This article was downloaded by: [Florida Atlantic University]

On: 10 November 2014, At: 11:46

Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK

Journal of Aquatic Animal Health

Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/uahh20

Detection and Identification of Fish-Pathogenic

Aphanomyces piscicida Using Polymerase Chain
Reaction (PCR) with Species-Specific Primers
a a a b b
Panarat Phadee , Osamu Kurata , Kishio Hatai , Ikuo Hirono & Takashi Aoki
a
Division of Fish Diseases , Nippon Veterinary and Animal Science University, Musashino ,
Tokyo, 180-8602, Japan
b
Laboratory of Genetics and Biochemistry , Tokyo University of Marine Science and
Technology, Minato-ku , Tokyo, 108-8477, Japan
Published online: 09 Jan 2011.

To cite this article: Panarat Phadee , Osamu Kurata , Kishio Hatai , Ikuo Hirono & Takashi Aoki (2004) Detection and
Identification of Fish-Pathogenic Aphanomyces piscicida Using Polymerase Chain Reaction (PCR) with Species-Specific Primers,
Journal of Aquatic Animal Health, 16:4, 220-230, DOI: 10.1577/H03-047.1

To link to this article: http://dx.doi.org/10.1577/H03-047.1

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the
Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and
should be independently verified with primary sources of information. Taylor and Francis shall not be liable for
any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of
the Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions
 Journal of Aquatic Animal Health 16:220–230, 2004
q Copyright by the American Fisheries Society 2004

Detection and Identification of Fish-Pathogenic

Aphanomyces piscicida Using Polymerase Chain Reaction (PCR)
with Species-Specific Primers
PANARAT PHADEE, OSAMU KURATA, AND KISHIO HATAI*
Division of Fish Diseases, Nippon Veterinary and Animal Science University,
Musashino, Tokyo 180-8602, Japan

IKUO HIRONO AND TAKASHI AOKI

Laboratory of Genetics and Biochemistry,
Tokyo University of Marine Science and Technology,
Minato-ku, Tokyo 108-8477, Japan
Downloaded by [Florida Atlantic University] at 11:46 10 November 2014

Abstract.—Aphanomyces piscicida is an important pathogen that plays a role in the morbidity

and mortality of various fish species around the world. The poor quality of current identification
techniques for this fungus led to the development of highly sensitive and specific polymerase chain
reaction (PCR) techniques. Primers derived from the ITS1 and ITS2 regions of A. piscicida NJM
0204 were designed for the detection and identification of fish-pathogenic A. piscicida, with the
potential for the diagnosis of mycotic granulomatosis (MG). Polymerase chain reaction amplifi-
cation showed that the primer set was specific only to fish-pathogenic A. piscicida, not to non-
fish-pathogenic Aphanomyces, Saprolegnia spp., Achlya spp., Dictyuchus spp., and Lagenidium spp.
In addition, PCR revealed an improved sensitivity sufficient to detect A. piscicida in artificially
infected goldfish Carassius auratus. Results demonstrated that the PCR method established in this
study is effective for the detection and identification of A. piscicida with MG.

Aphanomyces piscicida (also known as A. in-
vadans; David and Kirk 1997) is the causative
agent of mycotic granulomatosis (MG) in warm-
water fish from Japan (Egusa and Masuda 1971;
Miyasaki and Egusa 1972, 1973a, 1973b, 1973c;
Hatai 1980; Kurata et al. 2000). Aphanomyces pis-
cicida is responsible for a number of fungus-as-
sociated diseases, including epizootic ulcerative
syndrome (EUS) in freshwater and brackish-water
fish in the Asia–Pacific region (Miles et al. 2001),
red spot disease (RSD) in Australia (McKenzie and
Hall 1976; Callinan et al. 1989), and ulcerative
mycosis (UM) in the USA (Dykstra et al. 1989).
Infected fish usually present dermal ulcers and an
associated loss of scales, hemorrhage, edema, and
necrotic open ulcers on the body surface. It is usu-
ally found with aseptate hyphae extending to the
skeletal muscle within granulomas (Callinan et al.
1989; Viswanath et al. 1997; Lilley et al. 1998).
In some advanced lesions, fungal hyphae invade
the abdominal viscera, kidney, liver, or spinal cord
in several fish species and this almost certainly the
cause of death (Chinabut 1990; Wada et al. 1994;
Lilley et al. 1998; Viswanath et al. 1998)
In general, the taxonomy of the genus Aphan-
* Corresponding author: hatai@scan-net.ne.jp
Received September 16, 2003;; accepted August 4, 2004
omyces has been based on phenotype and char-
acteristics such as sexual versus asexual repro-
duction (Scott 1961). However, the isolation and
identification of pathogenic Aphanomyces spp. is
sometimes difficult (Willoughby and Roberts
1994) and time consuming (Einsele et al. 1997;
Bretagne et al. 1998; Zhao et al. 2001) because
the pathogen is poorly characterized and identifi-
cation requires a high level of technical expertise.
Pathogenicity to fish has been listed as one of the
distinguishing features of A. piscicida (A. inva-
dans); several variety of other species of Aphan-
omyces are incapable of sustained growth on fish
tissues (Blazer et al. 2002). However, this is not
a standard criterion for identification.
Recently, polymerase chain reaction (PCR)
methods have been used for the detection and iden-
tification of fungal diseases in humans, animals,
and plants (Glass and Donaldson 1995; Einsele et
al. 1997; Bretagne et al. 1998; Weiland 2000; Bak-
an et al. 2002). Fungal fish diseases have also been
investigated, and PCR techniques are used to iden-
tify fish pathogenic A. invadans (A. piscicida; Lil-
ley et al. 2003; Phadee et al. 2004) but have not
reported the effectiveness of PCR for detecting A.
piscicida in diseased fish. Only a limited number
of species-specific primers have been described.
Their specificity and sensitivity must be evaluated
before they can be widely accepted for routine use.

220
 APHANOMYCES PISCICIDA DETECTION USING PCR
In the present study, the primer pairs from ITS
regions were designed for the detection and iden-
tification of A. piscicida. The ribosomal DNA
(rDNA) ITS genes are the widely sequenced DNA
regions used for taxonomic analyses of fungi. It has
typically been used for molecular systematics at the
species level, and even within species (Carbone and
Kohn 1993; LoBuglio et al. 1993; Hopple and Vil-
galys 1994; Rehner and Uecker 1994; Berbee et al.
1995; Liu et al. 1995, 1997; Yen et al. 1995; Shi-
nohara et al. 1999), because of its higher degree of
variation and because ITS-based sequences have
less selective pressure than other genetic regions of
Downloaded by [Florida Atlantic University] at 11:46 10 November 2014
rDNA (Molina et al. 1995; Shinohara et al. 1999).
To supplement available information and to design
species-specific primers for the identification of A.
piscicida, we sequenced the ITS1, 5.8S, and ITS2
genes of A. piscicida NJM 0204 (AY283640). The
sequence showed that the ITS regions of fish-path-
ogenic Aphanomyces (AY283640) genes differed
from those of non-fish-pathogenic Aphanomyces
spp. 84-1240 (AF396683). Therefore, the species-
specific primers were designed based on noncon-
served regions for PCR to develop fast, practical
methods for the detection and identification of
Aphanomyces spp. 84-1240 that cause MG in gold-
fish Carassius auratus.
Methods
Fungal strains and culture.—Forty-two isolates
of Aphanomyces spp., 10 isolates of Achlya spp.,
1 isolate of Dictyuchus sp., 4 isolates of Lageni-
dium spp., and 18 isolates of Saprolegnia spp. were
used in this study (Tables 1, 2). The isolates of
Aphanomyces spp. were divided into 20 fish-path-
ogenic strains and 22 non-fish-pathogenic strains.
All fungi were inoculated onto glucose–yeast ex-
tract agar (GY agar) containing 1% glucose, 0.25%
yeast extract (Difco Laboratory), and 1% agar, and
incubated at 258C.
Laboratory induction of mycotic granulomato-
sis.—Twelve strains of A. piscicida (NJM 9510,
RF 6, NJM 9701, NJM 9801, NJM 9901, NJM
0003, NJM 0006, NJM 0008, NJM 0014, NJM
0015, NJM 0202, and NJM 0204) were injected
into goldfish 9–12 g in body weight and 65–70
mm in body length. Goldfish were divided into an
experimental and a control group, with five fish in
each group.
All Aphanomyces strains were induced for zoo-
spore formation according to Wada et al. (1996).
One hundred microliters of zoospore suspension,
approximately 1 3 104 to 1 3 105 spores/mL, were
inoculated into the left dorsal trunk muscle under
221
the dorsal fin with a 25-gauge sterilized needle and
1 mL syringe. Control fish were inoculated with
100 mL of sterilized water at the same site as in
test fish. The inoculated fish were reared at room
temperature (258C) and were collected for DNA
extraction after the fish showed clinical signs of
infection. The fungal-infected fish samples were
also confirmed by wet mount observation, reiso-
lation, and histopathological examination to en-
sure that the disease was caused by A. piscicida.
DNA preparation.—For DNA extraction of cul-
tured hyphae, fungi were incubated at 258C in GY
broth for 4 d (Aphanomyces spp.), 3 d (Saprolegnia
and Achlya spp.), or until young mycelia reached
0.5–1.0 cm in diameter. Mycelia were washed
twice with phosphate buffered saline (PBS). They
were then placed onto tissue paper for drying, and
20–50 mg of mycelia was each transferred to 1.5
mL microcentrifuge tubes. The samples were fro-
zen at 2858C for DNA extraction. DNA extraction
from all samples was performed using a genomic
prep cell and tissue DNA isolation kit (Amersham
Pharmacia Biotech, Inc.) according to manufac-
turer’s instructions. The DNA was dissolved in
sterilized, double-distilled water and DNA con-
centrations were calculated by the GeneQuant
proRNA/DNA calculator (Amersham Pharmacia
Biotech, Cambridge, England). The DNA solution
was stored at 48C until used.
Experimentally infected fish were dissected, and
infected tissues invaded by A. piscicida were re-
moved for DNA isolation. Tissue from the lesions
was dissected, and 25–50 mg of each slice was
transferred into 1.5 mL microcentrifuge tubes and
frozen at 2858C for DNA extraction. In addition,
control tissues (noninjected fish and control fish
injected with sterilized water) were subjected to
DNA extraction. The DNA isolation methods were
carried out following the manufacturer’s instruc-
tions as described above.
DNA sequencing and primer design.—The se-
quences of ITS1, 5.8S, and ITS2 genes of the A.
piscicida NJM 0204 (AY283640) fragment were
amplified using the primers designed from the
rDNA sequence of Aphanomyces sp. (84–1240)
isolated from menhaden (AF396683). Primer ITS-
F and ITS-R (59-GTCGTAACAAGGTTTCCGTA-
39 and 59-TAGCTTAAGTTCAGCGGGTA-39)
were designed from the end of the 18S and the
beginning of the 28S gene, respectively. Poly-
merase chain reaction mixtures were performed in
a 50 mL reaction volume using TaKaRa Ex Taq.
Polymerase chain reaction products were purified
using GFX PCR DNA and gel purification kit
 222 PHADEE ET AL.

TABLE 1.—Aphanomyces spp. (42 isolates) used in this study.

Species Strains Host Year Location

Fish-pathogenic Aphanomyces
A. invadans 3 PS 45 Striped mullet Mugil cephalus 1998 Australia
A. invadans 4 PS 48 Yellowfin bream Acanthopagrus australis a 1998 Australia
A. piscicida NJM 9201 Dwarf gourami Colisa lalia 1992 Singapore
A. piscicida NJM 9510 Spiny eel Macrognatus siamensis 1995 Thailand
A. invadans RF 6 Striped snakehead Channa striata b 1992 Thailand
A. piscicida NJM 9701 Ayu Plecoglossus altivelis 1997 Japan
A. piscicida NJM 9801 Striped snakehead 1998 Philippines
A. piscicida NJM 9803 Ayu 1998 Japan
A. piscicida NJM 9901 Mrigal Cirrhinus mrigala 1999 Bangladesh
A. piscicida NJM 9903 Puntius Puntius ticto c 1999 Bangladesh
A. piscicida NJM 9907 Golden gourami Trichogaster trichopterus d 1999 Japan
A. piscicida NJM 0002 Ayu 2000 Japan
Downloaded by [Florida Atlantic University] at 11:46 10 November 2014

A. piscicida NJM 0003 Ayu 2000 Japan

A. piscicida NJM 0004 Ayu 2000 Japan
A. piscicida NJM 0006 Atlantic menhaden Brevoortia tyrannus 2000 USA
A. piscicida NJM 0008 Atlantic menhaden 2000 USA
A. piscicida NJM 0014 Striped snakehead 2000 Thailand
A. piscicida NJM 0015 Striped snakehead 2000 Thailand
A. piscicida NJM 0202 Striped snakehead 2002 Thailand
A. piscicida NJM 0204 Striped snakehead 2002 Thailand
Non-fish-pathogenic Aphanomyces
Aphanomyces sp. 84-1240 Atlantic menhaden 1984 USA
Aphanomyces sp. RAR 35-9 Atlantic menhaden 1984 USA
A. astaci FDL 458 European crayfish Astacus astacus 1984 England
A. astaci D3 European crayfish ND Germany
Aphanomyces sp. NJM 9121 Japanese pond turtle Mauremys japonicus 1991 Japan
A. frigidophilus NJM 9500 Japanese char egg Salvelinus leucomaensis 1995 Japan
Aphanomyces sp. NJM 9525 Soft-shell turtle Pelodiscus sinensis 1995 Singapore
Aphanomyces sp. NJM 9619 Soft-shell turtle 1996 Japan
Aphanomyces sp. NJM 9622 Soft-shell turtle 1996 Japan
A. cochlioides MAFF 305548 Sugar beet Beta vulgaris 1984 Japan
A. euteiches MAFF 305549 Pea Pisum sativum 1984 Japan
A. iridis MAFF 305628 White potato Iris japonicus 1986 Japan
A. stellatus IA 0029 Water 1997 Japan
A. stellatus IA 0178 Soil 1997 Japan
A. stellatus IA 1651 Soil 1999 Japan
A. stellatus IA 1927 Soil 2000 Japan
A. laevis IA 0475 Water 1997 Japan
A. laevis IA 1498 Soil 1999 Japan
A. laevis IA 1833 Mud 2000 Japan
A. laevis IA 1876 Soil 2000 Japan
Aphanomyces sp. IA 0252 Water 1997 Japan
Aphanomyces sp. IA 1040 Water 1998 Japan
a Also known as surf bream.
b Also known as chevron snakehead.
c Also known as ticto barb.
d Also known as threespot gourami.

(Amersham Pharmacia Biotech, Inc., New Jersey,
USA) and cloned into the plasmid vector pCR2.1
using the TA cloning kit according to the manu-
facturer’s protocol (Invitrogen, Leek, Nether-
lands). Five clones of A. piscicida were sequenced
using automatic sequencing (400 L; LI-COR) with
protocols supplied by the manufacturer. The com-
puter program Genetyx was employed to analyze
the sequences. The primers for use in this study
were designed from the ITS1 and ITS2 regions of
A. piscicida NJM 0204 following the method de-
scribed by Sambrook and Russell (2001). The
primer ITS11 (59-GCCGAAGTTTCGCAAGAA
AC-39) and ITS23 (59-CGTATAGACACAAGCA
CACCA-39) were designed as forward and reverse
primers, respectively.
Polymerase chain reaction amplification.—Poly-
merase chain reaction amplification of pure cul-
tured fungal DNA was carried out in a TaKaRa
PCR thermal cycler personal (TaKaRa Biochem-
icals) using primers ITS11 and ITS23. Polymerase
chain reaction mixtures were performed in a 25
 APHANOMYCES PISCICIDA DETECTION USING PCR 223

TABLE 2.—Achlya spp. (10 isolates), Dictyuchus spp. (1 isolate), Lagenidium spp. (4 isolates), and Saprolegnia spp.
(18 isolates) used in this study; ND 5 no data.

Species Strain Host Year Location

Achlya
A. diffusa NJM 9228 Water 1992 Japan
A. prolifera NJM 9315 Shortnose gar Lepisosteus platostomus 1993 Japan
A. diffusa NJM 9617 Soft-shell turtle 1996 Japan
A. prolifera NJM 9621 Soft-shell turtle 1996 Japan
A. bisexualis NJM 9905 Guppy Poecilia reticulata 1999 Thailand
A. sp. NJM 0101 Guppy 2001 Japan
A. sp. NJM 0104 Guppy 2001 Thailand
A. sp. NJM 0203 Nile tilapia Oreochromis niloticus 2002 Thailand
A. sp. NJM 0205 Striped snakehead 2002 Thailand
A. prolifera 11398 ND ND ND
Dictyuchus spp. NJM 9682 Striped snakehead 1996 Thailand
Downloaded by [Florida Atlantic University] at 11:46 10 November 2014

Lagenidium
L. thermophilum NJM 9833 Mud crab Scylla serrata 1998 Indonesia
L. thermophilum NJM 0031 Black tiger shrimp larva Penaeus monodon 2000 Thailand
L. thermophilum NJM 0144 Black tiger shrimp larva 2001 Thailand
L. myophylum NJM 8601 Kuruma shrimp P. japonicus 1986 Japan
Saprolegnia
S. diclina NJM 0009 Ayu 1985 Japan
S. parasitica ATCC 90213 Brook trout Salvelinus fontinalis 1986 Japan
S. diclina ATCC 90215 Coho salmon Oncorhynchus kisutch 1986 Japan
S. diclina NJM 9101 Ayu 1991 Japan
S. salmonis NJM 9851 Sockeye salmon O. nerka 1998 Japan
S. australis NJM 9852 Chum salmon egg O. keta 1998 Japan
S. salmonis NJM 9858 Masu salmon O. masou 1998 Japan
S. parasitica NJM 9868 Sockeye salmon 1998 Japan
S. parasitica NJM 9877 Chum salmon 1998 Japan
S. parasitica NJM 9880 Sockeye salmon 1998 Japan
S. diclina NJM 9908 Masu salmon 1999 Japan
S. sp. NJM 0001 Guppy 2000 Thailand
S. sp. NJM 0128 Ayu 2001 Japan
S. parasitica NJM 0129 Ayu 2001 Japan
S. sp. NJM 0201 Striped snakehead 2002 Thailand
S. semihypogyna IA 1424 Water 1998 Japan
S. ferax ATCC 36146 Water ND England
S. hypogyna ATCC 52721 Water ND England

mL reaction volume containing 0.6 U Taq DNA
polymerase (Promega), 1.5 mM MgCl2, 0.2 mM
deoxynucleotide triphosphate, 0.5 mM of each
primer, and 20 ng of DNA template (final amount
5 1 ng) in thermophilic DNA polymerase 10 3
buffer supplied with commercially available Pro-
mega Taq. The resulting mixture was adjusted to
a final volume of 25 mL by the addition of ster-
ilized, double-distilled water. Conditions for PCR
involved preheating for 5 min at 948C, followed
by 25 cycles of amplification of the target se-
quence. Each cycle consisted of a denaturation at
948C for 30 s, annealing at 658C for 30 s, and
primer extension at 728C for 1 min, with 5 min
for a final extension at 728C. Additionally, control
amplifications using primers only were performed
to ensure that reagents used were not contaminated
with extraneous template DNA (negative control).
One nanogram of plasmid clone containing the ITS
genes of A. piscicida, identified by sequence
matching 100%, was included to ensure that the
PCR reaction occurred properly (positive control).
The PCR products were electrophoresed on a 2%
agarose gel containing 0.5 mg/mL of ethidium bro-
mide. Five microliters of PCR product and 1 mL
of loading dye were loaded into each lane. A DNA
marker was also run in parallel to approximate the
size of the PCR products and gels were photo-
graphed on an ultraviolet illuminator.
DNA amplification of fungal-infected fish tis-
sues involved two replicates of each strain of A.
piscicida. All amplification procedures were as
pure cultured fungus, except that for the DNA tem-
plate, the final amount used was 5 ng and 35 cycles
of PCR amplification were carried out. Also, a
positive control was performed with pure cultured
DNA of A. piscicida NJM 0204, and negative con-
trols consisted of reaction components without
 224 PHADEE ET AL.

FIGURE 1.—Agarose gel showing the PCR products of amplified genomic DNA from Aphanomyces spp. using
a specific primer (ITS11 and ITS23). The numbers in the left margin indicate the positions of size markers in base
pairs (bp). The leftmost lane shows a 100-bp ladder; lane C is the negative control, with no DNA template; lanes
1–23 correspond to the detection of the following strains of Aphanomyces spp.: (1) 3 P, (2) 4 P, (3) NJM 9201, (4)
NJM 9510, (5) RF 6, (6) NJM 9701, (7) NJM 9801, (8) NJM 9803, (9) NJM 9901, (10) NJM 9903, (11) NJM
9907, (12) NJM 0002, (13) NJM 0003, (14) NJM 0004, (15) NJM 0006, (16) NJM 0008, (17) NJM 0014, (18)
NJM 0015, (19) NJM 0202, (20) NJM 0204, (21) 84–1240, (22) RAR 35–9, and (23) D3.
Downloaded by [Florida Atlantic University] at 11:46 10 November 2014

template DNA from noninjected goldfish tissue
and DNA from goldfish injected with sterilized
water.
Minimal concentration of fungal DNA.—The
minimal amount of fungal DNA for successful de-
tection by PCR amplification with the primer set
(ITS11 and ITS23) was determined. This was done
using the DNA of A. piscicida from cultural hyphae
and the muscle tissues of goldfish experimentally
infected with A. piscicida NJM 0204. Polymerase
chain reaction amplification was performed as de-
scribed above, with a selected DNA concentration
between 1 fg and 1 ng of cellular DNA and 2.5 fg
to 5 ng of infected tissues.
Results
Sequence Analysis of Aphanomyces piscicida
Sequencing of A. piscicida NJM 0204 resulted
in a 735-base-pair (bp) fragment including a partial
sequence of an 18S rDNA fragment (bp 1–43);
complete sequences of ITS1 (bp 44–201), 5.8S
rDNA (bp 202–361), and ITS2 (bp 362–692); and
a partial sequence of 28S rDNA (bp 693–735). The
GenBank accession number of the sequence is
AY283640. The present sequence was aligned with
non-fish-pathogenic Aphanomyces spp. 84-1240,
accession number AF396683; the alignment score
showed 78.3% homology. It was found that the
sequence in this study corresponded to the ITS1
and ITS2 regions of a non-fish-pathogenic strain,
although their 5.8S sequence slightly differed,
while the 18S and 28S rDNA regions were highly
conserved. Therefore, the primer pair used in this
study was designed from the nonconserved area
of the ITS1 and ITS2 regions, designed as ITS11
and ITS23, respectively.
Specificity of Polymerase Chain Reaction Primers
The DNA from 76 strains of Saprolegniaceae
were used as DNA templates for PCR amplifica-
tion of the primer pairs (ITS11 and ITS23), in-
cluding 20 isolates of fish-pathogenic and 22 iso-
lates of non-fish-pathogenic Aphanomyces, 10 iso-
lates of Achlya, 1 isolate of Dictyuchus, 4 isolates
of Lagenidium, and 18 isolates of Saprolegnia spe-
cies. The PCR procedure using ITS11 and ITS23
detected only 20 isolates of fish-pathogenic
Aphanomyces spp. that exhibited homologous am-
plicons of 550 bp as same as the positive control.
Two strains of Aphanomyces isolated from men-
haden Brevoortia spp. (84-1240 and RAR 35-9)
were not detected (Table 3; Figure 1). However,
there were no PCR products from the negative con-
trol, the non-fish-pathogenic Aphanomyces spp.,
Achlya spp., Dictyuchus sp., Lagenidium spp., and
Saprolegnia spp. (Table 3).
Detection of Aphanomyces piscicida in Goldfish
Muscle
The experimental goldfish began to develop
clinical signs of MG 5–10 d after inoculation with
A. piscicida, although the time to lesion develop-
ment varied among strains. The diseased fish grad-
ually became ulcerated and scale displacement was
observed. The lesions around the injected area
were generally necrotic and there was hemorrhage;
fungal hyphae were seen erupting from lesions at
the site of injection among scales, and dermal ul-
cers were seen in some fish (Figure 2a). External
fungus was not observed, and no fish injected with
sterilized water developed lesions. After the ex-
perimentally infected fish developed severe lesions
(10–20 d postinjection), tissues were collected.
Fungal hyphae from fish muscle and morpholog-
 APHANOMYCES PISCICIDA DETECTION USING PCR 225

TABLE 3.—Detection of the five genera of fungi in Sap- TABLE 3.—Continued.

rolegniaceae using the ITS11 and ITS23 primer set.
Species Isolate Resulta
Species Isolate Resulta
Saprolegnia
Fish-pathogenic Aphanomyces S. diclina NJM 0009 2
A. invadans 3 PS 45 1 S. parasitica NJM 8604 2
A. invadans 4 PS 48 1 S. diclina NJM 0005 2
A. piscicida NJM 9201 1 S. diclina NJM 9101 2
A. piscicida NJM 9510 1 S. salmonis NJM 9851 2
A. invadans RF 6 1 S. australis NJM 9852 2
A. piscicida NJM 9701 1 S. salmonis NJM 9858 2
A. piscicida NJM 9801 1 S. parasitica NJM 9868 2
A. piscicida NJM 9803 1 S. parasitica NJM 9877 2
A. piscicida NJM 9901 1 S. parasitica NJM 9880 2
A. piscicida NJM 9903 1 S. diclina NJM 9908 2
A. piscicida NJM 9907 1 Saprolegnia sp. NJM 0001 2
Downloaded by [Florida Atlantic University] at 11:46 10 November 2014

A. piscicida NJM 0002 1 Saprolegnia sp. NJM 0128 2

A. piscicida NJM 0003 1 S. parasitica NJM 0129 2
A. piscicida NJM 0004 1 Saprolegnia sp. NJM 0201 2
A. piscicida NJM 0006 1 S. semihypogyna IA 1424 2
A. piscicida NJM 0008 1 S. ferax ATCC 36146 2
A. piscicida NJM 0014 1 S. hypogyna ATCC 52721 2
A. piscicida NJM 0015 1 a Plus signs indicate detection of the fungus, minus signs no detec-
A. piscicida NJM 0202 1
tion.
A. piscicida NJM 0204 1
Non-fish-pathogenic Aphanomyces
Aphanomyces sp. 84-1240 2 ical characteristics identified the fungus as A. pis-
Aphanomyces sp. RAR 35-9 2 cicida (Figure 2b). Histological signs were ob-
2
A. astaci FDL 458 served by Grocott and periodic acid2Schiff re-
A. astaci D3 2
Aphanomyces sp. NJM 9121 2 action staining showed granuloma formation sur-
A. frigidophilus NJM 9500 2 rounding fungal hyphae as a feature of MG (Figure
Aphanomyces sp. NJM 9525 2 2c). We also found that fungal hyphae had ex-
Aphanomyces sp. NJM 9619 2
Aphanomyces sp. NJM 9622 2 tended to the opposite side of the injection site and
A. cochlioides MAFF 305548 2 internal organs.
A. euteiches MAFF 305549 2 Polymerase chain reaction analysis of A. pisci-
A. iridis MAFF 305628 2
A. stellatus IA 0029 2
cida–infected goldfish using ITS11 and ITS23 was
A. stellatus IA 0178 2 visualized clearly as a band in the 12 strains of A.
A. stellatus IA 1651 2 piscicida with goldfish DNA (Figure 3). The same
A. stellatus IA 1927 2 size homologous PCR amplicon (550 bp) as found
A. laevis IA 0475 2
A. laevis IA 1498 2 with pure cultured fungus, NJM 0204, were seen
A. laevis IA 1833 2 in reactions from each sample but not with control
A. laevis IA 1876 2 and noninjected fish DNA (Table 4; Figure 3).
Aphanomyces sp. IA 0252 2
Aphanomyces sp. IA 1040 2
Minimal Concentration of Fungal DNA
Achlya
The minimal concentrations of template mate-
A. diffusa NJM 9228 2
A. prolifera NJM 9315 2 rials for PCR detection using the primer set (ITS11
A. diffusa NJM 9617 2 and ITS23) for cultural fungus and fungal infec-
A. prolifera NJM 9621 2 tious fish tissue were 250 fg and 500 fg, respec-
A. bisexualis NJM 9905 2
Achlya sp. NJM 0101 2
tively.
Achlya sp. NJM 0104 2
Achlya sp. NJM 0203 2 Discussion
Achlya sp. NJM 0205 2 Aphanomyces piscicida (David and Kirk 1997)
A. prolifera 11398 2
is the major causative agent of MG in freshwater
Dictyuchus spp. NJM 9682 2
and brackish-water fish in many parts of the world
Lagenidium and has spread across Asia (Roberts et al. 1994),
L. thermophilum NJM 9833 2 the USA (Blazer et al. 1999), Australia (Callinan
L. thermophilum NJM 0031 2
L. thermophilum NJM 0144 2 et al. 1989), and the Middle East (Shaheen et al.
L. myophylum NJM 8601 2 1999). The fungus is difficult to isolate and iden-
tify, even in cases of severe infection. While pre-
 226 PHADEE ET AL.
Downloaded by [Florida Atlantic University] at 11:46 10 November 2014
FIGURE 2.—Experimentally infected goldfish 14 d af-
ter an injection of fungal zoospores. Panel (a) shows a
goldfish with a mycotic granulomatosis lesion; panel (b)
is a wet mount of fungal hyphae in muscle tissue (bar
5 150 mm); and panel (c) shows a granuloma formation
surrounding the A. piscicida hyphae within goldfish mus-
cle (bar 5 60 mm; Grocott stained).
venting infection remains the ultimate goal, iden-
tifying individuals at greatest risk through rapid
diagnosis would provide an alternative since these
are the most likely to benefit from early treatment.
The PCR method would be very useful for con-
trolling, monitoring, or identifying fungal patho-
gens. Polymerase chain reactions developed for
the detection of fungal DNA have been designed
to amplify a single gene from an individual spe-
cies, such as Candida albicans (Crampin and Mat-
thews 1993; Haynes at al. 1995) and Aspergillus
fumigatus (Reddy et al. 1993). Haynes et al. (1995)
demonstrated the first use of a PCR-based detec-
tion and identification system for various genera
of pathogenic fungi, and Fell et al. (1992) de-
scribed a system for the detection of marine fungi,
with the potential for clinical applications. With
plant pathogens, A. cochlioides has also been suc-
cessfully differentiated and identified using PCR
amplifications of the actin coding sequence and
the ITS region of the rRNA gene (Weiland 2000).
In fungal fish disease, Lilley et al. (2003) used a
PCR technique to identify fish pathogenic Aphan-
omyces invadans using FP1 and FP2 primers which
designed from the ITS1 region (201 bp DNA se-
quence). The primer pair produced a product of 98
bp, but there are no reports concerning detection
of the pathogen within diseased fish. These primers
have also been used to detect A. invadans isolated
from Atlantic menhaden B. tyrannus in fixed tissue
(Blazer et al. 2002). Recently, we have used primer
pairs from mRNA of A. piscicida by PCR. The
primers permitted the detection and identification
of only fish-pathogenic Aphanomyces isolates and
are also available for the detection of diseased fish
(Phadee et al. 2004).
The rDNA genes are relatively well known and
their sequences are well characterized. It is known
that the ITS region exhibits more variability than
18S rDNA and differences between related species
(Paul 2000). Molina et al. (1995) suggest that the
highly conserved nature of 18S rDNA in Sapro-
legnia is indicated by the high coefficient of sim-
ilarity within a genus, while the ITS and 5.8S
rDNA exhibited more variability between species.
Leclerc et al. (2000) indicated that LSU and ITS
sequences gave many nucleotide signatures char-
acteristic of different genera and species. In the
present study we have designed primer pairs based
on ITS sequences. The sequences showed that ITS
regions of A. piscicida NJM 0204 differed from
ITS genes of Aphanomyces sp. 84–1240, a non-
fish-pathogenic pathogen. Therefore, we designed
species-specific primers from nonconserved areas
of ITS1 and ITS2.
Our results show that the PCR procedure using
ITS11 and ITS23 produced positive results for
DNA recovered from fish-pathogenic Aphanomy-
ces isolates but not for other strains, including non-
fish-pathogenic Aphanomyces spp., Dictyuchus sp.,
Achya spp., Lagenidium spp., and Saprolegnia
 APHANOMYCES PISCICIDA DETECTION USING PCR 227

FIGURE 3.—Agarose gel showing the PCR products of amplified genomic DNA of A. piscicida2infected goldfish
tissue using a specific primer (ITS11 and ITS23). The numbers in left margin indicate the positions of size markers
in base pairs. The leftmost lane shows a 100-bp ladder; lane C1 is the negative control, with no DNA template;
lane C2 is the positive control, with genomic DNA of A. piscicida NJM 0204; lane 1 is the genomic DNA of
Downloaded by [Florida Atlantic University] at 11:46 10 November 2014

noninjected goldfish; lane 2 is the genomic DNA of goldfish injected with sterilized water; lanes 3–14 correspond
to the following strains of A. piscicida with goldfish tissue DNA: (3) NJM 9510, (4) RF 6, (5) NJM 9701, (6) NJM
9801, (7) NJM 9901, (8) NJM 0003, (9) NJM 0006 (10) NJM 0008, (11) NJM 0014, (12) NJM 0015, (13) NJM
0202, and (14) NJM 0204.

spp., where amplification of the characteristic band cicida (seven isolates) from various countries and
was not seen. We found, however, that these prim- non-fish-pathogenic Aphanomyces strains (13 iso-
ers were unable to detect some fish isolated strains lates) were examined (accession numbers
(84-1240 and RAR 35–9). These strains are sap- AY283641-8 and AY455771-7). It was found that
rophytic Aphanomyces spp. based on the morpho- the ITS1, 5.8S, and ITS2 regions of all A. piscicida
logical, biological, genetic and serological char- strains were identical, and that the non-fish-path-
acteristics of these strains, which were different ogenic Aphanomyces isolates differed from the
from other strains of Aphanomyces isolated from fish-pathogenic Aphanomyces and also from each
infected fish (Dykstra et al. 1989; Willoughby et other (data not shown). These results suggest that
al. 1995; Lilley and Roberts 1997; Lilley et al. A. piscicida can be differentiated from other oom-
1997; Kurata et al. 2002, Lilley et al. 2003). How- ycetes by sequencing the ITS1, 5.8S, and ITS2
ever, these strains did not show pathogenicity to regions. Lilley et al. (2003) have differentiated A.
goldfish, a fish species susceptible to A. piscicida invadans from other fungi and have shown it as a
(Phadee et al., Nippon Veterinary and Animal Sci- single fish-pathogenic species by restriction frag-
ence University, unpublished data). Furthermore, ment length polymorphism analysis of rDNA and
to confirm the results, DNA sequences of A. pis- by sequencing the ITS1 regions.
Polymerase chain reaction was effective in de-
TABLE 4.—Detection of goldfish with experimentally tecting A. piscicida from goldfish tissue DNA and
induced mycotic granulomatosis using the ITS11 and showed homologous amplicons with pure cultured
ITS23 primer set. A. piscicida. In addition, we have been successful
Species Isolate Resulta in using PCR to detect the pathogen in naturally
occurring MG in Ayu (unpublished data). Fur-
Intact fish 2
Control b 2 thermore, the primer set (ITS11 and ITS23) de-
Aphanomyces piscicida NJM 9510 1 tected 250 fg and 500 fg as the minimum concen-
A. invadans RF 6 1 tration of pure culture fungi and fungus with fish
A. piscicida NJM 9701 1
A. piscicida NJM 9801 1 DNA, respectively. In previous studies, 1 pg of
A. piscicida NJM 9901 1 primer set P1 and P2 was sufficient to detect A.
A. piscicida NJM 0003 1 astaci cellular DNA (Oidtmann et al. 2002). Our
A. piscicida NJM 0006 1
A. piscicida NJM 0008 1 results indicate that the primer set in this study is
A. piscicida NJM 0014 1 useful in detecting MG and can differentiate fish-
A. piscicida NJM 0015 1 pathogenic Aphanomyces spp. from other similar
A. piscicida NJM 0202 1
A. piscicida NJM 0204 1 fungi. However, at the onset of disease or low lev-
a
els of infection, this procedure may not be suitable
Plus signs indicate detection of the condition, minus signs no
detection. because PCR analysis depends on the amount of
b Goldfish injected with sterilized distilled water. fungal DNA template. Nested PCR or some other
 228 PHADEE ET AL.
rapid diagnosis method (such as fluorescent anti-
body technique) may be necessary for the detec-
tion and identification of MG in experimentally
and naturally infected fish. Miles et al. (2003) have
successfully used an immunofluorescent antibody
technique (IFAT) to diagnose A. invadans.
This method required several procedures and is
time consuming. We plan to assess the general
suitability of the nested PCR optimized under ex-
perimental conditions by using MG, epizootic ul-
cerative syndrome, red spot disease, or ulcerative
mycosis and field trials for the direct detection of
A. piscicida (A. invadans) zoospores and naturally
Downloaded by [Florida Atlantic University] at 11:46 10 November 2014
infected fish as well as in fish tissues preserved in
a fixative.
Acknowledgments
We would like to thank K. Wakita and P. Sri-
saphoom for their technical help. We would also
like to thank the following for the provision of
fungal isolates: L. G. Willoughby, S. Chinabut, J.
H. Lilley, R. B. Callinan, M. J. Dykstra, M. G.
Bondad-Reantaso, H. Ohkubo, B. Oidtmann, and
S. Inaba.
References
Bakan, B., C. Giraud-Delville, L. Pinson, D. Richard-
Molard, E. Fourier, and Y. Brygoo. 2002. Identi-
fication by PCR of Fusarium culmorum strains pro-
ducing large and small amounts of deoxynivalenol.
Applied and Environmental Microbiology 68:5472–
5479.
Berbee, M. L., A. Yoshimura, and J. Sugiyama. 1995.
Is Penicillium monophyletic? An evaluation of phy-
logeny in the family Trichocomaceae from 18S,
5.8S, and ITS ribosomal DNA sequence data. My-
cologia 87:210–222.
Blazer, V. S., J. H. Lilley, W. B. Schill, Y. Kiryu, C. L.
Densmore, V. Panyawachira, and S. Chinabut. 2002.
Aphanomyces invadans in Atlantic menhaden along
the east coast of the United States. Journal of Aquat-
ic Animal Health 14:1–10.
Blazer, V. S., W. K. Vogelbein, C. L. Densmore, E. B.
May, J. H. Lilley, and D. Z. Zwerner. 1999. Aphan-
omyces as a cause of ulcerative skin lesions of men-
haden from Chesapeake Bay tributaries. Journal of
Aquatic Animal Health 11:340–349.
Bretagne, S., J. M. Costa, E. Bart-Delabesse, N. Dhedin,
C. Rieux, and C. Cordonnier. 1998. Comparison of
serum galactomannan antigen detection and com-
petitive polymerase chain reaction for diagnosing
invasive aspergillosis. Clinical Infectious Diseases
26:1407–1412.
Callinan, R. B., G. C. Fraser, and J. L. Virgona. 1989.
Pathology of red spot disease in sea mullet, Mugil
cephalus L., from eastern Australia. Journal of Fish
Diseases 12:467–479.
Carbone, I., and L. M. Kohn. 1993. Ribosomal DNA
sequence divergence within internal transcribed
spacer 1 of the Sclerotiniaceae. Mycologia 85:415–
427.
Chinabut, S. 1990. The histopathology of EUS in Asia.
Page 75 in M. J. Phillips and H.G. Kiddie, editors.
Regional Research Program on Relationships be-
tween Epizootic Ulcerative Syndrome in Fish and
Environment: a report on the Second Technical
Workshop, 13–26 August 1990. Network of Aqua-
culture Centres in Asia2Pacific, Bangkok, Thai-
land.
Crampin, A. C., and R. C. Matthews. 1993. Application
of polymerase chain reaction to the diagnosis of
candidosis by amplification of an HSP-90 gene frag-
ment. Journal of Medical Microbiology 39:233–
238.
David, J. C., and P. M. Kirk. 1997. Index of fungi IMI.
CAB International, Willingford, UK.
Dykstra, M. J., J. F. Levine, E. J. Noga, J. H. Hawkins,
P. Gerdes, W. J. Hargisjr, H. J. Grier, and D. Testake.
1989. Ulcerative mycosis: a serious menhaden dis-
ease of the southeastern coastal fisheries of the Unit-
ed State. Journal Fish Diseases 12:175–178.
Egusa, S., and N. Masuda. 1971. [A new fungal disease
of Plecoglossus altivelis.] Fish Pathology 6:41–46.
(In Japanese.)
Einsele, H., H. Hebart, G. Roller, J. Löffier, I. Rothen-
höfer, C. A. Müller, R. A. Bowden, J. van Burik,
D. Engelhard, L. Kanz, and U. Schumacher. 1997.
Detection and identification of fungal pathogens in
blood by using molecular probes. Journal of Clinical
Microbiology 35:1353–1360.
Fell, J. W., A. Statzell-Tallman, M. J. Lutz, and C. P.
Kutzman. 1992. Partial rRNA sequence in marine
yeasts: a model for identification of marine eukary-
otes. Molecular Marine Biology and Biotechnology
1:176–186.
Glass, N. L., and G. C. Donaldson. 1995. Development
of primer sets designed for use with the PCR to
amplify conserved genes from filamentous asco-
mycetes. Applied and Environmental Microbiology
61:1323–1330.
Hatai, K. 1980. Studies on pathogenic agents of sap-
rolegniasis in freshwater fishes. Nagasaki Prefec-
tural Institute of Fisheries, Special Report 8, Na-
gasaki, Japan..
Haynes, K. A., T. J. Westerneng, J. W. Fell, and W.
Moens. 1995. Rapid detection and identification of
pathogenic fungi by polymerase chain reaction am-
plification of large subunit ribosomal RNA. Journal
of Medical and Veterinary Mycology 33:319–325.
Hopple, J. S., and R. Vilgalys. 1994. Phylogenetic re-
lationships among coprinoid taxa and allies based
on data from restriction-site mapping of nuclear
rDNA. Mycologia 86:96–107.
Kurata, O., H. Kanai, and K. Hatai. 2000. Hemagglu-
tinating and hemolytic capacities of Aphanomyces
piscicida. Fish Pathology 35:29–30.
Kurata, O., K. Sanpei, K. Hikiji, and K. Hatai. 2002. A
galactose-binding protein revealed as hemaggluti-
nin in Aphanomyces piscicida. Fish Pathology 37:
1–6.
 APHANOMYCES PISCICIDA DETECTION USING PCR
Leclerc, M. C., J. Guillot, and M. Deville. 2000. Tax-
onomic and phylogenetic analysis of Saprolegni-
aceae (Oomycetes) informed from LSU rDNA and
ITS sequence comparisons. Antonie van Leeuwen-
hoek 77:369–377.
Lilley, J. H., R. B. Callinan, S. Chinabut, S. Kanchan-
akhan, I. H. MacRae, and M. J. Phillips. 1998. Epi-
zootic ulcerative syndrome (EUS) technical hand-
book. Aquatic Animal Health Research Institute,
Bangkok, Thailand.
Lilley, J. H., D. Hart, V. Panyawachira, S. Kanchanak-
han, S. Chinabut, K. Söderhäll, and L. Cerenius.
2003. Molecular characterization of the fish-path-
ogenic fungus Aphanomyces invadans. Journal of
Fish Diseases 26:263–275.
Downloaded by [Florida Atlantic University] at 11:46 10 November 2014
Lilley, J. H., and R. J. Roberts. 1997. Pathogenicity and
culture studies comparing the Aphanomyces in-
volved in epizootic ulcerative syndrome (EUS) with
other similar fungi. Journal of Fish Diseases 20:
135–144.
Lilley, J. H., K. D. Thompson, and A. Adams. 1997.
Characterization of Aphanomyces invadans by elec-
trophoretic and Western blot analysis. Diseases of
Aquatic Organisms 30:187–197.
Liu, Y., J. F. Ammirati, and S. O. Rogers. 1997. Phy-
logenetic relationships in Dermocybe and related
Cortinarius taxa based on nuclear ribosomal RNA
internal transcribed spacers. Canadian Journal of
Botany 75:519–532.
Liu, Y., S. O. Rogers, J. F. Ammirati, and K. Keller.
1995. Dermocybe, section Sanguineae: a look at
species relationships within the Sanguineae com-
plex. Sydowia X:142–154.
LoBuglio, K. F., J. I. Pitt, and J. W. Taylor. 1993. Phy-
logenetic analysis of two ribosomal DNA regions
indicates multiple independent losses of a sexual
Talaromyces state among asexual Penicillium spe-
cies in subgenus Biverticillium. Mycologia 85:592–
604.
McKenzie, R. A., and W. T. K. Hall. 1976. Dermal ul-
ceration of mullet (Mugil cephalus). Australian Vet-
erinary Journal 52:230–231.
Miles, D. J., S. Kanchanakhan, J. H. Lilley, K. D.
Thompson, S. Chinabut, and A. Adams. 2001. Ef-
fect of macrophages and serum of fish susceptible
or resistant to epizootic ulcerative syndrome (EUS)
on the EUS pathogen, Aphanomyces invadans. Fish
and Shellfish Immunology 11:569–584.
Miles, D. J., K. D. Thompson, J. H. Lilley, and A. Ad-
ams. 2003. Immunofluorescence of the epizootic
ulcerative syndrome pathogen, Aphanomyces inva-
dens, using a monoclonal antibody. Diseases of
Aquatic Organisms 55:77–84.
Miyasaki, T., and S. Egusa. 1972. [Study on mycotic
granulomatosis in freshwater fish, I. Mycotic gran-
ulomatosis prevailed in goldfish.] Fish Pathology 7:
15–25. (In Japanese.)
Miyasaki, T., and S. Egusa. 1973a. Study on mycotic
granulomatosis in freshwater fish, II. Mycotic gran-
ulomatosis prevailed in ayu. Fish Pathology 7:125–
133.
Miyasaki, T., and S. Egusa. 1973b. Study on mycotic
229
granulomatosis in freshwater fish, III. Mycotic gran-
ulomatosis in bluegill. Fish Pathology 8:41–43.
Miyasaki, T., and S. Egusa. 1973c. Study on mycotic
granulomatosis in freshwater fish, IV. Mycotic gran-
ulomatosis in some wide fishes. Fish Pathology 8:
44–47.
Molina, F. I., S. C. Jong, and G. Ma. 1995. Molecular
characterization and identification of Saprolegnia
by restriction analysis of genes coding for ribosomal
RNA. Antonie van Leeuwenhoek 68:65–74.
Oidtmann, B., S. Bausewein, L. Holzle, R. Hoffmann,
and M. Wittenbrink. 2002. Identification of the
crayfish plague fungus Aphanomyces astaci by poly-
merase chain reaction and restriction enzyme anal-
ysis. Veterinary Microbiology 85:183–194.
Paul, B. 2000. Pythium contiguanum nomen novum (syn.
Pythium dreschleri Paul), its antagonism to Botrylis
cinerea, ITS1 region of its nuclear ribosomal DNA,
and its comparison with related species. FEMS Mi-
crobiology Letters 183:105–110.
Phadee, P., O. Kurata, and K. Hatai. 2004. A PCR meth-
od for detection of Aphanomyces piscicida. Fish Pa-
thology 39:25–31.
Reddy, L. V., A. Kumar, and V. P. Kurup. 1993. Specific
amplification of Aspergillus DNA by polymerase
chain reaction. Molecular Cell Probes 7:121–126.
Rehner, S. A., and F. A. Uecker. 1994. Nuclear ribo-
somal internal transcribed spacer phylogeny and
host diversity in the coelomycete Phomopsis. Ca-
nadian Journal of Botany 74:1666–1674.
Roberts, R. J., G. N. Frerichs, K. Tonguthai, and S.
Chinabut. 1994. Epizootic ulcerative syndrome of
farmed and wild fishes. Pages 207–239 in J. F. Muir
and R. J. Roberts, editors. Recent advances in aqua-
culture V. Blackwell Scientific Publications, Ox-
ford, UK.
Sambrook, J., and D. W. Russell. 2001. Molecular clon-
ing: a laboratory manual, 3rd edition. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New
York.
Scott, W. W. 1961. A monograph of the genus Aphan-
omyces. Virginia Agricultural Experiment Station,
Technical Bulletin 151, Blacksburg, Virginia.
Shaheen, A. A., E. El-Sayed, and M. Faisal. 1999. Iso-
lation of Aphanomyces sp(p). associated with skin
lesions and mortalities in the striped (Mugil ce-
phalus) and the thin lip (Liza ramada) grey mullet.
Bulletin of the European Association of Fish Pa-
thologists 19:79–82.
Shinohara, M. L., K. F. LoBuglio, and S. O. Rogers.
1999. Comparison of ribosomal DNA ITS regions
among geographic isolates of Cenococcum geophil-
um. Current Genetics 35:527–535.
Viswanath, T. S., C. V. Mohan, and K. M. Shankar. 1997.
Clinical and histopathological characterization of
different types of lesions associated with epizootic
ulcerative syndrome (EUS). Journal of Aquaculture
in the Tropics 21:35–42.
Viswanath, T. S., C. V. Mohan, and K. M. Shankar. 1998.
Epizootic ulcerative syndrome (EUS), associated
with a fungal pathogen, in Indians fishes: histopa-
 230 PHADEE ET AL.
thology ‘‘a cause for invasiveness.’’ Aquaculture
165:1–9.
Wada, S., S. Rha, T. Kondoh, H. Suda, K. Hatai, and H.
Ishii. 1996. Histopathological comparison between
ayu and carp artificially infected with Aphanomyces
piscicida. Fish Pathology 31:71–80.
Wada, S., K. Yuasa, S. Rha, K. Nakamura, and K. Hatai.
1994. Histopathological of Aphanomyces infection
in dwarf gourami (Colisa lalia). Fish Pathology 29:
229–237.
Weiland, J. J. 2000. Differentiation and detection of
sugar beet fungal pathogens using PCR amplifica-
tion of actin coding sequences and the ITS region
of the rRNA gene. Plant Diseases 84:475–482.
Willoughby, L. G., and R. J. Roberts. 1994. Improved
Downloaded by [Florida Atlantic University] at 11:46 10 November 2014
methodology for isolation of the Aphanomyces fun-
gal pathogen of epizootic ulcerative syndrome
(EUS) in Asian fish. Journal of Fish Diseases 17:
541–543.
Willoughby, L. G., R. J. Roberts, and S. Chinabut. 1995.
Aphanomyces invadaris sp. nov., the fungal patho-
gen of freshwater tropical fishes affected by epi-
zootic ulcerative syndrome (EUS). Journal Fish Dis-
eases 18:273–275.
Yen, Z. H., S. O. Rogers, and C. J. K. Wang. 1995.
Assessment of Phialophora species based on ribo-
somal DNA internal transcribed spacers and mor-
phology. Mycologia 87:72–83.
Zhao, J., F. Kong, R. Li, X. Wang, Z. Wan, and D. Wan.
2001. Identification of Aspergillus fumigatus and
related species by nested PCR targeting ribosomal
RNA internal transcribed space regions. Journal of
Clinical Microbiology 39:2261–2266.