1 gene from Erwinia pyrifoliae WT ＃ 3, novel pathogen that affects Asian pear trees}

본 발명은 신규한 가지검은마름병원균 WT#3(Erwinia pyrifoliaeWT#3, KCCM 10283) 유래 WT#3-1 유전자를 이용한 새로운 생물농약에 관한 것으로서, 더욱 상세하게는 국내에서만 존재하는 신종 가지검은마름병원균 WT#3(KCCM 10283)를 분리 동정하고 기존의 화상병원균(Erwinia amylovora)에서 분리한 식물과민반응 단백질(Harpin) 보다 식물병 저항성 및 식물생장촉진 효과가 우수한 새로운 유도체를 발견함으로써 생물학적 식물병 방제제 및 식물생장 촉진제 등의 생물농약으로 이용할 수 있는 신규한 가지검은마름병원균 WT#3(KCCM 10283)에 관한 것이다.The present invention relates to a novel biopesticide using WT # 3-1 gene derived from a novel eggplant black rot pathogen WT # 3 ( Erwinia pyrifoliae WT # 3, KCCM 10283), and more specifically, a new eggplant black berry that exists only in Korea. Isolation and identification of pathogen WT # 3 (KCCM 10283) and control of biological plant diseases by finding new derivatives with better plant disease resistance and plant growth promoting effects than plant sensitization protein (Harpin) isolated from the existing Erwinia amylovora The novel eggplant black pathogen WT # 3 (KCCM 10283) which can be used as a biopesticide, such as an agent and a plant growth promoter, is related.

인류가 당면한 여러 문제 중에서 식량부족 현상은 세계가 그 문제의 심각성을 공히 인정하고 있는 측면이다. 그러나, 식량증대를 위해 재배하고 있는 작물은 병충해에 의해 그 양이 크게 감소하고 있어 가장 큰 문제점으로 지적되고 있다. 현재 병충해를 방제하기 위한 방법으로는 여러 방법 중에 살균제와 살충제를 직접 살포하여 병해충의 확산을 저지하거나 사멸시키는 화학적 방제가 널리 이용되고 있다. 이러한 살균제나 살충제의 경우, 병해충을 직접 죽이므로 가식적인 효과가 빨리 나타나고 이용방법이 용이하나, 지속적인 과다 사용으로 인해 각종 병해충의 약제 저항성을 유도하게 된다. 이는 새로운 농약개발의 필요성을 증대시키며 토양오염 및 수질오염 등의 환경문제를 야기시키는 우선 순위가 되고 있다. 따라서, 전 세계적으로 환경 친화적이고 병해충의 약제 저항성 유도의 염려가 없으면서도 방제에 효과적인 생물학적 방제제의 개발이 절실한 상태이다.Among the problems facing humanity, the food shortage is an aspect that the world recognizes the seriousness of the problem. However, the amount of crops grown for food growth has been pointed out as the biggest problem because the quantity is greatly reduced by pests. Currently, as a method for controlling pests, chemical control for preventing or killing the spread of pests by spraying fungicides and insecticides is widely used among various methods. In the case of such fungicides or insecticides, the pests are killed directly, so that the edible effect appears quickly and is easy to use, but due to continuous overuse, drug resistance of various pests is induced. This increases the need for new pesticide development and becomes a priority for causing environmental problems such as soil pollution and water pollution. Therefore, there is an urgent need for the development of biological control agents that are environmentally friendly and effective in controlling pests without fear of inducing drug resistance.

생물학적 방제제는 지구상 모든 생물은 그들이 살고 있는 환경으로부터의 신호에 대해 감지하고 반응할 수 있는 복잡한 생화학적 경로를 가지고 있다는 것에서부터 출발하였다. 이는 생물학적 방제제로서 사용할 수 있는 미생물 그 자체를 식물에 살포하여 방제효과를 얻고자 하였다. 그러나, 이러한 방법은 그 효능면에서 인간이 원하는 수준에 도달하지 못하였다. 이에 미생물이 생산하는 물질을 이용하여 식물의 자체 방어시스템을 자극하는 방법으로 병충해 방제에 응용하려는 연구로 전환되었다. 다시 말해서 생물학적 방제제란 병원균을 직접 죽이는 대신 식물체 자체의 면역기능을 활성화시켜주는 물질을 이용하여 병해충의 발생 및 확산을 방지하고자 하는 것이다.Biological control agents start from the fact that every living thing on Earth has a complex biochemical pathway that can sense and respond to signals from the environment in which they live. This was to obtain a control effect by spraying the microorganism itself that can be used as a biological control agent to the plant. However, this method has not reached the level desired by humans in terms of efficacy. Therefore, the research was applied to the control of pests by stimulating the plant's own defense system using materials produced by microorganisms. In other words, biological control agents are intended to prevent the development and spread of pests by using substances that activate the immune function of the plant itself instead of killing pathogens directly.

식물병저항성(plant disease resistance)이란, 우선 식물 표피세포의 큐틴질, 왁스층 및 기공의 모양 등 구조적 장벽과 병원균이 식물 세포내로 침입하더라도 식물 자체적으로 사포닌(saponin)이나 렉틴(lectin)과 같은 다양한 화학물질을 분비하여 침입한 병원균들의 확산을 저지하는 일차적 방어시스템에 의해 이루어진다[Agrios, G. N. 1997. Plant Pathology.4th ed. Academic Press, New York; Dong, X. 1998. SA. JA, ethylene, and disease resistance in plants. Curr. Opin. Plant. Biol.1:316-323; Feys, B. J. & Parker, J. E. 2000. Interplay of signaling pathways in plant disease resistance. Trends Genet. 16:339-455].Plant disease resistance refers to various chemicals such as saponins and lectins in the plant itself, even if structural barriers and pathogens invade the plant cells, such as the cuticle of the epidermal cells, the shape of the wax layer and the pores. Made by a primary defense system that prevents the spread of invading pathogens by secreting materials [Agrios, GN 1997. Plant Pathology. 4th ed. Academic Press, New York; Dong, X. 1998. SA. JA, ethylene, and disease resistance in plants. Curr. Opin. Plant. Biol. 1: 316-323; Feys, B. J. & Parker, J. E. 2000. Interplay of signaling pathways in plant disease resistance. Trends Genet. 16: 339-455.

그러나, 궁극적으로 식물체 자체에 면역기능을 활성화시키는 식물 저항성이란 감염부위 내 작은 지역으로 병원균의 확산을 국한시키는 것을 말한다. 이러한 경우를 기주조직의 국지적 사멸이라 하며, 또한 병원균의 확산 저지 및 기주조직의 국지적 사멸은 과민성 반응(hypersensitive response; HR)에 의해 유도된다[Richberg, M. H., Aviv, D. H. & Dangl, J. L. 1998. Dead cells do tell tales. Curr. Poin. PlantBiol. 1:480-485]. 이는 주변 세포에 대한 조기경보 시스템을 발동하게 되어 주변 세포들도 병원균에 대한 저항능력을 증강시키게 되는데 이러한 현상을 LAR(local acquired resistance)라고 한다. 또한, 많은 식물들은 감염되지 않은 부분에서도 방어시스템이 활성화되어 이차감염에 대해 식물전체에서 더 강한 방어시스템이 작동하게 된다. 이러한 방어시스템을 SAR(systemic acquired resistance)이라고 명명하며, 이는 여러 주 혹은 그 이상 유지될 수 있으며 종종 관련되지 않은 다른 종류의 병원균에 대해서도 저항성을 갖게된다[Hunt, M. D., Neuenschwander, U. H., Delaney, T. P., Weymann, K. B., Friedrich, L. B., Lawton, K. A., Steiner, H. Y. and Ryals, J. A. 1996. Recent advances in systemic acquired resistance research-a review. Gene 179:89-95]. 이밖에 ISR(induced systemic resistance)과 해충에 대한 상처반응(wound response) 등이 식물저항성으로 보고되었다[Pieterse, C. M., van Wees, S. C., van Pelt, J. A., Knoester, M., Laan, R., Gerrits, H., Weisbeek, P. J. and van Loon, L. C. 1998. A novel signaling pathway controlling induced systemic resistance inArabidopsis. Plant Cell 10:1571-1580; Ryan, C. A. and Pearce, G. 1998. SYSTEMIN: a polypeptide signal for plant defensive genes. Annu. Rev.Cell Dev. Biol. 14:1-17].However, plant resistance, which ultimately activates the immune function in the plant itself, means limiting the spread of pathogens to small areas within the infected area. This is called local killing of host tissues, and the inhibition of the spread of pathogens and local killing of host tissues is induced by hypersensitive response (HR) [Richberg, MH, Aviv, DH & Dangl, JL 1998. Dead cells do tell tales. Curr. Poin. Plant Biol. 1: 480-485]. This triggers an early warning system for surrounding cells, which also enhances the ability of the surrounding cells to resist pathogens. This phenomenon is called local acquired resistance (LAR). In addition, many plants activate their defenses even in the uninfected areas, resulting in a stronger defense system throughout the plant against secondary infection. This defense system is called systemic acquired resistance (SAR), which can be maintained for several weeks or more and is often resistant to other unrelated pathogens [Hunt, MD, Neuenschwander, UH, Delaney, TP]. , Weymann, KB, Friedrich, LB, Lawton, KA, Steiner, HY and Ryals, JA 1996. Recent advances in systemic acquired resistance research-a review. Gene 179: 89-95. In addition, induced systemic resistance and wound response to pests were reported as plant resistance [Pieterse, CM, van Wees, SC, van Pelt, JA, Knoester, M., Laan, R., Gerrits, H., Weisbeek, PJ and van Loon, LC 1998. A novel signaling pathway controlling induced systemic resistance in Arabidopsis . Plant Cell 10: 1571-1580; Ryan, CA and Pearce, G. 1998. SYSTEMIN: a polypeptide signal for plant defensive genes. Annu. Rev. Cell Dev. Biol. 14: 1-17.

이들 식물병 저항성(plant disease resistance) 기작들은 모두 식물생체 방어시스템을 유도하는 것을 유도물질(elicitor; inducer)에 의해 이루어지게 된다[Kessmann, H., Staub, T., Hofmann, C., Maetzke, T., Herzog, J., Ward, E., Uknes, S. and Ryals, J. 1994. Induction of systemic acquired disease resistance in plants by chemicals. Annu. Rev. Phytopathol. 32:439-459]. SAR의 경우 페놀계의 신호전달 물질인 SA(salicylic acid)에 의해 유도되어지는데, 이밖에 병원균에서 분리된 엘리시틴(elicitin)과 하르핀(Harpin) 등이 대표적이다[Ponchet, M., Panabieres, F., Milat, M. L., Mikes, V., Montillet, J. L., Suty, L., Triantaphylides, C., Tirilly, Y. and Blein, J. P. 1999. Are elicitins cryptograms in plant-oomycete communications? Cell Mol. Life Sci. 56:1020-1047]. 특히, 하르핀의 경우 화상병원균인Erwinia amylovora의 약 40kb의hrp유전자 집단 내hrpN유전자에서 생성된 단백질로서 기주식물에는 병을 일으키는 병원성요인으로 작용하나, 비기주식물에 있어서는 외부침입자로 인식되어 과민반응(HR)인 저항성기작을 나타내게 하는 요인이다. 하르핀은 산성이며, 열(100 ℃)에 대해 안정적이고 44 kda의 분자량을 가지며 글리신 함량이 많으나 시스테인은 없는 것으로 나타났다[Zhong-Min, W., Laby, R. J., Zumoff, C. H., Bauer, D. W., He, S. Y., Collmer, A. and Beer, S. V. 1992. Harpin, elicitor of the hypersensitive response produced by the plant pathogenErwinia amylovora. Science 257:85-88; 미국 특허 제 6,001,959호; 미국 특허 제5,850,015호; 미국 특허 등록번호 제 6,172,184호; 미국 특허 등록번호 제 6,174,717호; 미국 특허 제 5,849,868 호; 미국 특허 제 6,977,060호; 미국 특허 제 5,859,324호; 미국 특허 제 5,776,889호; 대한민국 특허 공개번호 제1999-022577호; 대한민국 특허 공개번호 제2000-075771호; 대한민국 특허 공개번호 제2000-070495호; 대한민국 특허 공개번호 제 2000-057395호].All of these plant disease resistance mechanisms are driven by elicitors (inducers) to induce plant biologic defense systems [Kessmann, H., Staub, T., Hofmann, C., Maetzke, T., Herzog, J., Ward, E., Uknes, S. and Ryals, J. 1994. Induction of systemic acquired disease resistance in plants by chemicals. Annu. Rev. Phytopathol. 32: 439-459. SAR is induced by salicylic acid (SA), a phenolic signaling substance, and other representatives are elicitin and harpin isolated from pathogens [Ponchet, M., Panabieres]. , F., Milat, ML, Mikes, V., Montillet, JL, Suty, L., Triantaphylides, C., Tirilly, Y. and Blein, JP 1999. Are elicitins cryptograms in plant-oomycete communications? Cell Mol. Life Sci. 56: 1020-1047]. In particular, harpin is a protein produced by the hrp N gene in the hrp gene population of about 40 kb of Erwinia amylovora , which acts as a pathogenic agent to host plants, but is recognized as an external invader in non-host plants. It is a factor that causes the resistance mechanism, which is a response (HR). Harpin is acidic, stable to heat (100 ° C), has a molecular weight of 44 kda, high glycine content, but no cysteine [Zhong-Min, W., Laby, RJ, Zumoff, CH, Bauer, DW, He, SY, Collmer, A. and Beer, SV 1992. Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora . Science 257: 85-88; US Patent No. 6,001,959; US Patent No. 5,850,015; US Patent No. 6,172,184; US Patent No. 6,174,717; US Patent No. 5,849,868; US Patent No. 6,977,060; US Patent No. 5,859,324; US Patent No. 5,776,889; Korean Patent Publication No. 1999-022577; Korean Patent Publication No. 2000-075771; Korean Patent Publication No. 2000-070495; Korean Patent Publication No. 2000-057395].

이들 식물생체 방어시스템을 유도하는 물질을 이용하여 현재 각종 제재가 시판되고 있다. 그 중 대표적인 것이 SA에 의해서 SAR이 유도된다는 사실이 밝혀진 후, SA와 구조가 유사한 INA(2,6-dichloroisonicotinic acid)와 BTH (benzothiadiazole)도 SAR을 유도한다는 것을 발견하여 식물활성물질로 등록된 후, ActigardTM과 BION이라는 이름으로 관엽채소, 토마토, 담배의 병에 대한 식물보호제로 미국과 유럽 등지에서 판매되고 있다. 또한, 일본에서는 PBZ (probenazole)를 Oryzemate 라는 이름으로 벼 도열병과 흰잎마름병의 방제약제로 사용하고 있다[Yoshioka, K., Nakashita, H., Klessig, D. F. and yamaguchi, I. 2001. Probenazole induces systemic acquired resistance inArabidopsiswith a novel type of action. Plant J. 25:149-157].Various sanctions are currently on the market using substances that induce these plant biological defense systems. Among them, SA was induced by SA, and INA (2,6-dichloroisonicotinic acid) and BTH (benzothiadiazole), which are similar in structure to SA, were found to induce SAR. , ActigardTMAnd BIONAs a plant protection agent against diseases of houseplants, tomatoes and tobacco under the name, it is sold in the United States and Europe. In Japan, PBZ (probenazole) Oryzemate It is used as a control agent for rice blast and leaf blight [Yoshioka, K., Nakashita, H., Klessig, D. F. and yamaguchi, I. 2001. Probenazole induces systemic acquired resistance inArabidopsiswith a novel type of action. Plant J. 25: 149-157.

특히, 그램음성 세균인 화상병원균(Erwinia amylovora)에서 그 존재가 처음 밝혀진 하르핀의 경우, 식물에 직접 분무하여 SAR을 유도하고 몇몇 종류의 곤충, 응애, 선충에 대한 방충제의 효능을 가지고 있으며, 광합성과 영양분 흡수를 촉진시켜 식물의 영양생장 및 생식생장을 증진시키는 식물생장촉진(PGP; plant growthprompting) 효과가 있는 것으로 보고되었다[Dong, H. S., Delaney, T. P., Bauer, D. W. and Beer, S. V. 1999. Harpin induces disease resistance inArabidopsisthrough the systemic acquired resistance pathway mediated by salicylic acid and theNIM1gene. Plant J. 20:207-215]. 또한, 하르핀은 독성이 거의 없고, 단백질이어서 사용 후 빨리 분해되어 환경오염의 염려가 없는 것이 특징이며, 고온처리(100 ℃)하여도 파괴되지 않아 제형화에 장점을 가지고 있는 것으로 나타났다[Zhong-Min, W., Laby, R. J., Zumoff, C. H., Bauer, D. W., He, S. Y., Collmer, A. and Beer, S. V. 1992. Harpin, elicitor of the hypersensitive response produced by the plant pathogenErwinia amylovora. Science 257:85-88].In particular, in the case of Harpin, which was first identified in the Gram-negative bacterium Erwinia amylovora , it is sprayed directly on plants to induce SAR and has the effect of insect repellent against several types of insects, mites and nematodes. It has been reported to have the effect of plant growthprompting (PGP), which promotes nutrient absorption and reproductive growth of plants by promoting nutrient absorption [Dong, HS, Delaney, TP, Bauer, DW and Beer, SV 1999. Harpin induces disease resistance in Arabidopsis through the systemic acquired resistance pathway mediated by salicylic acid and the NIM1 gene. Plant J. 20: 207-215. In addition, harpin is almost non-toxic, it is a protein, so it decomposes quickly after use, and there is no concern of environmental pollution, and it does not break down even at high temperature (100 ° C.). Min, W., Laby, RJ, Zumoff, CH, Bauer, DW, He, SY, Collmer, A. and Beer, SV 1992. Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora . Science 257: 85-88.

그래서 미국의 신생 벤처회사인 엔덴 바이오사이언스(Eden Bioscience) 사에서 화상병원균(E. amylovora)에서 유래한 하르핀을 이용하여 Messenger라는 이름으로 상품화하여 2000년부터 면화, 토마토, 담배, 고추, 오이, 딸기, 밀 등의 작물을 대상으로 미국에서 시판되고 있다. 이는 특히 진균제, 세균제, 바이러스제, 방충제, 그리고 식물생장 촉진제로서의 생물농약으로 인정받고 있다[미국 특허 등록번호 제 6,174,717호; 미국 특허 제 5,849,868호; 미국 특허 제 6,977,060호; 미국 특허 제 5,859,324호; 미국 특허 제 5,776,889호; 대한민국 특허 공개번호 제 1999-022577호; 대한민국 특허 공개번호 제 2000-075771호; 대한민국 특허 공개번호 제 2000-070495호; 대한민국 특허 공개번호 제 2000-057395호].That's why a messenger using Harpin from E. amylovora at Eden Bioscience, a new startup in the United States. Since 2000, it has been marketed in the United States for crops such as cotton, tomato, tobacco, pepper, cucumber, strawberry, and wheat. It is particularly recognized as a biopesticide as fungicides, bacteria, viruses, insect repellents, and plant growth promoters (US Pat. No. 6,174,717; US Patent No. 5,849,868; US Patent No. 6,977,060; US Patent No. 5,859,324; US Patent No. 5,776,889; Korean Patent Publication No. 1999-022577; Korean Patent Publication No. 2000-075771; Korean Patent Publication No. 2000-070495; Korean Patent Publication No. 2000-057395].

이에, 본 발명자들은 사과, 배 재배지에서 화상병과 유사한 병징을 발견하여 그 병원균을 분리, 동정하여 연구한 결과, 공지된 화상병원균 및 독일에서 최근에 보고된 가지검은마름병원균과 형태적으로 다른 신규한 가지검은마름병원균 WT#3(KCCM 10283)임을 알아내고, 이 신균주로부터 식물과민반응 유도 단백질을 코딩하고 있는 유전자 및 유도 단백질을 추출하여 식물병 저항성 및 식물생장 촉진 효과에 탁월함을 밝힘으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors have found similar symptoms to burn disease in apple and pear plantations, and have isolated and identified the pathogens. As a result, the present inventors have found a novel novel morphologically different from known burn pathogens and eggplant black germs recently reported in Germany. The present invention was found that the eggplant black rye pathogen WT # 3 (KCCM 10283), and extracting genes and inducing proteins encoding plant hypersensitivity inducing protein from the new strain, and excellent in plant disease resistance and plant growth promoting effect To complete.

따라서, 본 발명은 가지검은마름병원균 WT#3(KCCM 10283) 및 이를 이용한 생물농약을 제공하는데 그 목적이 있다.Accordingly, an object of the present invention is to provide a branched black blight pathogen WT # 3 (KCCM 10283) and a biopesticide using the same.

도 1은 본 발명에 따른 가지검은마름병원균 WT#3(KCCM 10283), 국내 가지마름병원균(E. pyrifoliaeEp16T) 및 화상병원균(E. amylovoraATCC 15580T) TEM 사진을 나타낸 것이다.Figure 1 shows a branched black blight pathogen WT # 3 (KCCM 10283), domestic bough blight pathogen ( E. pyrifoliae Ep16 T ) and burn pathogen ( E. amylovora ATCC 15580 T ) TEM picture according to the present invention.

도 2는 바이오로그 시스템을 이용하여 본 발명에 따른 가지검은마름병원균 WT#3(KCCM 10283)를 분류한 것이다.Figure 2 is a classification of the branched black blight pathogen WT # 3 (KCCM 10283) according to the present invention using a biolog system.

도 3은 본 발명에 따른 가지검은마름병원균 WT#3(KCCM 10283)의 16S rRNA 유전자를 분석하여 계통분류학적 위치를 나타낸 것이다.Figure 3 shows the phylogenetic location by analyzing the 16S rRNA gene of the branched black blight pathogen WT # 3 (KCCM 10283) according to the present invention.

도 4는 본 발명에 따른 가지검은마름병원균 WT#3(KCCM 10283)의 ITS지역 중 tRNAAla을 코딩하고 있는 지역을 분석한 결과를 나타낸 것이다.Figure 4 shows the results of analyzing the region encoding the tRNAAla of the ITS region of the eggplant black blight pathogen WT # 3 (KCCM 10283) according to the present invention.

도 5는 본 발명에 따른 가지검은마름병원균 WT#3(KCCM 10283)의 ITS지역 중 tRNAGlu을 코딩하고 있는 지역을 분석한 결과를 나타낸 것이다.Figure 5 shows the results of analyzing the region encoding the tRNAGlu of the ITS region of the eggplant black blight pathogen WT # 3 (KCCM 10283) according to the present invention.

도 6은 여러 균의 플라스미드 프로필 분석 결과를 나타낸 것이다.Figure 6 shows the results of plasmid profile analysis of several bacteria.

도 7은 본 발명에 따른 가지검은마름병원균 WT#3(KCCM 10283)의 식물과민반응 유도 단백질을 코딩하는 유전자의 유사도를 분석한 것이다.Figure 7 is an analysis of the similarity of the gene encoding the plant hypersensitivity reaction protein of eggplant black blight pathogen WT # 3 (KCCM 10283) according to the present invention.

도 8은 pKEP3에 클로닝 된 WT#3-1 유전자에서 발현된 식물과민반응 유도 단백질을 나타낸 것이다.Figure 8 shows a plant hypersensitivity induction protein expressed in the WT # 3-1 gene cloned in pKEP3.

도 9는 본 발명에 따른 가지검은마름병원균 WT#3(KCCM 10283)의 식물과민반응 유도 단백질의 유사도를 분석한 것이다.9 is an analysis of the similarity of the plant hypersensitivity reaction protein of eggplant black blight pathogen WT # 3 (KCCM 10283) according to the present invention.

도 10은 담배 엽맥에 가지검은마름병원균 WT#3(KCCM 10283) 유래 식물과민반응 유도 단백질을 접종하여 나타난 식물과민반응을 나타낸 것이다Figure 10 shows the plant hypersensitivity reaction inoculated with plant hypersensitivity induction protein derived from eggplant black blight pathogen WT # 3 (KCCM 10283) to tobacco leaf veins

도 11은 어린 배 과실표면에 버퍼만 처리한 대조구와 본 발명에 따른 가지검은마름병원균 WT#3의 식물과민 반응 단백질을 처리한 후 병징을 나타낸 사진이다.11 is a photograph showing the symptom after treatment of the control control protein of eggplant black blight pathogen WT # 3 according to the present invention and the control treated only buffer on the surface of young pear fruit.

도 12는 배추노균병(Peronospora brassica)에 대한 방제효과를 나타낸 사진이다.Figure 12 is a photograph showing the control effect on the Chinese cabbage ( Peronospora brassica ).

도 13은 참외노균병(Pseudoperonospora cubensis)에 대한 방제효과를 나타낸 사진이다.Figure 13 is a photograph showing the control effect against Pseudoperonospora cubensis .

도 14는 오이 흰가루병(Sphaerotheca fuliginea)에 대한 방제효과를 나타낸 사진이다[오이 흰가루병은 발병도를 소, 중, 다, 심, 0으로 구분하였으며, 그림의 오른쪽부터 심, 다, 중, 소, 0의 순이다].Figure 14 is a photograph showing the control effect on cucumber powdery mildew ( Sphaerotheca fuliginea ) [in the case of cucumber powdery powder is divided into small, medium, many, heart, 0, the incidence of In order.

도 15는 오이의 수확량을 나타낸 사진이다.15 is a photograph showing the yield of cucumbers.

도 16은 고추의 수확량을 나타낸 사진이다.16 is a photograph showing the yield of pepper.

도 17은 콩나물 성장촉진을 나타낸 사진이다.17 is a photograph showing growth of bean sprouts.

본 발명은 가지검은마름병원균 WT#3(KCCM 10283) 및 이를 이용한 식물병 방제제 및 식물생장 촉진제와 같은 생물농약을 그 특징으로 한다.The present invention is characterized by biopesticides such as eggplant black blight pathogen WT # 3 (KCCM 10283) and plant disease control agents and plant growth promoters using the same.

또한, 상기 균주 유래의 유전자를 포함한 재조합 pKEP3(KCCM 10282) 및 이를 이용한 식물과민반응 유도 단백질을 대량 생산하는 방법도 포함한다.In addition, recombinant pKEP3 (KCCM 10282) including the gene derived from the strain and a method for mass production of plant hypersensitivity induction protein using the same.

이와 같은 본 발명을 상세하게 설명하면 다음과 같다.The present invention will be described in detail as follows.

본 발명에 따른 신균주는 춘천의 사과, 배 재배단지에서 화상병(Erwinia amylovora)과 유사한 병징을 발견하여, 병원균을 분리·동정한 결과,Erwinia속에 속하는 신종(사과배가지검은마름병;Erwinia pyrifoliae, 독일팀에서 1999년 보고)과 같은 종으로 동정되어졌으나, 화상병원균과 독일팀에서 보고한 가지검은마름병원균 모두 여러 개의 편모를 가지고 있는 주생모인 반면, 신균주는 편모(flagella)를 가지고 있지 않는 무편모(non-flagella)로 국내에만 존재하는 가지검은마름병원균과도 형태적으로 큰 차이점을 나타낸다. 상기 신균주를 가지검은마름병원균 WT#3(Erwinia pyrifoliaeWT#3)으로 명명하고, 한국미생물보존센터에 2001년 6월 11일자로 기탁하였으며, 기탁번호는 KCCM 10283이다.The new strain according to the present invention finds a symptom similar to burn disease ( Erwinia amylovora ) in the apple and pear cultivation complex of Chuncheon, and isolates and identifies the pathogens, resulting in a new species belonging to the genus Erwinia (apple goji blight; Erwinia pyrifoliae , The pathogen was identified by the German team (reported 1999), but the burn pathogen and the branched black pathogen reported by the German team were both main hairs with multiple flagella, whereas the new strain did not have flagella. Eggplant black (non-flagella), which exists only in Korea, differs in morphology from blight germs. The new strain was named as the black blight pathogen WT # 3 ( Erwinia pyrifoliae WT # 3), and was deposited with the Korea Microorganism Conservation Center on June 11, 2001, and the accession number is KCCM 10283.

특히, 가지검은마름병원균 WT#3으로부터 유도체(elicitor; inducer)를 코딩하고 있는 유전자와 유도체 자체를 미국 코넬 대학에서 발견하여 에덴 바이오사이언스 사에서 시판하고 있는 하르핀과 비교, 분석한 결과, 유전자의 경우hrpN유전자와 유사도가 낮은 새로운 종류의 유전자를 클로닝하여 얻을 수 있다. 또한, 이 유전자로부터 생산되는 단백질의 경우, 하르핀 펩타이드와 유사도가 낮은 아미노산 배열을 가지고 있다.Especially, eggplant black blight pathogen Genes encoding derivatives from WT # 3 and the derivatives themselves were found by Cornell University and compared with Harpin commercially available from Eden Bioscience.hrpNThis can be achieved by cloning a new class of genes that are less similar to genes. In addition, the protein produced from this gene has an amino acid sequence that is less similar to the harpin peptide.

한편, 상기 균주 유래의 유전자, WT#3-1을 포함하는 재조합 플라스미드 pKEP3를 제작하고 이를 대장균에 형질전환시켜 이 형질전환체를 한국미생물에 2001년 6월 11일자로 기탁하였으며, 기탁번호는 KCCM 10282이다.Meanwhile, a recombinant plasmid pKEP3 containing the strain-derived gene, WT # 3-1, was prepared and transformed into Escherichia coli, and the transformant was deposited on Korean microorganisms as of June 11, 2001, and the accession number was KCCM. 10282.

상기 플라스미드를 함유한 형질전환체를 이용하여 기존의 하르핀보다 식물병저항성(plant disease resistance)과 식물생장 촉진(plant growth prompting)효과가 우수한 식물과민반응 유도 단백질을 대량 생산할 수 있다.By using the transformant containing the plasmid, it is possible to mass-produce a plant hypersensitivity inducing protein having better plant disease resistance and plant growth prompting effects than conventional harpins.

상기 단백질의 효과를 검정한 결과, 배추노균병, 참외노균병, 오이 흰가루병, 피망 역병, 피망 탄저병, 고추 역병, 벼 잎도열병에 대한 유도내성에 의한 방제효과가 화상병원균 유래 식물과민반응 단백질(Harpin)보다 우수하게 나타났고,오이, 피망, 딸기, 고추, 콩나물 수확량 증대 실험에서도 화상병원균 유래 식물과민반응 단백질(Harpin)보다 가지검은마름병원균 WT#3 유래 식물과민반응 유도 단백질이 더 우수한 증대효과를 가져왔다.As a result of assaying the effect of the protein, the control effect by the induced resistance to Chinese cabbage fungus, melon germ disease, cucumber powdery mildew, green pepper blight, green pepper anthrax, red pepper blight, rice leaf blast disease is more effective than plant-derived protein It was found to be excellent, and in the experiments to increase yields of cucumbers, green peppers, strawberries, peppers and bean sprouts, eggplant black blight pathogens were higher than burn-induced plant hypersensitivity protein (Harpin). WT # 3-derived plant hypersensitivity inducing protein had a better augmentation effect.

이하, 본 발명은 실시예에 의거하여 더욱 상세히 설명하겠는바, 본 발명이 다음 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples.

실시예 1: 신균주 분리 및 동정Example 1: Isolation and Identification of Mycobacteria

1) Schaad의 지침서 및 Bergey's manual에 준한 생리·생화학 실험1) Physiological and biochemical experiments according to Schaad's guidelines and Bergey's manual

춘천근교 배 재배지에서 화상병(Erwinia amylvora)과 유사한 병징을 발견하여 분리된 병원세균을 분리, 동정하기 위해 Schaad의 지침서[Schaad, N. W. 1988. Initial identification of common genera. In:Laboratory Guide for Identification of Plant Pathogenic Bacteria, ed. by N. W. Schaad. American Phytopathological Society., Minnesot. pp. 44-59] 및 Bergey's manual[Lelliott, R. A. and Dickey, R. S. 1984. GenusErwinia. In: Bergey's Manual of Systemic Bacteriology. vol. 1, pp. 469-476, Williams and Willkins Co., Baltimore/London]을 참고로 하여 본 병원세균이 포함되는Erwinia속의 생리·생화학 실험들을 수행한 결과, 다음 표 1과 같다.In order to isolate and identify isolated pathogens by finding a symptom similar to Erwinia amylvora in Chuncheon cultivated fields, Schaad 's guidelines [Schaad, NW 1988. Initial identification of common genera. In: Laboratory Guide for Identification of Plant Pathogenic Bacteria , ed. by NW Schaad. American Phytopathological Society., Minnesot. pp. 44-59 and Bergey's manual [Lelliott, RA and Dickey, RS 1984. Genus Erwinia . In: Bergey's Manual of Systemic Bacteriology. vol. 1, pp. 469-476, Williams and Willkins Co., Baltimore / London], the results of the physiological and biochemical experiments of the genus Erwinia containing the pathogen, as shown in Table 1 below.

상기 분리 균주는 젤라틴 액화, 3% 한천에서의 운동성 및 펙테이트(pectate)분해 등 생리적 실험에서는 전반적으로 가지검은마름병원균으로 공인된E. pyrifoliae와 잘 일치하였다.The isolated strains were in good agreement with E. pyrifoliae , which is generally recognized as a branched black pathogen in physiological experiments such as gelatin liquefaction, motility in 3% agar and pectate degradation.

반면, 탄소원의 이용도를 알아보기 위한 생화학적 요구도 실험에서는 트레할로스(Trehalose)와 L-아라비노스(L-arabinose)에서 다른 결과를 얻을 수 있었다. 즉, 이는 본 분리균주의 경우, 공인된 가지마름병원균과도 생리·생화학적 특성이 다르다는 것을 알 수 있다.On the other hand, biochemical demand experiments for the utilization of carbon sources showed different results in Trehalose and L-arabinose. That is, it can be seen that the physiological and biochemical characteristics of the isolated strains are different from those of the recognized eggplant pathogen.

2) 분리균주의 형태적 특성2) Morphological characteristics of separated strains

본 실험실에서 분리한 가지마름병원균 중 최근 공인된 국내 가지마름병원균(E. pyrifoliae)과 같은 그룹으로 분리되는 상기 분리 균주의 형태적 특성을 TEM (transmitted electro microscoph)을 이용하여 관찰한 결과, 가지마름병원균과 상이한 형태를 확인하였다[도 1].As a result of observing the morphological characteristics of the isolated strain which is separated into the same group as the recently recognized domestic eggplant pathogen ( E. pyrifoliae ) among the eggplant pathogens isolated in this laboratory using TEM (transmitted electro microscoph), Different forms were identified with the pathogens [FIG. 1].

즉, 가지마름병원균과 화상병원균이 속해 있는Erwinia속은 주생모(peritrichous flagella)의 간균형이다. 그러나, 상기 분리 균주의 경우, 간균형에 편모를 가지고 있지 않는 대표적인 차이점을 관찰할 수 있었다.In other words, the genus Erwinia , which belongs to eggplant and burn pathogens, is a hepatitis of the peritrichous flagella. However, in the case of the isolated strain, a representative difference that does not have flagella in the liver balance could be observed.

3) 바이오로그 시스템(Biolog System)을 이용한 분리 균주의 특성3) Characteristics of Isolated Strains Using Biolog System

분리 균주의 생화학적 특성을 세부적으로 알아보기 위해 96가지의 서로 다른 탄소원 및 질소원의 이용여부를 알아보는 바이오로그 시스템[BIOLOG, Hayward, CA 94545, USA]을 이용하였다. 먼저, TSA(triptic soy agar) 배지에서 28 ℃ 24시간 배양된 균체를 0.4% 염화나트륨, 0.03% 플루로닉(Pluronic) F-68, 0.01% 젤란 검(Gellan Gum) 용액에 63%의 현탁도로 현탁시킨 후, 96가지의 탄소원 및 질소원이 들어있는 wells에 치상하였다. 그 후 35 ∼ 37 ℃ 항온배양기에서 24시간 동안 배양하여 보라색으로 변하는 탄소원 및 질소원의 이용여부를 판독기를 이용하여 표시하고 그 값을 수리분류학적으로 구분하였다.The biolog system [BIOLOG, Hayward, CA 94545, USA] was used to examine the use of 96 different carbon and nitrogen sources in order to examine the biochemical properties of the isolates in detail. First, cells incubated at 28 ° C. for 24 hours in TSA (triptic soy agar) medium were suspended with 63% suspension in 0.4% sodium chloride, 0.03% Pluronic F-68, and 0.01% Gellan Gum solution. After incubation, it was wounded in wells containing 96 carbon and nitrogen sources. Thereafter, the cells were incubated for 24 hours in a 35-37 ° C. incubator to indicate the use of carbon and nitrogen sources that turned purple using a reader, and the values were classified by hydraulic classification.

도 2와 같이, 화상병원균인 ATCC15580, LMG2068, LMG1877, LMG1946 및ea246(미국) 균주만이 동일한 그룹으로 나타났다. 반면, 상기 분리 균주는 가지검은마름병원균(Ep4, Ep8, Ep16)과 같은 그룹으로 나타났다. 즉, 분리 균주는 화상병원과 다른 종임을 알 수 있었다.As shown in Fig. 2, only the ATCC15580, LMG2068, LMG1877, LMG1946, and ea246 (US) strains of the burn pathogens appeared in the same group. On the other hand, the isolated strain was found in the same group as the eggplant black blight pathogens (Ep4, Ep8, Ep16). That is, it was found that the isolated strain is a species different from burn hospital.

4) 분리 균주의 16S rRNA 유전자 분석4) 16S rRNA Gene Analysis of Isolated Strains

상기 분리 균주의 계통분류학적 위치를 알아보기 위해, 생명현상에 있어 필수적이고 그 염기서열이 비교적 잘 보존되어 있으며, 통계학적으로 분석하기에 용이한 16S rRNA 유전자의 염기서열을 분석하였다. 먼저, 서열번호 1로 나타낸 fD1 프라이머와 서열번호 2로 나타낸 rP2 프라이머를 이용하여 PCR 방법으로 증폭하여 얻었다. 그 후 pGEM-T 벡터 시스템을 이용하여 클로닝하여 그 염기서열을 분석하였다.In order to determine the phylogenetic location of the isolated strain, we analyzed the nucleotide sequence of 16S rRNA gene which is essential for life phenomena, its nucleotide sequence is relatively well preserved, and is easy to analyze statistically. First, the fD1 primer shown in SEQ ID NO: 1 and the rP2 primer shown in SEQ ID NO: 2 were amplified by PCR. Then, the nucleotide sequence was analyzed by cloning using the pGEM-T vector system.

상기 분석된 염기서열은 메가 프로그램[Kumar, S., Tamura, K. and Nei, M. 1993. MEGA: molecular evolutionary genetics analysis, version 1.0. The Pennsylvania State University, University Park.]에 의해 그 계통도를 작성한 결과 도 3과 같으며, 그 유사도로 볼 때 분리 균주는 가지검은마름병원균(E. pyrifoliaeEp16)과 98.9%의 유사도로 화상병원균보다 더 유사하게 나타났다.The analyzed nucleotide sequence is a mega program [Kumar, S., Tamura, K. and Nei, M. 1993. MEGA: molecular evolutionary genetics analysis, version 1.0. The Pennsylvania State University, University Park.] Shows that the schematic diagram is shown in Fig. 3, and the similarity of the isolate is higher than that of burn pathogen with 98.9% similarity to E. pyrifoliae Ep16. Similarly appeared.

각 균주간 16S rRNA 유전자의 유사도는 다음 표 2와 같다.The similarity of 16S rRNA gene between each strain is shown in Table 2 below.

또한, 16s rRNA 유전자 분석결과, 분리 균주는E. pyrifoliae와 같은 그룹으로 나타난 반면, 화상병원균인E. amylovora및 몇 해 전 국내의 사과 배에 발생하는 것으로 보고된 사과갈색잎점무늬병원균(Enterobacter pyrinus)과도 다른 그룹으로 나타났다.In addition, 16s rRNA gene analysis showed that the isolated strains were found in the same group as E. pyrifoliae , while E. amylovora , a burn pathogen, and apple brown leaf spot pathogen ( Enterobacter pyrinus) reported to occur in apple pears in Korea a few years ago . And appeared in a different group.

5) 분리 균주의 16S-23S ISR(Intergenic Spacer Region) 분석5) 16S-23S Intergenic Spacer Region Analysis of Isolated Strains

국내 가지마름병원균 및 국외 화상병원균의 ISR을 분석하기 위해, 서열번호 3로 나타낸 R16-1F과 서열번호 4로 나타낸 R23-1R 프라이머를 이용하여 PCR 방법으로 증폭하여 얻었다. 그 후 pGEM-T 벡터시스템을 이용하여 클로닝한 후, 그 염기서열을 분석하였다. 그 결과, 두 그룹으로 분리되었다. 즉, 화상병원균(E. amylovora)은 약 1215, 970, 720 bp 크기의 3종류의 밴드 패턴이었으며, 가지마름병원균(E. pyrifoliae)은 970, 720 bp의 2종류의 밴드 패턴만이 나타났다.In order to analyze ISR of domestic eggplant pathogens and foreign burn pathogens, R16-1F shown in SEQ ID NO: 3 and R23-1R primers shown in SEQ ID NO: 4 were amplified by PCR. Then, after cloning using the pGEM-T vector system, the base sequence was analyzed. As a result, it was divided into two groups. In other words, E. amylovora had three types of band patterns of about 1215, 970, and 720 bp size, while E. pyrifoliae had two types of band patterns of 970 and 720 bp.

현재, 이 두 그룹의 모든 균주가 970 bp는 약 70 bp크기의 tRNAAla지역을 가지고 있는 것으로, 720 bp크기는 tRNAGlu지역을 포함하고 있는 것으로 밝혀졌으며,E. amylovoraATCC15580과 분리 균주의 염기서열을 분석하여 계통도를 작성한 결과 도 4와 같다.Currently, all strains of these two groups were found to have a tRNAAla region of about 70 bp in size and 970 bp to include a tRNAGlu region, and analyzed the nucleotide sequences of E. amylovora ATCC15580 and isolates. The result of the schematic diagram is as shown in FIG. 4.

ITS지역 중 tRNAAla지역을 코딩하고 있는 970 bp 밴드의 염기서열을 분석하여 계통도를 작성한 결과, 분리 균주는 화상병원균 그룹과는 다른 그룹으로 나타났으며, 유사도에서 분리 균주는 화상병원균과 47.4%와 80.3%의 값을 나타났다. 그러나, 독일팀에 의해 가지검은마름병원균이라 명칭된Erwinia pyrifoliae의 tRNAAla지역은 등록되지 않아 비교하지 못하였다.As a result of analyzing the sequencing of the 970 bp band encoding the tRNAAla region of the ITS region, the isolated strains were found to be different from those of the burn pathogen group, and the similar isolates were found to be 47.4% and 80.3. Showed a value of%. However, the tRNAAla region of Erwinia pyrifoliae , known as the eggplant black blight pathogen, was not registered and could not be compared.

tRNAGlu지역을 코딩하고 있는 720 bp의 염기서열을 분석한 결과, 분리 균주는 독일팀에서 발표한 가지검은마름병원균(Erwinia pyrifoliae)과 85.2 ∼ 92.7%의 유사도를 나타내어 같은 그룹으로 나타났다[도 5].As a result of analyzing the sequence of 720 bp encoding the tRNAGlu region, the isolated strains showed similarity of 85.2-92.7% with the eggplant black blight pathogen ( Erwinia pyrifoliae ) published by the German team [Fig. 5].

6) 플라스미드 프로필에 의한 가지마름병원균의 분석6) Analysis of Eggplant Pathogens by Plasmid Profile

국내 가지검은마름병원균 및 국외 화상병원균의 플라스미드를 분리 그 양상을 분석한 결과, 2종류의 그룹으로 분리되었다[도 6].As a result of separating and analyzing the plasmids of domestically black blight and foreign burn pathogens, they were separated into two groups [FIG. 6].

Group I - 화상병원균E. amylovora(ATCC15580, LMG1877, 10296) (>29 kb)Group I-burn pathogen E. amylovora (ATCC15580, LMG1877, 10296) (> 29 kb)

Group Ⅱ - 가지검은마름병원균E. pyrifoliae(ep1, ep16, WT#3) (>29 kb, 5 kb, 2 ∼ 4 kb내에 3개)Group II-Eggplant black blight pathogen E. pyrifoliae (ep1, ep16, WT # 3) (> 29 kb, 5 kb, 3 to 2 kb)

즉, 화상병원균 그룹은 29 kb보다 큰 플라스미드 한 개만 존재하나, 분리 균주를 포함하는 가지검은마름병원균(E. pyrifoliae) 그룹은 5개의 플라스미드가 존재하는 것으로 나타났다.That is, only one plasmid larger than 29 kb exists in the burn pathogen group, but five plasmids exist in the E. pyrifoliae group including the isolated strain.

7) DNA-DNA 하이브리다이제이션(hybridization)에 의한 DNA 유사도 분석7) DNA similarity analysis by DNA-DNA hybridization

국내 가지검은마름병원균 및 국외 화상병원균의 전체 지놈 유사도를 조사하였다. 그 방법으로는 순수하게 분리된 전체 DNA를 1 ng/㎕로 정량하여 10 N NaOH를 첨가한 후, 80 ℃에서 10분간 끓여 DNA를 변성시켰다. 그 후 DIG-하이 프라임 시스템[Roche Molecular Biochemicals, Sandhofer Strasse 116, Germany]을 이용하여 탐침으로 사용할 DNA를 라벨링하여 나일론 막에 부착된 DNA와 함께 49℃에서 3시간동안 프리하이브리다이제이션을 시켰으며, 그 후 같은 온도에서 16시간동안 하이브리다이제이션 반응을 유도하였다. 반응이 끝난 막은 DIG 냉광 검출 킷[Roche Molecular Biochemicals, Sandhofer Strasse 116, Germany]를 이용하여 그 반응을 검정하였다.The overall genome similarity between domestic and black blight and foreign burn pathogens was investigated. In this method, purely isolated whole DNA was quantified at 1 ng / μl, 10 N NaOH was added, and the DNA was boiled at 80 ° C. for 10 minutes to denature the DNA. Then, the DNA to be used as a probe was labeled using a DIG-high prime system (Roche Molecular Biochemicals, Sandhofer Strasse 116, Germany), followed by prehybridization at 49 ° C. for 3 hours with DNA attached to a nylon membrane. The hybridization reaction was then induced at the same temperature for 16 hours. The reaction membrane was assayed using DIG cold light detection kit (Roche Molecular Biochemicals, Sandhofer Strasse 116, Germany).

그 결과, 그룹 I에는 국외 화상병원균(E. amylovoraATCC15580, LMG1877, LMG1946, LMG2068, 10296, 10297)들이 속하였으며, 그룹 Ⅱ에는 가지검은마름병원균(E. pyrifoliaeEp4, Ep8, Ep16, WT#3)이 해당되었다. 즉, 국외 화상병원균 및 최근 일본 요꼬하마 식물 보호 담당팀에 의해 화상병원균의 biovar로 재명명된 일본균주가 그룹 I이었고, 국내 가지마름병원균들은 그룹 Ⅱ로 분리되었다. 그 결과를 유사도로 표현하면 다음 표 3과 같다.As a result, group I belonged to foreign burn pathogens ( E. amylovora ATCC15580, LMG1877, LMG1946, LMG2068, 10296, 10297), and group II included branched black pathogens ( E. pyrifoliae Ep4, Ep8, Ep16, WT # 3). This was the case. In other words, group I was a group I, which was renamed as a biovar of burn pathogen by the foreign burn pathogen and the Japanese Yokohama plant protection team. The results are expressed in similarity as shown in Table 3 below.

이상의 분리 균주의 생화학적, 생리적, 유전적 특성을 종합해 보면, 분리 균주는 Schaa의 지침서 및 Bergey's manual에 준한 생리·생화학 특성, 바이오로그 시스템, 16S rRNA유전자, 16S-23S ISR, 플라스미드 프로필, 전체 DNA 유사도 실험 결과, 모두 최근 1999년 국내에만 존재하는 것으로 보고된 가지검은마름병원균(E. pyrifoliae)과 같은 그룹으로 나타났으나 몇몇 다른 특성을 가지고 있었다. 특히, 형태적으로 편모를 가지고 있지 않은 무편모(non-flagella)의 특성으로 보아이는 가지마름병원균과도 차별화 되는 병원균으로 생각된다. 이에, 상기 분리 균주를 가지검은마름병원균 WT#3(Erwinia pyrifoliaeWT#3)로 명명하였다. 이를 한국미생물보존센터에 2001년 6월 11일자로 기탁하였으며, 기탁번호는 KCCM 10283이다.Comprehensive biochemical, physiological and genetic characteristics of the isolated strains, the isolated strains were characterized by physiological and biochemical characteristics, biolog system, 16S rRNA gene, 16S-23S ISR, plasmid profile, whole according to Schaa's manual and Bergey's manual. As a result of DNA similarity test, all of them appeared in the same group as E. pyrifoliae , which was reported to exist only in Korea in 1999, but had some other characteristics. In particular, the characteristics of non-flagella, which do not have flagella, are considered to be pathogens that are differentiated from eggplant pathogens. Thus, the isolated strain was named eggplant black blight pathogen WT # 3 ( Erwinia pyrifoliae WT # 3 ) . It was deposited with the Korea Microorganism Conservation Center on June 11, 2001. The accession number is KCCM 10283.

실시예 2: 가지검은마름병원균 WT#3의 식물과민성 반응을 유발하는 특이 단백질 특성과 이를 코딩하는 유전자Example 2 Specific Protein Characteristics and Genes Encoding Plant Sensitization Responses of WT # 3

1) 가지검은마름병원균 WT#3의 식물과민반응 유도 유전자와 화상병원균의1) Genes of Plant Hypersensitivity Reactions of WT # 3 hrpNhrpN 유전자 유사도 분석Genetic Similarity Analysis

식물과민성 반응을 유도하는 특이 단백질을 코딩하는 유전자를 분석하기 위해, 가지검은마름병원균 WT#3의 전체 DNA를Sau3AI 효소로 부분 분해가 되도록 37 ℃에서 배양하였다. 그 후 1시간 별로 1 ㎕을 전기영동하여 DNA가 분해된 정도를 조사하여 약 6시간 배양 후가 가장 적절한 배양시간으로 나타났다. 이렇게 조작된 전체 DNA를 삽입 DNA로 준비하였으며,BamHI 효소로 분해된 클로닝 벡터에 DNA 리가제를 이용하여 14 ℃에서 12시간 배양하여 연결(ligation)을 유도하였다. 삽입 DNA와 벡터가 붙은 하나의 완전한 플라스미드 DNA를 HB101(E. coli)에 화학적 형질전환방법에 의해 세균 내로 전송하였다. 그 후 테트라사이클린(30 ㎎/㎖)이 함유된 Luria 한천 배지에 37 ℃에서 24시간 배양하여 형성되어지는 2,000개의 콜로니를 선발하였다.In order to analyze genes encoding specific proteins that induce plant hypersensitivity reactions, the whole DNA of WT # 3 in black blight pathogen was incubated at 37 ° C. to be partially digested with Sau 3AI enzyme. After that, 1 μl electrophoresis was performed every hour, and the degree of DNA degradation was examined. Thus prepared whole DNA was prepared as insert DNA, and the ligation was induced by incubating for 12 hours at 14 ° C. using DNA ligase in a cloning vector digested with Bam HI enzyme. One complete plasmid DNA with the inserted DNA and the vector was transferred into bacteria by chemical transformation method to HB101 ( E. coli ). Thereafter, 2,000 colonies were formed, which were formed by incubating at 37 ° C. for 24 hours in Luria agar medium containing tetracycline (30 mg / ml).

그런 다음, 화상병원균(E. amylovoraATCC15580)에서 유래한 식물과민성 반응을 유발하는 단백질(Harpin)을 코딩하고 있는 것으로 알려진hrpN유전자를 탐침으로 사용하여 써든 블랏팅(southern blotting) 방법으로 가지검은마름병원균 WT#3의 식물과민성 반응 유도 특이 단백질을 코딩하고 있는 유전자를 선발하였다.Then, using a hrpN gene known to encode a plant-induced hypersensitivity reaction (Harpin) derived from the burn pathogen ( E. amylovora ATCC15580) as a probe using a southern blotting method Genes encoding the plant-sensitive response inducing specific proteins of WT # 3 were selected.

또한, 선발된 유전자의 염기서열을 분석하여 화상병원균(E. amylovoraATCC15580)의hrpN유전자와 유사도를 비교한 결과는 도 7과 같다.In addition, the result of comparing the similarity with the hrpN gene of the image pathogen ( E. amylovora ATCC15580) by analyzing the nucleotide sequence of the selected gene is shown in FIG.

본 발명에 따른 가지검은마름병원균 WT#3로부터 식물과민성 반응 유도 단백질을 코딩하는 유전자를 분석한 결과, 1287 bp 크기를 얻을 수 있었다. 이에. 1287 bp를 WT#3-1로 명명하여 화상병원균(E. amylovoraATCC15580)의hrpN유전자(1212 bp)와 유사도를 조사하였다. 그 결과, WT#3-1은 83.2% 나타났다. 즉, 가지검은마름병원균 WT#3의 식물과민반응 유도 단백질을 코딩하는 유전자는 기존 화상병원균 유래 유전자와 차이가 있음을 알 수 있었다. 또한, 가지검은마름병원균 WT#3의 식물과민반응 유도 단백질을 코딩하는 유전자(WT#3-1)의 염기서열은 서열번호 5로 나타낸 바와 같다.As a result of analyzing the gene encoding the plant sensitization response inducing protein from the eggplant black blight pathogen WT # 3 according to the present invention, a size of 1287 bp was obtained. Therefore. 1287 bp was named WT # 3-1 and examined for similarity with the hrpN gene (1212 bp) of E. amylovora ATCC15580. As a result, WT # 3-1 was 83.2%. In other words, the gene encoding the plant hypersensitivity reaction protein of the eggplant black blight pathogen WT # 3 was found to be different from the gene derived from the existing burn pathogen. In addition, the nucleotide sequence of the gene (WT # 3-1) encoding the plant hypersensitivity reaction protein of the eggplant black rye pathogen WT # 3 is as shown in SEQ ID NO: 5.

2) 가지검은마름병원균 WT#3의 식물과민반응 유도 단백질 발현벡터 제작2) Preparation of plant hypersensitivity reaction protein expression vector of eggplant black blight pathogen WT # 3

상기 선발된 가지검은마름병원균 WT#3의 식물과민반응 유도 단백질을 코딩하는 유전자로부터 많은 양의 식물과민반응 단백질을 추출, 정제하기 위해 선발된 유전자를 포함하는 재조합 플라스미드 pKEP3를 다음과 같이 제작하였다.The selected branched black plasmid pKEP3 was prepared as follows to extract and purify a large amount of plant hypersensitivity protein from the gene encoding the plant hypersensitivity induction protein of Bacterial pathogen WT # 3.

재조합 플라스미드 pKEP3를 제작하기 위해서 대장균 재조합 단백질 발현 시스템[Novagen, Inc. Madison, WI53711 USA]을 이용하였다. 본 시스템은 pBR322플라스미드에서 근원하였으며, 여기에 외부 유전자 삽입 자리 앞에 T7 프로모터와lac레프레서(repressor)가 붙을 수 있는 오퍼레이터(operator)가 위치하고 있다. 이는 숙주로 사용되는 대장균의 게놈에서 만들어지는 T7 RNA 중합효소(polymerease)에 의해 많은 양을 발현하고자하는 외부 삽입 유전자의 발현을 용이하게 한다. 특히, 기질로 사용되는 IPTG를 배양 3시간 후에 첨가하면lacI유전자에서 생성되는lac레프레서와 이 IPTG가 결합하여 T7 중합효소의 역할을 제어하지 못하게 되므로 많은 양의 단백질이 합성되어지게 된다. 또한, 연결(ligation)되지 않았거나, 대장균 내로 형질전환되지 않은 것과 구별하기 위해 암피실린(ampicillin) 저항성 유전자가 포함되어 있다. 이는 완성된 형질전환체를 선별할 때, 배지에 암피실린을 첨가하여 선택형 마커로 사용할 수 있다.E. coli recombinant protein expression system [Novagen, Inc. for the production of recombinant plasmid pKEP3. Madison, WI53711 USA. The system is derived from the pBR322 plasmid, which has an operator to which the T7 promoter and lac repressor can be attached before the external gene insertion site. This facilitates the expression of an external insertion gene to be expressed in large amounts by T7 RNA polymerase produced in the genome of Escherichia coli used as a host. In particular, when IPTG, which is used as a substrate, is added after 3 hours of culture, a large amount of protein is synthesized because the lac repressor generated from the lacI gene and the IPTG do not control the role of the T7 polymerase. Also included are ampicillin resistance genes to distinguish from those that are not ligation or untransformed into E. coli. It can be used as a selective marker by adding ampicillin to the medium when selecting the finished transformants.

가지검은마름병원균 WT#3 유래 식물과민반응 유도 단백질을 코딩하고 있는 유전자(WT#3-1)와 위 대장균 재조합 단백질 시스템의 플라스미드를 제한효소NdeI과BamHI으로 37 ℃에서 12시간 배양하여 DNA의 5'와 3'말단이 동일한 형태로 형성되게 하였다. 그 후 DNA 리가제를 이용하여 14 ℃에서 16시간 배양하여 연결을 유도하였으며, 화학적 형질전환방법으로 대장균 내로 전송하였다.Genes encoding plant hypersensitivity reaction inducing protein (WT # 3-1) and plasmids of the recombinant E. coli recombinant protein system were tested for 12 hours at 37 ° C with restriction enzymes Nde I and Bam HI. The 5 'and 3' ends are formed in the same shape. After that, the DNA ligase was used to incubate for 16 hours at 14 ° C to induce ligation and transfer to E. coli by chemical transformation.

상기 pKEP3는 암피실린 저항성 유전자를 보유하고 있어 선택형 마커로 사용되며, His 태그를 가지고 있어 정제시 용이한 특성을 가지며, 강력한 T7 lac 프로모터를 가지고 있어 연결된 삽입 DNA로부터 많은 양의 단백질을 생산할 수 있다.The pKEP3 possesses an ampicillin resistance gene and is used as a selective marker, and has a His tag, which is easy to purify, and has a strong T7 lac promoter, thereby producing a large amount of protein from the inserted DNA.

상기 플라스미드를 대장균에 도입하여 형질전환된 대장균(pKEP3)을 한국미생물보존센터에 2001년 6월 11일자로 기탁하였으며 기탁번호는 KCCM 10282이다.The plasmid was introduced into Escherichia coli and transformed Escherichia coli (pKEP3) was deposited with the Korea Microorganism Conservation Center on June 11, 2001. The accession number is KCCM 10282.

3) 식물과민반응 유도 단백질 발현3) Plant hypersensitivity induction protein expression

형질전환된 대장균(pKEP3)(KCCM 10282)으로부터 단백질을 대량 생산하기 위해, 선택형 마커로 암피실린 50 ㎍/㎖과 대장균 자체의 게놈에서 만들어지는 다른 단백질들의 합성을 저해하기 위해 클로람프니콜(chloramphnicol) 33 ㎍/㎖을 첨가한 LB 액체배지에 형질전환체(KCC 10282)를 접종하여 37 ℃에서 12시간 2차 배양(subculture)한 후, 동일한 배지에 30 ℃에서 7시간 본 배양을 실시하였다. 이때 약 3시간 후 형질전환체의 OD값이 0.6이 되었을 때, IPTG 0.4 mM을 첨가하여 30 ℃로 온도를 낮추어 4시간동안 배양하였다. 총 7시간동안의 본 배양을 한 후, 원심분리(6,000 rpm, 15분)를 실시하여 상층액은 버리고 펠렛만을 5 mM MES 버퍼와 0.1 mM PMSF를 넣어 잘 혼합하였다. 혼합된 형질전환체(KCCM 10282)를 혼합액이 투명해질때까지 초음파 분쇄(sonication)하여 100 ℃에서 10분간 끓였다. 그 후, 15,000 rpm 10분간 원심분리하여 상층액만을 단백질 저해 칵테일(protein inhibitory cocktail)을 1/1,000의 비율로 첨가하여 0.45 ㎛ 필터로 여과한 후 추출된 단백질의 양을 정량하였다.To mass produce proteins from transformed E. coli (pKEP3) (KCCM 10282), chloramphnicol 33 to inhibit the synthesis of 50 μg / ml of ampicillin as an optional marker and other proteins made in the genome of E. coli itself 33 The transformant (KCC 10282) was inoculated into LB liquid medium to which μg / ml was added, and then subcultured at 37 ° C. for 12 hours, and then cultured in the same medium at 30 ° C. for 7 hours. At this time, when the OD value of the transformant was about 0.6 hours after 3 hours, IPTG 0.4 mM was added, and the temperature was lowered to 30 ° C. and incubated for 4 hours. After incubation for 7 hours in total, centrifugation (6,000 rpm, 15 minutes) was performed, and the supernatant was discarded. Only pellets were mixed with 5 mM MES buffer and 0.1 mM PMSF. The mixed transformant (KCCM 10282) was ultrasonically sonicated until the mixture became clear and boiled at 100 ° C. for 10 minutes. Thereafter, centrifugation was performed at 15,000 rpm for 10 minutes, and only the supernatant was added with a protein inhibitory cocktail at a ratio of 1 / 1,000, filtered through a 0.45 μm filter, and the amount of extracted protein was quantified.

상기 과정을 통해 생산된 가지검은마름병원균 WT#3(KCCM 10283) 유래 식물과민반응 유도 단백질을 코딩하고 있는 유전자를 함유한 재조합 플라스미드 pKEP3을 도입한 형질전환체(KCCM 10282)로부터 생산된 식물과민반응 유도 단백질을 Pioneer로 명명하였다.Plant hypersensitivity produced from the transformant (KCCM 10282) which introduced the recombinant plasmid pKEP3 containing the gene encoding the plant hypersensitivity induction protein derived from eggplant black rot pathogen WT # 3 (KCCM 10283) produced through the above process The inducing protein was named Pioneer.

그 결과, 클론 100 ㎖을 배양시 1,000 ㎍/5 ㎖의 단백질을 얻었다. 또한, 도 8에서 보는 바와 같이, 발현벡터인 pKEP3에 클로닝된 화상병원균(E. amylovoraATCC15580)과 본 발명에 따른 가지검은마름병원균 WT#3의 식물과민반응 유도 단백질을 코딩하는 유전자 모두 많은 양의 식물과민반응 단백질이 합성되는 것을 확인할 수 있었다.As a result, 1,000 µg / 5 ml of protein was obtained when 100 ml of the clone was cultured. In addition, as shown in Fig. 8, both genes encoding the plant sensitization-inducing protein of E. amylovora ATCC15580 cloned into the expression vector pKEP3 and the eggplant black blight pathogen WT # 3 according to the present invention It was confirmed that the plant-sensitive protein was synthesized.

4) 가지검은마름병원균 WT#3의 식물과민반응 유도 단백질 유사도 분석4) Analysis of plant hypersensitivity reaction protein similarity of WT # 3

정제된 식물과민반응 유도 단백질의 아미노산 서열을 분석하여 화상병원균 (E. amylovoraATCC15580)의 식물과민반응 단백질과 비교하여 그 유사도를 조사하였다[도 9].The amino acid sequence of the purified plant hypersensitivity inducing protein was analyzed and its similarity was compared with that of the plant hypersensitivity protein of E. amylovora ATCC15580 [Fig. 9].

그 결과, 상기 단백질은 85.9%의 유사도로 화상병원균의 식물과민반응 단백질(Harpin)과 차이가 있는 단백질임을 알 수 있었다.As a result, the protein was found to be a protein different from the plant hypersensitivity protein (Harpin) of the burn pathogen with a similarity of 85.9%.

5) 식물과민반응 유도 단백질의 과민반응(HR)5) Hypersensitivity of plant-induced reaction protein (HR)

현재까지 식물과민반응 유도 단백질은 기주식물에서는 병원성요인으로 작용하며, 비기주식물에서 HR을 유도하는 물질로 알려져 있다. 즉, 과민반응 유도란 기주식물에서는 병원성을 가지고 있다는 것으로 간접 해석할 수 있는 것이다. 이에, 화상병원균(E. amylovoraATCC15580) 유래 식물과민반응 단백질, 가지검은마름병원균 WT#3 유래 식물과민반응 유도 단백질 및 대조구로 단백질 용해 버퍼, 발현벡터 수용체 세균(E. coli) 및 벡터만을 비기주식물인 담배 엽맥에 주사기를 이용하여 접종하였다[도 10].To date, plant hypersensitivity-inducing proteins act as pathogenic agents in host plants and are known to induce HR in non-host plants. In other words, hypersensitivity induction can be indirectly interpreted as having pathogenicity in host plants. Thus, burn pathogens (E. amylovoraATCC15580) Derived Plant Hypersensitivity Protein, Eggplant Black Bacterial Pathogen WT # 3-derived plant hypersensitivity inducing protein and control protein lysis buffer, expression vector receptor bacteria (E. coli) And only the vector was inoculated using a syringe to the tobacco leaf veins, which are non-host plants [FIG. 10].

그 결과, 가지검은마름병원균 WT#3의 식물과민반응 유도 단백질과 화상병원균(ATCC15580)의 식물과민반응 단백질(Harpin)은 모두 HR을 유도한 반면, 대조구에서는 HR이 유도되지 않았다. 특히, WT#3-1의 식물과민반응 유도 단백질은 최소 0.25 ㎍/㎖농도에서 12시간 경과 후 HR이 유도되었으며, ATCC15580의 하르핀은 최소 0.5 ㎍/㎖농도에서 18시간 경과 후 HR이 유도되었다[표 4]. 즉, WT#3-1 유전자의 식물과민반응 유도 단백질은 저농도에서 더 빠른 시간내에 과민반응을 유도하며 이는 주요작물의 여러 병에 대한 과민반응 유도 내성을 더 빠르고 저농도에서 유발할 수 있다는 장점을 의미한다.As a result, the plant hypersensitivity-induced protein of WT # 3 and the plant hypersensitivity protein (Harpin) of burn pathogen (ATCC15580) induced HR, while the control group did not induce HR. In particular, the plant hypersensitivity inducing protein of WT # 3-1 induced HR after 12 hours at a concentration of 0.25 μg / ml, and the harpin of ATCC15580 induced HR after 18 hours at a concentration of 0.5 μg / ml. TABLE 4 In other words, the plant hypersensitivity inducing protein of the WT # 3-1 gene induces hypersensitivity reactions at lower concentrations in a shorter time, which means that the hypersensitivity induction resistance to various diseases of major crops can be induced faster and at lower concentrations. .

6) 가지검은마름병원균 WT#3 유래 식물과민반응 유도 단백질의 배에 대한 병원성 검정6) Pathogenicity test of embryos of WT # 3-derived plant hypersensitivity protein

정제된 가지검은마름병원균 WT#3 유래 식물과민반응 유도 단백질을 500 ㎍/㎖농도로 어린 배 과실표면에 직경 0.5 mm, 깊이 10 mm의 홈을 파고 접종하였다.Purified eggplant black pathogen WT # 3-derived plant hypersensitivity inducing protein was inoculated with a 500 µg / ml concentration of 0.5 mm diameter and 10 mm deep grooves on the surface of young embryos.

도 11에서 나타난 바와 같이, 버퍼만을 처리한 대조구에 비해 4일 경과 후 어린과육이 검게 변하는 병징을 관찰할 수 있었다. 즉, 기주식물에서는 강력한 병원성이 입증되었다.As shown in FIG. 11, it was observed that the young flesh became black after 4 days compared to the control treated with the buffer only. In other words, host plants have demonstrated strong pathogenicity.

이상의 가지검은마름병원균 WT#3의 식물과민성 반응 유도 단백질 특성과 이를 코딩하는 유전자에 대한 특성을 종합해 보면, 가지검은마름병원균 WT#3의 식물과민반응 유도 단백질 및 이를 코딩하고 있는 유전자는 화상병원균의 식물과민반응 단백질(Harpin) 및 이를 코딩하고 있는 유전자(hrpN유전자)와 매우 다른 유사도를 나타내었다. 특히 많은 양의 식물과민반응 유도 단백질을 얻기 위해 개발된 pKEP3 벡터 시스템은 향후 다른 유용 유전자원의 확보를 위해서 매우 중요한 방법이 될 것으로 사료된다. 또한, 가지검은마름병원균 WT#3의 식물과민반응 유도 단백질은 화상병원균 유래 단백질보다 저농도에서 빠른 시간내에 과민반응을 유도하여 향후 식물과민 반응 유도 내성 개발에 적합한 것으로 사료된다.To summarize the characteristics of the plant-sensitized response inducing protein of WT # 3 and the genes encoding the same, the plant-sensitized response-inducing protein of WT # 3 and the gene encoding the same are The plant hypersensitivity protein (Harpin) and the gene encoding the same ( hrpN gene) showed a very different similarity. In particular, the pKEP3 vector system developed to obtain a large amount of plant hypersensitivity inducing protein is considered to be a very important method for securing other useful gene sources in the future. In addition, the plant hypersensitivity-induced protein of WT # 3 of eggplant black blight pathogens was inducible to hypersensitivity reactions at low concentrations faster than burn-derived bacteria and is suitable for future development of plant hypersensitivity reactions.

실시예 3 : 가지검은마름병원균Example 3: Eggplant black blight pathogen WT#3의 식물과민반응 유도 단백질을 이용한 생물활성 연구Bioactivity Study Using Plant Hypersensitivity Induction Protein of WT # 3

1) 배추노균병(1) Chinese cabbage Peronospora brassicaPeronospora brassica ) 방제효과 검정Control effect test

가지검은마름병원균 WT#3와 화상병원균의 식물과민반응 유도 단백질을 코딩하고 있는 유전자를 pKEP3 벡터에 클로닝하여 단백질을 정제하였다.The genes were purified by cloning the genes encoding the plant hypersensitivity induction proteins of the black blight pathogen WT # 3 and the burn pathogen into the pKEP3 vector.

처리농도는 가지검은마름병원균 WT#3로부터 유래한 단백질을 40, 20, 10 ㎍/㎖ 농도로 처리하였으며 그 처리구를 각각 P40, P20, P10으로 명명하였다. 또한, 대조구로 화상병원균(E. amylovoraATCC15580)으로부터 유래한 단백질을 동일한 농도로 처리하여 비교하였으며 이를 AT40, AT20, AT10으로 명명하였다.The treatment concentration was 40, 20, 10 ㎍ / ㎖ protein was derived from the eggplant black rot pathogen WT # 3 and the treatment was named as P40, P20, P10, respectively. In addition, as a control, the protein derived from E. amylovora ATCC15580 was treated at the same concentration and compared and named as AT40, AT20, AT10.

처리방법은 다음과 같다;The treatment method is as follows;

① 침지방법 : 식물과민 반응 유도 단백질을 MES(2-N-[Morpholino]ethaanesulfonic acid) 버퍼에 각각의 농도로 희석하여 배추종자를 28 ℃에서 24시간 동안 담가 두어 이를 침지 처리구라 명명하였다. 그 후 상토가 담긴 화분에 파종하여 30일 동안 생육시켜 노지에 정식하였다.① Soaking method: Diluted plant hypersensitivity reaction protein in MES (2-N- [Morpholino] ethaanesulfonic acid) buffer at different concentrations and soaked cabbage seeds at 28 ℃ for 24 hours. After that, they were sown in pots containing topsoil, grown for 30 days, and planted in open field.

② 침지 + 분무방법 : 상기 ①의 침지방법과 동일하게 처리되어 노지에 정식된 후, 14일, 24일 후에 각각의 농도로 가지검은마름병원균 WT#3와 화상병원균 유래 단백질을 분무하였다.② immersion + spraying method: After treating in the same manner as the immersion method of ① and formulated in the open field, after 14 days, 24 days after spraying the eggplant black WT # 3 and burn pathogen-derived protein at respective concentrations.

③ 분무방법 : 정식 후, 14일, 24일 후에 각각의 농도로 가지검은마름병원균 WT#3와 화상병원균 유래 단백질을 분무하였다.③ Spraying method: After the formulation, 14 days and 24 days after spraying the eggplant black WT # 3 and burn pathogen-derived protein at respective concentrations.

상기와 같이 처리된 배추에 자연발생된 배추노균병의 발병지수를 55일 후에 표시하였다.The incidence index of cabbage neutrophil naturally occurring in the cabbage treated as above was displayed after 55 days.

상기 표 5에서와 같이, 적정 농도는 ATCC15580 균주의 경우 40 ㎍/㎖과 가지검은마름병원균 WT#3 10 ㎍/㎖로 나타났으며, 처리방법별로는 분무 + 침지구에서 효과가 있는 것으로 나타났다. 특히, 가지검은마름병원균 WT#3의 식물과민반응 유도 단백질이 더 효과적으로 나타남을 알 수 있었다[도 12].As shown in Table 5, the appropriate concentration of the ATCC15580 strain was 40 ㎍ / ㎖ and eggplant black blight pathogen WT # 3 10 ㎍ / ㎖, it was found that the treatment is effective in spray + immersion. In particular, it can be seen that the plant hypersensitivity reaction protein of eggplant black rot pathogen WT # 3 appears more effectively [Fig. 12].

2) 참외노균병(2) Melon fungal disease ( Pseudoperonospora cubensisPseudoperonospora cubensis ) 방제효과 검정Control effect test

가지검은마름병원균 WT#3와 화상병원균의 식물과민반응 유도 단백질을 코딩하고 있는 유전자를 pKEP3 벡터에 클로닝하여 단백질을 정제하였다.The genes were purified by cloning the genes encoding the plant hypersensitivity induction proteins of the black blight pathogen WT # 3 and the burn pathogen into the pKEP3 vector.

처리농도는 가지검은마름병원균 WT#3로부터 유래한 단백질을 40, 20, 10 ㎍/㎖ 농도로 처리하였으며 그 처리구를 각각 P40, P20, P10으로 명명하였다. 또한, 대조구로 화상병원균(E. amylovoraATCC15580)으로부터 유래한 단백질을 동일한 농도로 처리하여 비교하였으며 이를 AT40, AT20, AT10으로 명명하였다.The treatment concentration was 40, 20, 10 ㎍ / ㎖ protein was derived from the eggplant black rot pathogen WT # 3 and the treatment was named as P40, P20, P10, respectively. In addition, as a control, the protein derived from E. amylovora ATCC15580 was treated at the same concentration and compared and named as AT40, AT20, AT10.

처리방법은 다음과 같다.The treatment method is as follows.

① 침지방법 : 식물과민반응 유도 단백질을 MES 버퍼에 각각의 농도로 희석하여 참외종자를 28 ℃에서 24시간 동안 담가 두어 이를 침지 처리구라 명명하였다. 그 후 상토가 담긴 화분에 파종하여 30일 동안 생육시켜 직경 25 cm 화분에 정식하였다.① Soaking method: Diluted plant hypersensitivity induction protein to each concentration in MES buffer soaked melon seeds for 24 hours at 28 ℃ was named immersion treatment. After that, the seedlings were planted in pots containing the topsoil, grown for 30 days, and planted in pots of 25 cm in diameter.

② 침지 + 분무방법 : 상기 ①의 침지방법과 동일하게 처리되어 화분에 정식된 후, 17일, 28일 후에 각각의 농도로 가지검은마름병원균 WT#3와 화상병원균 유래 단백질을 분무하였다.② immersion + spraying method: After treating in the same manner as the immersion method of ① and planted in a pollen, after 17 days, 28 days after spraying the eggplant black WT # 3 and burn pathogen-derived protein at respective concentrations.

③ 분무방법 : 정식 후, 17일, 28일 후에 각각의 농도로 가지검은마름병원균 WT#3와 화상병원균 유래 단백질을 분무하였다.③ Spraying method: After the formulation, 17 days, 28 days after the eggplant black germ pathogen WT # 3 and burn pathogen-derived protein was sprayed at each concentration.

상기와 같이 처리된 참외에 자연발생된 참외노균병의 발병지수를 55일 후에 표시하였다.The incidence index of naturally occurring melon fungal disease in addition to the melon treated as described above was displayed after 55 days.

상기 표 6에 나타난 바와 같이, 적정 농도는 ATCC 15580 균주의 경우 40 ㎍/㎖과 가지검은마름병원균 WT#3 40 ㎍/㎖이 가장 우수하였으며 처리방법은 분무 + 침지방법이 가장 효과적인 것으로 나타났다[도 13].As shown in Table 6, the appropriate concentration is 40 ㎍ / ㎖ and eggplant black blight pathogen WT # 3 for the ATCC 15580 strain 40 ㎍ / ㎖ was the best and the treatment method was the most effective spray + immersion method [Fig 13].

3) 오이 흰가루병(3) Cucumber powdery mildew ( Sphaerotheca fuligineaSphaerotheca fuliginea ) 방제효과 검정Control effect test

가지검은마름병원균 WT#3와 화상병원균의 식물과민반응 유도 단백질을 코딩하고 있는 유전자를 pKEP3 벡터에 클로닝하여 단백질을 정제하였다.The genes were purified by cloning the genes encoding the plant hypersensitivity induction proteins of the black blight pathogen WT # 3 and the burn pathogen into the pKEP3 vector.

처리농도는 가지검은마름병원균 WT#3로부터 유래한 단백질을 40, 20, 10 ㎍/㎖ 농도로 처리하였으며 그 처리구를 각각 P40, P20, P10으로 명명하였다. 또한 대조구로 화상병원균(E. amylovoraATCC15580)으로부터 유래한 단백질을 동일한 농도로 처리하여 비교하였으며 이를 AT40, AT20, AT10으로 명명하였다.The treatment concentration was 40, 20, 10 ㎍ / ㎖ protein was derived from the eggplant black rot pathogen WT # 3 and the treatment was named as P40, P20, P10, respectively. In addition, as a control, the protein derived from E. amylovora ATCC15580 was treated at the same concentration and compared, and these were named as AT40, AT20, AT10.

처리방법은 다음과 같다;The treatment method is as follows;

① 침지방법 : 식물과민반응 유도 단백질을 MES 버퍼에 각각의 농도로 희석하여 오이종자를 28℃에서 24시간 동안 담가 두어 이를 침지 처리구라 명명하였다. 그 후 상토가 담긴 화분에 파종하여 30일 동안 생육시켜 노지에 정식하였다.① Soaking method: Dilute plant-induced reaction protein to each concentration in MES buffer and soak cucumber seeds at 28 ℃ for 24 hours. After that, they were sown in pots containing topsoil, grown for 30 days, and planted in open field.

② 침지 + 분무방법 : 상기 ①의 침지방법과 동일하게 처리되어 노지에 정식된 후, 14일, 24일 후에 각각의 농도로 가지검은마름병원균 WT#3와 화상병원균 유래 단백질을 분무하였다.② immersion + spraying method: After treating in the same manner as the immersion method of ① and formulated in the open field, after 14 days, 24 days after spraying the eggplant black WT # 3 and burn pathogen-derived protein at respective concentrations.

③ 분무방법 : 정식 후, 14일, 24일 후에 각각의 농도로 가지검은마름병원균 WT#3와 화상병원균 유래 단백질을 분무하였다.③ Spraying method: After the formulation, 14 days and 24 days after spraying the eggplant black WT # 3 and burn pathogen-derived protein at respective concentrations.

상기와 같이 처리된 오이에 자연발생된 오이흰가루병의 발병지수를 47일 후에 표시하였다.The incidence index of cucumber powdery mildew naturally occurring in cucumbers treated as described above was displayed after 47 days.

상기 표 7에 나타낸 바와 같이, 적정 농도는 ATCC15580 균주의 경우 20 ㎍/㎖과 가지검은마름병원균 WT#3의 경우 20 ㎍/㎖였으며, 처리방법은 분무처리방법이 가장 효과적인 것으로 나타났다. 특히, 이는 오이 흰가루병의 경우도 가지검은마름병원균 WT#3의 식물과민반응 유도 단백질이 더 효과적이었으며, 처리 방법 중 분무처리구에서 가장 좋은 방제가를 얻을 수 있었는데, 이는 흰가루병이 지상부병이며, 병전반이 포자에 의해 공기 중으로 이루어지는 특성상 분무의 방법이 적절한 것으로 사료된다[도 14].As shown in Table 7, the appropriate concentration was 20 ㎍ / ㎖ for ATCC15580 strain and 20 ㎍ / ㎖ for eggplant black blight pathogen WT # 3, spray treatment method was the most effective method. In particular, in case of cucumber powdery mildew, plant hypersensitivity-induced protein of WT # 3 was more effective, and the best control value was obtained from the spray treatment of powdery mildew disease. It is thought that the method of spraying is appropriate in view of the properties of the spores in the air [Fig. 14].

4) 오이 수확량 증대 검정4) Cucumber yield increase test

오이 수확량증대를 알아보기 위해 정식 후, 34일, 40일, 44일, 47일 경과 후에 4회 조사하였다.In order to determine the yield of cucumber, four surveys were conducted after 34 days, 40 days, 44 days, and 47 days after the establishment.

그 결과, 가장 많은 오이가 수확된 적정 농도는 ATCC15580 균주의 경우 40 ㎍/㎖과 가지검은마름병원균 WT#3의 경우 20 ㎍/㎖로 나타났다.As a result, the optimum concentration for harvesting the most cucumbers was 40 µg / ml for the ATCC15580 strain and 20 µg / ml for the branched black blight pathogen WT # 3.

오이 수확량의 경우도 가지검은마름병원균 WT#3의 식물과민반응 유도 단백질이 더 효과적으로 나타났으며, 1차 조사에서는 ATCC15580 40 ㎍/㎖과WT#320 ㎍/㎖의 농도에서 조기착과가 이루어지는 것을 관찰할 수 있었는데, 이는 약 일주일의 수확이 앞당길 수 있을 것으로 사료된다. 특히, 모든 처리구에서 수확한 오이는 정상크기보다 비대하게 큰 것이 많이 존재하였다[도 15].In the case of cucumber yield, the plant-induced hypersensitivity protein of WT # 3 of eggplant black bacterium was more effective. In the first investigation, early fruiting was carried out at the concentration of 40cc / ml of ATCC15580 and 20µg / ml of WT # 3. Observations could be made, which is expected to accelerate the harvest of about a week. In particular, the cucumbers harvested from all treatments were much larger than normal size [Fig. 15].

5) 피망 역병(5) green pepper plague ( Phytophthora capsiciPhytophthora capsici ) 방제효과 검정Control effect test

가지검은마름병원균 WT#3와 화상병원균의 식물과민반응 유도 단백질을 코딩하고 있는 유전자를 pKEP3 벡터에 클로닝하여 단백질을 정제하였다.The genes were purified by cloning the genes encoding the plant hypersensitivity induction proteins of the black blight pathogen WT # 3 and the burn pathogen into the pKEP3 vector.

처리농도는 가지검은마름병원균 WT#3로부터 유래한 단백질을 40, 20, 10 ㎍/㎖ 농도로 처리하였으며 그 처리구를 각각 P40, P20, P10으로 명명하였다. 또한, 대조구로 화상병원균(E. amylovoraATCC15580)으로부터 유래한 단백질을 동일한 농도로 처리하여 비교하였으며 이를 AT40, AT20, AT10으로 명명하였다.The treatment concentration was 40, 20, 10 ㎍ / ㎖ protein was derived from the eggplant black rot pathogen WT # 3 and the treatment was named as P40, P20, P10, respectively. In addition, as a control, the protein derived from E. amylovora ATCC15580 was treated at the same concentration and compared and named as AT40, AT20, AT10.

처리방법은 다음과 같다;The treatment method is as follows;

① 침지방법 : 식물과민반응 유도 단백질을 MES 버퍼에 각각의 농도로 희석하여 피망종자를 28 ℃에서 24시간 동안 담가 두어 이를 침지 처리구라 명명하였다. 그 후 상토가 담긴 화분에 파종하여 30일 동안 생육시켜 직경 25 cm 화분에 정식하였다.① Soaking method: Diluted plant hypersensitivity induction protein to each concentration in MES buffer and soaked bell pepper seeds at 28 ℃ for 24 hours and named it as soaking treatment. After that, the seedlings were planted in pots containing the topsoil, grown for 30 days, and planted in pots of 25 cm in diameter.

② 침지 + 분무방법 : 상기 ①의 침지방법과 동일하게 처리되어 화분에 정식된 후, 8일 후에 각각의 농도로 가지검은마름병원균 WT#3와 화상병원균 유래 단백질을 1회만 분무하였다.② immersion + spraying method: After treating in the same manner as the immersion method of ① and planted in a pollen, after 8 days, each of the concentrations of eggplant black WT # 3 and burn pathogen-derived protein was sprayed once.

③ 분무방법 : 정식 후, 8일 후에 각각의 농도로 가지검은마름병원균 WT#3와 화상병원균 유래 단백질을 1회만 분무하였다.③ Spraying method: After 8 days, eggplant-derived pathogen WT # 3 and burn pathogen-derived protein were sprayed only once in each concentration after 8 days.

상기와 같이 처리된 피망에 피망역병균 2 ×106cells/㎖을 접종한 후, 발병지수를 45일 후에 표시하였다.After inoculating 2 x 10 6 cells / ml of green pepper disease bacteria into the green peppers treated as above, the onset index was displayed after 45 days.

상기 표 9와 같이, 가지검은마름병원균 WT#3에서만 40 ㎍/㎖농도의 분무 + 침지방법이 가장 효과적인 것으로 나타났다.As shown in Table 9, it was found that the spray + immersion method of 40 ㎍ / ㎖ concentration only in eggplant black blight pathogen WT # 3.

6) 피망 탄저병(6) bell pepper anthrax ( Colletotrichum orbiculareColletotrichum orbiculare ) 방제효과 검정Control effect test

상기 5)의 피망 처리방법과 동일하게 실시하였고, 이와 같이 처리된 피망에 자연발생된 피망탄저병의 발병지수를 45일 후에 표시하였다.It was carried out in the same manner as the bell pepper treatment method of 5), the incidence index of the bell pepper anthrax spontaneously generated in the treated bell pepper was displayed after 45 days.

상기 표 10에 나타낸 바와 같이, 가지검은마름병원균 WT#3에서만 40 ㎍/㎖에서 효과가 있는 것으로 나타났다.As shown in Table 10, the eggplant black was found to be effective at 40 μg / ml only for the pathogen WT # 3.

7) 피망 수확량 증대 검정7) Bell pepper yield increase test

피망 수확량 증대를 알아보기 위해 정식 후, 45일 경과 후에 조사하였다.In order to investigate the increase in the yield of the bell pepper, it was investigated 45 days after the establishment.

피망 수확량 증대는 가지검은마름병원균 WT#3에서만 40 ㎍/㎖에서 가장 많았으며, 이는 피망 탄저병과도 일치하는 결과였다.The yield of green pepper was the highest at 40 ㎍ / ml of eggplant black rot fungi WT # 3, which is consistent with the pepper anthrax.

8) 딸기 분무처리에 대한 수확량 증대 검정8) Yield test for strawberry spray treatment

딸기 수확량 증대를 알아보기 위해 정식 후, 30, 33, 38, 41, 48, 55일 경과 후에 총 6회에 걸쳐 조사하였다.In order to determine the increase in strawberry yield, a total of six surveys were conducted 30, 33, 38, 41, 48, and 55 days after the establishment.

식물과민반응 유도 단백질을 처리한 모든 구에서 전체적으로 조금씩 양이 증대되었으나, 특히 가지검은마름병원균 WT#3 10 ㎍/㎖ 처리구에서 딸기 수확량이 가장 많이 증대되었다.The amount increased slightly in all groups treated with plant hypersensitivity-induced protein, but the yield of strawberries was the highest in 10 ㎍ / ml treatment of eggplant black bacterium WT # 3.

9) 고추 역병(Phytophthora capsici) 방제효과 검정9) Test for Control Effect of Pepper Plague ( Phytophthora capsici )

가지검은마름병원균 WT#3와 화상병원균의 식물과민반응 유도 단백질을 코딩하고 있는 유전자를 pKEP3 벡터에 클로닝하여 단백질을 정제하였다.The genes were purified by cloning the genes encoding the plant hypersensitivity induction proteins of the black blight pathogen WT # 3 and the burn pathogen into the pKEP3 vector.

처리농도는 가지검은마름병원균 WT#3로부터 유래한 단백질을 40, 20, 10 ㎍/㎖ 농도로 처리하였으며 그 처리구를 각각 P40, P20, P10으로 명명하였다. 또한, 대조구로 화상병원균(E. amylovoraATCC15580)으로부터 유래한 단백질을 동일한 농도로 처리하여 비교하였으며 이를 AT40, AT20, AT10으로 명명하였다.The treatment concentration was 40, 20, 10 ㎍ / ㎖ protein was derived from the eggplant black rot pathogen WT # 3 and the treatment was named as P40, P20, P10, respectively. In addition, as a control, the protein derived from E. amylovora ATCC15580 was treated at the same concentration and compared and named as AT40, AT20, AT10.

처리방법은 다음과 같다;The treatment method is as follows;

① 침지방법 : 식물과민반응 유도 단백질을 MES 버퍼에 각각의 농도로 희석하여 피망종자를 28 ℃에서 24시간 동안 담가 두어 이를 침지 처리구라 명명하였다. 그 후 상토가 담긴 화분에 파종하여 46일 동안 생육시켜 직경 25 cm 화분에 정식하였다.① Soaking method: Diluted plant hypersensitivity induction protein to each concentration in MES buffer and soaked bell pepper seeds at 28 ℃ for 24 hours and named it as soaking treatment. Thereafter, seedlings were planted in pots containing the soil, and grown for 46 days, and planted in pots of 25 cm in diameter.

② 침지 + 분무방법 : 상기 ①의 침지방법과 동일하게 처리되어 화분에 정식된 후, 8, 12일 후에 각각의 농도로 가지검은마름병원균 WT#3와 화상병원균 유래 단백질을 분무하였다.② Immersion + spraying method: The same treatment as in the above method of immersion ① and planted in pots, after 8, 12 days after each of the concentrations were sprayed eggplant black WT # 3 and burn pathogen-derived proteins.

③ 분무방법 : 정식 후, 8, 12일 후에 각각의 농도로 가지검은마름병원균 WT#3와 화상병원균 유래 단백질을 분무하였다.③ Spraying method: After 8, 12 days of formulation, eggplant-derived pathogen WT # 3 and burn pathogen-derived protein were sprayed at respective concentrations.

상기와 같이 처리된 고추에 고추역병균 2 ×106cells/㎖을 접종한 후, 발병지수를 63일 후에 표시하였다.After inoculating 2 × 10 6 cells / ml of pepper blight bacteria into the pepper treated as described above, the onset index was displayed after 63 days.

상기 표 13과 같이, 가지검은마름병원균 WT#3에서 10 ㎍/㎖농도의 분무가 화상병원균유래 단백질보다 효과적인 것으로 나타났다.As shown in Table 13, it was shown that the spray of 10 ㎍ / ㎖ concentration of eggplant pathogen WT # 3 is more effective than burn pathogen-derived protein.

10) 고추 수확량 증대 검정10) Pepper yield increase test

고추 수확량 증대를 알아보기 위해 정식 후, 38, 42, 46, 52, 55, 62, 69일 경과 후 7회 조사하였다.In order to investigate the increase in pepper yield, 7 times after the establishment, 38, 42, 46, 52, 55, 62, 69 days were examined.

고추 수확량은 전체 처리구에서 모두 증대되는 것을 알 수가 있었으며, 특히 가지검은마름병원균 WT#3 20 ㎍/㎖에서 가장 많은 수확을 할 수 있었다. 특히, 모든 처리구에서 고추의 개화시기 및 착과 시기가 빨라져 조기에 고추를 수확할 수 있었다[도 16].The yield of red pepper was increased in all the treatments, and the highest yield was obtained from 20 ㎍ / ml of eggplant black pathogen WT # 3. In particular, the flowering time and fruiting time of the pepper in all the treatment was early to harvest the pepper early [Fig. 16].

11) 벼 도열병(Magnaporthe grisea)의 방제효과 검정11) Control of the control effect of Magnaporthe grisea

가지검은마름병원균 WT#3와 화상병원균의 식물과민반응 유도 단백질을 코딩하고 있는 유전자를 pKEP3 벡터에 클로닝하여 단백질을 정제하였다.The genes were purified by cloning the genes encoding the plant hypersensitivity induction proteins of the black blight pathogen WT # 3 and the burn pathogen into the pKEP3 vector.

처리농도는 가지검은마름병원균 WT#3로부터 유래한 단백질을 40, 20, 10 ㎍/㎖ 농도로 처리하였으며 그 처리구를 각각 P40, P20, P10으로 명명하였다. 또한, 대조구로 화상병원균(E. amylovoraATCC15580)으로부터 유래한 단백질을 동일한 농도로 처리하여 비교하였으며 이를 AT40, AT20, AT10으로 명명하였다.The treatment concentration was 40, 20, 10 ㎍ / ㎖ protein was derived from the eggplant black rot pathogen WT # 3 and the treatment was named as P40, P20, P10, respectively. In addition, as a control, the protein derived from E. amylovora ATCC15580 was treated at the same concentration and compared and named as AT40, AT20, AT10.

처리방법은 다음과 같다;The treatment method is as follows;

① 침지방법 : 식물과민 반응 유도 단백질을 MES(2-N-[Morpholino]ethaanesulfonic acid) 버퍼에 각각의 농도로 희석하여 벼종자를 28 ℃에서 24시간 동안 담가 두어 이를 침지 처리구라 명명하였다. 그 후 모판에 파종하여 16일 동안 생육시켜 모내기를 실시하였다.① Soaking method: Diluted plant hypersensitivity induction protein in MES (2-N- [Morpholino] ethaanesulfonic acid) buffer at different concentrations and soaked rice seeds at 28 ℃ for 24 hours. Thereafter, seedlings were seeded and grown for 16 days to carry out seedlings.

② 침지 + 분무방법 : 상기 ①의 침지방법과 동일하게 처리되어 논에 이앙된 후, 45일, 52일 후에 각각의 농도로 가지검은마름병원균 WT#3와 화상병원균 유래 단백질을 분무하였다.② immersion + spraying method: After the same process as in the above immersion method ① was transferred to the rice paddy, 45 days, 52 days after the eggplant black WT # 3 and burn pathogen-derived protein was sprayed at each concentration.

③ 분무방법 : 이앙 후, 45일, 52일 후에 각각의 농도로 가지검은마름병원균 WT#3와 화상병원균 유래 단백질을 분무하였다.③ Spraying method: After transplanting, 45 days and 52 days after the injection of the eggplant black rot pathogen WT # 3 and burn pathogen-derived protein at each concentration.

상기와 같이 처리된 벼에 자연발생된 벼 도열병의 발병지수를 85일 후에 표시하였다.The incidence index of the rice blast naturally occurring in the rice treated as described above was displayed after 85 days.

상기 표 15와 같이, 가지검은마름병원균 WT#3에서 10 ㎍/㎖농도의 분무+침지구가 화상병원균유래 단백질보다 효과적인 것으로 나타났다.As shown in Table 15, the spray + immersion of 10 ㎍ / ㎖ concentration in eggplant black blight pathogen WT # 3 was shown to be more effective than the burn pathogen-derived protein.

12) 콩나물 성장촉진 효과 검정12) Test of bean sprouts growth promoting effect

가지검은마름병원균 WT#3와 화상병원균의 식물과민반응 유도 단백질을 코딩하고 있는 유전자를 pKEP3 벡터에 클로닝하여 단백질을 정제하였다.The genes were purified by cloning the genes encoding the plant hypersensitivity induction proteins of the black blight pathogen WT # 3 and the burn pathogen into the pKEP3 vector.

처리농도는 가지검은마름병원균 WT#3으로부터 유래한 단백질을 40, 20, 10 ㎍/㎖ 농도로 처리하였으며 그 처리구를 각각 P40, P20, P10으로 명명하였다. 또한, 대조구로 화상병원균(E. amylovoraATCC15580)으로부터 유래한 단백질을 동일한 농도로 처리하여 비교하였으며 이를 AT40, AT20, AT10으로 명명하였다.Treatment concentration was 40, 20, 10 ㎍ / ㎖ concentration of the protein derived from the eggplant black rot pathogen WT # 3 and the treatment was named as P40, P20, P10, respectively. In addition, as a control, the protein derived from E. amylovora ATCC15580 was treated at the same concentration and compared and named as AT40, AT20, AT10.

처리방법은 식물과민 반응 유도 단백질을 MES(2-N-[Morpholino]ethaanesulfonic acid) 버퍼에 각각 8 ㎍, 25 ㎍, 50 ㎍/㎖농도로 희석하여 콩나물용 콩에 25 ℃에서 24시간 동안 담가 두어 습실처리된 멸균된 병에 파종하여 7일 동안 생육시켜 성장촉진효과를 관찰하였다[도 17].The treatment method was diluted with 8 µg, 25 µg and 50 µg / mL concentrations of the plant hypersensitivity induction protein in MES (2-N- [Morpholino] ethaanesulfonic acid) buffer, respectively, and soaked in soybean sprouts for 24 hours at 25 ° C. It was sown in a sterilized bottle treated with wet plants and grown for 7 days to observe the growth promoting effect [FIG. 17].

그 결과, 가지검은마름병원균 WT#3의 8 ㎍/㎖ 농도에서 3 ∼ 4 cm 성장이 촉진된 것을 확인하였다.As a result, it was confirmed that 3-4 cm growth was promoted at the concentration of 8 µg / ml of the branched black pathogen WT # 3.

이상에서 설명한 바와 같이, 본 발명은 신규한 가지검은마름병원균 WT#3(KCCM 10283)을 분리 동정하였고, 가지검은마름병원균 WT#3(KCCM 10283)으로부터 새로운 단백질을 정제하여 이 단백질을 분석한 결과, 화상병원균 유래 단백질보다 식물병 저항성 및 식물생장 촉진 효과가 우수하여 저농도에서 빠른 시간내에 과민반응을 유도하여 향후 식물과민 반응 유도 내성 개발에 적합하다.As described above, the present invention was isolated and identified a novel eggplant black germ pathogen WT # 3 (KCCM 10283), and purified the new protein from eggplant black germ pathogen WT # 3 (KCCM 10283) and analyzed the protein In addition, plant disease resistance and plant growth promoting effect are superior to those of burn pathogen-derived proteins, so it is suitable for the development of resistance to induction of plant hypersensitivity reactions by inducing hypersensitivity reactions at low concentrations in a short time.

따라서, 본 발명에 따른 가지검은마름병원균 WT#3 또는 이로부터 유래된 식물과민반응 유도 단백질을 함유한 식물병 방제제 및 식물생장 촉진제 등의 생물농약으로 사용이 기대된다.Therefore, the eggplant black bacterium according to the present invention is expected to be used as biopesticides such as plant disease control agent and plant growth promoter containing WT # 3 or plant hypersensitivity inducing protein derived therefrom.

Claims (8)

식물병 방제 및 식물생장 촉진에 유효한 가지검은마름병원균 WT#3(Erwinia pyrifoliaeWT#3, KCCM 10283).Eggplant black rot fungi WT # 3 ( Erwinia pyrifoliae WT # 3, KCCM 10283), effective for controlling plant diseases and promoting plant growth. 서열번호 1의 염기서열을 가지며, 가지검은마름병원균 WT#3(KCCM 10283)의 식물과민반응(HR) 유도 단백질을 코딩하는 유전자.A gene having a nucleotide sequence of SEQ ID NO: 1 and encoding a plant hypersensitivity (HR) inducing protein of the branched black rye pathogen WT # 3 (KCCM 10283). 서열번호 2의 아미노산 서열을 가지며, 가지검은마름병원균 WT#3(KCCM 10283) 유래의 식물과민반응(HR) 유도 단백질.Plant hypersensitivity (HR) inducing protein having an amino acid sequence of SEQ ID NO: 2 and derived from branched black pathogen WT # 3 (KCCM 10283). 청구항 2의 유전자를 포함하는 재조합 플라스미드 pKEP3.Recombinant plasmid pKEP3 comprising the gene of claim 2. 재조합 플라스미드 pKEP3를 대장균에 도입하여 형질전환시킨 것임을 특징으로 하는 대장균(pKEP3)(KCCM 10282).E. coli (pKEP3) (KCCM 10282), characterized in that the recombinant plasmid pKEP3 was introduced into E. coli and transformed. 대장균(pKEP3)(KCCM 10282)을 배양하여 분비한 식물과민반응 유도 단백질을 추출, 정제하는 것임을 특징으로 하는 식물과민반응 유도 단백질의 대량 생산방법.A method for mass-producing plant hypersensitivity inducing protein, characterized in that to extract and purify the plant hypersensitivity inducing protein secreted by culturing Escherichia coli (pKEP3) (KCCM 10282). 가지검은마름병원균 WT#3(KCCM 10283) 또는 이로부터 유래된 식물과민반응 유도 단백질을 함유하는 식물병 방제제.Plant disease control agent containing the black blight pathogen WT # 3 (KCCM 10283) or a plant hypersensitivity-inducing protein derived therefrom. 가지검은마름병원균 WT#3(KCCM 10283) 또는 이로부터 유래된 식물과민반응 유도 단백질을 함유하는 식물생장 촉진제.Eggplant black growth pathogen WT # 3 (KCCM 10283) or a plant growth promoter containing a plant hypersensitivity inducing protein derived therefrom.
KR1020010049047A 2001-08-14 2001-08-14 New biopesticide using WT#3-1 gene from Erwinia pyrifoliae WT#3, novel pathogen that affects Asian pear trees KR20030015010A (en)

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