中果塊菌 ( Tuber mesentericum Vitt. ) 之分離、菌落生長及半無菌菌根合成

Abstract

本研究之目的為：(1)嘗試自台灣二葉松 ( Pinus taiwanensis Hay. ) 與中果塊菌( Tuber mesentericum Vitt. ) 形成之外生菌根分離出中果塊菌菌絲；(2)以半無菌合成確定分離出之純中果塊菌菌絲能與台灣二葉松形成外生菌根；(3)確定最適合中果塊菌菌落生長之培養基及溫度組合。 實驗結果顯示，以0.25% 次氯酸鈉 ( NaClO ) 浸泡菌根約3分鐘進行表面殺菌後，能夠成功分離出中果塊菌之純菌絲。將培養之洋菜菌絲體接種於半無菌培育之二葉松苗木，於一至二個月後可形成外生菌根，其在立體解剖顯微鏡下所觀察之特徵為：單根狀或分叉形兩種形態；顏色為淡黃色至黃褐色，老化後轉為深褐色；菌根幼時頂端附近被覆密集、微細之淡黃色至金黃色短剛毛，成熟後四周分布著淡黃色之棉花狀延伸菌絲，乃至逐漸平坦、前端膨大；老化後剛毛消失，呈現略為皺縮之形態。 於光學顯微鏡下觀察成功合成之菌根特徵為：菌根之延伸菌絲及剛毛表面平坦、基部較為寬大，先端偶有圓形突起之泡囊；延伸菌絲及剛毛內部並有明顯之隔膜出現，亦有發現分叉的情形。 由掃瞄式電子顯微鏡所觀察到之菌根特徵為：菌毯結構為不規則密實菌絲結構，厚度約17~25μm；菌絲表面平坦、無扣子體，多為單根狀，直徑為1.5~2.5μm，基部較為寬大，先端偶有發現膨大之囊狀體，也有發現菌絲分叉的情形。哈替氏網侵入至皮層細胞之第二層；皮層細胞內偶可發現澱粉粒的存在，未發現單寧之累積。 於光顯微鏡下觀察，發現洋菜培養基中之純培養菌絲頂端及周圍有許多團塊聚集之桑椹狀菌絲囊，菌絲上亦偶能發現膨大之囊狀體存在。為確定中果塊菌菌落生長試驗中，兩種培養基及四個溫度層級之最佳組合，使用完全逢機因子設計進行統計分析；結果顯示YMT培養基較MMN為佳，而最佳之培養基與溫度組合為YMT及25℃。

The purposes of this study were: (1) to isolate Tuber mesentericum Vitt. from the ectomycorrhizae of Pinus taiwanensis Hay. associated with T. mesentericum; (2) approve the isolated pure hyphae could form ectomycorrhizae with P. taiwanensis. by semi-aseptic synthesis; and (3) find the best growth medium and optimum temperature to the colony growth of T. mesentericum. Pure hyphae of T. mesentericum were isolated successfully, when the ectomycorrhize of P. + T. mesentericum were sterilized with 0.25 % NaClO for about three minutes. After inoculating the agar mycelial inocula on the rootlets of semi-aseptic cultivated Pinus taiwanensis seedlings, ectomycorrhizae could be formed in 1~2 months. The characteristics of ectomycorrhizae observed under stereomicroscope were as followings: monopodial or dichotomous; yellow brown to light brown when young, dark brown when wilted; fungal mantle with dense yellowish-golden setae when young, smooth or surrounded by cottony yellowish hyphae when mature, shrinking when aged. The characteristics of ectomycorrhizae observed under light microscope were: extending hyphal and setal surface from mycorrhizae smooth, a little swollen at the setal base, both with septa; occasionally with vesicle at the setal top or with forked hyphae. The characteristics of ectomycorrhizae observed under scanning electron microscope were: fungus mantle 17~25μm thick, irregular synenchyma; setal hyphae smooth, without clamp connection, monopodial, diameter 1.5~2.5μm, a little swollen at the setal base, occasionally with vesicle at the top or with forked hyphae. Hartig nets extended to the second layer of cortex cells; starch grains seldom found, and tannins not observed. Lots of mulberry-like gathered vesicles were found around and at the top of the hyphae cultured on agar medium under light microscope; expanded vesicles were occasionally found on hyphae. To ascertain the best combination of two different agar media and four different temperature levels on the colony growth of T. mesentericum, the complete randomized design with factorial test was used. The results showed that YMT agar medium was better than that of MMN, and the best combination of agar medium and temperature was YMT and 25℃.