원형질체와 몇 가지 살균제 효과 검정 방법을 이용한 고추 탄저병 살균제 계열별 효과검정 = Assessment of fungicidal activity against pepper anthracnose caused by Colletotrichum acutatum through protoplasts and several assay systems

다국어 초록 (Multilingual Abstract)

Colletotrichum acutatum, is a major devastating pathogen in South Korea causing anthracnose disease of pepper. In order to identify a suitable fungicide for the disease management and to find out the development of fungicide resistance, seven groups of fungicides with different mode of actions were evaluated against mycelial growth (poisoned food technique) and spore germination. Similarly, the tests were also carried out with the protoplasts isolated from C. acutatum on PDA amended with an osmotic stabilizer sorbitol and different concentrations of various fungicides. Further, 10 fungicides belong to 3 major groups namely Ergosterol biosynthesis inhibitor (3), preventive fungicides (3) and strobilurins (4) were tested on inoculated pepper fruits in vitro and under field conditions. All the seven fungicides tested against the mycelial growth on PDA amended with different concentrations were grouped in to 3 types based on their activity as 1. fungicides only effective against mycelial growth (tebuconazole, carbendazim, carbendazim + diethofencarb), 2. fungicide only effective against spore germination (propineb, trifloxystrobin and kresoxim methyl) and 3. fungicides ineffective against both mycelial growth and spore germination (polyoxin-B). Cellophane paper laid on PDA plates were smeared with 100 ㎍ of C. acutatum JC24 spores (1x106 spores/ml) and incubated for protoplast isolation at 25 C for 20 h. After incubation, the cellophane paper was added with 2% lysing enzyme, 1.2 M sorbitol and 0.02 M phosphate buffer (pH 7.0) and shaken at 70 rpm for 2 to 3 hours. The contents were filtered through cheese cloth and centrifuged at 3000 rpm for 10 min. Protoplast deposited at the bottom of centrifuge tube were collected and used for further studies. The regeneration of isolated protoplast was evaluated on plates amended with various fungicides at different concentrations. The regeneration was totally inhibited by propineb at a concentration of 10 ㎍/㎖ Similarly, the fungicide tebuconazole also exhibited 100% efficacy against protoplast regeneration at 0.5 ㎍/㎖, while the same showed 85.1 and 75.7% control value against the mycelial growth at the concentrations of 0.5 and 0.1 ㎍/㎖, respectively. The fungicides kresoxim methyl and trifloxystrobin showed 0, 21.9 and 15.1, 2.1% control value when used alone and 91.8, 75.3% and 64.7, 20.7% control value when used in combination with SHAM against protoplast regeneration at a concentrations of 10 and 1 ㎍/㎖, respectively, while the efficacy of carbendazim against both mycelial growth and protoplast regeneration was same at all concentrations tested. The optimum media and incubating temperature for the protoplast regeneration test on fungicides amended plates were evaluated. Regardless of the difference in regeneration on the plates amended with fungicides alone and in combination with SHAM, all the temperatures tested were on par with each other. Similar results were obtained in the experiments done with different types of media viz., PDA, CM (Complete Medium) and MM (Minimal Medium). Over all observations revealed that the CM (complete media) amended with fungicides and SHAM, incubated at 25 ℃ was found optimum for the evaluation of protoplast regeneration. But the plates amended with different media and fungicides when smeared with 100 ㎍ of spores at a concentration of 1x103/ml comparatively showed very low effect. The inoculation methods and effect of different fungicides against the pathogen were evaluated in vitro and in the field as well. Pepper fruits inoculated with 1x106/ml spore load on injured (pin pricking) and uninjured fruits incubated at 30℃ for 10 days showed 90% incidence on all the fruits. To evaluate the efficacy of fungicides against the disease, they were sprayed on inoculated fruits 24hours before (preventive) and 24hours after (curative) the pathogen inoculation. Azoxystrobin was highly effective and showed 97.3 and 94.6% control value while the kresoxim methyl showed 45.9 and 44.0% control value in preventive and curative application, respectively on the uninjured fruits inoculated with the pathogen. A field trial was conducted during 2008 to evaluate the effect of various fungicides. The fungicides were sprayed for 4 times at 10 days interval starting from 150days after planting. Among the fungicides tested, pyraclostrobin was highly effective and recorded a control value of 95.3%, while the folpet recorded the least (46.5%). In nut shell, the strobilurin fungicides are the effective against the anthracnose pathogen C. acutatum under both in vitro (azoxystrobin) and field assessments (pyraclostrobin). Evaluation of fungicides with different mode of actions against C. acutatum revealed that the tebuconazole was effective against mycelial growth while propineb and strobilurins fungicides were effective against the spore germination. Evaluation of strobilurins fungicide against the mycelial growth was not so good rather going for evaluation of protoplast regeneration. The fungicides used so far showed differential responses under different evaluation system like in vitro and in vivo tests, has to be given due importance and needed thorough investigation. Development of optimum evaluation parameters to assess fungicide resistance in vitro and in vivo is in progress.

목차 (Table of Contents)

• Ⅰ. 서 론 1
• Ⅱ. 재료 및 방법 5
• 1. 균주보관 5
• 2. 계열에 따른 살균제 효과 검정 5
• 1) 균사생장 억제 5
• 2) 포자 발아 억제 6
• 3. 원형질체 나출 및 살균제 효과 검정 7
• 1) 원형질체 나출 7
• ① 포자배양 시간 7
• ② 세포벽 분해효소 7
• ③ 삼투압 안정제 및 pH 8
• 2) 원형질체 재생 8
• 3) 살균제에 대한 원형질체 효과 8
• 4. 탄저병균 원형질체를 이용한 strobilurin계 살균제의 효과 검정 10
• 1) 살균제 효과에 미치는 온도의 영향 10
• 2) 배지의 종류가 살균제 효과에 미치는 영향 10
• 5. 고추 열매를 이용한 실내 검정에서 다양한 살균제의 방제 효과 11
• 6. 포장에서 각 살균제의 효과 검정 12
• Ⅲ. 결 과 14
• 1. 살균제의 균사생장 및 포자발아 효과 검정 14
• 1) 균사 생장 억제 효과 14
• 2) 포자발아 억제 효과 17
• 2. 원형질체 나출 및 살균제 효과 검정 19
• 1) C. acutatum JC24 원형질체 생성에 미치는 요인 19
• ① 포자배양 시간 19
• ② 세포벽분해 효소 19
• ③ 삼투압안정제 23
• ④ pH 23
• 2) C. acutatum JC24 원형질체의 재생 26
• 3) C. acutatum JC24 원형질체의 재생에 미치는 살균제의 영향 29
• 3. 호흡 저해 살균제의 저항성 조사를 위한 조건 탐색 36
• 1) 온도에 따른 효과 36
• 2) 배지종류에 따른 효과 36
• 3) 포자에서의 호흡저해 살균제 반응 36
• 4. 고추열매와 포장에서의 효과 검정 41
• 1) 포장에서의 발병 증가율 41
• 2) 고추열매에서의 병 방제 효과 43
• 3) 포장에서의 살균제 효과 46
• Ⅳ. 고 찰 49
• Ⅴ. 요 약 58
• Ⅵ. 인용문헌 62