버섯파리에 살충활성을 나타내는 새로운 Bacillus thuringiensis 균주를 분리 동정과 균주의 활용가능성 검토 및 생물학적 특성을 밝히며, 균주의 내독소단백질과 균주를 활성화 시키는 알려지지 않은 버섯파리의 단백질 분해효소를 분석하였고 활성을 나타내는 plasmid DNA의 분자유전학적 특성을 구명하고자 다양한 실험을 통해 다음과 같은 결과를 얻었다.

1. 버섯파리 재배사의 난방제 해충으로 알려진 버섯파리를 방제하기 위하여 토양으로부터 Bacillus thuringiensis 균주를 분리 동정하였다. B. thuringiensis subsp. kyushuensis CAB464로 동정되었으며, 파리목 해충인 버섯파리에 대해 높은 독성으로 속효성 살충효과를 확인할 수 있었다. B. thuringiensis CAB464 균주는 버섯파리의 섭식 및 용화와 우화를 저해하는 것을 확인할 수 있었으며, 해충에 직접 작용하기도 하지만, 섭식을 저해하여 아사시키는 살충기작도 가지고 있음을 알 수 있었다. 버섯농가에 이용가능성 및 방제효과를 보기 위하여 포트 검정한 결과, B. thuringiensis CAB464 균주는 80.1%의 방제가로 무처리구보다 약 50% 이상의 살충활성을 나타내며 생충율도 무처리구보다 약 38% 이상의 감소를 나타내고 있는 반면에 무처리구는 점점 증가하고 있는 것으로 나타났다. 버섯파리의 전령기기 혼재된 버섯 재배사에서 방제효과를 검정한 결과, 방제가는 1회 처리 후 75.6%, 2회 처리 후에는 81.7%를 나타내었고, 1차 처리보다 2회 처리에서 방제가는 더 높아졌으며, 생충율은 점점 감소하는 것을 확인할 수 있었다. 이를 통해 B. thuringiensis CAB464 균주는 버섯파리를 방제할 수 있는 신규 미생물살충제로서의 이용가능성을 가지는 것으로 나타난다.

2. 버섯파리에 살충활성을 나타내는 B. thuringiensis CAB464 균주의 내독소단백질의 특성을 확인하고 내독소단백질을 용해하는 알려지지 않은 버섯파리의 단백질분해효소를 분석하였다. B. thuringiensis CAB464 균주는 약 150, 70, 65, 50, 27 kDa 크기의 주요 단백질 밴드 패턴을 형성하였고, 활성독소 양상을 확인하기 위해 소화효소인 trypsin을 처리하였을 때 약 65, 60, 50, 27 kDa의 단백질 밴드를 형성하는 것을 확인할 수 있었다. B. thuringiensis CAB464 균주의 활성독소는 7일차까지 유지하다가 점점 옅어지기 시작하여 15일차에는 밴드가 거의 보이지 않았으며, 이를 통해 활성 독소가 버섯파리 유충에도 지속될 수 있음을 예상할 수 있었다. 버섯파리의 protease 효소도를 측정한 결과, 100, 45, 37, 27 kDa 밴드를 나타났으며, 단백질 분해효소 활성은 pH 7.5~9.5, 37℃에서 최적의 활성을 나타내었다. 버섯파리의 protease는 tannic acid 반응에서 약 80.3%로 가장 효과적으로 억제활성을 나타내었고, 이는 버섯파리 중장액이 serine protease로 속하는 것으로 판단된다. Protease의 특성을 밝히기 위해 높은 기질 특이성을 이용한 결과, trypsin의 기질인 BPVApNA는 91.1±1.9%를 나타내었으며, 버섯파리의 protease는 밝혀진 바가 없기 때문에 버섯파리의 protease 연구에 기초 자료가 될 것으로 사료된다.

3. B. thuringiensis CAB464 균주의 crystal protein에 관여하는 plasmid DNA의 분자유전학적 특성을 구명하고자 하였다. B. thuringiensis CAB464 균주가 가지고 있는 cry genes을 확인해본 결과, cyt2Aa 유전자를 가지고 있음을 알 수 있었다. 모기 유충에 살충활성이 있다고 보고되어 있으며, cyt2Aa 유전자로 인해 버섯파리에 대한 살충활성이 높음을 추측할 수 있었다. Crystal protein이 생성되지 않는 mutant 균주는 spore만이 관찰되어 crystal protein을 생성하는 능력이 소실되었으며, 버섯파리에 생물활성을 검정한 결과, 107(cfu/ml) 배양액에서 최대 16.7%의 사충률을 나타내며, 살충활성 능력을 가지지 않는 것으로 판단된다. Crystal protein을 형성하지 않기 때문에 균주가 갖는 crystal protein band에서 결실이 나타남을 확인할 수 있으며, cyt gene이 존재하지 않음을 의미한다. 따라서, B. thuringiensis CAB464의 mutant 균주는 살충성 단백질이 완전히 존재하지 않음이 확인 되었다. B. thuringiensis CAB464 균주가 가지고 있는 cyt2Aa 유전자의 cloning을 통해서 cyt2Aa 유전자는 버섯파리의 살충활성에 관여함을 확인하였다.

A new Bacillus thuringiensis strain that shows insecticidal activity to Lycoriella ingenua was separated and identified, the usability of the strain was reviewed, the biological characteristics of the strain were determined, the endotoxin protein of the strain and an unknown protease of L. ingenua that activates the strain were analyzed, and diverse experiments were conducted to identify the molecular genetic characteristics of the plasmid DNA that shows the activity. Through the experiments, the following results were obtained.
1. To control L. ingenua, which is known to be an intractable pest in mushroom cultivation facilities, a Bacillus thuringiensis strain was separated and identified from soil. The relevant strain was identified as B. thuringiensis subsp. kyushuensis CAB464 and its fast-acting insecticidal effect could be identified with its high toxicity to L. ingenua, which is a Diptera pest. It could be identified that B. thuringiensis strain CAB464 hinders the food intake, pupation, and emergence of L. ingenua and it could be seen that although strain CAB464 directly acts on pests, it also has the insecticidal mechanism to hinder food intake leading to starvation to death. Pot tests were conducted to examine its usability to mushroom farms and its control effects and according to the results, B. thuringiensis strain CAB464 showed a control value of 80.1%, which was higher by around 50% than that of the untreated control plot, and a pest survival rate, which was lower by around 38% than that of the untreated control plot. On the contrary, the untreated control plot showed gradually increasing survival rates. When the control effects of B. thuringiensis strain CAB464 were tested in a mushroom cultivation facility in which all age groups of L. ingenua were mixed, the control value was shown to be 75.6% after one time of treatment and 81.7% after two times of treatment. Therefore, the control value, which increased after the first treatment, increased further after the second treatment and it could be seen that the pest survival rate gradually decreased indicating that B. thuringiensis strain CAB464 is usable as a new microbial insecticide that can control L. ingenua.

2. The characteristics of the endotoxin protein of B. thuringiensis strain CAB464 that shows nsecticidal activity to L. ingenua were identified and an unknown protease of L. ingenua that dissolves the endotoxin protein was analyzed. B. thuringiensis strain CAB464 formed major protein band patterns of around 150, 70, 65, 50, and 27 kDa sizes and when B. thuringiensis strain CAB464 was treated with trypsin, which is a digestive enzyme, it could be identified that protein bands of around 65, 60, 50, and 27 kDa sizes were formed. The active toxin of B. thuringiensis strain CAB464 was maintained under day 7 and began to become thinner thereafter so that almost no band could be seen on day 15. Therefore, it could be expected that the active toxin can be maintain in the larva of L. ingenua too. The zymogram of the protease of L. ingenua was measured and the results showed bands of 100, 45, 37, and 27 kDa and the protease showed the optimum activity at pH 7.5~9.5, 37℃. The protease of L. ingenua showed the most effective activity reaching around 80.3% in tannic acid reactions indicating that the midgut fluid of L. ingenua belongs to serine proteases. The high substrate specificity of the protease was used to identify the characteristics of the protease and according to the results, BPVApNA, which is the substrate of trypsin, showed a specificity of 91.1±1.9%. This is considered to become basic data for studies of the protease of L. ingenua because no specificity or the protease of L. ingenua has been identified earlier.

3. Attempts were made to identify the molecular genetic characteristics of the plasmid DNA that is involved in the crystal protein of B. thuringiensis strain CAB464. On checking the cry genes of B. thuringiensis strain CAB464, it could be seen that B. thuringiensis strain CAB464 has cyt2Aa gene. Since cyt2Aa gene has been reported to have insecticidal activity to mosquito larvae, it could be assumed that B. thuringiensis strain CAB464 has high insecticidal activity to L. ingenua thanks to cyt2Aa gene. In the case of mutant strains in which no crystal protein is generated, only spores were observed indicating that the ability to generate crystal proteins was lost and the biologic activity of L. ingenua was tested and according to the results, a mortality rate of 20% at the maximum was shown in a 107(cfu/ml) culture medium indicating that the mutant strains have no insecticidal activity. Since no crystal protein was formed, it could be identified that deletion appeared in the crystal protein bands of the strains, which means that no cyt gene exists. Therefore, it was identified that no insecticidal protein exists in B. thuringiensis CAB464 mutant strains. Through cloning of the cyt2Aa gene of B. thuringiensis CAB464 showed that the cyt2Aa gene was involved in the insecticidal activity of L. ingenua.