KR100566264B1 - 3101 Biocontrol of plant diseases using novel endophytic isolate of Pseudomonas fluorescence MC3101 - Google Patents
3101 Biocontrol of plant diseases using novel endophytic isolate of Pseudomonas fluorescence MC3101 Download PDFInfo
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Abstract
본 발명은 광범위한 항진균 활성을 가지는 신규 내생 세균 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101 및 이를 함유하는 미생물 살균제에 관한 것이다. 본 발명의 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101는 식물에 내생하는 미생물로써 분리 균주들 중 항균 스펙트럼을 통해 선발되었고, 생리 생화학적인 시험과 16S rDNA 염기서열 분석결과에 의해 동정되었다. 슈도모나스 플루오레슨스((Pseudomonas fluorescence) MC3101은 대부분의 시설재배 및 노지재배에서 문제시되는 병원 진균인 오이류의 줄기마름병, 시들음병, 덩굴 쪼김병, 입고병, 인삼 뿌리썩음병, 입고병, 반점병, 벼의 잎집무늬마름병, 도열병, 이삭마름병, 적미병에 대하여 억제 효과를 가지며, 특히 잿빛곰팡이병(토마토, 가지, 장미, 딸기, 인삼), 탄저병(고추, 수박, 오이, 가지, 토마토, 장미 및 화훼류), 역병(고추, 수박, 오이, 가지, 토마토, 감자)에 강한 항진균 활성 스펙트럼을 가지는 미생물이다.The present invention relates to a novel endogenous bacterium Pseudomonas fluorescence MC3101 having a wide range of antifungal activity and a microbial fungicide containing the same. Pseudomonas fluorescence of the present invention ( Pseudomonas fluorescence ) MC3101 is an endogenous microorganism in the plant was selected through the antimicrobial spectrum of the isolates, was identified by physiological biochemical test and 16S rDNA sequence analysis. Pseudomonas fluorescence MC3101 is a fungus that causes problems in most plant and open field cultivation, cucumber stalk blight, wilting disease, vine splitting disease, rash, ginseng root rot, rash, spot disease and rice leaf. It has an inhibitory effect against pattern blight, heat blight, ear blight, and red rice, especially gray mold disease (tomato, eggplant, rose, strawberry, ginseng), anthrax (pepper, watermelon, cucumber, eggplant, tomato, rose and flower), It is a microorganism with a strong antifungal activity spectrum against late blight (pepper, watermelon, cucumber, eggplant, tomato, potato).
슈도모나스 플루오레슨스(Pseudomonas fluorescence), 균주, 미생물Pseudomonas fluorescence, strains, microorganisms
Description
본 발명은 미생물에 관한 것으로, 식물 병원성 진균에 대해 광범위한 길항능력을 가지는 신규의 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101 균주 및 이를 함유하는 항진균성 미생물 살균제에 관한 것이다.The present invention relates to a microorganism, and relates to a novel Pseudomonas fluorescence MC3101 strain having a wide range of antagonistic ability against plant pathogenic fungi and an 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 생물 자원 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 증식 이 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.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.
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.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 conducted in the 1980s, which is 30 to 50 years later than in foreign 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 bud rot), 사탕무우의 입고병(Damping-off), 벼의 도열병, 문고병 방제등에 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 bud rot, Damping-off of sugar beet, rice blast, paperback disease control, etc.
최근 작물을 재배하는데 있어 문제시되는 병해로는 벼에서의 도열병, 문고병, 이삭마름병이 있고, 시설재배에서는 시들음병, 잿빛 곰팡이병, 흰가루병, 탄저병 등이 문제시되고 있다.In recent years, the problem of growing crops is rice blast, paperback disease, ear blight, and facility cultivation is withering disease, gray mold, powdery mildew, anthrax.
본 발명의 목적은 미생물의 서식 및 적응된 위치가 토양이 아닌 식물내에 존재하면서 식물 병원균에 길항능력을 가지는 식물 내생 미생물인 슈도모나스 플루오레슨스((Pseudomonas fluorescence) MC3101을 제공하는데 있다.It is an object of the present invention to provide Pseudomonas fluorescence MC3101 which is a plant endogenous microorganism having antagonistic ability against plant pathogens while the habitat and adapted location of microorganisms are present in plants rather than soil.
본 발명의 다른 목적은 식물 병원성 진균에 대해 광범위한 길항능력을 가지며, 항진균 활성이 우수한 신규의 식물 내생 세균인 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101과 이를 함유하는 식물 병원성 진 균방제용 미생물 살균제를 제공하는데 있다.It is another object of the present invention to provide Pseudomonas fluorescence MC3101, a new plant endogenous bacterium having broad antagonistic ability against plant pathogenic fungi and excellent antifungal activity, and a microbial fungicide for plant pathogenic fungi containing the same. It is.
상기와 같은 목적을 달성하기 위한 본 발명은 식물 병원성 진균에 대해 광범위한 길항능력을 가지며, 항진균 활성이 우수한 신규의 내생 세균인 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101을 제공하는 것에 특징이 있다.The present invention for achieving the above object is characterized by providing a new endogenous bacteria Pseudomonas fluorescence ( Pseudomonas fluorescence ) MC3101 having a wide range of antagonistic ability against plant pathogenic fungi, excellent antifungal activity.
본 발명의 상기 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101은 국내의 식물 뿌리에서 분리한 세균으로서 각종 식물에 대해 비병원성 균주이다. Pseudomonas fluorescence of the present invention ( Pseudomonas fluorescence ) MC3101 is a bacterium isolated from plant roots in Korea is a non-pathogenic strain for various plants.
본 발명의 상기 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101 균주는 대부분의 시설재배 및 노지재배에서 문제시되는 병원 진균인 오이류의 줄기마름병(Alternaria alternata) , 시들음병(Fusarium oxysporum f. sp. lycolersici), 덩굴 쪼김병(Fusarium oxysporum), 입고병(Pythium ultinum), 인삼의 뿌리썩음병(Cylindrocarpon destructans), 입고병 (Rhizoctonia solani), 반점병(Alternaria panax), 벼의 잎집무늬마름병(Rhizoctonia solani), 도열병(Pyricularia grisea), 이삭마름병 (Fusarium rosem), 적미병(Fusarium moiliforme) 대하여 억제 효과를 가지며, 특히 토마토, 가지, 장미, 딸기, 인삼의 잿빛곰팡이병(Botrytis cinerea), 고추, 수박, 오이, 가지, 토마토, 장미 및 화훼류의 탄저병(Colletotrichum gloeosporioides), 고추, 수박, 오이, 가지, 토마토, 감자의 역병(Phytopthora capsici)에 강한 길항효과를 가진다.The Pseudomonas fluorescence MC3101 strain of the present invention is a fungus that is a problem in most plant cultivation and field cultivation, Cucumbers, Alternaria alternata , wilting disease ( Fusarium oxysporum f. Sp. Lycolersici ), vines Fusarium oxysporum , Pythium ultinum , Ginseng Root Rot Disease ( Cylindrocarpon destructans ), Ginseng Disease ( Rhizoctonia solani ), Spot Disease ( Alternaria panax ), Rhizoctonia solani ( Rhizoctonia solani ), Plague ( sea ) , Fusarium rosem , Fusarium moiliforme , have inhibitory effect, especially tomato, eggplant, rose, strawberry, ginseng, Botrytis cinerea , pepper, watermelon, cucumber, eggplant, tomato, rose And a strong antagonistic effect against anthrax ( Creetotrichum gloeosporioides ), pestilence of pepper, watermelon, cucumber, eggplant, tomato, potato ( Phytopthora capsici ).
또한, 본 발명은 상기 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101 균주와 미생물 담체인 바이오 그레뉼을 혼합하여 만든 미생물 살균제를 제 공하는 것에 특징이 있다.In addition, the present invention is characterized in providing a microbial fungicide made by mixing the Pseudomonas fluorescence MC3101 strain and a bio granule which is a microbial carrier.
본 발명의 상기 미생물 살균제는 종자코팅제, 바이오상토, 미생물영양제, 토양개량제, 퇴비부숙제, 엽면살포제 또는 관주살포제로 이용될 수 있다.The microbial fungicide of the present invention may be used as a seed coating agent, bio-topology, microbial nutritional agent, soil improving agent, composting agent, foliar spray or irrigation spray.
이하, 실시 예에 의하여 본 발명을 상세히 설명하면 다음과 같다. 단 이들 실시 예는 본 발명을 구체적으로 설명하기 위한 것으로, 본 발병의 범위가 이들 실시 예에 국한되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of 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 주요작물뿌리를 채취하였다. 채집한 뿌리는 표면 소독을 위해 1% Chloramine T 용액에서 15분간 침지한 뒤 glass bead를 넣고 20분간 교반하였으며, 이를 마쇄하여 10배수로 희석한 뒤 TSA(Difco) 배지에 접종하였다.In order to isolate plant endogenous bacteria with broad antagonistic ability against phytopathogenic fungi and excellent active effect, major crop roots of Jeolla and Chungcheong regions were collected. The collected roots were immersed in 1% Chloramine T solution for 15 minutes for surface disinfection, and then glass bead was added and stirred for 20 minutes. The roots were ground, diluted to 10-fold, and inoculated in TSA (Difco) medium.
그 결과로 425개의 대표세균이 분리되었고, 이 중 식물 병원성 진균에 강한 길항능력을 나타내는 균주 86개를 1차 선발하였다. 다시 광범위한 항진균 효과 시험 등을 통해 우수한 길항세균 중 세균학적 특성상 같은 종으로 생각되는 MC3101, MC3102, MC3103, MC3105, MC3106, MC3107, MC3108의 8균주를 선발하였다.As a result, 425 representative bacteria were isolated, and among them, 86 strains showing strong antagonistic ability against plant pathogenic fungi were selected first. Through the extensive antifungal effect test, 8 strains of MC3101, MC3102, MC3103, MC3105, MC3106, MC3107, and MC3108, which are considered to be the same species among the excellent antagonistic bacteria, were selected.
〈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 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101 균주를 1×107cfu/ml의 농도로 희석한 후 관주 접종하여 생장상에서 26℃, 14시간 광조건과 22℃, 10시간 암조건을 반복하면서 4-6주 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. Pseudomonas fluorescence MC3101 strain isolated from plant endogenous bacteria was diluted to a concentration of 1 × 10 7 cfu / ml, and then inoculated with irrigation to incubate at 26 ° C for 14 hours and 22 ° C for 10 hours in dark conditions. Repeatedly cultured until 4-6 weeks later to determine the pathogenicity.
본 발명 균주들인 MC3101, MC3102, MC3103, MC3105, MC3106, MC3107, MC3108은 잎, 줄기, 뿌리 모두 병징이 발생하지 않아 주요작물에 비병원성임이 확인되었다.MC3101, MC3102, MC3103, MC3105, MC3106, MC3107, MC3108 of the strains of the present invention, all of the leaves, stems, roots did not occur, it was confirmed that non-pathogenic to the main crop.
〈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
길항력과 균주의 활성 등의 기준으로 선발된 이들 균주는 Biolog 시험(표1 참조)과 20가지 이상의 세균학적인 추가적인 시험(표2 참조)을 수행하여 이들의 생리화학적 특성을 알아내고 동정하였다. 또한 선발된 길항 내생 세균을 16S rDNA염기서열 분석결과(표3 참조) MC3101, MC3102, MC3103, MC3105, MC3106, MC3107, MC3108 균주는 슈도모나스 플루오레슨스(Pseudomonas fluorescence)로 동정되었다.These strains selected on the basis of antagonism and activity of the strains were subjected to Biolog test (see Table 1) and more than 20 bacteriological additional tests (see Table 2) to identify and identify their physicochemical properties. In addition, the selected antagonistic endogenous bacteria 16S rDNA base sequence analysis results (see Table 3) MC3101, MC3102, MC3103, MC3105, MC3106, MC3107, MC3108 strains were identified as Pseudomonas fluorescence ( Pseudomonas fluorescence ).
이들 균주 중 표준균주인 MC3101을 농업생명공학연구원 농용미생물보존센터에 2003년 9월 4일자로 수탁번호 KACC 91062호로 기탁하였다.Among these strains, MC3101, a standard strain, was deposited in the Agricultural Biotechnology Conservation Center, Agricultural Microorganism Conservation Center, on September 4, 2003 under Accession No. KACC 91062.
〈실시예 4〉 각종 식물 병원균에 대한 생육저지 효과 검정<Example 4> Growth inhibition effect assay for various plant pathogens
식물병원균으로는 오이류의 줄기마름병, 시들음병, 덩굴 쪼김병, 입고병, 인삼의 뿌리썩음병, 입고병, 반점병, 벼의 잎집무늬마름병, 도열병, 이삭마름병, 적미병에 대하여 억제 효과를 가지며, 특히 잿빛곰팡이병(토마토, 가지, 장미, 딸기, 인삼), 탄저병(고추, 수박, 오이, 가지, 토마토, 장미 및 화훼류), 역병(고추, 수박, 오이, 가지, 토마토)등의 34종을 사용하였다. 병원성 진균류가 도말된 PDA(Difco Co) 배지에 길항 내생 세균을 배양한 배양액을 페이퍼 디스크(직경 6mm)에 묻힌 후 진균이 도말된 배지 위에 올려놓고 각 병원균의 생육이 잘 되는 온도에서 6-7일 동안 배양하면서 조사하였다. 길항 내생세균이 각 종 병원균에 미치는 영 향을 저지원의 직경을 조사한 결과는 표4와 같다.Plant pathogens have inhibitory effect against stem blight, wilted disease, vine pecking disease, wearing disease, ginseng root rot disease, wearing disease, spot disease, leaf blight of rice, heat blight, ear blight, red rice disease of cucumber, especially ash gray 34 kinds of fungal diseases (tomato, eggplant, rose, strawberry, ginseng), anthrax (pepper, watermelon, cucumber, eggplant, tomato, rose and flower), late blight (pepper, watermelon, cucumber, eggplant, tomato) . Culture medium containing antagonistic endogenous bacteria in PDA (Difco Co) medium with pathogenic fungi smeared on a paper disc (diameter 6 mm), and placed on the medium with smeared fungi. Irradiation while incubating. The effect of antagonistic endogenous bacteria on various pathogens was investigated in Table 4.
표4에서 보는 바와 같이, 본 발명의 신규 길항 식물 내생세균 슈도모나스 플루오레슨스(Pseudomonas fluorescence)로 동정된 균주들과 식물 병원진균의 길항효과에 대한 검정결과 대부분의 시설재배 및 노지재배에서 문제시되는 병원 진균인 토마토의 줄기마름병, 시들음병, 오이의 덩굴 쪼김병, 잘록병, 인삼의 뿌리썩음병, 입고병, 반점병, 벼의 잎집무늬마름병, 도열병, 이삭 마름병 등 대부분의 병원진균에 대하여 억제 효과를 가지며, 특히 토마토, 가지, 장미, 딸기, 인삼의 잿빛곰팡이병, 고추, 수박, 오이, 가지, 토마토, 장미 및 화훼류의 탄저병, 고추, 수박, 오이, 가지, 토마토, 감자의 역병에 강한 길항 효과를 나타내었다. 또한 이들 균주들 중에서도 MC3101균주가 억제능력이 가장 강하여 표준균주로 선발하였다.As shown in Table 4, as a result of the assay for the antagonistic effect of the strains and plant pathogens identified by the novel antagonistic plant endogenous bacteria Pseudomonas fluorescence of the present invention, hospitals in question in most plant cultivation and open field cultivation It has an inhibitory effect against most pathogenic fungi, such as fungus, tomato stem blight, wilted disease, cucumber vine splitting, buckwheat disease, ginseng root rot disease, wearing disease, spot disease, leafy blight of rice, heat blight and ear blight. It has a strong antagonistic effect on the blight of eggplant, rose, strawberry, ginseng's gray mold, pepper, watermelon, cucumber, eggplant, tomato, rose and flower anthracnose, pepper, watermelon, cucumber, eggplant, tomato, potato. Among these strains, the MC3101 strain was selected as the standard strain because it had the strongest inhibitory ability.
〈실시예 5〉미생물 살균제 제조용 바이오 그레뉼 제조<Example 5> Preparation of bio granules for producing microbial fungicides
항진균 활성 세균이 처리된 생태계에서 생존을 유지하면서 정착하고 번식하여 목적하는 항진균 활성을 나타내기 위해서는 중단기적으로 이용가능한 영양분, 보호소제 등이 필요하다. 따라서 이와 같은 작용 특성을 가지는 바이오 그레뉼(Bio-granule)을 제조하였다.Antifungal Actives Nutrients, protective agents, etc., which are available in the medium and long term are needed to establish and propagate while maintaining survival in a treated ecosystem. 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 표5와 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 producing bio granules are shown in Table 5.
〈실시예 6〉 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101를 함유하는 미생물 살균제의 제조<Example 6> Preparation of microbial fungicide containing Pseudomonas fluorescence MC3101
먼저, 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101를 배양하여 농축하였다. 이어서, 농축된 세균을 영양제와 참가제를 혼합액에 혼합하여 준비한 후 흡습제는 식물성 오일과 혼합하여 준비한 후 준비된 세균과 영양제 혼합액와 혼합한다. 마지막으로, 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101의 균체수가 바이오 그레뉼 그람당 108∼109정도되게 혼합하여 여기에 코팅제를 혼합하여 교반기를 이용하여 균일하게 섞는다.First, Pseudomonas fluorescence MC3101 was cultured and concentrated. 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, Pseudomonas fluorescence ( Pseudomonas fluorescence ) The number of cells of MC3101 is mixed to about 10 8 ~ 10 9 per gram of bio granules, the coating agent is mixed and mixed uniformly using a stirrer.
이렇게 만들어진 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101과 바이오 그레뉼 복합체를 건조하여 적당한 크기의 분말로 제조하여 미생물 살균제를 만들었다. Pseudomonas fluorescence ( Pseudomonas fluorescence ) MC3101 and the bio granules composite thus prepared was dried 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 7〉 미생물 살균제를 이용한 코팅 종자의 제조Example 7 Preparation of Coating Seeds Using Microbial Sterilizers
본 발명의 미생물 살균제를 이용하여 간편하게 코팅된 종자를 제조하였다. 실시예 6에서 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 the electrodeposition (sticker) and the extender (filler) to the microbial disinfectant prepared in Example 6, the seed coating machine was used for pellet coating, film coating or coated with 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 6에서 제조한 본 발명의 미생물 살균제를 0, 2, 4, 6, 8, 10 및 12개월을 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 6 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 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101 균주의 엽면, 토양생존률(2) Leaf surface and soil survival rate of Pseudomonas fluorescence MC3101 strain during microbial fungicide treatment
바이오 그레뉼화된 미생물 살균제내의 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101 균주에 대하여 배추 엽면, 배추 근권 토양, 일반 토양에서 1주 간격으로 생존율을 조사한 결과 조기의 생존륜에 비해서 2주 후에 2∼18배, 4주 후에는 10∼80배까지 증가하였다.The Pseudomonas fluorescence MC3101 strain in bio-granulated microbial fungicides was examined at 1 week intervals in cabbage leaf, cabbage root zone soil, and general soil. After 4 weeks, it increased to 10-80 times.
(3) 미생물 살균제가 처리된 코팅 종자의 발아율 조사(3) Investigation of germination rate of coated seed treated with microbial fungicide
실시예 6에서 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 6. As a result, the 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 6에서 제조한 미생물 살균제의 UV에 대한 안정성을 조사하였다. 햇볕에 6, 12, 24시간 노출시킨 미생물 살균제 내에 함유되어 있는 상기 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101 균주의 생존율은 감소하지 않았다.The stability against UV of the microbial fungicide prepared in Example 6 was investigated. The viability of the Pseudomonas fluorescence MC3101 strain contained in the microbial fungicide exposed to sun for 6, 12 or 24 hours was not reduced.
본 발명의 미생물 살균제는 종자코팅제, 바이오상토, 미생물영양제, 토양개량제, 퇴비부숙제, 엽면살포제 또는 관주살포제로 이용될 수 있다.The microbial fungicide of the present invention may be used as a seed coating agent, a biosoil, a microbial nutrient, a soil improving agent, a composting agent, a foliar spray or an irrigation spray.
상기한 바와 같이 본 발명의 슈도모나스 플루오레슨스(Pseudomonas fluorescence) MC3101은 식물 병원성 진균에 대해 광범위한 길항능력을 가지며, 항진균 활성이 우수하고, 대부분의 시설재배 및 노지재배에서 문제시되는 병원 진균 인 오이류의 줄기마름병, 시들음병, 덩굴 쪼김병, 입고병, 인삼의 뿌리썩음병, 입고병, 반점병, 벼의 잎집무늬마름병, 도열병, 이삭마름병, 적미병에 대하여 억제 효과를 가지며, 특히 잿빛곰팡이병(토마토, 가지, 장미, 딸기, 인삼), 탄저병(고추, 수박, 오이, 가지, 토마토, 장미 및 화훼류), 역병(고추, 수박, 오이, 가지, 토마토, 감자)에 강한 길항력을 나타내며, 이를 포함하는 미생물 제제를 이용하여 광범위한 진균에 의한 병을 효과적으로 방제할 수 있다.As described above, the Pseudomonas fluorescence MC3101 of the present invention has a wide range of antagonistic ability against plant pathogenic fungi, has excellent antifungal activity, and is a pathogenic fungus that is a problem in most plant cultivation and open field cultivation. It has an inhibitory effect on stem blight, wilting disease, vine splitting, wearing bottle, ginseng root rot, wearing bottle, spot disease, rice leaf blight, heat bottle, ear blight, and red rice disease, especially gray mold disease (tomato, eggplant, Rose, strawberry, ginseng), anthrax (pepper, watermelon, cucumber, eggplant, tomato, rose and flower), and antagonism (pepper, watermelon, cucumber, eggplant, tomato, potato) The disease can be effectively controlled by a wide range of fungi.
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