褐根病菌Phellinus noxius檢測用專一性引子對之開發
Other Title
Development of specific primers for Phellinus noxius
Journal
植物病理學會刊
Journal Volume
16
Journal Issue
4
Pages
193-202
Date Issued
2007-12
Date
2007-12
Author(s)
Abstract
以分離堵質之Phellinus noxius、Phellinus aplahynus、Phellinus gilvus、Phellinus laevlgatus、Phellinus hoehnelii、Phellinus igniarius、Phellinus lnermis、Phellinus mombranaceus Kretzchmaria spp.、Ganoderma australe、Ganoderma tropicum、Rosellinia necatrix、Phytophthora paracitica及Sclerotium rolfsii等菌株的genonuo DNA，應用聚合酵素連鎖反應技術(polymerase chain reaction, PCR)，以通用性引子對ITS1/ITS4增幅核醣體核酸(ribosomal DNA, rDNA)之內轉錄區間ITS1/5.8S/ITS2之基因序列，經解序及多重序列比對後，依據P. noxius ITS區域之相同序列處設計出專一性引子一對，正向引子PN-1F(5'-agtttgcgctcatccatctc-3')，反向引PN-2R ( 5'-agccgacttacgccagcag-3')。應用PN-1F/PN-2R專一性引子對進行PCR反應，對供試之P. noxius菌株均可坤編出414 bp或422 bp長度之序列片段；而供試之其它菌株，皆無法增幅出任何片段。此專一性引子對偵測P. noxius之DNA時，靈敏度可達0.01 ng。以相同之引子對進行PCR反應，偵測罹病之龍眼樹及相思樹根部組織，亦可坤福出預期之片段。因此本研究所設計之引子對PN-1F/PN-2R能用來快速診斷褐根病與輔助鑑定P. noxius。
Phellinus noxius is a very destructive pathogen which attacks hundred species of woody plants in Taiwan in recent years. In order to develop a pair of specific primers for rapid and accurate diagnosis of the disease associated with the pathogen in the fields, the ribosomal DNA (rDNA) sequences in internal transcript spacer (ITS, including ITS1/5.8S/ITS2) regions from P. noxius and several other soil-borne fungi were studied. The test fungi included P. noxius (18 isolates), other Phellinus species (P. aplahynus, P. gilvus, P. laevlgatus, P. hoehnelii, P. igniarius, P. lnermis, P. mombranaceus), Ganoerma australe, Ganoderma tropicum, Rosellinia necatrix, Kretzchmaria spp., Phytophthora paracitica and Sclerotium rolfsii. The universal PCR primers ITS1/ITS4 were used to analyze the DNA sequences of ITS regions for these fungi. Based on the alignments of ITS sequences, a set of primers PN-1F/PN-2R was designed. The forward primer PN-1F sequence is 5'-agtttgcgctcatccatctc-3' and the reverse primer PN-2R is 5'-agccgacttacgccagcag-3' . Using PN-1F/PN-2R primers for PCR amplification under a setup condition and the fungal genomic DNA as templates, a distinct 414 bp or 422 bp fragment were amplified form P. noxius, whereas no PCR products can be amplified from other soil-borne fungi. The PCR could amplify the fragments recognizable within 3-4 hr when concentration of purified DNA was higher or equal to 0.01 ng. Meanwhile, similar amplified fragments were obtained when the diseased root tissues of Dimocarpus longana and Acasia confuse from P. noxius infested fields were used as templates. Results presented here suggest that the primer pairs PN-1F/PN-2R can be efficiency in auxiliary identifying P. noxius and accurately and rapidly detecting diseases caused by the pathogen in the field.
Subjects
褐根病菌
引子對
Phellinus noxius
primer
Publisher
中華民國植物病理學會
Type
journal article
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