초록

○ 연구결과
1. P. penetrans의 분류 및 조기동정을 실시하여 뿌리혹선충의 생물학적 방제제로 이용
2. P. penetrans의 제형화를 실시하여 장기간 보존할수 있는 동시에 안전하게 뿌리 혹선충을 방제하여 상품성을 극대화 할 수 있음
3. P. penetrans을 대량 배양하여 농가에서의 뿌리혹선충 생물학적 방제에 이용
4. 뿌리혹선충의 조기동정기술개발에 성공하여 조기에 농가에서의 뿌리혹선충의 종류를 확인하여 효과적인 방제에 이용

Abstract ▼

Ⅳ. Results and suggestions
1. Classification of Pasteuria spp. and development of rapid identification technique
a. Due to stagnancy in the horticultural cultivation areas, nematode accumulation in the soils has occurred predominantly in the recent years. The biological control of nematodes us

Ⅳ. Results and suggestions
1. Classification of Pasteuria spp. and development of rapid identification technique
a. Due to stagnancy in the horticultural cultivation areas, nematode accumulation in the soils has occurred predominantly in the recent years. The biological control of nematodes using Pasteuria spp. is an eco-friendly approach for their control. The method of their classification and identification was established.
b. The genetic homology between two local Pasteuria spp. kw-1 and kw-2 was found to be 91%, where as that of two species deposited in GenBank, (P. penetrans, AF375881 and AF 077672) was found to be 99%. This implies that the local Pasteuria spp. KW-1 and KW-2 were genetically heterogeneous to that of the previously deposited species.
c. In order to prepare specific probe from Pasteuria spp., the 16s rRNA gene was amplified from chromosomal DNA of Pasteuria and 'Pas F-R' sequence was used as prospective probe for its identification.
d. Identification of Pasteuria spp. directly from soil was attempted, but failed to obtain any results. However, the detection of Pasteuria spp. using the probe is possible if there would be an easy method for Pasteuria spp. isolation from soil samples.
2. Development of formulation for P. penetrans and Safety evaluation
a. P. penetrans can be successfully suppressed Meloidogyne spp. in the melon, lettuce and tomato and this suppression effect will increase in the repeated cultivation.
b. P. penetrans was viable under wide range of pH levels and temperatures, and this bacteria can be used for biological control of M. arenaria.
c. The suppression effect of P. penetrans KW-1 after formulation was above 77%, it is similarly the suppression effect of P. penetrans KW-1 endospores.
d. When administration of 1.7×10⁸ endospores P. penetrans KW-1 WP to rat (SD), there were no residue in the small intestine, large intestine and paunch, no phenomena of congestion, decoloration, bleeding around the rat's mouth, congestion, and no discoloration near the anus and genital organ after 7, 14, 21 days.
e. After the administration of 1.7×10⁸ endospores P. penetrans KW-1 to SD rat, above 92% bacteria were exhausted in the feces at the first day, and no bacteria were found on the 7, 14, 21 day's feces. The LD50 value of P. penetrans KW-1 in rat was above 1.7×10⁸ endospores.
3. Analysis characteristic and mass production of P. penetrans
a. In order to mass produce P. penetrans with tomato in the field, firstly P. penetrans was activited for 60 min at 50℃ mixed with Meloidogyne spp., and the mixture of P. penetrans and root knot nematode was centrifuged (3000rpm, 5min), then shaking at 140rpm for 12hrs, effective attachment was obtained.
b. Meloidogyne spp. attached with P. penetrans were mixed with soil, and injected to 12-weeks-old tomato root at the level of 15,000 J2. After 40-50 days, mass P. penetrans were harvested.
c. Tomato hairy root were obtained with the method of transformation by Agrobacterium rhizogene (strain A4). After attachment by shaking at 140rpm for 12hrs, P. penetrans and root knot nematode were sterilized with 0.5% sodium hypochlorites solution, then injected into 12-weeks-old tomato root around, mass P. penetrans were harvested.
d. Proliferation of P. penetrans in vitro by hairy-root needed 35 days, it is faster for about 12 days than that proliferation in the field which needed 49 days.
e. Although it is possible to proliferate the P. penetrans with hairy root, there were several demerits such as contamination of medium, deficiency of nutrition after the decoloration of hairy root, and low re-appearance, high-cost, it is necessary to study deeply in the future.
4. Development of techniques for rapid and precise identification of root-knot nematodes
a. Survey was conducted on the distribution of root-knot nematodes (Meloidogyne spp.) in vegetable production areas in Korea. Soil samples were collected from greenhouses in Sungju (Kyungpook), Pusan (Kyungnam), Yeoju and Pyeongtaek (Kyungki), and Goheung (Chonnam) provinces. Using the female enzyme phenotypes of MDH and EST, the four major root-knot nematodes, M. incognita (MI), M. arenaria (MA), M. hapla (MH), and M. javanica (MJ) could be identified. The major species of root-knot nematode found in Sungju area were MA, MI, and few unknown species of Meloidogyne sp., and in Pusan area were MI and unknown species of Meloidogyne. In Goheung area, MH and MJ were identified. MH and MA were detected in Yeoju and MJ and MI were found in Pyeongtak area.
b. Isozyme phenotype analysis was conducted for the identification of major Meloidogyne species. Two to five fresh females of root-knot nematodes were isolated from infected plant roots and used for isozyme phenotype analysis. Electrophoresis was performed and esterase and malate dehydrogenase were stained. The phenotypic characteristics of esterase and malate dehydrogenase could separate the 4 major root-knot nematode species and unknown species of Meloidogyne isolates in Korea.
c. Partial mitochondrial DNA from single female or second stage juvenile (J2) of root-knot nematodes was amplified by PCR (Polymerase chain reaction), and the further analysis by RFLP (Restriction fragment length polymorphism) provided discriminatory profiles useful for 4 major Meloidogyne species, MA, MI, MJ, and MH in Korea. The sizes of PCR product (1.7 kb and 500 bp) and restriction patterns obtained from single female nematodes were consistent with the results from single J2 within the same species. MH was easily differentiated from the two other root-knot nematode species by the size of the PCR products. A fragment of 500 bp was generated from MH, while MA, MI, and MA produced an 1.7 kb fragment in PCR amplification. MA and MJ could be distinguished from MI by analysis of restriction enzyme digestion by Hinf I or Alu I. Hinf I had no digestion site in mitochondrial DNA of MA and MJ; however, it generated 1.3 kb and 400 bp fragments in MI. Alu I digestion resulted in 1 kb, 460 bp, and 250 bp fragments in MA and MJ, but showed different digestion patterns in MI by generating 800 bp, 460 bp, 250 bp, and 150 bp fragments. MA and MJ could be separated by Mse I digestion in that MJ produced the 85 bp while MA showed 160 bp in the biggest size of band.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 9
• CONTENTS ... 14
• 목차 ... 17
• List of figure ... 20
• List of table ... 23
• 제1장 연구개발과제의 개요 ... 25
• 제1절 연구개발의 필요성 ... 25
• 제2절 연구개발의 목적과 범위 ... 28
• 제2장 국내외 기술개발 현황 ... 29
• 제3장 연구개발 수행내용 및 결과 ... 30
• 제1절 P. penetrans의 분류 및 조기동정기술개발 ... 31
• 1. 서론 ... 32
• 2. 재료 및 방법 ... 34
• 1) 유전자분석을 통한 분류체계확립 ... 34
• 2) 단백질 분석에 의한 분류체계 확립 ... 36
• 3) DNA probes를 이용한 세균의 조기동정 및 monitoring 기술개발 ... 37
• 3. 결과 및 고찰 ... 38
• 1) 유전자분석을 통한 분류체계확립 ... 38
• 2) 단백질 분석에 의한 분류체계 확립 ... 47
• 3) DNA probes를 이용한 세균의 조기동정 및 monitoring 기술개발 ... 48
• 4. 결과 요약 ... 53
• 제2절 P. penetrans의 제형기술개발 및 안전성평가 ... 54
• 1. 서론 ... 55
• 2. 재료 및 방법 ... 57
• 1) 세균의 제형에 따른 방제효과 비교검정 ... 57
• 2) 세균의 제형이 세균의 물리 및 화학적 안정성에 미치는 영향 ... 59
• 3) 제형이 세균의 생존율에 미치는 영향 ... 60
• 4) 안전성평가 ... 61
• 2. 결과 및 고찰 ... 64
• 1) P. penetrans를 이용한 Meloidogyne spp. 방제효과 검정 ... 64
• 2) 제형이 P. penetrans의 물리 및 화학적 안정성에 미치는 영향 ... 70
• 3) 제형이 P. penetrans의 생존율에 미치는 영향 ... 72
• 4) P. penetrans KW-1 제형화에 따른 안전성평가 ... 75
• 4. 결과 요약 ... 78
• 제 3 절 P. penetrans의 생물학적 특성분석 및 대량 배양기술 개발 ... 79
• 1. 서론 ... 80
• 2. 재료 및 방법 ... 83
• 1) P. penetrans과 Meloidogyne spp.간의 생물학적 특성분석 ... 83
• 2) P. penetrans의 Meloidogyne spp.간의 효과적인 부착기술개발 ... 83
• 3) 토마토를 이용한 P. penetrans의 대량배양기술 개발 ... 84
• 4) 토마토의 모상근을 이용한 P. penetrans의 대량배양기술 개발 ... 87
• 3. 결과 및 고찰 ... 88
• 1) P. penetrans와 뿌리혹선충간의 생물학적 특성분석 ... 88
• 2) P. penetrans의 효과적인 선충부착기술 개발 ... 89
• 3) 토마토를 이용한 P. penetrans의 대량 배양기술개발 ... 90
• 4) 토마토의 모상근을 이용한 P. penetrans의 대량배양기술 개발 ... 99
• 4. 결과 요약 ... 102
• 제4절 뿌리혹선충의 분류 및 조기동정기술개발 ... 103
• 1. 서론 ... 104
• 2. 재료 및 방법 ... 106
• 1) 뿌리혹선충의 분포 조사 및 뿌리혹선충의 단백질 및 효소분석에 의한 분류 ... 106
• 2) 뿌리혹선충의 DNA 추출기술 개발 및 RAPD, RFLP 분석을 통한 유전자 진단체계의 개발 ... 107
• 3. 결과 및 고찰 ... 109
• 1) 뿌리혹선충 의 분포 조사 및 뿌리혹선충의 단백질 및 효소분석에 의한 분류 ... 109
• 2) 뿌리혹선충의 DNA 추출기술 개발 및 RAPD, RFLP 분석을 통한 유전자 진단체계의 개발 ... 114
• 3) 뿌리혹선충의 PCR-RFLP 분석을 통한 유전자 진단체계의 개발 ... 117
• 4. 결과 요약 ... 127
• 제4장 목표달성도 및 관련분야의 기여도 ... 128
• 1. 연차별 연구개발목표와 내용 및 달성도 ... 128
• 2. 연구개발목표의 관련분야의 기술발전에 대한 기여도 ... 129
• 제5장 연구개발결과의 활용계획 ... 131
• 제6장 참고문헌 ... 133
• 끝페이지 ... 139