衛生害蟲에 대한 無公害殺蟲劑開發에 관한 硏究(I) = Development of Microbial Insecticides Against Medically Important Insects Pests(I)

다국어 초록 (Multilingual Abstract)

I. Objective and Rationale
Chemical insecticides have been conventionally used over the past 40 years for the control of medically important insects, in particular, against disease vector mosquitoes such as Culex tritaeniorhynchus responsible for the transmission of Japanese encephalitis, Anopheles sinensis malaria and inland filariasis, SindAedes togoi malaysian filariasis in southern coastal island of Korean peninsula. However, the advant of insecticidal resistance in many species of disease vector species, particularly mosquitoes together with an increasing problems of environmental pollution and entoxication among man and animals through the direct contact or in medium of food chain brought about a serious consequences, the results of which can no longer be dependent upon solely the use of chemical insecticides. For the reasons previously mentioned, an increasing attention has been focused on the development of naturalistic bioagent the microbial insecticides pathogenic against mosquito vector species such as Bacillus thuringiensis subspecies israelensis known to be most potential with specific lethal activities against mosquitoes and black flies without any harmful side effects to other non-target aquatic insects or associated animals. Thus, it became evident that there, was urgent need for the development of research and investigation on the recovery of a new biological control agent which should possess the desirable properties as microbial pesticide in substitute of conventional insecticide usually requiring highly toxic to target vector species and also able to be mass produced economically on an industrial scale for the practical application in an integrated vector control strategies of our country. Since Goldberg and Margalit (1977) reported Bacillus thuringiensis var. israelensis to be an effective microbial insecticide in controlling mosquito larvae, a continuous efforts were directed to obtain more virulent and toxic strains and formulations by using techniques of mutation, gene cloning and genetic engineering by many scientists throughout the world and is still in progress even though the results were inconclusive or not much in satisfactory as had been initially expected. Present status of the research in our country has also been limited to the recovery of more potential strains or formulations with mostly imported B.t.i. isolates from the U.S.A. and other foreign countries. Therefore, the objectives of our research and investigation will be emphasized on the new recovery of microbial agent, particularly concentrated on Bacillus thuringiensis species and subspecies from the soil samples of different mosquito breeding habitats from different geographical localities, and subsequent B.t. isolation and serological identification will be conducted, with which a series of laboratory and field evaluations against various mosquito vector species to assess potentiality and effectiveness in controlling such target species. The further investigation will be concentrated on the development of more virulent strains and formulations and mass production of the potential ones through the use of different culture media for the operational control of not only mosquito vectors, but also other medically important flies and cockroaches in Korea.
II. The results of our first-year research and investigations were summarized as follows.
1. Recovery and Isolation of B.t. : A total of 1,145 colonies of Bacillus were recovered from soil samples of different survey areas covering 7 different provinces, of which 71 different B.t. isolations were made through the relevant biochemical characteristics tests, and the isolates were preserved in deep freezer at -70°C with each sample being serially numbered.
2. Bioassay of B.t. Isolates against Mosquito Vectors. : Laboratory bioassay of a total of 71 different B.t. isolates were conducted against Cx. tritaeniorhynchus and Cx, pipiens pallens larvae of the earlier 4th instar-stage to determine the level of susceptibility and efficacy for the lethal activities. Among the isolates tested, 51 were shown to be susceptible and, particularly, more effective 5 strains than the others were picked up for further study.
3. Production of Powdery Formulations and Evaluations. : Powdery formulations of each of the 5 selected strains of B.t.L were produced by the use of GYS culture medium (the serological identification of the strains were made through a kindly cooperation by Dr. Ohba in Japan): these were B.t. serotype H-14 of NE-1, NE-7, NE-52, NE-56, and NE-62. The level of susceptibility of 5 different formulations in comparison with the B.t.i. IPS standard were tested and the most susceptible strain was B.t. NE-62 with the value of LC₅₀ against Cx. tritaeniorhynchus and Cx. pipiens pallens were 0.0051 mg/1 and 0.0087mg/l respectively showing 2.8-4.9 folds more susceptible than that of B.t.i. IPS standard of 0.025mg/1 and 0.024mg/l against the cor­ responding species respectively.
4. Confined Field Evaluation of B.t丄 NE^l Powdery Formulation : The confined field treatment of powdery formulation of B.t.i. NE-1 at the dosage rate of 0.5mg/1 in the simulated rice paddy condition at the National Institute of Health outdoor premises produced 100% larval reduction of Cx. tritaeniorhynchus and Cx. pipiens pallens in 24 hours, and 98% control against Aedes togoi the principal vector of fdariasis, the overall results of the foregoing evaluation yielded a satisfactory level of mosquito larval control against 2 vector species.
5. Bioassay against Houseflies and Cockroaches : Laboratory bioassay of 71 different isolates of B.t. were also carried out against houseflies Musca domestica and cockroaches Blattella germanica by the use of baiting in the feed, however there appeared non significance in the adverse effects of the B.t. isolates on growth and reproduction of the tested insects compared with those of controls.

국문 초록 (Abstract)

I. 연구개발의 목적 및 중요성
각종 질병을 매개하는 위생해충을 구제하기 위하여 지난 40여년간 많은 화학살충제가 사용되어 왔으나 저항성해충의 출현, 환경오염, 인축독성 둥의 문제가 심각해져 기존 화학살충제에 의존한 구제는 그 한계에 이르렀다. 따라서 다른 수서곤충이나 동물에는 무해하고 모기유충이나 곱추파리 등에 대해서만 종 특이적 살충효과를 갖는 Bacillus thuringiensis var. israelensis 둥의 무공해 살충제 개발이 주목되고 있다. 이에 위생해충의 종합적 방제전략에 실용될 수 있는 이상적 생물제제의 개발과 대량 생산을 통한 경제성 향상 둥에 대한 조사연구가 시급하다. Goldberg 와 Margalit(1977)이 B. tkuringiensis var. israensis가 모기 유충에 대해 살충효과를 지님을 보고한 이래 전세계의 과학자들은 돌연변이, 유전자클로닝 등 유전공학기법을 통한 역가향상을 유도 하였으나 기대했던 효과를 거두지 못하였다. 또한 국내에서는 외국에서 도입된 B. thuringiensis var.israensis 를 이용한 연구 및 제제수입에 그치고 있는 실정이다. 따라서 본 연구의 목적은 국내 토양에서 각종 모기유충 및 위생 해충에 대해 우수한 독성을 지닌 B. thurirtgiensis 를 분리·선발 하여 이를 토대로 독성효과와 경제성을 향상시킬 수 있는 제조기술 공법을 개발하고자 한다.
II. 연구개발의 내용 및 범위
본 연구에서 수행한 개발내용 및 범위는 다음과 같다.
1. 새로운 균주의 분리·동정 : 전국 7 개도의 22 개 지역에서 토양을 채취하여 이로부터 배양된 1,145개의 Bacillus 집락을 위상차 현미경을 통해 관찰하였으며 동시에 생화학적 시험을 실시한 결과 71개의 B. thuringiensis를 분리하였고 분리된 균주는 一 70℃에 냉동 보관하였다.
2. 모기유충에 대해 독성을 지니는 균주선별 : 71 개 균주의 작은 빨간 집모기 Culex tritaeniorhynchus 빨간 집모기 Cx. piptens palens 유충에 대한 감수성 시험 결과 51 개 균주가 치사효과를 나타내었으며 이중 가장 강한 독성을 지닌 5 개 균주를 선발하였다.
3. 선택균주의 분말제제 제조 및 독성효과 조사 : NE—1 , NE-7, NE-52, NE-56 및 NE-62로 명명된 다섯 균주를 GYS 배지를 이용하여 분말제제화한 후 WHO 표준균주인 BTI — IPS 를 대조균주로 하여 반치사농도를 조사하였다. 다섯균주 중에서도 NE—62의 독성이 가장 강하여 Cx. tritaeniorhynchus 와 Cx. pipi ens pallens 유충에 대한 LC₅₀가 각각 0.0051 mg/l와 0.0087mg/l로 표준 균주의 0.025mg/l 와 0.024mg/l에 비해 2.8~4.9배 우수하였다
4. 분말제제의 야외 구제효과 조사 : 분말제제 NE—1을 처리한 인공야외실험 결과 0.5mg/l의 처리로써 Cx. tritaeni orhynchus와 Cx. pipiens pallens 유충에 대해 100 % 구제효과를 얻었으며 Aedes togoi 유충에는 98%의 구제효과를 나타냈다.
5. 분리균주가 파리 및 바퀴에 미치는 영향 : 71개 분리균주를 집파리 Musca domestica 유충과 독일 바퀴 Blatte germaica 성충이 섭식하도록 유도하여 그 영향을 살펴본 결과 생장과 생식을 저해하는 균주는 관찰되지 않았다.