褐根病菌Phellinus noxius檢測用專一性引子對之開發

Development of Specific Primers for Phellinus noxius

蔡志濃(J. N. Tsai) ； 謝文瑞(W. H. Hsieh) ； 安寶貞(P. J. Ann) ； 楊淨棉(C. M. Yang)

《植物病理學會刊》 16卷4期 (2007/12) Pp. 193-202

https://doi.org/10.6649/PPB.200712_16(4).0003

摘要

以分離培養之Phellinus noxius Phellinus aplahynus、Phellinus gilvus、Phellinus laevlgatus、Phellnus hoehnelii、Phellinus igniarius、Phellinus lnermis、Phellinus mombranaceus、Kretzchmaria spp.、Ganoderma australe、Ganoderma tropicum、Rosellinia necatrix、Phytophthora paracitica及Sclerotium rolfsii等菌株的genomic 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對，正向引子PN-1F(5-'agtttgcgctcatccatctc-3')，反向引子PN-2R(5'-agccgacttacgccagcag-3')。應用PN-1F/PN-2R專一性引子對進行PCR反應，偵測罹病之龍眼樹及相思樹根部組織，亦可增幅出預期之片段。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。

關鍵字

褐根病菌 ； 引子對

並列摘要

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, Kreizchmaria 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'-agtttgcgctcatccatctc3'” 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 us 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.

並列關鍵字

Phellinus noxius ； primer