植物棲居之梅奇氏酵母菌群 (Metschnikowia spp.) 易於植物、花、果實與相關的昆蟲樣本中被分離。梅奇氏酵母菌為異宗交配型 (heterothallic)，可產生細長接合管與子囊且內含1-2個針狀的子囊孢子。本研究利用本實驗室自2005至2016年間，於台灣各地區所採集之植物、土壤、菇類及水樣本中，所分離出174株之Metschnikowia屬及相關Candida屬酵母菌，包含土壤類菌株33株、植物類菌株102株、昆蟲類菌株9株、菇類菌株23株及水樣本類菌株7株。所有菌株利用傳統鑑定及分子鑑定的方法進行菌種確認、分類及親緣關係研究。藉由大單元核醣體D1/D2區域序列進行初步鑑定，並據此繪製親緣關係樹狀圖，共可分為12群 (A1-A12 clade)，並鑑定出11種已知菌種，包含C. berkhoutiae, C. dosseyi, C. fructus, C. pseudointermedia, M. cibodasensis, M. kofuensis, M. koreensis, M. rancensis, M. vitivola, M. reukaufii, M. sinensis，以及39種未被描述種。其中大部分未描述菌種歸群於A1-A5, A10-A12支群，且以A10-A12支群最多，分別有17個未描述種。其中39個可能新種之所有菌株除了進行形態觀察、生理生化試驗與分子特徵分析外，並分別進行種內交配實驗。結果發現85種內菌株間產生交配芽管，另有2種會產生Metschnikowia典型之針狀子囊孢子。另本實驗亦將其174株酵母菌以核醣體外轉錄區間ITS (internal transcribed spacers) 序列進行分子鑑定，共可劃分9群 (B1-B9 clade)，其結果與D1/D2區域序列分群結果類似，但因其在ITS序列在此菌屬之鑑定中，種間或種內的序列有很大的差異，而造成這些差異可能是因為菌株間的交配產生了基因取代的現象，使親緣關係樹狀圖與D1/D2區域序列為基礎之樹狀圖有所差異，故本實驗以D1/D2區域序列作為分子鑑定主要依據，而僅將ITS序列做為參考數據。本研究所分離之Metschnikowia屬之39種新種菌株，為台灣各地且大多由植物樣本所分離，在世界各地均尚未被發現。在植物樣本中分離的酵母菌主要是Metschnikowia rancensis, Metschnikowia reukaufii和Metschnikowia koreensis，這三個物種為世界性普遍分佈的物種，且廣泛在植物的花蜜所分離，而其餘大部分菌種多為區域性物種。由於植物中的花蜜被認為是動物主要覓食標的，並且許多新描述的物種都與昆蟲所覓食的植物相關，故 Metschnikowia屬之物種在寄主和棲息地具有高度專一性。

The plant-associated yeasts clustered in the Metschnikowia clade were commonly isolated from phylloplanes, flowers, fruits and plant-associated insects. The yeasts are heterothallic and demonstrated typical teleomorphic characteristics with mating tubes between two individual cells and slender ascus containing 1-2 needle-shaped ascospores in persistent or evanescent ascus. In this study, one hundred and seventy four strains of yeasts belonging to Metschmikowia genus and Candida genus were previously isolated from 110 samples which collected from plants, soil, mushrooms and water in Taiwan during 2005 to 2016 in our laboratory. Of these strains, thirty three were isolated from soil, one hundred two from plants, nine from insects, twenty three from mushrooms and seven from water. All of 174 yeast strains were identified and phylogenetically clustered by traditional and molecular approaches. Based on the sequences of the D1/D2 domain of the large subunit (LSU) rRNA gene, the phylogenic tree was constructed and showed 12 clades (A1-A12), indicating 11 recognized species, including C. berkhoutiae, C. dosseyi, C. fructus, C. pseudointermedia, M. cibodasensis, M. kofuensis, M. koreensis, M. rancensis, M. vitivola, M. reukaufii, M. sinensis and 39 tentative novel species, most of the tentative novel species are clustered into the A1-A5 (8 species) and A10-12 clade (17 species). All the strains belonging to tentative new species were idenfied by morphological, physiological and sequence analysis of LSU D1/D2 domain and ITS fragment. Also mating among intraspecific strains were tested and showed that 8 groups of them produced germ tubes, two of the groups even showed the typically needle-shaped ascospored of Metschnikowia genus. All the 174 yeast strains were also analyzed by the internal transcribed spacers (ITS) of rRNA gene sequences in this study, and 9 clades (B1-B9 clade) showed in the phylogenetic tree. The ITS sequences of yeast strains showed more significantly different than those of D1/D2 sequences within other intraspecies strains. ITS differences could be proposed that some species appear considerable substitution of genetic material from other strains. Therefore, the ITS phylogenic tree is not precise enough in classification of Metschnikowia genus. At present, many scientists are also exploring the issue, so all the yeast strains were identified based on sequences of the LSU D1/D2 domain, but ITS fragment sequences as a reference data. The 39 tentative new species of the Metschnikowia genus were isolated from Taiwan and mostly from plant samples, not found anywhere in the world. The species isolated in plant samples are mainly Metschnikowia rancensis, Metschnikowia reukaufii and Metschnikowia koreensis, which are ubiquitous species and are widely isolated from the nectar of plants. Because the nectar in plants is considered to be the major visiting target by respect insects, and many of the newly described species are associated with some of the plants visited by insects. Therefore, species of the genus Metschnikowia could be highly specific in their host and habitat

摘要……………………………………………………………………………………………I
Abstract………………………………………………………………………………………………II
誌謝辭…………………………………………………………………………………………IV
目錄…………………………………………………………………………………………………X
表目錄…………………………………………………………………………………………XIII
圖目錄……………………………………………………………………………………………XIV
壹、前言………………………………………………………………………………………1-15
貳、材料與方法…………………………………………………………………16-35
參、結果與討論………………………………………………………………………36-110
肆、討論……………………………………………………………………………………108-110
伍、參考文獻………………………………………………………………………………111-117
附錄一、…………………………………………………………………………………118-156
附錄二、…………………………………………………………………………………157-163

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