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標題: | 深脈塊菌之分離、培養及與臺灣松屬植物外生菌根合成 Tuber elevatireticulatum: its isolation, in vitro cultivation and formation of ectomycorrhiza with Taiwanese indigenous pine species |
作者: | Wing-Yi Cheung 張詠怡 |
指導教授: | 蔡明哲(Ming-Jer Tsai) |
共同指導教授: | 傅春旭(Chuen-Hsu Fu) |
關鍵字: | 深脈塊菌,塊菌菌株分離,培養基篩選,菌絲接種,分子生物鑑定,松屬樹種, Tuber elevatireticulatum,Tuber sp. isolation,media selection,mycelia inoculation,molecular identification,Pinus spp., |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 臺灣第一種被正式發表的原生白塊菌—深脈塊菌於2017年被發現,並以此作為實驗標的進行相關的研究。利用1/2MS培養基作深脈塊菌的菌種分離,並進行培養基篩選,以找出較合適培養、繼代深脈塊菌的培養基。深脈塊菌於1/2 MS培養基下呈現透明、稀疏的菌落、生長速度慢,培養兩個月菌落直徑約6.4 cm,菌絲微觀特徵為部份菌絲聚集成團或形成環狀形態、有菌絲膨大體、菌絲間有H形聯會。將菌種作培養基篩選,菌株於1/2 MS培養基中添加1 % PDA、1 % YEA或 1 %蔗糖,培養兩個月後,菌落直徑雖不及1/2 MS,但有較為密集的菌落,其中 1% 蔗糖生長最為密集,培養兩個月直徑為5.4 cm,故視為較合適的培養基。
獲得菌種後,將塊菌菌絲塊貼於受試驗的臺灣二葉松、臺灣五葉松及臺灣華山松苗木的根系上作接種。接種3個月後發現臺灣二葉松、臺灣五葉松每棵苗木均有合成出菌根,並以分子生物鑑定,確認共生的菌根菌為深脈塊菌。臺灣華山松則有半數的苗木合成出菌根,其菌根形態雖與臺灣二葉松、臺灣五葉松所合成出的菌根近乎相同,但無法透過分子生物方法鑑定出共生真菌的物種。故深脈塊菌的宿主範圍除了已知的江南油杉外,本研究亦另外發現臺灣二葉松及臺灣五葉松。 深脈塊菌於松屬樹木的菌根形態為單軸棒型、二歧型或珊瑚型的菌根排列;顏色呈淡黃至淡褐色、菌根根尖膨大、末端渾圓;菌毯表面披短刺、錐狀密集的剛毛體;剛毛體透明、不分岔;具有迷宮的紋路的哈替氏網。 由於深脈塊菌的菌落及與臺灣二葉松、臺灣五葉松合成所得的菌根沒有明顯區別性特徵,不可以單憑形態作出準確的鑑定,故有關深脈塊菌的研究需依賴分子生物鑑定的方法。 The first published Taiwan indigenous whitish truffle, Tuber elevatireticulatum, was discovered in 2017 and research on T. elevatireticulatum was carried out in this study. 1/2 MS agar medium was used for the isolation of T. elevatireticulatum. Colony of T. elevatireticulatum on 1/2 MS was transparent and sparse with slow growth. Some of the hyphae aggregated together or coiled to form a loop. Hyphal swelling and H-shape anatomses between two hyphae were observed under light microscope. Different agar media were tested in order to find out a more suitable medium for sub-culturing T. elevatireticulatum. Colonies of T. elevatireticulatum grew more slowly when 1 % PDA, 1 % YEA or 1% sucrose was added to the 1/2 MS agar, but formed denser mycelia. 1/2 MS agar medium with 1 % sucrose gave the densest colony among the other culturing media with slightly smaller colony diameter when compared with 1/2 MS agar medium (5.4 cm for 2 months instead of 6.4 cm for 2 months). It is regarded as the most suitable medium for culturing T. elevatireticulatum in this study. Cultivated mycelium was placed onto the root systems of Pinus taiwanensis, P. morrisonicola and P. armandii var. masteriana seedlings for inoculation. After 3 months’ inoculation, mycorrhizae were formed on all P. taiwanensis and P. morrisonicola seedlings. The mycobiont of the mycorrhizae was identified as T. elevatireticulatum by molecular identification. Mycorrhizae were formed by half of the P. armandii var. masteriana seedlings. The morphology of the mycorrhizae was very similar to the mycorrhizae formed in P. taiwanensis and P. morrisonicola seedlings. However, molecular identification of P. armandii var. masteriana mycorrhizae failed. Except Keteleeria fortunei var. cyclolepis, the host plants of T. elevatireticulatum confirmed in this study were P. taiwanensis and P. morrisonicola. The morphology of Pinus spp. mycorrhizae formed with T. elevatireticulatum was a simple, dichotomous or coralloid ectomycorrhizal system with the colour of pale yellow to pale brown. Root tips were swollen with rounded ends. The mantle was covered with short spiky, awl-shaped and dense cystidia. Cystidia were transparent and without branching. The Hartig net within the root cells showed labyrinthine patterns. Since there was no distinctive feature being observed from T. elevatireticulatum colony and mycorrhizae formed on P. taiwanensis and P. morrisonicola, precise morphological identification was difficult to carry out. Molecular identification was a necessary tool to confirm the fungal species obtained in the study. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72666 |
DOI: | 10.6342/NTU201902140 |
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顯示於系所單位: | 森林環境暨資源學系 |
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