Characterization of Saprolegnia isolates from Persian sturgeon (Acipencer persicus) eggs based on physiological and molecular data

Journal de Mycologie Médicale (2010) 20, 1—7

ORIGINAL ARTICLE/ARTICLE ORIGINAL

Characterization of Saprolegnia isolates from Persian sturgeon (Acipencer persicus) eggs based on physiological and molecular data ´ risation des souches de Saprolegnia isole ´ es d’œufs de l’esturgeon Caracte ´ es physiologiques et persan (Acipencer persicus) sur la base de donne ´ culaires mole M. Ghiasi a, A.R. Khosravi b,*, M. Soltani b, M. Binaii a, H. Shokri b, Z. Tootian c, M. Rostamibashman d, H. Ebrahimzademousavi d a

Aquatic Animal Health and Disease Department, Ecology Research Center of Caspian Sea, Khazar, Abad, Sari, Iran Mycology Research Center, Faculty of Veterinary Medicine, University of Tehran, P.O. Box 14155-6453, Tehran, Iran c Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran d Department of Aquatic Health and Disease, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran b

Received 3 May 2009; accepted 1 November 2009 Available online 28 December 2009

KEYWORDS Sturgeon; Acipencer persicus; ¨uf; Saprolegnia; RAPD-PCR; Zoospore

Summary Objective. — To investigate the physiological characteristics and genetic variations of Saprolegnia species isolated from Persian sturgeon (Acipencer persicus) eggs. Materials and methods. — One hundred and fifty fish eggs covered with fungal hyphae were collected from propagation center of sturgeon and teleost fish Shahid Rajaii in Mazandaran province between March and May 2007. The samples were cultured into the specific media and the colony hyphal radial growth rates were determined at 18 8C and 12 8C. Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) was applied for genetic variations of the isolates. Results. — All samples were positive for Saprolegnia species. Most fungal isolates grew well at 18 8C but no isolates grew at 12 8C. Repeated zoospore emergence was found in isolates of Saprolegnia species, which were genetically linked to reference S. parasitica. This phenomenon was not seen in isolates that genetically liked to reference S. diclina. The RAPD analysis among Saprolegnia isolates was counteracted from 385 amplified products in 21 separable positions and indicated that the fungal isolates were composed of four genetically distant groups.

* Corresponding author. E-mail address: [email protected] (A.R. Khosravi). 1156-5233/$ — see front matter # 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.mycmed.2009.11.005

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M. Ghiasi et al. Conclusion. — We found different physiological characteristics and genetic variations of Saprolegnia isolates from Acipencer persicus eggs. Although repeated zoospore emergence was clearly not specific for pathogenic Saprolegnia species, it was present in certain groups of isolates defined by genotype and can be useful in distinguishing subgroups of pathogenic Saprolegnia species. # 2009 Elsevier Masson SAS. All rights reserved.

MOTS CLÉS Esturgeon ; Apicenter persicus ; ¨uf ; Saprolegnia ; RAPD-PCR ; Zoospores

Re ´sume ´ Objectif. — Évaluer les caractéristiques physiologiques et les variations génétiques des espèces de Saprolegnia isolées d’œufs d’esturgeon (Apicenter persicus). Mate´riel et me´thodes. — Entre les mois de mars et de mai 2007, les œufs de 150 poissons présentant des amas d’hyphes ont été récoltés au centre d’élevage de l’esturgeon et des téléostéens de Shahid Rajaii situé dans la province de Mazandaran. Les échantillons sont ensemencés sur des milieux spécifiques et après incubation à 12 8C et 18 8C, le taux de mycélium est évalué sur la base de la croissance radiale. L’ADN génomique des Saprolegnia est ensuite extrait et amplifié par PCR (RAPD-PCR) pour la détermination des variations génétiques des isolats. Re ´sultats. — Tous les échantillons présentent des Saprolegnia en culture. La majorité de ces isolats montrent une croissance à 18 8C, en revanche, elle n’est pas observée à 12 8C pour certains d’entre eux. L’apparition répétée de zoospores est présente au sein des espèces de Saprolegnia qui sont génétiquement liées à l’espèce S. parasitica. Ce phénomène n’est pas observé chez les isolats génétiquement liés à S. diclina. L’analyse RAPD à partir des isolats de Saprolegnia a mis en évidence 385 produits amplifiés correspondants à 21 positions distantes, ce qui indique que le genre Saprolegnia est constitué de quatre groupes génétiquement bien différenciés. Conclusion. — Des différences sont observées au niveau des caractères physiologiques et des variations génétiques des espèces de Saprolegnia isolées d’œufs d’Apicenter persicus. Bien que l’apparition répétée de zoospores n’est pas clairement spécifique de l’espèce pathogène de Saprolegnia, ce caractère est présent dans certains groupes d’isolats définis par le génotype et peut se révéler utile dans les sous-groupes distinctifs d’espèces de Saprolegnia pathogènes. # 2009 Elsevier Masson SAS. Tous droits réservés.

Introduction Fungal infections of fish by oomycetes, commonly known as water molds, are widespread in fresh water and represent the most important fungal group affecting wild and cultured fish. The Saprolegniacea, in particular members of the genus Saprolegnia, are responsible for significant infections involving both living and dead fish and eggs especially in aquaculture facilities [23,24]. Infections due to Saprolegnia species were reported from many commercial fish hatcheries especially in salmonid species [8,15,16,23] and channel cat fish (Ichtalurus punctatus) [4,12]. Acipencerid fish species, which live mainly in the basins of the Black Sea and the Caspian Sea, provide valuable meat and eggs (known as caviar). Sturgeon is a migratory two environmental fish, which only occasionally live in one environmental river or lake [6]. The studies on occurrence of zoosporic fungi in acipenserid go back to the end of the 19th century. In the years that followed, this subject was not investigated. Only when the intensive breeding of acipenserid fishes started in artificial condition, where a great loss of eggs due to aquatic fungi infection occurred, the attention to saprolegniasis of acipencerid increased [18,19]. A number of reports have been issued on the occurrence of zoosporic fungi on incubated eggs in the basin of the Caspian Sea, referring mainly to such sturgeons as great sturgeon (Huso huso), Russian sturgeon (Acipenser guldenstaedti) and stellate sturgeon (Acipencer stellatus) [11,18,19].

In the past, Saprolegnia species just were identified based on morphology of reproductive structure, i.e. antheridia, oogonia and oospore [22]. However, many pathogenic Saprolegnia isolates usually do not develop any sexual stage when cultured in vitro and therefore cannot be identified [13]. Characterization of fish pathogenic Saprolegnia is useful for the furtherance of epidemiological studies of the source of infection, disease transmission, disease spreading and control of the disease. Some morphological and physiological studies have made it possible to identify different species and subgroups of Saprolegnia isolates. Variation in esterase isoenzyme pattern [3], difference in radial growth rate [14,28] and different biochemical characteristics [17] have been used for determination of distinct species and groups of Saprolegnia isolates. Today, molecular methods, specially polymerase chain reaction coupled with restriction fragment length polymorphism analysis (PCR—RFLP) is one of the most important methods to distinguish different Saprolegnia species and it could identify S. parasitica from S. diclina [20]. Also, RAPDPCR has been applied for analysis of the fish pathogenic Saprolegnia genome [1,2,8,26,27]. Persian sturgeon (Acipencer persicus Brorodin 1897) is the most important native sturgeon fish in the southern Caspian Sea. As a result of eutrophication and pollution of river water and river dams, the possibility of immigration and natural reproduction of this species is decreasing rapidly from year to year. To maintain their population, every year the brood

Characterization of Persian sturgeon (Acipencer persicus) eggs fish are captured from Caspian Sea basins and they are propagated in artificial conditions. Then, fertile eggs are incubated in hatcheries to changed fry and then to fingerling. The cultivated fingerlings are released to a supported location. In Iran, exact studies unfortunately have not been conducted on Saprolegnia species in Persian sturgeon eggs, and our investigation is the first study on this subject. In the present study, the RAPD—PCR technique and some ecophysiological parameters, such as presence or absence of repeated zoospore emergence and mycelial growth rates in specific temperature, were used to characterize genetic and physiological variations within Saprolegnia species isolated from Persian sturgeon eggs.

Materials and methods Isolation of Saprolegnia strains In this study, 150 eggs covered with fungal hyphae were collected from the hatching trays during three sampling phases (in each phase 50 eggs) from propagation center of sturgeon and teleost fish Shahid Rajaii in Mazandaran province during March to May 2007. On sampling time, water temperature was 17—19 8C. The samples were washed several times in sterile distilled water, and egg membranes with attached fungal hyphae were separated and washed again in sterile distilled water three times. Then, the egg membranes were placed in glass Petri dishes (5—10 eggs per plate) containing 25—40 ml of sterilized distilled water with Chloramphenicol and Gentamycin at a concentration of 100 mg/ml to prevent of bacterial contamination and maintained in hatcheries at 18 8C for one day. Then, the egg membranes directly were inoculated in peptone — glucose agar plates and incubated in the same temperature for 3—5 days. The samples G1, G2, G3, G4, G6 and G7 in the first phase, G8, G9, G11, G12, G13, G14, G15 and G16 in the second phase and G17, G18, G19, G20, G21, G22 and G23 in the third phase were sampled. All chemicals used, unless otherwise stated, were obtained from Merck Company (Darmstadt, Germany).

Hyphal radial growth rate in specific temperature The inoculum was prepared by cutting at the advancing edges of a young colony grown on PG-1 agar with a 6 mm diameter border. The different Saprolegnia isolates were inoculated on a fresh PG-1 agar dish. The colony hyphal radial growth rates were determined on the method used by Czeczuga et al. [7] at 18 8C and 12 8C.

Repeated zoospore emergence Repeated zoospore emergence was performed according to the method described by Diéguez-Uribendo et al. [10]. Briefly, the mycelia were grown in PG-1 drop cultures for three days at 20 8C. To trigger sporulation, the mycelia were washed three times with sterile well water and then incubated in Petri dishes containing well water for 14 h at 20 8C to allow the release of zoospores. The swimming zoospores were transferred to the test tubes, which were then agitated in a vortex mixer for 45 s to obtain synchronous encystment. The release of secondary

3 zoospores was observed under the microscope after incubating the cyst suspension at 20 8C for 150 min.

Standard Saprolegnia strains Saprolegnia parasitica (ACTT = 200048) and S. diclina (ATCC = 4206) were kept in the mycological collection of the Mycology Research Center of University of Tehran and selected for this study. All isolates were maintained on PG -1 agar medium.

DNA isolation A small piece of mycelium (approximately 1.5 mg dry weight) grown in PG-1 drop culture was inoculated in a 100 ml flask containing 25 ml PG-1 medium and cultured by shaking at 18 8C. After three days, the mycelia were harvested, washed with sterile distilled water and ground in liquid nitrogen with a mortar. The total genomic DNA was extracted as described by Diéguez-Uribeond et al. [10]. The DNA extracts were stored at 20 8C.

RAPD-PCR The PCR was carried out in 25 ml volumes containing 100 ng mycelia DNA (approximately 1.2 ml), 12.5 ml master mix (10 mM Tris-HCl (pH 8.60), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.2 mM of each dNTP and 2.5 units of Taq DNA polymerase) (Sinagen Company), 0.4 mM prime (approximately 1.3 ml) (Sinagen Company) and 10 ml distilled water. Amplification was performed in a Biotech Termalcycler system programmed for one cycle of initial denaturation at 95 8C for 5 min, 40 cycle of denaturation 94 ?C, annealing at 38 8C for 45 s and extension at 72 8C for 1.5 min and final extension at 72 8C for 10 min. Ten ml of PCR product were separated on 1.5% agarose gel and 100 bp ladder DNA (Fermentans) was used as the molecular weight marker. Gels were stained with ethidium bromide, visualized UV illumination and photographed with a Polaroid camera. The following primers were used: A10 (50 - GTG — ATC — GCA — G -30 ); OPU13 (50 - GGC —TGG — TTC — C -30 ); P10 (50 - GAC — AGA — CGC — G -30 ); OPT20 (50 - GAC — CAA — TGC — C -30 ) and FM1 (50 - AGC —CGC — CTC — CAT — GGC — CCC — AGG -30 ).

Data analysis The RAPD— PCR was performed at least twice. The presence or absence of a marker was scored as 1 or 0, respectively. Similarity coefficient between two isolates were calculated according to the formula of Nei and Li [21] as F = 2Nxy/Nx + Ny, where Nxy was the number of common fragments between two isolates and Nx and Ny were the number of fragments in isolates X and Y, respectively. The genetic distance was evaluated through euclidean distance. The dendrogram was constructed based on the unweighted pair-group method using arithmetic averages (UPGMA) [25]. Pooled data from five primers were used for this calculation.

Statistical analysis Statistical analysis was performed using the chi-square (X2) test. A P value less than 0.05 was considered significant.

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M. Ghiasi et al.

Results Hyphal redial growth The hyphal redial growth rates of all Saprolegnia isolates were investigated at different temperatures. At 18 8C, all of the isolates grew and no significant differences between them. No isolate grew at 12 8C (Fig. 1).

Repeated zoospore emergence Repeated zoospore emergence is the capacity of an encysted spore to release a new zoospore generation ingested of germinating. The G3, G4, G5, G6, G8, G11, G17, G18 and G21 isolates produced about 90 to 95% secondary zoospores of the primary cysts, and these isolates developed 3 generations of zoospores. These isolates had 92.4% similarity average to G5 (Group 1) according to RAPD data analysis. However, the G7, G9, G12, G13, G14 and G15 isolates underwent germinations instead. In those isolates, we found that the germinations of encysted spores formed short hyphae, about three to five diameters in length, and then terminated their growth. These isolates had 93.3% similarity average to G10 (Group 3) according to RAPD data analysis. In other isolates such as G1, G2, G16, G19, G20, G22 and G23, we found a combination of germinated cysts and second generation zoospore together in about equal proportions after they were encysted mechanically.

Amplification of Saprolegnia isolates genomic DNA Five primers gave consistent results and produced a reasonable number of identification and polymorphic pattern of the PCR products from independent genomic DNA preparation of each isolate. The number of amplified DNA fragments generated by each RAPD primer ranged from three (OPT20 and OPU13) to five (A10 and P10). Pooled data from five primers gave a total of 385 clearly amplified PCR bands in 21 different positions; the average number of band per isolate was 16.7. The size of the fragments ranged from 328 to 2981 bp, and 47.6% bands were larger than 1 kb. An example of an RAPD pattern generated by Primer P10 is shown in Fig. 2.

Figure 2 Amplification of genomic DNA from different Saprolegnia species isolated using primer P10. From the center (A) and right (B), 100 bp ladder size marker (M) and from number 1 to 23 were samples (A) G1, G2, G3, G4 and G5 (Saprolegnia parasitica ACTT = 200048), G6, G7, G8, G9 and G10 (Saprolegnia diclina ATCC = 4206), G11, G12, G13, G14 and G15, (B) G16, G17, G18, G19, G20, G21, G22 and G23. ´ nomique de diffe ´ rentes espe ` ces de Amplification de l’ADN ge Saprolegnia `a l’aide du primer P10.

The RAPD profiles obtained by using the P10 primer on 20 Saprolegnia isolates gave three distinguishable PCR markers (Fig. 2A, B). Of these, two fragments, i.e.1889 and 1233 were consistently present in all isolates whereas a 518 bp product was present in all isolates except the G10 (Saprolegnia diclina ATCC = 4206), G1, G2, G9, G13, G14, G15, G16, G19 and G20. It is possible that the 518 bp product could be useful for the development of diagnostic tools within the S. parasitica — diclina complex.

Genetic variation among Saprolegnia isolates based on analysis of RAPD pattern

Figure 1 Schematic diagram of based on colony diameter the end of 6th day of incubation. ´ sentant le diame ` tre des colonies `a la fin du Diagramme repre ` me jour de culture. sixie

All PCR fragments obtained were used for genetic distance analysis. The dendrogram and similarity matrix constructed from the pair wise similarity among all Saprolegnia isolates demonstrated that the tested isolates of Saprolegnia were grouped into four distinct groups (Fig. 3 and Table 1). All the samples were divided based on genetically characteristics within each group, members shared more than 86% similarity

Characterization of Persian sturgeon (Acipencer persicus) eggs

Figure 3 Saprolegnia species; dendrogram of 23 samples based on RAPD data set. Dendrogramme de 23 isolats de Saprolegnia selon les profils en RAPD.

of the RAPD band: i.e. the average similarity of Group 1 was 92.4%. The average similarities were shown in Table 1. Furthermore, within the groups, a cluster of 100% identity was found: i.e. cluster 1, G6 and G11. Groups 1, the largest group, contains 8 of 20 isolates, which were sampled from all sampling phase. The next large group is 3, contains 6 of 20 isolates, most of which were sampled from 2 and 3 phase sampling. Groups 2 and 4 were minor groups containing 3 or 4 Saprolegnia isolates per group. Group 2 was the smallest group with 93.3% average similarity. The members of this group were obtained from 2 and 3 phase sampling. Group 4 contained 4 isolates, 2 isolates in the first phase and 2 more in the third phase of sampling. The average similarity was from 82.7 to 79.9% among the groups (Table 2).

Discussion The RAPD profiles obtained in this study revealed genetic variations among fungal isolates from Persian sturgeon eggs and separated the isolates into four different groups. The largest group was group 1. One of the reference strains (G5 = S. parasitica; ACTT = 200048) fell in this group and its members had more than 90% similarity among themselves. The next large group was 3. One of the reference strains (G10 = S. diclina ATCC = 4206) fell in this group and its members had more than 90% similarity among themselves. No isolate in this study reproduced sexually in the laboratory, and thus could not be distinguished to species level. Therefore, it is possible that four distinct RAPD groups did not belong to the same species. We propose according to Bangyeekun et al. [1,2] that the closely related isolates of group 1 were plausibly S. parasitica and the closely related isolates of group 3 may be S. diclina. Czeczuga et al. [6] isolated S. declina, S. ferax, S. mixata, S. monica, S. parasitica, S. australis, S. shikotsuensis and S. unispora from five acipenserid fish eggs (Acipenser

5 guldenstadti, A. persicus, A. rudiventris, A. ruthenus, A. stellatus and Huso huso). S. parasitica is one of the most common and destructive pathogenic oomycetes to all fresh water and economical fish hatcheries [1,27]. It was also found in acipenserid fishes [18,19]. The study on saprolegniasis in acipencer fish hatcheries revelead that S. parasitica is dominant among Saprolegnia species [6]. This is in agreement with our study, because the most isolates have been in group 1 and obtained from all sampling time. S. diclina was like to S. parasitica reported from Great sturgeon (Huso huso) and Persian sturgeon hatcheries [6]. Identification of S. diclina from S. parasitica is very difficult because many pathogenic Saprolegnia isolates (especially S. parasitica) do not develop any sexual stages when cultured in vitro. In addition, surprisingly, they could be derived from a single sample. Today, molecular methods are the best for recognizing of Saprolegnia species [1,9]. To our knowledge, this study was conducted using molecular method to identify Saprolegnia isolates from Persian sturgeon eggs for the first time in Iran to our knowledge. Physiological properties of Saprolegnia were used for the classification of different subgroups within a species. Studies on esterase isoenzyme pattern [3], the relationships between growth rate, temperature [14,28] and the biochemical test including carbohydrate and amino acid assimilation [17] identified different groups. In this study, there was no difference between the hyphal radial growth rates of Saprolegnia isolates at 18 8C. In addition, no isolate grew at 12 8C. These results indicated that growth characteristics in the present study cannot be detected by RAPD—PCR. This is perhaps not too surprising considering that growth rate may be caused by a single or a few genes and thus not likely to be detected by the RAPD—PCR analysis. The same situation already has been reported [2]. The G3, G4, G5, G6, G8, G11, G17, G18 and G21 isolates (group 1) produced about 90 to 95% secondary zoospores of the primary cysts, and these isolates developed three generations of zoospores. However, the G7, G9, G12, G13, G14, and G15 isolates (group 3) underwent germinations instead. In other isolates such as G1, G2, G16, G19, G20, G22 and G23 (groups 2 and 4), we found a combination of germinated cysts and second generation zoospore together in about equal proportions after they were encysted mechanically. Cerenius and Söderhäll [5] demonstrated that repeated zoospore emergence is a specific mechanism for pathogenic Aphanomyces species and proposed this mechanism to be an adaptation to a parasitic mode of life. The trout isolates of S. parasitica as reported by Diéguez—Uribendo et al. [8] did not exhibit repeated zoospore emergence. Thus, repeated zoospore emergence is not a specific mechanism for pathogenic Saprolegnia isolates since some fish-pathogenic isolates exhibit germination after encystment. Bangyeekhun et al. [2] reported that S. parasitica isolated from crayfish and catfish isolates exhibited repeated zoospore emergence, whereas the S. diclina isolated did not. He proposed that the presence of repeated zoospore emergence in Saprolegnia isolates may be useful for colonizing fish skin. Then, he reported a relationship between zoospore behavior and host-specificity. He mentioned that the zoospore of salmonid isolates exhibited germination, whereas the zoospore of non-salmonid (catfish and crayfish) isolates performed zoospore emergence. Diéguez—Uribendo et al. [9]

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Table 1 Similarity matrix of Saprolegnia isolates based on Random amplification of polymorphic DNA (RAPD) profiles. Matrices de similitude des isolats de Saprolegnia selon les profils en random amplification of polymorphic DNA (RAPD). Isolates G1

G2

G3

G4

G5

G6

G7

G8

G9

G10

G11

G12

G13

G14

G15

G16

G17

G18

G19

G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15 G16 G17 G18 G19 G20 G21 G22 G23

0.788 0.75 0.848 0.857 0.895 0.824 0.882 0.944 0.857 0.914 0.889 0.882 0.857 0.813 0.824 0.889 0.903 0.875 0.813 0.75 0.71

0.903 0.938 0.941 0.865 0.909 0.788 0.8 0.941 0.824 0.857 0.848 0.824 0.839 0.909 0.914 0.8 0.839 0.903 0.839 0.8

0.903 0.848 0.833 0.938 0.688 0.765 0.848 0.727 0.824 0.75 0.848 0.8 0.875 0.882 0.828 0.8 0.867 0.867 0.828

0.882 0.865 0.909 0.788 0.857 0.882 0.824 0.857 0.848 0.824 0.839 0.848 0.914 0.867 0.774 0.903 0.839 0.8

0.923 0.914 0.857 0.865 1 0.889 0.919 0.914 0.889 0.848 0.914 0.973 0.813 0.909 0.909 0.848 0.813

0.895 0.895 0.95 0.923 0.923 0.95 0.895 0.923 0.833 0.895 0.95 0.8 0.833 0.833 0.833 0.8

0.765 0.833 0.914 0.8 0.889 0.824 0.914 0.875 0.941 0.944 0.839 0.875 0.938 0.875 0.839

0.944 0.857 0.971 0.889 0.941 0.857 0.875 0.824 0.833 0.774 0.813 0.813 0.813 0.839

0.865 0.973 0.947 0.944 0.919 0.882 0.833 0.895 0.848 0.824 0.824 0.824 0.788

0.889 0.919 0.914 0.889 0.848 0.914 0.973 0.813 0.909 0.909 0.848 0.813

0.919 0.971 0.889 0.909 0.857 0.865 0.813 0.848 0.848 0.848 0.813

0.944 0.973 0.882 0.833 0.947 0.848 0.882 0.824 0.824 0.788

0.914 0.938 0.824 0.889 0.839 0.875 0.875 0.875 0.839

0.909 0.857 0.919 0.813 0.909 0.848 0.848 0.813

0.875 0.824 0.828 0.867 0.933 0.933 0.897

0.889 0.774 0.875 0.938 0.875 0.839

0.848 0.882 0.882 0.824 0.788

0.828 0.828 0.867 0.828 0.8 0.933 0.786 0.759 0.897 0.966

0.875 0.839 0.733 0.774 0.848 0.833 0.813 0.875 0.824 0.848 0.848 0.824 0.813 0.788 0.8 0.875 0.824 0.828 0.867 0.8 0.733 0.759

G20

G21

G22

M. Ghiasi et al.

Characterization of Persian sturgeon (Acipencer persicus) eggs Table 2 Grouping of Saprolegnia species according to the average similarity of isolates based on Random amplification of polymorphic DNA (RAPD) profiles. ` ces de Saprolegnia selon les pourcentages de Groupes d’espe similarite´ des isolats selon les profils en random amplification of polymorphic DNA (RAPD). Group number

Members

Similarity average

Group 1

G6, G11, G18, G3, G5, G17, G21, G8, G4 G22, G23, G16 G12, G14, G10, G9, G13, G15, G7 G2, G19, G1, G20

92.4

Group 2 Group 3 Group 4

93.3 93.3 87

demonstrated that repeated zoospore emergence is a variable phenomenon and belongs to S. parasitica. In this study, members of group 1 (S. parasitica) produced repeated zoospore emergence but this potential was not seen in group 2 (S. diclina) and this phenomenon were emphasized by another study. It seems that the structure of the internal membrane of the eggs plays a significant role [18]. The eggs of Great sturgeon (Houso huso) and Persian sturgeon (Acipenser persicus) could be examples, they hosted the smallest number of fungi, compared to other acipencerid fish in the same condition [6,19]. In summary, RAPD—PCR analysis suggested that there are at least four groups of Saprolegnia playing a pathogenic role in saprolegniasis in Persian sturgeon hatcheries in Mazandaran province. The dominance of group 1 may represent a high virulent clone of Saprolegnia. Although repeated zoospore emergence was clearly not specific for pathogenic Saprolegnia species, it was present in certain groups of isolates defined by genotype and can be useful in distinguishing subgroups of pathogenic Saprolegnia.

Acknowledgments This work was supported by the Research Council of University of Tehran.

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