KR20050034000A - Biocontrol of plant diseases using novel endophytic isolate of burkholderia vietnamensis mc1404 - Google Patents

Biocontrol of plant diseases using novel endophytic isolate of burkholderia vietnamensis mc1404 Download PDF

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KR20050034000A
KR20050034000A KR1020030069750A KR20030069750A KR20050034000A KR 20050034000 A KR20050034000 A KR 20050034000A KR 1020030069750 A KR1020030069750 A KR 1020030069750A KR 20030069750 A KR20030069750 A KR 20030069750A KR 20050034000 A KR20050034000 A KR 20050034000A
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송완엽
이숙경
이정숙
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Abstract

An endophytic microorganism Burkholderia vietnamensis MC1404 and an antifungal composition comprising the same microorganism are provided, which microorganism has strong antifungal activity and resides within the plants, so that the microorganism can effectively control fungus residing within the plants. The microorganism Burkholderia vietnamensis MC1404(KACC 91060) having antifungal activity is provided, wherein the Burkholderia vietnamensis MC1404(KACC 91060) is isolated from the roots of plants; and the Burkholderia vietnamensis MC1404(KACC 91060) has antifungal activity against Botrytis cinerea, Alternaria alternata, Fusarium oxysporum f. sp. lycolersici, Fusarium oxysporum, Pythium ultinum, Colletotrichum lagenarium, Colletotrichum gloeosporioides, Phytophtora drechsien, Phytopthora capsici, Phytopthora infestans, Rhizoctonia solani, Cylindrocarpon destructans, Alternaria panax, Rhizoctonia solani, Pyricularia grisea, Fusarium rosem and Fusarium moniliforme.

Description

병원성 진균에 길항력을 가지는 신규의 식물 내생 세균인 버크홀더리아 비에나멘시스 엠씨1404 및 이를 함유하는 항진균성 미생물 살균제 {Biocontrol of plant diseases using novel endophytic isolate of Burkholderia vietnamensis MC1404}Burkholderia viennamensis MC1404, a novel plant endogenous bacterium with antagonism against pathogenic fungi, and an antifungal microbial fungicide containing the same {Biocontrol of plant diseases using novel endophytic isolate of Burkholderia vietnamensis MC1404}

본 발명은 미생물에 관한 것으로, 더욱 상세하게는 식물 병원성 진균에 대해 광범위한 길항능력을 가지는 신규의 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주 및 이를 함유하는 항진균성 미생물 살균제에 관한 것이다.The present invention relates to microorganisms, and more particularly to a novel Burkholderia vietnamensis MC1404 strain having a wide range of antagonistic ability against plant pathogenic fungi and antifungal microbial fungicide containing the same.

원래 자연 생태계에는 어떤 한 가지 생물 집단이 지나치게 커지는 것을 막을 수 있는 여러 가지 견제 장치가 있다. 그들은 포식자이거나 기생자, 경쟁자, 길항자, 억제자 등으로 존재하고 같은 종끼리는 서로 모이게 하거나 분산시키는 통신물질도 가지고 있다.Originally, natural ecosystems have a number of containment mechanisms that can prevent a single biomass from growing too large. They exist as predators, parasites, competitors, antagonists, suppressors, etc., and they also have communication materials that bring together or disperse the same species.

생물의 종류로 보면 바이러스, 세균, 곰팡이, 원생동물 같은 미생물과 곤충, 거미류, 선충, 조류 등 다양한 생물이 있다. 이러한 생물자원 중에서 억제능력이 우수한 미생물을 선발하여 화학농약을 대신하여 병이나 해충의 발생을 효과적으로 방제할 수 있도록 개발한 것이 미생물 살균제다.The types of organisms include microorganisms such as viruses, bacteria, fungi and protozoa, and various organisms such as insects, arachnids, nematodes and birds. Among these biological resources, microbial fungicides are developed to select microorganisms with excellent suppression ability and to effectively control the occurrence of diseases and pests in place of chemical pesticides.

미생물 살균제는 자연계에 이미 존재하였던 것이어서 그들의 생존이나 증식이 자연의 질서에 의해서 조절되므로 더 이상의 해나 잔류의 문제도 없다. 또한 기주와 기생자, 먹이와 포식자, 경쟁자의 관계가 성립될 때만 방제가 가능하므로 목적 외의 생물에게는 피해가 없다.Microbial fungicides have already existed in nature, so their survival or proliferation is controlled by natural order, so there are no further harm or residual problems. In addition, since control is possible only when the relationship between host and parasitic, prey, predator, and competitor is established, there is no damage to non-purpose creatures.

농업이 발전된 대부분의 국가들이 지난 50년 동안 화학농약에 전적으로 의존해 왔지만 환경을 보존하고 가꾸는 일이 농산물로 얻은 경제적 이익보다 더 중요시되는 오늘에 와서는 생물농약에 더 무게를 두고 보다 많은 작물에 적용하려고 노력하고 있다.Although most countries with advanced agriculture have relied solely on chemical pesticides for the past 50 years, today, where conservation and care is more important than economic benefits from agricultural products, they are now trying to put more weight on biopesticides and apply them to more crops. Trying hard.

외국의 경우 1927년 미국에서 감자 더뎅이병 방제용으로 방선균을 이용한 것이 최초이며, 1960년 이후부터 농약의 형태로 실용화되기 시작하였고, 일본에서는 1962년에 담배 허리마름병에 Trichoderma 생균 제제를 시초로 이후에 많은 제품이 미국에서 개발되었으며 대부분의 실용화 제품이 육묘중 발생되는 모잘록병 방제용으로 개발되었다.In the case of foreign and it is the first with actinomycetes for potatoes deodengyi disease control in the United States in 1927, after the Trichoderma live agents in tobacco waist blight began to be commercialized in the form of pesticides since 1960, in Japan in 1962 as the beginning Many products have been developed in the United States, and most of the commercial products have been developed for the control of mosslock disease during seedling.

병해 방제용 생물농약 중 가장 획기적인 성공 제품으로는 각종 작물 특히 다년생 목본류의 뿌리에 발생되고 있는 뿌리혹 세균병(Crown gall)에 길항미생물인 Agrobacterium radiacter strain 84 및 K1026균주를 이용한 Galltrol, Dygall, Nogall, Bakuterozu 제품이 있다. 현재의 유기합성농약이 모든 세균병에 효과가 저조하나 특히 뿌리혹 세균병은 유기합성농약 효과를 전혀 볼 수 없는데 비해, 이 미생물농약은 확실한 효과를 나타내어 우수한 방제제로 이용되고 있다.The most significant success products among biopesticides for controlling diseases are Galltrol, Dygall, Nogall, Bakuterozu using Agrobacterium radiacter strain 84 and K1026 strains, which are antagonistic microorganisms to the root gall, which occurs in the roots of various crops, especially perennial woody roots. There is a product. Although the present organic synthetic pesticides are less effective against all bacterial diseases, especially the root-knot bacterial diseases do not show the organic synthetic pesticides at all, but the microbial pesticides have a definite effect and are used as excellent control agents.

국내의 경우 미생물 자체를 이용하거나, 미생물이 생산하는 생리활성물질에 대한 국내의 연구 및 개발은 외국에 비해 30~50년 뒤늦은 1980년대에 기초적 연구가 수행되어 왔으며, 전문가가 부족한 실정과 체계가 갖춰져 있지 않은 상황에 의해 실용화되고 있는 제품은 개발되어 있지 않다.In Korea, the research and development of bioactive substances produced by microorganisms or microorganisms have been carried out in the 1980s, which is 30 to 50 years later than in other countries. The product which is put to practical use by the situation which does not exist is not developed.

그러나, 최근에 체계적인 연구실과 전문가들에 의해 꾸준한 노력에 따라 연구역사에 비해 연구수준은 많이 향상되어 좋은 결과가 이루어지고 있다. 미생물자체를 이용하여 병해방제용으로 연구된 것으로는 1985년부터 국가연구기관 및 대학의 병리학자를 주축으로 담배의 모자이크병(TMV), 세균성마름병(Bacterial wilt), 오이의 시들음병(Fusarium wilt), 고추의 역병(Phytophthora blight), 딸기의 시들음병, 눈마름병(Rhizoctonia budrot), 사탕무우의 입고병(Damping-off), 벼의 도열병, 문고병 방제 등에 관한 연구가 보고되어 있다Recently, however, the research level has been improved much compared to the research history by the systematic laboratories and experts. It has been studied for the prevention of diseases using microorganisms. Since 1985, pathologists of national research institutes and universities have focused on tobacco mosaic disease, bacterial wilt, cucumber wilting disease, and pepper. Research has been reported on Phytophthora blight, wilting strawberry, Rhizoctonia budrot, Damping-off of sugar beet, rice blast, paperback disease control, etc.

최근 작물을 재배하는데 있어 문제시되는 병해로는 벼에서의 도열병, 문고병, 잎마름병이 있고, 시설재배에서는 시들음병, 잿빛 곰팡이병, 흰가루병, 탄저병 등이 문제시되고 있다.In recent years, the problem of growing crops is rice blast, paperback disease, leaf blight, and facility cultivation is withering disease, ash fungus, powdery mildew, anthrax.

또한, 최근들어 식물성장촉진 근류세균(plant growth-promoting rhizobacteria, PGPR)의 중요성이 점차 확대되고 있다. 이 세균 그룹은 형광성 슈도모나드(fluorescent pseudomonad)로 주로 구성되어 있었으나, Bacillus spp., Burkholderia sp.을 비롯한 많은 근권세균에서 보고되고 있으며 주로 뿌리내외부에서 서식한다. 식물성장촉진 근류미생물에 의한 토양성 식물병의 생물학적 방제에 대한 중요한 생태학적 요소는 세포 외로 분비되는 사이드로포어의 산물이다.In recent years, the importance of plant growth-promoting rhizobacteria (PGPR) is gradually increasing. This group of bacteria consisted mainly of fluorescent pseudomonads, but has been reported in many rhizosphere bacteria including Bacillus spp. And Burkholderia sp. And lives mainly inside and outside roots. Plant growth-promoting An important ecological factor in the biological control of soil-borne plant diseases by mycorrhizal microorganisms is the product of the extracellular secreted sidelopores.

사이드로포어는 근권내에서 미생물학적 상호작용에 있어서, 필수적인 역할을 담당하여 식물 성장과 수율을 향상시킨다. 근권에서 식물성장촉진 근류미생물에 의한 질병 감소와 성장 촉진 매카니즘은 철에 대한 사이드로포어 매개 경쟁에 의한 대립 상호작용의 하나이다.Sideropores play an essential role in microbial interactions in the rhizosphere, improving plant growth and yield. Plant growth-promoting diseases in the rhizosphere The disease-reducing and growth-promoting mechanisms by microorganisms are one of the opposing interactions caused by siderophore-mediated competition for iron.

식물성장촉진 근류세균의 사이드로포어의 보고에 따르면, 식물 근권내로 식물성장촉진 근류미생물의 도입에 의한 식물질병 억제 또는 식물 성장촉진에 대한 연구이다.According to the report of the siderophore of plant growth promoting mycorrhizal bacteria, the study on the inhibition of plant diseases or the promotion of plant growth by introducing the plant growth promoting myocytes into the plant root zone.

본 발명의 목적은 미생물의 서식 및 적응된 위치가 토양이 아닌 식물내에 존재하면서 식물 병원균에 길항능력을 가지는 식물 내생 세균 균주인 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404를 제공하는데 있다.SUMMARY OF THE INVENTION An object of the present invention is to provide a Burkholderia vietnamensis MC1404, a plant endogenous bacterial strain which has the ability to antagonize plant pathogens while the habitat and adapted location of the microorganisms are present in the plant rather than the soil.

본 발명의 다른 목적은 식물 병원성 진균에 대해 광범위하게 길항능력을 가지며, 식물성장을 촉진하는 신규의 식물 내생 세균인 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404와 이를 함유하는 식물 병원성 진균방제용 미생물 살균제를 제공하는데 있다.Another object of the present invention is a new plant endogenous bacterium Burkholderia vietnamensis MC1404, which has a wide range of antagonistic ability against plant pathogenic fungi, and promotes plant growth, and for plant pathogenic fungi containing the same. To provide a microbial fungicide.

상기와 같은 목적을 달성하기 위한 본 발명은 식물 병원성 진균에 대해 광범위한 길항력을 가지며, 항진균 활성이 우수한 신규의 미생물 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주를 제공하는 것에 특징이 있다.The present invention for achieving the above object is characterized by providing a novel microbial Burkholderia vietnamensis ( Burkholderia vietnamensis ) MC1404 strain having a wide range of antagonism against plant pathogenic fungi, and excellent antifungal activity.

본 발명의 상기 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주는 국내의 식물 뿌리에서 분리한 세균으로서 각종 식물에 대해 비병원성이다. Burkholderia vietnamensis ( Burkholderia vietnamensis ) MC1404 strain of the present invention is a non-pathogenic to various plants as bacteria isolated from domestic plant roots.

본 발명의 상기 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주는 식물 병원성 진균에 의해 광범위한 기주 식물에서 발생하는 딸기, 토마토, 가지, 장미, 인삼의 잿빛곰팡이병(Botrytis cinerea), 토마토의 줄기마름병(Alternaria alternata), 시들음병(Fusarium oxysporum f. sp. lycolersici), 수박의 탄저병(Colletotrichum lagenarium), 역병(Phytophtora drechsien), 오이의 덩굴쪼김병(Fusarium oxysporum), 입고병(Pythium ultinum), 고추의 탄저병(Colletotrichum gloeosporioides), 역병(Phytopthora capsici), 인삼의 입고병(Rhizoctonia solani), 뿌리썩음병(Cylindrocarpon destructans), 반점병(Alternaria panax), 감자 역병(Phytopthora infestans), 벼의 잎집무늬마름병(Rhizoctonia solani), 도열병(Pyricularia grisea), 이삭마름병(Fusarium rosem), 적미병(Fusarium moniliforme) 등에 우수한 길항력을 나타낸다. Burkholderia vietnamensis ( Burkholderia vietnamensis ) MC1404 strain of the present invention is caused by plant pathogenic fungi, strawberries, tomatoes, eggplant, roses, ginseng fungus ( Botrytis cinerea ), stems of tomatoes Alternaria alternata , wilting disease ( Fusarium oxysporum f. Sp. Lycolersici ), watermelon anthrax ( Colletotrichum lagenarium ), late blight ( Phytophtora drechsien ), cucumber vines ( Fusarium oxysporum ), wearing disease ( Pythium ultinum ) ( Colletotrichum gloeosporioides ), late blight ( Phytopthora capsici ), ginseng ( Rhizoctonia solani ), root rot ( Cylindrocarpon destructans ), spot disease ( Alternaria panax ), potato blight ( Phytopthora infestans ), rice leaf blight ( Rhizoctonia Pyricularia grisea , Fusarium rosem , Fusarium moniliforme and the like have excellent antagonism.

본 발명의 상기 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주는 식물 종자의 발아율을 저해하지 않고 종자에 발생하는 병원성 진균에 대해 길항능력을 가진다. Burkholderia vietnamensis ( Burkholderia vietnamensis ) MC1404 strain of the present invention has the ability to antagonize pathogenic fungi occurring in seeds without inhibiting the germination rate of plant seeds.

본 발명의 상기 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주는 생태계 파괴가 없는 무공해 생물방제법에 사용할 수 있다. Burkholderia vietnamensis ( Burkholderia vietnamensis ) MC1404 strain of the present invention can be used in pollution-free biocontrol without destruction of the ecosystem.

본 발명의 상기 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주는 항진균 활성에 더하여 식물 생육촉진 등의 능력을 가짐으로 토양 개량제, 퇴비부숙제, 엽면살포제 또는 관주살포제 등의 유효성분으로 이용될 수 있으며, 바이오상토의 제조에도 활용될 수 있다. Burkholderia vietnamensis ( Burkholderia vietnamensis ) MC1404 strain of the present invention has the ability to promote plant growth in addition to antifungal activity can be used as an active ingredient such as soil improver, composting agent, foliar spray or irrigation spraying agent. It can be used in the manufacture of biotope.

또한, 본 발명은 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주와 미생물 담체인 바이오 그레뉼을 혼합하여 만든 미생물 살균제를 제공하는 것에 특징이 있다.In another aspect, the present invention is characterized by providing a microbial fungicide made by mixing a Burkholderia vietnamensis MC1404 strain and a bio granule which is a microbial carrier.

본 발명의 상기 미생물 살균제는 수화제, 입제, 코팅 입제, 바이오 상토, 미생물영양제, 식물 생육촉진제, 토양 개량제, 종자 코팅제, 퇴비 부숙제, 엽면 살포제 또는 관주 살포제로 이용될 수 있다.The microbial fungicide of the present invention may be used as a hydrating agent, granules, coated granules, bio-topology, microbial nutritional agents, plant growth promoters, soil improvers, seed coatings, composting, foliar spraying or irrigation spraying.

이하, 실시예에 의하여 본 발명을 상세히 설명하면 다음과 같다. 단 이들 실시예는 본 발명을 구체적으로 설명하기 위한 것으로, 본 발병의 범위가 이들 실시예에 국한되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to Examples. However, these Examples are for illustrating the present invention in detail, and the scope of the present invention is not limited to these Examples.

〈실시예 1〉길항 내생 세균의 분리 및 식별Example 1 Isolation and Identification of Antagonistic Endogenous Bacteria

식물 병원성 진균에 대해 광범위한 길항능력을 가지며, 활성 효과가 우수한 식물 내생 세균을 분리하기 위하여 전라 및 충청 지역의 주요작물 뿌리를 채취하였다. 채집한 뿌리는 표면 소독을 위해 1% Chloramine T 용액에서 15분간 침지한 뒤 glass bead를 넣고 20분간 교반하였으며, 멸균수로 수세 후 이를 마쇄하여 10배수로 희석한 뒤 TSA(Difco) 배지에 도말 접종하였다.The roots of major crops in Cholla and Chungcheong regions were collected to isolate plant endogenous bacteria with broad antagonistic ability against plant pathogenic fungi and excellent active effect. The collected roots were immersed in 1% Chloramine T solution for 15 minutes for surface disinfection, then put into glass bead and stirred for 20 minutes. After washing with sterile water, the roots were ground and diluted to 10-fold and smeared onto TSA (Difco) medium. .

그 결과로 425개의 대표세균이 분리되었고, 이 중 식물 병원성 진균에 강한 길항력을 나타내는 균주 86개를 1차 선발하였다. 다시 광범위한 항진균 효과 시험 등을 통해 우수한 길항세균 중 세균학적 특성이 같은 MC1103, MC1303, MC1401, MC1402, MC1404, MC1405, MC1502, MC1506의 8균주를 선발하였다.As a result, 425 representative bacteria were isolated, and among them, 86 strains showing strong antagonism against plant pathogenic fungi were selected first. In addition, eight strains of MC1103, MC1303, MC1401, MC1402, MC1404, MC1405, MC1502, and MC1506 with the same bacteriological properties among the excellent antagonistic bacteria were selected through extensive antifungal effect tests.

〈실시예 2〉식물 내에서의 비병원성 검정Example 2 Nonpathogenic Assay in Plants

분리된 내생 세균이 비병원성임을 확인하기 위하여 주요 작물인 벼, 토마토, 가지, 오이, 고추에 대한 병원성 실험이 수행되었다. 기주 식물은 멸균 토양에 각 작물의 종자를 파종하여 생장상에서 2일 동안 보관하여 발아시킨 후, 26℃, 14시간 광조건과 22℃, 10시간 암조건으로 생육시키면서 준비하였다. 분리된 식물 내생 세균인 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주를 1×107cfu/ml의 농도로 희석한 후 관주 접종하여 생장상에서 26℃, 14시간 광조건과 22℃, 10시간 암조건을 반복하면서 7-8주 후까지 배양하면서 병원성 여부를 확인하였다.Pathogenicity experiments were conducted on the main crops of rice, tomatoes, eggplant, cucumbers and peppers to confirm that the isolated endogenous bacteria were non-pathogenic. The host plants were seeded in sterile soil, seeded in each crop and stored for 2 days in growth and germinated, and then grown under 26 ° C., 14 hours light condition and 22 ° C., 10 hours dark condition. Burkholderia vietnamensis MC1404 strain, an isolated plant endogenous bacterium, was diluted to a concentration of 1 × 10 7 cfu / ml, then inoculated with irrigation and grown at 26 ° C for 14 hours and 22 ° C for 10 hours. Repeated dark conditions and cultured until after 7-8 weeks to determine the pathogenicity.

본 발명 균주인 MC1404는 잎, 줄기, 뿌리 모두 병징이 발생하지 않아 주요작물에 비병원성임이 확인되었다.MC1404 strain of the present invention was confirmed that it is non-pathogenic to the main crop because no symptoms occur in the leaves, stems, roots.

〈실시예 3〉작물의 주요 병해를 일으키는 곰팡이에 대한 길항 내생 세균의 동정<Example 3> Identification of antagonistic endogenous bacteria against mold causing major diseases of crops

길항능력과 균주의 활성 등의 기준으로 선발된 이들 균주는 Biolog시험(표1 참조)과 20가지 이상의 세균학적인 추가적인 시험(표2 참조)을 수행하여 이들의 생리화학적 특성을 알아내고 동정하였다.These strains were selected on the basis of antagonistic ability and activity of the strains, and performed biolog tests (see Table 1) and 20 or more bacteriological additional tests (see Table 2) to identify and identify their physicochemical properties.

또한 선발된 길항 내생 세균을 16S rDNA 염기서열 분석결과(표3 참조) MC1103, MC1303, MC1401, MC1402, MC1404, MC1405, MC1502, MC1506 균주는 버크홀더리아 비에나멘시스(Burkholderia vietnamensis)로 동정되었다.In addition, the selected antagonistic endogenous bacteria 16S rDNA sequence analysis results (see Table 3) MC1103, MC1303, MC1401, MC1402, MC1404, MC1405, MC1502, MC1506 strains were identified as Burkholderia vietnamensis .

이들 균주 중 표본 균주인 MC1404를 농업생명공학연구원 농용미생물 보존센터에 2003년 9월 4일자로 수탁번호 KACC 91060호로 기탁하였다.MC1404, a sample strain of these strains, was deposited with the accession number KACC 91060 on September 4, 2003, at the Agricultural and Biotechnology Research Center for Agricultural Microorganisms.

표1. MC 균주의 Biolog 시험Table 1. Biolog test of MC strain

표2. MC 균주의 생리생화학적 특성Table 2. Physiological Biochemical Properties of MC Strains

표3. MC1404 균주의 16S rDNA의 염기서열Table 3. Nucleotide Sequences of 16S rDNA of MC1404 Strain

〈실시예 4〉각종 식물 병원균에 대한 생육저지 효과 검정<Example 4> Growth inhibition effect assay for various plant pathogens

식물병원균으로는 광범위한 기주 식물에서 발생하는 잿빛곰팡이병, 줄기마름병, 시들음병, 덩굴쪼김병, 입고병, 역병, 탄저병 등을 비롯하여 인삼의 입고병, 뿌리썩음병, 반점병, 벼의 잎집무늬마름병, 도열병, 이삭마름병, 적미병 등의 병원 진균 48종을 사용하였다.Phytopathogenic bacteria include gray mold, stem blight, wilting disease, vine splitting, rash, plague, anthracnose, ginseng, rot, spot, rice leaf, blight, ear, and ear. 48 fungal pathogens, such as blight and red rice, were used.

병원성 진균의 포자 또는 균사절편이 도말된 PDA(Difco Co.) 배지에 길항 내생 세균을 배양한 배양액을 페이퍼 디스크(직경 6mm)에 묻힌 후 진균이 도말된 배지위에 올려놓고 각 병원균의 생육이 잘 되는 온도에서 3∼4일 배양하여 조사하였다. 길항 내생세균이 각 종 병원균에 미치는 영향을 저지원의 직경을 조사한 결과는 표4와 같다.A culture medium containing antagonistic endogenous bacteria in PDA (Difco Co.) medium on which spores or mycelia fragments of pathogenic fungi are smeared is placed on a paper disc (diameter 6 mm), and placed on a medium on which the fungi are smeared, so that each pathogen grows well. Incubation was carried out for 3 to 4 days at temperature. The effect of antagonistic endogenous bacteria on various pathogens was investigated in Table 4.

표4. MC 균주의 항진균 활성 스펙트럼Table 4. Antifungal Activity Spectrum of MC Strains

표4에서 보는 바와 같이, 본 발명의 길항 식물 내생세균 버크홀더리아 비에나멘시스(Burkholderia vietnamensis)로 동정된 균주들과 식물 병원진균의 길항효과에 대한 검정결과 광범위한 기주 식물에서 발생하는 잿빛곰팡이병, 줄기마름병, 시들음병, 덩굴쪼김병, 입고병, 역병, 탄저병 외에도 인삼의 입고병, 뿌리썩음병, 반점병, 벼의 잎집무늬마름병, 도열병, 이삭마름병, 적미병 등에 우수한 길항능력을 나타내었다. 또한 이들 균주들 중에서도 MC1404 균주가 억제능력이 가장 강하여 표준균주로 선발하였다.As shown in Table 4, the results of assay for the antagonistic effect of the strains and phytopathogens identified as antagonistic plant endogenous bacteria Burkholderia vietnamensis of the present invention, asymptomatic disease occurring in a wide range of host plants In addition to stem blight, wilting disease, vine splitting, wearing disease, late blight, anthrax, ginseng's stocking disease, root rot disease, spot disease, rice leaf blight, rice blast, ear blight, red rice disease, etc. In addition, among these strains, MC1404 strain was selected as the standard strain because the strongest inhibitory ability.

〈실시예 5〉버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404의 배양Example 5 Culture of Burkholderia vietnamensis MC1404

상기 실시예 1에서 선발한 신규 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주를 대상으로 항진균 활성물질이 생성되는 최적 조건을 조사하였다. 최적 배지 조성은 펩톤 4g, 이스트 추출물 2g, 덱스트로스 7g, 황산 마그네슘 1g, 황산철 0.05g을 증류수 1L에 섞은 후 pH를 7.4로 조정하여 액체배지를 제조하였다. 배양은 30℃에서 180rpm으로 24시간 진탕 배양하였다.The new Burkholderia vietnamensis ( Burkholderia vietnamensis ) MC1404 strain selected in Example 1 was investigated for the optimal conditions for the production of antifungal actives. The optimum medium composition was prepared by mixing 4 g of peptone, 2 g of yeast extract, 7 g of dextrose, 1 g of magnesium sulfate, and 0.05 g of iron sulfate in 1 L of distilled water and adjusting the pH to 7.4. The culture was shaken for 24 hours at 180 ° C at 30 ° C.

〈실시예 6〉콩과 오이류 종자에서 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404의 항 진균 능력 조사Example 6 Investigation of Antifungal Activity of Burkholderia vietnamensis MC1404 in Soybean and Cucumber Seeds

본 발명의 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404는 콩나물 또는 오이류 종자에서 문제시되는 Fusarium sp., Pythium sp. 및 Rhizoctonia sp. 및 기타의 전염성 진균에 길항력이 있는지를 조사하였다. 무병 종자에 병원성 진균들의 포자 현탁액(1×105 cfu/ml)을 스프레이를 이용하여 살포시켜 25℃ 항온기에 2~3일간 배양하고, 여기에 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 현탁액(1×107cfu/ml)을 살포시켜 30℃ 항온기서 4∼5일간 배양하였다. Burkholderia vietnamensis MC1404 of the present invention is a problem in Fusarium sp., Pythium sp. And Rhizoctonia sp. And antagonistic activity against other infectious fungi. Spore suspension of pathogenic fungi (1 × 10 5 cfu / ml) was sprayed on disease-free seeds by spraying, and then incubated in a thermostat at 25 ° C. for 2-3 days, followed by Burkholderia vietnamensis MC1404 suspension. (1 × 10 7 cfu / ml) was sprayed and incubated at 30 ° C. for 4-5 days.

본 시험의 결과 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404는 콩나물 종자에 병을 일으키는 진균들의 생장을 억제시키는데 큰 효과를 가지고 있고, 종자의 발아율에도 영향을 미치지 않았다.As a result of this test, Burkholderia vietnamensis MC1404 has a great effect on inhibiting the growth of fungi causing disease in soybean sprout seeds, and did not affect seed germination rate.

이와 같이 본 발명의 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404는 포장에서 쓰이는 미생물 살균제 뿐 아니라 종자 수준에서도 진균에 탁월한 길항능력을 가진다.Thus, Burkholderia vietnamensis MC1404 of the present invention has excellent antagonistic ability against fungi at the seed level as well as microbial fungicide used in the packaging.

〈실시예 7〉식물 생육 촉진 효과 검정<Example 7> Plant growth promotion effect assay

버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주의 식물생장 촉진효과를 조사하기 위하여 무영양토와 시판되는 육묘용 상토에 MC1404 균주의 배양체를 접종하여 실시하였다.In order to investigate the plant growth promoting effect of Burkholderia vietnamensis ( Burkholderia vietnamensis ) MC1404 strain was carried out by inoculating the culture of MC1404 strain on non-nutrient soil and commercial seedling soil.

고추종자(품종: 노광), 오이종자(품종: 실버스타) 및 토마토 종자(품종: 서광)를 1% NaOCl 내에서 두차례 표면살균하고, 멸균증류수로 세척하여 준비하였고, 미생물 현탁액은 28℃에서 24 시간동안 TS 액체배지에서 교반(200 rpm) 배양시킨 후, 멸균시킨 0.1M MgSO4 용액 내에서 현탁하여 준비하였다.Pepper seeds (breed: exposure), cucumber seeds (breed: Silverstar) and tomato seeds (breed: Seogwang) were surface sterilized twice in 1% NaOCl, washed with sterile distilled water, and the microbial suspension was prepared at 28 ° C. Stirred in a TS liquid medium for 24 hours (200 rpm), then incubated in sterile 0.1M MgSO 4 solution to prepare.

폿트(6-11cm)에 토양을 담고 무처리 대조군을 제외하고 미생물 현탁액(5×108cfu/ml) 5ml을 포트의 토양에 관주 접종한 후, 준비된 고추, 오이 및 토마토 종자를 폿트당 3개씩 파종하였다. 접종된 종자를 28℃ 암조건과 65%의 상대습도로 조절된 생육상에서 2일 동안 발아시킨 후, 폿트를 25℃ 유리온실로 옮겨 28일간 생육시킨 후 초장, 생체중 및 건물중을 조사하였다. 대조구는 균주를 접종하지 않은 멸균시킨 0.1M MgSO4 용액만을 토양에 혼합하여 같은 조건으로 처리하여 조사하였다.Inoculate soil in a pot (6-11 cm) and inoculate 5 ml of microbial suspension (5 × 10 8 cfu / ml), except for the untreated control, into the soil in the pot, and then prepare three pepper, cucumber and tomato seeds per pot. Sowing was carried out. Inoculated seeds were germinated for 2 days under conditions controlled at 28 ° C. dark conditions and 65% relative humidity, and the pots were transferred to 25 ° C. glass greenhouse for 28 days and examined for height, live weight and dry weight. The control group was irradiated with the same conditions by mixing only the sterilized 0.1M MgSO 4 solution without inoculation into the soil.

무영양토에서의 실험결과는 표5에서와 같이, MC1404균주 처리구 에서는 무처리구에 비하여 초장, 생체중, 건물중이 고추는 각각 3.3배, 8.3배, 5.2배, 오이는 4.0배, 4.5배, 5.4배, 토마토는 2.0배, 2.6배, 2.5배의 생장촉진 효과를 보였다.As shown in Table 5, the MC1404 strain treatment group showed 3.3 times, 8.3 times, 5.2 times, cucumbers 4.0 times, 4.5 times, and 5.4 times, respectively. Tomato showed 2.0, 2.6, 2.5 times of growth promoting effect.

육묘용 상토에서의 실험결과는 표6에서 보는 바와 같이, MC1404 균주 처리구에서는 무처리구에 비하여 초장, 생체중, 건물중이 고추는 각각 2.1배, 1.9배, 2.7배, 오이에서는 1.7배, 1.5배, 1.6배, 토마토에서는 1.5배, 2.1배, 1.6배의 생장촉진 효과를 보였다.As shown in Table 6, the experimental results in seedling soils were 2.1, 1.9, 2.7, and 1.7, 1.5, and 1.6, respectively, for cucumber, live weight, and dry pepper in the MC1404 strain treated group, compared to the untreated group. Pear and tomato showed 1.5, 2.1, and 1.6 times of growth promoting effects.

표5. 무영양토에서 MC1404 균주의 식물생장 촉진효과Table 5. Promoting Plant Growth of MC1404 Strains in Non-nutrient Soils

표6. 육묘용 상토에서 MC1404 균주의 식물생장 촉진효과Table 6. Promoting Plant Growth of MC1404 Strains in Seedling Soils

〈실시예 8〉미생물 살균제 제조용 바이오 그레뉼 제조<Example 8> Preparation of bio granules for producing microbial fungicides

항진균 활성 세균이 처리된 생태계에서 생존을 유지하면서 정착하여 목적하는 항진균 활성을 나타내기 위해서는 중단기적으로 이용가능한 영양분, 보호소제 등이 필요하다. 따라서 이와 같은 작용 특성을 가지는 바이오 그레뉼(Bio-granule)을 제조하였다.Antifungal Active In order to maintain desired survival in the treated ecosystem and to exhibit the desired antifungal activity, nutrients, protective agents, etc. which are available in the interim period are necessary. Therefore, a bio-granule having such an action characteristic was prepared.

바이오 그레뉼은 흡습제에 식물성 오일류, 영양제, 첨가제 등을 혼합하여 만들었다. 흡습제로는 고흡습성 수지(acrylic acid-sodium acrylate copolymer)를 사용하였고, 식물성 오일류는 옥수수, 콩, 유채 및 기타의 식물성 종자로부터 추출된 오일을 이용하였으며, 영양제로는 설탕, 펩톤과 뮤기염류를 사용하였다. 첨가제로는 증량제, UV차단제, 계면활성제를 사용하였다. 바이오 그레뉼 제조용 조성성분과 성분량은 표7과 같다.Bio granules are made by mixing vegetable oils, nutrients, and additives with an absorbent. As the hygroscopic agent, a highly hygroscopic resin (acrylic acid-sodium acrylate copolymer) was used, and the vegetable oils were oil extracted from corn, soybean, rapeseed and other vegetable seeds, and the sugars, peptone and mugi salts were used as nutrients. It was. As additives, extenders, UV blockers, and surfactants were used. Composition and amount of ingredients for the preparation of bio granules are shown in Table 7.

표7. 바이오 그레뉼 조성성분Table 7. Bio granule composition

〈실시예 9〉버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404를 함유하는 미생물 살균제의 제조Example 9 Preparation of Microbial Fungicide Containing Burkholderia vietnamensis MC1404

먼저, 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404를 액체 배양하여 농축하거나 고체배지에 배양된 균체의 집락을 인산 완충액으로 수세하여 준비하였다. 이어서, 농축된 세균을 영양제와 참가제를 혼합액에 혼합하여 준비하고, 흡습제는 식물성 오일과 혼합하여 준비한 후 준비된 세균과 영양제 혼합액와 혼합한다. 마지막으로, 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404의 균체수가 바이오 그레뉼 그람당 108~109정도되게 혼합하여 여기에 코팅제를 혼합하여 교반기를 이용하여 균일하게 섞는다.First, Burkholderia vietnamensis ( Burkholderia vietnamensis ) MC1404 was prepared by concentration in liquid culture or colonies of cells cultured in a solid medium washed with phosphate buffer. Subsequently, the concentrated bacteria are prepared by mixing the nutrient and the participating agent in a mixed solution, and the hygroscopic agent is prepared by mixing the vegetable oil and then mixed with the prepared bacteria and the nutrient mixture. Finally, Burkholderia vietnamensis ( Burkholderia vietnamensis ) MC1404 cell number of 10 8 ~ 10 9 per gram of bio granules are mixed and mixed with a coating agent and mixed uniformly using a stirrer.

이렇게 만들어진 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404와 바이오 그레뉼 복합체를 건조하여 적당한 크기의 분말로 제조하여 미생물 살균제를 만들었다. Burkholderia vietnamensis ( Burkholderia vietnamensis ) MC1404 and the bio granule complexes were dried and made into a powder of a suitable size to make a microbial fungicide.

본 발명의 미생물 살균제를 이용하여 수화제, 입제, 코팅입제 등도 만들 수 있다.The microbial fungicide of the present invention can also be used to make a hydrate, granules, coating granules and the like.

〈실시예 10〉미생물 살균제를 이용한 코팅 종자의 제조Example 10 Preparation of Coating Seeds Using Microbial Sterilizers

본 발명의 미생물 살균제를 이용하여 간편하게 코팅된 종자를 제조하였다. 실시예 9에서 만든 미생물 살균제에 전착제(sticker), 증량제(filler)를 혼합한 후, 종자코팅기계를 이용하여 종자를 펠렛코팅, 필름코팅하거나 두 가지 기법을 동시에 이용하여 다양한 형태의 바이오 종자 코팅된 종자를 만들었다.The coated seeds were prepared simply using the microbial fungicide of the present invention. After mixing a sticker and a filler with the microbial disinfectant prepared in Example 9, the seed coating machine was used for pellet coating, film coating or coating various types of bio seeds using both techniques simultaneously. Made seeds.

본 발명의 종자코팅제의 경우 파종하면 흡습제가 토양 수분을 흡수하여 근권에 미생물이 정착하여 개체군 증식에 유리한 환경을 만들어주어 식물체의 뿌리와 함께 근권에 선점되어 미생물이 효율적으로 병원균의 침입을 막아주면서 유용물질을 분비하여 작물의 생육을 촉진하는 효과를 얻을 수 있다.In the case of seed coating agent of the present invention, the absorbent absorbs soil moisture, so that the microorganisms settle in the rhizosphere to create a favorable environment for the growth of the population, and preoccupied in the rhizosphere with the root of the plant is useful while effectively preventing the invasion of pathogens. By secreting substances, the effect of promoting the growth of crops can be obtained.

〈실험예 1〉 미생물 살균제의 안정성 및 성능 조사Experimental Example 1 Investigation of Stability and Performance of Microbial Disinfectants

(1) 미생물 살균제의 안정성(1) Stability of microbial fungicide

미생물 살균제의 약효 지속은 미생물의 생존기간과 관련이 있다. 따라서 실시예 9에서 제조한 본 발명의 미생물 살균제를 0, 2, 4, 6, 8, 10 및 12개월 동안 실온에서 방치한 다음 살아있는 미생물 수를 조사하였다.The duration of drug efficacy of microbial fungicides is related to the survival of microorganisms. Therefore, the microbial fungicide of the present invention prepared in Example 9 was left at room temperature for 0, 2, 4, 6, 8, 10 and 12 months and then the number of living microorganisms was examined.

그 결과 대조군에 비하여 건조되어 보관된 본 발명의 농약은 8개월 이후 기간 동안에도 10%이내의 차이로 회생되어 안정한 결과를 보인다. 항균활성의 결과에 있어서도 변함이 없는 결과를 보여주었다.As a result, the pesticide of the present invention, which is dried and stored as compared to the control group, regenerates within a 10% difference even after 8 months, and shows a stable result. The results of the antimicrobial activity showed no change.

(2) 미생물 살균제 처리시 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주의 엽면, 토양생존률(2) Leaf surface and soil survival rate of Burkholderia vietnamensis MC1404 strain during microbial fungicide treatment

바이오 그레뉼화된 미생물 살균제내의 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주에 대하여 배추 엽면, 배추 근권 토양, 일반 토양에서 1주 간격으로 생존율을 조사한 결과 초기의 생존률에 비해서 2주 후에 2~18배, 4주 후에는 10~80배까지 증가하였다. Burkholderia vietnamensis MC1404 strains in biogranulated microbial fungicides were examined at 1 week intervals in cabbage leaf, cabbage root zone soil, and general soil. It increased to 18-fold and 10-80 fold after 4 weeks.

(3) 미생물 살균제가 처리된 코팅 종자의 발아율 조사(3) Investigation of germination rate of coated seed treated with microbial fungicide

실시예 9에서 제조된 본 발명의 미생물 살균제를 이용하여 코팅된 콩, 오이, 수박, 토마토, 고추 종자들을 100개씩 이용하여 발아율을 조사하였다. 그 결과 코팅종자의 발아율 및 발아세는 10~30% 증가하였고, 발아된 유묘의 초세가 미처리된 종자보다 훨씬 강하였다.Germination rate was investigated using 100 seeds of soybean, cucumber, watermelon, tomato and pepper coated with the microbial fungicide of the present invention prepared in Example 9. As a result, germination rate and germination of coated seeds increased by 10 ~ 30%, and the germination of seedlings was much stronger than untreated seeds.

(4) 미생물 살균제의 UV에 대한 안정성(4) UV Stability of Microbial Fungicide

실시예 9에서 제조한 미생물 살균제의 UV에 대한 안정성을 조사하였다. 햇볕에 6, 12 및 24시간 노출시킨 미생물 살균제 내에 함유되어 있는 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404 균주의 생존율은 감소하지 않았다.The stability against UV of the microbial fungicide prepared in Example 9 was investigated. Survival of Burkholderia vietnamensis MC1404 strain contained in microbial fungicides exposed to sun for 6, 12 and 24 hours did not decrease.

본 발명의 미생물 살균제는 수화제, 입제, 코팅 입제, 바이오 상토, 미생물영양제, 식물 생육촉진제, 토양 개량제, 종자 코팅제, 퇴비 부숙제, 엽면 살포제 또는 관주 살포제로 이용될 수 있다.Microbial fungicides of the present invention can be used as a hydrating agent, granules, coated granules, bio-tops, microbial nutrition, plant growth promoters, soil improvers, seed coatings, compost debris, foliar spray or irrigation spray.

상기한 바와 같이 본 발명의 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404는 식물 병원성 진균에 대해서 광범위한 길항력을 나타내며, 이를 포함하는 미생물 제제를 이용하여 광범위한 진균에 의한 병을 효과적으로 방제할 수 있다.As described above, the Burkholderia vietnamensis MC1404 of the present invention exhibits a wide range of antagonism against plant pathogenic fungi, and can effectively control diseases caused by a wide range of fungi using microbial agents including the same. .

또한, 종자 내에서 발생하는 진균에 대해서도 우수한 억제능력을 가지고 있어 종자 소독제로서도 효과적이고, 근류미생물로서 식물 생육을 촉진시키는 기능을 가지고 있다.In addition, it has an excellent inhibitory ability against fungi occurring in seeds, and is effective as a seed disinfectant and has a function of promoting plant growth as a root microbe.

Claims (5)

병원성 진균에 대해 항진균 활성을 가지는 미생물 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404(수탁번호 : KACC 91060).Microorganism Burkholderia vietnamensis MC1404 (Accession No .: KACC 91060) having antifungal activity against pathogenic fungi. 제 1 항에 있어서,The method of claim 1, 상기 병원성 진균은 넓은 식물 병원성 진균에 의해 광범위한 기주 식물에서 발생하는 잿빛곰팡이병(Botrytis cinerea), 줄기마름병(Alternaria alternata), 시들음병(Fusarium oxysporum f. sp. lycolersici), 덩굴쪼김병(Fusarium oxysporum), 잘록병(Pythium ultinum), 탄저병(Colletotrichum lagenarium, Colletotrichum gloeosporioides), 역병(Phytophtora drechsien, Phytopthora capsici, Phytopthora infestans)을 포함하며, 인삼의 입고병(Rhizoctonia solani), 뿌리썩음병(Cylindrocarpon destructans), 반점병(Alternaria panax), 벼의 잎집무늬마름병(Rhizoctonia solani), 도열병(Pyricularia grisea), 이삭마름병(Fusarium rosem), 적미병(Fusarium moniliform) 중에서 선택되는 것을 특징으로 하는 버크홀더리아 비에나멘시스 MC1404.The pathogenic fungi are caused by a wide range of plant pathogenic fungi, Botrytis cinerea , Alternaria alternata , Fusarium oxysporum f. Sp. Lycolersici , Fusarium oxysporum (Pythium ultinum), anthracnose (Colletotrichum lagenarium, Colletotrichum gloeosporioides), late blight (Phytophtora drechsien, Phytopthora capsici, Phytopthora infestans) to include, and wearing bottle (Rhizoctonia solani), root rot (Cylindrocarpon destructans), banjeombyeong (Alternaria panax) ginseng Berkholderia viennamensis MC1404 , characterized in that the leaf selected from the blight ( Rhizoctonia solani ), blast ( Pyricularia grisea ), ear blight ( Fusarium rosem ), Fusarium moniliform . 제 1 항에 있어서,The method of claim 1, 상기 버크홀더리아 비에나멘시스 MC1404는 미생물농약, 종자코팅제, 바이오상토, 미생물영양제, 식물 생육 촉진제, 토양개량제, 퇴비부숙제, 엽면살포제 또는 관주살포제의 유효성분으로 이용되는 것을 특징으로 하는 버크홀더리아 비에나멘시스 MC1404.The Birkholderia viennamensis MC1404 is a buckholder, characterized in that it is used as an active ingredient of microbial pesticides, seed coating agent, biosoil, microbial nutritional agent, plant growth promoter, soil improver, composting agent, foliar spray or irrigation spraying agent. Leah Vienamensis MC1404. 제 1 항의 버크홀더리아 비에나멘시스(Burkholderia vietnamensis) MC1404를 흡습제인 고흡수성 수지, 식물성 오일류, 영양제, 첨가제와 코팅제를 혼합하여 바이오 그레뉼 복합체를 만들고 건조하여 제조된 미생물 살균제.A microbial fungicide prepared by mixing the Burkholderia vietnamensis MC1404 of claim 1 with a superabsorbent resin, a vegetable oil, a nutrient, an additive, and a coating agent as an absorbent to make a bio granule complex and drying it. 제 4 항에 있어서,The method of claim 4, wherein 상기 미생물 살균제는 수화제, 입제, 코팅 입제, 바이오 상토, 미생물영양제, 식물 생육촉진제, 토양 개량제, 종자 코팅제, 퇴비 부숙제, 엽면 살포제 또는 관주 살포제로 이용되는 것을 특징으로 하는 미생물 살균제.The microbial fungicide is a microbial fungicide, characterized in that it is used as a hydrating agent, granules, coating granules, bio-topology, microbial nutrients, plant growth promoting agents, soil improvers, seed coating agents, composting, foliar spraying or irrigation spraying.
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