請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/30980
標題: 廣葉杉萎凋病菌的偵測培養基研發與應用
Development of a Medium for Detecting Ophiostoma querci on Chinese Fir and Its Application
作者: 吳宜晏
Wu, Yi-Yan
關鍵字: LLCFC medium
選擇性培養基
ITS
microwave DNA extraction method
Ophiostoma querci
selective medium
廣葉杉萎凋病菌
Ophiostoma querci
LLCFC培養基
微波萃取核酸法
ITS
出版社: 植物病理學系
摘要: 中文摘要 廣葉杉萎凋病菌的偵測培養基研發與應用 吳宜晏 國立中興大學植物病理學系 評估14種培養基對廣葉杉萎凋病菌Ophiostoma querci OPH-106與OPH-110菌絲生長的影響,發現本菌在Leonian agar培養基平板上生長最為快速。進一步,以其去除碳、氮素源的配方作為基礎培養基,分別加入 19種碳素源與19種氮素源後,探討各種碳、氮素源對本菌生長的影響,結果顯示乳糖(lactose)及木糖(xylose)兩種碳素源可顯著促進菌絲的生長。在氮素源方面則以酪蛋白(casein) 搭配乳糖最具有顯著促進菌絲生長的效果。此外,比較不同碳氮素源比例對本菌生長的影響,結果發現在含有0.5%(w/v)乳糖及0.1%(w/v)酪蛋白之培養基(LLC)平板上,本菌的菌絲生長最好。利用1N鹽酸及1N氫氧化鈉將LLC液體培養基之酸鹼值調整為3至10間,發現本菌在酸鹼值4時生長最佳。比較13種化學藥劑對本菌菌絲生長及孢子發芽的影響,結果證明200ppm福多寧(flutolanil)可促進本菌的生長。至於在抗生素方面,鏈黴素與亞胺環己酮兩種各別在100 ppm以下不會抑制本菌菌絲的生長。將麥芽抽出物6.25公克、磷酸氫鉀1.25公克、硫酸鎂0.625公克、乳糖5公克、酪蛋白1公克、洋菜粉20公克及蒸餾水1公升均勻混合,高溫高壓滅菌(121℃,15 lb,15 min.)後,逐一加入福多寧200 mg/L、亞胺環己酮(cycloheximide)100 mg/L及鏈黴素(streptomycin sulfate) 100 mg/L,製成偵測本病菌的Leonian-乳糖-酪蛋白-福多寧-亞胺環己酮(LLCFC)培養基。將接種病原菌的木屑分別於LLCFC、添加200 mg/L鏈黴素的馬鈴薯葡萄糖瓊脂培養基(PDAS)、2%MEA添加100mg/L亞胺環己酮及Reay et al所採用的4號:添加200 mg/L 氯黴素及100 mg/L鏈黴素的YM medium(YMCS)及6號:添加200 mg/L 氯黴素 、100 mg/L鏈黴素及400 mg/L亞胺環己酮的YM medium(YMCSC)等五種培養基平板上,進行本病原菌的分離效率評估,其中以LLCFC培養基最具有偵測到病原菌的效果。此外,以微波快速萃取核酸及修正的Dellaporta Plant DNA Miniprep法搭配ITS專一性引子對,確實可快速檢測本病原菌的存在。以高溫(50℃)處理病原菌孢子懸浮液或帶菌木片,可有效抑制病原菌的生長。又貝芬替500 mg/L、免賴得500 mg/L、腐絕1000 mg/L、撲克拉錳250 mg/L及撲克拉400 mg/L等五種化學藥劑處理帶菌木片一小時後,亦可有效抑制病原菌的存活。
ABSTRACT Development of a Medium for Detecting Ophiostoma querci on Chinese Fir and Its Application Wu Yi-Yan Department of Plant Pathology National Chung Hsing University A medium for detecting Ophiostoma querci, the causal agent of Chinese fir wilt, was developed in this study. Among 14 basal media, best growth rates of isolates OPH-106 and OPH-110 of the pathogen were observed on Leonian agar. By separately introducing 19 different carbon sources into the Leonian basal media, lactose and xylose were identified to be conducive to the mycelial growth of the pathogen. On the other hand, with lactose as a carbon source, casein was found to be most effective in enhancing mycelial growth among the 19 nitrogen sources tested. Moreover, optimal growth for O. querci was observed on the Leonian-lactose-casein (LLC) medium, which included 0.5%(w/v) lactose and 0.1%(w/v) casein at pH 4. Comparing the effects of 13 pesticides on mycelial growth and spore germination, it was found that 200 ppm flutolanil effectively favored the pathogen. Both streptomycin sulfate and cycloheximide at 100 ppm were obviously not effective in inhibiting growth of the pathogen. The Leionian-lactose-casein- flutolanil-cyclohexmide (LLCFC) medium consisting of 6.25g malt extract, 1.25g K2HPO4, 0.625g MgSO4, 5g lactose, 1g casein, 20g agar, 200mg/L flutolanil, 100mg/L cycloheximide and 100mg/L streptomycin sulfate in 1L distilled water was hence formulated for detecting O. querci. Efficacy of the pathogen recovery was evaluated by introducing spore-infested sawdust into five testing media, namely, PDAS, YMCS, YMCSC, MEAC, and LLCFC. LLCFC medium was found to be most effective in detecting the pathogen from the infested sawdust. In addition, rapid detection and identification of O. querci could be achieved by microwave DNA extraction method and amended Dellaporta Plant DNA Miniprep Method with specific ITS primer. In this study, two treatments including that the infested wood chips were baked in the oven at 50℃ for 1 hour and treated with each of 5 pesticides for 1 hour had been developed for control of the pathogen.
URI: http://hdl.handle.net/11455/30980
顯示於類別:植物病理學系

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