謝煥儒臺灣大學：植物病理與微生物學研究所陳其敏Chen, Chi-MinChi-MinChen2007-11-272018-06-292007-11-272018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/58051使用採自台北、宜蘭、花蓮、新竹、苗栗、台中、台南、南投、嘉義、雲林、高雄、屏東的土樣樣品453個以及水樣18個，以一齡斑蚊幼蟲做釣餌，從中分離出五株對斑蚊幼蟲有病原性的水生菌，此五株均分離自花蓮縣兆豐農場的土樣，經鑑定後，其中有四株是腐黴菌(Pythium spp.)以及一株Saprolegnia megasperma。土壤分離率為1%，由人工耕地分得的分離率為4%，天然林地及水邊土樣之分離率為0%。將五株水生菌作植物病源性測試，發現Saprolegnia megasperma接種於青梗白菜種子則會造成青梗白菜種子不發芽以及造成褐根，接種四株腐黴菌青梗白菜及莧菜種子的發芽率為80~100%。 四株腐霉菌菌絲最適生長溫度分佈在24-32℃，而Saprolegnia megasperma的最適生長溫度為16℃-32℃，在光線為全暗的狀況下，五株水生菌在4℃以下及40℃以上停止生長，四株腐霉菌也以全暗的情況下生長較佳。Saprolegnia megasperma在不同光照下，則以全光時生長勢最佳。在不同水質中，以魚缸水作處理腐黴菌菌絲塊，四株腐黴菌都可獲得最大游走子產量。除90028外，另外三株腐黴菌培養七天之菌落可獲得最大游走子數。游走子產量隨天數增加而增加，腐黴菌90028則剛好相反，培養七天的菌落游走子產量最少，培養三天的游走子產量最多。90027對一齡埃及斑蚊以及白線斑蚊的半致死濃度分別為7.8 zoospores/μl以及11.9 zoospores /μl，90028對一齡埃及斑蚊以及白線斑蚊的半致死濃度分別為18.1 zoospores /μl以及16.6 zoospores /μl，90034對一齡埃及斑蚊以及白線斑蚊的半致死濃度分別為36. 5 zoospores /μl以及30.7 zoospores /μl，90037對一齡埃及斑蚊以及白線斑蚊的半致死濃度分別為27.1 zoospores /μl以及35.6 zoospores /μl。 觀察接種24小時的一齡斑蚊幼蟲，死亡的斑蚊幼蟲在肛節腮有大量團狀菌絲，夾雜著部分靜止子。接種48小時後觀察，大多數被感染斑蚊幼蟲體軀長出孢囊，嚴重者整隻被白色菌絲包圍住，只露出部分身體。在掃描式電子顯微鏡下觀察接種2小時的斑蚊幼蟲，僅少數一、兩隻可在體表觀察到少量靜止子附著，接種四小時，已可在斑蚊幼蟲肛節腮處觀察到有靜止子大量聚集附著的現象，觀察接種六小時的斑蚊幼蟲，除觀察到有靜止子大量聚集，還可發現多數靜止子都已經發芽。The first-instar larvae of Aedes aegypti and Aedes albopcitus were used as baits for isolation of mosquito-parasitic fungi from soil and water samples collected from different locations in Taiwan. Totally, 453 soil samples and 18 water samples collected from Taipei, Ilan, Hualien, Hsinchu, Miaoli, Taichung, Tainan, Nanto, Chiayi, Yunlin, Kaohsiung, and Pintung, respectively were tested. Mosquito-parasitic fungi were isolated from 5 soil samples, all from cultivated sites (cultivated fields, managed grasslands, gardens). Four isolates of Pythium spp. and one isolate of Saprolegnia ( Saprolegnia megasperma ) were isolated from the soil samples collected from the farm in Hualien. In the pathogenicity test on Amaranthus mangostanus and Brassica chinensis seedlings and seeds, it showed brown roots on seedlings and 0% germination rate on seeds when Saprolegnia megasperma were inoculated. The four Pythium spp. won’t cause brown root on seedlings and showed 80%~100% germination rate on seeds. Optimum temperature range for mycelial growth of the four Pythium spp. was 24-32℃and the Saprolegnia megasperma was 16-32℃. In the dark, the four Pythium spp. won’t growing when temperature below 4℃ and above 40℃. The Saprolegnia megasperma grows better in the light. Treating the four Pythium spp. agar blocks with different kinds of solution, we had maximum zoospore production with tap water that has been put into a fish tank for 7 days. The zoospore production were increase with the period of hyphae growing but the zoospore production of 90028 was reverse. The LC50 of the four Pythium spp. were 7.8 -36. 5 zoospores/μl of Aedes aegypti and 11.9-35.6 zoospores /μl of Aedes albopictus. Using zoospores of Pythium spp. as inocula to infect the first-instar larvae, scanning electron micrographs shows cyst attaching to the anal gill and few between head and thorax in 4 hours. Photomicrograph of inoculation Pythium sp. zoospores on first-instar larvae in 24 hours, hyphae growth from the anal gill and some cyst aggregate either. Some infected larvae were surrounded by hyphae and some formed sporangia. It shows potential to introduce Pythium spp. as biocontrol agents against larvae of Aedes aegypti and Aedes albopcitus.目 錄 中文摘要 二 英文摘要 四 目錄 六 圖目錄 七 表目錄 八 壹、緒論………………………………………………………...….1 貳、往昔研究………………………………………………….….3 一、孑孓之生物防治……………………………………..…3 二、腐黴菌游走子之產量促進、趨性與致病活性…..….9 參、材料與方法………………………………..……………….14 一、土壤樣品及水樣的採集與處理……...……………...…..14 二、水生菌之分離……………….…………………...……...….14 三、具寄生性水生菌之鑑定………………….…………….....15 四、水生菌對青梗白菜及莧菜病原性測試….………….…16 五、水生菌在不同條件下生長速率測定………….….….....16 六、不同條件對腐霉菌游走子產生之影響………...……....16 七、實驗蟲源及飼養……………………….………….…...…...17 八、腐黴菌游走子的製備…………………..………..……..….18 九、半致死濃度之測定…………………………….…………..18 十、掃描式電子顯微鏡(SEM)材料之製備………….....…..18 肆、結果…………………..…………………….………………...19 一、水生菌的分離及純化………………………………..…..…19 二、水生菌在不同條件下生長速率測定………………....27 三、水生菌對青梗白菜及莧菜之病原性測試…..……..…33 四、不同水質以及菌株生長日數對游走子產生之影 響………………………….………………….…...…..35 五、半致死濃度之測定…………………………………………45 六、寄生情形之觀察…………….……….……..……………….47 伍、討論………………………….….……….……….…..…52 陸、參考文獻………………………………………..….……58702283 bytesapplication/pdfen-US水生菌斑蚊幼蟲生物防治watermoldsbiocontrolotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/58051/1/ntu-93-R91633002-1.pdf