結合依賴培養法、不依賴培養法與伺機採集之方法研究台灣沿海植物濱刺草之內生與非內生真菌

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楊均威(Jun-Wei Yang) 查詢紙本館藏

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論文名稱

結合依賴培養法、不依賴培養法與伺機採集之方法研究台灣沿海植物濱刺草之內生與非內生真菌
(Study of endophytic and non-endophytic fungi from the coastal grass Spinifex littoreus in Taiwan by combining culture-dependent, culture-independent and opportunistic collection approaches)

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摘要(中)

內生真菌是生活在無症狀的植物組織內並不會引起顯著疾病的微生物。非內生真菌生長在植物表面並可能引起植物病。濱刺草是一個沿岸植物，具有穩定沙岸與保護沿岸地區的重要的功能，研究也表明濱刺草具有抗發炎之潛力，然而在研究上很少有與濱刺草有關之微生物的文獻發表。本研究中內生真菌之調查使用依賴培養法與不依賴培養法，在非內生真菌部分則使用傳統的伺機採集方法進行調查。經由依賴培養法與不依賴培養法，108個植物片段與600個克隆分別鑑定了19與12個內生菌。其中只有3個種類在這兩個方法被同時鑑定到，結果顯示結合兩種方法去鑑定植物之內生菌會比只使用一種方法來的更有效率。使用伺機採集方法，7個非內生真菌被鑑定，其中2種真菌也被發現與內生真菌相同。在本研究中利用了以上三種方法並依據RNA polymerase II second largest subunit DNA 序列與型態學之特徵，同時也發現了Neostagonospora 屬的一個世界新種，Neostagonospora spinificis。產孢細胞之形狀與分生孢子橫隔數目、大小都是有用的特徵來區分Neostagonospora 屬之不同種。

摘要(英)

Endophytic fungi are microorganisms that live asymptomatically within plant tissues without causing apparent disease. Non-endophytic fungi grow on the plant surface, some of them may cause disease. Spinifex littoreus is an important coastal grass for stabilizing and protecting sand coastal areas and it has been shown to have anti-inflammatory properties, however, few has been published about the organisms associated with this plant. In this study, endophytic fungi were investigated by culture-dependent and culture-independent approaches and non-endophytic fungi were investigated by the opportunistic collection approach. By the culture-dependent and culture-independent approaches, 108 plant segments and 600 colonies yielded 19 and 12 species of endophytic fungi, respectively. Only 3 species were identical in both approaches, indicating that the combination of two approaches to detect endophytic fungi is more effective than a single approach. By the opportunistic collection approach, 7 species of non-endophytic fungi were identified; two of them were conspecific with endophytes. In this study, a new species of Neostagonospora was found by these approaches according to characterization by RNA polymerase II second largest subunit (RPB2) DNA sequences and micromorphology. The shape of conidiogenous cells as well as numbers of septa and size of conidia are useful characters for distinguishing between species of Neostagonospora.

關鍵字(中)

★ 內生真菌
★ 濱刺草
★ 依賴培養法
★ 不依賴培養法
★ 伺機採集法

關鍵字(英)

★ endophytic fungi
★ Spinifex littoreus
★ culture-dependent
★ culture-independent
★ opportunistic collection

論文目次

中文摘要 iv
英文摘要 v
誌謝 vi
目錄 vii
圖目錄 x
表目錄 xii
List of abbreviations xiii
1 Introduction 1
1-1 Endophytic fungi and non-endophytic fungi 1
1-2 Description of Spinifex littoreus 2
1-3 The methods to study the fungi associated with plants 2
1-4 The genus of Neostagonospora 4
1-5 Research aims 4
2Materials and Methods 5
2-1 Collection and preparation of plants 5
2-2 Culture-dependent approach 5
2-2-1 Isolation of fungi 6
2-2-2 Surface sterilization 6
2-2-3 Media 6
2-2-4 DNA isolation 7
2-2-4-1 Tissue Dissociation 7
2-2-4-2 Lysis 8
2-2-4-3 DNA Binding 8
2-2-4-4 Wash 9
2-2-4-5 DNA Elution 9
2-2-5 PCR 10
2-2-6 Agarose gel electrophoresis 12
2-2-7 Purification of PCR products 12
2-2-7-1 Processing PCR Amplifications 12
2-2-7-2 Binding of DNA 12
2-2-7-3 Washing 13
2-2-7-4 Elution 13
2-2-8 DNA sequencing 14
2-2-9 Identification 14
2-2-10 Morphological identification 14
2-3 Culture-independent approach 15
2-3-1 Pretreatment and DNA isolation 15
2-3-2 PCR 16
2-3-3 Gel extraction 16
2-3-4 Ligation 17
2-3-5 Lysogeny broth (LB) media 18
2-3-6 Preparation of transformation competent cells 19
2-3-7 Transformation 19
2-3-8 Colony PCR 20
2-3-9 Agarose gel electrophoresis 21
2-3-10 Restriction fragment length polymorphism (RFLP) analysis 21
2-4 Opportunistic collection approach 22
2-5 Molecular identification 23
2-6 Phylogenetic analysis 23
3 Results 26
3-1 Species richness of endophytic fungi by the culture-dependent approach 26
3-2 Species richness of endophytic fungi by culture-independent approach 26
3-3 Comparison of the endophytic fungi detected by culture-dependent and culture-independent approaches 27
3-4 Comparison of endophytic species and non-endophytic species 27
3-5 Description of species 28
3-5-1 Cladosporium oxysporum 29
3-5-2 Fusarium equiseti 31
3-5-3 Hortaea werneckii 33
3-5-4 Lacellina macrospora 34
3-5-5 Myrmecridium schulzeri 36
3-5-6 Neostagonospora spinificis 37
3-5-7 Nigrospora sphaerica 40
3-5-8 Phaeosphaeria sp. 42
3-5-9 Rhizopycnis vagum 43
4 Discussion 44
5 References 54
6 Appendixes 88

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指導教授

羅南德(Roland Kirschner)

審核日期

2015-11-24

推文