Botanical Studies (2006) 47: 379-387.
These authors contributed equally to this study.
Corresponding author: E-mail: firstname.lastname@example.org; Tel:
+886-2-3366-5208; Fax: +886-2-2362-0271.
Due to their beauty both as cutting flowers and potted
flowering plants, orchids (Orchidaceae L.) have become
the most important floriculture crops of Taiwan in recent
years. Fungal diseases known to attack orchids include
anthracnose, Botrytis petal blight, and Southern blight
(Leu, 1994; Huang and Lee, 1994). Besides, some species
of Phytophthora, which belong to the Oomycete group
of Stramenopiles, were found to cause severe black
rot in orchids, including P. cactorum
(Leb. and Cohn)
(Burnett, 1974) , P. erythroseptica Pethybridge
(Hall, 1989), P. parasitica Dastur (=P.
nitcotianae Breda de Haan) (Ann, 1995), P. palmivora
(Butler) Butler (Ann, 1995; Yehm et al., 1998), and P.
multivesiculata Ilieva, Man in ¡¥t Veld, Veenbaas-Rijks
et Pieters. sp. nov. (Ilieva et al., 1998). In Taiwan, P.
palmivora and P. parasitica are known to attack a wide
variety of orchids, including Cattleya, Cymbidium,
Dendrobium, Oncidium, and Phalaenopsis, to mention
only the most important ones (Ann, 1995; Yehm et al.,
1998), while P. multivesticulata was reported only in
one case, infecting C. tracyanum
(Chern and Ann, 1996;
Ilieva et al., 1998). Diagnosis of orchid Phytophthora
disease is complicated by the observation that symptoms
caused by Phytophthora are hard to distinguish with those
caused by the bacterial pathogen Erwinia carotovora
subsp. carotovora (Su and Leu, 1992), and even worse,
these pathogens might infect orchids simultaneously.
Traditionally, diagnosis of the orchid Phytophthora disease
was performed by isolation of Phytophthora pure culture
from diseased plants, followed by identification based
on morphological characteristics, which might take more
than one week to identify a pathogen. In the present study,
a nested polymerase chain reaction (PCR) method was
developed in order to simplify and speed up the procedure
for disease diagnosis.
PCR is now used extensively for detection of plant
pathogens due to advantages of sensitivity, speed, and high
sample throughput (Martin et al., 2000). The key step for
development of a PCR method is to design oligonucleotide
primers with good specificity. For P. parasitica, primers
have been designed based on a variety of sequences,
including the sequence of a P. parasitica-specific DNA
Detection of orchid Phytophthora disease by nested
, Li-Chun HUANG
, Pao-Jen ANN
, and Ruey-Fen LIOU
Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 106, TAIWAN
Plant Pathology Division, Agricultural Research Institute, Council of Agriculture, Executive Yuan, Wufong 403, TAIWAN
(Received February 7, 2006; Accepted April 14, 2006)
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.
Keywords: Internal transcribed spacer (ITS); Nested PCR; Orchid Phytophthora disease; Phytophthora
palmivora; Phytophthora parasitica; Rapid detection.