BioMed Research International

Volume 2014 (2014), Article ID 439197, 11 pages

http://dx.doi.org/10.1155/2014/439197

Research Article

Diversity and Enzymatic Profiling of Halotolerant Micromycetes from Sebkha El Melah, a Saharan Salt Flat in Southern Tunisia

Atef Jaouani,¹ Mohamed Neifar,¹ Valeria Prigione,² Amani Ayari,¹ Imed Sbissi,¹ Sonia Ben Amor,¹ Seifeddine Ben Tekaya,¹ Giovanna Cristina Varese,² Ameur Cherif,³ and Maher Gtari¹

¹Laboratoire Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis, Université Tunis El Manar, Campus Universitaire, 2092 Tunis, Tunisia

²Dipartimento di Scienze della Vita e Biologia dei Sistemi, Università degli Studi di Torino, Viale Mattioli 25, 10125 Torino, Italy

³Laboratoire Biotechnologie et Valorisation des Bio-Géo Ressources, Institut Supérieur de Biotechnologie de Sidi Thabet, Université La Manouba, 2020 Sidi Thabet, Tunisia

Received 2 May 2014; Accepted 28 June 2014; Published 16 July 2014

Academic Editor: Sara Borin

Copyright © 2014 Atef Jaouani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Twenty-one moderately halotolerant fungi have been isolated from sample ashes collected from Sebkha El Melah, a Saharan salt flat located in southern Tunisia. Based on morphology and sequence inference from the internal transcribed spacer regions, 28S rRNA gene and other specific genes such as β-tubulin, actin, calmodulin, and glyceraldehyde-3-phosphate dehydrogenase, the isolates were found to be distributed over 15 taxa belonging to 6 genera of Ascomycetes: Cladosporium (), Alternaria (), Aspergillus (), Penicillium (), Ulocladium (), and Engyodontium (). Their tolerance to different concentrations of salt in solid and liquid media was examined. Excepting Cladosporium cladosporioides JA18, all isolates were considered as alkali-halotolerant since they were able to grow in media containing 10% of salt with an initial pH 10. All isolates were resistant to oxidative stresses and low temperature whereas 5 strains belonging to Alternaria, Ulocladium, and Aspergillus genera were able to grow at 45°C. The screening of fungal strains for sets of enzyme production, namely, cellulase (CMCase), amylase, protease, lipase, and laccase, in presence of 10% NaCl, showed a variety of extracellular hydrolytic and oxidative profiles. Protease was the most abundant enzyme produced whereas laccase producers were members of the genus Cladosporium.

1. Introduction

Sebkhas are salt flats occurring on arid coastline in North Africa, Arabia, Baja California, and Shark Bay Australia [1]. They are considered among the most poikilotopic environments and characterized by extreme salt concentrations and electromagnetic radiation exposure together with low water and nutrient availabilities [2]. Regarded as detrimental to “normal subsistence,” organisms copying such conditions to survive and thrive are designed extremophiles [3]. Beside halophytes plants and algae, the mostly diverse dwellers of sebkhas being unveiled are members of bacterial, archaeal, and fungal ranks [4–8]. Members of fungi kingdom recovered from extreme environments such as sebkhas’ have shed light on two promising viewpoints: first, as model for deciphering stress adaptation mechanisms in eukaryotes [9] and secondary, as novel and largely unexplored materials for the screening of novel bioactive natural products [10]. Over the past decade, there is an increased awareness for new hydrolytic enzymes useful under nonconventional conditions [11].

Sebkha El Melah, a Saharan salt flat of southern Tunisia, has an area of approximately 150 km² and the level is slightly below the sea. Fluvial basin excavation of Sebkha El Melah appeared at the beginning of the Würmian Quaternary period [12]. Around 40,000 BP the lagoon was highly desalinated by freshwater arrivals. At the upper Würm, seawater withdrew and the basin evolves to a temporary lake or continental sebkha. More recently, around 8000 years BP, the lagoon evolved into an evaporite basin. The sebkha sediments are composed of several saliferous layers of rock salt and gypsum (calcium sulfate) and/or polyhalite (sulfate of potassium, calcium, and magnesium) [12]. Here we report the isolation of moderately halotolerant fungi from Sebkha El Melah. Strains have been identified based on morphological and molecular markers and their resistance to salt, thermal, alkaline, and oxidative stresses was assessed. Their ability to produce different hydrolytic and oxidative enzymes under salt stress was also evaluated.

2. Material and Methods

2.1. Sampling Site Description and Fungal Isolation

Three locations from the Sebkha El Melah margins (L1: 33°23′01.1′′N 10°54′56.8′′E; L2: 33°21′42.1′′N 10°55′05.5′′E; and L3: 33°23′37.7′′N 10°53′40.2′′E) were chosen for sampling (Figure 1). From each location, a composite sample was prepared aseptically from five subsamples (1–10 cm deep) and collected from the arms and center of an X (each arm was 1 m in length) [13]. One cm soil from the ground surface was firstly removed to avoid contamination during sampling procedure. Samples were then transported to the laboratory in a cool box and stored at 4°C prior to processing.

Figure 1: Map of Sebkha El Melah (Google Earth). L1, L2, and L3 indicate locations of sampling.

Fungi were isolated on potato dextrose agar (PDA) containing 10% of NaCl and 0.05% of chloramphenicol using the soil plate method where few milligrams of sample were directly spread on the agar medium. This method has a slight edge over the dilution plate method since it allows higher total number of isolates and limits invasion by species which sporulate heavily [14].

2.2. Morphological and Molecular Identification

Isolated fungi were identified conventionally according to their macroscopic and microscopic features. After determination of their genera [15–17], they were transferred to the media recommended of selected genus monographs for species identification.

DNA extraction was achieved as described by Liu et al. [18]; the amplification of the internal transcribed spacer regions (nuclear-encoded 18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA) was performed using the couple of universal primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) [19] and the thermal cycler conditions according to Luo and Mitchell [20]. PCR was carried out in 25 μL volumes containing 2.5 μL of 1X PCR reaction buffer (100 mM Tris-HCl, 500 mM KCl, pH 8.3), 1.5 μL MgCl₂, 0.2 μmol/L (each) primer, 0.2 μmol/L (each) dNTP, and 2.5 units of Taq polymerase (Dream Taq, Fermentas) and 1 μL of DNA template. Depending on the fungus genus, different gene sequences were amplified. For the Aspergillus flavus group, the calmodulin gene was amplified using the primers CL1 (5′-GARTWCAAGGAGGCCTTCTC-3′) and CL2A (5′-TTTTGCATCATGAGTTGGAC-3′) according to Rodrigues et al. [21]; for the Cladosporium genus, the actin gene was amplified using the primers ACT-512F (5′-ATGTGCAAGGCCGGTTTCGC-3′) and ACT-783R (5′-TACGAGTCCTTCTGGCCCAT-3′) according to Bensch et al. [22]; for Alternaria genus, the glyceraldehyde-3-phosphate dehydrogenase gene was amplified using the primers GPD1 (5′-CAACGGCTTCGGTCGCATTG-3′) and GPD2 (5′-GCCAAGCAGTTGGTTGTGC-3′) according to Berbee et al. [23]; for Penicillium and Aspergillus genera, the β-tubulin gene was amplified using the primers Bt2a (5′-GGTAACCAAATCGGTGCTGCTTTC-3′) and Bt2B (5′-ACCCTCAGTGTAGTGACCCTTGGC-3′) according to Glass and Donaldson [24].

The PCR products were purified with QIAquick Wizard PCR purification Kit (Promega) according to the manufacturer’s instructions, and the sequences were determined by cycle sequencing using the Taq Dye Deoxy Terminator Cycle Sequencing kit (Applied Biosystems, HTDS, Tunisia) and fragment separation in an ABI PrismTM 3130 DNA sequencer (Applied Biosystems, HTDS, Tunisia). The sequences obtained were compared reference sequences in the NCBI GenBank database using the BLASTN search option [25].

2.3. Effect of pH, Salinity, Temperature, and Oxidative Stress

PDA medium was used to study the effect of different stresses on solid media. For oxidative stresses, H₂O₂ or paraquat was filter sterilized and added separately to melted PDA medium previously autoclaved. Paraquat is a redox-cycling agent widely used to generate reactive oxygen species and induce oxidative stress in bacteria [26] and fungi [27]. For pH stress, PDA medium was buffered with 100 mM Glycine-NaOH to pH 10 before autoclaving. Salt stress in solid media was studied in PDA medium containing different concentrations of salts. The inoculated plates with 3 mm cylindrical mycelial plugs were then incubated at 30°C for oxidative, salt, and pH stresses and at 4°C and 45°C for thermal stresses, and radial growth was measured daily. Results were expressed as relative growth of fungal strains under different stresses as follows: (Colony diameter under stress/colony diameter without stress after 7 days incubation) 100.

The effect of salinity in liquid medium was carried out in Biolog system, a commercially redox based test (Biolog Inc., Hayward, CA). Malt extracts (2%) containing 0%, 5%, 10%, 15%, and 20% of salt were inoculated by a suspension of spores and fragmented mycelium according to the supplier’s instructions in 96-well microtiter plates. After 15 days incubation at 30°C, the numeric results were extracted using PM Data Analysis 1.3 software. The fungal growth was assimilated to the reduction of the redox indicator. The ability of the fungus to grow in the presence of salt was expressed as the ratio of kinetic curve surface under stress versus without stress.

2.4. Extracellular Enzymes Production Profiling

The capacity of fungal isolates to produce extracellular enzymes, namely, amylase, cellulase, protease, laccase and lipase, was assayed in the presence of 10% of NaCl. Inoculation was made by transferring 3 mm of cylindrical mycelial plugs on the corresponding media. Amylase production was assayed on PDA containing 1% soluble starch. Enzyme production is shown by the presence of clear halo when iodine was poured onto the plates. Cellulase production was tested on PDA medium containing 1% of carboxymethylcellulose. The presence of activity is reflected by a clear halo on red background after flooding the plates with 0.2% Congo red for 30 min. Protease production was revealed on skim milk agar by the appearance of a clear zone corresponding to casein hydrolysis/solubilization surrounding the microbial colony. The laccase production was detected on PDA medium containing 5 mM of 2,6 dimethoxyphenol (DMP). Oxidation of the substrate is indicated by the appearance of brown color. Lipase production was tested on PDA medium containing 10 mL/L of Tween 20 and 0.1 g/L of CaCl₂. Positive reaction is accompanied by the presence of precipitates around the fungal colony. The enzymes production was expressed as activity ratio (PR) which corresponds to the activity diameter (halo of enzymatic reaction) divided by the colony diameter after 7 days incubation at 30°C.

2.5. Statistical Analysis

The data presented are the average of the results of at least three replicates with a standard error of less than 10%.

3. Results

3.1. Isolation and Identification of Halotolerant Fungi

Twenty-one fungal isolates were obtained on halophilic medium containing 10% NaCl and subjected to morphological and molecular identification. Seventeen strains were identified at genus level based on 28S rRNA gene sequences, while four were identified based on ITS regions. Final assignment was based on combination of morphological and β-tubulin, actin, calmodulin, and glyceraldehyde-3-phosphate dehydrogenase genes sequencing (Table 1). The 21 strains have been identified as Cladosporium cladosporioides (), Cladosporium halotolerans (), Cladosporium sphaerospermum (), Alternaria tenuissima (), Aspergillus flavus (), Aspergillus fumigatiaffinis (), Aspergillus fumigatus (), Penicillium canescens (), Penicillium chrysogenum (), Penicillium sp. (), Alternaria alternata (), Ulocladium consortiale (), Ulocladium sp. (), Engyodontium album (), and Embellisia phragmospora () species. All the strains have been deposited at the Mycotheca Universitatis Taurinensis (MUT) in the University of Turin.

Table 1: Identification of fungal isolates.

Strain code
28S identification
ITS identification
Identification based on specific primers
Morphological identification
Final identification and accession number in NCBI

JA1
Penicillium
Penicillium flavigenum JX997105 (100%) 

P. confertum JX997081 (100%) 

P. dipodomyis JX997080 (100%) 

P. commune KC333882 (100%) 

P. chrysogenum KC009827 (100%) 

P. griseofulvum JQ781833 (100%)
Penicillium  chrysogenum 

(β-tubulin)
Penicillium  chrysogenum
Penicillium  chrysogenum Thom 

28S KF417559 

ITS KF417577

JA2
Cladosporium
Cladosporium sp. GU017498 (100%) 

Hyalodendron sp. AM176721 (100%) 

C. sphaerospermum AB572902 (99%) 

C. cladosporioides EF568045 (99%)
Cladosporium  sphaerospermum (Actin)
nd
Cladosporium sphaerospermum Penzig 

28S: KF417560 

ITS: KF417578

JA3
Penicillium chrysogenum
Penicillium canescens HQ607858(99%)
Penicillium  chrysogenum 

(β-tubulin)
Penicillium  chrysogenum
Penicillium  chrysogenum Thom 

28S: KF417561 

ITS: KF417579

JA4
Aspergillus
Aspergillus aureofulgens EF669617 (100%)
Aspergillus flavus 

(calmodulin)
nd
Aspergillus flavus Link

28S: KF417562 

ITS: KF417580

JA5
nd
Penicillium desertorum JX997039 (100%) 

P. chrysogenum KC009826 (99%)
Penicillium canescens group 

(β-tubulin and calmodulin)
nd
Penicillium canescens Sopp 

ITS: KF417581

JA6
Alternaria
Alternaria triticimaculans JN867470 (100%) 

A. tenuissima JN867469 (100%) 

A. mali JN867468 (100%) 

A. alternata JQ690087 (100%)
Alternaria tenuissima 

(GPD)
Alternaria alternata
Alternaria tenuissima (Nees)  

Wiltshire 

28S: KF417563 

ITS: KF417582

JA7
nd
Engyodontium album HM214540 (100%)

Engyodontium  album
Engyodontium album (Limber) de Hoog 

ITS: KF417583

JA8
Cladosporium
Cladosporium cladosporioides EF568045 (100%) 

C. sphaerospermum AM176719 (100%) 

C. halotolerans JX535318 (99%)
Cladosporium halotolerans 

(Actin)
Cladosporium  cladosporioides/halotolerans
Cladosporium halotolerans Zalar, de Hoog, and Gunde-Cimerman 

28S: KF417564 

ITS: KF417584

JA9
Embellisia/Chalastospora
Embellisia phragmospora JN383493 (100%)
Alternaria tenuissima similarity  

Alternaria arborescens  

Alternaria alternata (GPD)
Embellisia  phragmospora
Embellisia phragmospora (Emden) E.G.  

28S: KF417565 

ITS: KF417585

JA10
Aspergillus
Aspergillus lentulus JN943567 (99%) 

A. aff. fumigatus JN246066 (99%) 

A. fumigatiaffinis HF545316 (99%) 

A. novofumigatus FR733874 (99%)
Aspergillus fumigatus 

(β-tubulin)
Aspergillus fumigatiaffinis
Aspergillus fumigatus Fresenius 

28S: KF417566 

ITS: KF417586

JA11
nd
Aspergillus aff. fumigatus JN246066 (100%) 

A. fumigatiaffinis KC253955 (99%)
Aspergillus fumigatiaffinis 

(β-tubulin)
nd
Aspergillus fumigatiaffinis Hong, Frisvad, and Samson 

ITS: KF417587

JA12
Ulocladium
Ulocladium consortiale JQ585682 (100%) 

Alternaria radicina HM204457 (99%)
Ulocladium consortiale (GPD)
Ulocladium tuberculatum/consortiale
Ulocladium consortiale (Thümen) E.G. Simmons 

28S: KF417567 

ITS: KF417588

JA13
Cladosporium
Cladosporium cladosporioides JX868638 (99%) 

C. sphaerospermum HM999943 (99%)
Cladosporium  sphaerospermum group 

(Actin)
Cladosporium  sphaerospermum
Cladosporium sphaerospermum Penzig 

28S: KF417568 

ITS: KF417589

JA14
Cladosporium/Davidiella
Cladosporium cladosporioides KC009539 (99%) 

Davidiella tassiana GU248332 (98%)
nd
nd
Cladosporium cladosporioides (Fresenius) G.A. de Vries 

28S: KF417569 

ITS: KF417590

JA15
Penicillium
Penicillium spinulosum KC167852 (100%) 

P. glabrum KC009784 (100%)
nd
Penicillium glabrum
Penicillium sp.  

28S: KF417570 

ITS: KF417591

JA17
Ulocladium
Ulocladium consortiale JQ585682 (100%) 

U. chartarum JN942881 (99%)
nd
Ulocladium sp.
Ulocladium sp.  

28S: KF417572 

ITS: KF417593

JA18
Cladosporium
Cladosporium cladosporioides HQ380770 (100%)
nd
Cladosporium cladosporioides
Cladosporium cladosporioides (Fresenius) G.A. de Vries 

28S: KF417573 

ITS: KF417594

JA19
Alternaria
Alternaria sp. KC139473 (100%) 

A. arborescens JQ781762 (100%) 

A. alternata JN107734 (100%)
Alternaria tenuissima  

Alternaria arborescens  

Alternaria alternata  

(GPD)
Alternaria alternata
Alternaria alternata Keissler 

28S: KF417574 

ITS: KF417595

JA20
Alternaria
Alternaria brassicae JX290150 (100%) 

A. porri HQ821479 (100%)
Alternaria tenuissima  

Alternaria arborescens  

Alternaria alternata  

(GPD)
Alternaria alternata
Alternaria alternata Keissler 

28S: KF417575 

ITS: KF417596

JA22
Penicillium
Penicillium chrysogenum KC341721 (99%) 

P. dipodomyicola JX232278 (99%) 

P. rubens JX003126 (99%) 

P. commune JN676122 (99%)
Penicillium  chrysogenum 

(β-tubulin)
Penicillium  chrysogenum
Penicillium  chrysogenum Thom 

28S: KF417576 

ITS: KF417597

JA23
nd
Alternaria alternata JQ809324 (100%) 

A. quercus KC329620 (100%) 

A. tenuissima KC329619 (100%) 

A. atrans KC329618 (100%)
Alternaria tenuissima  

Alternaria arborescens  

Alternaria alternata  

(GPD)
Alternaria alternata
Alternaria alternata Keissler 

ITS: KF417598

3.2. Salt Tolerance of Fungal Isolates

Salt tolerance of the fungal isolates was assessed on solid and liquid media for NaCl content ranging from 5 to 20%. In solid media, salt tolerance was estimated as relative growth represented by the ratio of colony diameter under salt stress to that without salt stress. As illustrated in Table 2, all the isolated strains succeeded to grow in the presence of 10% of salt. While 19 isolates remain able to grow under 15% NaCl, only 7 isolates tolerated 20% NaCl: Penicillium chrysogenum JA1 and JA22, Cladosporium halotolerans JA8, Cladosporium sphaerospermum JA2, Cladosporium cladosporioides JA18, Aspergillus flavus JA4, and Engyodontium album JA7.

Table 2: Effect of salt concentration on fungal growth in solid and liquid media.

Strain code
Strain
Solid media (1)
Liquid media (2)

5% NaCl
10% NaCl
15% NaCl
20% NaCl
5% NaCl
10% NaCl
15% NaCl
20% NaCl

JA1
Penicillium  chrysogenum
74
72
60
18
83
54
10
0

JA3
Penicillium  chrysogenum
100
72
37
0
96
46
11
0

JA22
Penicillium  chrysogenum
100
82
41
25
90
46
0
0

AJ5
Penicillium canescens
70
30
20
0
79
44
0
0

JA15
Penicillium sp.
83
70
34
0
53
18
0
0

JA8
Cladosporium halotolerans
80
68
32
18
30
0
0
0

JA2
Cladosporium  sphaerospermum
76
64
34
22
47
19
11
0

JA13
Cladosporium sphaerospermum
100
49
25
0
81
79
12
0

JA14
Cladosporium cladosporioides
40
30
10
0
0
0
0
0

JA18
Cladosporium cladosporioides
58
40
24
8
63
61
4
0

JA4
Aspergillus flavus
90
80
48
26
56
16
0
0

JA10
Aspergillus fumigatus
100
76
35
0
100
57
11
0

JA11
Aspergillus fumigatiaffinis
100
46
25
0
52
30
12
0

JA19
Alternaria alternata
52
38
24
0
57
9
0
0

JA20
Alternaria alternata
60
40
0
0
37
24
0
0

JA23
Alternaria alternata
100
68
20
0
65
12
0
0

JA6
Alternaria tenuissima
100
60
22
0
80
55
17
0

JA9
Embellisia phragmospora
94
50
10
0
78
26
0
0

JA12
Ulocladium consortiale
72
28
0
0
32
10
0
0

JA17
Ulocladium sp.
100
70
28
0
67
20
0
0

JA7
Engyodontium album
56
36
14
10
43
10
0
0

(1) Relative growth on solid media after 7 days incubation = (⌀ colony under salt stress/⌀ colony without salt stress) × 100. (2) Relative growth in liquid media after 7 days incubation = (kinetic curve surface under salt stress/kinetic curve surface without salt stress) × 100.

When liquid cultures were used, fungal isolates seemed to become more sensitive to salt stress. Indeed, none of the strains was able to grow in the presence of 20% NaCl, whereas only 8 strains and 19 strains tolerated 15% and 10% NaCl, respectively (Table 2).

3.3. Alkaline, Temperature, and Oxidative Stress

Excepting Cladosporium cladosporioides JA18, all tested strains were able to grow at pH 10. All isolates were able to grow at 4°C while only five strains Aspergillus fumigatus JA10, Aspergillus fumigatiaffinis JA11, Alternaria alternata JA23, Ulocladium consortiale JA12, and Ulocladium sp. JA17 showed a significant growth at 45°C. All 21 strains tolerated oxidative stress generated by 10 mM H₂O₂ and 500 μM paraquat (Table 3).

Table 3: Effect of alkaline, thermal, and oxidative stresses on fungal growth.

Strain code
Strain
Alkaline stress (1)
Thermal stress (2)
Oxidative stress (3)

pH 10
4°C
45°C
H₂O₂  10 mM
Paraquat 500 µM

JA1
Penicillium  chrysogenum
43
39
—
66
74

JA3
Penicillium  chrysogenum
42
50
—
84
71

JA22
Penicillium  chrysogenum
47
45
—
68
53

JA5
Penicillium canescens
26
28
—
59
63

JA15
Penicillium sp.
43
100
—
100
100

JA8
Cladosporium halotolerans
34
26
—
44
40

JA2
Cladosporium  sphaerospermum
21
24
—
52
48

JA13
Cladosporium sphaerospermum
21
43
—
55
44

JA14
Cladosporium cladosporioides
34
38
—
20
31

JA18
Cladosporium cladosporioides
—
41
—
18
16

JA4
Aspergillus flavus
46
22
—
47
39

JA10
Aspergillus fumigatus
89
41
61
100
100

JA11
Aspergillus fumigatiaffinis
94
26
100
100
100

JA19
Alternaria alternata
49
35
—
69
89

JA20
Alternaria alternata
58
48
—
100
100

JA23
Alternaria alternata
100
83
40
57
52

JA6
Alternaria tenuissima
57
30
—
81
100

JA9
Embellisia phragmospora
58
67
—
100
100

JA12
Ulocladium consortiale
44
37
36
56
100

JA17
Ulocladium sp.
93
28
100
81
100

JA7
Engyodontium album
34
18
—
66
53

Relative growth of fungal strains under different stresses after 7 days incubation was expressed as follows: (1) (⌀ colony at pH 10/⌀ colony at pH 5) × 100; (2) (⌀ colony at 45°C or 4°C/⌀ colony at 30°C) × 100; (3) (⌀ colony with H₂O₂ or paraquat/⌀ colony without stress) × 100. —: not significant growth.

3.4. Enzymatic Profiling of Isolates

Among the 21 strains tested, 13 strains displayed at least one of the five-screened activities: protease, amylase, cellulase, lipase, and laccase, in the presence of 10% NaCl (Table 4). Protease and amylase were the most abundant activities shown by 9 and 6 strains, respectively. Four strains belonging to Cladosporium and Penicillium genera produced laccase while Cladosporium sphaerospermum JA2, Aspergillus flavus JA4, and Engyodontium album JA7 were able to produce lipase. Cellulase activity was detected only in Penicillium sp. JA15.

Table 4: Enzymes activities of fungal isolates in the presence of 10% NaCl.

Strain code
Strain
Protease
Amylase
Cellulase
Lipase
Laccase

JA1
Penicillium  chrysogenum
++
+
−
−
−

JA3
Penicillium  chrysogenum
++
−
−
−
−

JA22
Penicillium  chrysogenum
+
+
−
−
−

AJ5
Penicillium canescens
−
−
−
−
+

JA15
Penicillium sp.
−
−
+
−
−

JA8
Cladosporium halotolerans
+
−
−
−
+

JA2
Cladosporium  sphaerospermum
+++
−
−
+
+

JA13
Cladosporium sphaerospermum
−
+
−
−
+

JA14
Cladosporium cladosporioides
+
+
−
−
−

JA18
Cladosporium cladosporioides
−
−
−
−
−

JA4
Aspergillus flavus
+
−
−
+
−

JA10
Aspergillus fumigatus
−
−
−
−
−

JA11
Aspergillus fumigatiaffinis
−
−
−
−
−

JA19
Alternaria alternata
−
−
−
−
−

JA20
Alternaria alternata
−
+
−
−
−

JA23
Alternaria alternata
−
−
−
−
−

JA6
Alternaria tenuissima
+
−
−
−
−

JA9
Embellisia phragmospora
−
−
−
−
−

JA12
Ulocladium consortiale
−
−
−
−
−

JA17
Ulocladium sp.
−
−
−
−
−

JA7
Engyodontium album
+
+
−
++
−

AR: activity ratio = (⌀ activity/⌀ colony). −: no activity; +: AR < 1; ++: 1 < AR < 2; +++: 2 < AR < 3.

4. Discussion

With regard to bacteria that have been well explored in southern desert region of Tunisia [28–31], data related to fungi are scarce and are limited to truffle and mycorrhiza, so far considered as real specialists of desert environments [32, 33]. To the best of our knowledge, this is the first report on the isolation and characterization of fungi from Tunisian desert and particularly from salt flat. A collection of 21 fungi isolates have been established from samples ashes collected from Sebkha El Melah. These alkalihalotolerant fungi have been assigned to 15 taxa belonging to 6 genera of Ascomycetes. Several studies showed that fungi belonging to Cladosporium, Alternaria, and Ulocladium genera were clearly predominant under desert and salty environments [34, 35]. These fungi have in common thick-walled and strongly melanized spores which are important for UV, radiation, and desiccation tolerance [10]. On the other hand, Molitoris et al. [36] reported that other halotolerant and halophilic fungi such as Aspergillus and Cladosporium spp. are predominant in saline desert soil of Dead Sea. Many Aspergillus species have been also reported to constitute dominant fungi in desert of Saudi Arabia and Libya [37, 38], and halotolerant species, including Aspergillus spp., Penicillium spp., and Cladosporium sphaerospermum, have been consistently isolated from hypersaline environments around the globe [39]. In this study, contrary to many reports on hypersaline environments, no species belonging to the genera Eurotium, Thrimmatostroma, Emericella, and Phaeotheca [9] have been obtained, probably because of the initial alkaline pH of the Sebkha El Melah salt lake. Actually, the effect of pH on the fungal diversity is controversial. Misra [40] observed that fungal diversity varies with the pH while other investigators found no significant effect of pH values of water and soil habitats on fungal occurrence [41]. It is more likely that the number of the isolated fungi is directly correlated to the organic matter content of water, mud, and soil samples [42].

Beside the identification of the recovered fungal isolates from Sebkha El Melah, the second goal of the current study was the detection of some of their physiological and biochemical features. This allows understanding ecological adaptation to extreme environment and predicts some biotechnological usage. The 21 strains have been screened for tolerance to extreme NaCl concentrations, basic pH, temperature, and oxidative stress and for the production of important enzymatic activities in presence of 10% NaCl.

Excepting Cladosporium cladosporioides JA18, all isolates obtained in this study can be considered as moderately haloalkaliphilic fungi as deduced from their ability to grow at pH 10 and 10% of NaCl. However, the isolates were able to grow when salt was not added to their growing media. Excepting some Wallemia ichthyophaga the most strictly halophilic fungus [43], all other fungal strains known to date are able to grow without salt, a fact confirmed in our study. However, gradual decrease in fungal growth was observed with the increasing of salt concentration in the culture medium. Nineteen strains remain able to grow under 15% of NaCl, whereas 7 strains were able to tolerate 20% of NaCl. This result was confirmed by salt tolerance assay in liquid media as estimated by Biolog system. It is noteworthy that fungi were more sensitive to salt stress in liquid media than in solid media. This could be explained by the alteration of the osmotic gradient, forcing the fungi to expend more energy in the osmoregulatory processes, resulting in slower growth [44]. Moreover, at higher salt concentration death occurs.

Regarding the stress of pH, the capacity of the majority of isolates to growth at pH 10 implies firstly that some habitats in the salt lake may have a varying pH and secondly that fungi can tolerate a wide pH range. Prima facie, the overall results in solid and liquid media showed that Penicillium chrysogenum JA1 and JA3, Cladosporium sphaerospermum JA2 and JA13, Cladosporium cladosporioides JA18, Aspergillus fumigatus JA10, Aspergillus fumigatiaffinis JA11, and Alternaria tenuissima JA6 are the most alkalihalotolerant isolates in this study.

The tolerance of the strains to extreme 45°C was tested and results indicated that Aspergillus fumigatus JA10, Alternaria alternata JA23, Ulocladium sp. JA17, and Aspergillus fumigatiaffinis JA11 were able to grow. Of particular interest, the latter two strains retained 100% of the growth rate and biomass production as estimated by colony diameter. Moreover, their ability to grow at low temperature may allow them to better adapt to the big temperature fluctuation in desert environments. Additionally, exposure to substrates generating oxidative stress such as H₂O₂ at 10 mM and paraquat at 500 μM did not alter significantly the growth of almost tested strains demonstrating their ability to tolerate oxidative stress. These findings may explain their presence in desert regions that are considered amongst the most stressful environments on Earth because of the high UV radiation, desiccation, increased salinity, low nutrient availability, seasonal and daily temperature variation, and solar irradiation [6, 10].

It has been postulated that microorganisms sharing a rich and particular extracellular enzymatic activities are common in harsh conditions characterizing their ecological habitat including high level of aridity, temperature, ionic strength, and particularly the low nutrient availability [31]. This implies the need by microorganisms for an effective utilization of each possible available organic compound [45]. Moreover, fungal isolates from hot desert were revealed to play an important role in seeds germination by breaking dormancy and increasing water uptake [46]. In the present study, the capacity of fungal isolates to produce extracellular enzymes was assayed in the presence of 10% of NaCl. Enzymes tested were the following: amylase for degradation of starch, abundant carbohydrate polymer in many plant tissues; protease for degradation of plant and animal proteins; cellulase which hydrolyses the cellulose, the main component of wood, ubiquitous substrate for fungi; and finally the laccase involved in plant material delignification and in the synthesis of the melanin and related compounds to protect fungi against radiation. Thirteen strains displayed high productions at least for one of the five-screened activities while no clear correlation of enzyme production profile with fungal systematic groups was noted. The abundance of protease activity is in line with previous data on fungal isolates from extreme environments showing high caseinase activities with little effect of salinity and temperature on enzyme production [36]. The relative limited number of isolates displaying cellulase, amylase, lipase, and laccase activities suggests that high concentration of salt may have an adverse effect on enzyme production and/or activity. Their energy was probably oriented to avoid salt stress due to 10% NaCl rather than the production of bioactive extrolites [47]. However, not detecting the enzyme is not absolute confirmation of an isolate inability to produce it. It could also mean that the enzyme was not released from the mycelium or that the medium is inadequate for its detection [48]. Laccase production in the presence of 10% of salt by the Cladosporium group may be of biotechnological interest, for example, in mycoremediation of high salty environments contaminated by organic pollutants.

In conclusion, fungal community described in this study was similar to those reported in inhospitable habitats characterized by limitation of nutrients, moisture deficit, and exposure to high solar radiation. Further studies are needed in order to elucidate their biogeochemical roles in such an extreme environment and to exploit their promising potential to produce new biomolecules such as enzymes and protective agents against oxidative stress.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

The authors acknowledge the financial support from the European Union in the ambit of the Project BIODESERT (EU FP7-CSA-SA REGPOT-2008-2, Grant agreement no. 245746) and the Tunisian Ministry of Higher Education and Scientific Research in the ambit of the laboratory Project LR MBA20. Atef Jaouani wants to thank the Tunisian Society for Microbial Ecology (ATEM) for supporting publication fees of this work.

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