自萵苣露菌罹病葉直接解序全長段露菌ITS與其親緣性分析

Direct Sequencing for Phylogenetic Analysis of Whole ITS Sequence of Bremia lacfucae from the Infected Lettuce Leaves

本研究針對萵苣露菌（Bremia lactucae）核糖體轉錄區間DNA（internal transcribed spacer region of the ribosomal DNA，ITS rDNA）之解序及偵測，設計專一性引子對，並探討引子對之靈敏度、田間罹病葉及臘葉標本之偵測應用與直接自罹病葉增幅露菌ITS之方法可行性，並依據ITS分析萵苣露菌菌株間之親緣性。以廣效引子對ITS1/ITS4解序之B. lactucae ITS特定片段，篩選設計引子對BL1（5'-GTTGCATTGCCTTGAATTTG-3'）及BL2（5'-AACCGAAGCTAAACTGCG-3'），BL1/BL2引子對專一性之靈敏度，每次反應中只要有10pg B.lactucae DNA即可增幅清楚之特定產物，即使在250ng萵苣DNA背景值情況下，也可增幅出萵苣露菌特定DNA片段。田間萵苣感染露菌後，不論葉片是否產生病徵，BL1/BI2引子對確實增幅出B. lactucae專一片段，證實其感染狀況；又以2006年苣露菌病臘葉標本為偵測對象，亦可增幅出萵苣露菌之特定片段。若於田間萵苣罹病葉直接增幅露菌ITS，易受萵苣及微生物干擾，然利用ITS1/BL2及BL1/ITS4兩組引子對直接擴增出2段萵苣露菌ITS片段，經解序組合成全長段之萵苣露菌ITS序列，本研究證實專一位及廣效性引子對之組合模式，可增幅並組合出全長段萵苣露菌ITS序列，此一引子對組合方法，可做為直接自罹病葉上增幅出絕對寄生菌ITS模式及鑑定工具。以ITS為萵苣露菌菌株親緣性之分析標的，其結果顯示大部分萵苣露菌被分成同群，不因區域而有差別，而同屬不同種之Bremia spp.則依菊科寄主植物而各自成群，顯示菊科露菌具有高寄主專一位，據此建議將來自同屬或鄰近種寄主之露菌菌株歸為同種較為妥適。

"The specific primers, BLI (5'-GTTGCATTGCCTTGAATTTG-3') and BL2 (5'AACCGAAGCTAATTAAACTGCG-3'), were designed from the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) of Bremia lactucse for sequencing and detection. The specificity of BL1/BL2 used in detection of the ITS of B. lactucae from the infected lettuce leaves of field or herbarium specimens was evaluated. The determined whole ITS sequences were also used for phylogenetic analyses in this study. The DNA fragment could be easily amplified from 10 pg of B. lactucae DNA using BLl/BL2 in polymerase chain reaction (PCR), even the lettuce DNA was as high as 250 ng included in the DNA mixture. The ITS fragment could also be amplified from the symptomless leaves to verify B. lectucee infection. Furthermore, the BLl/BL2 primers were also used to amplify the specific ITS fragment from the B. lactucae-infected herbarium specimen collected in 2006. The specific BLl/BL2 primers combined with the common ITS primers, such as ITS1/BL2 and BLlIITS4, were used to amplify the ITS sequences of B. lactucae from the infected lettuce leaves, and the two amplified fragments can be merged into the whole ITS sequence. The combination of ITSl/BL2 and BLllITS4 primers can be an useful tool in directly amplifying the whole ITS sequence for diagnosis of B. lsctuaee species. Phylogenetic analyses based on the ITS sequence reveal that most of B. lectucee isolates can be clustered in the same clade, and no relationships between the isolates and the locations of isolate origins were observed. Other Bremis species on Compositae were clustered in different clades, and each clade corresponded to the individual host plant, indicating that those downy mildews from Compositae were highly host specific. It suggests that the isolates of Bremia spp. from the same genus or the related species of hosts should be classified as a species."