Fungal contamination in rainbow trout eggs in Kermanshah province propagations with emphasis on Saprolegniaceae

Abstract

Fungal infection in the eggs of freshwater fish is well known as a problematic disease. The aim of the present study was to isolate and identify parasitic and saprophytic fungi from affected eggs of rainbow trout at two fish hatchery in Kermanshah province. The sample were inoculated in culture media (SDA,CMA,GPA and stilled water with cotton seed culture) at room temperature (18-24°C).17 species of fungi isolated from the fungal eggs. Five fungi species that isolated in this study were belonged to the saprolegniaceae family including

Full text

Iranian Journal of Fisheries Sciences 9(1) 151-160 2010
Fungal contamination in rainbow trout eggs in Kermanshah
province propagations with emphasis on Saprolegniaceae
Shahbazian N.¹; Ebrahimzadeh Mousavi H.A.¹*; Soltani M.¹;
Khosravi
A.R.¹; Mirzargar S.¹ and Sharifpour I.
2
Received: May 2009 Accepted: July 2009
Abstract
Fungal infection in the eggs of freshwater fish is well known as a problematic disease. The
aim of the present study was to isolate and identify parasitic and saprophytic fungi from
affected eggs of rainbow trout at two fish hatchery in Kermanshah province. The sample were
inoculated in culture media (SDA,CMA,GPA and stilled water with cotton seed culture) at
room temperature (18-24°C).17 species of fungi isolated from the fungal eggs. Five fungi
species that isolated in this study were belonged to the saprolegniaceae family including
Saprolegnia paraitica, Saprolegnia lapponica ,Saprolegnia ferax, Saprolegnia hypogyna and
Saprolegnia diclina. Another fungi that isolated in this study were, Fusarium oxyparum,
Fusarium npoa, Fusarium sp., Penicilium citrinium, Penicillium expansum, Aspergillus
treuse, Aspergillus clavatus, Cladosporium sp., Alternaria sp., Helmintosporium sp.,
Pscilomyces sp., Mocur. It seems that Saprolegnia parasitica with 26.8 percent of isolation
was the most important fungal infestation of egg in kermanshah trout hatcheries. Mocur with
19.6% had the most frequency after S. parasitic and Fusarium, Aspergillus and Pescillomyces
with 2.45% had the lowest frequency. In this study S. ferax, S. hypogyna and S. diclina are
reported from Iran for the first time.
Keywords:
Saprolegnia, Rainbow trout egg, Fungal infestation
1- Faculty of Veterinary Medicine, University of Tehran, P.O.Box: 14155-6453 Tehran, Iran
2-Iranian Fisheries Research Organization, P.O.Box: 14155-6116 Tehran, Iran
*Corresponding author's email: hmosavi@ut.ac.ir

152 Shahbazian et al., Study of fungal contamination in rainbow trout eggs in…
Introduction
Over the past ten years, aquaculture has
increased on average by 11% per year.
Production has increased from 13 million
tons of fish in year 1990 to 37.9 million
tons in 2001 and 51.65 million tons in 2006
(West, 2006). The majority of global produc-
tion (58%) comes from freshwater aquaculture
(West, 2006). Aquaculture especially rainbow
trout fish farming has been developed in Iran
and fish culture now is becoming an
economically important industry. Water
mold infections cause losses of freshwater
fish and their eggs in both nature and
commercial fish farms (Bangyeekhun et al.,
2001). Unfertilized fish eggs are susceptible
to fungal infection particularly from the
family Saprolegniaceae. During egg
incubation, these fungi produce mycelia
which grow and spread from the nonviable
to the healthy eggs suffocating them and
causing mortality (Mousavi, 2009). During
recent years decreasing of rainbow trout
eggs in propagation center of Iran have
become common and evidences show that
about half of the produced egg loses due to
fungal infection (Ebrahimzadeh, 2006).
Oomycets contain some of the most
devastating pathogens of animals. Taxono-
mically Oomycetes are divided into three sub-
classes: Saprolegniomycetidae, Rhiomycetidae
and Peronospormycetidae. Most fish and
animal pathogen Oomycetides belong to the
Saprolegniomycetidae which has two order:
Saprolegniale and Leptomitales (West,
2006). Saproleniaceae genera particularly
Saprolegnia and Achlya are generally
opportunitic pathogen (Bruno & Wood,
1994) but some strains can be virulent and able
to cause primary infection in fish and their
eggs (Willoughby & Pickering, 1997).
Identification of Saprolegnia species
classically is based on morphology of the
reproductive structures, i.e. antheridia, oogonia
and oospores (Willoughby, 1978; Neish &
Hughes, 1980). There are some reports about
fungi isolation from fish and fish eggs from
Iran (Ghiassi, 2007; Dakhili, 2009; Kazemi,
2009; Bozorgnia, 2009).
In this survey, for the first time, isolation of
fungi along with emphasis of morphological
characteristics of Saprolegniaceae from eggs of
salmonid hatcheries in Kermanshah province
was studied.
Materials and methods
400 infected rainbow trout eggs were collected
from 2 hatcheries with temperature between
10.4-11.8°C and pH 7.5-7.8 in Kermanshah
province, west of Iran during winter of
2008. Fungus-contaminated eggs were
collected by sterile forceps and transferred
to screw capped bottle contained sterilized
tap water (STW). In laboratory the sample
were washed 3 times with sterile distilled
water and were placed in each egg sterilized
Petri dishes containing three halves of
sterilized cottonseeds and sterilized tap
water, STW (volume 40ml) at then
incubated at 18-24°C for 8 hours under
natural light. From the growth fungi
microscopic slide were taken and examined
under compound microscope. In order to
obtain sexual organs, some hyphae were
aseptically taken out with the help of sterile

Iranian Journal of Fisheries Sciences, 9(1), 2010 153
needles and transferred to GPA (glucose
peptone agar) containing 250µgr/ml
penicillin and 250µgr/ml chloramphenicol
for prevention of bacterial contamination at
18˚C for at least 48-72 hours (Willoughby
et al., 1984). All morphological characteristics
measurements and observation under
microscopic study were done. For isolation
of another saprophytic fungi, Sabourodexteros
agar (SDA) and Corn Meal Agar (CMA)
media were used. Culture media after
inoculation, incubated at 18-24˚C for 24-48
hours and from colonies wet smears were
done with used from lactophenol methylen
blue microscopic study were done. The
identification were followed by methods
described by Coker & Matthews (1937),
Johnson (1956), Seymour (1970), Beakes et
al. (1994) and Khulbe (2001). In table 2
some hatcheries condition during sampling
were shown. Water temperature, salinity and
pH were measured digitally (Multi 340i-
WTW) and eggs number, infested eggs
percent and infested broodstock percent were
obtained manually (observation).
Results
Based on fungal morphological characterists
17 species of fungus were isolated from the
infected eggs including Penicillium citrinum,
Penicillium expansum, fusarium poae,
Fusarium oxysporum, Fusarium sp.
Aspergillus clavatuse, Aspergillus treuse,
Cladosporium sp., Alternaria sp.,
Helmintosporium sp., Pcscilomyces sp.
and
mocur sp. (Table 1). In saprolegniaceae 5
isolated species were S. parasitica, S.
lapponica, S. diclina, S. hypogyna and
Saprolenia ferax. The morphological
characteristics of the isolated Saprolegnia
species are as fallow:
Withish cotton–like colonies were observed
which stout hyphe especially in place of hyphe
adhesion to clavate zoosporangia on GPagar.
After 18 days of culture in STW on the cotton
seeds at room temperature, sexual structures
were formed. Lateral oogonia and spherical
moderately thick, 35-45µm in diameter was
observed. Antheridia did not develop in the
entire culture (Fig. 1). Cotton-like whitish
colony, hyphae slender, aseptate and
cylindrical zoosporangia were formed on
GPA. Sexual structure was formed after 8 days
of culture on GPagar with cottonseeds.
Terminal pyriform oogonia with centric
oospore (45-70µm in diameter). Anthridia
were present diclinous and laterally appresssed
to the oogonial wall (Fig. 2). Whitish cotton-
like colonies on GPagar, abundant cylindrical
zoosporangia, terminal spherical and
cylindrical oogonia (90-110µm in diameter)
were formed on main hyphae. Anthridia
(arrow) was observed (diclinous) (Fig. 3).
Whitish cotton-like colonies on GPA and
CMA (Fig. 4), hyphae moderately stout,
aseptate branched (30-75µm in diameter) were
observed. Zoosporangium were abundant with
different shape (cylindrical, pyriform and the
other) sexual structure were not observed on
cottonseeds culture in STW, gemmae
abundant, variable in shape, spherical and
pyriforme or irregular (Fig. 5). Hyphae
slender, aseptate and branched, zoosporangia
filiform or clavate; oogonia terminal, lateral
spherical or pyriform (50-55µm in diameter)
and oospores centric (Fig. 6).

154 Shahbazian et al., Study of fungal contamination in rainbow trout eggs in…
Figure1: Oogonia of Saprolegnia lapponica (100x) Figure 2: Oogonia of Saprolegnia diclina (100x)
Figure 3: Oogonia of Saprolegnia ferax (100x) Figure 4: Oogonia of Saprolegnia hypogyna (100x)
Figure 5: Pyriform zoosporangium of S. parasitica (40x) Figure 6: Cotton like colony of S. parasitica

Iranian Journal of Fisheries Sciences, 9(1), 2010 155
Table 1: Absolute and relative frequency of isolated fungi from rainbow trout eggs
Fungi Farm
1
Farm
2
frequency Percentage
S. parasitica 7 4 11 26.8
S. lapponica 1 - 1 2.45
S. diclina 1 - 1 2.45
S.
ferax
1
-
1
2.4
5
S.
hypogyna
1
-
1
2.4
5
Fusarium
oxyparum
1
1
2
4.89
Fusar
ium
poa
-
1
1
2.4
5
Fusarium sp. - 1 1 2.45
Penicilium citrinium 2 - 2 4.89
Penicillium expansum - 2 2 4.89
Aspergillus treuse - 1 1 2.45
Aspergillus
clavatus
-
1
1
2.4
5
Cladosporium
3
2
5
12.2
Alternaria
1
-
1
2.4
5
Helmintosporium 1 - 1 2.45
Pscilomyces 1 - 1 2.45
Mocur 5 3 8 19.6
Absolute frequency
2
5
16
41
100
Relative frequency
61
39
100
100
Table 2: Some hatchery conditions during sampling
3
-
5
Broodstock age (year)
49
-
65
Crowding of eggs (Number/cm
2
)
10.4-11.5
Water temperature (°C)
0.4-0.6 Salinity
7.5-7.8
pH
20-42
Eggs infestation (%)
30-45
Broodstock infestation (%)
Spring & river
Water resource

156 Shahbazian et al., Study of fungal contamination in rainbow trout eggs in…
Discussion
In saprolegniacae family 5 isolates were S.
parasitica, S. laponica, S. diclina, S.
hypogyna and S. ferax. In the study in
cultivated cyprinids in Iran, thirty-nine
species of fungi were isolated from the gill
lesions. The most important pathogenic
fungi were Branchiomyces, Saprolegnia,
Fusarium, Phoma and Exophiala. The most
frequency isolated fungi were Fusarium
(46.4%) and Saprolegnia (25%), whereas
Exophiala (3.6%) was shown the lowest
frequency (Firouzbakhsh et al., 2005). In
this study, like previous ones, the highest
rate of infestation was related to
Saprolegnia genus with 36.6% frequency.
Among different isolates from eggs, S.
parasitica was the most important with
26.8% frequency. It has been established
that the greatest losses on fish eggs,
however, are due to saprolegnia species
(Willoghby, 1970; Czeczuga & Kiziewicz,
1999 ; Hussein et al., 2001). Saprolegnia
ferax was reported from different places
under various ecological condition in the
world (Khulbe, 2001). In this study S. ferax,
S. hypogyna and S. diclina were reported
from Iran for the first time. S. frax cause
great losses in acipenserid hatcheries in
Russia (Lartseva, 1986). Kitanchareon
(1997) reported S. ferax and S. hypogyna in
aquatic system and on infected salmonid
fish and fish eggs from Japan. Ghiassi et al.
(2007) reported S. parasitica from Rutillus
frisii kutum in hatcheries in Mazandaran
province, Iran. Kazemi (2009) reported five
species of fungi were isolated from the
Acipenser persicus larva
that were included
of Alternaria spp., Cladosporium spp.,
paecilomyces spp., penicillium spp. and
chrysosporium spp.
In cases that Fusarium mostly were
isolated, lower saprolegnia infestation were
observed. The same result was reported by
Ebrahimzadeh in rainbow trout propagation in
Mazandaran province (Ebrahimzadeh et al.,
2007). In the study by Ebrahimzadeh
(2007) in Mazandaran province, twelve
species of fungi isolated from the fungal
salmonid eggs. Three of the isolated fungi
were belonged to the saprolegniaceas'
family including: Saprolegnia parasitica,
Saprolegnia sp., Achlya sp. Other nine
recognized fungi were: Penicillium,
Aspergillus
,
Paeciliomyces, Acremonium,
Fusarium oxysporum, F. solani, Alternaria,
Mucor and Helminthosporium. In this study
three species of pathogenic fungi in aquatic
were isolated, including:
Saprolegnia parasitica, Saprolegnia sp.,
Achlya sp. It suggested that saprolegnia
parasitica with 13.18% of isolation was the
most important of fungal egg infestation
Mazandaran salmonid hatcheries.
Mueller in a same study showed that in
water culture media Fusarium prevents
growth of Saprolegnia (Pickering et al.,
1994). Totally in this study, 17 fungi
species were isolated respectively with
higher infestation by Saprolegnia. There
were some noticeable differences between
studied hatcheries. Frequency of fungal
infestation in farm 1 and 2 were 61 and
39% respectively that the same ratio (2/1)
was shown macroscopically. It can be

Iranian Journal of Fisheries Sciences, 9(1), 2010 157
concluded that collecting fungal eggs (can
prevent from spread of contamination to
healthy eggs), inhibiting high density of
eggs in incubators, maintaining physical
and chemical quality of water, brood stock
age and health are the main factors to
control fungal diseases in fish farms.
Ecological differences resulting from different
hatcheries conditions (chemical factors, age of
bloodstock, eggs crowded, ect) may have
played a role of the fungi that developed on
rainbow trout eggs in the present study.
Although environmental variables were not
studied directly, they are known to influence
the growth, reproduction and intensity of
aquatic fungal infection (Richards &
Pickering, 1978; Willoughby, 1994). Alabi
(1971) observed that the occurrence of
Saprolegniaceae correlated with some
parameters of water (i.e., temperature, pH,
ionic concentration and organic content).
There is no doubt that ecological differences
play an important role in the species
diversity of the fungi that develop on both
fish and eggs (Willoughby, 1986; Hussein
et al., 2001). There are some report that
showed water quality, crowded hatchery
conditions, pollution and water temperature
changes can causes of Saprolegniasis in fish
and fish eggs (Pickering, 1994; Bruno,
1999; Snisezco, 1974; Beakes et al., 1994).
Higher infestation in farm 2 might be
related to lower age of brood stocks but
there was not any relation with infestation
in eggs. It is note worthy that in farm 2 all
infected eggs were collected daily but in
farm 1 it was done once a week. So by
keeping in mind that Saprolegnia genus
was the most important one it can be
concluded that Saprolegniacea is the
pioneer in affecting rain bow trout eggs.
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Iranian Journal of Fisheries Sciences 9(1) 151-160 2010
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   .                  
             )  (   .  
                
   
:
        
  .          :    
          
   
 .       
 /                   .
 /             
 /     .
                
  .
١ -  قو ،نا اد اد اد :٣ -١١
٢ -  قو نا ،ناا ت ت  :١١ -١١
*ل  وا : hmosavi@ut.ac.ir

Iranian Journal of Fisheries Sciences, 9(1), 2010 161
 ت:
       