Aphanomyces frigidophilus, fungus-like organisms isolated from water of springs in Białystok, Poland

Investigations into occurrence of fungus Aphanomyces frigidophilus in water of springs Dojlidy Górne, Jaroszówka and Pietrasze within the town Białystok in Podlasie Province, Poland were conducted in Winter, Spring, Summer and Autumn of the year 2005. Samples were processed in the laboratory by routine methods commonly used to isolate these organisms. Bait method with the use of hemp seeds Cannabis sativa, small pieces of snake skin Natrix natrix and exuviae of shrimp Gammarus sp. as bait was applied to isolate the fungus Aphanomyces frigidophilus from the springs. The isolate was maintained on Potato Dextrose Agar PDA and stored in the culture collection of the Real Jardín Botánico CSIC Madrid, Spain. Aphanomyces frigidophilus occurred in 18 [(6)16.7%) in Winter, 3(8.3%) in Spring, 2(5.6%) in Summer, 7(19.4%) in Autumn, 2005] of the examined water samples. In Spring Dojlidy Górne it was very common and was found in all research seasons. The isolate was characterized by studding sequencing the internal transcribed spacer of nuclear DNA (ITS1+5.8S+ITS2). The results indicated the sequence comparisons of two ITS nuclear DNA for species identification: Aphanomyces frigidophilus 18S ribosomal RNA gene, partial sequence, internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence and 28S ribosomal RNA gene, partial sequence. The results indicated the sequence of our isolate corresponded to the species Aphanomyces frigidophilus (AY647192, version AY647192.1; GI: 48766837).


INTRODUCTION
Water mould constitute a common group of organisms found in a variety of water ecosystems. Some of them are animal or human parasites. In favorable conditions, water mold acting as saprobionts can assume pathogenic properties, being a potential source of infection (Dick, 2001;Czeczuga et al., 2004a;Kiziewicz and Kurzatkowska, 2004;Kirk et al., 2008). In spring of rivers, a lot of representatives of water moulds of the class Oomycetes are met. The phylogenetic relationship of Oomycetes (watermolds) to fungi has been debated for many years. Oomycota (oomycota means egg fungus) or Oomycetes have been for a long time recognized as significantly different from the organisms classified as the Phylum Oomycetes in the Kingdom Fungi (True fungi) (Alexopoulos et al., 1996). Scientific studies have shown that some organisms may look like fungi yet are not really members of the Kingdom fungi. A cladistic classification based on modern insights supports a relatively close *Correponding author. E-mail: bozena.kiziewicz@umb.edu.pl. relationship between Oomycetes with photosynthetic organisms such as brown algae and diatoms, within the heterokonts. The Oomycetes have been differently classified by numerous taxonomists, for instance they have been classified as heterokont organisms in the Kingdom Stramenopila, Phylum Heterokonta, Class Peronosporomycetes (Alexopoulos et al., 1996;Dick, 2001).
De Bary in 1860 (de Bary, 1860) established a new genus Aphanomyces to include a number of saprotrophic and parasitic plants observed during this period. The genus Aphanomyces comprising approximately 35-40 species is a smaller and less frequently encountered genera of the Saprolegniales order, Oomycetes Class (Scott, 1961;Uribeondo et al., 2009). A few species are parasites and responsible for economical important diseases affecting agriculture and aquaculture crops as well as wildlife populations of freshwater animals (Papavizas and Ayers, 1974;Sӧderhall and Cerenius, 1999).
The main task of the present study was to assess distribution of fungus-like organisms Aphanomyces frigidophilus from water of three limnocrenic springs situated in Białystok, Podlasie Province, Poland.

Microbial analyses
Microbial analyses was made in the laboratory, The Real Jardín Botánico CSIC, Madrid, Spain of the year 2006. For the microbial analysis of fungi samples of water were collected from each sampling site of research springs of rivers. The water collected from the respective reservoirs was poured in sterile conditions into plastic bottles, and placed in the laboratory. All containers were incubated at 13 ±2°C, with access to daylight resembling natural conditions and following the recommended instructions (Seymour and Fuller, 1987). Water limnokrenic type of springs in Poland have almost constant temperature throughout the year. The water temperature the research springs was about 9°C throughout the period of the study. pH of springs of water was neutral close to the acid.
Samples were processed in the laboratory by routine methods commonly used to isolate these organisms. Bait method with the use hemp seeds Cannabis sativa L., small pieces of snake skin Natrix natrix and exuviae of shrimp Gammarus sp. as bait was applied to isolate the fungus A. frigidophilus from the springs. Water samples (100 ml) of each site were homogenized and four aliquots of 25 ml were placed in Petri dishes of 9 cm diameter with sterile baits. Dishes were stored in the laboratory at room temperature 13 ±2°C for 4-5 days (Seymour and Fuller, 1987). The colonized fragments of hemp-seeds, shrimp and snake skin were transferred to new Petri dishes which contained sterilized, filtered spring or distilled water and crystalline penicillin (2000 units per litre of water) to inhibit bacterial growth. Dishes were microscopically examined weekly for up to three weeks in order to identify the genus or species. The isolate was maintained on Potato Dextrose Agar (PDA) (Merck Cat. No. 1.10130.0500, Merck KCaA 64271 Damstadt, Germany) (Unestam, 1965) and stored in the culture collection of the Real Jardín Botánico CSIC, Madrid, Spain. Morphological characters of asexual structures and measurements were made microscopically on material mounted in water. Fungi were successively observed under an optic microscope Olympus BX 51 (100, 400 and 1000x magnification). The respective stages of the fungal development were evaluated by means of an ocular micrometer. Several microscopic preparations were made from each sample.

Isolates and internal transcribed spacer (ITS) sequences
Sequences were obtained from pure cultures, ITS-polymerase chain reaction (PCR)-based specific tests from GenBank representing a total of 12 Aphanomyces spp. All the isolates were growing on PDA (Merck Cat. No. 1.10130.0500, Merck KCaA 64271 Damstadt, Germany) (Unestam, 1965) for 3-5 days at room temperature as described by Cerenius et al., (1987). Mycellia pellets were washed with sterile water collected in 1.5 ml microcentrifuge tubes and stored at 20°C before DNA extractions. The origin of the isolates and their reference numbers were coded with the initials SAP and are maintained in the Oomycetes culture collection of the Real Jardín Botánico CSIC, Madrid. In this study, was used the following sequences as reference for species A. frigidophilus Kitanch, and Hatai, AY647192.

DNA extraction and PCR amplification
For DNA extraction, mycelium was grown as drop cultures (Cerenius and Sӧderhall, 1985) and from them, genomic DNA was extracted using an Easy Nucleic Acid® (EZNA) (Fungal DNA Miniprep Kit (Omega Biotek, Doraville, USA) as described in the study of Martín and García-Figueres (1999). DNA fragments containing internal transcribed spacers ITS1 and ITS2 including 5.8S gene of the nuclear DNA was amplified with primer pairs ITS5/ITS4 (White et al., 1990) primers as described in Martin et al., (2004). Nucleotide BLASTN searches with option Standard nucleotide BLAST and BLASTN 2.6 were used to compare the sequence obtained against the sequences in the National Centre of Biotechnology Information (NCBI) nucleotide databases.

Hyphal growth rates and repeated zoospore emergence
Selected isolates of parasitic and saprotrophic/opportunistic species were inoculated in Potato Dextrose Agar PDA (Unestam, 1965) and incubated at 20°C. Colony diameter was measured every 24 h during maximum period of 3 days. Briefly, mycelia were grown in PDA drop cultures for 3 days at 20°C. To trigger sporulation, mycelia were washed thee times with sterile water and incubated in petri dishes containing sterile water for 14 at 15°C to allow zoospore release. The release of new zoospores was observed under the microscope after incubating the cyst suspension at 15°C for 150 min.

Production of sexual structures
Aphanomyces isolates were cultivated on sterile substrates including hemp seeds (C. sativa L.), exuviae of Gammarus sp., and snake skin N. natrix in petri dishes containing autoclaves, sterilized water (one bait per dish) and incubated in the dark at 20°C. Dishes were examined periodically (for 3 weeks) for evidence of growth with a stereo microscope and to verify the presence or absence of sexual structures. All isolates were characterized and identified according to the study of Scott (1961). Taxonomic identifications were made according to the study of Scott (1961), Batko (1975), Pystina (1994) and Seymour and Fuller (1987).
Ap. frigidophilus has the hyphae with rounded hyphal tips, hyphe thin in 5-7 μm in diameter, aseptate, sporangia long with a single row. Primary zoospores near the orifice emerged in this manner, whereas those emerging later became elongate with rounded ends during the passage through zoosporangium. Secondary zoospores were reniform, laterally biflagellate. Oogonia were abundant on short oogonial stalk, 16-25 μm diameter. Young oogonia with rough-ended outer contours, elongated oogonia between hyphae. Mature oogonium showing single subcentric oospore 14-22 μm diameter, with a large shiny vesicle surrounded by fine granules, the outer contours have short papillate or irregular (Kitancharoen and Hatai, 1997).
However, fungi in the genus Aphanomyces have been occasionally reported on fish eggs. Ap. laevis is only species of this genus that has been reported on eggs of rainbow trout Onchorhynchus mykiss (Scott and O' Bier, 1962) and vendance Coregonus albula (Czeczuga and Woronowicz, 1993), although an unidentified Aphanomyces was also reported on rainbow trout (Scott and O'Bier, 1962). The first A. frigidophilus has been reported only from Japan. Ap. frigidophilus was detected on the eggs of Japanese char Salvelinus leucomaenis from Tochigi Prefectural Fisheries Experimental Station, Utsonomiya, Japan Hatai, 1997, 1998). The first in Poland and in Europe Ap. frigidophilus have been described only on the basic morphological studies by Czeczuga et al., (2004a; in fish eggs of European Whitefish Coregonus lavaretus holsatus obtained from Lake Wdzydze in Kaszuby (Poland). The next in Europe Ap. frigidophilus was described on crayfish cuticule Austropotamobius pallipers by Ballesteros et al. (2006), too. Dead crayfish were collected in river Tajuňa, Guadalajara (Spain). The fungus was grown on fish eggs of European Whitefish in water of Cypiesk Spring and Akcent Pond in Białystok, Poland. Worth a special note was the finding Ap. frigidophilus, new to Polish freshwaters (Czeczuga et al., 2004a;. The fungus was growing also on the eggs of sea trout Salmo trutta m. trutta in running water from River Biała, Krasna and Supraśl near Białystok (Poland) (Czeczuga et al., 2005). Ap. frigidophilus was the most common straminipilous organisms found on the eggs of Atlantic salmon Salmo salar. The investigated eggs were collected from 60 females of Atlantic salmon caught during their spawning migration in Darłowo town on the River Wieprza (wild form), and Świbno town on the River Vistula (wild form), and from those bred in fresh water in hatcheries at Miastko town (farmed form). Ap. frigidophilus was found growing on the eggs of Atlantic salmon from 14 females from research rivers such as Wieprza and Vistula and from hatcheries in Miastko (Czeczuga et al., 2011).
Molecular phylogenetic relationships among 12 species of Aphanomyces de Bary were analyzed based on 108 ITS sequence s of nuclear DNA by Uribeondo et al., (2009). Within Aphanomyces clade, three main lineages were found: plant parasitic, animal parasitic and saprotrophic or oportunic parasitic. The animal parasitic lineage had low support and comprised sequences from Aphanomyces spp. that are parasites of animals, or have been isolated from animals, and isolates of the saprotrophic species Ap. stellatus. This lineage contained the reference sequences for Aphanomyces astaci, Ap. frygidophilus, Ap. invadans, Ap. piscicida and Ap. stellatus. This lineage comprised species Aphanomyces that thrive in freshwater or estuarine aquatic environments, and included all species parasitic to animals, the animal associated species Ap. frigidophilus, and saprotrophic species Ap. stellatus. The Ap. frigodophilus clade comprised the sequences of refe-rence for A. frigidophilus (AY647192). Isolates originated from Japan and Spain with a wide host range that included salmonids (Japan) and freshwater crayfish (Spain) (Kitanchaoren and Hatai, 1997;Ballesteros et al., 2006;Uribeondo et al., 2009). Although, isolates of this species have always been obtained from diseased aquatic animals, it is uncertain whether this species is a parasitic or opportunistic (Uribeondo et al., 2009).
The results indicated the sequence comparisons of two ITS nuclear DNA for species identification. The results indicated the sequence of our isolate corresponded to the species Ap. frigidophilus (AY647192, version AY647192.1; GI: 48766837). GenBank sequence AY647192.1 corresponding to strain NJM9500 of Ap. frigidophilus directly submitted by Phadee et al. (2004). Thus, this study represents the first isolation of Ap. frigidophilus in fresh waters in Poland. This parasitic fungus considered among one hundred worst invasive species (Global Invasive Species Database, 2005) and is responsible for the decline of the indigenous European freshwater animals not only to crayfish, and also fish which are currently endangered in Europe and specially in Poland (Czeczuga et al., 2004a;b, 2005;Ballesteros et al., 2006;Uribeondo et al., 2009).

Conclusion
The study conducted in springs of rivers located in Białystok, Poland showed the occurrence of fungus-like organisms Aphanomyces frigidophilus Kitanch. and Hatai in samples of water reservoirs. The results indicated the sequence comparisons of two ITS nuclear DNA for species identification. The results indicated the sequence of our isolate corresponded to the species Ap. frigidophilus. Thus, this study represents the first isolation (on the basic molecular studies) of Ap. frigidophilus in fresh waters in Poland.