利用聚合?連鎖反應偵測百合鐮孢菌萎凋病菌

Detection of Fusarium oxysporum f. sp. lilii by PCR

高衛婷( W. T Kao) ； 施儼育( Y. Y Shih) ； 謝文瑞( W. H Hsieh)

《植物病理學會刊》 11卷3期 (2002/09) Pp. 112-122

https://doi.org/10.6649/PPB.200209_11(3).0002

摘要

由病原菌Fusarium ozysporum Ssblecht f.sp. Lilii Imle 所引起之百合鐮孢菌萎凋病是目前台灣百合栽培的主要限制因子之一。一般分離鑑定百合鐮孢菌萎凋病病原菌(F. oxysoporum f. sp liill)之方法，是先將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，或取罹病百合植根圈土壤，利用五氯硝基苯(pentachloronitrbenzene, PCNB)選還性培養基分離。將罹病組嬂或帶菌土壤中分離出之Fusarium spp.經由形態之鑑定為F.oxysporum 再以接種百合的方式確定其病原性，以確認所分得為F. oxysoporum f. sp lilli。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 reastion ,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、埔里及豐原等也百合栽培田之罹病植株與土壤中取樣，以PCNB選擇性棓養基分離，經形態上鑑定後共集F. oxysoporum之菌株74株，將此74個供試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(nucleoties)之核酸引子進行篩選。以此選出編號OPAT-04(5'-TTGCTCGCC-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'-GCTTTCGGCCGCCTCTCATAG-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(grnomic DNA)可得大小為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(Rhizoctoniasolani、Pythium aphanidermatum、Phytophthora parasitica、及Scleerotium orlfsii等）、在百合上常分離到的Fusarium 屬真菌如F.solani及F.moniliforme 等、與在不同作物如絲瓜、薑、胡瓜、豇豆、洋香瓜、苦瓜、香蕉、唐菖浦等上之F.moniliforme分化種，結果皆無此386bp 專一性DNA片段產生。推論此專一性DNA片段只可在對百合有病原性的F.oxysporum f.sp.lilii之基因體核中增幅產生，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菌是否為F.rcysporum f.sp.lilii。此外，利用此專一性久子對進行PCR增幅，其敏感度可達200pg/μl之基因體核酸的濃度。

關鍵字

百合 ； 百鐮孢菌萎凋病 ； 病原菌偵測 ； 核酸探針 ； 聚合?連鎖反應 ； 隨機增幅核酸多型性分析

並列摘要

The basal rot, root rot and wilt of lilies caused by Fusarium oxysporum f. sp. lilli are the most destructive disease during its growing period in Taiwan. F. oxysporum f.sp. lilii is difficult to identify because of the various morphological characteristics and virulence. For the reason using the molecular techniques to provide a fast and precise method in identification of F. oxysporum f. sp. Lila is needed. Total 74 F. oxysporum isolates isolated from the diseased bulbs, plants, and the soil in the field of His-u, Fengyuan, and Pu-li were used in the experiment. For the pathogenicity tests, bulbs were dipped in the spore suspension (106 du/mI) of each isolate then planted in the steriIized sandy Ioam soil. The pathogenicity tests revealed that 30 isolates of F. oxysporum were pathogenic on lily and the other 44 isolates were nonpathogenic. Random amplified polymorphic DNA (RAPD) was used to screen 100 rand m primers with pathogenic. Random amplified polymorphic DNA (RAPD) was used to screen 100 random primers with genomic DNA of 3 pathogenic F. oxysporum isolates (F016, F025, and G0I6) 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 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 Fusalamplified specific DNA fragment was recovered, coined, and sequenced. And the specific primers set Fusal-5(5'-GCTTTCGGCCGCCTCTCATAG-3') andFusal-６(5'-GCTTTCGGCCGCCTCTCATAG-3') were designed from the nucleotide sequence of the above specific DNA fragment for polymerase chain reaction designed from the nucleotide sequence of the above specific DNA fragment for polymerase chain reaction (PCR). As expected, a 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 forementioned. 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 1i1y, and same different formae speciales isolates of F. oxysporum, tesed i.e. F. oxysporum f. sp. Luffae, F. oxysporum f. sp. cucumerinum, F. axysporum f. sp. tracheiphilum, F. oxysparum f. sp. Me/anis, F. oxysporum f. sp. monodricae, F. oxysporum f. sp. cubense, and F. oxysporum f. sp. gladioli. The results fungal pathogens when the genomic DNA of the cultured mycelia was used for PCR. And the minimal DNA concentration could be detected is about 200 pg / μl.

並列關鍵字

Lily ； Fusarium oxysporum f. sp. lilii ； detection ； polymerase chain reaction ； probe ； random amplified polymorphic DNA