有害木層孔菌配對型之研究

Abstract

摘要 本研究以嘉義樹木園染病羅望子樹頭所產生之有害木層孔菌子實體，搜集空飄孢子，將個別單孢分離培養，進行螢光染色菌絲前端以計算細胞核數目及太空包實驗確認能否出菇，並將單孢分離株作自體與異體配對實驗及分子鑑定，使用ITS4/5引子對進行PCR實驗，將核酸序列結果以Mega 6.0做分析，觀察單孢分離株與配對後的菌株序列是否有關係，作為檢定有害木層孔菌配對型的方法。 實驗結果則為，培養於馬鈴薯瓊脂培養基 (PDA) 上之單孢分離菌落與一般病組織分離菌落，在外觀上並無顯著的差異，菌落生長快慢與褐化及產生網紋、皮革狀組織並無絕對相關，在繼代培養中也多有變異；細胞核染色實驗中的單孢菌絲細胞核主要為兩個核，少部分有單核及三核，年老菌絲則沒有觀察到細胞核；太空包出菇實驗之大部分菌株無法產生可供開包的健康菌絲，開包的28包太空包中只有15包長出類似子實體的構造，但仍沒有產生具有可供辨識之特徵，如菌孔及孢子；在配對實驗裡，可以發現不同菌株配對時，兩個菌落間會產生相當明顯之交界帶，自交時則無，但仍有部分自體配對產生明顯交界帶與二級菌絲，因此單依此現象無法用做確認配對型的結果；而於分子技術鑑驗中，經Mega 6.0的Neighbor-joining tree分析，大略將單孢分為兩個群落，而將不同群落之單孢分別選出，進行單孢分離株異體配對，切下交界帶的菌塊放大培養，重複做序列分析後只表現出一種單孢基因型，沒有產生第三型的基因，顯示兩個單孢接觸後並不會產生核的融合。 依照目前結果，大部分有害木層孔菌之單孢菌絲主要為兩核，配對後產生之基因不會產生核融合的現象，但因未能確立配對型的細胞核染色與太空包實驗皆缺乏和子實體的對照，因此無法確認有害木層孔菌的配對型為何者，只能就目前的結果推測應比較傾向同核配對型。

In this study, we would like to find out whether the mating type in Phellinus noxius is homothallic or heterothallic. We collected the infected tissue which developed fruiting bodies in Chiayi Arboretum, using airborne spores, and isolated single spores then cultured on PDA. Then dyed the top of hyphae with DAPI to calculate the number of nuclear, and applied bulk bags as confirmation of single spore isolation fruiting. Afterwards, the single spore isolation was taken to make the pairing test in autologous and allogeneic. For the molecular identification, we used ITS 4 / 5 primers to carry out PCR experiments, the sequence were analyzed by Mega 6.0, and then observed the difference between single spore isolation and tissue. If the results were related, it would be the consultation of disease control in the future. The form of colonies on PDA were not different between the single spore isolation and tissues isolation, the growth rate and browing effect were no significant differences in mycelium , neither the leather-like tissue. Nuclear staining shows that the nuclei in hyphae in single spore isolation were two nuclei, and few were one nuclear and triple nuclei, old hyphae was not observed in nuclei, but due to lack of the data with fruiting body or tissue isolations, we can’t compare the difference in sexual generation and asexual generation, so the mating type can’t be confirmed. Part of the fruiting experiment can’t be generated for the mycelium, there were 28 packages can open and only 15 packages to appear the similar structure to fruiting body, but still had not produce for identification of features such as holes and spores. In pairing experiment, it could be found obvious shape when two colonies reached each other, but there were still some significant autologous pairing with the secondary mycelium cross the intermingling zone, thus the phenomenon can’t be so sure to confirm the mating type. In molecular techniques, through the Mega6.0 (neighbor-joining tree) analysis, will be roughly divided into two communities, then we paired each one of two communities to analyzed the mating mycelium, the sequence showed only one genotype appear, and it represented there were no nuclear fusion when two isolations paired together. According to the results, the majority of hyphae in single spore isolations were two nuclei, and there were no nuclear fusion after mating, the bulk bag experiments and dyeing nuclei were lacking data with fruiting body, so we can’t confirm the mating type were homothallic or heterothallic, if there will be a more perfect experiments which can confirm the mating type, it should be against characteristics, and may develop corresponding control methods.