臺灣博碩士論文加值系統

黑殭菌( M. anisopliae )為真菌中之不完全菌，具有昆蟲病原性，可供作發展作為真菌性殺蟲劑。探討黑殭菌菌株之DNA指模(DNA fingerprinting)及找出特殊分子標記(molecular marker)作為商品化註冊及菌種鑑定之依據為重要課題。由台灣及各國共收集黑殭菌15株菌株，寄主範圍分佈於鱗翅目、鞘翅目、直翅目、蜚蠊目及同翅目；另亦將擬青霉菌(Paecilomyces javanica) PJ-H菌株作為比較。以黑殭菌外觀形態、抗 UV 光、抗藥（benomyl）性及昆蟲致病性之試驗結果，各分生孢子大小以 HO194菌株的分生孢子顯著較小，2575菌株的分生孢子顯著較大；抗UV光效果以538及MA-126兩菌株顯著較好；抗藥性以MAUZ-1及MA-126最佳；對於昆蟲致病力而言，所有菌株對斜紋夜盜之致病力皆低，對蚜蟲致病力及埃及斑紋之致病力各菌間有顯著性差異；另外將各菌株培養於SDA液體培養基，經25℃、7天的振盪懸浮培養(130rpm)後收集菌絲，用改良式SDS方法由菌絲萃取genomic DNA作為RAPD-PCR供試材料。所有菌株利用40種random primers(Operon)經RAPD-PCR作用後之結果分析，發現寄主間親緣關係愈接近，其基因多形性相似性愈高，尤其為同一種寄主所分離出來的菌株相似性最高；不同地緣所分離的菌種具差異基因多形性；從抗 UV 光、抗藥性及昆蟲致病性之試驗與RAPD-PCR 之結果分析得知，黑殭菌MA-126菌株是由MA-1誘變所得之抗藥性耐UV菌株，兩種具有差異之基因多形性，故證實MA-126為突變菌株，另外，菌株MAUZ-1及KCAL為MA-126所篩選出之菌株，在抗 UV 光、抗藥性及昆蟲致病性之試驗結果以和MA-126有所差異，在RAPD-PCR分析結果亦顯示有特異的DNA多形性

Green muscardine fungi, Metarhizium spp., are entomopathogenic deuteromycetes and potentially valuable for developing into myco-insecticides. Determining the DNA fingerprinting and locating molecular markers of the different isolates of Metarhizium spp. are of vital importance to the registration of fungal products as myco-insecticides or to the identification of fungal species. There were a total of 15 isolates of Metarhizium anisopliae and a PJH isolate of Paecilomycetes javanicus collected from various places in Taiwan and other countries. The hosts from which the fungi were isolated included Lepidoptera, Coleoptera, Orthoptera, Blattaria and Homoptera. Based on the morphological characteristics, UV resistance, chemical resistance against benomyl, and entomopathogenicity, a comparison was made among the 15 isolates. The results indicated that the condiospores of the isolate HO194 were the smallest and those of the isolate 2575 were the largest. Of the 15 isolates, 14 were determined as mutants of M. anisopliae var. anisopliae on the basis of the characteristics of their conidia. The two isolates, 538 and MA-126, were significantly more resistant against UV light at 250 nm and MAUZ-1 and MA-126 were more resistant against the fungicide, benomyl. In terms of entomopathogenicity, all isolates had low virulence against Pseudaletia unipuncta. There were, however, significant differences in pathogenicity against aphids and Aedes aegypti. In addition, all of the isolates were cultured in SDB (Sabouraud Dextrose Broth) liquid media at 25C and agitated at 130 rpm for 7days before the mycelia were collected. The mycelia were then extracted with the modified SDS method. The extracted genomic DNA was analyzed by RAPD-PCR with 40 different random primers (operons). The results indicated that the closer the genetic relationship among the hosts was the higher the similarity of the DNA polymorphism; e.g., MA-1 and 35520. On the contrary, the isolates from different geographical origins had different genotypings; e.g., 683, 2575 and 3604. When comparing the UV light resistance, chemical resistance, and entomopathogenicity as well as the results of the RAPD-PCR analysis, it was determined that the isolate MA-126 was a mutant strain induced from the isolate MA-1. This determination was confirmed by the differences found in the DNA polymorphism between MA-126 and MA-1. Although MAUZ-1 and KCAL were selected from MA-126, their UV resistance, chemical resistance and entomopathogenicity were different from that of MA-126. This was also confirmed by the unique DNA polymorphism using the RAPD-PCR analysis. Therefore, the RAPD-PCR analysis could be used not only in identifying fungal isolates from different hosts basing on their genetic similarity or their geographical origins, but also in determining their physiological and biochemical differences in UV resistance, chemical resistance and entomopathogenicity.

壹、 前言-----------------------------------------------------1
貳、 前人研究-------------------------------------------------7
一、 蟲生真菌資源---------------------------------------------7
二、 黑殭菌(Metarhizium spp.)之歷史---------------------------9
三、 黑殭菌的形態與分類地位----------------------------------12
四、 黑殭菌之生物學------------------------------------------13
五、 黑殭菌之致病作用機構------------------------------------22
六、 黑殭菌對哺乳動物的毒性及其他非標的生物的毒性及影響------28
七、 RAPD-PCR在蟲生真菌分類與鑑定之應用----------------------31
參、 材料與方法----------------------------------------------40
一、 菌種來源------------------------------------------------40
二、 分生孢子外部形態之掃描式電子顯微鏡觀察------------------40
三、 孢子對UV之抵抗性試驗------------------------------------42
四、 菌落對農藥免賴得(benomyl)之抗藥性檢測-------------------43
五、 供試昆蟲致病力試驗之菌株接種源培養----------------------44
六、 斜紋夜盜(Spodoptera litura Fabricius)之致病力試驗-------45
七、 對棉蚜(Aphis gossypii)之致病力試驗----------------------46
八、 對埃及斑蚊(Aedes aegypti Linnaeus)之致病力試驗----------47
九、 菌株培養與菌株塊收集------------------------------------48
十、 染色體DNA(genomic DNA)萃取------------------------------49
十一、 DNA定量-----------------------------------------------51
十二、 RAPD-PCR----------------------------------------------52
肆、 結果----------------------------------------------------55
一、 黑殭菌分生孢子外部形態之掃描式電子顯微鏡(SEM)觀察-------55
二、 黑殭菌分生孢子對UV之抵抗性比較--------------------------60
三、 不同黑殭菌菌株在SDA平板培養基之生長比較-----------------63
四、 不同黑殭菌菌株對農藥免賴得(benomyl)之抵抗性比較---------65
五、 不同黑殭菌菌株對斜紋夜盜(Spodoptera litura)之致病力比較-----------------------------------------------------------------68
六、 不同黑殭菌菌株對棉蚜(Aphis gossypii)之致病力比較--------74
七、 不同黑殭菌菌株對埃及斑蚊(Aedes aegypti)之致病力比較-----77
八、 不同黑殭菌菌株及擬青黴菌利用RAPD-PCR擴增DNA之圖譜分析---81
伍、討論----------------------------------------------------111
陸、參考文獻------------------------------------------------122

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