Ecological diversity and economical importance of species from Aphanomyces genus

Species from Aphanomyces genus were investigated in 225 limnologically and trophically different water bodies (springs, rivers, ponds and lakes) as well as in humid soils of northeastern Poland. Distribution of particular species and their role in diverse ecosystems has been investigated. Thirty taxa, including: 2 species of cultivated parasitic plants, 3 species of parasitic animals, 6 saprotrophic species and 5 species of saprotrophic/ opportunistic species were recorded. Some of Aphanomyces species occurred also as parasites of algae, straminipiles and invertebrates. Among parasitic species: A. cochlioides, A. euteiches (cultivated plant parasite), A. astaci (crayfish parasite), A. piscicida (fish parasite) playing economically important role were found. Amino acid, carbohydrate and urease assimilation tests were used.


INTRODUCTION
The number of fish species bred in the control conditions increased in recent years according to Food and Agriculture Organization (FAO, 2012).On the other hand, death on a large scale of the particular fish populations may occur due to bacterial and mycotic infections (Bruno and Wood, 1988).Straminipila from the Saprolegniales order, especially Saprolegnia, Achlya and Aphanomyces species are responsible for eggs infection in the fish species.For example Hatai and Hoshiai (1992) dealt with saprolegniosis of Oncorhynchus kisutch demonstrating heavy losses caused by Saprolegnia parasitica in the breeding of this species, even up to 50%.Achlya flagellata and A. prolifera are known to have caused total damage to the incubated eggs of Tor tor Lac. in India (Sati and Khulbe, 1981).Aphanomyces astaci is known to cause so-called "plague" of crayfish (Schikora, 1903) and A. laevis known in mass deaths of rainbow trout during reproduction on the Taiwan (Chien, 1981).Since the mid-1980s epizootic ulcerative syndrome (EUS) have been described as a disease affecting wild and farmed freshwater and estuarine fish (Chinabut, 1998).It has been reported from Australia, North America, Asia and Africa (OIE, 2007).The agent associated with this disease is the straminipiles organism belonging to Aphanomyces genus.In 1997 Kitancharoen and Hatai (1997) described also A. frigidophilus as a parasite of the Japan charr eggs.Whereas, such species of the Aphanomyces genus as A. cochlioides and A. euteiches because of their agriculturally importance, are specialized to parasitize roots of sugar beet and Fabaceae species, respectively (Bangsund and Leistritz, 1993;Sauvage et al., 2007;Dieguez-Uribeondo et al., 2009).
On account of that, authors have decided to find out which of the already known straminipiles organisms belonging to Aphanomyces genus is responsible for the mycotic diseases in plants and animals and can grow in different ecosystems of the north-eastern Poland.

Occurrence environment of Aphanomyces species
Material was collected in north-eastern part of Poland within 1985 to 2012.Water samples were collected from 225 limnologically and trophically different water bodies-springs (31), rivers (45), ponds (21) and lakes (128).Oligotrophic, mesotrophic, eutrophic and dystrophic types of lakes were investigated.Water samples were collected at a depth of 10 (spring)-30cm (other water bodies) and at a distance of 0.5m from the bank, at three sites.Nineteen water parameters were determined (Table 1, only as representatives for particular types of water bodies) according to generally accepted methods (Golterman and Clymo, 1969;APHA, 2005).
The soil species was collected from roots of common bean, common serradella, common vetch, pea and sugar been (Table 4) at farmed land of north-eastern Poland.The amino acid, carbohydrate and urease assimilation tests were performed according to Yuasa and Hatai (1996) and Kitancharoen and Hatai (1998).

Determination of Aphanomyces species
Thickness, height and branches of the hyphae were measured in Aphanomyces species.The diameter of zoosporangia, zoospores, cystospores, oospores, oogonia and antheridia were defined.Morphological structure of the oogonia, antheridia and their number were also studied.It was investigated, if the oogonia are mono-, diclinous or androgynous.Some parasitic Aphanomyces species showed the repeated zoospore emergence (RZE) as adaptation to the parasitic mode of life (Cerenius and Söderhäll, 1985).For parasitic species (plant and animal) general principles of culture procedures were used (Seymour and Fuller, 1987;Roberts et al., 1993;Willoughby and Roberts, 1994;Paterson and Bridge, 1994;Watanabe, 2002).The parasitic species of the sugar beet and of some species of the legumes (soil species) were isolated according to Dyer et al. (2004) and Sauvage et al. (2007) methods.The isolates collected from the necrotic roots of the investigated plants were transferred to the corn meal agar (CMA, Difco, Detroit, MI) supplemented with rifampicin (38 mg/l).For each isolate, a single hyphal tip was transferred to a new CMA plate to insure only one genotype per isolate.The cultures were stored in sterile distilled water and were replaced in storage by culturing on CMA with rifampicin to repeat the process for storage (Parke and Grau, 1992).The taxa of Aphanomyces species were identified using the keys of Scott (1961), Batko (1975), Pystina (1994), Johnson et al. (2002) and Petrini and Petrini (2013).

RESULTS
The water samples used for the analysis differed in nutrient content and other parameters (Table 1).The highest nitrogen, sulphates and chlorides content was found in Spring Cypisek, Akcent and Fosa, Komosa Pond.The lowest content of nitrogen, sulphates and chlorides was noted in Hancza Lake.30 taxa from Aphanomyces genus were found in the water bodies and soil of north-eastern Poland (Table 2).Same species occurred in all types of the investigated water reservoirs.A. astaci, A. laevis, A. irregularis, A. parasiticus and A. stellatus belong to this group.Such species as A. apophysii, A. coniger, A. ovidostruens, A. polysporus, A. scaber and A. volgensis were found only on the plant debris, other species also on animals' substratum (Table 3). A. cochlioides and A. euteiches are the parasit on cultivated plants (Table 4).6 Aphanomyces species including: A. frigidophilus, A. invadans and A. piscicida and others were present on the fish eggs (Table 5).Investigated Aphanomyces species assimilated only three amino acids: alanine, glutamine and cysteine and from carbohydrates only glucose and starch, but they did not assimilate urease (Table 6).

DISCUSSION
Thirty taxa Aphanomyces were found in the water bodies of north-eastern Poland (Table 2).Five species occurred in all types of investigated water bodies, two species-A.cochloides and A. euteiches occurred in humid soils.A. cochlioides is a parasite of roots of sugar beet, A. euteiches of pea and other legumes species roots.The infected roots of plants ranged from 31.8% (common bean) to 50.7% (pea) during humid years.Therefore, soil species of the Aphanomyces genus are economically important, especially for the food industry (pea, sugar beet) (Levenfors and Fatehi, 2004) and for some plant species used as animal foodstuff for domestic animals (Papavizas and Ayers, 1974;Holub et al., 1991;Brantner and Windels, 2001).Most Aphanomyces species were found in such eutrophic reservoirs of the stagnant water bodies as ponds ( 16) and eutrophic lakes (14 species), (Table 2).Only a few Aphanomyces species were found in water with a small content of the biogenic substances.There are oligotrophic, α-mesotrophic and dystrophic lakes (5-6 species).In the flowing waters (springs, streams, rivers) the authors found many (12-13) Aphanomyces species and such species as A. astaci, A. irregularis, A. laevis, A. parasiticus and A. stellatus were found in all types of the investigated water bodies.According to this fact they are called the eurytrophic species and have a wide range of the ecological tolerance.There are various organic compounds of carbon, free saccharides, free amino acids, numerous enzymes and other compounds (Hoagland et al., 1993) called extracellular products (being excreted by the phytoplankton and makrophytes) which exert the influence on the mycotal species in water.They are a medium for different heterotrophic organisms including mycotal species.As it is known, in oligotrophic, αmesotrophic and dystrophic lakes there is a little amount of biogenic compounds, the phytoplankton and the macrophytes excreting less extracellular products serving as nutrients for heterotrophic organisms.Such species as A. apophyscii, A. coniger, A. ovidostruens, A. polysporus, A. scaber and A. volgensis were found only on the plant substratum, other species also on the animal substratum.The 9 Aphanomyces species present in fish, A. invadans and A. piscicida occurred only on fish eggs. A. euteiches is the parasite of Fabaceae family (Papavizas and Ayers, 1974).A. astaci, A. invadans and A. piscicida are animal parasites.A. astaci, which has been described by Schikora (1903), infects freshwater crayfish (Söderhäll and Cerenius, 1999).Both A. invadans and A. piscicida devastated natural and cultured stocks of freshwater and estuarine fish (Chinabut, 1998;Johnson et al., 2004).Some authors confirmed this both species as synonym (Dieguez-Uribeondo et al., 2009), others-as separate species (Phadee et al., 2004a, b). A. piscicida was described by Hatai (1980) and causes mycotic granulomatosis in fish (MG) (Egusa and Masuda, 1971), whereas A. invadans was described by Willoughby et al. (1995) and is an agent of EUS (Epizootic ulcerative syndrome).Kitancharoen and Hatai (1997) described new species of A. frigidophilus from eggs of Japan charr.In Europe, first A. frigidophilus was described on eggs of some species of coregonide fishes (Czeczuga et al., 2004).Two years later, Ballesteros et al. (2006) found this species in cuticule of dead crayfish Austropotamobius pallipes in Spain and Kiziewicz et al. (2013) observed its occurrence in water from the some springs of north-eastern Poland.Growth of A. frigidophilus was also stated on eggs of some species from Salmo genus (Czeczuga et al., 2004(Czeczuga et al., , 2011a)), sturgeonids, Chinook salmon (Czeczuga et al., 2011b) and African catfish (Czeczuga et al., 2013).
As shown in Table 3, such species as: A. irregularis, A. laevis, A. parasiticus and A. stellatus were found on plant debris and on fish eggs.They all belong to opportunistic pathogens, which are sapro-and necrotrophic species similar to many species belonging to Achlya and Saprolegnia genera.Accourding to our study such saprotrophic species as A. amphiginus and A. helicoides were growing on decaging invertebrate animals and plant debris.According to Dieguez-Uribeondo et al. (2009).A. helicoides belongs to saprotrophic-opportunistic group.The most numerous pathogens from this group on fish eggs are: A. laevis (41 species) and A. stellatus (29). A. irregularis was found on eggs of 8 fish species, A. parasiticus-on 7. Mentioned species were also present on the eggs of lamprey (Czeczuga, 1997).According to Batko (1975) and Pystina (1994) A. irregularis is saprotrophic species and A. parasiticus-parasite on other fungal species, especially from Achlya genus.A. laevis both-eggs and adult individuals of many economically valuable fish species (Lartzeva, 1986;Dudka et al., 1989).A. bosminae and A. daphniae (occurring on fish eggs) were found in some of investigated water bodies.A. bosminae was firstly described by Scott (1961) and first investigated fungus in America.It is a parasite of freshwater crustacean from the Bosmina genus.The authors found it on the eggs of Leuciscus leuciscus in water from River Suprasl (Czeczuga and Muszynska, 1999).A. daphniae is known to be a parasite of the plankton crustacean Daphnia hyalina (Leydig) (Prowse, 1954).In our study its growth was revealed on the eggs of Cobitis aurata (Filippi) in River Suprasl water (Czeczuga and Muszynska, 1997).
The occurrence of Aphanomyces species has been registered in water bodies of African continent by El-Sharouny and Badram (1985), Shaheen et al. (1999), El-Hissy et al. (2004) and Ali (2009).Aphanomyces species have been associated with skin lesions and mortality of some fish species.This phenomenon has been observed in two species of fishes from excavated earthen pond at the western shore of the Suez Canal, Egypt, during the winter of 1971 (Shaheen et al., 1999).According to Lilley et al. (2003) this invasive Aphanomyces fish infection has been reported by Shaheen et al. (1999) and has been caused by A. invadans species.It has also been observed on skin-muscles on aquarium species of Labeo bicolour Smith specimens and on the alevins of the Nile tilapia-Oerochromis niloticus L.
The assimilation of the amino acids, carbohydrate and urease by two investigated Aphanomyces species was very small.Specimens of A. frigidophilus from fish eggs have assimilated only 3 amino acids, 3 carbohydrates, whereas A. laevis assimilated only one amino acid and 2 carbohydrates.Whereas specimens of Saprolegnia, Achlya or Pythium assimilated 4 to 5 amino acids, 9 to 16 carbohydrates and urease (Kitancharoen and Hatai, 1998).The specimens

Table 1 .
Chemical and physical properties of water in represented of particular types of the water bodies (in mg l -1 ).

Table 2 .
Aphanomyces species founded in particular types of the environment.

Table 6 .
Amino acid, carbohydrate and urease assimilated by two Aphanomyces species.