楊樹銹病病原鑑定及PCR快速檢測技術開發

近年來，隨著國內楊樹種引進與造林面積增加，病蟲害發生情形也隨之增加，其中首見楊樹銹病，此病是侵害楊樹幼樹和苗木的主要病害之一，病原菌為Melampsora larici-populina，主要為害葉片，也為害葉柄、嫩梢和冬芽。此病亦好發於苗木，在外來苗木的引進上造成一定風險。而到目前為止，防檢局也尚未將其列入檢疫項目。本研究將台南採集之楊樹樣本進行核酸抽取與ITS序列分析，檢測出之銹病病原與中國與瑞典等地所發表的銹病病原核酸序列相同，確定此病原為M. larici-populina。由於銹病菌屬絕對寄生菌，必須在活在寄主組織上才能生長繁殖，因此無法進行病原菌分離，且銹病孢子在初感染宿主葉片之非產孢時期不易由外部病徵進行觀察鑑定，故本實驗室針對病原菌已定序之ITS片段核酸序列設計三組引子對，三組引子對皆能對楊樹銹病菌有效增幅出特定大小之核酸片段，且無雜訊產生，在進一步測試三組引子對之專一性後，結果以第一組引子對為最佳，該引子對同時具有高靈敏度，在病原核酸稀釋1,000,000倍後仍能有效增幅，靈敏度可達1.5 pg。本研究開發之專一性引子對可針對該病原進行具專一性及靈敏度且快速的分子診斷，未來可提供作為此病原防檢疫之應用。

關鍵字

楊樹；銹病；聚合酶鏈鎖反應

並列摘要

Recently, the introduction and afforestation of the poplar species were increased and the risks of pests and dieases carried by poplar were highly increased. Among the diseases, poplar rust caused by Melampsora larici-populina, is the main threaten to poplar sapling and seedling. This pathogen can damage leaves, petioles, young shoots, and winter buds. The disease usually happens in seedlings, which causes risk on the introduction of exotic seedlings. However, the fungal pathogen is not yet cataloged in the BAPHIQ quarantine items. In this study, we extracted the nucleic acid of infected poplar samples from Tainan and accomplished the ITS sequence analysis. The ITS sequence showed that sequence homology was similar with the rust pathogen species of M. larici-populina published in China and Sweden regions. The pathogen is difficult to identify due to obligation parasite. Moreover, the external symptoms of the rust spores in host leaves are difficult to observe and identify in the early infection stage. In this study, the ITS region was amplified and sequenced, then three primer sets were designed to examine the specificity to M. larici-populina. The results of specificity test of three primer sets showed that the F1/R1 primer pairs posses the best specificity and high sensitivity, the pathogen nucleic acid could still be detected under 1.5 pg.

並列關鍵字

Populus sp. ； rust ； polymerase chain reaction (PCR)

國際替代計量

楊樹銹病病原鑑定及PCR快速檢測技術開發

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