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標題: 台灣三種瓜類萎凋病菌 Fusarium oxysporum 之病原性及分子親緣關係
Pathogenicity and phylogeny of Fusarium oxysporum causing three cucurbits wilting in Taiwan
作者: 鍾井辰
Ching-Chen Chung
關鍵字: 瓜類;萎凋病菌;交互病原性;嫁接苦瓜;親緣關係;SIX6;Cucurbitaceae;Fusarium oxysporum;cross-pathogenicity;grafted bitter gourd;phylogeny;SIX6
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葫蘆科植物為廣泛分佈於世界各地之作物;在台灣,以絲瓜、苦瓜及胡瓜為田間常見之果菜類作物。然於作物生長季中,常會發生嚴重的真菌性病害,其中以 Fusarium oxysporum (Fo) 所造成的瓜類萎凋病為瓜類栽培最主要限制因子。Fo在侵染作物時具有寄主專一性,不同菌株可以分化型 (Forma specialis) 加以區分。儘管如此,交互病原性 (cross-pathogenicity) 的現象在瓜類作物與萎凋病菌之間發生普遍,且交互感染現象受多種因子影響,如品種、溫度、植齡、接種方式等。因此,輔以分子親緣分析可以區分不同的分化型。在台灣,由於苦瓜萎凋病發生嚴重,農民普遍以苦瓜嫁接絲瓜根砧方式防治苦瓜萎凋病,然近年卻於田間觀察到嫁接苦瓜仍有萎凋現象,此現象是否為交互感染所引發,有必要進一步釐清。本研究自田間嫁接苦瓜穗與絲瓜根砧中分離到萎凋病菌,以生物分析法 (bioassay) 確定其分化型;另於不同條件下測試絲瓜、苦瓜及胡瓜萎凋病菌於3種瓜類上之交互感染情形。此外亦以嫁接苦瓜植株進行接種試驗,瞭解嫁接植株發病與萎凋病菌於植體內分布情況。最後,以核糖體DNA (rDNA) 基因間隔區 (intergenic spacer region, IGS)、轉譯延長因子 (translation elongation factor-1α, EF-1α) 及 secreted in xylem 6 (SIX6) 核酸序列,分析3種瓜類萎凋病菌之親緣關係。於病原性測試結果指出,於田間嫁接植株中所分離出萎凋病菌菌株皆可感染絲瓜。於20°C環境下,將胡瓜、絲瓜及苦瓜以剪根方式接種3種瓜類萎凋病菌後,胡瓜與苦瓜之萎凋病菌可感染農民自留種 (open pollination, OP) 絲瓜,而絲瓜萎凋病菌可感染萬吉品種胡瓜;另於28°C下,苦瓜萎凋病菌 Fomo33仍可感染部份絲瓜植株,而絲瓜萎凋病菌 Fomhl6則不使絲瓜植株產生病徵,僅纏據於植體中。以浸根方法接種真葉未展開之幼齡植株時,交互感染之現象更明顯;然以帶菌土壤 (infested soil) 接種時,3種萎凋病菌無明顯交互感染。此外,嫁接植株接種測試中,得知植株僅受絲瓜萎凋病菌危害,萎凋病菌可分布於全株,然苦瓜萎凋病菌僅侷限於下胚軸或根砧部位。於分子親緣關係分析,指出IGS與EF-1α 序列無法有效區別胡瓜與絲瓜萎凋病菌;而以與病原性相關之 SIX6 基因序列分析,則顯示SIX6基因序列能有效區別胡瓜與絲瓜萎凋病菌,具有輔助區分不同分化型之潛力。

Cucurbitaceae is an important crop distributed over the world widely. In Taiwan, loofah, bitter gourd and cucumber are three vegetables common in the field. However, several infectious fungal diseases are severe during growth season. Among the diseases, the Fusarium wilt caused by Fusarium oxysproum (Fo) is one of the most important diseases in cucubit production area. Currently, more than 150 formae speciales have been recorded throughout the world. Although formae speciales show the pathogenic specificity on original host, the cross-pathogenicity has been found generally within cucurbitaceae Fusarium wilt. Besides, there are several factors affecting the cross-pathogenicity such as cultivar, temperatuer, plant stage, and method of inoculation. Therefore, combining pathogenicity tests with molecular phlogeny analysis would help the identification of formae speciales. In Taiwan, grafted bitter gourd (with loofah stock) is widely used to prevent Fusarium wilt in the filed, however, grafted bitter gourd still wilt in recent years. It is necessary to clarify if F. oxysproum f. sp. momordicae (Fomo) is cross pathogenic to loofah stock or F. oxysproum f. sp. luffae (Folu) already arrive at the soil. In this study, the F. oxysproum obtained from cucumber, bitter gourd, and loofah were used to examine the pathogenicty and cross-pathogenicty with three cucurbits and phylogeny analysis was carried out . The results indicated that F. oxysproum isolates from cucumber and bitter gourd could infect loofah (open pollination, OP) and F. oxysporum isolates from loofah could infect cucumber (cv. Vantage) at 20°C based on root dipping method. In addition, some of loofah (cv. Merit) individuals could be infested by F. oxysporum isolates from bitter gourd at 28°C with root dipping method, but one isolate from loofah (Fomhl6) did not cause symptom on loofah under the same condition. Fomhl6 isolate colonized in loofah only. When we inoculated young stage plant (seedling with 2 cotyledons) by root dipping method, the symptom of cross-infection was even more severe. But when we inoculated plants with infested soil, cross-pathogenicity was not obvious. Also, we did the inoculation of grafted (loofah as stock, bitter gourd as scion) plants to mimic the plant infested in the field. The results revealed the main influence isolates on grafted plant was Folu. For the distribution of isolates in grafted plant, we found that Folu could be distributed in the whole plant, however, Fomo was limited in hypocotyl (or stock). Finally, results of phylogeny analysis indicated the F. oxysporum isolates from three cucurbits did not associate with host specificity based on rDNA intergenic spacer (IGS) and translation elongation factor-1α (EF-1α) sequences. However, the sequences of secreted in xylem 6 (SIX6) gene, which associated with pathogenicty, could separate the F. oxysporum isolates from three cucurbits.
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