Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31511
標題: 台灣中草藥內生真菌生物多樣性之研究及 Appoharknessia sp.在蔬菜病害防治上之應用
The study of biodiversity of endophytic fungi from Chinese herb and its application on control of vegetable disease
作者: 何旻遠
Ho, Min-Yuan
關鍵字: endophytic fungi;內生真菌;bioactiveity;secondary metabolite compounds;biocontrol;生物活性;二次代謝產物;生物防治
出版社: 植物病理學系所
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摘要: 
摘要
植物內生真菌 (endophytic fungi) 為一群生長於健康植物組織內之真菌,許多研究指出它們可產生多種具生物活性的二次代謝產物,能夠抑制有害微生物或腫瘤細胞之功效。本研究自福山植物園、台中新社種苗繁殖場及溪頭森林遊樂區蒐集到 21 科 32 屬 49 種中草藥植物,並分離出 197 株內寄生真菌。藉由形態學的觀察及分子生物學的分析,可將這些菌株歸類為 42 屬,以 Phomopsis、Phomatospora、Guignardia、Nigrospora、Xylaria 及 Hypoxylon 六三屬真菌分離比例最高。此外,本研究針對 33 株於分類上屬於炭角菌科 (Xylariaceae) 的菌株,增幅 rDNA internal transcribed spacer (ITS) 與 large-subunit (LSU) D1/D2 region 基因核苷酸序列,並以近鄰結合法 (Neibor-joining method, NJ) 進行分子親緣性的分析。結果顯示,ITS 與 LSU D1/D2 核苷酸序列可將臺灣內生性炭角菌科的真菌區分成 Hypoxylon 與 Xylaria 二個大分子群,Hypoxylon 屬的菌株可形成一的獨立分子群,並被高的 Bootstrap值 (>90%) 所支持;而 Xylaria 屬的菌株則分散為於不同的小分子群,指出 Xylaria 屬菌株於 ITS 與 LSU D1/D2 的核苷酸序列變異較高。
於測試內生真菌對植物病原拮抗能力測試,顯示 197 株菌株中有 6 株菌株對供試病原的生長可表現不同抑制效果,其中Apoharknessia sp.(CB10)、Ophioceras tenuisporum (CI02) 及 Stictis radiata(TR09)菌株對大部份病原菌的生長具有較明顯的抑制效果。本研究選取生長較快速的菌株 CB10,進行活性代謝產物之分析實驗。在測試培養條件的實驗當中,分別針對不同的溫度、酸鹼值及接種源濃度,對 CB10 菌株分泌生物活性物質之多寡進行分析,結果指出在 30℃、pH 6 及接種源為 106 spore/ml 的環境下培養 18 天,菌株培養液具有最高的抑菌活性。在溫室防治實驗中,施用 CB10 菌株的菌絲萃取物與發酵濾液防治不結球白菜炭疽病。結果顯示發酵濾液可促使炭疽病發生更嚴重,而菌絲萃取物則能有效降低不結球白菜炭疽病的罹病度,施用 10 和100 μg/ml 可降低不結球白菜15.6 及 25 % 的罹病度,而在接種前 4 天處理 10 和 100 μg/ml 菌絲萃取物的組別,則可降低 34.4 與 37.5 % 的罹病度,證實菌絲萃取物具有誘導不結球白菜抗病的能力。另於溫室中防治不結球白菜幼苗立枯病的實驗中,使用 10 與 100 μg/ml 菌絲萃取物浸泡處理種子,可提高種子之存活率達 25.9 與 16.7 %。
在生物活性的代謝產物分析方面,以正向管柱層析 (填充物為 Silica gel ) 收集 CB10 菌株萃取物,並對各分層進行活性測試,分析結果以 100 % 甲醇分層中的抗菌活性最明顯,指出具有拮抗作用的物質屬於中低極性。以 LC/MS/MS 進行活性產物的分析,並以 SDBS 資料庫進行比對,得知自菌絲萃取的物質包括醯胺、羧酸、偶氮洋紅及類黃酮等物質,其中類黃酮類物質 diosmin 具有生物活性,可誘導植物產生抗病反應與抑制真菌生長。未來應可進一步開發成植物保護製劑。

Abstract
Endophytic fungi residing in live in medical plants; can produce many kinds of the secondary metabolites. Many reports indicated that many of them could inhibit the growth of pathogens and tumor cells. A total of 197 endophytic fungi isolates from 21 family 32 genus 49 species of the different parts of the medical plants were collected from Fushan, Shinshe and Chitou. We classified as 42 genera base on morphological characteristics and molecular analysis. Among the endophytic flora, Phomopsis、Phomatospora、Guignardia、Nigrospora、Xylaria and Hypoxylon were the dominant fungal endophytes in Taiwan. For Molecular analysis, rDNA sequence of ITS and LSU D1/D2 region were used to evaluate phylogenetic relationship between Xylariaceae isolates by neighbor-joining method (NJ). Results indicated that 33 Xylariaceae isolates could be divided into two major molecular groups, including Hypoxylon and Xylaria. Hypoxylon isolates form a phylogenetic group and support with high bootstrap values (>90%). Xylaria isolates were scattered into different phylogeny clades, they had higher diversity than Hypoxylon isolates in ITS and LSU D1/D2 region. Six isolates was evaluated for its ability to inhibit mycelial growth of plant pathogenic fungi. On the antagonistic activity test, there were six isolates showed antagonistic activity. According to inhibition spectrum, Apoharknessia sp.(CB10) , Stictis. radiata (TR09) and Ophioceras tenuisporum (CI02) showed maximum in vitro inhibition of mycelial growth of plant pathogenic fungi and cell growth of plant pathogenic bacteria. This study selected isolate CB10 for further study because of the higher growth rate. After investigating the impact of temperature, PH and inoculum density in liquid culture, the result reveal that the optimum conditions for bioactivity was added 106 spore/ml cultured in pH 6 PDB medium at 30 ℃, and the best antagonistic activity wound be detect after 18 days. A preliminary test on the disease control of Rhizoctoniasolani AG4 and Colletotrichum higginisianum in greenhouse indicated that the infection was reduced by soaking and spraying application of the mycelial extraction. The application of spray 100 μg/ml mycelial extraction 4 days before inoculum pathogen reduce more disease index percentage of anthracnose infection than 0 dpi before inoculum. The seed soaking 10 μg/ml mycelial extraction is much effective than 100 μg/ml mycelial extraction on controlling damping-off disease. The result may indicated that mycelial extraction can induce resistant of plant.
After analysis of bioactive compounds of mycelium extraction, a flavonoid compound, diosmin, had bioactivity to inhibit pathogenic mycelial growth and induce
Plant resistance. It may develop bioagent used in plant protection.
Key words:endophytic fungi, bioactiveity, secondary metabolite compounds, biocontrol.
URI: http://hdl.handle.net/11455/31511
其他識別: U0005-3101201110202400
Appears in Collections:植物病理學系

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