Isolation and molecular identification of Ascomycetes in sediments and waters of the Gulf of Aqaba, Red Sea

Basem M. Jaber, Rola Al-Silawi, Tariq Al-Najjar

Department of Biological Sciences, The University of Jordan, Amman, Jordan.
Department of Marine Biology, Faculty of Marine Sciences, The University of Jordan/Aqaba Branch, Aqaba, Jordan.

DOI: 10.4236/ns.2012.48074                

Abstract

Using molecular approach, we aimed to identify fungal phylotypes that exist in the Gulf of Aqaba, Red sea. Several samples were taken from sediments and seawater of three locations along 26 kilometers at 5 m depth. 18S small subunit rRNA gene was targeted for PCR amplification and sequencing. Partial sequences introduced as query in BLASTN phylogenetic analysis revealed 100% identity with Ascomycota, namely, Aspergillus sp. Penicillium sp. and its closely related Eupenicillium sp. The top scorer species in this analysis were Aspergillus sydowii, Aspergillus wentii, Aspergillus flocculosus, Penicillium expansum and Eupenicillium javanicum with 98% - 100% identity. Phylogenetic analyses demonstrates close relatedness among isolated fungi and potential association with Ascomycetes. This study reports a new geographical location in which facultative marine Ascomycetes exist in, and sheds some light on fungal diversity in Gulf of Aqaba.

Keywords

Marine Fungi; Ascomycete; 18S ssu rRNA; Gulf of Aqaba; Red Sea

1. INTRODUCTION

Fungi are found ubiquitously in the environment. They execute a wide range of important ecological functions especially ones associated with decomposition of organic substrates in both terrestrial as well as marine environments, and occasionally in extreme conditions [1-5]. The magnitude of fungal biodiversity was estimated to be 1.5 million species [6,7]. However, some estimation exceeded this number to 5.1 M [8]. Among the estimated 1.5 million, only 5% - 10% has been formally described [3,8]. Fungi that live in the sea are defined as obligate marine or facultative. Obligate marine fungi are those that grow and sporulate exclusively in a marine or estuarine habitat and are permanently or intermittently submerged in water. Whereas, facultative marine fungi are those that normally occupy freshwater habitats or terrestrial surroundings but are able to grow (and possibly to sporulate) in the marine environment [9,10]. Currently there are 444 known recognized species of obligate and facultative marine fungi. Ascomycota represented by 360 species whereas Basidiomycota are represented by 10 species and 74 species of mitosporic fungi [11]. Facultative marine fungi can be transferred to seawater by wind, rain or runoff soil. Some facultative marine fungi were able to evolve adaptation to marine environment and eventually became obligate marine [12].

Many reasons have posed the need to examine the extent of marine biodiversity of fungi. For example, the emergence marine life diseases to corals, sponges, sea fan corals and fish [3,13-15]. Some marine fungi are reported as sources of enzymes used in bioremediation [16-18]. New biosynthetic products have been revealed in marine-derived fungi [19-21]. Moreover, marine fungi can be exploited for monitoring marine environment pollution [22].

Literature search revealed no studies examining fungal biodiversity in Gulf of Aqaba, a semi-enclosed water body located in the most southern part of Jordan and at the northern end of the Red Sea. Only one study examined manglicolous fungal diversity in of the Red Sea in the upper Egypt [23].

The Gulf of Aqaba is a sub-tropical arid area between longitude 34˚25' to 35˚00'E and latitude 28˚00' to 29˚33'N (Figure 1). The Gulf is unique for its great depth (about 1830 m) in proportion to its width (maximum 25 km); the mean depth of the Gulf is about 800 m. Most of the rainfall in the region of the Gulf occurs during the period November-May. The average rainfall in Aqaba town is about 35 mm/yr. Daily temperature ranges between 14˚C in January to 45˚C in summer and evaporation rates of

Figure 1. Location of the sampling sites along the coast of the Gulf of Aqaba.

seawater between 200 and 365 cm/yr [24]. The aim of this study is to investigate the existence of fungal life in seawater and sediments of the Gulf of Aqaba and determine their phylotypes. Using molecular identification approach, we report the isolation and identification of 5 Ascomycete sp. Partial sequences of the 18S small subunit (ssu) rRNA gene were used as queries in BLASTN search that revealed 98% - 100% identity. Phylogenetic analyses revealed close relationship among the isolated fungi and their potential affiliation with Ascomycetes. We propose further studies examining fungal biodiversity in this unique marine environment.

2. MATERIALS AND METHODS

2.1. Sampling Sites

Water samples were collected using 100 ml screw-cap sterilized bottle, from three selected locations, namely; Marine Science Station (MSS); Phosphate Loading Berth (PLB) and Industrial Complex area (IC) by snorkeling, or by SCUBA diving at a depth of 5 m, during Fall 2010.

The sampling sites are shown in Figure 1. Meanwhile sediment samples were collected from the same location using similar 100 ml sterilized plastic bottle. The sampling was performed twice during 2 months period in the fall 2010. All samples were transferred to the laboratory by using icebox and cultured within 40 hours of arrival.

2.2. Fungal Isolation and Culture Conditions

Seawater samples were diluted 1/10 using 0.5 M sterile NaCl solution, whereas sediment samples were first washed with sterile NaCl solution to prepare wash off solution. From both preparations, 100 μl of either diluted sample or wash off were taken to spread on 2.5% (w/v) Sabouraud Dextrose agar plates containing 50 μg/μl ampicillin. To rule out any possible contamination during manipulation, negative control plates received only sterile NaCl washing solution and were exposed to circulating air inside the laminar flow during culturing. All plates were incubated for 3 days at 30˚C. After incubation, cultured plates were screened to select isolated fungal colonies. Each selected colony was sub-cultured several times to ensure obtaining pure culture. Colony selection was primarily based on morphological differences between colonies. Although some colonies exhibited only slight differences, yet they were selected for further analysis. Nine colonies were selected for further identification analysis. Spores were harvested using sterile cotton swab pre-wetted with salt-tween [0.1% NaCl plus 0.1% tween-80 (vol/vol)] then released in salt-tween solution. After that, spores were counted using hemocytometer. Approximately 6 million spores from each isolate/colony were inoculated into Erlenmeyer flasks containing 25 mL of Sabouraud Dextrose Broth 2.5% (w/v) 50 μg/μl ampicillin. The flasks were incubated for 48 h at 30˚C and 150 rpm shaking. All inoculations and fungal handling were done under ClassII BIOAIR Laminar flow (Euroclone division, Italy) to avoid any possible air contamination.

2.3. Genomic DNA Extraction

Fungal mycelia of each isolate were collected by centrifugation at 10,000 rpm using regular bench-top centrifuge. Prior to that, microscopic examination was done to test the purity of the culture. Genomic DNA was extracted from 2 - 3 mg of mycelia using recombinant lyticase (Sigma, USA) and according to the protocol in wizard genomic DNA purification kit (Promega, USA).

2.4. Primers and 18S rRNA Gene PCR Amplification and Sequencing

Forward Primer 5’-ATT GGA GGG CAA GTC TGG TG-3’ and reverse primer 5’-CCG ATC CCT AGT CGG CAT AG-3’ bind to conserved regions of the fungal 18S ssu rRNA gene and enclosing containing the variable regions V7 and V9 as described before [25]. PCR amplification was carried out using ReadyMix^TM Taq PCR Reaction Mix (Sigma, USA) which contains all required reagents except primers. PCR amplification was carried out in reaction volume of 50 µl of which 25 μl of ready mix Taq PCR which contained 1.5 units Taq DNA polymerase, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin and 0.2 mM dNTP, in addition to 2 µl of 10 μM of each primers and 5 µl (0.25 µg/µL) DNA templates were added to each reaction mixture. Blank that contained only water instead of DNA, was used in each PCR run to check for DNA contamination.

DNA amplification was carried out by thermal cycler (Biorad, USA). The samples were denatured at 94˚C for 2 minutes, followed by 30 cycles of repeated denaturation (30 s at 94˚C), annealing (45 s at 55˚C), enzymatic chain extension (60 s at 72˚C) and a final extension at 72˚C for 10 minute. Following amplification, PCR products were analyzed by 1% agarose gel electrophoresis in 1X TBE buffer (9 mM Tris-borate, 0.2 mM EDTA) and analyzed after staining with ethidium bromide.

2.5. Sequence Analysis

PCR products were sequenced at the Macrogen Company sequencing facility using Applied Biosystems 3730 mXL automated DNA sequencer (Macrogen, Korea). For identification, sequences were submitted to BLA-STN (National Center for Biotechnology Information; (http://www.ncbi.nlm.nih.gov). The 18S rRNA gene sequences from each species and the matched sequences from GenBank were aligned using ClustalW2 (EMBLEuropean Bioinformatics Institute-EBI; http://www.ebiac.uk). Phylogenetic analyses were performed using Phylogeney.fr [25]. Phylogentic tree was reconstructed using the maximum likelihood method implemented in the PhyML program (v3.0 alRT) and graphed using TreeDyn (V198.3) [26].

3. RESULTS

3.1. Phenotypic Characterization

Fungi were cultured from form the sediment and seawater of the Gulf of Aqaba, Red seas.

All isolates grew equally well on Sabouraud Dextrose agar plates at 30˚C. Most colonies grew up to 3 - 4 cm in diameter after 48 h incubation at 30˚C. No colonies grew on the negative control plates. According to their growth characteristics and colony morphology, 9 well-isolated colonies were selected for further characterization and identification.

3.2. PCR Amplification of the 18S ssu rRNA

The primers target a consensus sequence of the 18S ssu rRNA gene containing the variable regions V7 and V9. Amplification with the primers described above is expected to yield a 482- to 503-bp fragment, depending on the fungus tested. Figure 2 shows the amplicons resulted from PCR amplification. The figure does not reveal detectable size variation among amplicons.

3.3. 18S rRNA Gene Sequence Analysis

Table 1 summarizes the results of the 18S ssu rRNA gene sequence analysis for these fungal isolates. The amplified fungal sequences were used as BLASTN queries against the NCBI database. The sequences of the PCR amplicons were found to be 98% - 100% similar to the sequences of 18S rRNA regions of the respective genera and species of closely related fungi. For example, the fungal sequence amplified from Marine Science Station sediment isolate no. 5 (MSS_S5) showed 100% identity with an Aspergillus sydowi (Acc. No. JN938975) which was previously sequenced from Aspergillus sydowii strain DAOM 213727 18S ssu ribosomal RNA

Conflicts of Interest

The authors declare no conflicts of interest.

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