仙草的可培養性內生真菌之多樣性

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謝仲韋(Chung-Wei Hsieh) 查詢紙本館藏

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論文名稱

仙草的可培養性內生真菌之多樣性
(Diversity of cultivable endophytic fungi of Chinese mesona (Platostoma palustre))

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摘要(中)

仙草於台灣是一種重要的經濟作物，過往研究具有抗發炎及利尿的功效，然而目前世界上並無任何對於仙草的真菌記錄，包括內生及非內生真菌。於此研究中我們從經由表面消毒後的健康宿主之根、莖和葉中分離了內生真菌，以DNA序列分析及形態學鑑定菌種。我們從15株無病徵仙草，共810個植物切片中分離出154個菌株，其中Cercospora、Colletotrichum 和Fusarium 為最常見的屬，此三屬一般被認為是低專一性之植物病原菌，我們經由感染實驗證實了其中一支內生真菌Corynespora cassiicola 的潛在致病性，我們假設這些病原菌是潛伏於宿主中，待其年老或環境不利於生長時，第一時間感染並腐生宿主本體，我們還利用了感染實驗來驗證從實驗中所分離的植物病原菌可以再次感染回健康的宿主。Hypoxylon 是本研究中出現頻率第四的屬，並且Hypoxylon cf. investiens 是唯一一種Hypoxylon 菌種。

摘要(英)

Platostoma palustre is an important economic crop in Taiwan. Previous studies have shown its anti-inflammatory and diuretic effects. However, there is no fungal record in the world for this plant, including endophytic and non-endophytic fungi. In this study, we isolated endophytic fungi from surface sterilized healthy roots, stems, and leaves. Pure cultures were identified by DNA sequence analysis and morphology. We obtained 154 isolates from 810 fragments of 15 symptomless plants of P. palustre. The most common species belonging to the genera Cercospora, Colletotrichum, and Fusarium were generally considered to be low-specific plant pathogens. We confirmed the latent pathogenicity with an infection experiment with an endophytic strain of Corynespora cassiicola. We hypothesize that these fungi are latent pathogens in the host and may get the initiative to infect the host when the host becomes weakened by senescence or changing environment. Hypoxylon was the fourth most common genus and represented by H. cf. investiens was as the single Hypoxylon species.

關鍵字(中)

★ 仙草
★ 內生真菌
★ 多樣性
★ 感染實驗

關鍵字(英)

★ Chinese mesona
★ Platostoma palustre
★ mesona chinensis
★ endophyte
★ endophytic fungi

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
......................................................................................................
viii
List of abbreviations
........................................................................................
ix
1
Introduction................................................................................
1
1-1
Endophytic fungi and application.................................................
1
1-2
Platostoma palustre......................................................................
1
1-3
History of the methods to identify fungi......................................
2
1-4
Internal transcribed spacer............................................................
2
1-5
Culture-dependent approach for the detection of endophytic fungi from host plant used in this study.......................................
3
1-6
Research aims...............................................................................
3
2
Materials and Methods..............................................................
4
2-1
Collection and preparation of plants............................................
4
2-2
Culture-dependent approach.........................................................
4
2-2-1
Surface sterilization......................................................................
4
2-2-2
Media............................................................................................
5
2-3
DNA isolation...............................................................................
6
2-3-1
Tissue Dissociation.......................................................................
6
2-3-2
Lysis.............................................................................................
6
2-3-3
DNA Binding...............................................................................
7
2-3-4
Wash.............................................................................................
7
2-3-5
DNA Elution.................................................................................
8
2-4
PCR..............................................................................................
8
2-4-1
Reaction mix................................................................................
9
2-4-2
PCR programs..............................................................................
10
2-5
Agarose gel electrophoresis..........................................................
11
2-6
Purification of PCR products.......................................................
11
2-6-1
Sample capture.............................................................................
11
2-6-2
Sample binding.............................................................................
11
2-6-3
Wash and dry................................................................................
12
2-6-4
Elution..........................................................................................
12
2-7
DNA sequencing...........................................................................
12
2-8
Molecular analysis........................................................................
13
2-9
Identicication................................................................................
13
2-10
Host plant infection experiments..................................................
13
3
Results..........................................................................................
15
3-1
Species richness of endophytic fungi...........................................
15
3-2
Comparison of endophytic species composition among the plant organs..................................................................................
15
3-3-1
Cercospora spp.............................................................................
16
3-3-2
Colletotrichum spp.......................................................................
19
3-3-3
Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei............
23
3-3-4
Fusarium spp................................................................................
25
3-3-5
Hypoxylon cf. investiens (Schwein.) M.A. Curtis........................
28
3-3-6
Trichoderma koningii Oudem......................................................
29
3-3-7
Basidiomycetous yeasts................................................................
30
4
Discussion....................................................................................
32
5
References...................................................................................
36

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指導教授

羅南德(Roland Kirschner)

審核日期

2016-10-31

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