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姓名 陳冠菱( Kuan-Ling Chen)  查詢紙本館藏   畢業系所 生命科學系
論文名稱
(Fungi on leaves of Nelumbo nucifera Gaertn.)
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摘要(中) 蓮花為多年生水生植物 (真雙子葉植物;龍眼目;蓮科;蓮屬),主要分布在亞洲的熱帶和亞熱帶地區。它的葉子具有蓮葉效應因此有自我清潔的能力。到目前為止,已經發現了一些寄生真菌,如Alternaria nelumbii, Colletotrichum gloeosporioides, Pseudocercospora nymphaeacea,但是內生真菌尚未有任何記錄、發現。本研究的目的是探測和鑑定真菌,包括外部真菌和內生真菌。我們在台灣不同的地方採集蓮花葉片,每一片葉子分為葉片和葉柄。將植物片段用95%乙醇和6%次氯酸鈉進行表面消毒,再用無菌水清洗。表面消毒的有效性用imprint technique來驗證。從約1002個植物片段中獲得了476株菌株,可被分為33個種類。大多數菌株由葉片分離出,佔71%,而葉柄只有29%。從植物組織中分離的內生真菌利用菌種型態顯微鏡觀察以及DNA分析來鑑定種類。其中外生真菌Pseudocercospora sp. nov. 和Dissoconium sp. nov. 為新種類。
摘要(英) Nelumbo nucifera is an herbaceous, perennial aquatic plant belonging to Nelumbonaceae (order Proteales, in the clade eudicots, genus Nelumbo), and is found in tropical and subtropical Asia. The self-cleansing property of its leaves is called lotus effect. So far, some parasitic fungi of N. nucifera have been reported, like Alternaria nelumbii, Colletotrichum gloeosporioides, Pseudocercospora nymphaeacea, but the endophytic fungi have not been recorded. The purpose of this study is to detect and identify fungi from leaves of N. nucifera including ectophytic and endophytic fungi. We collected leaves of N. nucifera at different places in Taiwan. Each leaf was divided into leaf lamina and petiole. These parts were surface-sterilized with 95% ethanol and 6% sodium hypochlorite and then rinsed in sterile water. The effectiveness of surface sterilization was verified by the imprint technique. From 1002 plant segments, 476 isolates were produced in culture, comprising 33 typical terrestrial species. Most isolates were from leaves (71%) compared to those from petiole (29%). Among ectophytic fungi, the species Pseudocercospora sp. nov. and the genus Dissoconium are recorded for Taiwan for the first time.
關鍵字(中) ★ 蓮花
★ 蓮葉效應
★ 內生真菌
關鍵字(英)
論文目次 中文摘要 ………………………………………………………………… i
英文摘要 ………………………………………………………………… ii
誌謝 ………………………………………………………………… iii
目錄 ………………………………………………………………… iv
圖目錄 ………………………………………………………………… vi
表目錄 ………………………………………………………………… vii
Abbreviation Index ………………………………………………………… viii
1 Introduction…………………………………………………… 1
1-1 Nelumbo nucifera Gaertn.……………………………………… 1
1-1-1 Systematics…………………………………………………….. 1
1-1-2 Organ systems…………………………………………………. 1
1-1-3 Lotus effect…………………………………………………….. 1
1-1-4 Application…………………………………………………….. 2
1-2 Evolutionary relationship of Nelumbo and Nymphaea……….... 2
1-3 Fungi…………………………………………………………… 3
1-3-1 Fungi on Nelumbo nucifera (external)…………………………. 3
1-3-2 Endophytic fungi…………………………………………….…. 4
1-4 Research aims………………………………………………….. 4
2 Materials and Methods………………………………………… 5
2-1 Plants and collection sites……………………………………… 5
2-2 Isolation of endophytic fungi…………………………………... 5
2-3 Surface sterilization……………………………………………. 6
2-3-1 Leaves………………………………………………………….. 6
2-3-2 Rhizomes……………………………………………………….. 6
2-3-3 Seeds…………………………………………………………… 7
2-4 Study of evtophytic fungi………………………………………. 7
2-5 Media…………………………………………………………... 8
2-6 DNA isolation………………………………………………….. 9
2-6-1 Tissue Dissociation…………………………………………….. 9
2-6-2 Lysis……………………………………………………………. 9
2-6-3 DNA Binding…………………………………………………... 10
2-6-4 Wash…………………………………………………………… 10
2-6-5 DNA Elution…………………………………………………… 10
2-7 PCR…………………………………………………………….. 11
2-7-1 Material………………………………………………………… 11
2-7-2 Protocol for PCR……………………………………………….. 11
2-8 Agarose gel electrophoresis……………………………………. 13
2-9 Purification of PCR products…………………………………... 13
2-9-1 Processing PCR Amplifications………………………………... 13
2-9-2 Binding of DNA………………………………………………... 13
2-9-3 Washing………………………………………………………... 14
2-9-4 Elution………………………………………………………….. 14
2-10 DNA sequencing……………………………………………….. 14
2-11 Identification…………………………………………………… 15
2-11-1 Morphological identification…………………………………... 15
2-11-2 Molecular identification………………………………………... 15
2-12 Phylogenetic analysis…………………………………………... 16
2-13 Isolation frequency and colonization rate……………………… 17
2-14 Statistical analysis……………………………………………… 17
3 Results………………………………………………………….. 18
3-1 Species richness of endophytic fungi…………………………... 18
3-2 Comparison of endophytic species composition among the plant organs……………………………………………………..
18
3-3 Comparison of endophytic species composition among sampling sites…………………………………………………..
18
3-4 Comparison of endophytic species and ectophytic species……. 19
3-5 Description of species………………………………………….. 19
3-5-1 Alternaria alternata……………………………………………. 20
3-5-2 Cladosporium oxysporum……………………………………... 21
3-5-3 Colletotrichum siamense………………………………………. 22
3-5-4 Dissoconium sp. nov.………………………………………….. 23
3-5-5 Fusarium solani……………………………………………….. 25
3-5-6 Nigrospora oryzae……………………………………………... 26
3-5-7 Phomopsis sp.………………………………………………….. 27
3-5-8 Pseudocercospora sp. nov.…………………………………….. 28
3-5-9 Sporobolomyces odoratus……………………………………... 30
3-5-10 Xylariaceae sp.………………………………………………… 31
4 Discussion……………………………………………………… 33
5 References……………………………………………………… 38
6 Appendixes……………………………………………………... 63
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指導教授 羅南德(Roland Kirschner) 審核日期 2014-2-24
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