pg_0001

Botanical Studies (2007) 48: 79-89.

Corresponding author: E-mail: kowh@dragon.nchu.edu.tw;

Tel: +886-4-23302301; Fax: +886-4-2333-8162.

INTRODUCTION

Peronophythora litchii Chen ex Ko et al. is unique in

its ability to produce Peronospora-like sporangiophores

on artificial media (Chen, 1961; Ko et al., 1978). This

causal organism of litchi fruit rot produced differentiated

sporangiophores with dichotomous branchlets (Figure

1A). The growth of sporangiophores was terminated at

maturation by the synchronous formation of sporangia

on the tapering tips of branchlets (Figure 1B). The

sporangia on each sporangiophore enlarged and matured

at approximately the same rates (Figure 1C, D). Although

Pp. litchii produced sporangiophores characteristic of

the species of the Peronosporaceae, its sex organs and

ease of culture on artificial media resembled those in

the Pythiaceae. A new family Peronophythoraceae was,

therefore, established to accommodate the species with

characteristics of both Pythiaceae and Peronosporaceae

(Ko et al., 1978). The transitional nature of Pp. litchii was

further supported by the observation of the characteristics

of both Peronospora and Phytophthora in its oospore

germination (Ann and Ko, 1980).

In 1982, Chi et al. (1982) noted the growth renewal

of sporangiophores of Pp. litchii and re-named the

organism Phytophthora litchii. Subsequently, Huang et

al. (1983) and Ho et al. (1984) re-examined the asexual

reproduction of the organism to ascertain its taxonomic

status. Their studies substantiated the findings of Chi

et al. (1982) that occasionally, the sporangiophore

was capable of growth renewal, but also showed that

in general, the sporangisphore of Peronophythora is

basically determinate and sometimes indeterminate. Since

the asexual reproduction of Peronophythora had the

characteristics of both Peronospora and Phytophthora,

Huang et al. and Ho et al. considered it unique enough

to justify Peronophythoraˇs retention as a distinct genus,

transitional between Phytophthora and Peronospora

(Ann and Ko, 1980; Chen, 1961; Ko et al., 1978).

It is also not known whether the growth renewal of

conidiophores on artificial media will also occur in

members of Peronosporaceae when their culture on media

becomes possible in the future. In recent years, partial

DNA sequences have been shown to be useful in the

Evaluation of the rearrangement of taxonomic position

of Peronophythora litchii based on partial DNA

sequences

Zheng-Guang ZHANG

, Xiao-Bo ZHENG

, Yuan-Chao WANG

, and Wen-Hsiung KO

Department of Plant Protection, Nanjing Agricultural University, Nanjing, P.R. China

Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan

(Received March 16, 2006; Accepted April 26, 2006)

ABSTRACT.

Similarity levels of ribosomal ITS sequences and 28S ribosomal DNA sequences among

members of the Oomycota were analyzed to determine the validity of the suggestion of transferring

Peronophythora litchii to Phytophthora based on their close phylogenetic relationship. The validity of the

suggestion is dependent on the accuracy of the assumption of the existence of a negative correlation between

the order of the taxonomic ranks and the level of the sequence similarity of these two DNA fragments.

Included in this report are sequences obtained in this study and sequences retrieved from the GenBank.

Phytophthora sojae and Plasmopara viticola showed a higher level of ITS sequence similarity than all the

species pairs of Phytophthora and several isolate pairs of Peronospora parasitica tested, indicating the lack

of validity of transferring the taxonomic rank based on the ITS sequence relationships. The isolates AR 246

and HV 656 of Bremia lactucae showed a lower level of 28S sequence similarity than some species pairs

of the same genus of Peronospora, Phytophthora, Pythium, Albugo and Achlya, and most species pairs of

the different genera tested indicated also the lack of validity of transferring the taxonomic rank based on

the 28S sequence relationships. The taxonomic statuses of Peronophythora as a distinct genus transitional

between Peronospora and Phytophthora and of Peronophythoraceae as a distinct family transitional between

Peronosporaceae and Pythiaceae, therefore, remain valid.

Keywords: 28S rDNA; ITS rDNA; Peronophythora litchii; Phytophthora; Sequence similarity.

MICROBIOlOgy

pg_0002

Botanical Studies, Vol. 48, 2007

phylogenetic studies of Oomycota (Lee and Taylor, 1992;

Crawford et al., 1996; Matsumoto et al., 1999; Hudspeth et

al., 2000; Forster et al, 2000; Cooke et al, 2000; Petersen

and Rosendahl, 2000; Riethmuller et al., 2002; Martin and

Tooley, 2003; Voglmayr, 2003; Ko et al., 2006). When

Riethmuller et al. (2002) and Voglmayr (2003) reported

the phylogenetic relationships of members in Oomycota

using 28S ribosomal DNA sequences and ribosomal ITS

sequences, respectively, the authors also suggested the

transfer of Peronophythora litchii to Phytophthora because

of its close phylogenetic relationship to Phytophthora

palmivora and Ph. arecae. Pernophythora litchii, with

well differentiated sporangiophore and synchronous

formation of sporangia at maturity (Figure 1A-D), is

morphologically and physiologically distinct from species

o f Phytophthora, which usually form undifferentiated

sporangiophore producing sporangia as they grow (Figure

2A-D). We therefore undertook to evaluate the suitability

of using these two types of phylogenic relationships to

change the traditional taxonomic status, which was based

on morphological and physiological characteristics.

The use of DNA-fragment-based phylogenetic

relationships to determine the taxonomic status of an

organism is dependent on the assumption that for that

fragment of DNA, different isolates of the same species

have higher levels of DNA homology than different

species of the same genus, which in turn have higher levels

of DNA homology than different species from different

genera. The accuracy of this assumption has been tested

for neither 28S nor ITS DNA. We, therefore, sequenced

the ribosomal ITS DNA of 20 isolates belonging to

thirteen species from six genera in Peronosporaceae,

Peronophythoraceae and Pythiaceae, and analyzed their

sequence homology in this study. The ITS DNA sequences

retrieved from GenBank or published by Vaglmayr (2003)

and the 28S DNA sequences published by Riethmuller

et al. (2002) were also included in the analysis to test the

accuracy of the assumption for these two DNA fragments.

F igu re 1. Diffe rent s tages of development of s porangia on differentiated s porangiophores of P eronophythora litc hii . A,

sporangiophore with dichotomous branchlets; B, initiation of synchronous formation of sporangia; C, the early stage of sporangial

development; D, the mature stage of sporangia.

pg_0003

ZHANG et al.  Taxonomic position of

Peronophythora litchi

MATERIAlS AND METHODS

Fungal material

The organisms with ITS sequenced in this study are

listed in Table 1 Each isolate used was derived from a

single zoospore (Ho and Ko, 1997; Zheng and Ko, 1997;

Ann and Ko, 1989) and maintaimed on 10% V-8 juice agar

(Wu et al., 2003). Those organisms with ITS sequences

available in GenBank for comparison are listed in Table 2.

To obtain mycelia for genomic DNA extraction, 20 pieces

of agar culture (approximately 1⊙1⊙2 mm) cut from the

advancing margin of a 3-day-old colony growing on a

plate of lima bean agar for species of Phytophthora and

Peronophythora, or on potato dextrose agar for species

of Pythium, were placed in a 250-ml flask containing

100 ml Plichˇs liquid medium (Zhang et al., 2004). After

incubation at 25C in darkness on a shaker for 6 days,

mycelia were collected on a filter paper and stored at

-20C until use. Sporangia of Peronospora parasitica,

Pseudoperonospora cubensis, and Plasmopara viticola

obtained directly from infected leaves of their respective

hosts (Table 1) were also stored at -20C.

DNA preparation

DNA was prepared according to Panabieres et al. (1989)

with the following modification. Approximately 10 mg

of frozen mycelia or sporangia were ground in liquid

nitrogen, and the resulting powder was suspended in 0.5

ml of NIB buffer consisting of 100 mM NaCl, 10 mM

EDTA, 10 mM -mercaptoethanol, 0.5% NP-40, and 30

mM Tris-HCl, pH 8.0. The suspension was centrifuged at

12,000 g for 1 min. After removal of the supernatant, the

pellet was resuspended in NIB buffer, and the suspension

was centrifuged as above. The resulting pellet was then

resuspended in 0.8 ml of homogenization buffer consisting

of 0.1 M NaCl, 0.2 M sucrose and 10 mM EDTA before

adding 0.2 ml of lysis buffer containing 2.5% sodium

dodeyl sulphate, 0.25 M EDTA, and 0.5 M Tris, pH 9.2.

Figure 2. Development of sporangia on undifferentiated sporangiophores of Phytophthora palmivora. A, re-growth of sporangiophore

and initiation of a new sporangium on its tip after maturation of the first sporangium; B, enlargement of the second sporangium; C, re-

growth of sporangiophore and formation of a new sporangium on its tip after; maturation of the second sporangium; D, re-growth of

sporangiophore and formation of a new sporangium on its tip after maturation of the third sporangium.

pg_0004

Botanical Studies, Vol. 48, 2007

After incubation at 55C for 30 min, the homogenate

was extracted twice with the same volume of phenol-

chloroform-isoamyl alcohol (25:24:1) and centrifuged at

12,000 g for 15 min. The aqueous phase was extracted

with an equal volume of chloroform-isoamyl alcohol

(24:1), precipitated with two volumes of cold absolute

alcohol for 10 min, and centrifuged at 12,000 g for 15 min.

The pellet was washed with 70% ethanol, air-dried, and

resuspended in 50 l TE buffer consisting of 1 mM EDTA

and 10 mM Tris-HCl, pH 7.4. The A260 nm / A280 nm

ratios of these DNA preparations were between 1.7 and

2.0, and their A260 nm / A230 nm ratios were between

1.6 and 2.0, suggesting that they were essentially free of

proteins and carbohydrates. All DNA preparations were

kept at -20C.

DNA amplification and sequencing

The 5.8S rRNA gene and the two flanking internal

transcribed spacers (ITS1 and ITS2) were amplified

with primers ITS1 and ITS4 (White et al., 1990). The

50 l reaction mixtureconsisting of 10 ng template

DNA, 1 M each primer, 100 M each dNTP, 5 l 10 X

polymerase chain reaction (PCR) buffer, 1.5 mM MgCl

and 2.5 units Taq DNA polymerase (Promega Corp.,

Madison, WI, USA)was subjected to thermal cycling

in a PTC-200 DNA Engine Cycler (MJ Research, MA,

USA). The thermal cycling parameters were as follows:

initial denaturation for 5 min at 94C, then 1 min at 55C,

3 min at 72C, and 1 min at 94C for 30 cycles, and a final

elongation at 72C for 10 min. Amplification products

were run on 1.5% agarose gel, stained with ethidium

bromide, and visualized under UV illumination in order

to determine the number and size of DNA products

amplified in the PCR. PCR products were subcloned

using the pGEM-T Easy Vector System of Promega, and

these clones from each isolate were sequenced using the

Table 1. Collection data and GenBank accession number for ITS sequenced in the present study.

Species and present study

Associated habitat

Location

Source GenBank accession no.

Peronospora parasitica

Brassica oleracea

China

NAU

AY269996

Pseudoperonospora cubensis

Cucumis sativus

China

NAU

AY744946

Plasmopara viticola

Vitis vinifera

China

NAU

AY742739

Peronophythora litchii

Litchi chinensis

Taiwan

P. J. Ann

AY269997

Phytophthora capsici

98110 (A1)

Capsicum frutescens

Taiwan

P. J. Ann

AY742735

20081 (A1)

Capsicum frutescens

Taiwan

P. J. Ann

AY742736

Ph. infestans

TD1 (A1)

Solanum tuberosum

China

Q. H. Chen

AY269995

TD2 (A2)

Solanum tuberosum

China

Q. H. Chen

AY922974

Ph. nicotianae

991 (A1)

Soil

Taiwan

G. A. Zentmyer

AY208131

6134 (A2)

Solanum melongena

Taiwan

P. J. Ann

AY208128

Ph. palmivora

8829

Citrus sp.

Taiwan

P. J. Ann

AY744949

88108 (A1)

Carica papaya

Taiwan

P. J. Ann

AY742734

Ph. sojae, S317

Glycine max

China

C. Y. Shen

AY245092

Ph. tropicalis

23047 (A1)

Prunus persica

Taiwan

P. J. Ann

AY742737

129F-1 (A0)

Dianthus caryphyllus

Taiwan

W. H. Ko

AY742738

Pythium aphanidermatum

Cucumis sativus

China

NAU

AY269999

Py. splendens

117 (-)

Soil

Taiwan

M. Aragaki

AY269993

461 (+)

Soil

Taiwan

M. Aragaki

AY269994

Py. vexans

Pyv 6-1

Soil

Taiwan

W. H. Ko

AY269998

Pyv 6-2

Soil

Taiwan

W. H. Ko

AY922975

*Used in the comparison with those available in GenBank.

NAU=Nanjing Agricultural University.

pg_0005

ZHANG et al.  Taxonomic position of

Peronophythora litchi

Table 2. Similarity of ITS sequenced in this study with those available in GenBanka.

Species and isolate Associatedd habitat

Location

Source

GenBank

accession no. Similarity (%)

Peronospora parasitica

SMK 13558

Arabis glabra

Korea

H. D. Shin AY 210983

94.6

SMK 17785

Bararea orthoceras

Korea

H. D. Shin AY 210984

95.1

SMK 16040

Brassica campestris

Korea

H. D. Shin AY 210986

99.4

SMK 13731

Brassica campestris

Korea

H. D. Shin AY 210985

99.6

SMK 15691

Capsella bursa-paestoris

Korea

H. D. Shin AY 210987

84.8

SMK 18835

Capsella bursa-paestoris

Korea

H. D. Shin AY 210988

84.6

HV 746

Capsella bursa-paestori

AY 198254

82.8

SMK 18819

Cardamine flexuosa

Korea

H. D. Shin AY 210989

75.9

SMK 17273

Cardamine leucaretha

Korea

H. D. Shin AY 210990

95.1

SMK 17298

Cardamine leucaretha

Korea

H. D. Shin AY 210991

95.1

SMK 17322

Cardamine leucaretha

Korea

H. D. Shin AY 210992

95.2

SMK 18539

Cardamine leucaretha

Korea

H. D. Shin AY 210993

95.2

SMK 18833

Cardamine scutata

Korea

H. D. Shin AY 210994

76.7

SMK 17319

Darba nemorosa

Korea

H. D. Shin AY 210995

81.9

SMK 17270

Darba nemorosa

H. D. Shin AY 210996

81.9

SMK 18842

Darba nemorosa

Korea

AY 210999

81.9

SMK 18831

Darba nemorosa

Korea

H. D. Shin AY 210998

81.6

SMK 18811

Darba nemorosa

Korea

H. D. Shin AY 210997

81.8

Maks 9

H. D. Shin AY 578093

84.2

SMK 15604

Raphanus sativus

Korea

H. D. Shin AY 211002

90.2

SMK 15363

Raphanus sativus

Korea

H. D. Shin AY 211001

89.3

SMK 14315

Raphanus sativus

Korea

H. D. Shin AY 211000

90.2

SMK 11409

Rorippa islandica

Korea

H. D. Shin AY 211003

76.6

SMK 12789

Rorippa islandica

Korea

H. D. Shin AY 211005

75.5

SMK 18713

Rorippa islandica

Korea

H. D. Shin AY 211004

76.6

SMK 18859

Rorippa islandica

Korea

H. D. Shin AY 211007

75.6

SMK 18834

Rorippa islandica

Korea

H. D. Shin AY 211006

75.4

SMK 18194

Sisymbrium letewm

Korea

H. D. Shin AY 211008

84.9

Thlaspi arvense

Sweden O. Constantinescu AF 465759

76.8

SMK 17271

Thlaspi arvense

Korea

H. D. Shin AY 211009

83.5

SMK 18832

Thlaspi arvense

Korea

H. D. Shin AY 211010

83.6

Pseudoperonospora cubensis

H. D. Shin

HV 222

Cucumis sativus

Austria

H. Voglmayr AY 198306

99.8

Plasmopara viticola

Peronophythora litchii

90113

Litchi chinensis

Taiwan

L. C. Huang AY 251666

100

CBS 100.81

Litchi chinensis

H. Voglmayr AY 198308

100

Phytophthora capsici

H599

Capsicum annuum

New Mexico M. Aragaki AY 208129

99.9

21170

Capsicum annuum

Taiwan

L. C. Huang AY 251662

99.9

KACC 40157

Capsicum annuum

Korea

S. B. Hong AF 228078

92.2

H210

Lycopersicon esculentum

Hawaii

M. Aragaki AY 208130

100

KACC 40177

Lycopersicon esculentum

Korea

S. B. Hong AF 228079

99.1

P 141

Lycopersicon esculentum

K. L. Ivors AY 423292

99.9

pg_0006

Botanical Studies, Vol. 48, 2007

Table 2. (Continued)

Species and isolate

Associatedd habitat

Location

Source

GenBank

accession no. Similarity (%)

Pepper

Italy

I. Camele

AJ 555612

99.6

IMI 352321

Piper nigrum

India

D. E. Cooke AF 266787

99.3

IMI 325900

Theobroma cacao

Brazil

A. A. Appiah AF 167083

97.6

IND 44

Theobroma cacao

India

A. A. Appiah AF 467085

97.7

IMI 304412

Theobroma cacao

Cote dˇIvoire A. A. Appiah AF 467084

97.5

Ph. infestans

KACC 40706

Lycopersicon esculentum

Korea

S. B. Hong AF 228084

100

P6.4CIP-C

G. Z. Abad AF 489701

100

KACC 40707

Solanum tuberosum

Korea

S. B. Hong AF 228083

100

IMI 66006

Solanum tuberosum The Netherlands D. E. Cooke AF 266779

99.5

Ph. nicotianae

KACC 40403

Epiphyllum trucatum

Korea

S. B. Hong AF 228085

99.8

KACC 40407

Lilium lonegiflorum

Korea

S. B. Hong AF 228086

99.9

UQ 848

Australia

D. E. Cooke AF 266776

99.9

IMI 325395

A. R. Crawford L 41383

97.3

331

Nicotiana tabacum

USA

K. L. Ivors AY 423299

100

IMI 354462

Theobroma cacao

Malaysia

A. A. Appiah AF 467087

99.9

Ph. palmivora

21162

Carica papaya

Taiwan

P. J. Ann

AY 208126

99.9

93105

Cattleya spp. (orchid)

Taiwan

L. C. Huang AY 251647

99.7

9257

Cattleya spp. (orchid)

Taiwan

L. C. Huang AY 251648

99.9

KACC 40167

Chrysalidocarpus lutescens ND

S. B. Hong

AF228087

99.7

KACC 40409

Cymbidium sp.

Korea

S. B. Hong AF 228088

98.7

UQ 1294

Papus New Guinea D. E. Cooke AF 266780

99.7

P 80

A. R. Crawford L 41384

99.2

VR 13

Ghana

A. A. Appiah AF 467091

97.8

20230

Persea americana

Taiwan

P. J. Ann

AY 208127

100

94P30

Theobroma cacao

Indonesia

M. Ducamo AJ 517463

99.9

PPaP 33

Theobroma cacao

Taiwan

A. A. Appiah AF 467093

99.7

TRI 1

Theobroma cacao

Trinidad

M. Ducamp AJ 517462

99.7

GHWR 48

Theobroma cacao

Ghana

A. A. Appiah AF 467090

99.9

PNG 14

Theobroma cacao Papus New Guinea M. Ducamp AJ 517464

99.5

4C7

Theobroma cacao

Cuba:Baracao M. Ducamp AJ 517465

99.7

PPaC 55

Theobroma cacao

Taiwan

A. A. Appiah AP 467094

99.4

TP 1

Theobroma cacao

Ghana

A. A. Appiah AP 467092

99.9

PI-10

Theobroma cacao

Costa Rica

K. L. Ivors AY 423300

99.9

IMI 325923

Theobroma cacao

Costa Rica A. A. Appiah AF 467088

99.6

94P43

Theobroma cacao

Indonesia

A. A. Appiah AF 467089

99.6

Ph. sojae

R 25

Glycine max

USA

M. Catal

AY 590277

99.8

R 1

Glycine max

USA

M. Catal

AY 590274

99.8

R 7

Glycine max

USA

M. Catal

AY 590276

99.8

MSU 85

Glycine max

USA

M. Catal

AY 590271

99.8

MSU 88

Glycine max

USA

M. Catal

AY 590273

99.6

MSU 84

Glycine max

USA

M. Catal

AY 590270

99.6

pg_0007

ZHANG et al.  Taxonomic position of

Peronophythora litchi

Species and isolate Associatedd habitat

Location

Source

GenBank

accession no. Similarity (%)

KACC 40412

Glycine max

S. B. Hong AF 228089

99.6

MSU 72

Glycine max

USA

M. Catal

AY 590266

99.6

UQ 1200

Glycine max

Australia

D. E. Cooke AF 266769

99.6

SOY 324

Glycine max

D. E. Cooke AF 403501

99.6

MSU 86

Glycine max

USA

M. Catal

AY 590272

99.6

Glycine max

USA

M. Catal

AY 590275

99.8

ATCC 48068

Glycine max

K. L. Ivors AY 423301

99.8

MSU 76

USA

M. Catal

AY 590268

99.8

MSU 75

USA

M. Catal

AY 590267

99.8

MSU 83

USA

M. Catal

AY 590269

99.8

Q. Chen

AY 256844

99.6

P 2-3

Q. Weng

AY 277278

99.6

Pg 2

Soil

USA

Z. Zhang

AY 242606

99.8

Pg 1

Soil

USA

Z. Zhang

AY 242605

99.6

Pg 3

Soil

USA

Z. Zhang

AY 242607

99.8

Ph. tropicalis

066

Cyclamen persicum

R. Schubert AJ 299733

99.2

H 778-1

Dianthus caryophyllus

R. Schubert AJ 299734

99.2

H 213

Leucosperinum sp.

Hawaii

M. Aragaki AY 207010

99.3

H 352

Theobroma cacao

New Guinea M. Aragaki AY 208125

98.3

Pythium aphanidermatum

100439R

G. W. Moorman AF 452149

100

346952

G. W. Moorman AF 452151

100

340458

G. W. Moorman AF 452148

100

Egypt

Y. H. Gherbawy AJ 628984

100

141749R

G. W. Moorman AF 452146

100

135

France

A. M. Schurko AY 151180

100

P 36-3

K. Kageyama AB 095052

100

UQ 2071

Soil

Australia

D. E. Cooke AF 271227

100

Zen 97-378-U

G. W. Moorman AF 452152

100

TOc 159

G. W. Moorman AJ 233438

100

363669R

G. W. Moorman AF 452150

100

P 12

G. W. Moorman AF 452153

99.6

DC 16

B. G. Lou

AY 278109

99.2

F-1245

India

B. Paul

AY 207380

98.7

Py. splendens

OPU 591

Pachiva aguatica

Japan

M. Tojo

AY 375242

99.5

Py. vexans

80936-95

G. W. Moorman AF 452137

96.8

81708-98

G. W. Moorman AF 452136

96.8

UQ 2074

Soil

Australia

D. E. Cooke AF 271224

94.1

Data were retrieved from GenBank on November 2, 2004.

ND=No data.

Table 2. (Continued)

pg_0008

Botanical Studies, Vol. 48, 2007

dideoxy method of Sanger (Sanger and Coulson, 1975) by

Takara Biotechnology Co., Kyoto, Japan. Three clones for

each isolate were sequenced.

Data analysis

The nucleotide alignments were carried out using

the optimal alignment method of DNAMAN software

(Version 4.0, Lynnon BioSoft, Quebec, Canada).

RESUlTS AND DISCUSSION

The similarity of the overall ITS sequences between

two isolates of the same species sequenced in this study

was all very high, ranging from 98.9 to 100% (data not

shown). Therefore, only one isolate from each species

(Table 1) was used for comparison with isolates of the

same species with ITS sequences available in GenBank in

the homology tests. The similarity of the ITS sequence of

Peronospora parasitica on Brassica oleracea sequenced

in this study with the 31 isolates of the same species on

different hosts in the Brassicaceae retrieved from the

GenBank ranged from 75.4 to 99.6% (Table 2). Those

with more than 95% ITS sequence similarity were on

Brassica campestris, Barbarea orthoceras, and Cardamine

leucaretha while those with less than 77% similarity were

o n Cardamine flexuosa, Cardamine scutata, Rorippa

islandica, and Thlaspi arvense. The ITS sequence

similarity of Phytophthora capsici and Pythium vexans

sequenced in this study with those available in GenBank

was 92.2 to 100% and 94.1 to 96.8%, respectively. The

similarity of ITS sequences of all other species sequenced

in this study with those of the same species retrieved from

GenBank was more than 97% (Table 2).

Voglmayr (2003) suggested transferring Pp. litchii

t o Phytophthora because of its close ITS sequence

relationship with Ph. palmivora and Ph. arecae. The

validity of the suggestion is dependent on the assumption

of a negative correlation between the order of the

taxonomic ranks and the level of ITS sequence similarity.

To test the assumption using the isolates sequenced in

this study, the ITS sequences of Phytophthora sojae and

Pseudoperonospora cubensis were compared with species

of the same or different genus. The similarity levels of

99.3% and 89.5% between Ph. sojae and Plasmopara

viticola and Ph. sojae and Ph. tropicalis, respectively,

(Table 3) are inconsistent with the assumption. The

accuracy of the assumption further diminished when these

data were compared with the 75.4% similarity between

Pe. parasitica o n B. oleracea and the same species on

R. islandica (SMK 18834) (Table 2), thus invalidating

the suggestion to transfer Pp. litchii to Phytophthora

based on the ITS sequence relationship. The result of the

88.1% similarity between Ps. cubenis and Pp. litchii in

comparison with 72.8% between Ps. cubensis and Pe.

parasitica, 83.5% between Ps. cubensis and Pl. viticola,

and 83.8% between Ps. cubensis and Ph. sojae (Table 3)

also invalidated the transfer of Pp. litchii to Phytophthora.

The ITS sequence analysis of data published by Voglmayr

(2003) also failed to support the transfer of taxonomic

ranks in Oomycota based on the sequence relationship of

this DNA fragment. Among the four species pairs of the

same genus compared, three have lower levels of sequence

similarity than the four species pairs of different genera

tested (Table 4).

Riethmuller et al. (2002) suggested that Pp. litchii

should be transferred to Phytophthora because of its close

28S sequence relationship with Ph. arecae. The validity

of the suggestion is also dependent on the assumption

that different isolates of the same species have higher

levels of 28S sequence similarity than different species

of the same genus, which in turn have higher levels of

Table 3. Similarity of ITS sequences of Phytophthora sojae and Pseudoperonospora cubensis to those of other species used in this

study.

Species compared

Similarity (%)

Ph. sojae

Ps. cubensis

Peronospora parasitica

70.4

72.8

Pseudoperonospora cubensis

83.8

100

Plasmopara viticola

99.3

83.5

Peronophythora litchii

86.9

88.1

Phytophthora capsici

88.8

88.4

Ph. infestans

84.6

87.3

Ph. nicotianae

85.0

87.1

Ph. palmivora

86.5

88.0

Ph. sojae

100

83.8

Ph. tropicalis

89.5

88.8

Pythium aphanidermatum

59.5

58.9

Py. splendens

61.8

61.3

Py. vexans

64.9

66.0

pg_0009

ZHANG et al.  Taxonomic position of

Peronophythora litchi

similarity than different species from different genera.

Comparison of 28S sequence similarity between isolates

of the same species, between species of the same genus,

and between species of different genera based on the data

published by Riethmuller et al. (2002) fails to support

this assumption. The 92.1% 28S sequence similarity

level between isolates AR246 and HV656 of Bremia

lactucae was lower than some different species pairs of

Peronorpora, Phytophthora, Pythium, Albugo, and Achlya

(Table 5). This is inconsistent with the assumption. Also

undermining the assumption is the lower similarity levels

between Peronospora avensis and Pe. niessleana (75.0%),

Table 4. The ITS sequences similarity between species of the same genus and different genus based on the data published by

Voglmayr (2003).

Same genus

Different genus

Species compared

Similarity (%)

Species compared

Similarity (%)

1. Pe. sparsa

Pe. alta

82.5

Ph. palmivora

92.1

2. Pe. aestivalis

2. Ps. cubensis

Pe. lepidii-sativi

72.9

Pe. sherardiae

92.4

3. Ph. nicotianae

3. Ps. cubensis

Ph. cactorum

94.6

Ph. nicotianae

88.2

4. Py. irregulare

4. Pe. sparsa

Py. ultimum

72.2

Pp. litchii

91.9

Table 5. The 28S sequence similarity between isolates of the same species, between species of the same genus, and between species

of different genus based on the data published by Riethmuller et al. (2002).

Isolate/species compared

No. of data available

Similarity (%)

Between different isolates of the same species

Bremia lactucae

92.1-99.4

Between different species of the same genus

Peronospora

75.0-99.4

Phytophthora

96.0-100

Pythium

82.6-98.4

Albugo

65.2-94.4

Achlya

92.6

Between species of different genus

1. Saprolegnia ferax

Scoliolegnia asterophora

94.2

2. Calyptralegnia achlyoides

Aplanes androgynus

96.2

3. Pachymetra chaunorhiza

Aphanomyces stellatus

95.4

4. Dictyuchus monosporus

Brevilegnia megasperma

95.7

5. Bremiella baudysii

Plasmopora sii

98.4

6. Peronophythora litchii

Phytophthora multivesticulata

96.9

7. Peronospora potertillae-sterilis

Phytophthora nicotianae

95.8

9. Pythium middletonii

Lagenidium chthamalophilum

87.0

pg_0010

Botanical Studies, Vol. 48, 2007

Pythium undulatum and Py. monospermum (82.6%), and

Albugo portulacae and Al. tragoponis (MG9-4) (65.2%)

in comparison with the similarity levels of all the eight

species pairs of different genera tested (87.0 to 98.4 %)

(Table 5). The inaccuracy of the assumption invalidates

the suggestion to transfer Pp. litchii to Phytophthora based

on the 28S sequence relationship.

Results from this study show that although ITS and

28S sequences are useful in phylogenetic studies, they

are not valid in the determination of the taxonomic

status of Oomycota. Therefore, the taxonomic status

of Peronophythora as a distinct genus transitional

between Peronospora and Phytophthora, and that of

Peronophythoraceae as a distinct family transitional

between Peronosporaceae and Pythiaceae (Ann and Ko,

1980; Chen, 1961; Ho et al., 1984; Huang et al., 1983)

remain valid. The reason for the lack of correlation

between the taxonomic ranks and sequence similarity

of ITS and 28S DNA in Oomycota is not known. It is

possible that these two DNA fragments are not related

to the morphological and physiological traits and the

nucleotide substitutions in these regions are, therefore,

independent of the taxonomical characteristic changes in

this group of organisms. Further study is needed to test

this hypothesis.

Acknowledgements. This work was supported in

part by the National Basic Research in Development

Program (No. 2002 CB111402) and the National

Science Foundation of China (No. 30270055). A grant

from the National Science Council of Taiwan (NSC

95-2811-B-055-001) to W. H. Ko is also acknowledged.

We thank M. Aragaki, P. J. Ann, Q. H. Chen, C. Y. Shen

and G. A. Zentmyer for supplying the cultures used in this

study.

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