보고서 정보
주관연구기관 |
경상남도농업기술원 |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2013-02 |
과제시작연도 |
2012 |
주관부처 |
농촌진흥청 Rural Development Administration(RDA) |
등록번호 |
TRKO201300014056 |
과제고유번호 |
1395026697 |
사업명 |
농업현장실용화기술개발 |
DB 구축일자 |
2013-12-21
|
DOI |
https://doi.org/10.23000/TRKO201300014056 |
초록
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Ⅲ. 연구개발의 내용 및 범위
○ 큰느타리(새송이)버섯 액체종균 유해 미생물 분리 및 피해증상 확인
- 새송이버섯 농가 오염액체종균 및 오염 배지 수거
- 오염 원인균에 대한 미생물동정
- 오염 유발 미생물 분리 및 재접종을 통한 피해증상 확인
○ 분리미생물 동정에 기초한 종간 또는 속내 공통 특이마커 개발
- Gram (+) 및 Gram(-) 세균 증폭 가능 degenerancy primer 제작
- Bacillus sp. 및 Pseudomonas sp. 의 속간 특이 유전자 및 속내 공통유전
Ⅲ. 연구개발의 내용 및 범위
○ 큰느타리(새송이)버섯 액체종균 유해 미생물 분리 및 피해증상 확인
- 새송이버섯 농가 오염액체종균 및 오염 배지 수거
- 오염 원인균에 대한 미생물동정
- 오염 유발 미생물 분리 및 재접종을 통한 피해증상 확인
○ 분리미생물 동정에 기초한 종간 또는 속내 공통 특이마커 개발
- Gram (+) 및 Gram(-) 세균 증폭 가능 degenerancy primer 제작
- Bacillus sp. 및 Pseudomonas sp. 의 속간 특이 유전자 및 속내 공통유전자 이용 판별 마커 분리
- 분리 유전자 염기서열에 기초한 특이 primer 제작
○ PCR 기술 이용 액체종균 유해미생물 진단 기술 개발
- 종 특이 증폭가능 primer 조합에 의한 multiplex PCR 기술 이용 진단 kit 개발
- Bacillus sp. 및 Pseudomonas sp. 등 오염유발 미생물 판별 특이 프라이머에 대한 비특이적 증폭 가능성 확인
- 특이 primer 조합에 의한 Multiplex PCR 반응 적정 조건 확립
- 농가시료에 대한 전처리 적정 방법 확립
○ 종균배양실 오염가능 미생물 분리 • 동정 및 액체탱크의 비개폐방법에 의한 비노출식 접종방법 확립
- 노출식 접종에 의한 오염 가능 미생물 분리 및 동정
- 오염 미생물에 의한 액체종균 피해 증상 구명
- 종균 투입 전용 배관 또는 호스를 이용한 액체탱크의 완전 비개폐 방식에 의한 종균접종 시스템 확립
○ 액체탱크 비노출식 접종방법의 농가적용에 따른 오염경감 효과 구명
- 접종원과 액체탱크 간 종균 전달이 가능한 line을 이용하여 탱크내부로 종균 투입
- 접종과정에 필요한 커플러 및 line 규격 정립
- Line 접종에 의해 만들어진 액체종균 이용에 따른 버섯 배양특성 및 생육특성 조사
- Line 접종에 의해 만들어진 액체종균에 대한 미생물 오염 조사
Abstract
▼
This study was conducted to identify and detect the mushroom pathogens in liquid spawn of P leurotus eryngii, using simultaneous multiplex polymerase chain reaction (multiplex PCR) assay. Also. we were try to develop the inoculation system in liquid spawn of Pleurotus eryngii for the stable producti
This study was conducted to identify and detect the mushroom pathogens in liquid spawn of P leurotus eryngii, using simultaneous multiplex polymerase chain reaction (multiplex PCR) assay. Also. we were try to develop the inoculation system in liquid spawn of Pleurotus eryngii for the stable production of mushroom. Bacterial isolates from liquid spawn during the mushroom cultivation and their diversity were examined using a PCR-based approach. A total of 98 strains were isolated from different contaminated liquid spawn and 50 out of isolated strains which seriously inhibit the mycelial growth of P . eryngii were examined by 16S rDNA sequence analysis. Based on sequence analysis of rDNA and sequence identity values, a total of 12 different genus were found at the contaminated liquid spawn; Bacillus, Bacterium, Caulobacter, Ewingella, Lactococcus, Microbacterium, Paenibacillus, Pseudomonas, Rahnella, Salmonella, Serratia, Staphylococcus. Bacillus of the phylum Firmicutes was the most dominant genus (60.0%) in contaminated substrates followed by Ewingella sp., Serratia sp., Staphylococcus sp., and M icrobacterium sp. (6.0%), P aenibacillus sp (4.0%), P seudomonas sp., Bacterium sp., Caulobacter sp., Lactococcus sp. and R ahnella sp.(2.0%). A total of 79 strains were isolated from different contaminated liquid spawn room and 10 out of isolated strains which seriously inhibit the mycelial growth of P leurotus eryngii were examined by 16S rDNA sequence analysis. Based on sequence analysis of rDNA and sequence identity values, a total of 3 different genus were found at the contaminated liquid spawn; Pseudomonas, Bacillus, Pantoea. Pseudomonas of the phylum Firmicutes was the most dominant genus (60.0%) in contaminated substrates followed by Bacillus sp. (30.0%) and P antoea sp. (10.0%). In the case of the contaminated media and fruit bodies, a total of 28 strains were isolated from different contaminated substrates and 21 out of isolated strains which seriously inhibit the mycelial growth of P . eryngii were examined by 16S rDNA sequence analysis. Based on sequence analysis of rDNA and sequence identity values, a total of 11 different genus were found at the contaminated media and fruit bodies; Enterobactrer, Serratia, Bacillus, Pseudomonas, Ewingella, Raoultella, Uncultured bacterium, Bacterium, Leclercia, Microbacterium, Pantoea, Rahnella. Serratia of the phylum Firmicutes was the most dominant genus (19.0%) in contaminated substrates followed by P seudomonas sp. (14.3%), Bacillus sp. Ewingella sp., and Uncultured bacterium sp. (9.5%), Ewingella sp. and Uncultured bacterium sp. (4.8%). Fungal isolates from liquid spawn during the mushroom cultivation and their diversity were examined using a PCR-based approach. A total of 6 strains were isolated from different contaminated liquid spawn and 4 out of isolated strains which seriously inhibit the mycelial growth of P . eryngii were examined by ITS sequence analysis. Based on sequence analysis of ITS and sequence identity values, a total of 6 different genus were found at the contaminated liquid spawn; Rhizopus, Geotrichum, Ascomycota, Coprinellus, Aspergillus, Peniophora. R hizopus sp. was the most dominant genus (40.0%) in contaminated liquid spawn followed by Ascomycota sp., Aspergillus sp., and P eniophora sp. (20.0%). However, Geotrichum sp. and Coprinellus sp. did not disturb the mycelial growth and formation of fruit body. Fungal isolates from spawn room during the mushroom cultivation and their diversity were examined using a PCR-based approach. A total of 85 strains were isolated from different contaminated liquid spawn and 39 out of isolated strains which seriously inhibit the mycelial growth of P leurotus eryngii were examined by ITS sequence analysis. Based on sequence analysis of ITS and sequence identity values, a total of 8 different genus were found at the contaminated liquid spawn; Penicillium, Aspergillus, Trichoderma, Gibberella, Alternanthera, Rhizopus, Phanerochaete, Trichothecium. Penicillium was the most dominant genus (40.0%) in contaminated liquid spawn followed by Aspergillus (23.1%) , Trichoderma(10.3%), Gibberella (5.1%), Alternanthera, Rhizopus, Phanerochaete, and Trichothecium (2.6%). However, Cladosporium sp., Uncultured fungus, Talaromyces sp., Ascomycete sp., Davidiella sp., M ucor sp. did not disturb the mycelial growth and formation of fruit body. In the case of the contaminated media and fruit bodies, a total of 3 strains were isolated from different contaminated substrates and 2 out of isolated strains which seriously inhibit the mycelial growth of P . eryngii were examined by ITS sequence analysis. Based on sequence analysis of ITS and sequence identity values, a total of 3 different genus were found at the contaminated media and fruit bodies; Gelatoporia, Trichoderma, Penicillium. Although, Gelatoporia sp. did not disturb the normal growth, Trichoderma sp. and P enicillium sp. seriously inhibit the mycelial growth and formation of fruit body of P . eryngii. These results show that a number of microorganisms have the ability to cause growth inhibition in the cultivation of P . eryngii. In the base of these results, we designed four Genus-specific primer pairs for multiplex PCR from 27 different fungi species, about 200 differnet bacteria scpecies, 16 different Bacillus sp., 15 different Pseudomonas sp. Their amplicon size were 400bp, 1,580bp, 380bp, and 300bp, respectively. Non specific amplicons by Genus-specific primer pairs were not observed in genomic DNA of pathogenic bacteria and fungi of P. eyngii. The extracted crude DNA of liquid spawn including pathoges was detected as PCR template successfully. To protect the contamination of liquid spawn in the inoculation process of mycelium. we developed the line inoculation system using the filtered air pressure. For the mass production of liquid spawn, pre-culture was conducted in a 500ml glass bottle. The bottle is comprised of the screw cap including the transmission air line, emission air line, and inoculation line. The mycelium in bottle was transferred to the tank through the sterilized tube by the filtered air pressure. The line inoculation system against to the tank was successfully operated without contamination in manufacturing process of liquid spawn. These results would be an efficient tool to detect and proetect a pathogenic bacteria and fungi in liquid spawn of P . eryngii.
목차 Contents
- 제출문 ... 1
- 요약문 ... 2
- SUMMARY ... 5
- 제1장 서론 ... 7
- 제2장 국내외 기술개발 현황 ... 8
- 제3장 연구개발수행 내용 및 결과 ... 9
- 제1절 연구수행 방법 ... 9
- 제2절 연구결과 ... 13
- 제3절 적요 ... 59
- 제4장 연구개발목표 달성도 및 대외기여도 ... 62
- 제1절 : 목표대비 대외달성도 ... 62
- 제2절 : 정량적 성과 ... 63
- 제5장 연구개발결과의 활용계획 ... 64
- 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 65
- 제7장 기타 중요 변동사항 ... 66
- 제8장 국가과학기술종합정보시스템에 등록한 연구장비 현황 ... 67
- 제9장 참고문헌 ... 68
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