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題名 | 利用聚合酶連鎖反應偵測百合鐮孢菌萎凋病菌=Detection of Fusarium Oxyporum f. sp. lilii by PCR |
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作者姓名(中文) | 高衛婷; 施儼育; 謝文瑞; | 書刊名 | 植物病理學會刊 |
卷期 | 11:3 2002.09[民91.09] |
頁次 | 頁112-122 |
分類號 | 435.456 |
關鍵詞 | 百合; 百合鐮孢菌萎凋病; 病原菌偵測; 核酸探針; 聚合酶連鎖反應; 隨機增幅核酸多型性分析; Lily; Fusarium oxysporum f. sp. lilii; Detection; Polymerase chain reaction; Probe; Random amplified polymorphic DNA; |
語文 | 中文(Chinese) |
中文摘要 | 由病原菌Fusarium oxysporum Schlecht f. sp. Lilii Imle所引起之百合鐮孢菌萎凋病是目前臺灣百合栽培的主要限制因子之一。一般分離鑑定百合鐮孢面萎凋病病原菌(F. oxysporum f. sp. Lilii)之方法,是先將擬似罹患鐮孢菌萎凋病之百合植株其種球莖基部、鱗片及底根等部位做組織分離,或取罹病百合植株根圈土壤,利用五氯硝基苯(pentachloronitrobenzene, PCNB)選擇性培養基分離。將罹病組織或帶菌土壤中分離出之Fusarium spp.經由形態上之鑑定為F. oxysporum後再以接種百合的方式確定其病原性,以確認所分得為F. oxysporum f. sp. Lilii。上述之病原性測定需耗時長達四星期以上,耗日費時,故本試驗嘗試應用聚合?連鎖反應(polymerase chain reaction, PCR)技術以開發一快速、方便且準確的病原菌偵測方法。首先由溪湖、埔里及豐原等地百合栽培田之罹病植株與土壤中取樣,以PCNB選擇性培養基分離,經形態上鑑定後共收集F. oxysporum之菌株74株,將此74個供試菌株以上述之接種方式做病原性的測定,結果有30株菌株對百合具病原性,另44株則否。選取3株病原性菌株(F016、F025、G016)與6株無病原性菌株(F032、F036、F038、F040、F140、F141),利用隨機增幅核酸多型性分析(random amplified polymorphic DNA, RAPD),以100個長度為10個核?酸(nucleotides)之核酸引子進行篩選。以此篩選出編號OPAT-4 (5'-TTGCCTCGCC-3')之引子,所增幅產生之一特殊去氧核醣核酸(DNA)片段能區別病原性與非病原性菌株,經回收此具鑑別性之DNA片段後加以解序,並設計出專一性核酸引子對Fusal-5(5'-GCTTTCGGCCGCCTCTCATAG-3')與Fusal-6(5'-TTGCCTCGCCGATAGTGGAT-3')。以此專一性核酸引子對再利用PCR增幅病原性菌株之基因體核酸(genomic DNA)可得大小為386bp的DNA片段。利用Fusal-5與Fusal-6專一性引子對及PCR增幅方式測試先前所分得之74個菌株,結果與以接種方法所測定之病原性結果相符合,相同的30株具病原性菌株可增幅出此386bp專一性DNA片段,而另44株無病原性菌株則否,且以此386bp DNA片段進行南方雜合反應也有相同結果。另外以此專一性引子對測試常見百合土壤病原菌(Rhizoctonia solani、Pythium aphanidermatum、Phytophthora parasitica、及Sclerotium rolfsii等)、在百合上常分離到的Fusarium屬真面如F. solani及F. moniliforme等、與在不同作物如絲瓜、薑、胡瓜、虹豆、洋香瓜、苦瓜、香蕉、唐菖浦等上之F. oxysporum分化種,結果皆無此386bp專一性DNA片段產片。推論此專一性DNA片段只可在對百合具有病原性的F. oxysporum f. sp. Lilii之基因體核酸中增幅產生,故所開發之核酸引子對Fusal-5與Fusal-6可成功應用在鑑別分離培養之真菌是否為F. oxysporum f. sp. lilii。此外,利用此專一性引子對進行PCR增幅時,其敏感度可達200pg/μ1之基因體核酸的濃度。 |
英文摘要 | The basal rot, root rot and wilt of lilies caused by Fusarium oxysporum f. sp. lilii are the most destructive disease during its growing period in Taiwan. F. oxysporum f. sp. lilli is difficult to identify because of the various morphological characteristics and virulence. For the reason using the molecular teachniques to provide a fast and precise method in identification of F. oxysporum f. sp. lilii is needed. Total 74 F. oxysporum isolates isolated form the diseased bulbs, plants, and the soil in the field of His-hu, Feng-yuan, and Pu-li were used in the experiment. For the pathogenicity tests, bulbs were dipped in the spore suspension (106 cfu/ml) of each isolate then planted in the sterilized sandy loam soil. The pathogenicity tests revealed that 30 isolates of F. oxysporum were pathogenic on lily and the other 44 isolates were nonpathogenic. Radom amplified polymorphic DNA (RAPD) was used to screen 100 random primers with genomic DNA of 3 pathogenic F. oxysporum isolates (F016,F025, and G016) and 6 non-pathogenic F. oxysporum isolates (F032,F036, F038, F040, F140, and F141). The primer OPAT-4 (5'-TTGCCTCGCC-3) was found to amplify a specific DNA fragment only in the genomic DNA of the pathogenic isolates. The amplified specific DNA fragment was recovered, cloned, and sequenced. And the specific primers set Fusal-5 (5'-GCTTTCGGCCGCCTCTCATAG-3') and Fusal-6 (5'-TTGCCTCGCCGATAGTGGAT-3') were designed from the nucleotide sequence of the above specific DNA fragment for polymerase chain reaction (PCR). As expected, as 386-bp specific DNA fragment was produced in PCR. Using the specific primers to detect the 74 F. oxysporum isolates, the same results were obtained as the pathogenicity test forementioined. The specific DNA fragment was not amplified in the genomic DNA of the common pathogens Rhizoctonia solani, Pythium aphanidermatum, Phytophothora parasitica, Sclerotium rolfsii, and F. moniliforme found on lily, and some different formae speciales of F. oxysporum, tesed i.e. F. oxysporum f. sp. luffae, F. oxysporum f. sp. cucumerinum, F. oxysporum f. sp. tracheiphilum, F. oxysporum f. sp. melonis, F. oxysporum f. sp. monodricae, F. oxysporum f. sp. cubense, and F. oxysporum f. sp. gladioli. The results showed that the specific primer set developed can successfully detect F. oxysporum f. sp. lilii from other fungal pathogens when the genomic DNA of the cultured mycelia was used for PCR. And the minimal DNA concentration could be detected is about 200pg/μ1. |
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