KR20140105261A - Novel Bacillus ASC-31, the culture medium producing therefor and the composition comprising the same - Google Patents
Novel Bacillus ASC-31, the culture medium producing therefor and the composition comprising the same Download PDFInfo
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- KR20140105261A KR20140105261A KR1020130019284A KR20130019284A KR20140105261A KR 20140105261 A KR20140105261 A KR 20140105261A KR 1020130019284 A KR1020130019284 A KR 1020130019284A KR 20130019284 A KR20130019284 A KR 20130019284A KR 20140105261 A KR20140105261 A KR 20140105261A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/12—Powders or granules
- A01N25/14—Powders or granules wettable
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
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본 발명은 고추탄저병 방제활성을 갖는 신규 바실러스 속 ASC-31 균주, 이의 생산을 위한 배지조성물 및 이를 포함하는 조성물에 관한 것이다.The present invention relates to a novel Bacillus sp. ASC-31 strain having pepper anthrax control activity, a medium composition for the production thereof and a composition comprising the same.
구체적으로, 본 발명은 분리균주 바실러스속 ASC-31 (Bacillus sp. ASC-31)를 이용하여 고추병해인 고추탄저병 (콜렉토트리쿰 글로에스포리디스, Colletotrichum gloesporides) 의 생물학적 방제에 관한 것이다. 더욱 상세하게는 고추의 전 생육기간에 걸쳐 발생하여 가장 심한 피해를 일으키는 탄저병을 방제하는 신규 미생물 균주인 바실러스 속 ASC-31 (Bacillus sp. ASC-31)를 이용하여 화학 농약을 대체하여 친환경생물제제에 관한 것이다.More specifically, the present invention is in isolated strain Bacillus ASC-31 (Bacillus sp. ( Colletotrichum gloesporides ), a red pepper disease, which is caused by the use of ASC-31 (ASC-31). More particularly, the present invention relates to a new microorganism strain Bacillus sp. ASC-31 ( Bacillus sp. sp. ASC-31) to replace environmental chemical pesticides.
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[문헌 14]Kwon, C.S., 1999. Studies on occurrence of anthracnose of red pepper in northen kyeong-buk province. Department of Agricultural Biology , Graduate school Andong national University . [14] Kwon, CS, 1999. Studies on the anthracnose of red pepper in northen kyeong-buk province. Department of Agricultural Biology , Graduate school Andong national University .
[문헌 15]Lee, Y.S., Choi, J.W., Kim, S.D., Baik, H.S., 1999 Isolation of Antagonistic Bacteria to Phytophthora capsici for Biological Control of Phytophthora blight of Red Pepper, p. 1-2. [Literature 15] Lee, YS, Choi, JW, Kim, SD, Baik, HS, 1999 Isolation of Antagonistic Bacteria to Phytophthora capsici for Biological Control of Phytophthora blight of Red Pepper, p. 1-2.
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[문헌 18]Lee, S.Y., Lee, S.B., Kim, Y.K., Kim, H.G., 2004. Effect of agrochemicals on mycelial growth and spore germination of a hyperparasite, Ampelomyces quisqualis 94013 for controlling cucumber powdery mildew, Kor . J. Pesti . Sci. 8, 71-78.Lee, SY, Lee, SB, Kim, YK, Kim, HG, 2004. Effect of agrochemicals on mycelial growth and spore germination of a hyperparasite, Ampelomyces quisqualis 94013 for controlling cucumber powdery mildew, Kor . J. Pesti . Sci . 8, 71-78.
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[문헌 20]Okayama, K., Tsujimeto, A., 1994. Occurrence of strawberry anthracnose caused by Glomerella cingulata (Stoneman) Spaulding et Schrenk and pathogenicity of the fungus, Ann . Phytopatho . Soc . 60, 617-623.[Patent Literature 20] Okayama, K., Tsujimeto, A., 1994. Occurrence of strawberry anthracnose caused by Glomerella cingulata (Stoneman) Spaulding et Schrenk and pathogenicity of the fungus, Ann . Phytopatho . Soc . 60, 617-623.
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고추에 발생하는 병해는 26여종으로 고추의 주요 생산 제한 요인 중 하나인 탄저병은 오래 전부터 우리 나라 뿐만 아니라 전 세계적으로 고추 뿐만아니라 다양한 작물에 큰 피해를 주고 있다. (Okayama and Tsujimeto, 1994). 우리나라에서 경제적 작물인 고추재배시 발생하는 탄저병은 역병과 함계 경제적으로 큰피해를 주고 있는 병이다. 국내에서 분리된 고추 탄저병균 간에 분생포자 형태, 강모, 균총 색깔, 균사생장, 병원성의 특징에 따라 Colletotrichum gloeosporioides, Colletotrichum acutatum, Colletotrichum coccodes, Colletotrichum dematium, Glomerella cingulata 로서 모두 5 종이 분리 동정되었다(Park and Kim, 1992). Anthracnose, one of the major production limitations of pepper, has been causing harm to various crops as well as pepper as well as our country for a long time. (Okayama and Tsujimeto, 1994). Anthracnose caused by the cultivation of red pepper, which is an economical crop in Korea, is a disease that causes economic damage due to plague. Five isolates were identified as Colletotrichum gloeosporioides, Colletotrichum acutatum, Colletotrichum coccodes, Colletotrichum dematium and Glomerella cingulata according to the characteristics of conidia, bristles, mycelium, and pathogenicity , 1992).
그중 고추열매에 주로 발생하는 탄저병은 C. gloeosporioides와 C. acutatum 에 의해 해마다 탄저병이 발생하고 있으며, 우리나라에서 장마기부터 시작하여 수확기 까지 지속적으로 탄저병이 발생하여 농업경제에 큰 피해를 끼치고 있다. Kim 등(1986)은 C. gloeosporioides 풋고추와 붉은 고추에 대한 병원성, 분생포자 형태, PDA 배지에서 배양적 특성에 따라 G, R계통으로 분리하였다. G 계통은 풋고추와 붉은 고추에서 탄저병을 일으키며, PDA 배지상에 분생포자를 형성하고, 자낭반과 강모는 형성하지 않는다. 분생포자는 한쪽 끝이 좁거나 둥글고, 균사생장 적온은 26~28℃이며, PDA배지에서 자라는 균총의 색깔은 흰색에서 회색빛을 형성한다고 하였다. R 계통은 붉은 고추에서 병징을 나타내며, PDA 배지에서 분생포자와 자낭반을 형성하고 양끝이 둥글고 무딘 분생포자를 형성하며 균총의 색깔은 회색 내지 어두운색을 나타낸다. Among them, anthracnose caused mainly by C. gloeosporioides and C. acutatum is anthracnose caused by the anthracnose per year. In Korea, anthrax disease occurs continuously from the rainy season to the harvest season, which causes serious damage to the agricultural economy. Kim et al. (1986) isolated G, R strains according to their culture characteristics in pathogenicity, conidium form, and PDA medium for C. gloeosporioides green pepper and red pepper. G system causes anthrax in green pepper and red pepper, forms conidiospore on PDA, and does not form sacs and bristles. Min and the roundness of myopia was 26 ~ 28 ℃, and the color of the growth on the PDA medium was white to gray. R line shows symptoms in red pepper and forms conidia and cerulean in PDA medium, round ends of both ends and blunt spores are formed, and the color of pistil is gray to dark color.
이와 같이 고추탄저병은 잎, 줄기, 과실 등에 발생하여 수량감소의 직접적인 원인으로 작용하며 해마다 발생정도에 차이를 나타내지만 온도가 높고 비가 많은 해에 발생이 심한 것으로 알려져 있다. 강수량이 많고 일조량이 낮았든 2011년에 경북 북부에서 평균 15%의 발병율을 나타내었고 반대로 강수량이 적었던 2001년에는 발병율이 0.3%로 극히 적었게 발병하였다. 일반적으로 농가에서는 고추탄저병 방제를 위해 저항성 품종재배, 이병 식물 제거 등 경종적 방법을 하거나 살균제에 의한 화학적 방제를 이용하고 있다. 최근들어 농작물의 안전에 대한 문제가 대두 되면서 미생물 농약 및 천연물 농액 등 생물학적 방제제를 이용한 방제에 대한 연구가 진행되어있어 병원균에 대한 길항성을 나타내는 미생물을 이용하는 방제가 관심을 모으고 있다. 길항미생물을 이용하는 생물학적 방제는 세포벽을 분해하는 용균작용 Bacillus속 (Kim and Kim, 1997; Woo et al.,2007), Penicillium속 (Imamura et al., 2000), Pseudomonas속 (Jung and Kim, 2004), Streptomyces속(Jeong et al ., 2004; Lee et al., 1990)등이 생산하는 항생물질에 의해 직접 식물병원균의 생육을 저해하는 항생작용, 식물병원균에 기생하면서 식물에 대한 진균의 병원성을 억제하는 기생작용 (Lee et al., 2004) 생육공간에서 영양분과 같은 생육에 필요한 인자를 경쟁함으로써 병원균의 생육 및 증식을 억제하는 경쟁적 길항작용(Jung et al., 2006; Neilands, 1984; Paulitz and Loper, 1991; Scher and Bake, 1982)-참고문헌 없음, 그리고 미생물이 생산하는 exopolysaccharide(EPS), lipopolisaccaride(LPS), salicylic acis(SA), hydrogen cyanide(HCN), 2,3-butanediol 등의 물질들에 의해서 식물의 면역기능을 활성하여 병에 대한 저항성을 유도하는 유도저항성 작용 (Lee, 1997; Liu et al., 1995; Ping and Boland 2004) 등을 들 수 있다. As described above, anthracnose of pepper occurs in leaves, stems, fruits and the like, and acts as a direct cause of decrease in yield. It shows difference in occurrence every year, but it is known to occur in a high temperature and a heavy rain. In 2011, when the amount of precipitation was high and the amount of sunshine was low, the average incidence rate was 15% in North Gyeongbuk Province. In contrast, when the amount of precipitation was low, the incidence rate was extremely low, 0.3%. Generally, farmers use a countermeasure such as cultivation of resistant cultivars, removal of diseased plants, or chemical control by bactericide to control pepper anthracnose. Recently, as a problem of the safety of crops has emerged, researches on the control using biological control agents such as microbial pesticides and natural material concentrates have been proceeding, and therefore, there has been an interest in using microorganisms that exhibit antagonism to pathogens. Biological control using antagonistic microorganisms is a bacterium that degrades cell walls. Bacillus genus (Kim and Kim, 1997; Woo et al ., 2007), Penicillium genus (Imamura et al ., 2000), Pseudomonas genus (Jung and Kim, 2004), Streptomyces genus (Jeong et al . , 2004; Lee et al., 1990), etc. The production of antibiotic directly to inhibit the growth of plant pathogens by the antibiotics which, parasitic and parasitic in plant pathogen inhibiting the virulence of the fungi on the plant operation (Lee et al ., 2004) Competitive antagonism that inhibits the growth and proliferation of pathogens by competing factors necessary for growth such as nutrients in the growth space (Jung et al ., 2006; Neilands, 1984; Paulitz and Loper, 1991; Scher and Bake, 1982) - No reference, and by plants such as exopolysaccharide (EPS), lipopolisaccaride (LPS), salicylic acis (SA), hydrogen cyanide (HCN) and 2,3-butanediol produced by microorganisms (Lee, 1997; Liu et < RTI ID = 0.0 > al ., 1995; Ping and Boland 2004).
생물농약(Biopesticide)에는 미생물을 이용하여 식물병, 해충 및 잡초를 방제하는 미생물농약과, 식물 혹은 미생물이 생산하는 천연물을 이용하는 생화학농약이 있다. 미생물농약의 경우, 제품의 최적화를 통해 대량생산 및 제제화가 어려우며, 제품의 보존 안정성이 낮아 화학농약과 같이 표준화, 규격화, 유통상의 용이성이 확보되지 않아 시장이 확대되지 않고 있다. 그리고, 미생물농약을 실제 포장에 사용하였을 경우, 처리한 후 미생물이 상당한 양으로 증식한 후에야 식물병을 방제 효과를 볼 수가 있어, 빠르게 식물병을 방제할 수 없는 문제점이 있다. 또한, 적용 병해의 스펙트럼이 폭 넓지 않아 작물에 발생하는 식물병원 종류에 따라 여러 생물농약을 처리해야 하므로 현실적으로 농민에게 경제적인 부담이 되고 있다.Biopesticides include microbial pesticides that use microorganisms to control plant diseases, insects and weeds, and biochemical pesticides that use natural products produced by plants or microorganisms. In the case of microbial pesticides, it is difficult to mass-produce and formulate the product through optimization of product, and since the storage stability of the product is low, standardization, standardization, and ease of distribution are not secured like chemical pesticides. When the microbial pesticide is used in actual packaging, the microorganism can not be effectively used for controlling the plant disease since the microorganism can propagate the microbial cells only after the microbial cell has grown to a considerable amount. In addition, since the spectrum of applied diseases is not wide, various bio-pesticides must be treated according to the types of plant hospitals generated in the crops, which realistically puts an economic burden on the farmers.
그러나 이와 같은 많은 연구 사례와 세계적인 노력에도 불구하고, 다양한 식물병원균을 효과적으로 방제할 수 있는 생물 농약은 아직 정착되지 않은 실정이어서 새로운 친환경적인 생물학적 방제제의 개발이 요구되고 있다.However, in spite of the many research examples and global efforts, the pesticides that can effectively control various plant pathogens have not yet been established and development of new environmentally friendly biological control agents is required.
이에, 본 발명자들은 고추 병원균에 뛰어난 길항력을 가지며, 과피에 안정적인 집락형성(colonization)이 가능한 균주를 분리하였으며, 동정한 결과 바실러스 속 ASC-31 (Bacillus sp. ASC-31)임을 밝혔고, 이를 이용한 미생물제제가 고추병해인 탄저병에 항진균 활성을 가짐을 밝혀 친환경 생물제제로의 가능성을 확인함으로 본 발명을 완성하였다.
Thus, the present inventors isolated strains capable of stably colonizing colonies of peanut pathogens and having excellent gastric motility, and as a result, identified Bacillus sp. ASC-31 ( Bacillus sp. sp. ASC-31), and that the microbial agent using the microbial agent has an antifungal activity against anthrax, which is a pathogen of the red pepper, confirming the possibility of an eco-friendly biological agent, thereby completing the present invention.
상기 목적을 수행하기 위하여, 본 발명은 고추탄저병 방제활성을 갖는 신규 바실러스 속 ASC-31 균주 (Bacillus sp. ASC-31) (기탁기관: 한국생명공학연구원, 기탁일: 2013년 2월 20일, 기탁번호: KCTC 12370BP)를 제공한다.In order to accomplish the above object, the present invention provides a novel Bacillus sp. ASC-31 strain ( Bacillus sp. ASC-31) (deposited by Korea Research Institute of Bioscience and Biotechnology, deposited on Feb. 20, 2013, Accession No .: KCTC 12370BP).
본 발명에 따른 신규 바실러스 속 ASC-31 균주는 대한민국 경북 청송, 영양, 또는 안동 지역 고추재배 지역으로부터 분리 및 동정한 바실러스 속의 신규한 균주임을 특징으로 한다. The novel strain of Bacillus sp. ASC-31 according to the present invention is a novel strain of Bacillus sp. Isolated and identified from Cheongsong, Nutrition, orchid cultivation area in Andong, Gyeongbuk, Korea.
본원에서 정의되는 고추탄저병의 원인균은 콜레토트리쿰 글로에오스포리오데스(Colletotrichum gloeosporioides), 콜레토트리쿰 아쿠타툼 (Colletotrichum acutatum), 콜레토트리쿰 코코데스 (Colletotrichum coccodes), 콜레토트리쿰 데마티움(Colletotrichum dematium,) 또는 글로메엘라 신굴라타 (Glomerella cingulata), 바람직하게는 콜레토트리쿰 글로에오스포리오데스(Colletotrichum gloeosporioides), 보다 바람직하게는, 콜레토트리쿰 글로에오스포리오데스(Colletotrichum gloeosporioides, KACC 40003)를 포함한다.The causative organism of pepper anthracnose, as defined herein, is Colletotrichum < RTI ID = 0.0 > gloeosporioides ), Collet sat tree glutamicum Aqua tatum (Colletotrichum acutatum), Collet sat tree glutamicum coco des (Colletotrichum coccodes), Collet sat tree glutamicum having Marty Titanium (Colletotrichum dematium,) or article Romero Ella singul rata (Glomerella cingulata), preferably Collet Sat Colletotrichum gloeosporioides , and more preferably, Colletotrichum gloeosporioides ( KACC 40003).
본 발명은 고추탄저병 방제활성을 갖는 신규 바실러스 속 ASC-31 균주를 산업적으로 생산하기 위한 순수 배양용 배지조성물을 제공한다.The present invention provides a culture medium for pure culture for industrially producing a novel strain of Bacillus sp. ASC-31 having pepper anthracnose control activity.
상기 배지조성물은 통상적인 LB 배지, 바람직하게는, NaCl 5 내지 15g/L, 트립톤(Tryptone) 2 내지 10g/L, 및 효모 추출물(Yeast extract) 5 내지 20g/L의 농도, 보다, 바람직하게는 NaCl 8 내지 12g/L, 트립톤(Tryptone) 3 내지 7g/L, 및 효모 추출물(Yeast extract) 8 내지 12g/L의 농도로 구성됨을 특징으로 한다.The above-mentioned culture medium composition preferably has a concentration of 5 to 20 g / L of a conventional LB medium, preferably 5 to 15 g / L of NaCl, 2 to 10 g / L of Tryptone and 5 to 20 g / L of yeast extract, Is characterized by a concentration of 8-12 g / L of NaCl, 3-7 g / L of Tryptone, and 8-12 g / L of yeast extract.
본 발명의 고추탄저병 방제활성을 갖는 신규 바실러스 속 ASC-31 균주 (Bacillus sp. ASC-31) 의 분리방법, 균학적 성질 및 용도는 하기와 같으며, 이하 상세하게 설명한다.The isolation method, mycological properties, and uses of the novel Bacillus sp. ASC-31 strain ( Bacillus sp. ASC-31) having the anthracnose control activity of the present invention are as follows and will be described in detail below.
본 발명의 고추탄저병 방제활성을 갖는 신규 바실러스 속 ASC-31 균주 (Bacillus sp. ASC-31) 는 고추재배지역 토양으로부터 분리하였으며 (상기 미생물을 ASC-31 균주 (Bacillus sp. ASC-31로 명명하고 한국생명공학연구원(KCTC)에 2013년 2월 20일자로 기탁하였다).Novel Bacillus ASC-31 strain having a red pepper anthracnose antifungal activity of the present invention (Bacillus sp. ASC-31) was isolated from the pepper cultivation area the soil (and named the microorganism in ASC-31 strains (Bacillus sp. ASC-31 Korea Biotechnology Research Institute (KCTC) on Feb. 20, 2013).
균주 특성은 다음과 같다.The characteristics of the strain are as follows.
NaCl 10g/L, 트립톤(Tryptone) 5g/L, 및 효모 추출물(Yeast extract) 10g/L을 함유한 LB 액체 배지에서 30℃, 48시간 진탕배양할 때 활성을 나타낸 균의 형태로서, 세포의 형상은 2.5~3.0 × 1.5㎛ 크기의 내생포자를 형성하는 그램 양성 간균으로서 동일한 조건의 배지에서 균의 형태로서 나타난 집락의 형태는 볼록한 원형의 우유빛 집락을 형성한다. 생리학적 성질로는 20 내지 60℃, 바람직하게는 30 내지 55℃ 이하의 생육 온도, pH 3.0 내지 11.0, 바람직하게는 4.5 내지 10의 생육 pH 조건에서 성장하며, 산소의 영향은 호기성이다.
The cells were cultured in an LB liquid medium containing 10 g / L of NaCl, 5 g / L of Tryptone and 10 g / L of yeast extract and cultured at 30 DEG C for 48 hours. The shape is a gram-positive bacterium that forms endogenous spores of 2.5 to 3.0 × 1.5 μm in size. The form of the colonies, which appeared as the form of bacteria in the medium of the same conditions, forms a convex circular milky colony. The physiological characteristics are growth at a growth temperature of 20 to 60 DEG C, preferably 30 to 55 DEG C, pH of 3.0 to 11.0, preferably 4.5 to 10, and the effect of oxygen is aerobic.
본 발명은 고추탄저병 방제활성을 갖는 신규 바실러스 속 ASC-31 균주 (Bacillus sp. ASC-31) 또는 상기 ASC-31 균주 배양액을 유효성분으로 포함하는 고추병해 방제용 생물 농약제제 및 이를 이용하는 것을 특징으로 하는 고추병해의 방제방법을 제공한다.
The present invention relates to a novel Bacillus sp. ASC-31 strain ( Bacillus sp. ASC-31) or an ASC-31 The present invention provides a method for controlling red pepper disease characterized by using a culture broth of a strain as an active ingredient and using the pesticide preparation for controlling red pepper disease.
본 발명의 일 구체예에 있어서, 본 발명은 신규 바실러스 속 ASC-31 균주 (Bacillus sp. ASC-31) 또는 상기 ASC-31 균주 배양액을 유효량 함유하고 농화학적으로 허용되는 담체를 포함하는 고추 탄저병에 대한 예방 및 치료용 조성물을 제공한다. In one embodiment of the present invention, the present invention provides a novel Bacillus sp. ASC-31 strain ( Bacillus sp. ASC-31) or ASC-31 A composition for prevention and treatment of anthrax anthracnose containing an effective amount of a strain culture broth and containing an agrochemically acceptable carrier is provided.
본 발명 조성물은 당업계에 공지된 방식의 미생물 농약제제화 방식에 따라, 예컨대 배양된 균체의 배양액, 동결건조 균체와 전분, 조단백질 및 암석분 등을 포함하도록 분말, 펠렛 또는 과립, 마이크로캡슐 등으로 제형화하여 그대로 사용할 수 있다. 상기 조성물은 표면 활성화제, 비활성 담체, 보존제, 습윤제, 공급 촉진제, 유인제, 캡슐화제, 결합제, 유화제, 염료, U.V. 보호제, 완충제 등 목적 병원균 및 작물에 따라 적용하기 용이한 다른 성분을 부가함으로 수득할 수 있다. 본 발명의 조성물은 적용하기 전에 적당한 양의 물 또는 다른 희석제로 희석을 하여 직접 적용하기에 적합한 형태 또는 농축액 또는 1차 조성물일 수있다. 살선충성 농도는 특히 농축 또는 직접 사용하거나 하는 것은 특정 제제의 성질, 적용 식물의 종류, 재배지역의 토양 및 기후 등에 따라 다양할 수 있음은 당업계에 명백히 이해될 것이다.The composition of the present invention may be formulated into powders, pellets or granules, microcapsules or the like to contain, for example, culture broth of the cultured cells, lyophilized cells and starch, crude protein and rock particles, etc. according to a microbial pesticide formulation method known in the art And can be used as it is. The composition may further comprise a surface active agent, an inert carrier, a preservative, a wetting agent, a feed promoter, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, Protecting agents, buffers, and other components that are easy to apply depending on the intended pathogens and crops. The compositions of the present invention may be in a form or concentrate or a primary composition suitable for direct application by dilution with an appropriate amount of water or other diluent prior to application. It will be clearly understood by those skilled in the art that the pesticidal concentration may vary depending on the nature of the particular formulation, the type of application plant, the soil and the climate in the cultivation area, and the like, particularly concentrated or direct use.
본 발명의 미생물 농약 조성물에 포함되는 미생물의 양에 관해서는, 본 발명의 효과를 손상하지 않는 한 특별히 제한은 없지만, 미생물, 및 무기염 등의 합계량 전체에 대하여, 5 내지 50중량%(건조 중량)로 하는 것이 바람직하고, 10 내지 30중량%로 하는 것이 보다 바람직하다. 또한, 본 발명의 미생물 농약 조성물에 포함되는 미생물의 농도는 그 콜로니형성 단위로서, 바람직하게는 1×10 5 내지 1×1015 cfu/g, 보다 바람직하게는 1×10 9 내지 1×1012 cfu/g로 할 수 있다. The amount of the microorganism contained in the microbial pesticide composition of the present invention is not particularly limited as long as it does not impair the effect of the present invention, but it is preferably 5 to 50% by weight (dry weight ), More preferably from 10 to 30% by weight. The concentration of the microorganism contained in the microbial pesticide composition of the present invention is preferably 1 × 10 5 to 1 × 10 15 cfu / g, more preferably 1 × 10 9 to 1 × 10 12 cfu / g.
본 발명에 이용하는 무기염로서는 본 발명의 효과를 방해하지 않는 한 특별히 제한은 없고, 예컨대, 탄산염, 황산염, 인산염 및 질산염 등을 들 수 있지만,탄산염 및 황산염이 바람직하고, 탄산염으로서는 탄산수소나트륨이, 또한 황산염으로서는 황산나트륨이 특히 바람직하다.The inorganic salt used in the present invention is not particularly limited so long as it does not interfere with the effect of the present invention. Examples of the inorganic salt include carbonate, sulfate, phosphate and nitrate, and carbonate and sulfate are preferable. As the carbonate, As the sulfate, sodium sulfate is particularly preferable.
본 발명의 미생물 농약 조성물에 포함되는 무기염의 양은 본 발명의 효과를 손상하지 않는 한 특별히 제한은 없지만, 미생물, 및 무기염 등의 합계량에 대하여 10 내지 50중량%로 하는 것이 바람직하고, 20 내지 45중량%로 하는 것이 보다 바람직하다. 이들의 범위로 무기염을 함유시키면 식물 병해에 대한 방제 효과, 미생물 농약 조성물의 수화성, 및 미생물 농약 조성물의 살포에 의해 생기는 작물 표면의 약반 저감의 관점에서 균형이 잘 맞는 미생물 농약 조성물이 얻어지기 때문이다.The amount of the inorganic salt contained in the microbial pesticide composition of the present invention is not particularly limited so long as the effect of the present invention is not impaired, but it is preferably 10 to 50% by weight, more preferably 20 to 45% by weight based on the total amount of microorganism, By weight. When an inorganic salt is contained in these ranges, a microbial pesticide composition with a well-balanced balance can be obtained from the viewpoint of the control effect against plant disease, the water-solubility of the microbial pesticide composition, and the reduction of the surface area of the crop caused by the application of the microbial pesticide composition Because.
또한, 본 발명의 미생물 농약 조성물은 계면활성제를 포함하고 있지 않더라도 좋지만 포함하는 것이 더욱 바람직하다. 계면활성제를 함유시키면, 미생물 농약 조성물을 희석하여 살포액을 조제한 경우에, 유효 성분인 미생물의 살포액 중에서의 분산성 및 현수성이 향상하기 때문이다. Further, the microbial pesticide composition of the present invention may contain no surfactant, but it is more preferable to include the surfactant. When the surfactant is contained, dispersibility and hygroscopicity of the microorganism as an effective ingredient in the spray solution are improved when the spray solution is prepared by diluting the microbial pesticide composition.
본 발명에 이용하는 계면활성제로서는, 본 발명의 효과를 방해하지 않는 한 특별히 제한은 없고, 음이온형, 양이온형 등, 어느 유형의 계면활성제이더라도 좋지만, 음이온형의 계면활성제가 바람직하다.The surfactant to be used in the present invention is not particularly limited as long as it does not interfere with the effect of the present invention, and may be any type of surfactant such as an anionic type or a cationic type, but an anionic type surfactant is preferable.
본 발명의 미생물 농약 조성물에 함유시키는 계면활성제의 양은 본 발명의 효과를 손상하지 않는 한 특별히 제한은 없지만, 미생물 농약 조성물 전량에 대하여 5 내지 30중량%로 하는 것이 바람직하고, 8 내지 20중량%로 하는 것이 보다 바람직하다. 이들의 범위로 계면활성제를 함유시킨 경우는, 미생물의 수화성·현수성이 향상하여, 미생물 농약 조성물의 보존성이 충분히 확보되며, 미생물 농약 조성물의 살포에 의한 식물에의 약해가 거의 생기지 않기 때문이다.The amount of the surfactant to be contained in the microbial pesticide composition of the present invention is not particularly limited as long as it does not impair the effect of the present invention, but is preferably 5 to 30% by weight, more preferably 8 to 20% . When the surfactant is contained in these ranges, the water-solubility and hygroscopicity of the microorganism are improved, the preservation of the microbial pesticide composition is sufficiently ensured, and the microbial pesticide composition scarcely causes damage to the plant by spraying .
또한, 본 발명의 미생물 농약 조성물은 상기의 물질 이외에, 예컨대 수용성의 물질 등 본 발명의 효과를 방해하지 않는 것이면, 그것을 함유하고 있더라도 좋다.In addition, the microbial pesticide composition of the present invention may contain, in addition to the above-mentioned substances, water-soluble substances such as water-soluble substances as long as they do not hinder the effects of the present invention.
본 발명의 미생물 농약 조성물의 제형으로서는 특별히 제한은 없고, 입상, 분상, 액상 등이라도 좋지만, 입상, 분상이 바람직하다. 그 경우의 평균 입경은, 1μm 내지 100 μm의 범위 내인 것이 바람직하고, 1μm 내지 50μm의 범위 내인 것이 보다 바람직하다. 평균 입경이 이러한 범위 내인 경우는, 미생물 농약 조성물의 취급이 용이하고, 또한, 1μm이하의 입경으로 한 경우는, 미생물의 포자 직경을 하회할 경우가 있기 때문에, 미생물이 사멸하여 식물 병해에 대한 방제 효과가 충분히 발휘되지 않는 경우가 있기 때문이다.The formulation of the microbial pesticide composition of the present invention is not particularly limited and may be granular, powder, liquid or the like, but it is preferably granular or powdery. In this case, the average particle diameter is preferably in the range of 1 to 100 mu m, more preferably in the range of 1 to 50 mu m. When the average particle size is within this range, it is easy to handle the microbial pesticide composition, and when the particle size is 1 μm or less, the spore diameter of the microorganism may be lowered. Therefore, microorganisms die, The effect may not be fully exhibited.
본 발명의 미생물 농약 조성물의 사용 방법에 관해서는 특별히 제한은 없지만, 예컨대 1000배, 2000배, 4000배 등, 농약등록시에 정해진 소정의 희석 배율이되도록 물로 희석하여 살포액을 제작하고, 그 살포액을 동력 분무기 등을 사용하여 식물 전체에 안개 형상으로 분무 처리하는 등으로 이용할 수 있다.The method of using the microbial pesticide composition of the present invention is not particularly limited, but it may be diluted with water to a predetermined dilution magnification determined at the time of pesticide registration such as 1000 times, 2000 times, 4000 times, For example, spraying the entire plant in the form of mist using a power sprayer or the like.
또한 본 발명의 미생물제제는 본 발명의 균주 또는 균주의 배양액 또는 배양물을 포함하는 다양한 제제의 형태, 즉 액제, 수화제, 분제, 입제, 유제 등으로 제조할 수 있다. 그 제조방법은 당업계의 일반적인 제조방법에 따른다.
In addition, the microorganism preparation of the present invention can be produced in the form of various preparations including the culture or culture of the strain or strain of the present invention, that is, a liquid preparation, a wetting agent, a powder, a granule, The production method thereof is according to a general production method in the art.
본 발명에 따른 신규 바실러스 속 ASC-31 균주는 고추탄저병의 원인균, 특히 콜레토트리쿰 코코데스와 콜레토트리쿰 글로에오스포리오이데스에 대한 항균활성이 우수하면서도 고추에 유해한 영향을 미치지 않고 과실의 생육을 촉진시킬 수 있다.The novel strain of Bacillus sp. ASC-31 according to the present invention has excellent antimicrobial activity against causative microorganisms of pepper anthracnose, particularly choletotrichum cucodez and choletotrichum gloeosporioides, It is possible to promote growth.
도 1은 SEM에 의한 ASC-31의 형태학적 특징을 나타낸 도이며;
도 2은 길항성 박테리아 균주 ASC-31의 시간경과에 따른 세포성장 저해활성을 나타낸 도이며;
도 3은 ASC-31의 길항 활성을 나타낸 도이며;
도 4은 ASC-31 균주 배양액의 희석배율에 따른 항진균 활성을 나타낸 도이다.1 is a diagram showing morphological characteristics of ASC-31 by SEM;
FIG. 2 is a graph showing the cell growth inhibitory activity of the antagonistic bacterial strain ASC-31 over time; FIG.
Figure 3 shows the antagonistic activity of ASC-31;
Fig. 4 shows antifungal activity according to the dilution ratio of the ASC-31 strain culture solution. Fig.
본 발명은 다음의 실시예에 의거하여 더욱 상세히 설명되나, 본 발명이 이에 의해 제한되지는 않는다.
The present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.
실시예 1. 길항균주의 분리 및 선발Example 1. Isolation and selection of antagonistic strains
다양한 고추재배 지역(경북 청송, 영양, 안동)으로부터 sampling한 토양 균원 시료로부터 10 g을 멸균수 90 ml에 넣어 30℃에서 24시간 진탕배양 후 LB (Luria Bertani) agar 배지(NaCl 10g/L, Tryptone 5g/L, Yeast extract 10g/L), Difco, 244620)에 단계희석법으로 도말하여 30℃에서 3일간 배양한 후 증식한 균주들 선별 분리하였다. 선별 분리된 균주들을 순수 분리한 후, 1차 스크리닝을 위해 고추탄저병을 방제할 수 있는 균주를 선발하기 위해 PDA(Potato Dextrose Agar) plate (Difco, 254920)에서 대치배양(pairing culture)을 하여, C. gloeosporioides 의 생육을 저해하는 균주들을 일차적으로 선발하였다. 고추 탄저병 병원 균주인 C. gloeosporioides는 농촌진흥청 국립농업과학원 농업유전자원센터(Korea Agricultural Culture Collection KACC)로부터 Colletotrichum gloeosporioides KACC 40003 분양 받아 사용하였다. Lactobacillus (Luria Bertani) agar medium (NaCl 10 g / L, Tryptone (10 g / L), Tryptone (10 g / L) 5g / L, Yeast extract 10g / L), Difco, 244620) by the stepwise dilution method and cultured at 30 ° C for 3 days. After the pure separation of screening the isolated strain, to a PDA (Potato Dextrose Agar) plate ( Difco, 254920) replacing the culture (pairing culture) in order to selecting a strain capable of controlling the red pepper anthracnose for primary screening, C The strains inhibiting the growth of gloeosporioides were first selected. C. gloeosporioides , an anthracnose pathogenic strain of pepper, was purchased from the Korean Agricultural Culture Collection (KACC) of the National Institute of Agricultural Science and Technology (RDA) under the name Colletotrichum gloeosporioides KACC 40003.
일차적으로 선별된 균주로부터 고추탄저병 저해 활성이 우수한 균주를 선발하기 위해 LB 액체 배지에 선발 균주를 접종한 뒤 48시간, 30℃에 진탕해양한 후, 배양액을 8,000 rpm, 15분간 원심분리하여, 배양 상등액을 이용하여 문헌에 기재된 페이퍼 디스크 확산법(paper disc diffusion) 방법으로 항진균 활성을 보이는 균주 중 활성이 우수한 균주를 최종적으로 분리하여 본 연구에 사용하였다.
In order to select strains with excellent antioxidative activity against pepper anchovy, the selected strains were inoculated into LB broth and shaken at 30 ° C for 48 hours. The culture broth was centrifuged at 8,000 rpm for 15 minutes, Using the supernatant, the paper disk diffusion method described in the literature was used to isolate the strain with the highest activity among the strains showing antifungal activity.
고추탄저병에 대해 강한 항진균활성을 갖는 길항세균을 분리 및 선발을 위해 총 100여점의 토양시료로부터 균주를 분리하였고, 분리 균주 중 항진균 활성을 조사하여 탄저병에 대하여 높은 항진균 활성이 가장 높게 나타난 #31을 최종 선발하여 ASC-31로 명명 후 실험에 사용하였다.
In order to isolate and select antagonistic bacterium having strong antifungal activity against pepper anthracnose, isolates were isolated from 100 total soil samples. The antifungal activities of isolates were investigated and the highest antifungal activity against anthracnose The final selection was named ASC-31 and used for the experiment.
실험예 1. 길항세균 배양학적 특성 및 생장 특성Experimental Example 1. Cultural characteristics and growth characteristics of antagonistic bacteria
최종 선발된 길항균의 배양시간에 따른 생장 및 항진균 활성물질의 생산특성을 조사하기 위하여 NaCl 10g, Tryptone 5g, Yeast extract 10g 에 균주를 접종하고 30℃, 120 rpm으로 진탕배양 하여 생육활성은 550nm에서 흡광도를 측정하여 균주의 배양학적 특성을 조사하였다.
In order to investigate the growth and antifungal activity of the selected antagonistic bacteria, 10 g of NaCl, 5 g of Tryptone and 10 g of yeast extract were inoculated with the strain and incubated at 30 ° C. and 120 rpm with shaking at a growth rate of 550 nm And the culture characteristics of the strains were examined.
최종 선발된 항진균 활성을 나타내는 균주의 정확한 분류학적 동정을 위하여 주사전자현미경 검경을 통해 형태학적인 특성을 조사하였다.
The morphological characteristics of the strains were investigated by scanning electron microscopy (SEM).
최종 선발된 길항세균의 동정을 위하여 분리 균주인 ASC-13을 LB 배지를 이용하여 30℃에서 순수 배양한 후 생화학적 특성을 조사한 결과, 내생포자를 형성하는 그램 양성(gram positive)으로 세포크기는 2.5~3.0 × 1.5㎛ 으로 짧은 간균으로(도 1), 최종적으로 Bacillus sp. ASC-31로 명명하였다.
In order to identify the finally selected antagonistic bacteria, ASC-13 was cultivated in pure medium at 30 ° C using LB medium, and its biochemical characteristics were examined. As a result, gram-positive 2.5 to 3.0 x 1.5 占 퐉 in short bacilli (Fig. 1), and finally Bacillus sp. ASC-31.
실험예 2. 길항세균의 항진균성 활성물질의 생산Experimental Example 2. Production of antifungal active substance of antagonistic bacteria
길항균이 생산하는 항진균성 활성물질에 의한 고추 탄저병균 생육의 길항정도를 측정하기 위해 먼저 길항세균을 NaCl 10g, Tryptone 5g, Yeast extract 10g 에 30℃에서 2일간 전 배양한 후 8,000 rpm에서 15분간 원심분리한 후, paper disc diffusion method를 이용하여 C. gloeosporioides 가 접종되어진 PDA plate에 길항세균 배양 상등액 100 ul를 점적한 paper disc(Ø8mm)를 올려, 28℃에서 5일간 배양하여 항진균 활성을 조사하였다.
In order to measure the antagonistic effect of antifungal agents produced by antagonistic antifungal agents, antagonistic bacteria were preincubated in 10 g of NaCl, 5 g of Tryptone, and 10 g of yeast extract for 2 days at 30 ° C., centrifuged at 8,000 rpm for 15 minutes After separating, paper disc (Ø8mm) was placed on a PDA plate inoculated with C. gloeosporioides in 100 μl of antagonistic bacterial culture supernatant and cultured at 28 ° C for 5 days.
본 실험 결과, ASC-31의 최적 pH와 최적 온도 배양시간에 따른 생장 및 항진균 활성물질의 생산특성을 조사한 결과, 도 2와 같이 배양 24시간에 가장 높은 O.D. 2.65로 나타났으며, 최대 항진균활성은 배양시간 36시간에 14 mm로 나타났다. 이러한 결과로 보아 ASC-31은 대수성장기를 지나 생산되는 2차 대사산물에 의해 항진균 활성을 나타냄을 추정할 수 있었다.(도 3 내지 5)
As a result, the highest OD of 2.65 was obtained at 24 hours of incubation as shown in Fig. 2, and the maximum antifungal activity The incubation time was 14 mm at 36 hours. As a result, ASC-31 could be estimated to exhibit antifungal activity by the secondary metabolites produced through the logarithmic growth phase (Figures 3 to 5).
실험예 3. 길항세균의 탄저병포자 발아 억제Experimental Example 3. Inhibition of anthrax spore germination of antagonistic bacteria
선발된 길항균의 포자발아 억제율을 조사하기 위하여 탄저병 병원균의 포자현탁액(1×106cfu/㎖)과 5종의 길항미생물 1×106～107cfu/㎖ 정도로 배양된 배양액을 ×50 농도에서 병원균에 대한 포자발아 억제율을 조사하였다.Spore suspension of the anthracnose pathogen to investigate the spore germination inhibition of the starter gilhanggyun (1 × 10 6 cfu / ㎖ ) and five kinds of antagonistic micro-organisms 1 × 10 6 ~ 10 7 cfu / ㎖ so the cultured broth in the × 50 concentration of The inhibition rate of spore germination on pathogen was examined.
병원균 포자현탁액과 길항미생물 배양액을 농도별로 섞어서 24시간 동안 배양한 다음에 현미경(Nikon, ECLIPSE E200)으로 병원균의 포자발아 여부를 관찰하였으며, 현미경 하에서 한 시료 당 3반복 관찰하였다. 발아관이 포자 두께 이상으로 자라 나왔거나 포자의 정상모양인 장타원형에서 변형된 것만을 발아한 것으로 간주하였다.The pathogen spore suspension and the antagonistic microorganism culture were mixed for 24 hours, and then germination of the pathogen was observed with a microscope (Nikon, ECLIPSE E200) and observed three times per microscope under a microscope. Germination was considered to be germinated only when the germ tube was grown above the spore thickness or deformed from the long elliptical shape of the spore.
병원균 포자현탁액을 길항미생물 배양액과 농도별 섞어 24시간 배양결과, 표 1에서와 같이 72.7%의 포자 발아 억제율이 조사 되었다. 이러한 결과로 보아 고추재배지에 ASC-31 배양액을 살포하였을 경우에 고추 탄저병의 포자를 억제하여 생물학적 방제가 가능할 것으로 사료된다.As shown in Table 1, the inhibition rate of spore germination was 72.7% as a result of incubation of the pathogen spore suspension with the antagonistic microorganism culture solution for 24 hours. These results suggest that the application of ASC-31 culture medium to red pepper cultivars may inhibit the sporulation of red pepper anthracnose by biological control.
Antagonic bacteria strainsPhytopathogenic Fungus
Antagonic bacteria strains
본 발명의 바실러스 속 ASC-31 균주을 포함하는 방제 조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.
The preparation examples of the controlling composition comprising the Bacillus sp. ASC-31 strain of the present invention will now be described, but the present invention is not intended to be limited thereto but is only specifically described.
제제예Formulation example 1. 방제 조성물 1. Control composition
바실러스 속 ASC-31 균주 배양액은 콜로이달 밀러(colloidal miller)를 이용하여 균체를 분쇄한 후, 액상형태의 계면활성제인 폴리옥시에틸렌 노닐페닐 에테르(polyoxyethylene nonylphenyl ether : CAS No. 68412-54-4)를 배양액 전체 중량에 대하여 7중량% 첨가하고, 보조제로 비타민 C(ascobic acid)와 글리세롤(glycerol)을 배양액 전체 중량에 대하여 각각 0.2중량%, 1중량%를 첨가하는 방법으로 액상수화제 형태로 제제화 하였다
The culture broth of the Bacillus sp. ASC-31 strain was prepared by pulverizing the cells using a colloidal miller, and then adding polyoxyethylene nonylphenyl ether (CAS No. 68412-54-4) as a liquid surfactant, Was added in an amount of 7% by weight based on the total weight of the culture medium and added with 0.2% by weight and 1% by weight of vitamin C (ascorbic acid) and glycerol as auxiliary agents to the total weight of the culture liquid in the form of a liquid wettable powder
상기의 성분을 가지고 대한약전 산제제법에 준하여 제조한다.The above components are prepared according to the Korean Pharmacopoeia.
상기의 혼합물의 조성비는 비교적 사료조성물에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 사료조성물 제조방법에 따라 상기의 성분을 혼합한 다음, 대한약전 산제제법에 준하여 제조에 사용할 수 있다.Although the composition ratio of the above-mentioned mixture is relatively mixed with the components suitable for the feed composition, it is also possible to arbitrarily modify the blending ratio thereof. The above components are mixed according to a conventional method for preparing a feed composition, It can be used for manufacturing according to an acid production method.
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