Semi-aseptic Mycorrhiza Synthesis of Tuber magnatum (Pico) and Effects of Different pH Media on It’s Mycorrhiza Formation and Macro-nutrient Elements in Leaves of Cyclobalanopsis glauca ( Oerst )

意大利白塊菌(Tuber magnatum Pico)已知能與白楊、白柳、毛櫟等林木形成外生菌根，在適當之自然條件下，能在這些林分中生產出世界最有價值之菇體。本研究之主要目的為(1)將意大利之白塊菌與青剛櫟(Cyclobalanopsis glauca )利用孢子懸浮液接種進行半無菌菌根合成；(2)比較不同pH介質對意大利白塊菌菌根感染率與苗木葉部養分濃度之關係。結果顯示意大利白塊菌孢子懸浮液能與青剛櫟形成典型外生菌根，其菌根特徵為：單根、羽狀型根系，菌根淡褐至暗褐色，常形成淡褐色之剛毛，在菌根根尖之剛毛能形成囊狀體菌絲，菌毯厚10-15µm，偽薄壁細胞結構、延伸菌絲黃褐色。單株苗木在不同pH介質中之菌根感染率以在pH 7.5者最高，可達78%；但就整體平均感染率而言則是以pH 6.5介質菌根感染率最佳。所有pH介質接種的苗木皆能形成意大利白塊菌之菌根。連續在不同月份觀察菌根之動態生長，從生長初期至老化期最快約為2-3個月，慢可達10個月以上。連續觀察菌根感染7個月後之結果，發現在pH 5.9與pH 6.0介質中菌根感染率呈S曲線型，在達到感染高峰後則略有呈現下降趨勢。pH 6.5、pH 7.0、pH 7.5介質在苗木培養之較早期即可達到良好之感染率，所以此三種介質可縮短菌根合成時間。由本次試驗中結果顯示介質之pH與菌根感染率間呈顯著正相關。不同pH介質對菌根形態之形成並無明顯差異。除此之外，不同pH介質對苗木葉片養分濃度及生長上除了P、K與Mg對其具有些微影響外，其餘則大多無顯著之關係。在添加養分之人工介質中，接種菌根與否對苗木葉片養分及生長上亦無明顯差異(p>0.05)。Tuber magnatum could form ectomycorrhizae on Populus alba, Salixalba , Quercus sp., and Cistus sp.. It could produce the most valuable ascomata in these stands only under very appropriate natural conditions so far. In this study the spore suspension of Tuber magnatum was used as inoculum and the inoculated seedlings were cultured in artificial soils adjusted to different pH levels by adding different amounts of vermiculite and peat. The aims of this study were (1) to perform semi-aseptic mycorrhiza synthesis by spores suspension of Tuber magnatum on Cyclobalanopsis glauca seedlings, and (2) to compare the mycorrhiza infection percentage and the relationship of nutrient concentrations in leaves when the seedlings were cultured in artificial soils of different pH levels . The results showed that the spore suspension of Tuber magnatum could form typical ectomycorrhizae on C. glauca . It’s mycorrhizal characteristics was described as follows: monopodial , club-shaped , glat , light brown , yellowish brown to dark brown when aging , with or without setae , the setal hyphae hyaline or yellowish brown and sometimes with vesicle at the middle or near the end , with irregularly pinnate root system ; fungus mantle 10-15 µm thick , pseudoparenchymatous structure , extraradical hyphae light brown to yellowsch brown in color . The mycorrhizal infection percentage was the highest for the treatment grown in artificial soils of pH 7.5 , in terms of individual seedlings , but it was the best for the treatment grown in pH 6.5 artificial soils when all the treated seedlings were counted. All inoculated seedlings could form ectomycorrhizae in spite of different pH levels of artificial soils . By observing the mycorrhizal development dynamics continuously , it was found that it took about 2 months for the earliest mycorrhizal formation and the aging of mycorrhizae could happen in 2-3 months or over ten months. The mycorrhizal infection percentage for continuous 7 months was with S-shaped curve, and then fell down slightly for those grown in pH 5.9 and 6.0 artificial soils . The seedlings cultured in artificial soils of pH 6.5 , pH 7.0 and pH 7.5 could reach high mycorrhizal infection percentage at the earlier stage of cultivation. Therefore , it could reduce mycorrhiza-forming time. Significantly positive relationship existed between different pH medium and mycorrhizal infection percentage. Mycorrhzial morphology showed no difference, when the seedlings were cultured in different artificial soils. Different pH artificial soils had little influence on the leaf nutrient concentrations , except P, K and Mg. Either those inoculated or uninoculated seedlings showed no obvious difference( p <0.05) in leaf nutrient concentrations , when the artificial soils were added with MMN nutrient solution.摘要 i Abstract ii 目錄 iv 表目錄 vii 圖目錄 viii 一 前言 1 1.1 塊菌之介紹與其經濟價值 1 1.2 天然塊菇不當採收與破壞 2 1.3 塊菇人工栽培之開始及其面對之問題 3 1.4 塊菌菌根苗商業化 3 1.5 塊菇在台灣之研究及未來可應用之方向 4 1.6 研究目的 5 二 前人研究 6 2.1 塊菌之菌根合成 6 2.1.1 塊菌資源之地理分佈及生育地性質 6 2.1.2 菌根合成之寄主植物 8 2.1.3 合成菌根之接種源 9 2.1.4 外生菌根之鑑定 11 2.1.5 Tuber屬之菌根鑑定 13 2.2 不同pH值對菌根形成之影響 18 2.3 菌根與苗木生長及葉部養分 20 三 材料與方法 23 3.1 菌根合成 23 3.1.1 苗木培育 23 3.1.2生長栽培介質之處理與苗木之移植 23 3.1.3 塊菌之接種源 24 3.1.4 青剛櫟與白塊菌之半無菌合成 24 3.1.5 立體顯微鏡之觀察 25 3.1.6 光學顯微鏡之觀察與水膜片之製備 25 3.1.7 掃瞄式電子顯微鏡觀察 26 3.1.8 菌根之鑑定 27 3.2 不同pH介質對菌根形成及與葉部主要養分之關係 27 3.2.1 介質pH之調整與測定 27 3.2.2 菌根感染率之計算 27 四 結果 30 4.1 青剛櫟苗木與意大利白塊菌半無菌合成菌根試驗 30 4.1.1 菌根在立體顯微鏡下之形態特徵 30 4.1.2菌根在光學顯微鏡下之形態特徵 31 4.1.3 菌根在掃瞄式電子顯微鏡之形態特徵 32 4.2 不同pH介質處理對苗木菌根形成之影響 36 4.2.1 各連續月份中苗木感染率與菌根感染動態 36 4.2.2 不同pH介質處理之菌根感染動態 37 4.2.3 不同pH介質對菌根感染率之影響 40 4.3 不同pH介質與苗木葉片主要養分之關係 45 4.3.1不同pH介質對苗木葉片養分濃度之影響 45 4.3.2 不同pH介質與苗木葉片養分及主要養分間之相關分析 49 4.3.3 接種塊菌對苗木葉片養分含量與苗高及根頸之影響 50 4.4 不同pH介質與苗高及根頸關係 50 4.4.1 不同pH介質下苗木之苗高與根頸之生長情形 50 4.4.2 不同pH介質、苗高與根頸、菌根感染率之相關分析 51 4.5 不同pH介質中之菌根生長形態 52 五 討論 54 5.1 青剛櫟苗木與意大利白塊菌之半無菌合成菌根 54 5.2 不同pH介質對幼苗菌根形成及葉片養分影響 58 六 結論 64 七 參考文獻 6