KR20020086154A - Pseudomonas aeruginosa 17S and their mutants promoted plant growth and showed antagonistic effect on phytopathogens - Google Patents

Pseudomonas aeruginosa 17S and their mutants promoted plant growth and showed antagonistic effect on phytopathogens Download PDF

Info

Publication number
KR20020086154A
KR20020086154A KR1020010025922A KR20010025922A KR20020086154A KR 20020086154 A KR20020086154 A KR 20020086154A KR 1020010025922 A KR1020010025922 A KR 1020010025922A KR 20010025922 A KR20010025922 A KR 20010025922A KR 20020086154 A KR20020086154 A KR 20020086154A
Authority
KR
South Korea
Prior art keywords
pseudomonas aeruginosa
phytopathogens
growth
tomato
strain
Prior art date
Application number
KR1020010025922A
Other languages
Korean (ko)
Other versions
KR100419783B1 (en
Inventor
임재욱
이상우
서명훈
이수연
심상연
이용환
Original Assignee
경기도(농업기술원)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 경기도(농업기술원) filed Critical 경기도(농업기술원)
Priority to KR10-2001-0025922A priority Critical patent/KR100419783B1/en
Publication of KR20020086154A publication Critical patent/KR20020086154A/en
Application granted granted Critical
Publication of KR100419783B1 publication Critical patent/KR100419783B1/en

Links

Classifications

    • 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
    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas
    • C12R2001/385—Pseudomonas aeruginosa

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

PURPOSE: Provided is Pseudomonas aeruginosa 17s and its mutant promoted plant growth and showed antagonistic effect on phytopathogens, such as Pythium aphanidermatum, P. ultimum, Fusarium oxysporum and the like. CONSTITUTION: Pseudomonas aeruginosa 17s(KCTC 0978BP) is treated with Streptomycin, thus has Streptomycin resistance. It shows antagonistic effect on phytopathogens by increasing the production of salicylic acid, wherein the phytopathogens include Mycosphaerella melonis, Sclerotinia sclerotitorum, Rhizoctonia solani, Verticillium dahliae, Didymella bryoniae, Bipolaris coicis, Alternaria solani, Botrytis elliptica, Sclerotinia sclerotitorum, Sclerotinia sp., Botrytis cinerea, Phytophthora capsici, Magnaporthe grisea, Phytophthora infestans and the like.

Description

식물의 생장을 촉진시키며 식물병원균에 대하여 길항력을 갖는 슈도모나스 애루지노사 17Ｓ 및 그 돌연변이주{Pseudomonas aeruginosa 17S and their mutants promoted plant growth and showed antagonistic effect on phytopathogens}Pseudomonas aeruginosa 17S and their mutants promoted plant growth and showed antagonistic effect on phytopathogens} which promotes plant growth and antagonizes phytopathogens

본 발명은 식물병원균에 대한 길항력을 나타내고 식물생장촉진 효과가 있는 슈도모나스 애루지노사(Pseudomonas aeruginosa) 17S 균주에 관한 것이다. 보다 상세하게는 토마토 줄기썩음병균에 대해 길항력을 가지는 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S)에 관한 것으로써, 토마토 뿌리에서 토마토 줄기썩음병균에 대해 길항력을 나타내는 균주들을 분리한 후, 그 중 토마토 생육을 촉진하는 균주들을 다시 분리하여 그 중 17번째 분리된 슈도모나스 애루지노사(Pseudomonas aeruginosa)에 항생물질을 처리하여 항생물질에 대해 저항성을 나타내는 것을 특징으로 하는 변이주인 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa 17S)에 관한 것이다.The present invention shows a antagonistic activity against phytopathogens and Pseudomonas aeruginosa having a plant growth promoting effect (Pseudomonas aeruginosa) Relates to the 17S strain. More specifically tomatoes Pseudomonas aeruginosa 17S having antagonism against stem rot bacteriaPseudomonas aeruginosa17S), the strains showing antagonism against tomato stem rot bacterium from tomato roots were isolated, and among them, the strains promoting tomato growth were separated again and Pseudomonas aeruginosa (17th) was isolated.Pseudomonas aeruginosa) Pseudomonas aeruginosa 17S ()Pseudomonas aeruginosa 17S).

본 발명과 관련하여 식물 생장 촉진의 기작에 대한 연구결과를 살펴보면 다음과 같다. 식물생장촉진 미생물이 식물체의 병원균에 대한 전신성 저항성을 유도하는 리포다당류(lipopolysaccharide) 등의 엘리씨터(elicitor)를 생산함으로써 식물체의 병원균에 대한 저항성을 높이고 작물의 생장을 촉진한다(Leeman 등, 1995). 식물 병원균의 생장을 억제하는 유기산이나 항생물질을 생산한다(Carroll 등, 1995; Moon 등, 1996; Rosales 등, 1995). 그리고 사이드로포어(siderophore)를 생산, 분비함으로써 토양 내의 철 이온의 농도를 급감시켜 식물 병원균이 자라지 못하도록 한다(Buysens 등, 1996; Carroll 등, 1995; Seong 등, 1991; Seong 등, 1992). 위의 연구결과를 토대로 자명한 것은 식물생장촉진 미생물의 작용 기작 중 여러 부분이 식물 병원균의 생육 및 침입 억제와 관련되어 있으므로 식물 생장 촉진과 식물 병원균의 생물학적 방제는 밀접한 관계를 갖는다는 것이다.Looking at the results of research on the mechanism of plant growth promotion in relation to the present invention. Plant growth-promoting microorganisms increase plant resistance to pathogens and promote crop growth by producing elicitors such as lipopolysaccharides that induce systemic resistance to plant pathogens (Leeman et al., 1995). ). It produces organic acids and antibiotics that inhibit the growth of plant pathogens (Carroll et al., 1995; Moon et al., 1996; Rosales et al., 1995). By producing and secreting siderophores, the concentration of iron ions in the soil is drastically reduced to prevent plant pathogens from growing (Buysens et al., 1996; Carroll et al., 1995; Seong et al., 1991; Seong et al., 1992). Based on the above findings, it is clear that since many of the mechanisms of plant growth promoting microorganisms are involved in the growth and inhibition of invasion of plant pathogens, the promotion of plant growth and the biological control of plant pathogens are closely related.

본 발명의 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa 17S)와 관련한 문헌을 살펴보면 다음과 같다.Pseudomonas aeruginosa 17S of the present invention (Pseudomonas aeruginosa 17S) Looking at the literature as follows.

슈도모나스 애루지노사(Pseudomonas aeruginosa)는 여타의Pseudomonasspp.와 같이 토양에 서식하는 토양 세균이며 식물병의 생물학적 방제원으로서 보고되고 있다(Busens 등, 1995; Buysens 등, 1996; Kurmar 등, 1992; Moon 등, 1996; Seong 등, 1991; Seong 등, 1992). 특히 피티움 아파니더마튬(Pythium aphanidermatum), 피티움 울티움(P.ultimum), 푸사리움 옥시스포리움(Fusarium oxysporum) 등의 다양한 병원균에 대하여 모두 길항능을 보이는 균주에 대한 보고도 있으며 그 기작으로 사이드로포어(siderophore)와 항생 물질 생산이 거론되고 있다(Gamliel 등, 1993). 이러한 생물학적인 재배방법에 의해 작물의 병충해 피해발생을 방지하는 방제원의 처리는 수확량을 증가시키나, 현재 양액 재배에서 근권 미생물의 이용에 대한 연구는 미진한 편이다. 펄라이트와 피트모스를 7:3으로 섞은 배지에 토마토를정식하고 슈도모나스 플로리센스(Pseudomonas fluorescens)의 세균을 처리한 결과 10% 정도의 상품 과중이 증가하였다고 보고하였다(Gagne 등, 1993). 식물 생장촉진 근권 세균을 오이에 처리했을 때 푸사리움(Fusarium) 시들음병에 대하여 전신저항성을 유도시켰다(Liu 등, 1995). 또한 플러그 육묘시 광합성 세균과 세포융합에 의한 융합균주를 토마토와 오이에 처리하였을 때 식물체 생장이 촉진되었으나 슈도모나스(Pseudomonas) 균주는 생육을 억제하는 경향이 있었다(Cho 등, 1998).Pseudomonas aeruginosa , like other Pseudomonas spp., Is a soil bacterium that has been reported to be a biological control agent of plant diseases (Busens et al., 1995; Buysens et al., 1996; Kurmar et al., 1992; Moon; Et al., 1996; Seong et al., 1991; Seong et al., 1992). In particular, there are reports of strains showing antagonism against various pathogens such as Pythium aphanidermatum , P. ultimum , and Fusarium oxysporum . Siderophore and antibiotic production have been discussed (Gamliel et al., 1993). The treatment of the control agent to prevent the pest damage caused by the biological cultivation method increases the yield, but the study of the use of the rhizosphere microorganisms in nutrient cultivation is currently insufficient. Tomatoes were grown in a medium of 7: 3 mixed with pearlite and pitmoss and treated with Pseudomonas fluorescens , which reported a 10% increase in product weight (Gagne et al., 1993). Plant growth-promoting myobacteria were treated with cucumber to induce systemic resistance to Fusarium wilt disease (Liu et al., 1995). But also the plant growth promoting when processing the fusion strains by photosynthetic bacteria and cell fusion when the plug seedlings in the tomato and cucumber Pseudomonas (Pseudomonas) strains has been a tendency to inhibit the growth (Cho et al., 1998).

또한, 본 발명과 관련한 양액재배에서의 병발생 원인은 작업농기구, 신발의 흙, 장비를 매개로 하거나 그리고 역경 또는 사경 재배시 자갈이나 모래에 있는 병원균이 재배되는 작물에 전반되어 병이 발생하기도 한다. 또한 종자, 초파리 등의 곤충에 의해 병발생이 가능하다(Paulitz, 1997). 일단 병원균이 전염되면, 순환식 양액 재배의 경우 병의 전반 속도가 빨라 방제가 힘들며, 채소류 양액 재배의 경우 잔류 독성 등의 문제로 농약에 의한 방제가 곤란하여 길항균을 이용한 식물병 방제에 대한 연구를 필요로 하고 있다. 토마토 암면 양액재배시 푸사리움 옥시스포리움(Fusarium oxysporium)에 의한 청고병을 방제하기 위하여 길항성 트리초더마(Trichoderma)를 처리한 결과 발병도를 70% 감소시키는 등(Rattink, 1993), 생물학적 방제에 대한 다양한 연구가 진행되고 있으므로(Paulitz, 1997), 유용미생물을 근권에 안정적으로 정착시켜 병발생을 억제함과 동시에 작물의 수량을 증대시킬 목적으로 근권세균을 선발하여 이용할 수 있는 연구를 필요로 한다.In addition, the cause of the disease in the nutrient solution cultivation associated with the present invention may be caused by working farm equipment, shoes soil, equipment, and spread to crops in which pathogens in gravel or sand are grown during adversity or landscape cultivation. . Disease can also be caused by seeds and fruit flies (Paulitz, 1997). Once the pathogens are transmitted, it is difficult to control the circulating nutrient cultivation due to the rapid propagation of the disease, and in the case of vegetable nutrient cultivation, it is difficult to control the pesticides due to the problem of residual toxicity. I need it. Treating tomato rock wool nutrient solution to Fusarium oxysporium ( Fusarium oxysporium ) in order to control the antagonist Trichoderma (70%) reduced the incidence (Rattink, 1993). Since various studies are underway (Paulitz, 1997), studies are needed to select and use rhizosphere bacteria for the purpose of stably establishing useful microorganisms in the rhizosphere to suppress disease occurrence and increase the yield of crops.

이에 본 발명자들은 슈도모나스 애루지노사(Pseudomonas aeruginosa) 17S의 배양여액을 식물병원균인 토마토 시들음병균(Fusarium oxysporum f. sp. radis-lycopersici)의 포자발아의 62%를 억제시키고 인삼근부병(Nectria radicicola)의 포자발아의 97.4%를 억제시키며, 또한 가지 육묘시 배양현탁액을 처리하면 생체중과 건물율을 증가시켜 토마토 시들음병과 고추 역병을 100% 방제하는 것을 확인하였다.Therefore, the present inventors suppressed 62% of spore germination of Fusarium oxysporum f. Sp. Radis-lycopersici of Pseudomonas aeruginosa 17S culture filtrate and inhibited the growth of Ginseng Root Disease ( Nectria radicicola ). Inhibition of 97.4% of spore germination and treatment of eggplant seedlings increased the fresh weight and dry matter ratio, and it was confirmed that 100% control of tomato wilting disease and pepper blight was observed.

상기 본 발명의 문제점을 해결하기 위하여, 토마토 뿌리에서 토마토 줄기썩음병균에 대하여 길항력을 나타내는 균주를 일차 선발하였고, 토마토의 생육을 촉진하는 균주에 17번째 균을 분리하고, 상기 분리한 균주가 항생물질에 대해 저항성을 가지는 변이주임을 확인하였다.In order to solve the problems of the present invention, the first selected strains showing antagonism against tomato stem rot bacteria in the tomato root, isolate the 17th bacteria to promote the growth of tomatoes, the isolated strain is antibiotic It was confirmed that the strain is resistant to the material.

따라서, 본 발명은 식물병원균에 대한 길항력과 식물생장촉진 효과가 있는 슈도모나스 애루지노사(Pseudomonas aeruginosa) 17S를 제공하는데 특징이 있다.Therefore, the present invention is characterized in providing Pseudomonas aeruginosa 17S having an antagonistic activity against plant pathogens and promoting plant growth.

본 발명은 식물병원균에 대한 길항력을 나타내고 식물생장촉진 효과가 있는 슈도모나스 애루지노사(Pseudomonas aeruginosa) 17S 균주에 관한 것이다. 보다 상세하게는 토마토 줄기썩음병균에 대해 길항력을 가지는 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S)에 관한 것으로써, 보다 구체적으로는 토마토 뿌리에서 토마토 줄기썩음병균(Pseudomonas corrugata)에 대해 길항력이 있는 균주들을 분리한 후, 토마토의 생육을 촉진하는 균주를 다시 분리하여 그 중 가지의 생육을 촉진하는 아래 표 1의 특성을 지니는 슈도모나스 애루지노사(Pseudomonas aeruginosa) 17번 균주를 분리하였다.The present invention shows a antagonistic activity against phytopathogens and Pseudomonas aeruginosa having a plant growth promoting effect (Pseudomonas aeruginosa) Relates to the 17S strain. More specifically tomatoes Pseudomonas aeruginosa 17S having antagonism against stem rot bacteriaPseudomonas aeruginosa17S), more specifically tomato stem rot bacteria (Pseudomonas corrugataPseudomonas aeruginosa having the characteristics shown in Table 1 below to isolate the strain antagonistic against), and to isolate the strain that promotes the growth of tomatoes again to promote the growth of eggplant (Pseudomonas aeruginosa) Strain 17 was isolated.

슈도모나스 애루지노사(Pseudomonas aeruginosa) 17번 균주는 길항균 중에서 17번째 분리한 것으로, 이를 이용하여 항생물질인 스트랩토마이신(streptomycin) 200ppm에서 저항성을 나타내는 돌연변이주를 유기하였다. 따라서 이를 도모나스 애루지노사 17S로 명명하였다.Pseudomonas aeruginosa strain 17 was isolated from the antagonistic 17th, using the strain mutant strain resistant to 200ppm of the antibiotic streptomycin. Thus it was named Domonas Aruginosa 17S.

이러한 연구에서 식물병원균에 대한 길항력과 식물생장촉진 작용을 하는 슈도모나스 애루지노사(Pseudomonas aeruginosa17S) 균주, 그리고 상기 균주는 항생물질인 스트랩토마이신에 대해 저항성을 가지는 변이주임을 확인하였다.In this study, it was confirmed that Pseudomonas aeruginosa 17S strain, which acts as an antagonist against plant pathogens and promotes plant growth, and the strain was resistant to the antibiotic strapomycin.

이하 미생물 분리 및 동정, 정착성, 효과검정, 저항성 등을 실시예를 통하여 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 청구범위를 실시예에 내재된 기술적 사상에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, microorganism isolation and identification, fixation, effect test, resistance, and the like will be described in detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the claims of the present invention are not limited to the technical spirit inherent in the examples.

실시예 1. 길항세균 및 식물생장촉진세균의 분리와 동정Example 1 Isolation and Identification of Antagonistic Bacteria and Plant Growth Promoting Bacteria

토마토 근면에서 정착한 세균 중 토마토 줄기썩음병균에 대하여 길항력을 보이는 세균을 선발하기 위하여 토마토 뿌리 5g을 200㎖ 플라스크에 살균수 100㎖를 넣고 5분간 진탕하여 근권 토양을 제거한 후, 뿌리 5g을 살균수 200㎖를 넣고 마쇄하여 순차 희석한 후 킹스(King's) B배지에 1일간 배양하였다. 하루 후 24시간 150rpm으로 영양배지(nutrient broth)에서 배양한 줄기썩음병균(Pseudomonas corrugata)을 킹스(King's) B배지 표면에 분무하여 병원균과 콜로니가 형성된 세균이 같이 자라게 한 후 병원균의 생장을 억제한 세균 콜로니를 선발하였다. 선발된 38균주의 길항세균 중에 식물 생장촉진세균을 분리하고자 '하우스 모모타로' 토마토 종자를 파종하고, 본엽이 나기 전에 길항세균을 영양(nutrient) 배지에 접종한 후 100rpm, 25℃에서 48시간 배양한 후 5,000rpm으로 원심분리하였다. 10mM-인산 완충용액(10mM-phospate buffer)을 사용하여 배양액을 108cell/㎖로 희석하고 토마토묘의 배축을 절단 후 길항세균 배양 희석액에 2시간 침지한 다음 살균토양에 삽목하여 토마토 생체중을 조사하여 17번 균주를 선발하였다. 선발된 균주를 동정하기 위하여 생명공학연구소 유전자원센터 유전자은행에 동정을 위탁하여 세균의 형태 및 배양 특성, 생화학적 특성을 API20NE 키트(kit)로 조사한 결과 85%의 신뢰도로서 슈도모나스 애루지노사(Pseudomonas aeruginosa)로 동정할 수 있었다(표 1). 버기스 매뉴얼(Bergey's manual)에 기재된 표준균주와 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa117S)의 형질을 비교해 보면 모든 특징이 일치하였지만 젤라티나아제 생성과 탈질산능은 불일치하였다. 상기의 두 가지 특성은 모두 배양 조건에 따라서 민감하게 달라지는 부분으로서 동정의 신뢰도를 떨어뜨릴 만큼 큰 차이는 아니었다(표 1). 항생제에 대한 저항성을 조사해 본 결과 앰피실린에 대해서 저항성을 보였다.In order to select the bacteria showing antagonism against tomato stem rot bacteria among the bacteria settled in the tomato diligent, 5 g of tomato roots were put in 100 ml of sterilized water in a 200 ml flask and shaken for 5 minutes to remove root root soil. 200 ml of water was added, ground, and diluted sequentially, followed by incubation for 1 day in King's B medium. Stem rot bacteria grown on nutrient broth at 150 rpm for 24 hours after a dayPseudomonas corrugata) Was sprayed onto the surface of King's B medium so that pathogens and colony-forming bacteria grew together, and bacterial colonies that inhibited the growth of pathogens were selected. To isolate plant growth bacteria among 38 strains of selected strains, sowing the seed of 'Homo Momotaro' tomato, inoculating the antagonistic bacteria into the nutrient medium before incubating the leaves and incubating for 48 hours at 100rpm, 25 ℃ After centrifugation at 5,000rpm. 10 mM-phosphate buffer was used to8After diluting with cell / ml and cutting the embryo seedlings of tomato seedlings, they were immersed in the antagonist culture diluent for 2 hours, and then inserted into sterile soil to examine tomato live weight, and strain 17 was selected. In order to identify the selected strains, they were entrusted to the Genetic Resource Center Genetic Bank of the Biotechnology Research Institute. The bacterial morphology, culture characteristics, and biochemical characteristics were investigated with the API20NE kit. Pseudomonas aeruginosaPseudomonas aeruginosaI was able to identify () (table 1). Standard strain and Pseudomonas aeruginosa 17S described in the Burgy's manual (Pseudomonas aeruginosa117S) traits were consistent, but gelatinase production and denitrification were inconsistent. Both of these characteristics were sensitive enough to be changed depending on the culture conditions and were not large enough to reduce the reliability of identification (Table 1). Investigating resistance to antibiotics showed resistance to ampicillin.

실시예 2. 균주의 정착성 조사를 위한 마커 측정Example 2 Marker Determination for Investigation of Settleability of Strains

항생제 저항성 균주를 유기하기 전에, 각각의 항생제에 대하여 원래의 균주가 가지고 있는 저항성을 조사하기 위하여 앰피실린, 스트렙토마이신, 카나마이신, 네오마이신, 클로람페니콜, 테트라싸이클린, 리팜피신을 각각 50ppm씩 처리한 5㎖ 영양(nutrient) 배지에 첨가하고 각각의 균주를 접종한 다음 30℃, 150rpm에서 16시간 동안 배양하여 혼탁도를 흡광도 550㎚에서 측정하였다. 17번 균주로부터 항생제 내성 균주를 선발하기 위하여 두 가지 방법을 이용하였다. 첫번째, 각 균주를 5㎖ 영양(nutrient) 배지에서 30℃, 150rpm으로 16시간 동안 현탁 배양한 다음 이로부터 50㎕ 접종원을 취하여 동일한 조건에서 2∼4시간 동안 배양하며 550㎚에서의 흡광도가 0.3∼0.5가 되도록 하였다. 배양체 1.5㎖를 5,000rpm에서 5분간 원심분리하여 상층액 1.2㎖를 버린 후 여액에 세균 침전을 재현탁하고 이에 사면을 주어 항생제 농도 구배를 둔 스트렙토마이신, 카나마이신, 네오마이신, 테트라싸이클린, 리팜피신 50ppm을 첨가한 영양(nutrient) 한천 배지에 항생제 농도 구배 방향에 대하여 직각으로 접종하였다. 7일간 30℃, 암조건에서 배양하여 생장을 보이는 균주를 다시 해당 항생제 50ppm의 영양(nutrient) 한천 배지에 접종하여 항생제 내성을 안정화한 후 농도를 100ppm, 200ppm으로 조절한 한천 배지에 접종하여 항생제 내성이 강화된 균주를 분리하였다. 두번째, 상기와 동일한 방법으로 17번 균주의 550㎚에서의 흡광도가 0.3∼0.5인 접종원을 준비한 다음 스트렙토마이신, 카나마이신, 네오마이신, 테트라싸이클린, 리팜피신을 5ppm씩 첨가한 50㎖ 영양(nutrient) 배지에 500㎕를 접종하고 30℃, 150rpm에서 7일간 배양하여 혼탁도를 조사하였다. 혼탁이 관찰되면 이에서 다시 접종원 500㎕를 취하여 항생제 농도를 2배로 증가시킨 50㎖ 영양(nutrient) 배지에 접종하고 30℃, 150rpm에서 16시간 배양하여 생장 여부를 관찰하였다. 이 과정에서 항생제의 농도가 100ppm, 200ppm이 될 때까지 반복한 후 생장을 보이는 균주를 분리하였다. 두 가지 방법으로 분리된 균주를 항생제가 첨가되지 않은 영양(nutrient) 배지에서 4차례 계대 배양한 다음 다시 항생제200ppm을 첨가한 영양(nutrient) 배지에서 현탁배양하여 생장 여부를 확인하였다. 항생제 내성의 안정성을 확인한 후 생장이 확인된 균주만을 선발하여 마커로 이용하였다. 유용세균의 근권 내 정착성과 밀도를 조사하기 위하여 앰피실린 저항성의 특성은 확인하였으나 보다 정확한 자료를 얻기 위하여 다시 선발된 근권세균의 항생제 돌연변이주를 분리하였다. 17번 균주로부터 스트렙토마이신 200ppm에 저항성을 갖는 균주인 17S 균주를 분리할 수 있었으며, 항생제에 대해 저항성을 갖는 균주는 항생제를 처리하지 않은 배지에서 모두 4차례의 계대배양 후에도 항생제 저항성을 상실하지 않고 있었으며 또한 균주의 생장에 있어서도 원래의 균주와 차이가 없었다(표 2).In order to investigate the resistance of the original strain to each antibiotic, prior to the antibiotic resistance strains, 5 ml of 50 ml each of ampicillin, streptomycin, kanamycin, neomycin, chloramphenicol, tetracycline, and rifampicin were treated. nutrient) was added to each medium and inoculated with each strain and then incubated for 16 hours at 30 ℃, 150rpm to measure the turbidity at absorbance 550nm. Two methods were used to select antibiotic resistant strains from strain 17. First, each strain was incubated for 16 hours at 30 ° C. and 150 rpm in 5 ml nutrient medium, and then 50 µl inoculum was incubated for 2 to 4 hours under the same conditions, and the absorbance at 550 nm was 0.3 to 0.5. Centrifuge 1.5 ml of the culture for 5 minutes at 5,000 rpm, discard 1.2 ml of the supernatant, resuspend the bacterial precipitate in the filtrate, and give a slope to add 50 ppm of streptomycin, kanamycin, neomycin, tetracycline, and rifampicin with an antibiotic concentration gradient. One nutrient agar medium was inoculated at right angles to the antibiotic concentration gradient direction. After 7 days of incubation at 30 ° C. in a dark condition, the strains showing growth were inoculated again in the nutrient agar medium of 50 ppm of the corresponding antibiotic to stabilize the antibiotic resistance, and then inoculated in the agar medium adjusted to the concentration of 100ppm and 200ppm for antibiotic resistance. This enhanced strain was isolated. Second, prepare an inoculum having an absorbance of 0.3 to 0.5 at 550 nm of strain 17 in the same manner as above, and then, 500 ml of 50 ml nutrient medium containing 5 ppm of streptomycin, kanamycin, neomycin, tetracycline, and rifampicin. The inoculation was inoculated and cultured for 7 days at 30 ° C. and 150 rpm to check turbidity. When turbidity was observed, 500 µl of the inoculum was again taken and inoculated into a 50 ml nutrient medium which doubled the concentration of antibiotics, and cultured at 30 ° C. and 150 rpm for 16 hours to observe growth. In this process, the antibiotic concentration was repeated until the concentration of 100ppm, 200ppm was isolated strain showing growth. The strains isolated by the two methods were passaged four times in nutrient medium without antibiotics, and then suspended and cultured in nutrient medium containing 200 ppm of antibiotics. After confirming the stability of antibiotic resistance, only the strain whose growth was confirmed was selected and used as a marker. The characteristics of ampicillin resistance were determined to investigate the fixation and density of the useful bacteria, but the antibiotic mutant strains of the selected isolates were isolated to obtain more accurate data. The 17S strain, which is resistant to 200 ppm of streptomycin, was isolated from strain 17, and the strains resistant to antibiotics did not lose antibiotic resistance even after four passages in the medium without antibiotics. In addition, the growth of the strain was not different from the original strain (Table 2).

실시예 3. 식물병원균 균사생장억제 효과 검정Example 3. Phytopathogen mycelial growth inhibitory effect assay

참외덩굴마름병균(Mycosphaerella melonis), 가지균핵병균(Sclerotina sclerotiorum), 벼잎집무늬마름병균(Rhizoctonia solani), 가지시들음병균(Verticillium dahliae), 참외덩굴마름병균(Didymella bryoniae), 율무잎마름병균(Bipolaris coicis), 토마토겹무늬병균(Alternaria solani), 백합잎마름병균(Botrytis elliptica), 스토크 균핵병균(Sclerotinia screlotiorum), 상추균핵병균(Sclerotinia sp.), 상추잿빛곰팡이병균(Botrytis cinerea), 모잘록병균(Rhizoctonia solani), 고추역병균(Phytophthora capsici), 벼도열병균(Magnaporthe grisea), 토마토잎마름병균(Phytophthora infestans)에 대한 균사생장억제율을 조사하였다. 배지로는 PDA, YDA, V8한천배지를 사용하였으며 우선 병원균을 9㎝ 페트리디쉬(petridish) 가운데 접종하고 주변부에 슈도모나스애루지노사 17S(Pseudomonas aeruginosa17S)를 접종하여 균사생장 억제율[(대조구 균사길이-처리구 균사길이)/대조구 균사길이×100]을 조사하였다(표 3). 그 결과 배지에 따라서 또 균주 조합에 따라서 균사 생장의 억제 효과가 다르게 나타났다. 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S)의 균사생장억제효과를 보면 세 가지 배지에서 모두 15개의 식물병원균의 균사생장을 억제한 것으로 나타났으며 YDA배지에서도 다른 배지와 비교 시 높은 균사생장억제율을 보였다.Melon vine blight fungus (Mycosphaerella melonis), kind gyunhaekbyeong bacteria (Sclerotina sclerotiorum), rice sheath blight fungus (Rhizoctonia solani), kind of wilt fungi (Verticillium dahliae), a melon vine blight fungus (Didymella bryoniae), yulmu leaf blight fungus (Bipolaris coicis ), Alternaria solani , Botrytis elliptica , Sclerotinia screlotiorum , Sclerotinia sp. , Botrytis cinerea , Mozloctonia, Rhizoctonia Mycelial growth inhibition of solani , Phytophthora capsici , Magnaporthe grisea , and tomato leaf blight ( Phytophthora infestans ) were investigated. PDA, YDA, V8 agar medium was used as a medium. First, the pathogens were inoculated in 9 cm petridish, and Pseudomonas aeruginosa 17S was inoculated at the periphery to inhibit mycelial growth. Treatment mycelial length) / control mycelial length × 100] (Table 3). As a result, the inhibitory effect of mycelial growth was different depending on the medium and the strain combination. Inhibition of mycelial growth of Pseudomonas aeruginosa 17S by Pseudomonas aeruginosa 17S showed that it inhibited the mycelial growth of 15 phytopathogens in all three media and showed higher mycelial growth inhibition rate compared to other media in YDA medium. .

실시예 4. 육묘시 식물병 발생억제 효과 시험Example 4. Effect of inhibiting plant disease occurrence at seedling

슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S)를 액체영양배지(쇠고기추출물 3g, 글루코오스 2.5g, 펩톤 5g, 증류수 1ℓ)에서 28℃, 150rpm으로 72시간 진탕배양하여 사용하였다. 토마토(하우스모모타로)와 고추(여명)를 72공 플러그트레이에 서울농자재에서 생산한 과채류 비접목용 육묘용상토를 넣고 파종하여 육묘하였다. 본엽이 3매 나왔을 때, 증류수로 108cfu/㎖로 희석하여 관주하였다. 토마토 시들음병(F. oxysporumf. sp.lycopersici), 고추역병(Phytophthora capsici)은 호밀에 10% 자당(sucrose)을 첨가하고 2번에 걸쳐 121℃에서 살균 후 병원균에 접종하여 25℃에서 배양하였다. 사용 전 접종원 2g을 마쇄후 물 1ℓ에 혼합하여 슈도모나스 애루지노사(Pseudomonas aeruginosa25R, 17S)를 처리한 처리구를 다음날 관주하였다. 그 결과, 무처리구(Pseudomonas aeruginosa25R)에서는 토마토 시들음병이 53% 발생하였으나 처리구(Pseudomonas aeruginosa 17S)에서는 전혀 발생하지 않아서 방제가가 100%이었다. 고추 역병은 대조구에서 13% 발생되었으나 처리구에서는 병이 전혀 발생하지 않아 역시 가지역병에 대한 방제가도 100%이었다.Pseudomonas aeruginosa 17S ( Pseudomonas aeruginosa 17S) was used in a liquid nutrient medium (beef extract 3g, glucose 2.5g, peptone 5g, distilled water 1g) at 28 ℃, 150rpm for 72 hours. Tomatoes (House Momotaro) and red peppers (dying) were seeded and seeded in 72-hole plug trays with fruit seedlings for non-grafting seedlings produced by Seoul Agricultural Materials. When three main leaves came out, the mixture was diluted with 10 8 cfu / ml with distilled water and irrigated. Tomato wilting disease ( F. oxysporum f. Sp. Lycopersici ) and red pepper disease ( Phytophthora capsici ) were added to 10% sucrose in rye, sterilized twice at 121 ° C. and inoculated with pathogens and incubated at 25 ° C. After treatment, 2 g of inoculum was mixed with 1 L of water, and treated with Pseudomonas aeruginosa 25R and 17S. As a result, 53% of tomato wilted disease occurred in the non-treated group ( Pseudomonas aeruginosa 25R), but did not occur at all in the treated group ( Pseudomonas aeruginosa 17S ) was 100% control value. Red pepper plague occurred 13% in the control group, but no disease occurred in the control group.

실시예 5. 육묘시 생육촉진효과 검정 및 양액재배시 처리효과 규명Example 5 Growth Promotion Effect in Growth and Identification of Processing Effect in Nutrient Cultivation

17S 균주 액체영양배지에서 진탕배양 후 5,000rpm에서 원심분리하였고, 증류수 108cfu/㎖로 희석된 용액을 70공 플러그 트레이당 100㎖씩 피트모스에 혼입한 후에 '모모타로' 토마토와 '백다다기' 오이종자를 파종하였다. 생육조사는 파종 25일 후에 실시하였다. 또한 토마토 양액재배에서 17S 균주의 처리효과를 검정하기 위하여 17S 균주를 킹스(King's) B 액체배지를 이용하여 25℃에서 150rpm으로 36시간 배양 후 5,000rpm에서 원심분리하여 침전시켰으며, 증류수 108cfu/㎖로 희석한 후 사용하였다. 토마토 '모모타로T93' 품종(1998년 7월 11일)인 펄라이트와 피트모스를 7:3의 비로 혼합시 공시세균을 혼입하였으며, 상토는 직경 9㎝의 지피포트에 담은 후 종자를 파종하였다. 파종 후 야마자키 토마토전용액을 1/2 농도로 양액 육묘하였다. 육묘 후 본토에서는 펄라이트 단용배지에 미리 공시유용세균을 증류수 108cfu/㎖로 희석한 후에 처리하였다. 세균의 밀도조사는 정식전에 1회, 재배기간 중 1회, 재배 후 1회 조사하여 총 3회 조사하였다. 조사방법은 뿌리 1.5g을 취하여 100㎖의 살균수에 넣고 150rpm, 20분간 진탕한 후에 항생제를 첨가한 슈도모나스 아가(Pseudomonas agar, Difco사) 배지에 순차 희석한 시료를 도말한 후 42℃에서 배양하여 토마토 근면에 정착한 공시세균의 밀도를 조사하였다. 항생제로는 앰피실린 50㎍/㎖, 리팜피신 50㎍/㎖를 첨가하여 다른 미생물이 자라지 못하게 하였다.그 결과, 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S)는 펄라이트와 코코피트 혼합배지에서는 대조구와 비교시 큰 차이가 없었으나 원예용 상토에서는 고추와 가지의 생체중을 다소 증가시켰다. 또한 처리농도별로 살펴보면 펄라이트와 코코피트 혼합배지에서 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S) 108cfu/㎖로 처리하여 토마토 생육을 촉진시켰으며 원예용 상토에서는 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S) 106cfu/㎖로 처리하여 고추와 가지의 생체중을 증가시켰다.After shaking culture in 17S strain liquid nutrient medium, the solution was centrifuged at 5,000rpm, and the solution diluted with 10 8 cfu / ml of distilled water was mixed into 100 ml of pitmos per 70-hole plug tray, followed by 'Momotaro' tomato and 'White tea' cucumber. Seeds were sown. Growth survey was performed 25 days after sowing. In addition, in order to test the treatment effect of 17S strain in tomato cultivation culture, 17S strain was cultured at 150 rpm at 25 ° C for 36 hours using King's B liquid medium, and then precipitated by centrifugation at 5,000 rpm, and distilled water was 10 8 cfu. It was used after diluting to / ml. When mixed with pearlite and peat moss (Momotaro T93) varieties (July 11, 1998) at a ratio of 7: 3, the test bacteria were mixed, and the top soil was sown in a zippoport with a diameter of 9cm and sown seeds. After sowing, the Yamazaki tomato solution was grown in a half concentration. After seedling, mainland treated bacteria were diluted in distilled water with 10 8 cfu / ml beforehand in perlite medium. Bacterial density was investigated three times, once before planting, once during cultivation period, and once after cultivation. In the method of irradiation, 1.5 g of roots were taken in 100 ml of sterile water, shaken at 150 rpm for 20 minutes, and then serially diluted with Pseudomonas agar (Difco) medium containing antibiotics was incubated at 42 ° C. The density of test bacteria settled in tomato diligence was investigated. Antimicrobial agents were added 50 μg / ml of ampicillin and 50 μg / ml of rifampicin to prevent other microorganisms from growing. As a result, Pseudomonas aeruginosa 17S was compared with the control in the pearlite and cocoite mixture medium. There was no significant difference, but in horticultural soils, the fresh weight of red pepper and eggplant was slightly increased. In addition, by treatment concentration, Pseudomonas aeruginosa 17S ( Pseudomonas aeruginosa 17S) was treated with 10 8 cfu / ml in the mixed culture of pearlite and cocoite to promote tomato growth, and Pseudomonas aeruginosa 17S in horticultural soils. Treatment with 10 6 cfu / ml increased the fresh weight of red pepper and eggplant.

실시예 6. 유용미생물 처리에 의한 토마토 유도저항성 조사Example 6 Induction Resistance of Tomato by Useful Microorganism Treatment

시험작물로서는 모모타로T93과 서광을 실시예 1과 동일방법으로 육묘하여 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S)를 같은 방법으로 처리하였다. 분석방법은 라스킨(Laskin)(1989) 등이 이용한 방법을 변형하여 사용하였다. 처리 후 본엽 5매 정도 전개되면 잎을 취하여 살리실릭산(salicylic acid)을 분석하였다. 우선 시료 1g을 막자사발로 액체질소를 이용하여 잘 분쇄한 다음 90% 메탄올 10㎖를 넣고 다시 추출하였다. 추출 후 18,000g으로 15분간 원심분리한 다음 피펫으로 상등액을 취하였다. 침전물을 100% 메탄올 5㎖로 다시 추출후 원심분리하여 두 상층액을 모아 18,000rpm에서 원심분리하였다. 이 상층액을 40℃에서 감압 농축하고 농축잔재물을 5%(wt/vol) 트리클로로아세틱산(trichloroacetic acid) 5㎖로 용해시켰다. 10㎖의 에틸아세테이트-싸이클로펜탄-이소프로판올(ethylacetate-cyclopantane-isopropanol)(50:50:10)의 유기용매로 분액여과기에서 추출하여 상층액을 질소가스로 농축한 후 0.5㎖의 메탄올로 다시 용해시켜 시료로 사용하였다. 유동상으로 메탄올을 사용하였고, 유속은 0.6㎖/min이었고, 컬럼(colum) C18컬럼(Lichrosorb RP-18 10micron, 25㎝)을 사용하였고 스펙트로 플로르슨스 디텍터를 이용하여 검출하였다. 들뜸(excitation) 파장은 314㎚이었고 방사(emission)파장은 406㎚이었다.As a test crop, Momotaro T93 and fluorescence were seeded in the same manner as in Example 1, and Pseudomonas aeruginosa 17S was treated in the same manner. The analysis method was modified by the method used by Laskin (1989). After treatment, about 5 sheets of leaf were developed, and the leaves were taken and analyzed for salicylic acid. First, 1 g of the sample was pulverized well using liquid nitrogen with a mortar, and then extracted with 10 ml of 90% methanol. After extraction, the mixture was centrifuged at 18,000 g for 15 minutes, and the supernatant was collected by a pipette. The precipitate was extracted again with 5 ml of 100% methanol and centrifuged to collect the two supernatants and centrifuged at 18,000 rpm. The supernatant was concentrated under reduced pressure at 40 ° C., and the concentrated residue was dissolved in 5 ml of 5% (wt / vol) trichloroacetic acid. 10 ml of ethylacetate-cyclopantane-isopropanol (50:50:10) was extracted with a separatory filter using an organic solvent. The supernatant was concentrated with nitrogen gas and dissolved again with 0.5 ml of methanol. Used as a sample. Methanol was used as the fluidized bed, the flow rate was 0.6 ml / min, a column C 18 column (Lichrosorb RP-18 10 micron, 25 cm) was used and detected using a Spectro Florson's detector. The excitation wavelength was 314 nm and the emission wavelength was 406 nm.

상기 실시예 6은 전신획득저항성과 관련한 실험으로 이는 식물병원균이 침입하였을 때 나타나는 식물의 방어적 반응으로 이러한 반응은 생물적 또는 화학적 유기 인자에 의해 일어나는데 화학적 유기 인자는 살리실릭산(salicylic acid : SA) 등이 있다(White 등 1994). 이 SA는 병든 조직에 축적이 많이 되어 있으며, 이러한 SA의 농도는 전신획득 저항성 발현과 관련된 단백질(SAR marker protein)과 관련이 높은 것으로 보고되었다(Yalpani 등, 1991).Example 6 is an experiment related to systemic acquisition resistance, which is a defensive reaction of plants caused by the invasion of phytopathogens. The reaction is caused by biological or chemical organic factors, and the chemical organic factors are salicylic acid (SA). (White et al. 1994). These SAs are highly accumulated in diseased tissues, and the concentrations of these SAs have been reported to be associated with SAR marker proteins (Yalpani et al., 1991).

따라서 본 발명에서는 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S) 처리구와 무처리구(슈도모나스 애루지노사 25R 처리구)로 토마토에서 SA의 농도 차이를 실험하였다. 실험결과 모모타로 T93에서 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S) 처리구와 무처리구인 슈도모나스 애루지노사 25R(Pseudomonas aeruginosa25R)처리구와 비교시 SA농도는 무처리구(Pseudomonas aeruginosa25R 처리구)보다 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S) 처리구에서 SA농도가 높게 나타났다(표 5).Therefore, in the present invention, the concentration difference of SA in tomatoes was treated with Pseudomonas aeruginosa 17S ( Pseudomonas aeruginosa 17S) treatment and untreated ( Pseudomonas aeruginosa 25R treatment). Experimental results Pseudomonas Ke in Momotaro T93 Rouge labor 17S (Pseudomonas aeruginosa 17S) treated and untreated in Pseudomonas Ke Rouge labor 25R (Pseudomonas aeruginosa 25R) treatment as compared to SA concentration is untreated Pseudomonas Ke Rouge labor 17S than (Pseudomonas aeruginosa 25R treatment) ( Pseudomonas aeruginosa 17S) treatment showed a high SA concentration (Table 5).

이와 같은 실험결과로 볼 때 슈도모나스 애루지노사 17S(Pseudomonasaeruginosa17S)처리는 엽내 SA함량을 증가시켜 토마토가 식물병원균에 대한 저항성을 가지게 함을 확인하였다.Pseudomonas aeruginosa 17S ( Pseudomonas aeruginosa 17S) treatment was found to increase the SA content in the leaves to make tomatoes resistant to phytopathogens.

그리고, 상기 분리된 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S)가 신규한 미생물로 인정되어, 반복재현성을 입증하기 위해 미생물기탁기관(KCTC: KOREAN COLLECTION FOR TYPE CULTURES)에 기탁일(2001.3.30)자로 기탁하였다.(기탁번호: KCTC 0978BP)In addition, the separated Pseudomonas aeruginosa 17S ( Pseudomonas aeruginosa 17S) is recognized as a novel microorganism, and as a deposit date (2001.3.30) to a microorganism deposit institution (KCTC: KOREC COLLECTION FOR TYPE CULTURES) to prove repeatability. Deposited (KCTC 0978BP)

이상에서 상세히 설명하였듯이, 본 발명은 토마토 줄기썩음병균에 대해 길항력을 가지는 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S)를 제공한다. 본 발명의 슈도모나스 애루지노사 17S(Pseudomonas aeruginosa17S)를 육묘기에 처리시 토마토 시들음병과 고추역병의 병발생을 억제시키며 가지와 고추의 생체중을 증가시킨다. 특히 토마토의 엽내 살리실릭산(salicylic acid)의 함량을 증가시켜 식물병원균에 대한 전신저항성과 식물생장촉진을 증가시킨다.As described in detail above, the present invention provides Pseudomonas aeruginosa 17S having an antagonism against tomato stem rot bacteria. Pseudomonas aeruginosa 17S ( Pseudomonas aeruginosa 17S) of the present invention when the seedling treatment inhibits the disease of tomato wilting and pepper blight and increases the live weight of eggplant and pepper. In particular, increasing the content of salicylic acid in the leaves of tomatoes increases the systemic resistance to plant pathogens and promotes plant growth.

분리균주와 표준균주의 형태 및 배양 특성표Form and Culture Characteristics of Isolated and Standard Strains 배양특성Culture characteristics 분리균주(17S)Separated strain (17S) 표준균주x Standard strain x 옥시다아제Oxidase 양성positivity 양성positivity 카탈라아제Catalase 양성positivity 양성positivity 베타-갈락토시다아제Beta-galactosidase 음성voice 음성voice 베타-글루코시다아제Beta-glucosidase 음성voice 음성voice 아르기닌 디하이드로라아제Arginine Dehydrolase 양성positivity 양성positivity 트립토판 디아미나아제Tryptophan deaminase 음성voice 음성voice 라이신 디카르복실라아제Lysine decarboxylase 음성voice NDND 오르니틴 디카르복실라아제Ornithine decarboxylase 음성voice NDND 씨트릭산 사용Use of Citric Acid 양성positivity NDND 황화수소 생성Hydrogen sulfide generation 음성voice 음성voice 유레아제 생성Urease generation 음성voice NDND 젤라티나아제 생성Gelatinase production 음성voice 양성positivity 인돌 생성Indole generation 음성voice NDND VP반응VP reaction 음성voice NDND 질산 환원능Nitrate reducing ability 양성positivity 양성positivity 탈질산능Denitrification 음성voice 양성positivity 42℃ 생장42 ℃ growth 양성positivity 양성positivity 리파아제 생성(트윈 80 가수분해)Lipase Production (Twin 80 Hydrolysis) 양성positivity NDND 글루코오스,멜리보오스의 산화적 발효Oxidative Fermentation of Glucose and Melibose 양성positivity 양성positivity 아라비노오스의 산화적 발효Oxidative Fermentation of Arabinoses 음성voice NDND 만니톨, 이노시톨, 솔비톨, 람노오스,슈크로오스, 아미그달린, 아라비노오스 발효Mannitol, inositol, sorbitol, rhamnose, sucrose, amigdaline, arabinose fermentation 음성voice NDND 글루코오스, 아라비노오스, 만노오스,글루코네이트, 카프릭산, 말레이트, 만니톨,아세틸화글루코오스아민 집적Glucose, arabinose, mannose, gluconate, capric acid, malate, mannitol, acetylated glucoseamine accumulation 양성positivity 양성positivity 말토오스, 아디페이트, 페닐아세테이트 집적Maltose, Adipate, Phenyl Acetate Accumulation 음성voice 음성voice *ND는 버기스 매뉴얼(Bergey's manual)에 설명되어 있지 않은 형질* ND is a characteristic not described in the Burgy's manual.

토마토, 가지, 고추 육묘시 슈도모나스 애루지노사 17S 처리효과Pseudomonas aeruginosa 17S treatment effect for tomato, eggplant, and pepper seedlings 육묘용토Seedling soil 작 물Water 처 리(cfu/㎖)Treatment (cfu / ml) 초장(㎝)Extra long (cm) 경경(㎜)Diameter (mm) 생체중(g)Live weight (g) 건물율(%)% Building 균밀도(cfu/g)Uniform Density (cfu / g) 펄라이트:코코피트(7:3)Pearlite: coco pit (7: 3) 토마토tomato 108 10 8 34.9 b*34.9 b * 4.4 a4.4 a 32.5 b32.5 b 6.5 a6.5 a 4.5×105 4.5 × 10 5 106 10 6 36.5 b36.5 b 4.5 a4.5 a 27.1 c27.1 c 5.7 b5.7 b 5.0×105 5.0 × 10 5 104 10 4 32.9 b32.9 b 4.6 a4.6 a 27.4 c27.4 c 6.0 ab6.0 ab 7.5×104 7.5 × 10 4 대조구Control 40.9 a40.9 a 4.9 a4.9 a 38.2 a38.2 a 6.1 ab6.1 ab -- 가지Branch 108 10 8 21.1 a21.1 a 3.0 a3.0 a 20.2 a20.2 a 7.3 a7.3 a 5.0×106 5.0 × 10 6 106 10 6 19.6 a19.6 a 2.9 b2.9 b 16.6 b16.6 b 6.7 a6.7 a 1.3×105 1.3 × 10 5 104 10 4 19.9 a19.9 a 3.0 ab3.0 ab 20.8 a20.8 a 7.2 a7.2 a 2.5×105 2.5 × 10 5 대조구Control 21.1 a21.1 a 3.0 ab3.0 ab 16.1 b16.1 b 5.5 b5.5 b -- 고추pepper 108 10 8 18.5 b18.5 b 2.7 b2.7 b 8.1 b8.1 b 7.2 a7.2 a 1.8×106 1.8 × 10 6 106 10 6 18.9 a18.9 a 2.6 b2.6 b 8.1 b8.1 b 7.4 a7.4 a 6.0×105 6.0 × 10 5 104 10 4 21.2 a21.2 a 3.0 a3.0 a 11.9 a11.9 a 7.6 a7.6 a 6.0×105 6.0 × 10 5 대조구Control 21.3 a21.3 a 2.9 ab2.9 ab 10.6 a10.6 a 6.7 b6.7 b -- 원예상토Horticulture 토마토tomato 108 10 8 45.5 a45.5 a 4.5 a4.5 a 36.1 bc36.1 bc 10.1 a10.1 a 3.3×106 3.3 × 10 6 106 10 6 46.3 a46.3 a 4.7 a4.7 a 38.7 ab38.7 ab 9.8 a9.8 a 5.8×106 5.8 × 10 6 104 10 4 41.0 a41.0 a 4.7 a4.7 a 32.5 c32.5 c 9.4 a9.4 a 8.8×105 8.8 × 10 5 대조구Control 46.1 a46.1 a 4.7 a4.7 a 42.8 a42.8 a 10.0 a10.0 a -- 가지Branch 108 10 8 17.5 a17.5 a 2.8 ab2.8 ab 13.1 a13.1 a 10.4 a10.4 a 3.0×105 3.0 × 10 5 106 10 6 18.5 a18.5 a 2.8 ab2.8 ab 13.3 a13.3 a 10.4 a10.4 a 1.5×105 1.5 × 10 5 104 10 4 14.1 b14.1 b 2.7 b2.7 b 9.1 b9.1 b 10.9 a10.9 a 1.3×104 1.3 × 10 4 대조구Control 18.1 a18.1 a 3.0 a3.0 a 11.9 a11.9 a 10.1 a10.1 a -- 고추pepper 108 10 8 26.8 ab26.8 ab 3.2 a3.2 a 12.5 b12.5 b 10.1 a10.1 a 5.8×105 5.8 × 10 5 106 10 6 28.5 a28.5 a 3.4 a3.4 a 15.3 a15.3 a 9.9 a9.9 a 1.3×106 1.3 × 10 6 104 10 4 25.6 b25.6 b 3.3 a3.3 a 13.1 b13.1 b 7.6 a7.6 a -- 대조구Control 26.4 ab26.4 ab 3.2 a3.2 a 12.0 b12.0 b 8.9 a8.9 a -- * DMRT at 5% level* DMRT at 5% level

슈도모나스 애루지노사 17S의 식물병원균에 대한 균사생장억제율Mycelial Growth Inhibition of Plant Pathogens of Pseudomonas aeruginosa 17S 병원균명Pathogen name 균사억제율(%)Mycelial inhibition rate (%) PDAPDA YDAYDA V8V8 참외덩굴마름병(Mycosphaerella melonis)Melon Vine Blight ( Mycosphaerella melonis ) 4040 4242 3333 가지균핵병(Sclerotina sclerotiorum) Sclerotina sclerotiorum 2727 4040 2424 벼잎집무늬마름병(Rhizoctonia solani) Rhizoctonia solani 3636 3434 1414 가지시들음병(Verticillium) Verticillium 3939 4444 1212 참외덩굴마름병(Didymella bryoniae)Melon vine blight ( Didymella bryoniae ) 3030 3636 2222 율무잎마름병(Bipolaris coicis)Yule Radish ( Bipolaris coicis ) 4848 4141 3535 토마토겹무늬병(Alternaria solani)Tomato leaf disease ( Alternaria solani ) 3838 3939 2727 백합잎마름병(Botrytis elliptica)Lily of the Leaves ( Botrytis elliptica ) 4545 4444 3333 스토크균핵병(Sclerotinia screlotiorum) Sclerotinia screlotiorum 3333 3535 2222 상추균핵병(Sclerotinia sp.) Sclerotinia sp. 3737 2828 1919 상추잿빛곰팡이병(Botrytis cinerea)Lettuce Gray Mold Disease ( Botrytis cinerea ) 3333 3939 4040 모잘록병(Rhizoctonia solani) Rhizoctonia solani 3333 3838 3131 고추역병(Phytophthora capsici)Red pepper plague ( Phytophthora capsici ) 2323 3737 3232 벼도열병(Magnaporthe grisea) Magnaporthe grisea 2626 4040 5656 토마토잎마름병(Phytophthora infestans)Tomato leaf blight ( Phytophthora infestans ) 3737 3737 3333 *억제율 = (대조구 균사길이 - 처리구 균사길이) / 대조구 균사길이 ×100* Inhibition rate = (control mycelial length-treated mycelial length) / control mycelial length × 100

플러그 육묘시 유용미생물 처리에 의한 병발생 억제 효과Effect of microbial treatment on plug growth 작 물Water 처리*process* 초장(㎝)Extra long (cm) 생체중(g/주)Live weight (g / week) 건물율(%)% Building 발병주율(%)Incidence rate (%) 방제가Control 유용세균밀도(cfu/g)Useful bacterial density (cfu / g) 토마토tomato 17S17S 41.8 b41.8 b 6.0 ab6.0 ab 11.4 ab11.4 ab 00 100100 2.4×108 2.4 × 10 8 17S+F17S + F 46.3 a46.3 a 7.1 a7.1 a 10.9 b10.9 b 00 100100 1.2×108 1.2 × 10 8 FF 37.9 c37.9 c 5.7 b5.7 b 12.1 a12.1 a 5353 00 -- 고 추pepper 17S17S 34.7 a34.7 a 3.0 a3.0 a 13.0 ba13.0 ba 00 100100 2.1×105 2.1 × 10 5 17S+P17S + P 34.3 a34.3 a 3.1 a3.1 a 12.2 b12.2 b 00 100100 1.8×105 1.8 × 10 5 PP 344 a344 a 3.5 a3.5 a 13.8 a13.8 a 1313 00 -- * LSD at 5% level* 25R : 슈도모나스 애루지노사 25R17S : 슈도모나스 애루지노사 17SF : 토마토 시들음병(Fusarium oxysporumf.sp.lycopersici) P : 고추역병(Phytophthora capsici)* LSD at 5% level * 25R: Pseudomonas aeruginosa 25R17S: Pseudomonas aeruginosa 17SF: Tomato wilted bottle (Fusarium oxysporumf.sp.lycopersici) P: Red pepper plaguePhytophthora capsici)

슈도모나스 애루지노사 17S 처리시 엽내 SA함량 변화Changes in SA Contents in Leaves after Pseudomonas aeruginosa 17S Treatment 품 종kind 살리실산 함량(㎍/g 생체중)Salicylic Acid Content (µg / g Live Weight) 무처리No treatment 17S 처리구17S treatment slot 모모타로 T93Momotaro T93 5.55.5 7.07.0

Claims (5)

표 1의 특성을 가지는 슈도모나스 애루지노사 17S(KCTC 0978BP).Pseudomonas aeruginosa 17S having the properties of Table 1 (KCTC 0978BP). 제1항에 있어서,The method of claim 1, 스트랩토마이신에 대한 저항성을 보유하는 것을 특징으로 하는 슈도모나스 애루지노사 17S.Pseudomonas aeruginosa 17S, characterized by a resistance to straptomycin. 제1항 또는 제2항에 있어서,The method according to claim 1 or 2, 살리실릭산(salicylic acid) 생산을 증가시켜 식물병원균의 저항성을 향상시키는 슈도모나스 애루지노사 17S.Pseudomonas aeruginosa 17S, which increases salicylic acid production to improve the resistance of phytopathogens. 제1항의 미생물균주를 이용하여 유기된 돌연변이주.Mutant strains that were derived using the microbial strain of claim 1. 제1항의 미생물균주로부터 생산된 길항물질.Antagonist produced from the microbial strain of claim 1.
KR10-2001-0025922A 2001-05-11 2001-05-11 Pseudomonas aeruginosa 17S and their mutants promoted plant growth and showed antagonistic effect on phytopathogens KR100419783B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR10-2001-0025922A KR100419783B1 (en) 2001-05-11 2001-05-11 Pseudomonas aeruginosa 17S and their mutants promoted plant growth and showed antagonistic effect on phytopathogens

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR10-2001-0025922A KR100419783B1 (en) 2001-05-11 2001-05-11 Pseudomonas aeruginosa 17S and their mutants promoted plant growth and showed antagonistic effect on phytopathogens

Publications (2)

Publication Number Publication Date
KR20020086154A true KR20020086154A (en) 2002-11-18
KR100419783B1 KR100419783B1 (en) 2004-02-21

Family

ID=27704648

Family Applications (1)

Application Number Title Priority Date Filing Date
KR10-2001-0025922A KR100419783B1 (en) 2001-05-11 2001-05-11 Pseudomonas aeruginosa 17S and their mutants promoted plant growth and showed antagonistic effect on phytopathogens

Country Status (1)

Country Link
KR (1) KR100419783B1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019144249A1 (en) * 2018-01-25 2019-08-01 Am Ecological S.A. Microorganisms for biocontrol of botrytis
CN111518726A (en) * 2020-05-09 2020-08-11 中南林业科技大学 Pseudomonas aeruginosa and screening method and application thereof
CN112063558A (en) * 2020-09-17 2020-12-11 湖南农业大学 Pseudomonas strain and application thereof
CN112980744A (en) * 2021-04-28 2021-06-18 福建农林大学 Pseudomonas and application thereof in prevention and treatment of potato late blight

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019144249A1 (en) * 2018-01-25 2019-08-01 Am Ecological S.A. Microorganisms for biocontrol of botrytis
CN111518726A (en) * 2020-05-09 2020-08-11 中南林业科技大学 Pseudomonas aeruginosa and screening method and application thereof
CN111518726B (en) * 2020-05-09 2022-06-03 中南林业科技大学 Pseudomonas aeruginosa and screening method and application thereof
CN112063558A (en) * 2020-09-17 2020-12-11 湖南农业大学 Pseudomonas strain and application thereof
CN112980744A (en) * 2021-04-28 2021-06-18 福建农林大学 Pseudomonas and application thereof in prevention and treatment of potato late blight

Also Published As

Publication number Publication date
KR100419783B1 (en) 2004-02-21

Similar Documents

Publication Publication Date Title
KR100535912B1 (en) The novel bacillus amyloliquefaciens ktgb0202 and control method of plant pathogenic fungi using that
Trejo-Estrada et al. In vitro and in vivo antagonism of Streptomyces violaceusniger YCED9 against fungal pathogens of turfgrass
US6280719B1 (en) Antifungal biocontrol agents, a process for preparing and treating the same
CN112625970B (en) Burkholderia cepacia JT79 and application thereof
Dicklow et al. A novel Streptomyces species for controlling plant-parasitic nematodes
KR101199931B1 (en) Promotion of nitrogen mineralization of organic fertilizers and control of plant diseases using bacillus velezensis KRICT934
Spiegel et al. Evaluation of a newly isolated bacterium, Pseudomonas chitinolytica sp. nov., for controlling the root‐knot nematode Meloidogynejavanica
US4798723A (en) Agricultural products from Pseudomonas cepacia strains and methods of preparation
Hebbar et al. Rhizobacteria of maize antagonistic to Fusarium moniliforme, a soil-borne fungal pathogen: isolation and identification
KR102375337B1 (en) Composition for controlling plant diseases using Brevibacillus brevis HK544
US10327448B2 (en) Bacterium of Bacillus genus and uses thereof
KR101224485B1 (en) Novel Streptomyces geldanamycininus BS3283 and method for controlling plant pathogen diseases using the same
KR100747700B1 (en) Antagonistic soil bacterium bacillus subtillis and biological control and plant growth promotion using the same
KR100411185B1 (en) A new Streptomyces sp. AG-P(KCTC 8965P), and an agent for control of plant diseases using this strain
KR101107331B1 (en) Novel Streptomyces argenteolus strain having activity aganist plant pathogens
CN117165494A (en) Kiwi fruit canker biocontrol strain Wq-1 and application thereof
KR100419783B1 (en) Pseudomonas aeruginosa 17S and their mutants promoted plant growth and showed antagonistic effect on phytopathogens
Harris et al. Soil bacteria as selective biological control agents of winter annual grass weeds in winter wheat
CA1293701C (en) Agricultural products and methods
EP0160089A1 (en) Rhizobacterial plant protection
KR20070080775A (en) Serratia plymuthica a21-4, biological control method of phytophthora diseases using the a21-4 and antifungal compound produced by the a21-4
KR102143334B1 (en) Pseudomonas frederiksbergensis strain possessing antifungal activity against major pathogenic bacteria of plant and use thereof
US5968504A (en) Fungus Gliocladium catenulatum for biological control of plant diseases
KR20220001147A (en) Composition for controlling plant bacterium disease comprising culture solution of Penibacillus elgii JCK-5075 strain or extract thereof, manufacturing method thereof, and controlling method for plant bacterium disease
KR0153138B1 (en) Streptomyces moscow and antibiotics

Legal Events

Date Code Title Description
A201 Request for examination
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant
FPAY Annual fee payment

Payment date: 20121213

Year of fee payment: 10

FPAY Annual fee payment

Payment date: 20131217

Year of fee payment: 11

FPAY Annual fee payment

Payment date: 20150507

Year of fee payment: 12

FPAY Annual fee payment

Payment date: 20160321

Year of fee payment: 13

FPAY Annual fee payment

Payment date: 20170217

Year of fee payment: 14

FPAY Annual fee payment

Payment date: 20180305

Year of fee payment: 15

FPAY Annual fee payment

Payment date: 20190311

Year of fee payment: 16

FPAY Annual fee payment

Payment date: 20191209

Year of fee payment: 17