溫度對塊菌菌落生長之效應及夏塊菌之外生菌根合成

Abstract

塊菌為世界最珍貴之菌根食用菌，因經濟價值高，最近常被外生菌根研究者作為研究之材料。因此，本研究主要的目的為：(1)確定數種塊菌菌落生長之最適溫度及最高致死溫度；(2)確定夏塊菌(Tuber aestivum)與青剛櫟及台灣二葉松之半無菌苗共生形成的菌根形態。 首先將已分離成功之冬塊菌、印度塊菌、台灣塊菌、擬外孔塊菌、法國黑孢塊菌以及意大利白塊菌，6種塊菌菌落繼代培養作為培養基與溫度試驗之材料。將各塊菌菌落分別接種於YMT及YpSs培養基上，進行培養基之選定。接著，以YpSs培養基做為基質，將接種好之培養皿分別置於15℃、20℃、25℃、30℃以及34℃生長控制箱中培養至其中生長最快之一種菌落接近布滿整個培養皿時即終止此試驗；以33℃、34℃、35℃及36℃之溫度進行最高致死溫度之試驗，俾找出各塊菌菌落之最高致死溫度。以夏塊菌之孢子懸浮液分別接種台灣二葉松及青剛櫟之半無菌苗，待其菌根形成後，以立體顯微鏡、光學顯微鏡及掃描式電子顯微鏡進行菌根形態之觀察。 結果顯示不同培養基之間只有擬外孔塊菌菌落呈現顯著差異，即YMT中生長之菌落＞YpSs中生長之菌落；在溫度試驗中，各塊菌菌落的直徑生長均以25℃生長最佳，34℃生長最差，其溫度處理之菌落生長優劣順序為25℃≧20℃>30℃≧15℃>34℃。其中，冬塊菌(Tuber brumale)於各溫度下，其菌落生長最佳且快速，其他塊菌菌落在34℃下均停止生長；35℃為意大利白塊菌與印度塊菌菌落之最高致死溫度，36℃是法國黑孢塊菌與台灣塊菌之最高致死溫度。 就菌落顏色言，無論是培養基試驗或溫度試驗其菌落大部分為白色。YpSs培養基上大多數的塊菌菌落生長均勻且緻密，YMT培養媒質則較稀且薄；溫度試驗中，各溫度下的塊菌菌落，除了30℃之印度塊菌為橘褐色，其餘皆呈現白色菌落，所有菌落均會分泌色素；擬外孔塊菌可耐短期的高溫，冬塊菌於36℃中始停止生長。 立體顯微鏡下觀察到夏塊菌之外生菌根有稀疏纖毛狀或羊毛狀之剛毛，分布於菌根頂端或前半部；菌根之顏色變化由幼至老能從淡黃色轉變為黃褐色至深褐色；其菌根形態與台灣二葉松形成分叉或單根棒狀，而與青剛櫟則形成單根棒狀或規則羽狀，菌根長度為500~3000 μm。 以光學顯微鏡觀察，發現其自菌毯延伸出的菌絲為波浪狀、具隔膜、該菌絲上有突起及菌絲囊形成。在掃描式電子顯微鏡下，發現其自菌毯延伸出的菌絲與囊狀體有菌絲融合之現象，菌絲表面平坦、葱形菌絲的形成、具發育良好菌毯、哈替氏網侵入至第一層皮層細胞，哈替氏網於皮層細胞壁間能形成指狀菌絲。 夏塊菌菌根形態特徵於光學顯微鏡下發現之突起與掃描式電子顯微鏡下發現之特徵相符，其菌根直徑為116~300 μm，菌絲直徑為1~2.3 μm，菌毯厚度為12~20 μm，菌毯為多角形或略圓形之細胞組成，屬規則密絲組織。

Some truffles are the most precious edible ectomycorrhizal muschrooms and with considerable economic importance in the world. Recently they were often used as materials for ectomycorrhizal research. Therefore, the purposes of this study were (1) to ascertain the optimum and the highest deadly temperature to the colony growth of some Tuber species, and (2) to describe the ectomycorrhizal morphology formed by Tuber aestivum on Cyclobalanopsis glauca and Pinus taiwanensis with semi-aseptic synthesis. The materials used for culture medium and temperature test were the isolated Tuber brumale, Tuber indicum, Tuber formosanum, Tuber pseudo-excavatum, Tuber melanosoprum, and Tuber magnatum. The above-described six Tuber species were inoculated on the YMT and YpSs culture medium to compare which medium was better for their colony growth. The temperature treatment for each colony growth of each truffle species was set at 15℃, 20℃, 25℃, 30℃ and 34℃, respectively. Each treatment was triplicated. This test ended when one of the rapidly grown Tuber sp. colony reached to the edge of petri dish. The highest deadly temperature test was set at 33℃, 34℃, 35℃ and 36℃. The spore suspension of T. aestivum was used to inoculate the semi-aseptic seedlings of C. glauca and P. taiwanensis. The mycorrhizal morphology was observed under stereomicroscope, light microscope and scanning electron microscope. The results showed that only the colony growth of T. pseudo-excavatum had significant difference (P<0.05) between the YMT and YpSs medium. The most optimum colony growth temperature was at 25℃ for all Tuber species tested and at 34℃ the worst. The order for the colony growth magnitude at different temperatures was 25℃≧20℃>30℃≧15℃>34℃. Among which, the colony growth of T. brumale was the fastest. Except T. brumale, the colony growth of all other species ceased at 34℃. The highest deadly temperature was 35℃ for Tuber magnatum and Tuber indicum, and 36℃ for Tuber melanosoprum and Tuber formosanum. In terms of colony color, most of their colonies were white in spite of culture medium test or temperature test. The colony growth was uniform and dense on the YpSs medium, but sparse and thin on the YMT medium. The colony color of all Tuber sp. is white, except the colony color of T. indicum at 30℃ was orange-brown. All colonies could secrete pigment. T. pseudo-excavatum could endure short-period high temperature, and the colony growth of T. brumale ceased at 36℃. The ectomycorrhizal characteristics of Tuber aestivum observed under stereomicroscope were as followings: simple, regularly pinnate (for C. glauca only) or dichotomous (for P. taiwanensis only), 500~3000 μm in length, 114~300 μm in diameter; light yellow in the young age, yellow brown to brown when mature, dark brown in the wilted age; fungal mantle smooth or with sparse yellowish-golden or wooly yellowish setae on mycorrhizal top. Under light microscope, the ectomycorrhizal characteristics of Tuber aestivum were: with wave-like extending hyphae, with septal hyphae, setae with vesicle or wewithout, hyphae sometimes with small warts. The characteristics of ectomycorrhizae observed under scanning electron microscope re: fungus mantle 12~20 μm thick, regular synenchyma; hyphal surface smooth, 1~2.3 μm in diameter, occasionally with cystida at the top or with hyphal anastomoses. Hartig nets reached to the first layer of cortex cells, formed finger-like hyphae in the cortical cell wall.