IJMBR 2 (2014) 24-30 ISSN 2053-180X Two ascomycetes from different aquatic habitats Hussein Al-Nasrawi Florida State University – USA. E-mail: [email protected]; [email protected]. Article History ABSTRACT Received 23 May, 2014 Two species of the genus Halosphaeria Linder were isolated from dead plant Received in revised form 26 segments submerged in two lentic habitats. Halosphaeria cucullata (Kohlm.) was June, 2014 Accepted 15 July, 2014 isolated from a stem segment of a reed submerged in Al-Soda Marsh Lake in Iraq where water salinity exceeds 1.6% as a salty environment. This species was Key words: distinguished by a small ascocarp (150-258 µm) and ascospore (8-10 × 20-28 µm) Halosphaeria, with a gelatinous appendage in the terminal end of the ascospore. Halosphaeria Fungi, species was isolated from a segment of an unknown plant species submerged in Isolation, Lake Sevan in Armenia. It was distinguished by a large ascocarp (200-300 µm) Armenia. and ascospore (10-15 × 25-32 µm) with a sheath of hyaline gelatinous appendage surrounding the entire ascospore. The two isolates were described and Article Type: illustrated as new records and kept in the Basrah herbarium under the number Full Length Research Article BSRA 9001 and BSRA 9002, respectively. ©2014 BluePen Journals Ltd. All rights reserved INTRODUCTION In lakes with a typical littoral zone, macrophytes form dead plant segments submerged in lentic habitats in two three characteristic zones starting from the coast, different aquatic ecosystems, in particular, fungi with the emergent plants, floating leaved plants and submerged phenotypic characteristics of Halosphaeria fungi. plants (Bernatowicz and Wolny, 1974; Fare et al., 2001; Hutchinson, 1975). Fungi are common colonizers distributed on macro- MATERIALS AND METHODS phytes submerged in fresh water habitats. In particular, ascomycete fungi occur on a variety of submerged Description of study area substrates in both lotic (creeks, rivers, and streams) and lentic (bogs, lakes, ponds, and swamps) habitats Al-Soda marsh is a lentic aquatic ecosystm (shallow lake) (Shearer, 1993; Cai et al., 2003; Shearer et al., 2004, located in the Al-Huwaizah marshes in Southern Iraq. It is 2007; Raja et al., 2009). characterized by high salinity (1.6%) and is rich with the There are a number of fungal species appearing in common reed Phragmites australis Trin (Scott, 1995). marine and terrestrial environments. Meanwhile the same Lake Sevan is an aquatic ecosystem (natural fresh fungal species may also occur in both saline and fresh- water lake) located in the central part of the Republic of water environments (Gessner, 2001; Shearer, 1993). Armenia. It is the largest lake in Armenia and the Studies on freshwater ascomycetes have been Caucasus region. It is one of the largest high-altitude conducted at spatially limited sites in lentic habitats (Cai lakes in the world. The lake is part of the Sevan National et al., 2003). Park which consists of 4 protected reserve zones as well Halosphaeriaceae represents the largest and most as recreational and economic zones. diverse lineage of marine ascomycetes till date as The flora of the basin is highly diverse and about 1,600 proposed by Müller and von Arx (1962) and Eriksson species of vascular plants have been described. The (1984). Pinruan et al. (2004) isolated Halosphaeria dominant vegetation of the basin is mountain steppe, appendiculata Linder from decaying palm leaves sub- alpine and alpine vegetation with different species of submerged in a fresh-water environment. Astragalus and Acantholimon. On Lake Sevan, emergent The present study focused on isolation of fungi from vegetation exists only in a limited calm area and Int. J. Res. Environ. Stud. 25 Figure 1. Ascocarp of Halosphaeria cucullata. pondweeds (Potamogeton spp.) are abundant to depths Lundqvist (1970), Matsushima (1975), Mouchaccu of 2-5 m. (1986), Munk (1966), Pitt (1979), Raper and Fennell (1973) and Sivavanesan (1987). Sampling and morphological study Note: Practically, it was observed that the gelatinous appendages cannot be diagnosed by using Lactophenol Thirty (30) submerged plant samples were collected from stain due to dissolving of gelatin material, so we used Al-Soda Marsh Lake in Iraq; and another 30 submerged water instead of lactophenol to clarify appearance of the plant segments samples were collected from Sevan lake appendages. in Armenia, both of which were collected in 2008. The samples were randomly collected from freshwater and salty water habitats following the procedures described RESULTS by Shearer et al. (2004). The samples were placed in Ziplock© plastic bags lined Description with moist filter paper and were transported to the laboratory. In the laboratory, the samples were placed in moist chambers and were incubated at ambient Halosphaeria cucullata (Kohlm.) (Kohlmeyer, 1972): temperature (~25°C) at 12/12 h light and dark conditions. Afterward, they were subsequently examined with a Ascocarp is spherical with diameter of 150-258 µm, dissecting microscope within one week of collection and completely submerged, ostulate, grey to brown color, periodically over 6‒12 months (Shearer, 1993; Shearer et surrounded by a thick wall, distinguished by the presence al., 2004). of long neck of 200-250 µm and diameter of 30-50 µm, For isolation of ascomycetous fungi, the samples were centric, cylindrical and straight. incubated for more than 1 month in the moist chamber to Asci are of size range 50-70 µm and contain 8 permit ascocarp development. Ascocarps were picked ascospores. Ascospores’ size range from 8-10×18-20 out from the substrate under dissecting microscope, µm, unitunicate, septate, divided in two cells by crushed on glass microscope slides to open the ascocarp transverse septum, with hyaline colour, supplied with a and the dispersed ascospores properties were examined. thin gelatinous appendage, attached in the terminal end of the ascospore from the apex. The isolate resembles the species isolated by Kohlmeyer (1972); however the Identification process ascocarp neck in this new isolate is longer. The fungus is saprobic and has been previously isolated from Tamarix Isolated fungi were identified according to the following aphylla plant in California saline lake in Ohio, Brazil, keys: Ahmed et al. (1971), Arx (1975), Arx et al. (1986), India, Southern Africa, Australia, Japan and Mexico. This Arx (1988), Boerema (1993), Booth (1971), Cain (1961), new isolate is the first record of this fungus in Iraq, Cai et al. (2003), Ellis (1971), Ellis (1976), Garcia et al. isolated from dead reed stem (P. australis) submerged in (2004), Goh and Clement (2003), Hoog and Guarro Al-Soda Marsh Lake. The isolated fungus was kept in (1995), Horie and Udagawa (1973), Horie (1980), Hyde Basrah herbarium under the number BSRA 9001 and Sarma (2000), Kohlmeyer and Kohlmeyer (1979), (Figures 1 and 2). Al-Nasrawi 26 Figure 2. Ascospore of Halosphaeria cucullata. Halosphaeria species terrestrial origin (Spatafora et al., 1998) and marine ascomycetes could have migrated from terrestrial Ascocarp is spherical with diameter of 200-300 µm, habitats to freshwater and brackish water, and then to completely submerged, ostiolate black grey color, marine environments (Shearer, 1993; Jones, 1995). surrounded by a thick wall, distinguished by the presence Jones et al. (1986) and Jones (1995) suggested that of long neck of 120-200 µm and diameters of 20-30 µm, ascospore shape and ascospore appendage ontogeny is cylindrical and straight. of primary importance as taxonomic characters in the Asci are of size range 10-20×25-35 µm, and contain 8 delineation of genera in the Halosphaeriaceae. Ascos- ascospores. Ascospores’ size range from 8-10×20-28 pore septation is also variable; therefore there is no clear µm, unitunicate, septate and divided in two cells by pattern for ascospore morphology within the transverse septum. The ascospore is surrounded by a Halosphaeriaceae. thin gelatinous appendage which covers the whole body In the present study, two Halosphaeria species were of the ascospore. isolated from different aquatic environments and revealed The isolate of the present study resembles different morphological characteristics of the ascospore Halosphaeria galerita isolated by Tubaki (1968), except appendages. H. cucullata isolated from salty habitats in that this new isolate has a bigger ascocarp. The fungus is Iraq is distinguished by a long neck 200-250 µm and saprobic and also has been previously isolated from ascospores of size range of 8-10×18-20 µm, unitunicate, woody segment in Denmark, Mexico, Spain, United septate, divided in two cells by transverse septum, with States, Germany and France. The isolated fungus was hyaline colour, supplied with a thin gelatinous kept in Basrah herbarium under the number BSRA 9002 appendage, attached in the terminal end of the (Figures 3 and 4). Table 1 gives a comprehensive list of ascospore from the apex. the fungal species isolated from Al-Soda Marsh Lake, The second isolate, Halosphaeria sp., was isolated Iraq and Sevan Lake, Armenia. from fresh water habitats in Armenia. It is distinguished by having completely submerged ascocarp surrounded by a thick wall and the presence of long neck 120-200 µm DISCUSSION and unitunicate ascospores 8-10×20-28 µm, divided in two cells by transverse septum. The ascospore is The Halosphaerialean fungi are saprobic on decaying surrounded by a thin sheath of gelatinous appendage woody and herbaceous substrata in aquatic environ- which covers the entire ascospore. ments. The biodegradation process of submerged The results of the present study are similar to those of substrates occurs via fungal enzyme secretion onto the Gregory and Darryl (1979) who isolated ascomycetes substrate. In particular, cellulases degrade cell-tissue including two Halosphaeria species from wood blocks structures and pectinases decompose pectin and submerged in salty habitat of Minas Basin salt marshes protease which degrade cytoplasmic proteins (Chamier and fresh water in habitat of Wallis River, also in the and Dixon, 1982; Chamier, 1985). month of August.