Oogonia of Saprolegnia ferax (100x) 

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... 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. 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 needles and transferred to GPA (glucose peptone agar) containing 250 μ gr/ml penicillin and 250 μ gr/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). Based on fungal morphological characterists 17 species of fungus were isolated from the infected eggs including Penicillium citrinum, 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, 35-45 μ m 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 (45-70 μ m 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 (90-110 μ m 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 (30-75 μ m 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 (50-55 μ m in diameter) and oospores centric (Fig. ...