印度塊菌與福山植物園41種殼斗科樹木之共生關係

Abstract

本研究以子囊菌塊菌屬（Tuber）之印度塊菌（Tuber indicum）製成 20 ml 孢子濃度為 5×10^3個/ml之孢子懸浮液，接種至福山植物園41種殼斗科樹木，以探討不同殼斗科樹種與印度塊菌共生的可能性，期望以高寄主親和性的印度塊菌接種 於推薦樹種青剛櫟等殼斗科樹木之根系，得出印度塊菌在不同季節、樹種、培養介質及接種方法下形成菌根共生關係的最佳條件。實驗分為兩次地下根部接種及地上部樹冠接種，菌根收集後將以形態學觀察及記錄其合成情形、表面構造、排列、 菌氈、菌絲及剛毛等顯微形態特徵；另外亦輔以分子生物鑑定比對基因資料庫及製 作基因親緣樹以分析菌根菌的種類及親緣關係。 地下部根系接種的發根率佔總接種包數70-85%之間，菌根率在55-63%之間，其中第二次接種的菌根共生情況比第一次的效果較好，而菌根測序率約 43-47%； 而枝條高壓接種的發根率及菌根率比根系接種低，分別為 45 及 33 %，菌根測序率 約 1.5 %。第一次地下部根系接種中擔子菌佔 85 %之絕大多數，以硬皮馬勃科、牛 肝菌科及革菌科之菌根菌為主，其中擔子菌的 Tomentella 屬、Russula recondite Melera & Ostellari.，以及子囊菌 Lauriomyces bellulus Crous & M.J. Wingf 為台灣新 記錄種，惟未有發現塊菌科菌根菌；第二次地下部根系接種仍以擔子菌佔 76 %大多數，以革菌科、紅菇科及硬皮馬勃科之菌根菌為主，並有 6 個菌根樣本共 3 屬 5 種的殼斗科樹木顯示出與塊菌共生的可能，惟非本次實驗接種之印度塊菌，其中擔子菌的 Tomentella sublilacina (Ellis & Holw.) Wakef、Russula recondita Melera & Ostellari.、Russula heterophylla (Fr.) Fri.、Lactarius kesiyae Verbeken & K.D. Hyde、 Laccaria fulvogrisea Popa, K.H. Rexer & G. Kost、Entoloma inusitatum Noordel.、Sebacina flagelliformis Oberw.，以及子囊菌的 Neonectria 屬為台灣新記種；而與塊菌屬 Tuber thailandicum N. Suwannarach, J. Kumla & S. Lumyong 及 Tuber sp.11 相似之菌根菌則可能為世界新種，惟仍需待子囊果之發現以完成完整的形態學鑑定。地 上部枝條高壓接種的在發根率、菌根率及測序率均較地下部根系接種低，但卻可以 讓塊菌得到接近無雜菌且菌類多樣性較低的生長環境，避免與其他於土壤中擁有 高空氣傳播能力和菌絲快速生長的菌根菌競爭。 地下部根系接種之 6 個塊菌菌根樣本若單以形態學作鑑定符合前人於印度塊菌的菌根形態之描述。研究結果顯示形態學可以輔助區分菌根菌之有無及排除部分非塊菌屬之菌根菌，但如果要用作鑑別塊菌屬之菌根，需要另外再輔以分子生物 鑑定，才能得到較準確之鑑定。然而，分子生物技術於塊菌菌根的鑑定上亦有許多 的難題需要克服，如菌根菌測序率普遍偏低，亦無法以基因鑑定技術測出印度塊菌 之結果等；而另外進行之其他印度塊菌接種實驗亦得到與福山印度塊菌接種實驗 相似之結果，排除了個別偶然事件的可能性。因此，印度塊菌與寄主如青剛櫟是否 真的如前人研究般具有廣泛寄主而且具高親和性，仍然是值得全盤再深究、實驗、 證明與探討。最後，在實驗過程中以分子鑑定的方法能於未發現子實體的情況下， 得知臺灣可能存在有不少新記錄之真菌，包括類似於日本及泰國發現的一些白塊菌，若環境適合出菇，再輔以一些保護設置，應有不低機會可找到相關之子實體， 以完成完整的形態學鑑定。

The objective of this study is to analysis the symbiotic relationship of Tuber with Fagaceae tree species under different environmental condition, inoculation method and growing medium, as the basic information for further research on the cultivation of truffles. Based on researches by predecessors, Tuber indicum, with wide host range and affinity, were inoculated to 41 species of Fagaceae trees, reported as common host of truffles and with high affinity with Tuber species, in Fushan Botanical Garden of Taiwan. Experiments were carried out in two ways: (1) twice root inoculation and (2) once branch air-layering inoculation in one-year period. Mycorrhizae were then sampled for detailed morphological analysis and molecular identification, and some for further phylogenetic analysis.

Root inoculations experiments resulted in 70-85 % root induction rate, with mycorrihzae formation rate for 55-63 %. The second root inoculation experiment, in winter season, resulted in more mycorrhizae formations. Branch air-layering inoculation experiment obtained lower root induction rate and mycorrihzae formation rate than root inoculation, of 45 % and 33 % respectively. In the first root inoculation experiment, most of the mycorrhizae (85 %) were found to be associated with mycorrhizal fungi of Sclerodermataceae, Boletaceae and Thelephoraceae of basidiomycota, with Tomentella sp., Russula recondite Melera & Ostellari. and Lauriomyces bellulus Crous & M.J. Wingf as the new discovery species of Taiwan. In the second root inoculation experiment, most of the mycorrhizae (76 %) were found to be associated with mycorrhizal fungi of Sclerodermataceae, Russulaceae and Thelephoraceae of basidiomycota, with Tomentella sublilacina (Ellis & Holw.) Wakef, Russula recondita Melera & Ostellari., Russula heterophylla (Fr.) Fri., Lactarius kesiyae Verbeken & K.D. Hyde, Laccaria fulvogrisea Popa, K.H. Rexer & G. Kost, Entoloma inusitatum Noordel., Sebacina flagelliformis Oberw., Neonectria sp. as the new discovery species of Taiwan; Species similar to Tuber thailandicum N. Suwannarach, J. Kumla & S. Lumyong and Tuber sp.11 may be the new species of world. 6 mycorrhizae samples from 5 different Fagaceae tree species, were found to be associated with Tuber, but should with white truffles found in Japan and Thailand, not with the inoculated black truffle Tuber indicum. Branch air-layering inoculation experiment results in much lower root induction and mycorrhizae formation rate, but reviews much less contamination by other undesired mycorrhizal fungi. The 6 Tuber mycorrhizae samples from 5 different Fagaceae tree species were morphologically identical to mycorrhizae associated with Tuber indicum as sited in references. It reviews the difficulty and unreliability of identification of mycorrhizal fungi solely by mycorrhizae morphology, the assist of molecular analysis is necessary. However,there are still many unsolved problems in the detection and identification of Tuber species in mycorrhizae. Tuber indicum were detected neither from the experiment in Fushan Botanical Garden nor from the other experiment we have conducted in nurseries. The wide host range and affinity of Tuber indicum and Fagaceae host reported in previous research is doubted based on the result in this study. The possibility of truffles cultivation needs further experiments and discussion.