- 學位論文
歐亞主要塊菌在洋菜培養基生長之比較及其與青剛櫟形成菌根形態之研究
Study on Colony Growth in Agar Medium and Mycorrhiza Morphological Characteristics Formed on Cyclobalanopsis glauca (Thunb.) Oerst. of Five Asian and European Tuber species
摘要
塊菌的美味與芳香是美食家追求之高價值可食用性真菌類，亦是需求量逐漸增加之高經濟價值食品，已引起全球廣泛研究。台灣塊菌(Tuber formosanum)是台灣原生塊菌與青剛櫟(Cyclobalanopsis glauca)共生之菌種；印度塊菌(Tuber indicum)、擬外孔塊菌(Tuber pseudoexcavatum)是中國雲南、四川地區廣泛分佈之菌種，且已外銷至歐洲市場；此外，法國黑孢塊菌(Tuber melanosporum)及義大利紳士白塊菌(Tuber magnatum)則是歐洲地區極具經濟價值之菌種。 本研究目的為：(1)確定前述5種塊菌在YpSs洋菜培養基最適菌落 (colonization) 生長之溫度及pH；(2)觀察比較5種塊菌不同發育階段之菌根形態。 研究方法為(1)以6種溫度(10℃、15℃、20℃、25℃、30℃、33℃)及3種pH值(5.0、6.0、7.0)處理，採完全逢機複因子試驗設計，於YpSs洋菜培養基中確定其對菌落生長之效應；(2) 5種塊菌分別與青剛櫟合成之菌根，於立體顯微鏡及掃瞄電子顯微鏡觀察其3種不同生長期(幼期、成熟期、老化期)之形態，並予描述及建立簡易檢索表。 菌落生長試驗結果顯示：不同溫度對5種塊菌之菌落生長皆呈極顯著差異，紳士白塊菌、黑孢塊菌、擬外孔塊菌之最佳生長溫度皆為20℃及25℃，印度塊菌及台灣塊菌則為25℃；而最差溫度5種菌種皆為33℃。不同pH值對5種塊菌之菌落生長呈顯著或不顯著差異，於20℃、25℃時，除擬外孔塊菌呈顯著差異外，且以pH 6生長最佳，pH 7最差，其餘4種菌種皆不呈顯著差異。又5種菌種中，印度塊菌適應低溫之能力較其他菌種差，而台灣塊菌則對溫度之忍耐範圍較廣；黑孢塊菌、台灣塊菌於較低及較高溫時，對培養介質pH適應能力較敏感，而紳士白塊菌、擬外孔塊菌及印度塊菌則於較高溫度時，對培養介質pH適應能力較敏感；擬外孔塊菌於中性培養介質中，對溫度之耐度較大。5種塊菌於較低溫度時，較低pH之培養介質，對其菌落生長較為有利，反之，於較高溫度時，中性pH之培養介質，對其菌落生長則較為有利。 溫度對5種塊菌於洋菜培養基之菌落生長形態甚具影響，反之，pH則影響不大，各菌種內於不同溫度間呈明顯差異，但不同pH值則無明顯差異；菌種間於10℃及33℃時之菌落生長形態差異不大，但於其他溫度時則呈現不同差異。 依據5種塊菌於洋菜培養基之菌絲菌落生長差異，及與青剛櫟合成之菌根其形態特徵差異，建立簡易檢索表，可供為5種塊菌菌種初步鑑定之參考。但因不同生長期及地理分布差異，種內形態變異大，形態特徵之分類較不可靠，建議在塊菌菌種分類上應以形態及分子生物技術相輔應用，以達到可信賴之鑑定目的。 本研究之5種塊菌菌種在洋菜培養基中，最適宜之生長溫度為20-25℃，pH值為5.0、6.0、7.0，且均可與青剛櫟接種合成菌根，如此之條件將可應用於後續塊菌菌根苗合成與栽培之研究。目前台灣塊菌已成功地人工接種栽植並生產塊菌子實體，因此紳士白塊菌、黑孢塊菌、擬外孔塊菌及印度塊菌，在台灣地區接種栽植之可行性應是樂觀的，期能於台灣地區海拔800~ 1,500公尺區域之石灰質土壤或人工施用石灰改良土壤之生育地栽培之。
並列摘要
Some Tuber species are edible fungi that, given their distinctive taste and aroma, are highly valued by gourmets and are among the most prized foods with an increasing demand, have drawn extensive research interests worldwide. Tuber formosanum is a Taiwan endemic truffle and symbioses on Cycobalanopsis glauca . T. indicum and T. pseudoexcavatum are widespread truffle species in Yuan-Nan and Sz-Chuan provinces in China and together with T. sinense , they are all exported to European market with high commercial value. In addtion, T. melanosporum and T. magnatum are European truffles and they also possess great culinary and commercial value. Therefore, the purposes of this study were to(1) ascertain the optimum temperature and pH for the colony growth of five Tuber species in YpSs agar medium , and (2) to observe and compare the ectomycorrhizal morphology of five Tuber species at different growth stage. The methodology of the study can be given a result as follows: (1) six different temperatures (10°C、15°C、20°C、25°C、30°C、33°C) and 3 different pH (5.0、6.0、7.0) having been incorporated completely random complex factor design in this research, to confirmed that the colony growth effects on the YpSs argar medium, and (2) to observe and describe the ectomycorrhizal morphology formed by five Tuber species on C. glauca respectively at different growth stage ( infancy、maturity、agedness) under a stereomicroscope and scanning electron microscope, and to establish an index table for identification of five Tuber species of this study. The testing results showed that different temperatures will cause significant difference in the colony growth of five Tuber species, and the best temperatures were found to be 20°C and 25°C for T. magnatum, T. melanosporum and T. pseudoexcavatum respectively, and 25°C for both T. indicum and T. formosanum . In addition , the worst temperature was 33°C for five Tuber species of this study. Different pH treatments showed either significant or insignificant difference in the colony growth of five Tuber species. Then at 20 °C and 25 °C it showed a significant differences in the colony growth of T. pseudoexcavatum ; and its best pH for the colony growth was at pH 6.0 whereas the worst pH was at pH 7.0. Eventually, it showed no significant difference in the colony growth of other four Tuber species. Meanwhile, in five Tuber species, to the adaptability at lower temperature, T. indicum was the worst; to the tolerance on temperature , T. formosanum was in wider range than other species. For the adaptability to pH in agar culture media, it showed that T. melanosporum and T. formosanum were more sensitive at a lower and a higher temperature, whereas T. magnatum, T. indicum and T. pseudoexcavatum were more sensitive at a higher temperature. In addition T. pseudoexcavatum has a great endurance to the temperature when cultivated in pH 7.0 media. At the lower pH value and the lower temperature cultural media, it is more advantageous to the colony growth of five Tuber species. On the other hand, at the higher temperature and pH 7.0 cultural media, it is more beneficial to the colony growth . The influence of temperature is tremendous to the morphology of colony growth of five Tuber species in agar media. On the contrary, the pH value merely plays a minor role in this aspect. Meanwhile, different temperature will cause great variation to the morphology of colony growth in intra species although the pH value is not a crucial factor. However, it should be pointed out that the variation of inter species morphology of colony growth is not distinct for the temperature ranges from 10?C to 33?C whereas it becomes significant as the temperature outside the above range. According to differences of mycelia colony growth in agar media, and the ectomycorrhizal morphological characteristics formed by five Tuber species on C. glauca respectively, one can set up a simple index table, to provide a reference for the identification of five Tuber species. Due to the differences of growth stage and geographical distribution , the variation of intra specific morphological is enormous and the classification of morphological characteristics is not reliable. As a result, it is recommended that the truffles classification should be based on the applied techniques of morphology and molecular biotechnology together and eventually to achieve the goal of reliable species identification. The best colony growth conditions for T. melanosporum, T. magnatum , T. pseudoexcavatum , T. indicum and T. formosanum in agar medium were the temperature range from 20°C to 25°C, pH value varies from 5.0, 6.0, 7.0, and formed the ectomycorrhiza by five Tuber species on C. glauca. Meanwhile, the above condition can be used in the subsequent research for synthesis and cultivation of truffle mycorrhizal seedlings. Nowadays, T. formosanum has been successfully inoculated and planted artificialiy and the fruit badies were also generated. Therefore, it is optimistic for the feasibility of inoculation and planting of T. magnatum, T. melanosporum, Tuber pseudoexcavatum and T. indicumn and it is also expected that these Tuber species can be cultivated in a natural calcareoussoil soils or artificially limed soils situated at the altitude from 800 m to 1,500 m in Taiwan.