Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31038
標題: 引起蕙蘭與文心蘭葉斑病的鐮孢菌病原性測定與分子特性
Pathogenicity and molecular character of Fusarium spp. causing leaf spot on Cymbidium and Oncidium
作者: 簡苡琦
Jain, Yi-Chi
關鍵字: Fusarium black spot disease
Fusarium細斑病
Fusarium proliferatum
host range
molecular phylogenticity
Fusarium proliferatum
寄主範圍
分子親緣性
出版社: 植物病理學系所
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摘要: 台灣栽培之蘭花廣受全球喜愛,為外銷農產品中最重要的花卉作物,除蝴蝶蘭 (Phalanenopsis)以外,又以蕙蘭 (Cymbidium) 與文心蘭 (Oncidium),為外銷產值較高的兩種花卉,於台灣的蘭花產業占有重要的地位。自2010年1月至2011年2月於蕙蘭與文心蘭園進行病害之調查,並觀察到多種葉斑病徵,經分離與初步鑑定得知,由Fusarium所引起的葉部病害常於兩種蘭園被發現,進一步由形態及分子生物學鑑定,自蕙蘭21棵罹病植株中分離到64株真菌,其中43株為F. proliferatum、1株為F. verticillioides,且多分離自黑色細斑之病徵;而自文心蘭22棵罹病植株中分離出55株真菌,有8株皆屬F. proliferatum菌種,多分離自黑色細斑之病徵;然調查中並無分離到可引起日本蕙蘭葉斑病之F. fractiflexum,且於田間亦無發現帶有黃色病斑之蕙蘭葉片。將自蕙蘭與文心蘭分離的Fusariumrm菌株以107 conidia/ml孢子懸浮液,直接滴加於蕙蘭與文心蘭切離葉片以針穿刺造成的傷口,進行致病力測試。接種結果顯示,所有供試菌株均可引起黑色壞疽病斑,且來自蕙蘭與文心蘭所分離的F. proliferatum菌株可同時於此兩種蘭科花卉引起壞疽病斑。進一步挑選致病力較強之菌株,製成106 conidia/ml孢子懸浮液,噴灑於蕙蘭與文心蘭瓶苗植株做無傷口接種,結果顯示供試F. proliferatum可於蕙蘭瓶苗上引起與田間類似的細斑病徵。於寄主範圍測試中,來自蕙蘭與文心蘭罹病葉分離之菌株可造成非寄主的蘭科植物產生褐色病斑,然病原性較弱。將分離自水稻、蝴蝶蘭及石斛蘭之F. proliferatum進行對蕙蘭與文心蘭的致病力和瓶苗病原性測試,得知分離自其它作物的F. proliferatum菌株可引起蕙蘭與文心蘭切離葉片產生病斑,然其致病力表現與在蕙蘭瓶苗上之罹病度仍較來自蕙蘭或文心蘭的菌株低,此結果顯示F. proliferatum具廣寄主及伺機性之特色,但菌株間的致病能力仍具有差異。本研究增幅F. proliferatum菌株的Polyketide synthase (PKs)、translation elongation factor 1 alpha (EF 1α)及Intergenic spacer (IGS) 的基因序列,並以近鄰結合法 (Neighbor-joining method, NJ) 與最大簡約法 (Maximum parsimony method, MP) 進行分析,結果顯示,利用PKs與EF 1α分析所得之樹狀圖,顯示來自蕙蘭、文心蘭及石斛蘭之F. proliferatum菌株可形成一大分子群,支持度為>67 %,而來自蝴蝶蘭與水稻之F. proliferatum菌株則各自形成獨立單一分子群,支持度分別為>92 %與>65 %。然IGS序列分析之樹狀圖顯示,由石斛蘭所分離出之F. proliferatum菌株可自成一分子群,支持度為100 %與其餘菌株在分子層次上具有差異性。此外,來自水稻的F. fujikuroi與F. proliferatum菌株於三種基因序列分析所得系統樹中,均可形成同一個分子群,證實F. fujikuroi與水稻的F. proliferatum於PKs、EF 1α及IGS基因序列具有較高的親緣性。
Orchid produced in Taiwan is popular in the world. Recently, the orchid has become the most important products to export to different countries. Among the orchid products, Phalaenopsis is the number one, and Cymbidium and Oncidium are second and third. Total 64 fungi and 55 fungal isolates were obtained from 21 Cymbidium plants leaves and 22 Oncidium plants leaves showed black spot, respectively. Based on morphological and molecular characteristics, the 43 isolates from Cymbidium were identified as Fusarium proliferatum and one isolate was identified as F. verticillioides. The other sides, 8 of 55 isolates from Oncidium were identified as F. proliferatum. The results demonstrated that F. proliferatum is the major species on Cymbidium and also can cause leaves spot on Oncidium, however, the F. fractiflexum published in Japan could not be found in Cymbidium or Oncidium in Taiwan. For confirming the pathogenicity, the isolates of F. proliferatum from Cymbidium and Oncidium were examined. The results showed that all isolates of F. proliferatum can cause necrosis on detatched leaves after inoculated with 5 μl of 107 conidia/ml microspore suspension by dropped method. In addition, the isolates of F. proliferatum from Cymbidium and Oncidium could cause more severe symptom on young seeding of Cymbidium and Oncidium after inoculated with 106 conidia/ml microspore suspension by sprayed method. In this study, the pathogenicity of F. proliferatum from Cymbidium and Oncidium to different hosts was exam ined. The result indicated that the isolates of F. proliferatum from Cymbidium and Oncidium can not cause necrosis spot on detached leaves of different hosts. Moreover, the isolates of F. proliferatum from Phalaenopsis, Oryza, and Dendrobidium were tested their pathogenicity to Cymbidium and Oncidium by inoculated in detached leaves. The result demonstrated that the isolates of F. proliferatum from different hosts can infect and show variably pathogenicity in Cymbidium and Oncidium. Thus, the isolates of F. proliferatum have the characteristics of wide host range and opportunism. Three DNA regions, polyketide synthase (PKs)、translation elongation factor 1 alpha (EF 1α) and intergenic spacer (IGS) of ribosome, were sequenced and analyzed the phylogenetic relationship between the isolates of F. proliferatum. The result revealed that the isolates of F. proliferatum from Cymbidium and Oncidium formed a molecular group and did not support the characters of host, location and pathogenicity based on neighbor-joining method (NJ) and maximum parsimony method (MP) analysis. However, the isolates of F. proliferatum from Phalaenopsis, Oryza, and Dendrobidium could form monophyletic group and support by high bootstrap value, respectively. Moreover, the isolates of F. proliferatum and F. fujikuroi from Oryza could be grouped a cluster by high bootstrap value. Thus, the two species f F. proliferatum and F. fujikuroi have high molecular phylogentic relationship.
URI: http://hdl.handle.net/11455/31038
其他識別: U0005-2108201118041700
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2108201118041700
Appears in Collections:植物病理學系

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