Hyphoderma mutatum毒殺線蟲活性之分析

Abstract

Liou and Tzean的研究結果中發現，屬於擔子菌皮殼菌屬的Hyphoderma mutatum是所有測試的分離株中，毒殺線蟲能力最強的菌種。由於H. mutatum毒殺的對象包括松材線蟲及葉芽線蟲等重要的病原性線蟲，因此具有作為生物防治媒介的潛力。目前對於H. mutatum的研究相當少，造成該方面研究的停滯。我們希望藉由發展出研究H. mutatum毒殺線蟲機制之分析工具，建立起H. mutatum 與松材線蟲之模式系統，以期在未來能分離及鑑定出毒殺線蟲之物質，同時亦能用以探討其毒殺線蟲的機制。首先，我們調整培養 H. mutatum 的條件，以松材線蟲作為目標進行毒殺線蟲活性試驗，結果顯示在基礎培養基中僅添加 0.02% 蛋白胴(peptone)培養出毒殺能力最弱之菌絲；若再添加1% 甘露糖(D-mannose)，能培養出具最強毒殺能力之菌絲。之後，我們便使用此二種培養基培養H. mutatum進行後續實驗。由於H. mutatum屬於攝入性毒殺型的線蟲寄生真菌(nematophagous fungi)，因此需有一餵食系統來分析純化出之毒性物質。結果顯示在添加octopamine時，松材線蟲攝入螢光染劑的比例提升，且添加25 mM octopamine時，確實能成功餵食 H. mutatum的菌絲粗萃液，造成線虫死亡。此外，我們選用了聚乙二醇媒介轉殖法對H. mutatum的原生質體進行轉殖，並利用green fluorescence protein (GFP) 及hygromycin B phosphotransferase (Hpt) 的基因作為報導基因。進行轉殖得到的再生菌絲，均能以聚合酶連鎖反應偵測到gfp及hpt基因的存在，雖然後者在含有 hygromycin的培養基上可生長，但前者於螢光顯微鏡下卻無GFP訊號。這樣的結果表示，trpC 啟動子於H. mutatum中是有作用的，但為何無法在 H. mutatum中測得GFP訊號，仍待進一步的探討。

In a previous study, Liou and Tzean found that Hyphoderma mutatum which belongs to the subdivision of Basidiomycotina has the strongest nematicidal activity in all testing isolates. Bursaphelenchus xylophilus and Aphelenchoides besseyi, two of the most important plant nematodes, are affected by the nematicidal activity of Hyphoderma mutatum, making it a potential biocontrol agent of nematode diseases. There were not much researches on Hyphoderma mutatum thus far. Hence, we hope to develop an array of analytical tools in order to build up a H. mutatum-Bursaphelenchus model system to identify the nematicidal substances as well as to understand the mechanisms underlying the nematicidal activity of H. mutatum. Based on a previous study, we selected several media to grow H. mutatum for nematicidal activity assay against B. xylophilus. The results indicated that mycelia grown on minimal medium containing only 0.02% peptone possessed lower activity while supplementing with additional 1% mannose gave rise to higher activity. These two media were termed hypo- and hyper-toxigenic media, respectively, and were used for further experiments. (Hyphoderma mutatum is toxic to nematodes only when they ingest contents of the mycelia.) In the nematode feeding system we set up, the fluorescence dye FITC was successfully uptaken at high level in the presence of octopamine, and the highest nematode mortality was detected when 25 mM octopamine was added into the crude extracts of H. mutatum. We also tried to use polyethylene glycol (PEG)-mediated transformation and two reporter genes, gfp (green fluorescence protein) and hpt (hygromycin B phosphotransferase), to establish a transformation system for H. mutatum. The gfp or hpt gene can be detected in regenerated protoplasts using polymerase chain reaction, but no GFP signals can be observed under fluorescence microscopy although transformants containing the hpt gene could grow on hygromycin-containing media, indicating that trpC promoter is working in H. mutatum and it should be further elucidated why no GFP signal can be detected in transformed protoplasts.