Botanical Studies (2006) 47: 379-387.

3

These authors contributed equally to this study.

*

Corresponding author: E-mail: rfliou@ntu.edu.tw; Tel:

+886-2-3366-5208; Fax: +886-2-2362-0271.

INTRODUCTION

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)

Schroeter

(Burnett, 1974) , P. erythroseptica Pethybridge

var. erythroseptica

(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

MOLECULAR BIOLOGY

Detection of orchid Phytophthora disease by nested

PCR

Huei-Ling TSAI

1,3

, Li-Chun HUANG

1,3

, Pao-Jen ANN

2

, and Ruey-Fen LIOU

1,

*

1

Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 106, TAIWAN

2

Plant Pathology Division, Agricultural Research Institute, Council of Agriculture, Executive Yuan, Wufong 403, TAIWAN

(Received February 7, 2006; Accepted April 14, 2006)

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.

Keywords: Internal transcribed spacer (ITS); Nested PCR; Orchid Phytophthora disease; Phytophthora

palmivora; Phytophthora parasitica; Rapid detection.

380

Botanical Studies, Vol. 47, 2006

segment obtained from the genomic library (Ersek et

al., 1994), the parA1 gene which encodes the elicitin

(Kamoun et al., 1993; Lacourt and Duncan, 1997; Kong

et al., 2003), and ribosomal internal transcribed spacer

(ITS) sequence (Ippolito et al., 2002); the latter is a good

candidate for designing PCR primers due to its high copy

number, which was estimated to be 820 copies per diploid

nucleus in P. infestans

(Judelson and Randall, 1998).

Indeed, sequences of high copy number have been the

choice of many studies in order to enhance the sensitivity

of detection by PCR (Judelson and Tooley, 2000; Jyan et

al., 2002; Martin et al., 2004). Besides, sensitivity may

be improved by the use of nested PCR. In this method,

two primer pairs were designed based on the sequence

of a DNA segment, with one pair nested within the other.

PCR was first run with the outer primers. Afterwards, a

second PCR was performed using the inner pair as the

primer and the amplification product from the first PCR

as the template. Thus, in addition to sensitivity, specificity

of detection may be improved by nested PCR (Martin et

al., 2000). In this paper, a nested PCR method for rapid

detection of Phytophthora pathogens was developed in

order to assist early diagnosis of the orchid Phytophthora

disease.

MATERIALS AND METHODS

Fungal cultures and growth conditions

Isolates of Phytophthora spp. and other fungi used in

the study were listed in Table 1. Isolates of Phytophthora

spp. and Peronophthora litchii were provided by the

third author (Dr. P. J. Ann), Pythium myriotylum and

Py. ultimum by Dr. P. H. Wang (Dept. Life Science,

Tunghai University, Taichung, Taiwan), Fusarium spp.

and Rhizoctonia solani by Dr. S. S. Tzean (Dept. Plant

Pathology and Microbiology, National Taiwan University).

To prepare mycelia for isolation of DNA, isolates were

grown on liquid media, harvested by filtration, and frozen

at -80¢XC until use. Phytophthora infestans was grown

on Rye B medium at 20¢XC

for 20 days (Caten and Jinks,

1968), other Phytophthora spp. on 5% V8 agar (5%

Campbell¡¦s V8 juice, 0.02% CaCO

3

, and 2% Bacto agar)

at 25¢XC

for 10 days, Pythium spp. on 10% V8 agar (10%

Campbell¡¦s V8 juice, 0.02% CaCO

3

, and 2% Bacto agar)

at 30 ¢XC for 5 days, and o ther fungi o n potato dext ro se

broth (Difco Laboratories, Detroit, Michigan) at 25¢XC

for

8 days.

Isolation of DNA

DNA was isolated by using the DNeasy Plant Mini Kit

(Qiagen

R

, Basel, Switzerland). The concentration of DNA

was determined by spectrophotometry, using GeneQuant

II (Amersham Biosciences, Uppsala, Sweden).

Design of oligonucleotide primers for PCR

To design oligonucleotide primers for PCR, sequences

of 28S rRNA and ITS1-5.8S rRNA-ITS2 from a variety of

Phytophthora spp. were collected from the NCBI website

(http://www.ncbi.nlm.nih.gov/) and analyzed by multiple

sequence alignment using Clustal X (Thompson et al.,

1994). In addition, to confirm the accuracy of sequences,

ITS1-5.8S rRNA-ITS2 sequences of representative

Phytophthora spp. analyzed in this study were cloned by

PCR using primers ITS1 and ITS4 (White et al., 1990).

Table 1. Phytophthora spp. and other isolates analyzed in this study.

Species

Isolate

Location

Host

Ribosomal ITS

accession number

P. botryosa

ATCC26479

AY251664

ATCC52221

AY251665

P. cactorum

AY251663

P. capsici

21170 Yunlin

Capsicum annuum (sweet pepper) AY251662

P. cinnamomi

PC97 Chiayi

AY251661

94006 Wufeng, Taichung Persea Americana (Avocado)

AY251660

P. citricola

9023 Linluo, Pingtung Syzygium samarangese (wax apple)

9024 Linluo, Pingtung Syzygium samarangese (wax apple)

9025 Sinyuan, Pingtung Syzygium samarangese (wax apple)

9026 Sinyuan, Pingtung Syzygium samarangese (wax apple)

P. citrophthora

95004 Ilan

Fortunella sp. (kumquant)

AY251659

Annona squamosa (custardapple)

98165 Ilan

Fortunella sp. (kumquant)

AY251658

98167 Ilan

Citrus tankan (tankan)

95004 Ilan

Calamondin

97083 Taiping, Taichung Averrhoa carambola (star fruit)

TSAI et al. ¡X Rapid detection of orchid Phytophthora disease

381

Species

Isolate

Location

Host

Ribosomal ITS

accession number

P. colocasiae

9177 Minsyong, Chiayi Colocasie esculenta (taro)

AY251657

97066 Shueili, Nantow Colocasie esculenta (taro)

AY251656

20216 Kinmen

Colocasie esculenta (taro)

AY251655

98115 Houli, Taichung Colocasie esculenta (taro)

P. cryptogea

90130

Euphobia pulcherrima (poinsettia) AY251653

98067 Dounan, Yunlin Solanum tuberosum (potato)

98176

Lycopersicum esculentum (tomato)

94011 Yongjing, Changhua Gerbera jamesonii (transvaal daisy) AY251654

P. drechsleri (P. melonis)

98141 Pusin, Changhua Cucumis sativus (cucumber)

AY251650

96032

Benincasa hispida (wax gourd)

AY251651

98107 Tainan

Momordica charantia (bitter gourd)

CH-1

Momordica charantia (bitter gourd)

P. infestans

98029 Dounan, Yunlin Solanum tuberosum (potato)

20040 Rueiyen, Hawlian Lycopersicum esculentum (tomato)

99017 Houli, Taichung Solanum tuberosum (potato)

20060 Houli, Taichung Solanum tuberosum (potato)

P. meadii

NTU-01 Taipei

Zantedeschia aethiopica (white arum

lily)

AY251649

P. palmivora

93105 Dacheng, Changhua Cattleya sp. (orchid)

AY251647

9253 Taitung

Phalaenopsis aphrodite

AY251648

9257 Taitung

Cattleya sp. (orchid)

PpaA1-5 Madou, Tainan Persea Americana (Avocado)

9150

Hedera japonica (English ivy)

8829 Nansi, Tainan Citrus sinensis Osb. (sweet orange)

9097 Yuli, Hualien

Carica papaya (papaya)

P. parasitica

92171 Chiayi

Peperomia sp.

92033 Taitung

Saintpulia ionantha (African violet)

991-3 United States Citrus sp.

92145 Jhongpu, Chiayi Sinningia speciosa (glozimia)

92143 Jhongpu, Chiayi Sinningia speciosa (glozimia)

98161 Wufeng, Taichung Adenium obesum (Desert rose)

98151

Pandanus odorus

Peronophythora litchii

90113 Minsyong, Chiayi Litchi chinensis (litchi)

AY251666

Pythium aphanidermatum

Py. myriotylum

Py. splendens

Pys10 Puli, Nanto

Py. sylvaticum

CCRC33460

Py. ultimum

Fusarium moniliforme

CCRC 31492

F. oxysporum f. sp. melonis CCRC 32121 France

Cucumis melo (muskmelon)

F. oxysporum f. sp. momordicae CCRC 35046 Dahu, Miaoli Momordica charantia (bitter gourd)

F. roseum Link

CCRC 35115 Wufeng, Taichung Musa sapientum (banana)

F. solani (Martius) Saccardo CCRC 32448 New Zealand

F. verticillioides Saccardo CCRC 35113 Wufeng, Taichung Sorghum bicolor (sorghum)

Rhizoctonia solani

AG-3 021122

Table 1. (Continued)

382

Botanical Studies, Vol. 47, 2006

Table 2. PCR primers used in this study.

Target

Name Primer sequence (5¡¦ to 3¡¦)

Location in

rDNA

Expected size of

PCR product (bp)

Phytophthora spp. Phy1s ACT TTC CAC GTG AAC CGT ATC A

ITS1

~1000

Phy2a GCA CGA GCC ACT CAG GGA TG

28S

P. palmivora

Pal1s CAC GTG AAC CGT ATC AAA ACT

ITS1

648

Pal2a CAA TCA TAC CAC CAC AGC TGA

ITS2

P. parasitica

Par1s ACG TTT GGG CTT CGG CCT GAT T

ITS1

680

Par2a GAT GCA TAC CGA AGT ACA CAT TA

ITS2

Plant

Pl1s GGT CGT ACG CAC GAG CCA CT

18S

678

Pl2a ATT ACT CCG ATC CCG AAG G

18S

Nucleotide sequences of the recombinant clones were

determined on both strands of DNA, using the BigDye

terminator cycle sequencing ready reaction kit and an

ABI Prism 310 Genetic Analyzer apparatus (Applied

Biosystems, Foster City, CA, USA), followed by analyses

using programs in the GCG software package (Genetics

Computer Group, Wisconsin Package Version 10.0).

Genus-specific primers were designed based on the highly

conserved regions of ITS1 and 28S rRNA, while species-

specific primers based on the ITS1 and ITS2 sequences

which are highly diverged among species (Table 2).

Besides, a primer set was designed based on the conserved

sequences of plant 18S ribosomal DNA to serve as a

positive control for PCR while using DNA prepared from

plants as the template (Table 2).

Test of primer specificity by PCR

PCR was performed in a 20-£gl reaction, which

contained 10 ng of template DNA, 1.25 £gM of

oligonucleotide primers, 0.2 mM dNTP, 1X PCR

buffer, and 1 U of DyNazyme

TM

II DNA polymerase

(Finnzymes, Espoo, Finland). Amplification was initiated

by denaturation at 94¢XC for 5 min, followed by 25 cycles

of [95¢XC/30 sec-58¢XC/30 sec-72¢XC/1 min] and a 10-min

extension at 72¢XC in a thermocycler (GeneAmp PCR

System 2400, Perkin elmer, Foster City, CA, USA). PCR

amplified products were analyzed by 1.5% agarose gel

electrophoresis in 1¡Ñ TAE.

Nested PCR

To carry out the nested PCR, the first PCR was

performed using Phy1s/Phy2a as the primer pair. After

completion of the amplification reaction, the PCR mixture

was diluted 100¡Ñ with sterilized ddH

2

O, followed by the

initiation of a second PCR using 3 £gl of the diluted mixture

as the template and species-specific oligonucleotides as

the primers. The second PCR was performed according

to procedures described in the previous section except

that, instead of 58¢XC, the annealing temperature was set

at 67¢XC. PCR amplified products were analyzed by 1.5%

agarose gel electrophoresis in 1¡Ñ TAE.

Detection of orchid Phytophthora disease by

nested PCR

Diseased Oncidium sp. (Ramsey) was collected from

Ping Tong, Taiwan. Crude extract was prepared from

Oncidium tissues accroding to the method developed

by Wang et al. (1993) with some modifications. A small

piece of the Oncidium tissue (approximately

0.2 g) was

immersed in 500 £gl of 0.5 N NaOH and macerated with

a homogenizer. Following centrifuation at 17,000 g for 5

min, the supernatant was collected and mixed thoroughly

with 9 volumes of 0.1 M Tris (pH 8.0). Aliquots of

the mixture were then used as the template for PCR as

described in the previous section.

Isolation

of Phytophthora spp. from the culture

media of orchids

To find out whether the culture media were

contaminated, Phytophthora spp. were trapped according

to procedures describled by Grimm and Alexander (1973)

with some modifications. Culture media collected from

a orchid garden located on the Ping Tong area of Taiwan

were soaked in water for 3-4 days at room temperature,

with the addition of six leaf pieces (1¡Ñ1 cm

2

) excised from

the orchid. Afterwards, the baits were collected and used

for extraction of DNA and nested PCR as described in the

previous section.

RESULTS

Primer design and specificity

To design oligonucleotide primers for PCR, sequences

of 28S rRNA and ITS1-5.8S rRNA-ITS2 from a variety

o f Phytophthora spp. were collected from the NCBI

website and analyzed by multiple sequence alignment

using Clustal X. Besides, to ensure that Phytophthora

isolates analyzed in this study contained the same

ribosomal ITS sequences as those obtained from the NCBI

website, sequences encompassing the ITS1-5.8S rRNA-

ITS2 regions of representative isolates o f Phytophthora

spp. and Pe. litchii were cloned by PCR and analyzed.

The resultant data were deposited in the GenBank

TSAI et al. ¡X Rapid detection of orchid Phytophthora disease

383

with accession numbers AY251647- AY251651 and

AY251653-AY251666 (Table 1). Analysis by Blastn of

NCBI indicated that, with the exception of AY251649, the

best hits of all sequences were ribosomal ITS sequences

obtained from the same species by other laboratories,

and thus confirmed the identity of the aforementioned

Phytophthora and Pe. litchii isolates. Analysis by multiple

sequence alignment also indicated that sequences of

ITS1-5.8S rRNA-ITS2 were very homogeneous within

species of Phytophthora (data not shown). The sequence

of AY251649 was cloned from P. meadii, which was

known to infect white arum lily (Zantedeschia aethiopica)

(Liou et al., 1999). Thus far, there is no other ribosomal

sequence from this species available in the GenBank.

Analysis by multiple sequence alignment indicated

that some regions of ITS1 and 28S rRNA sequences were

conserved among different species o f Phytophthora.

Three primers were designed accordingly, including

Phy1s, Phy2a, and Phy2a-1, which would make up two

primer pairs for PCR: Phy1s/Phy2a and Phy1s/Phy2a-1,

respectively (Table 2). To evaluate the specificity of

the primers, PCR was performed using Phy1s/Phy2a

or Phy1s/Phy2a-1 as the primer set, and DNA from

13 Phytophthora spp. (a total of 47 isolates) and other

species as the template (Table 1). When PCR was

performed using Phy1s/Phy2a as the primers, analysis of

the amplified products indicated that DNA fragments of

approximately 1,000 bp in length were obtained from all

the Phytophthora spp. analyzed, including P. botryosa,

P. cactorum, P. capsici, P. cinnamomi, P. citricola, P.

citrophthora, P. colocasiae, P. cryptogea, P. drechsleri,

P. infestans, P. meadii, P. palmivora, and P. parasitica

(Figure 1). No amplification signal was detected, however,

when PCR was performed using DNA from Pythium

spp. or other fungal isolates as the templates, with the

exception of Pe. litchii (data not shown). PCR with Phy1s/

Phy2a-1, in contrast, gave rise to amplified products not

only from Phytophthora spp., but also from Pythium.

As a result, this primer set was not used in the following

experiments.

Specificity of the species-specific primer sets, Pal1s/

Pal2a and Par1s/Par2a for P. palmivora and P. parasitica,

respectively, was evaluated in a similar way. When PCR

was primed with Pal1s/Pal2a, DNA fragment of the

expected length (648 bp) was obtained only when DNA

from P. palmivora was used as the template (Figure 2A,

lane 15). When the same experiments were performed

using Par1s/Par2a as the primers, DNA fragment of the

expected length (680 bp) was obtained only when DNA

from P. parasitica was used as the template (Figure 2B,

lanes 16 and 22). These results confirmed the specificity

of these two primer sets toward their respective targets

and thus supported their applications for detection of P.

palmivora and P. parasitica, respectively.

Detection of P. palmivora and P. parasitica

by

nested PCR and sensitivity test

To detect P. palmivora and P. parasitica by nested

PCR, the first PCR reaction was primed with Phy1s/Phy2a,

while the second PCR with Pal1s/Pal2a or Par1s/Par2a.

To determine the minimal amount of the template DNA

that is enough for generation of the amplified product, the

first PCR reaction was performed using different amounts

of P. palmivora or P. parasitica DNA as the templates.

As shown in Figure 3, the PCR-amplified product from

Figure 1. Specificity test of the primer pair Phy1s/Phy2a. PCR

was performed using Phy1s/Phy2a as primers, and DNA from

representative Phytophthora spp. as the template. The amplified

products were analyzed by 1.5% agarose gel electrophoresis.

Lane 1: P. citricola 9025; 2: P. drechsleri (P. melonis); 3: P.

infestans 98029; 4: P. parasitica 92143; 5: P. cryptogea; 6: P.

colocasiae 98115; 7: P. palmivora 93105; 8: P. botryosa; 9: P.

citricola; 10: P. cinnamomi PC97; 11: P. citrophthora 95004; 12:

P. parasitica 991-3; 13: P. parasitica 92145; 14: P. citrophthora

97083; 15: P. colocasiae 9177; 16: P. infestans 20040; 17: P.

drechsleri 96032; 18: P. cryptogea 98176; 19: P. cinnamomi

94006; 20: P. cactorum; 21: P. citricola 9023; 22: P. colocasiae

20216; 23: P. parasitica 98151; 24: P. cryptogea 94011; 25:

P. capsici 21170; 26: P. citrophthora 98165; 27: P. parasitica

98161; 28: P. cryptogea 90130; 29: P. citrophthora 98167; 30:

P. meadii; 31: P. botryosa 52221; 32: P. citricola 9026; 33: P.

citrophthora; 34: P. colocasiae 97066; 35: P. citricola 9024; 36:

P. drechsleri 98141; M: 1 kb plus DNA ladder (Invitrogen).

384

Botanical Studies, Vol. 47, 2006

1 p g of P. palmivora DNA was detectable by ethidium

bromide staining of the agarose gel (Figure 3A, lane 5).

No signal was observed, however, as the amount of DNA

template decreased. When the second PCR was performed

using Pal1s/Pal2a as the primers, and diluted (1:100)

amplified product from the first PCR as the template,

amplification signal was detectable even when only 10 fg

of P. palmivora DNA was used as the template for the first

PCR (Figure 3B, lane 7).

The same experiments were performed using Par1s/

Par2a and P. parasitica DNA. As shown in Figure 4,

the PCR-amplified product from 0.1 pg of P. parasitica

DNA was detectable by ethidium bromide staining of the

agarose gel (Figure 4A, lane 6). No signal was observed

when the amount of DNA template decreased. When the

second PCR was performed using Par1s/Par2a as the

primers and diluted amplified product from the first PCR

as the template, amplification signal was detectable while

10 fg of P. parasitica DNA was used as the template for

the first PCR (Figure 4B, lane 7). It was thus obvious

that, with nested PCR, the sensitivity of detection was

enhanced by 10-100 folds.

Detection of orchid Phytophthora diseases by

nested PCR

To develop a method for rapid detection of orchid

Phytophthora diseases by nested PCR, sample tissues

were collected from leaves, pseudostems, and roots of

diseased Oncidium. Furthermore, Phytophthora pathogens

which might exist in the culture media were trapped with

leaf pieces of the orchid. Crude extract was then prepared

from each of these samples by the NaOH method and

analyzed by PCR. In addition to Phy1s/Phy2a, the primer

set Pl1s/Pl2a (Table 2), which was designed based on the

conserved sequences of plant 18S rRNA, was included

Figure 2. Specificity test of primer pairs Pal1s/Pal2a and Par1s/

Par2a. PCR was performed using Pal1s/Pal2a (A) or Par1s/Par2a

(B) as primers, and DNA from representative Phytophthora

and Pythium species as the template. The amplified products

we re analyzed by 1.5% agaros e gel ele ctrophores is . L ane

1: P. infestans; 2: P. citricola 9025; 3: Py. myriotylum; 4: P.

drechsleri (melonis); 5: Py. aphanidermatum; 6: P. cinnamomi;

7: P. capsici; 8: Py. splendens; 9: P. cryptogea 98067; 10: P.

colocasiae 9177; 11: P. citrophthora 95004; 12: Peronophythora

litchii; 13: Py. s ylvatic um; 14: P. cry ptoge a 98176; 15: P.

palmivora 93105; 16: P. parasitica 98151; 17: P. drechsleri

96032; 18: P. cactorum; 19: P. cryptogea 94011; 20: Py.

ultimum; 21: P. meadii; 22: P. parasitica 98151; 23: P. botryosa

52221; 24: ddH

2

O; M: 1 kb plus DNA ladder (Invitrogen).

Arrows indicated the location of the DNA fragments obtained

from P. palmivora (A, lane 15) and P. parasitica (B, lanes 16

and 22), respectively.

Figure 3. Sensitivity analysis of nested PCR for Phytophthora

palmivora. The first PCR was performed using Phy1s/Phy2a as

the primer pair, and indicated amounts of P. palmivora DNA as

the template (A). The second PCR was performed using Pal1s/

Pal2a as the primer pair and diluted amplified products from

the first PCR as the template (B). The amplified products were

analyzed by 1.5% agarose gel electrophoresis. Lane 1: 10 ng;

2: 1 ng; 3: 100 pg; 4: 10 pg; 5: 1 pg; 6: 100 fg; 7: 10 fg; 8: 1 fg;

9: 100 ag; 10: 10 ag; 11: 1 ag; 12: ddH

2

O; M: 1 kb plus DNA

ladder (Invitrogen).

Figure 4. Sensitivity analysis of nested PCR for Phytophthora

parasitica. The first PCR was performed using Phy1s/Phy2a as

the primer pair, and indicated amounts of P. parasitica DNA as

the template (A). The second PCR was performed using Par1s/

Par2a as the primer pair and diluted amplified products from

the first PCR as the template (B). The amplified products were

analyzed by 1.5% agarose gel electrophoresis. Lane 1: 10 ng;

2: 1 ng; 3: 100 pg; 4: 10 pg; 5: 1 pg; 6: 100 fg; 7: 10 fg; 8: 1 fg;

9: 100 ag; 10: 10 ag; 11: 1 ag; 12: ddH

2

O; M: 1 kb plus DNA

ladder (Invitrogen).

TSAI et al. ¡X Rapid detection of orchid Phytophthora disease

385

and used as a positive control to ensure quality of plant

DNA extraction. As shown in Figure 5A, DNA fragments

of approximately 1,000 bp long were observed with DNA

from the pseudostem, root, and leaf of Oncidium sp., as

well as that from the leaf baits (Figure 5A, lanes 1-4),

indicating the presence of Phytophthora pathogens in

these specimens. This fragment was also detected while

PCR was performed using DNA from P. palmivora as

the template (Figure 5A, lane 5), but not with DNA from

the orchid (Figure 5A, lane 6). Besides, DNA fragments

of 678 bp in length were amplified from genes encoding

plant 18S rRNA (Figure 5A, lanes 1-4 and 6). To identify

the Phytophthora species, a second PCR was performed

using diluted amplified product from the first PCR as

the template. Amplified products of the expected size

were observed only when PCR was primed with Pal1s/

Pal2a (Figure 5B), but not Par1s/Par2a (data not shown),

indicating the pathogen which caused disease in Oncidium

sp. was P. palmivora, rather than P. parasitica. DNA

fragments about 1,000 bp in length, which appeared as

bands of weaker intensity above the 648-bp fragments,

were the DNA templates obtained from the first PCR

(Figure 5B).

DISCUSSION

Both P. palmivora and P. parasitica are important

plant pathogens, able to cause severe diseases in a

wide variety of crops (Erwin and Ribeiro,

1996). While

infecting orchids, they caused severe orchid Phytophthora

disease, which has been a major threat for cultivation of

orchids in Taiwan (Ann, 1995). Traditional methods for

identification of Phytophthora spp. are time-consuming

and require considerable expertise to differentiate species

of Phytophthora based on characteristics of morphology.

In this study, a nested PCR assay was established for

rapid detection of Phytophthora pathogens of orchids.

Compared with conventional PCR, nested PCR has the

advantages of higher sensitivity and better specificity,

and thus has been used as the detection method in many

studies (Grote et al., 2002; Ippolito et al., 2002; Martin

et al., 2004). In our assay, oligonucleotides used to prime

the first PCR, Phy1s and Phy2a, were designed according

to the conserved sequences of ITS1 and 28S rRNA of

Phytophthora. As shown by the specificity test, this

primer set could amplify DNA from 13 Phytophthora

spp. as well as Pe. litchii, an Oomycete pathogen known

to cause blossom blight in litchi (Litchi chinensis) (Ann

and Ko, 1984), but not pathogens belonging to the genus

Pythium. Since Pe. litchii is known to infect only litchi,

its interaction with Phy1s/Phy2a has not been a major

concern. When PCR was primed with this primer set,

appearance of an amplification signal with the expected

size would indicate the presence of Phytophthora

pathogen(s). Following the first PCR, nested PCR was

then performed using species-specific oligonucleotides,

Pal1s/Pal2a or Par1s/Par2a, as the primer set to identify

the pathogen at the species level. Specificity of these two

primer pairs was verified by PCR using DNA prepared

from 13 Phytophthora spp., which represented species

isolated from diseased plants collected from different

areas of Taiwan in recent years with the exception of P.

botryose and P. cactorum. Phytophthora multivesticulata

was reported only once in 1996 (Chern and Ann, 1996),

and thus was not included in the analysis. To exclude the

possibility that Pal1s/Pal2a and Par1s/Par2a might interact

with DNA of P. multivesticulata, we search the GenBank

for the ribosomal ITS sequence of P. multivesticulata.

There is only one sequence (DQ335639) available.

Analysis of the sequence indicated that, because of

sequence divergence, the aforementioned concern should

not present a problem (data not shown).

The idea that nested PCR was carried out with the

addition of both primer sets (Pal1s/Pal2a and Par1s/Par2a)

simultaneously, namely by multiplex PCR, was very

attractive. Results obtained from the experiments (data not

shown), however, indicated that it is not applicable, due

to the similarity in the size of amplified DNA fragments

Figure 5. Detection of the orchid Phytophthora disease by

nested PCR. Samples from infected tissues of Oncidium sp. were

process by the NaOH method and analyzed by the nested PCR.

The first PCR was performed using Phy1s/Phy2a and Pl1s/pl2a

as the primer pairs (A), while for the second PCR, Pal1s/Pal2a

was included in the PCR reaction (B). The amplified products

were analyzed by 1.5% agarose gel electrophoresis. For both (A)

and (B), lane 1: the leaf baits from infested media; 2: diseased

orchid leaves; 3: dis eased orchid roots; 4: dis eased orchid

pseudostems; 5: Phytophthora palmivora. Lane 6 of (A): leaves

from a healthy orchid. M: 1 kb plus DNA ladder (Invitrogen).

Arrows indicated the location of the amplified DNA fragments.

386

Botanical Studies, Vol. 47, 2006

obtained with Pal1s/Pal2a (648 bp) and Par1s/Par2a (680

bp), which were hard to distinguish by the regular agarose

gel electrophoresis system used in this study. With nested

PCR, as have been demonstrated in other studies (Ippolito

et al., 2002; Martin et al., 2004), sensitivity of the test

was enhanced 10-100 folds, and as less as 10 fg of DNA

was enough for generating a significant amplification

signal. However, there are risks of contamination when

performing nested PCR in two rounds. Special precautions

must be taken while setting up the reactions (Takahashi

and Nakayama, 2006).

To detect the existence of Phytophthora pathogens

in the orchids, DNA was extracted from the diseased

plant tissues by the use of an alkali method (Wang et al.,

1993). It took only a couple of minutes to obtain DNA

to be used for PCR. Furthermore, to ensure that negative

results from nested PCR were indeed indicative of the

absence of Phytophthora pathogens, in addition to Phy1s/

Phy2a, a second primer set (Pl1s/Pl2a) was included in

the first PCR. This primer set was designed based on the

conserved sequences of plant 18S rDNA and thus might

serve as a positive control to check the quality of DNA,

which was prepared from diseased plants and used as the

template for PCR. Analysis by nested PCR indicated that

the tested orchids were infected only by P. palmivora.

The possibility that the absence of P. parasitica might

result from failure of Par1s/Par2a to interact with DNA

obtained from infected tissues was excluded, since it

has been demonstrated previously that this primer set

is useful for detection of P. parasitica in infected plants

(data not shown). With the nested PCR assay, presence of

Phytophthora spp. on diseased plants might be detected

within a few hours, and thus provided a very useful

tool for diagnosis of orchid Phytophthora disease. As

mentioned in the previous section, P. palmivora and P.

parasitica are important plant pathogens on numerous

crops, including citrus, tomato, and a wide variety of

ornamental crops (Erwin and Ribeiro,

1996). The method

described here can also be adopted for detection of other

plant diseases caused by these two pathogens.

Acknowledgements. We thank Professor S. S. Tzean

of National Taiwan University and Professor P. H. Wang

of Tunghai University for providing the fungal and

Pythium isolates, respectively. This study was supported

by the Bureau of Animal and Plant Health Inspection

and Quarantine, Council of Agriculture, Executive

Yuan, Taiwan (91AS-7.2.2-BQ-B1(6) and 92AS-1.8.2-

BQ-B1(6)).

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