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 China Fir and Its Application

吳宜晏(Yi-Yan Wu) ； 林宗俊(Tsung-Chun Lin) ； 姜保真(Israel Bau-Jen Jian) ； 廖天賜(Tien-Szu Liao) ； 黃振文(Jenn-Wen Huang)

《農林學報》 60卷3期 (2011/09) Pp. 243-262

https://doi.org/10.30089/JAF.201109.0006

摘要

本研究主要目的在於研發廣葉杉萎凋病菌Ophiostoma querci的偵測培養基，首先將麥芽抽出物6.25g、磷酸氫鉀1.25g、硫酸鎂0.625g、乳糖5g、酪蛋白1g、洋菜粉20g及蒸餾水1公升均勻混合，經高溫高壓滅菌（121℃，15lb，15 min.）後，逐一加入福多寧（flutolanil）200 mg/L、亞胺環己酮（cycloheximide）100mg/L及鏈黴素（streptomycin sulfate）100mg/L，即製成偵測本病菌的Leonian－乳糖－酪蛋白－福多寧－亞胺環己酮（LLCFC）培養基。將接種本病原菌的木屑分別置於LLCFC（Leonian－乳糖－酪蛋白－福多寧－亞胺環己酮培養基）、PDAS（在馬鈴薯葡萄糖瓊脂培養基添加200 mg/L鏈黴素）、MEAC（在麥芽萃取物瓊脂培養基添加100 mg/L亞胺環己酮）、YMCS（在酵母及麥芽萃取物瓊脂培養基添加200 mg/L氯黴素與100 mg/L 鏈黴素）及YMCSC（在酵母及麥芽萃取物瓊脂培養基添加200 mg/L氯黴素、100 mg/L鏈黴素及400mg/L亞胺環己酮）等五種培養基平板上，進行評估它們分離本病原菌的效果，結果發現LLCFC培養基偵測本病原菌的效果最佳。進一步利用LLCFC培養基評估乾熱與化學藥劑處理木片防治本病原菌的效果，發現以高溫處理病原菌孢子懸浮液（50℃）或帶菌木片（90℃），可有效抑制病原菌的生長。此外，以貝芬替500mg/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。此外，以修正之Dellaporta PlantDNA Minipreparation 法搭配ITS引子對（ITS1-F，5’-CTTGGTCATTTAGAGGAAGTAA-3’及ITS4，5’-TCCTCCGCTTATTGATATGC-3’），證實LLCFC培養基分離到的菌類確為O.querci。

關鍵字

選擇性培養基 ； 廣葉杉萎凋病菌 ； Ophiostoma querci ； LLCFC培養基 ； ITS

並列摘要

A semiselective medium, Leionian-lactosecasein-flutolanil-cyclohexmide (LLCFC) medium, for detecting Ophiostoma querci, the causal agent of China fir wilt, was developed in this study. The 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 formulated for detecting O. querci. Recovery efficacy of the pathogen was evaluated by introducing spore-infested sawdust into five testing media, namely PDAS, YMCS, YMCSC, MEAC, and LLCFC. LLCFC medium was most effective in detecting the pathogen from the infested sawdust. The infested wood chips baked in the oven at 90℃ for 1 hour or treated separately with five different pesticides for 1 hour were used for the evaluation of the treatment efficacy by the LLCFC medium. The names and concentrations of each pesticide used were Carbendazim 500 mg/L, Benomyl 500 mg/L, Mertect 1000 mg/L, Prochloraz-Mn 250 mg/L and Prochloraz 400 mg/L. Both treatment methods were proved to be effective in controlling the pathogen. In addition, the pathogens isolated by LLCFC medium, were identified and confirmed as O. querci by using a modified Dellaporta Plant DNA Minipreparatio method in combination with the ITS primers (ITS1-F, 5'-CTTGGTCATTTAGAGGAAGTAA-3' and ITS4, 5'-TCCTCCGCTTATTGATATGC-3').

並列關鍵字

LLCFC medium ； ITS ； Ophiostoma querci ； Selective medium