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Fungal flora in greenhouse of Phalaenopsis and thestudy of Fusarium solani associated withPhalaenopsis yellowing leaf disease
|關鍵字:||Phalaenopsis;蝴蝶蘭;fungal diversity;Fusarium solani;inoculation;molecular phylogenticity;真菌相;Fusarium solani;接種試驗;分子親緣性分析||出版社:||植物病理學系所||摘要:||
本研究自2007年4月27日至2008年1月9日為止，從臺中、彰化、雲林、嘉義、台南、高雄及屏東等共48家經農委會動植物防疫檢疫局檢查合格之輸美蝴蝶蘭園區分離到248株菌株。經形態學與分子生物學鑑定，可將248株菌株中的237株菌株鑑定為Alternaria、Annulohypoxylon、Ascomycetes sp.、Aspergillus、Bipolaris、Chaetomium、Colletotrichum、Curvularia、Exserohilum、Fusarium、Lasiodiplodia、Nodulisporium、Trichoderma及Penicillium等13屬真菌，而Fusarium屬有5種，Penicillium屬有7種，Aspergillus屬有3種，Trichoderma屬有4種， Alternaria屬有2種， Curvularia屬有2種，Bipolaris屬、Colletotrichum屬、Chaetomium屬、Exserohilum屬、Lasiodiplodia屬和Annulohypoxylon屬各1種。所有蒐集到菌株以菌絲塊接種後，依病斑直徑大小判斷，顯示248株菌株中具強毒性之菌株有62株，弱毒性之菌株138株，而無毒性之菌株有48株。將強毒性的菌株以105 conidia /ml孢子懸浮液接種蝴蝶蘭植株後，証實除F. solani菌株仍具毒性外，其餘菌株無法引起任何病徵。測試14種殺真菌劑對所分離菌株生長抑制效果，顯示50%撲克拉錳（Prochlorate manganece）可溼性粉劑對供試菌株之菌絲生長抑制效果較其他供試藥劑來得好，此外24.9%待克利（Difenoconazole）乳劑、23.6%百克敏（Pyraclostrobin）乳劑及5%菲克利（Hexaconazole）水懸劑等藥劑除對Fusarium屬真菌抑制效果較不明顯外，對其餘供試菌株皆有良好的抑制效果。比較施用殺真菌劑前後之蝴蝶蘭葉表真菌相變化，顯示百克敏、菲克利及撲克拉錳皆能降低蝴蝶蘭葉表真菌之種類與分離比率。本研究亦針對引起蝴蝶蘭黃葉病F. solani菌株的病原性及分子特性進行研究，結果得知引起蝴蝶蘭黃葉病之F. solani菌株無法引起虎頭蘭(Cymbidium hybrid)、四季蘭(Cymbidium ensifolium)、報歲蘭(Cymbidium sinense)、文心蘭(Oncidium sp.)、石斛蘭(Dendrobium sp.)及嘉德麗亞蘭(Cattleya sp.)產生病徵，此外將引起蝴蝶蘭黃葉病的F. solani菌株以孢子懸浮液澆灌方式接種於豌豆、洋香瓜和胡瓜的地基部與根部亦無法造成任何病徵，顯示引起蝴蝶蘭黃葉病之F. solani菌株可能具有寄主專一性。比較不同溫度對引起蝴蝶蘭黃葉病F. solani菌株病原性影響，結果指出當接種溫度超過20℃時，發病嚴重度高於16℃或18℃。於分子親緣性分析上，本研究增幅internal transcribed spacer(ITS)、intergenic spacer(IGS)及β-tubulin基因核苷酸序列，並利用近鄰結合法(Neighbor-joining method, NJ)和最大簡約法(Maximum parsimony method, MP) 進行分子系統親緣性分析，顯示引起蝴蝶蘭黃葉病之F. solani菌株可形成單一分子群(monophylogentic group)，並被高的bootstrap值所支持(>84%)，顯示引起蝴蝶蘭黃葉病F. solani菌株的分子親緣性與不同寄主來源的F. solani菌株有明顯差異。比較病原性測試及分子親緣性分析結果，初步認定引起蝴蝶蘭黃葉病F. solani菌株與其他來源之F. solani菌株有所不同。
A total of 248 isolates was obtained from 48 Phalaenopsis greenhouse including Taichung, Changhua, Yulin, Chiayi, Tainan, Kaohsiung and Pingtung. Based on morphological characteristics and ITS rDNA, 237 isolates were identified as Alternaria (2 species), Annulohypoxylon (1 species), Ascomycetes sp., Aspergillus (3 species), Bipolaris (1 species), Chaetomium (1 species), Colletotrichum (1 species), Curvularia (2 species), Exserohilum (1 species), Fusarium (5 species), Lasiodiplodia (1 species), Nodulisporium (1 species), Penicillium (7 species) and Trichoderma (4 species). All fungal isolates were examined for their virulence using mycelial disk. The inoculation results showed that 62 isolates were highly virulence (HV), 138 isolates were less virulence (LV), and 48 isolates were avirulence (AV). However, when 105 conidia/ml conidia suspension was used to inoculate Phalaenopsis, highly virulent isolates showed no symptom on Phalaenopsis except isolates of F. solani. To control the fungal pathogen, 14 different fungicides were examined for the efficacy of mycelial growth in Potato Dextrose Agar (PDA) medium. The results showed that the mycelial growth of fungal isolates could be inhibited when PDA was added with 10 mg/l Prochlorate manganese and Pyraclostrobin. Furthermore, 100 mg/l of Difenoconazole, Hexaconazole and Iminoctadine triacetate were effective in inhibiting mycelial growth of fungal isolates. However, other fungicides did not effectively inhibit mycelial growth of fungal isolates. Consequently, Prochlorate manganese, Pyraclostrobin and Hexaconazole were selected for further test on Phalaenopsis. The results demonstrated that 3 fungicides could reduce the fungal flora and population after spraying on Phalaenopsis lines of V3, V3-1 and Shiny-Yaun Golden Beauty. This work also studied the biological characters of F. solani associated with Phalaenopsis yellowing leaf disease. The isolates of F. solani caused Phalaenopsis yellowing leaf disease were to inoculate on five different Orchidaceae plants, including Cymbidium hybrid, Cymbidium ensifolium, Cymbidium sinense, Oncidium sp., Dendrobium sp. and Cattleya sp. The results revealed that F. solani obtained from Phalaenopsis didn't cause significant symptom on the test plants. Furthermore, the isolates of F. solani causing Phalaenopsis yellowing leaf disease did not cause any symptom on pea, cucumber and melon after inoculation. In this study, effect of temperature on disease severity of Phalaenopsis inoculated by F. solani was examined. The results indicated that the disease was more severe at 20℃ and 24℃ than those at 16℃ and 18℃. For molecular analysis, sequence of internal transcribed spacer (ITS), intergenic spacer (IGS) and β-tubulin gene were used to evaluate the phylogenic relationship by neighbor-joining method (NJ) and maximum parsimony method (MP). The results demonstrated that isolates of F. solani from Phalaenopsis formed an monophylogentic group and supported with high bootstrap values (>84%). Moreover, molecular phylogentic analysis also revealed that the isolates of F. solani causing Phalaenopsis yellowing leaf disease were molecular distinct from other F. solani from different plant hosts. According to the results of inoculation test and molecular phylogenicity, the isolates of F. solani causing Phalaenopsis yellowing leaf disease are significantly different from other F. solani isolates from other sources.
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