Detection of Orchid Phytophthora Disease by Nested PCR

以nested PCR檢測蘭花疫病

蔡惠玲(Huei-Ling Tsai) ； 黃麗君(Li-Chun Huang) ； 安寶貞(Pao-Jen Ann) ； 劉瑞芬(Ruey-Fen Liou)

《Botanical Studies》 47卷4期 (2006/10) Pp. 379-387

摘要

蘭花為蘭科(Orchidaceae)之草本花卉，除了可供切花外，也可做為盆花觀賞，是頗具特色與經濟價值之重要花卉。在台灣，可感染蘭花之疫病菌主要包括Phytophthora palmivora及P. parasitica，但偶爾也可發現P. multivesticulata。P. palmivora及P. parasitica可危害之蘭花種類繁多，且因蔓延十分迅速，常在管理較差之蘭園造成嚴重經濟損失。為縮短及簡化蘭花疫病檢測流程，本研究根據疫病菌核醣體核酸序列設計Phytophthora spp.廣效性引子對(Phy1s/Phy2a)可用於檢測台灣常見疫病菌種類。此外，我們也針對P. palmivora及P. parasitica分別設計種專一性引子對，並配合Phy1s/Phy2a之應用，開發以nested PCR快速檢測上述兩種疫病菌之技術，可快速確定蘭花罹患疫病及栽培介質之帶菌情形，並確認所感染之疫病菌種類，以便儘早擬定防治策略，減少不必要之經濟損失。

關鍵字

疫病菌 ； 蘭花疫病 ； 快速檢測 ； Internal transcribed spacer ITS ； Nested PCR

並列摘要

Orchid disease caused by Phytophthora has long been a major threat to cultivation of orchids in Taiwan. Phytophthora spp. known to infect orchids include mainly P. palmivora and P. parasitica. Identification of Phytophthora species by the conventional method includes the use of selective media to obtain Phytophthora isolates and examination by microscopy. The procedures are rather labor-intensive and time-consuming. In order to accelerate and simplify the process of diagnosis, we have developed a nested PCR assay for rapid and accurate detection of Phytophthora pathogens infecting orchids. After isolation of DNA from the plant tissue, PCR was performed using a primer set specific for Phytophthora. Amplification of DNA fragments of approximately 1 kb in length indicated the presence of Phytophthora pathogens. To identify the species, nested PCR was then performed using amplified product from the first PCR as the template and species-specific oligonucleotides as the primers. Amplification of specific DNA fragments would tell whether the orchids were infected by P. palmivora, P. parasitica, or both. Furthermore, the sensitivity of detection was greatly enhanced. This assay provides a rapid and sensitive method for detection of Phytophthora pathogens in infected orchids as well as infested media used for cultivation of orchids, and thus can assist growers in early diagnosis of the devastating orchid Phytophthora disease.

並列關鍵字

Internal transcribed spacer ITS ； Nested PCR ； Orchid Phytophthora disease ； Phytophthora palmivora ； Phytophthora parasitica ； Rapid detection