Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31012
標題: 偵測靈芝菌的選擇性培養基與專一性引子之開發與應用
Development of a Selective Medium and the Specific Primer for Detecting Ganoderma lucidum and Their Application
作者: 林竑廷
Lin, Hung-Ting
關鍵字: Ganoderma lucidum
Ganoderma lucidum
Ling-Chih
selective medium
specific primer
detection
compost
sawdust waste
urea
靈芝
選擇性培養基
專一性引子
偵測
堆肥
太空包
尿素
出版社: 植物病理學系所
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摘要: 靈芝 (Ganoderma lucidum (Leyss. ex Fr.) Karst.) 具有高度的藥用價值,因而廣受菇農的栽種。為了要滿足市場的需求,故以太空包木屑培育靈芝後,會產生大量的廢棄太空包基質。這些廢棄的太空包基質常被用來製作成堆肥;惟若是堆肥中含有靈芝菌的活體時,則果樹會有被感染的風險。所以本研究嘗試研發選擇性培養基與聚合酶連鎖反應搭配專一性引子兩種方法,用以偵測堆肥中是否有存活的靈芝菌。靈芝菌選擇性培養基係利用 10 % V-8 培養基作為基礎基質,並添加 2 g l-1 腐絕 (40% ai)、200 mg l-1 滅達樂 (35% ai)、200 mg l-1 鏈黴素、0.2 g l-1 氯黴素、1 mg l-1 五氯硝基苯 (75% ai)、1.25 g l-1 單寧酸、10 ml l-1 酒精 (95% v/v) 及 2 ml l-1 乳酸 (85% v/v) 配製而成。靈芝菌選擇性培養基可由堆肥中分離出靈芝菌,且尚可抑制其他雜菌之生長。培養基中添加之單寧酸會使靈芝菌落周圍出現褐色反應,有助於靈芝菌的辨識。利用多重土丸取樣法分析不同靈芝接種源濃度之太空包木屑基質;結果顯示,靈芝菌選擇性培養基可偵測到台中及嘉義之太空包木屑基質經過 1000 倍無菌太空包基質稀釋後仍有 G. lucidum 的存活。此外,進一步研發聚合酶連鎖反應搭配專一性引子對,用以驗證靈芝菌選擇性培養基偵測靈芝菌之準確度。本研究由NCBI (National Center for Niotechnology information,USA.) 資料庫中篩選78株靈芝菌株之原始核苷酸 ITS1, 5.8S 及 ITS2 序列,設計出正向引子 GanJwF (5’-GCCTGCGTTTATCACAAACTC-3’) 搭配反向引子 ITS4-B (5’-CAGGAGACTTGTACACGGTCCAG-3’, Gardes and Bruns, 1993) 偵測靈芝菌,結果 GanJwF 與 ITS4-B 可增幅出約 600 bp 之條帶,且包含有大部分 ITS1, 5.8S 及 ITS2。該專一性引子對可偵測到含量僅 100 pg 之靈芝菌 DNA 且不與其他栽培種之擔子菌以及堆肥中之雜菌作用。結合這兩種偵測靈芝菌之方法可準確的偵測出廢棄太空包基質是否存活有靈芝菌。此外,利用熱及添加尿素的方式評估除滅靈芝菌之效果;結果顯示,將堆肥加熱至 55 ℃ 處理 10 分鐘或是添加 0.5 % 之尿素搭配 0.1 % 之生石灰處理 14 天,均可有效地將堆肥中之靈芝菌除滅。
Ling-Chih, Ganoderma lucidum (Leyss. ex Fr.) Karst., is widely cultivated for its high medical value. In order to match the requirement of market, a large quantity of the sawdust is used for cultivation of Ling-Chih resulting in a lot of sawdust waste. The sawdust waste is often made as composts and used as fertilizers to supplement the nutrients for plants. However, if mycelia of Ling-Chih keep alive in composts, it may make high risk of fruit crops infected. Ganoderma spp. are the causal agents of basal stem rot (BSR) and mostly attack the root systems of the plants. In general, Ling-Chih is considered to be one of white-rot fungi and it is able to digest lignin, mainly. Large amounts of wood decay have an adverse effect on strength and stability, and make plants gradually weaken to die. Owing to the visible disease symptoms appear at a very late stage of infection, two detection methods, the selective medium and polymerase chain reaction (PCR) method based on taxon specific primer, were developed in this study to detect Ganoderma lucidum at early stage from compost. GLS medium was developed by amendment of 1 l 10% V-8 vegetable juice agar with 2 g thiabendazole (40% ai), 200 mg metalaxyl (35% ai), 200 mg streptomycin sulfate, 0.2 g chloramphenicol, 1 mg PCNB (75% ai), 1.25 g tannic acid, 10 ml ethanol (95% v/v), and 2 ml lactic acid (85% v/v). The GLS medium was able to rapidly isolate G. lucidum from the compost. Amendment of the medium with tannic acid could induce G. lucidum to produce brown halo surrounding the colony and make us easily recognize the fungus. To evaluate the efficacy of GLS medium, multiple-pellet soil-sampler method was used to assay the inoculum level of G. lucidum. The GLS medium was able to detect G. lucidum in the sawdust substrate from Taichung and Chiayi at 1000-fold dilution with sterilized sawdust substrate. The PCR method based on taxon specific primer was developed to confirm the detection results of GLS medium. The original nucleotide sequences of ITS1, 5.8S, and ITS2 of 78 isolates of G. lucidum were selected from NCBI GenBank (National Center for Biotechnology Information, USA). Based on the alignment results of ITS sequences, the forward primer GanJwF (5'-GCCTGCGTT-TATCACAAACTC-3') was designed and used with the reverse primer ITS4-B (5'-CAGG-AGACTTGTACACGGTCCAG-3', Gardes and Bruns, 1993) to detect G. lucidum. Approximate 600 bp PCR products containing most of the ITS1, 5.8S, and ITS2 regions could be amplified by primer pair of GanJwF and ITS4-B. PCR method based on GanJwF/ITS4-B specific primer pair is able to detect as low as 100 pg of G. lucidum DNA template and specific to G. lucidum, but not to other cultivated basidiomycetous or saprophytic fungi occurring in the compost. Two methods could used for for detecting the survival of G. lucidum in the compost and therefore be recommended for the manufacturer to determine if the compost is suitable as fertilizer. In order to eliminate G. lucidum from the compost, heat and urea treatments were used to suppress the growth of G. lucidum. The results showed that compost treated with 55 ℃ for 10 min or treated with 0.5% urea and 0.1% CaO for 10 days could completely eradicate the survival of G. lucidum.
URI: http://hdl.handle.net/11455/31012
其他識別: U0005-1908201116285200
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