(A) study on the development of biological control agent for Fusarium wilt on Cymbidium genus = 난 시들음병 방제를 위한 생물농약의 개발에 대한 연구

국문 초록 (Abstract)

본 연구에서는 화학농약으로 방제가 어려워 난 재배농가에 많은 피해를 주고 있는 난 시들음병을 방제하기 위한 생물농약을 개발하려고 하였다. 발병된 이병주의 잎과 벌브에서 병원균을 분리하였다. 분리된 병원균은 현미경 관찰을 통하여 Fusarium oxyform f.sp. cattleyae 로 판명되었으며, 병원성 검정을 통하여 분리된 병원균은 강한 병원성을 보여 심비디움속의 시들음병의 원인 병원균으로 판단하였다. 또한 심비디움 종과 품종에 따라 감수성이 상이함도 알 수 있었다. 특히 동양계 심비디움이 서양계 심비디움에 비해 높은 감수성을 보여주었다. 길항균의 분리는 제주도 양란 심비디움 재배지와 심비디움 자생지에서 64토양 샘플에서 15의 세균과 15의 방선균을 1차로 선발하였다. 선발된 균주들 중에 가장 높은 저해율을 갖는 심비디움 시들음병을 방제하기 위한 길항균으로 선발하였다.
선발된 길항균은 형태학적, 배양학적 특성과 세포벽 성분의 분석을 통하여 Streptomyces sp. 에 속하는 것으로 나타났다. 정확한 균주 동정을 위하여 16S rRNA 염기서열 분석을 통하여 Streptomyces kasugaensis로 동정되었으며, 신규한 균주로 인정되어 Streptomyces kasugaensis GBA-0927 로 명명하였다.
동정된 미생물은 다른 주요한 곰팡이성 병해의 병원균에도 높은 활성을 보였으며, 타작물의 적용시험결과 오이의 주요한 병 방제에도 높은 방제율을 보였다. 동정된 길항미생물의 산업적 생산을 위하여 최적의 발효공정은 SCF 100 ppm, EDTA 10 ppm, Mycophenolic acid 32 ppm을 첨가하고 pH up-shock을 주었을때, 2.5 L jar fermenter에서 가장 많은 포자수인 1.08 × 10^8과 55 % 의 높은 포자 전환율을 보였다.
동정된 미생물의 항곰팡이성 물질은 용매추출, 컬럼크로마토그래피, 분취크로마토그래피 등의 방법으로 순수한 물질로 정제하였다. 정제된 항곰팡이성 물질은 NMR, IR, Mass 등의 기기분석을 통하여 분자량 741의 polyene 계열의 물질로 판명되었다. 이 항생물질은 이전의 연구를 통하여 Streptoverticillium eurocidicum var asterocidicus S-822 에서 생산되는 YS-822A 라는 물질이었지만, Streptomyces kasugaensis 에서 생산 되는 것으로는 보고된 바가 없는 물질이었다. YS-822A 의 주요 식물병원균에 대한 최소저해농도를 측정한 결과 2∼4 ppm 의 높은활성을 나타내었으며, 처음 보고되었을 때 유사한 구조인 amphotericin B 보다 낮은 활성으로 관심을 끌지못하였으나, 주요 식물병원균에 대해서는 높은 활성을 갖고 있으므로 친환경적인 농업용 항생물질 (생물농약)으로서의 개발가능성이 높은 것으로 사료되었다.

다국어 초록 (Multilingual Abstract)

The new antifungal biological pesticides were exploited for wilt disease which causes extensive damage to Cymbidium orchid cultivation farmers. The phytopathogen was isolated from out broken leaves and bulbs. Screened pathogen was proven to be a Fusarium oxyform f.sp. cattleyae by microscope and it was a main pathogen against wilt disease showing strong pathogenic effect. Besides, different sensitivities were observed depending on various Cymbidium genus and species. Especially oriental Cymbidium had a higher sensitivity than western Cymbidium.
In this study, 15 bacteria and 15 actinomycetes were initially screened from Cymbidium cultivation area and 64 of habitant areas in Jeju island for the isolation of antifungal microbes showing antifungal activity against pathogens. Final antifungal microbe, GBA-12, was selected with the highest prevent power of 73.3%. Selected antifungal microbe belongs to Streptomyces sp. by the analysis of cultural characteristic and cell wall compositions. This microbe was finally identified as a Streptomyces kasugaensis by the analysis of DNA sequencing, 16S r DNA analysis. This microbe was named as Streptomyces kasugaensis GBA-0927.
Identified microbe showed high selective antifungal effect on other fungal pathogens, especially in cucumber. The optimal media condition for industrial production is composed of 100 ppm of SCF, 10 ppm of EDTA and 32 ppm of Mycophenolic acid. After pH up-shock, this media showed the largest number of spores, 1.08 × 10^(8) with the spore conversion rate of 55%.
Antifungal agent was purified by liquid extraction, column chromatography, and prep chromatography from identified microbe. Purified antifungal compound was identified as one of polyenes with molecular weight of 741 by NMR, IR and Mass Spectroscopy. Though it was known as YS-822A produced by Streptoverticillium eurocidicum var. asterocidicus S-822, it has not been reported to be found in Streptomyces kasugaensis. Even though the antifungal activity of YS-822A was lower than that of amphotericin B which has similar structure to YS-822A when it was first reported, it gives attractive interest as agricultural antibiotics with high antifungal effect showing high MIC value of 2∼4 (㎍/L) on several major plant pathogens. The effective antifungal microbe, Streptomyces kasugaensis GBA-0927, against Fusarium oxysporum f.sp. cattleyae could be applied to other fungal plant pathogens and its antibiotics leave nothing desired for the agricultural antifungal agents.

목차 (Table of Contents)

• Table of Contents = i
• List of Tables = v
• List of Figures = vii
• Abstract = xi
• 1. Introduction = 1
• 2. Materials and Methods = 16
• 2.1. Materials = 16
• 2.1.1. Microoragnisms = 16
• 2.1.2. Chemicals and Media = 16
• 2.1.3. Apparatus = 16
• 2.2. Methods = 17
• 2.2.1. The isolation and identification of pathogens causing wilt disease on Cymbidium orchid = 17
• 2.2.2. The test for pathogen and investigation of pathogenic mechanism = 17
• 2.2.3. The screening of antagonistic microorganism against the pathogen causing wilt disease on Cymbidium orchid = 20
• 2.2.3.1. The isolation of antagonistic microorganism and test of its effect = 20
• 2.2.3.2. Selection of antagonistic microorganisms and activity test = 21
• 2.2.4. Adaptability and preventive power test = 21
• 2.2.5. Application of other crop diseases = 22
• 2.2.6. The identification of selected antagonist against Fusarium oxysporum f.sp. cattleyae = 22
• 2.2.6.1. The analysis of morphological characteristics = 22
• 2.2.6.2. The analysis of cultural characteristics = 23
• 2.2.6.3. The analysis of cell wall characteristics = 23
• 2.2.6.4. 16S rRNA sequencing = 23
• 2.2.7. Optimization of fermentation condition for mass production = 24
• 2.2.8. Antibiotic activity test (paper disc method) = 24
• 2.2.9. Isolation and purification of antibiotic agents = 24
• 2.2.9.1. Stability of pH and heat sensitivity = 26
• 2.2.9.2. HP-20 column chromatography = 26
• 2.2.9.3. Solvent extraction = 26
• 2.2.9.4. Thin layer chromatography (TLC) = 27
• 2.2.9.5. Silica gel column chromatography = 27
• 2.2.9.6. Sephadex LH-20 column chromatography = 27
• 2.2.9.7. Prep-high performance liquid chromatography (Prep-HPLC) = 27
• 2.2.10. Structure identification of purified antibiotics = 28
• 2.2.11. Minimum inhibitory concentrations (MICs) = 28
• 3. Results and Discussion = 29
• 3.1. The isolation and identification of pathogens causing wilt disease on Cymbidium orchid = 29
• 3.2. Evaluation of infection mechanism and pathogenicity = 29
• 3.3. The screening of antagonistic microorganism against the pathogen causing fusarium wilt on Cymbidium orchid = 35
• 3.3.1. The isolation of antagonistic microorganism and test of its effect = 37
• 3.3.2. Selection of antagonistic microorganisms and activity test = 37
• 3.4. Adaptability and preventive power test = 37
• 3.5. Application of other crop diseases = 42
• 3.6. The identification of selected antagonist against Fusarium oxysporum f.sp. cattleyae = 42
• 3.6.1 The morphological characteristics = 52
• 3.6.2. The cultural characteristics = 52
• 3.6.3. The cell wall characteristics = 52
• 3.6.4. 16S rRNA sequencing = 56
• 3.7. Optimization of fermentation condition = 56
• 3.7.1. Determination of discrimination condition between mycelia and spore = 56
• 3.7.2. Confirmation of differentiation = 60
• 3.7.3. Change of beef extract = 60
• 3.7.4. Optimization medium composition = 60
• 3.7.5. Effect of initial pH = 66
• 3.7.6. Effect of initial dextrose concentration = 66
• 3.7.7. Effect of DPA and divalent cations = 66
• 3.7.8. Effect of mycophenolic acid = 72
• 3.7.9. Effect of spent culture fluid (SCF) = 72
• 3.7.10. Effect of EDTA = 72
• 3.7.11. Effect of antifoam = 77
• 3.7.12. Effect of adding SCF = 77
• 3.7.13. Fermentation = 77
• 3.8. Assay of antibiotics (paper disc method) = 83
• 3.9. The isolation and purification of antibiotic agent = 83
• 3.9.1. Stability test for pH and heat sensitivity = 83
• 3.9.2. Diaion HP-20 column chromatography = 85
• 3.9.3. Solvent extraction = 85
• 3.9.4. Thin layer chromatography (TLC) = 85
• 3.9.5. Silica gel column chromatography = 85
• 3.9.6. Sephadex LH-20 column chromatography = 92
• 3.9.7. Prep-high performance liquid chromatography (Prep-HPLC) = 92
• 3.10. The structural analysis of purified antibiotics = 92
• 3.11. Minimum inhibitory concentrations (MICs) = 100
• 4. Conclusion = 112
• References = 115
• 국문요약 = 123