Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31409
標題: 杏鮑菇細菌性腐敗病菌 Burkholderia gladioli 之鑑定及不同來源的 B. gladioli 菌株之表型特徵與遺傳特性分析
Characterization of the causal agent of bacterial decay on king oyster mushroom, Burkholderia gladioli, and phenotypic and genotypic analyses of B. gladioli strains isolated from different sources
作者: 林羿廷
Lin, Yi-Ting
關鍵字: king oyster mushroom;杏鮑菇;Burkholderia gladioli;phenotypic and genotypic characteristics;Burkholderia gladioli;表型特徵及遺傳特性
出版社: 植物病理學系所
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摘要: 
本研究於 2008 年自台中縣大里地區的杏鮑菇栽培場獲得一疑似細菌危害之菇體樣本,自罹病組織進行病原細菌分離純化後獲得菌株 MBg1、MBg2 及 MBg3,經杏鮑菇與煙草葉片接種試驗確認其病原性,再分析其生理生化特性,得知該菌可生長於 41℃,但不能生長於 4℃ 及含 5 % NaCl 之 NA 培養基上;此外,該菌具有明膠水解酵素、脂質分解酵素、幾丁質分解酵素、蛋白酶、氧化酶及觸酶等酵素活性。利用脂肪酸組成及 Biolog 系統分析,初步鑑定 MBg1、MBg2 及 MBg3 為 Burkholderia gladioli,相似值分別為 0.84、0.76 及 0.58。進一步利用 MBg1、MBg2 及 MBg3 之 16S rDNA 及 ITS 序列與 GenBank database 的核酸序列進行比對,結果顯示其與 B. gladioli 之相似度為 99%。綜合生理生化測試、脂肪酸組成、Biolog 鑑定系統、16S rDNA 及 ITS 序列分析結果,將杏鮑菇細菌性腐敗病的致病菌株 MBg1、MBg2 及 MBg3 鑑定為 B. gladioli。由於 B. gladioli 可生存的生態區位相當廣,且不同生態區位菌株的表型特徵及遺傳特性具差異性,因此,本研究利用 Biolog 系統、病原性測試、拮抗測試、ITS 序列及 ERIC/BOX-PCR DNA 指紋圖譜分析不同來源的 B. gladioli 菌株。根據代謝指紋圖譜分析結果,引起杏鮑菇細菌性腐敗病之菌株 MBg1、MBg2 及 MBg3 與鳳梨果腐病菌 PBg5 及土壤分離菌株 Bg1 較相近。利用 ITS 序列分析與 ERIC/BOX-PCR DNA 指紋圖譜所繪製之關係圖顯示杏鮑菇來源之 MBg1、MBg2 及 MBg3 菌株與土壤來源之菌株 Bg1、Bg2 較相近,其相似值分別為 97.4% 與 0.734。另外,植物接種測試的結果顯示杏鮑菇細菌性腐敗病菌 MBg1、MBg2 及 MBg3 與土壤分離菌株 Bg1、Bg2 對杏鮑菇皆具病原性,但對唐菖蒲及洋蔥之致病力較弱;鳳梨果腐病菌 PBg3、PBg5 對杏鮑菇、唐菖蒲及洋蔥皆具弱病原性;此外,自唐菖蒲分離的病原菌 B. glumae 菌株 HBG4、HBG10 對唐菖蒲及洋蔥皆具病原性,但在杏鮑菇菇體上不會造成組織崩解及凹陷的病徵;土壤來源之 B. cepacia complex 菌株 Bg3 對洋蔥具弱病原性,但對杏鮑菇及唐菖蒲則不具病原性;土壤來源之 B. cepacia complex 菌株 Bg4、Bg5 對杏鮑菇、唐菖蒲及洋蔥皆不具病原性,顯示 Burkholderia spp. 雖有廣泛的寄主範圍,但不同的菌株感染寄主時應有不同的致病因子與機制。根據真菌菌絲生長與細菌生長的抑制測試結果得知不同來源之 B. gladioli 菌株皆具有抑制杏鮑菇及 Rhizoctonia solani AG-4 菌絲生長與抑制 B. glumae 菌株 RBg9 生長的能力,但 B. gladioli 感染杏鮑菇之致病因子與抑制細菌 B. glumae 生長之因子則有待進一步分析。綜合上述結果,我們推論杏鮑菇細菌性腐敗病菌 MBg1、MBg2 及 MBg3 與土壤分離菌株 Bg1、Bg2 具有相似的表型特徵及遺傳特性。

In the year of 2008, 3 bacterial strains, MBg1, MBg2, and MBg3, were isolated from the decay tissues of king oyster mushrooms that were cultivated at a mushroom farm in Daili, Taichung. The 3 strains elicited decay symptom on king oyster mushrooms and necrosis in tobacco leaves in pathogenicity tests, indicating they might be the causative agents of bacterial decay disease of king oyster mushrooms. The MBg strains were assayed for their physiological and biochemical properties, and the results revealed that they can grow on nutrient agar medium at 41 ℃ but not at 4 ℃, and they are sensitive to the osmotic pressure exerted by 5% (w/v) NaCl. The MBg strains have multiple enzyme activities, including gelatinases, lipases, chitinases, proteases, oxidase, and catalase. The MBg strains were identified using fatty acid methyl ester analysis (Agilent Technologies, Santa Clara, CA) and SHERLOCK® Microbial Identification System, and in each instance, the bacterium was confirmed as Burkholderia gladioli. In addition, the Biolog system (Biolog, Hayward, CA) and sequence identity comparisons of 16S ribosomal DNA gene and 16S-23S internal transcribed spacer (ITS) were performed to characterize the bacteria isolated from king oyster mushroom. The bacteria were also confirmed as B. gladioli based on a similarity of 0.58 with Biolog and 99% sequence identity for 16S rDNA and ITS sequences. Because strains of B. gladioli are commonly found in diverse ecological niches, it is predicted that B. gladioli strains from different environments may have different phenotypic and genotypic characteristics. In this study, we characterized B. gladioli strains isolated from different sources by carbon source utilization, pathogenicity assays, antagonistic activities, ITS sequences, and ERIC/BOX-PCR DNA fingerprinting. Metabolic profiles showed that the MBg strains causing king oyster mushroom bacterial decay are similar to pineapple fruit rot pathogen PBg5 and soil isolate Bg1. ITS sequences and ERIC/BOX-PCR DNA fingerprinting analyses revealed that MBg strains are closely related to soil-isolated strains Bg1 and Bg2 with respective similarities of 97.4% and 0.734. Pathogenicity assays demonstrated that MBg strains and soil-isolated strains Bg1 and Bg2 are virulent to king oyster mushroom and weakly virulent to gladiolus (Gladiolus hybridus) and onions; the strains of pineapple fruit rot pathogen, PBg3 and PBg5, are weakly virulent to king oyster mushroom, gladiolus, and onions; the gladiolus-pathogenic strains HBG4 and HBG10 that were identified as B. glumae are pathogenic to gladiolus and onions but not to king oyster mushroom; the soil-isolated strain Bg3, classified as a member of B. cepacia complex, is weakly virulent to onioin but non-virulent to king oyster mushroom and gladiolus; the soil-isolated strains Bg4 and Bg5 that are also classified as members of B. cepacia complex are not virulent to king oyster mushroom, gladiolus, or onion. In addition, the antagonistic activities of B. gladioli strains were assayed by measuring the mycelium growth of king oyster mushroom and Rhizoctonia solani AG-4 and the bacterial growth of B. glumae RBg9, and the results revealed that all strains of B. gladioli used in this study have inhibitory abilities against the growth of king oyster mushroom, R. solani AG-4, and B. glumae strain RBg9 on cultured media. Taken together, our results indicated that B. gladioli MBg strains isolated from king oyster mushroom share similar phenotypic and genotypic characteristics to soil-isolated B. gladioli strains Bg1 and Bg2.
URI: http://hdl.handle.net/11455/31409
其他識別: U0005-1908201020200000
Appears in Collections:植物病理學系

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