동결건조 보호제 처리에 따른 Bacillus spp.를 이용한 생존 평가와 마늘 흑색썩음균핵병의 생물적 방제
Survival Assessment of Bacillus spp. Using Freeze-drying Protectant and Biological Control of Garlic White Rot Caused by Sclerotium cepivorum
- 주제(키워드) Garlic white rot , antifungal microorganism , freeze-drying , lyoprotectant , plant growth promoting
- 발행기관 강릉원주대학교 대학원
- 지도교수 김병섭
- 발행년도 2018
- 학위수여년월 2018. 2
- 학위명 석사
- 학과 및 전공 일반대학원 원예학과
- 세부전공 식물병리학
- 원문페이지 59
- 본문언어 한국어
- 저작권 강릉원주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Garlic white rot caused by Sclerotium cepivorum is major pathogenic fungi of Korea. Until now, garlic white rot is controlled by application of fungicide. In order to develop environment-friendly microbial pesticide, the experiments was carried out to evaluate the biocontrol control of 4 species bacteria of Paenibacillus sp. T-9, Bacillus spp. DM152, SG08-09, SG09-12 by both in vitro, in vivo experiment. Dual culture assay showed strong inhibitory effect of among 11 species phytopathogens, such as Botrytis cinerea, Colletotrichum acutatum, Rhizoctonia solani, Fusarium oxysporum, Magnaporthe grisea, Phytophthora capsici, S. cepivorum, Sclerotinia sclerotiorum, Sclerotinia minor, Pythium sp., Pythium ultimum. In sclerotial germination experiment, the application 10-1~10-8 dilution dipping method of 4 species antifungal microorganism showed an excellent effect of DM152, SG08-09, SG09-12 in spite of lower concentration except in T-9 but in inhibition ratio of mycelial growth using garlic seed bulb resulted in very low activity of SG08-09 (29.6%), T-9 (21.4%) against Boscalid (100%), Metconazole (100%), Bacillus subtilis QST 713 (100%), Paenibacillus polymyxa AC-1 (95.6%), DM152 (93.0%), SG09-12 (90.4%). In pot trials was performed to assess the disease control efficiency of 4 species bacteria against 2 fungicides, 2 microbial pesticide, which showed moderate effect T-9 (56.9%), SG08-09 (52.8%), DM152 (52.2%), SG09-12 (51.8%), Bacillus subtilis QST 713 (59.6%), Paenibacillus polymyxa AC-1 (55.3%) but strong effect against Boscalid (72.6%), Metconazole (88.7%) through in vivo assay. Finally, DM152, SG09-12 strain were chosen in order to freeze-drying experiment. two bacteria has been lyophilized via freeze-drying along with lyoprotectant such as skim milk, peptone (each 10%, 20%, 30% concentration). The freeze-dried powder of DM152, containing skim milk 20%(1.8x108CFU/ml) exhibited similar survival density against sterile water(1.6x109CFU/ml) and SG09-12, containin g skim milk 30%(1.1x107CFU/ml) showed high survival density against sterile water(1.5x106 CFU/ml). After that, in order to measure survival density of microorganism by 0℃, 25℃, 35℃ storage temperature, measured every 2 weeks for 6 weeks. In the result, it decreased as the temperature rises and the best storage temperature was found at 0℃. Freeze-dried two bacteria strains of selected final concentration enhanced all plant growth under greenhouse condition. In addition to control effect of the garlic white rot showed the best control efficacy in SG09-12 skim milk 30% 106CFU/ml, DM152 skim milk 20% 108CFU/ml in vivo experiment. Freeze-dried Bacillus spp. DM152, SG09-12 exhibited effective plant growth promoting and antifungal activity. In conclusion, a two bacteria strain is expected to use as a biofertilizer and microbial pesticide.
more목차
I. 서언………………………………………………………………………………………….....1
1. 마늘 재배현황………………………………………………………………………………1
2. 마늘 흑색썩음균핵병…………………………………..……..……..……..………………..2
3. 국내 친환경농산물의 시장규모와 전망…………………………………………………..3
4. 생물농약의 필요성…………………………………………………………………………..4
5. 연구 목적……………………………………………………………………………………..5
II. 재료 및 방법………………………………………………….…………………………....6
<실험1> 4종의 길항미생물을 이용한 마늘 흑색썩음균핵병 항균활성………………...6
1. 실험에 사용한 균주 및 배양……………………………………………………………....6
2. 다양한 식물병원균에 대한 항균활성 검정……………………………………………..6
3. 길항세균의 동정……………………………………………………………..........................7
4. 균핵 발아 억제효과………………………………………………………………………....8
- 4.1. S. cepivorum 균핵 형성 및 수확…………………………………………………8
- 4.2. 시료 준비……………………………………………………………………………..8
- 4.3. 처리방법………………………………………………………………………………9
5. In vitro, in vivo 스크리닝……………………………………………………………………..9
- 5.1. In vitro 스크리닝……………………………………………………………………..9
- 5.2. 처리방법……………………………………………………………………………...10
- 5.3. In vivo 스크리닝……………………………………………………………………..10
6. 통계분석……………………………………………………………………………………..11
<실험2> 최종 선발된 2종의 길항미생물을 이용한 동결건조 처리 후 생존 밀도 및 저장성 평가와 마늘생장촉진활성 및 흑색썩음균핵병 방제효과……………………….12
1. 동결건조 후 생존 밀도 및 저장성 평가………………………………………………..12
-1.1. 재료 및 시약 제조…………………………………………………………………....12
-1.2. 생존 밀도 평가………………………………………………………………………..12
-1.3. 저장성 평가……………………………………………………………………………13
2. 마늘생장촉진활성…………………………………………………………………………..13
-2.1. 재료 및 미생물 현탁액 처리……………………………………………………......13
-2.2. 생장촉진활성 효과 검정…………………………………………………………......13
3. 마늘 흑색썩음균핵병 방제효과………………………………………………………......15
-3.1. 재료 및 병원균 접종………………………………………………………………....15
-3.2. 처리방법……………………………………………………………………………......15
4. 통계분석…………………………………………………………………………………......16
Ⅲ. 결과 및 고찰. ……………………………………………………………………………...17
<실험1> 4종의 길항미생물을 이용한 마늘 흑색썩음균핵병 항균활성……………….17
1. 다양한 식물병원균에 대한 항균활성 검정…….………………………………….......17
2. 길항세균의 동정…………………………………………………………..........................21
3. 균핵 발아 억제효과………………………………………………………………………23
4. In vitro, in vivo 스크리닝……..……………………………………………………………..26
-4.1. In vitro 스크리닝……………………………………………………………………..26
-4.2. In vivo 스크리닝……………………………………………………………………...29
<실험2> 최종 선발된 2종의 길항미생물을 이용한 동결건조 처리 후 생존 밀도 및 저장성 평가와 마늘생장촉진활성 및 흑색썩음균핵병 방제효과……………………….32
1. 동결건조 후 생존 밀도 평가…………………………………………………..................32
-1.1. 저장성 평가…………………………………………………………………………....35
2. 마늘생장촉진활성 검정…………………………………………………………………....38
3. 마늘 흑색썩음균핵병 방제효과 검정…………………………………………………....41
Ⅳ. 종합 고찰…………………………………………………………………………………..44
Ⅴ. 참고 문헌……………………………………………………………………………..........46
감사의 글…………………………………………………………………………….................49
