Detection of orchid Phytophthora disease by nested PCR

Abstract

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.