Research ArticleDiversity and Enzymatic Profiling of Halotolerant Micromycetesfrom Sebkha El Melah a Saharan Salt Flat in Southern Tunisia

Atef Jaouani1 Mohamed Neifar1 Valeria Prigione2 Amani Ayari1

Imed Sbissi1 Sonia Ben Amor1 Seifeddine Ben Tekaya1 Giovanna Cristina Varese2

Ameur Cherif3 and Maher Gtari1

1 LaboratoireMicroorganismes et Biomolecules Actives Faculte des Sciences de Tunis Universite Tunis ElManar CampusUniversitaire2092 Tunis Tunisia

2 Dipartimento di Scienze della Vita e Biologia dei Sistemi Universita degli Studi di Torino Viale Mattioli 25 10125 Torino Italy3 Laboratoire Biotechnologie et Valorisation des Bio-Geo Ressources Institut Superieur de Biotechnologie de Sidi ThabetUniversite La Manouba 2020 Sidi Thabet Tunisia

Correspondence should be addressed to Atef Jaouani atefjaouaniissbatrnutn

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

Academic Editor Sara Borin

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

Twenty-one moderately halotolerant fungi have been isolated from sample ashes collected from Sebkha El Melah a Saharan saltflat located in southern Tunisia Based on morphology and sequence inference from the internal transcribed spacer regions 28SrRNA gene and other specific genes such as 120573-tubulin actin calmodulin and glyceraldehyde-3-phosphate dehydrogenase theisolates were found to be distributed over 15 taxa belonging to 6 genera of Ascomycetes Cladosporium (119899 = 3) Alternaria (119899 = 4)Aspergillus (119899 = 3) Penicillium (119899 = 5) Ulocladium (119899 = 2) and Engyodontium (119899 = 2) Their tolerance to different concentrationsof 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 inmedia containing 10 of salt with an initial pH 10 All isolates were resistant to oxidativestresses and low temperaturewhereas 5 strains belonging toAlternariaUlocladium andAspergillus generawere able to growat 45∘CThe screening of fungal strains for sets of enzyme production namely cellulase (CMCase) amylase protease lipase and laccase inpresence of 10NaCl showed a variety of extracellular hydrolytic and oxidative profiles Protease was the most abundant enzymeproduced whereas laccase producers were members of the genus Cladosporium

1 Introduction

Sebkhas are salt flats occurring on arid coastline in NorthAfrica Arabia Baja California and Shark Bay Australia [1]They are considered among the most poikilotopic environ-ments and characterized by extreme salt concentrations andelectromagnetic radiation exposure together with low waterand nutrient availabilities [2] Regarded as detrimental toldquonormal subsistencerdquo organisms copying such conditions tosurvive and thrive are designed extremophiles [3] Besidehalophytes plants and algae the mostly diverse dwellers ofsebkhas being unveiled are members of bacterial archaealand fungal ranks [4ndash8]Members of fungi kingdom recoveredfrom extreme environments such as sebkhasrsquo have shed light

on two promising viewpoints first as model for decipheringstress adaptation mechanisms in eukaryotes [9] and sec-ondary as novel and largely unexplored materials for thescreening of novel bioactive natural products [10] Overthe past decade there is an increased awareness for newhydrolytic enzymes useful under nonconventional conditions[11]

Sebkha El Melah a Saharan salt flat of southern Tunisiahas an area of approximately 150 km2 and the level is slightlybelow the sea Fluvial basin excavation of Sebkha El Melahappeared at the beginning of the Wurmian Quaternaryperiod [12] Around 40000 BP the lagoon was highly desali-nated by freshwater arrivals At the upper Wurm seawaterwithdrew and the basin evolves to a temporary lake or

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 439197 11 pageshttpdxdoiorg1011552014439197

2 BioMed Research International

continental sebkha More recently around 8000 years BPthe lagoon evolved into an evaporite basin The sebkhasediments are composed of several saliferous layers of rocksalt and gypsum (calcium sulfate) andor polyhalite (sulfateof potassium calcium and magnesium) [12] Here we reportthe isolation ofmoderately halotolerant fungi from Sebkha ElMelah Strains have been identified based on morphologicaland molecular markers and their resistance to salt thermalalkaline and oxidative stresses was assessed Their ability toproduce different hydrolytic and oxidative enzymes undersalt stress was also evaluated

2 Material and Methods

21 Sampling Site Description and Fungal Isolation Threelocations from the Sebkha El Melah margins (L133∘23101584001110158401015840N 10∘54101584056810158401015840E L2 33∘21101584042110158401015840N 10∘55101584005510158401015840Eand L3 33∘23101584037710158401015840N 10∘53101584040210158401015840E) were chosen for sampling(Figure 1) From each location a composite sample wasprepared aseptically from five subsamples (1ndash10 cm deep)and collected from the arms and center of an X (each armwas 1m in length) [13] One cm soil from the ground surfacewas firstly removed to avoid contamination during samplingprocedure Samples were then transported to the laboratoryin a cool box and stored at 4∘C prior to processing

Fungi were isolated on potato dextrose agar (PDA)containing 10 of NaCl and 005 of chloramphenicol usingthe soil plate method where few milligrams of sample weredirectly spread on the agar mediumThis method has a slightedge over the dilution plate method since it allows highertotal number of isolates and limits invasion by species whichsporulate heavily [14]

22 Morphological and Molecular Identification Isolatedfungi were identified conventionally according to theirmacroscopic and microscopic features After determinationof their genera [15ndash17] they were transferred to the mediarecommended of selected genus monographs for speciesidentification

DNA extraction was achieved as described by Liu etal [18] the amplification of the internal transcribed spacerregions (nuclear-encoded 18S rRNA-ITS1-58S rRNA-ITS2-28S rRNA) was performed using the couple of universalprimers ITS1 (51015840-TCC GTA GGT GAA CCT GCG G-31015840) andITS4 (51015840-TCC TCC GCT TAT TGA TAT GC-31015840) [19] andthe thermal cycler conditions according to Luo and Mitchell[20] PCRwas carried out in 25 120583L volumes containing 25 120583Lof 1X PCR reaction buffer (100mM Tris-HCl 500mM KClpH 83) 15 120583L MgCl

2 02 120583molL (each) primer 02 120583molL

(each) dNTP and 25 units of Taq polymerase (Dream TaqFermentas) and 1 120583L of DNA template Depending on thefungus genus different gene sequences were amplified Forthe Aspergillus flavus group the calmodulin gene was ampli-fied using the primers CL1 (51015840-GARTWCAAGGAGGCC-TTCTC-31015840) andCL2A (51015840-TTTTGCATCATGAGTTGGAC-31015840) according to Rodrigues et al [21] for the Cladosporium

L1L2

L3

Alg

eria Tunisia

Med

iterr

anea

n Se

aLi

bya

Figure 1 Map of Sebkha El Melah (Google Earth) L1 L2 and L3indicate locations of sampling

genus the actin gene was amplified using the primers ACT-512F (51015840-ATGTGCAAGGCCGGTTTCGC-31015840) and ACT-783R (51015840-TACGAGTCCTTCTGGCCCAT-31015840) according toBensch et al [22] for Alternaria genus the glyceraldehyde-3-phosphate dehydrogenase gene was amplified using theprimers GPD1 (51015840-CAACGGCTTCGGTCGCATTG-31015840) andGPD2 (51015840-GCCAAGCAGTTGGTTGTGC-31015840) according toBerbee et al [23] for Penicillium and Aspergillus generathe 120573-tubulin gene was amplified using the primers Bt2a(51015840-GGTAACCAAATCGGTGCTGCTTTC-31015840) and Bt2B(51015840-ACCCTCAGTGTAGTGACCCTTGGC-31015840) according toGlass and Donaldson [24]

The PCR products were purified with QIAquick WizardPCR purification Kit (Promega) according to the manu-facturerrsquos instructions and the sequences were determinedby cycle sequencing using the Taq Dye Deoxy Termi-nator Cycle Sequencing kit (Applied Biosystems HTDSTunisia) and fragment separation in an ABI PrismTM 3130DNA sequencer (Applied Biosystems HTDS Tunisia) Thesequences obtained were compared reference sequences inthe NCBI GenBank database using the BLASTN searchoption [25]

23 Effect of pH Salinity Temperature and Oxidative StressPDAmediumwas used to study the effect of different stresseson solid media For oxidative stresses H

2O2or paraquat was

filter sterilized and added separately to melted PDA mediumpreviously autoclaved Paraquat is a redox-cycling agentwidely used to generate reactive oxygen species and induceoxidative stress in bacteria [26] and fungi [27] For pH stressPDA medium was buffered with 100mM Glycine-NaOH topH 10 before autoclaving Salt stress in solid media wasstudied in PDA medium containing different concentrationsof salts The inoculated plates with 3mm cylindrical mycelialplugs were then incubated at 30∘C for oxidative salt andpH stresses and at 4∘C and 45∘C for thermal stresses andradial growth was measured daily Results were expressedas relative growth of fungal strains under different stressesas follows (Colony diameter under stresscolony diameterwithout stress after 7 days incubation) times 100

The effect of salinity in liquid medium was carried out inBiolog system a commercially redox based test (Biolog IncHayward CA) Malt extracts (2) containing 0 5 1015 and 20of salt were inoculated by a suspension of spores

BioMed Research International 3

and fragmentedmycelium according to the supplierrsquos instruc-tions in 96-well microtiter plates After 15 days incubationat 30∘C the numeric results were extracted using PM DataAnalysis 13 software The fungal growth was assimilated tothe reduction of the redox indicatorThe ability of the fungusto grow in the presence of salt was expressed as the ratio ofkinetic curve surface under stress versus without stress

24 Extracellular Enzymes Production Profiling The capacityof fungal isolates to produce extracellular enzymes namelyamylase cellulase protease laccase and lipase was assayedin the presence of 10 of NaCl Inoculation was madeby transferring 3mm of cylindrical mycelial plugs on thecorresponding media Amylase production was assayed onPDA containing 1 soluble starch Enzyme production isshown by the presence of clear halo when iodine waspoured onto the plates Cellulase production was testedon PDA medium containing 1 of carboxymethylcelluloseThe presence of activity is reflected by a clear halo on redbackground after flooding the plates with 02 Congo redfor 30min Protease production was revealed on skim milkagar by the appearance of a clear zone corresponding tocasein hydrolysissolubilization surrounding the microbialcolonyThe laccase productionwas detected on PDAmediumcontaining 5mM of 26 dimethoxyphenol (DMP) Oxidationof the substrate is indicated by the appearance of brown colorLipase production was tested on PDA medium containing10mLL of Tween 20 and 01 gL of CaCl

2 Positive reaction

is accompanied by the presence of precipitates around thefungal colony The enzymes production was expressed asactivity ratio (PR) which corresponds to the activity diameter(halo of enzymatic reaction) divided by the colony diameterafter 7 days incubation at 30∘C

25 Statistical Analysis The data presented are the average ofthe results of at least three replicates with a standard error ofless than 10

3 Results

31 Isolation and Identification of Halotolerant FungiTwenty-one fungal isolates were obtained on halophilicmedium containing 10 NaCl and subjected to morpho-logical and molecular identification Seventeen strains wereidentified at genus level based on 28S rRNA gene sequenceswhile four were identified based on ITS regions Finalassignment was based on combination of morphologicaland 120573-tubulin actin calmodulin and glyceraldehyde-3-phosphate dehydrogenase genes sequencing (Table 1) The 21strains have been identified as Cladosporium cladosporioides(119899 = 2) Cladosporium halotolerans (119899 = 1) Cladosporiumsphaerospermum (119899 = 2) Alternaria tenuissima (119899 = 1)Aspergillus flavus (119899 = 1) Aspergillus fumigatiaffinis (119899 = 1)Aspergillus fumigatus (119899 = 1) Penicillium canescens (119899 = 1)Penicillium chrysogenum (119899 = 3) Penicillium sp (119899 = 1)Alternaria alternata (119899 = 3) Ulocladium consortiale (119899 = 1)Ulocladium sp (119899 = 1) Engyodontium album (119899 = 1) andEmbellisia phragmospora (119899 = 1) species All the strains have

been deposited at the Mycotheca Universitatis Taurinensis(MUT) in the University of Turin

32 Salt Tolerance of Fungal Isolates Salt tolerance of thefungal isolates was assessed on solid and liquid media forNaCl content ranging from 5 to 20 In solid media salttolerance was estimated as relative growth represented bythe ratio of colony diameter under salt stress to that withoutsalt stress As illustrated in Table 2 all the isolated strainssucceeded to grow in the presence of 10 of salt While 19isolates remain able to grow under 15 NaCl only 7 isolatestolerated 20 NaCl Penicillium chrysogenum JA1 and JA22Cladosporium halotolerans JA8 Cladosporium sphaerosper-mum JA2 Cladosporium cladosporioides JA18 Aspergillusflavus JA4 and Engyodontium album JA7

When liquid cultures were used fungal isolates seemedto become more sensitive to salt stress Indeed none of thestrains was able to grow in the presence of 20NaCl whereasonly 8 strains and 19 strains tolerated 15 and 10 NaClrespectively (Table 2)

33 Alkaline Temperature and Oxidative Stress ExceptingCladosporium cladosporioides JA18 all tested strains wereable to grow at pH 10 All isolates were able to grow at 4∘Cwhile only five strains Aspergillus fumigatus JA10 Aspergillusfumigatiaffinis JA11 Alternaria alternata JA23 Ulocladiumconsortiale JA12 andUlocladium sp JA17 showed a significantgrowth at 45∘C All 21 strains tolerated oxidative stressgenerated by 10mMH

2O2and 500 120583M paraquat (Table 3)

34 Enzymatic Profiling of Isolates Among the 21 strainstested 13 strains displayed at least one of the five-screenedactivities protease amylase cellulase lipase and laccase inthe presence of 10 NaCl (Table 4) Protease and amylasewere the most abundant activities shown by 9 and 6 strainsrespectively Four strains belonging to Cladosporium andPenicillium genera produced laccase while Cladosporiumsphaerospermum JA2 Aspergillus flavus JA4 and Engyodon-tium album JA7 were able to produce lipase Cellulase activitywas detected only in Penicillium sp JA15

4 Discussion

With regard to bacteria that have been well explored insouthern desert region of Tunisia [28ndash31] data related tofungi are scarce and are limited to truffle and mycorrhiza sofar considered as real specialists of desert environments [3233] To the best of our knowledge this is the first report on theisolation and characterization of fungi from Tunisian desertand particularly from salt flat A collection of 21 fungi isolateshave been established from samples ashes collected fromSebkha El Melah These alkalihalotolerant fungi have beenassigned to 15 taxa belonging to 6 genera of AscomycetesSeveral studies showed that fungi belonging toCladosporiumAlternaria and Ulocladium genera were clearly predominantunder desert and salty environments [34 35] These fungihave in common thick-walled and strongly melanized sporeswhich are important for UV radiation and desiccation

4 BioMed Research International

Table1Identifi

catio

nof

fung

aliso

lates

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA1

Penicilliu

m

Penicilliu

mflavigenu

mJX997105

(100)

Pconfertum

JX997081

(100)

PdipodomyisJX9

97080(100)

Pcommun

eKC3

33882(100)

Pchrysogenu

mKC

009827

(100)

Pgrise

ofulvum

JQ781833

(100)

Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417559

ITSKF

417577

JA2

Cladosporiu

m

Cladosporiu

mspG

U017498

(100)

Hyalodend

ronspA

M176721

(100)

Csphaerosperm

umAB5

72902(99

)C

cladosporioidesEF

568045

(99

)

Cladosporiu

msphaerosperm

um(A

ctin)

nd

Cladosporiu

msphaerosperm

umPenzig

28SKF

417560

ITSKF

417578

JA3

Penicilliu

mchrysogenu

mPenicilliu

mcanescensH

Q607858(99

)Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417561

ITSKF

417579

JA4

Aspergillus

Aspergillus

aureofulgens

EF669617

(100)

Aspergillus

flavus

(calmodulin)

ndAspergillus

flavusL

ink

28SKF

417562

ITSKF

417580

JA5

ndPenicilliu

mdesertorum

JX997039

(100)

Pchrysogenu

mKC

009826

(99

)Penicilliu

mcanescensgroup

(120573-tu

bulin

andcalm

odulin)

ndPenicilliu

mcanescens

Sopp

ITSKF

417581

JA6

Alternaria

Alternariatriticim

aculan

sJN867470

(100)

Atenu

issim

aJN

867469

(100)

AmaliJN867468

(100)

AalternataJQ

690087

(100)

Alternariatenu

issim

a(G

PD)

Alternariaalternata

Alternariatenu

issim

a(N

ees)

Wiltshire

28SKF

417563

ITSKF

417582

JA7

ndEn

gyodontiu

malbum

HM214540

(100)

Engyodontiu

malbum

Engyodontiu

malbum

(Lim

ber)de

Hoo

gITSKF

417583

JA8

Cladosporiu

m

Cladosporiu

mcla

dosporioidesEF

568045

(100)

Csphaerosperm

umAM176719

(100)

Chalotoleran

sJX5

35318(99

)

Cladosporiu

mhalotoleran

s(Actin)

Cladosporiu

mcla

dosporioideshalotoleran

s

Cladosporiu

mhalotoleran

sZa

larde

Hoo

gand

Gun

de-C

imerman

28SKF

417564

ITSKF

417584

JA9

Embellisia

Chalasto

sporaEm

bellisia

phragm

ospora

JN383493

(100)

Alternariatenu

issim

asim

ilarity

Alternariaarborescens

Alternariaalternata(G

PD)

Embellisia

phragm

ospora

Embellisia

phragm

ospora

(Emden)

EG

28SKF

417565

ITSKF

417585

BioMed Research International 5

Table1Con

tinued

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA10

Aspergillus

Aspergillus

lentulusJN943567

(99

)A

afffum

igatus

JN2460

66(99

)A

fumigatiaffinisH

F545316(99

)A

novofumigatus

FR733874

(99

)

Aspergillus

fumiga

tus

(120573-tu

bulin

)Aspergillus

fumiga

tiaffinis

Aspergillus

fumiga

tus

Fresenius

28SKF

417566

ITSKF

417586

JA11

ndAspergillus

afffum

igatus

JN2460

66(100)

AfumigatiaffinisK

C253955(99

)

Aspergillus

fumiga

tiaffinis

(120573-tu

bulin

)nd

Aspergillus

fumiga

tiaffinis

Hon

gFrisv

adand

Samson

ITSKF

417587

JA12

Ulocla

dium

Ulocla

dium

consortia

leJQ

585682

(100)

Alternariaradicin

aHM2044

57(99

)Ulocla

dium

consortia

le(G

PD)

Ulocla

dium

tuberculatum

consortiale

Ulocla

dium

consortia

le(Th

umen)E

GSim

mon

s28SKF

417567

ITSKF

417588

JA13

Cladosporiu

mCladosporiu

mcla

dosporioidesJX868638

(99

)C

sphaerosperm

umHM999943

(99

)

Cladosporiu

msphaerosperm

umgrou

p(A

ctin)

Cladosporiu

msphaerosperm

um

Cladosporiu

msphaerosperm

umPenzig

28SKF

417568

ITSKF

417589

JA14

Cladosporiu

mD

avidiella

Cladosporiu

mcla

dosporioidesKC

009539

(99

)DavidiellatassianaGU248332

(98

)nd

nd

Cladosporiu

mcla

dosporioides

(Fresenius)G

AdeV

ries

28SKF

417569

ITSKF

417590

JA15

Penicilliu

mPenicilliu

mspinulosum

KC167852

(100)

Pgla

brum

KC00

9784

(100)

ndPenicilliu

mgla

brum

Penicilliu

msp

28SKF

417570

ITSKF

417591

JA17

Ulocla

dium

Ulocla

dium

consortia

leJQ

585682

(100)

Uchartarum

JN942881

(99

)nd

Ulocla

dium

sp

Ulocla

dium

sp

28SKF

417572

ITSKF

417593

JA18

Cladosporiu

mCladosporiu

mcla

dosporioidesHQ380770

(100)

ndCladosporiu

mcla

dosporioides

Cladosporiu

mcla

dosporioides

(Fresenius)G

AdeV

ries

28SKF

417573

ITSKF

417594

JA19

Alternaria

AlternariaspK

C139473(100)

AarborescensJQ781762

(100)

AalternataJN

107734

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

28SKF

417574

ITSKF

417595

6 BioMed Research International

Table1Con

tinued

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA20

Alternaria

Alternariabrassicae

JX290150

(100)

AporriH

Q821479

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

28SKF

417575

ITSKF

417596

JA22

Penicilliu

m

Penicilliu

mchrysogenu

mKC

341721

(99

)PdipodomyicolaJX232278

(99

)Prubens

JX003126

(99

)Pcommun

eJN676122

(99

)

Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417576

ITSKF

417597

JA23

nd

AlternariaalternataJQ

809324

(100)

AquercusK

C329620(100)

Atenu

issim

aKC

329619

(100)

Aatrans

KC329618

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

ITSKF

417598

BioMed Research International 7

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 0JA3 Penicillium chrysogenum 100 72 37 0 96 46 11 0JA22 Penicillium chrysogenum 100 82 41 25 90 46 0 0AJ5 Penicillium canescens 70 30 20 0 79 44 0 0JA15 Penicillium sp 83 70 34 0 53 18 0 0JA8 Cladosporium halotolerans 80 68 32 18 30 0 0 0JA2 Cladosporium sphaerospermum 76 64 34 22 47 19 11 0JA13 Cladosporium sphaerospermum 100 49 25 0 81 79 12 0JA14 Cladosporium cladosporioides 40 30 10 0 0 0 0 0JA18 Cladosporium cladosporioides 58 40 24 8 63 61 4 0JA4 Aspergillus flavus 90 80 48 26 56 16 0 0JA10 Aspergillus fumigatus 100 76 35 0 100 57 11 0JA11 Aspergillus fumigatiaffinis 100 46 25 0 52 30 12 0JA19 Alternaria alternata 52 38 24 0 57 9 0 0JA20 Alternaria alternata 60 40 0 0 37 24 0 0JA23 Alternaria alternata 100 68 20 0 65 12 0 0JA6 Alternaria tenuissima 100 60 22 0 80 55 17 0JA9 Embellisia phragmospora 94 50 10 0 78 26 0 0JA12 Ulocladium consortiale 72 28 0 0 32 10 0 0JA17 Ulocladium sp 100 70 28 0 67 20 0 0JA7 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) times 100 (2) Relative growth in liquid mediaafter 7 days incubation = (kinetic curve surface under salt stresskinetic curve surface without salt stress) times 100

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 [10mM] Paraquat [500120583M]

JA1 Penicillium chrysogenum 43 39 mdash 66 74JA3 Penicillium chrysogenum 42 50 mdash 84 71JA22 Penicillium chrysogenum 47 45 mdash 68 53JA5 Penicillium canescens 26 28 mdash 59 63JA15 Penicillium sp 43 100 mdash 100 100JA8 Cladosporium halotolerans 34 26 mdash 44 40JA2 Cladosporium sphaerospermum 21 24 mdash 52 48JA13 Cladosporium sphaerospermum 21 43 mdash 55 44JA14 Cladosporium cladosporioides 34 38 mdash 20 31JA18 Cladosporium cladosporioides mdash 41 mdash 18 16JA4 Aspergillus flavus 46 22 mdash 47 39JA10 Aspergillus fumigatus 89 41 61 100 100JA11 Aspergillus fumigatiaffinis 94 26 100 100 100JA19 Alternaria alternata 49 35 mdash 69 89JA20 Alternaria alternata 58 48 mdash 100 100JA23 Alternaria alternata 100 83 40 57 52JA6 Alternaria tenuissima 57 30 mdash 81 100JA9 Embellisia phragmospora 58 67 mdash 100 100JA12 Ulocladium consortiale 44 37 36 56 100JA17 Ulocladium sp 93 28 100 81 100JA7 Engyodontium album 34 18 mdash 66 53Relative growth of fungal strains under different stresses after 7 days incubation was expressed as follows (1) ( colony at pH 10 colony at pH 5) times 100 (2)( colony at 45∘C or 4∘C colony at 30∘C) times 100 (3) ( colony with H2O2 or paraquat colony without stress) times 100 mdash not significant growth

8 BioMed Research International

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

Strain code Strain Protease Amylase Cellulase Lipase LaccaseJA1 Penicillium chrysogenum ++ + minus minus minusJA3 Penicillium chrysogenum ++ minus minus minus minusJA22 Penicillium chrysogenum + + minus minus minusAJ5 Penicillium canescens minus minus minus minus +JA15 Penicillium sp minus minus + minus minusJA8 Cladosporium halotolerans + minus minus minus +JA2 Cladosporium sphaerospermum +++ minus minus + +JA13 Cladosporium sphaerospermum minus + minus minus +JA14 Cladosporium cladosporioides + + minus minus minus

JA18 Cladosporium cladosporioides minus minus minus minus minus

JA4 Aspergillus flavus + minus minus + minus

JA10 Aspergillus fumigatus minus minus minus minus minus

JA11 Aspergillus fumigatiaffinis minus minus minus minus minus

JA19 Alternaria alternata minus minus minus minus minus

JA20 Alternaria alternata minus + minus minus minus

JA23 Alternaria alternata minus minus minus minus minus

JA6 Alternaria tenuissima + minus minus minus minus

JA9 Embellisia phragmospora minus minus minus minus minus

JA12 Ulocladium consortiale minus minus minus minus minus

JA17 Ulocladium sp minus minus minus minus minus

JA7 Engyodontium album + + minus ++ minus

AR activity ratio = ( activity colony) minus no activity + AR lt 1 ++ 1 lt AR lt 2 +++ 2 lt AR lt 3

tolerance [10] On the other hand Molitoris et al [36]reported that other halotolerant and halophilic fungi suchas Aspergillus and Cladosporium spp are predominant insaline desert soil of Dead Sea Many Aspergillus species havebeen also reported to constitute dominant fungi in desert ofSaudi Arabia and Libya [37 38] and halotolerant speciesincludingAspergillus sppPenicillium spp andCladosporiumsphaerospermum have been consistently isolated fromhyper-saline environments around the globe [39] In this studycontrary to many reports on hypersaline environments nospecies belonging to the genera EurotiumThrimmatostromaEmericella and Phaeotheca [9] have been obtained probablybecause of the initial alkaline pH of the Sebkha El Melahsalt lake Actually the effect of pH on the fungal diversity iscontroversialMisra [40] observed that fungal diversity varieswith the pH while other investigators found no significanteffect of pH values of water and soil habitats on fungaloccurrence [41] It is more likely that the number of theisolated fungi is directly correlated to the organic mattercontent of water mud and soil samples [42]

Beside the identification of the recovered fungal isolatesfrom Sebkha El Melah the second goal of the current studywas the detection of some of their physiological and biochem-ical featuresThis allows understanding ecological adaptationto extreme environment and predicts some biotechnologicalusage The 21 strains have been screened for tolerance toextreme NaCl concentrations basic pH temperature andoxidative stress and for the production of important enzy-matic activities in presence of 10 NaCl

Excepting Cladosporium cladosporioides JA18 all isolatesobtained in this study can be considered as moderatelyhaloalkaliphilic fungi as deduced from their ability to grow

at pH 10 and 10 of NaCl However the isolates were ableto grow when salt was not added to their growing mediaExcepting some Wallemia ichthyophaga the most strictlyhalophilic fungus [43] all other fungal strains known todate are able to grow without salt a fact confirmed inour study However gradual decrease in fungal growth wasobserved with the increasing of salt concentration in theculture medium Nineteen strains remain able to grow under15 of NaCl whereas 7 strains were able to tolerate 20 ofNaCl This result was confirmed by salt tolerance assay inliquid media as estimated by Biolog system It is noteworthythat fungi were more sensitive to salt stress in liquid mediathan in solid mediaThis could be explained by the alterationof the osmotic gradient forcing the fungi to expend moreenergy in the osmoregulatory processes resulting in slowergrowth [44] Moreover at higher salt concentration deathoccurs

Regarding the stress of pH the capacity of the majority ofisolates to growth at pH 10 implies firstly that some habitats inthe salt lake may have a varying pH and secondly that fungican tolerate a wide pH range Prima facie the overall resultsin solid and liquid media showed that Penicillium chryso-genum JA1 and JA3 Cladosporium sphaerospermum JA2 andJA13 Cladosporium cladosporioides JA18 Aspergillus fumi-gatus JA10 Aspergillus fumigatiaffinis JA11 and Alternariatenuissima JA6 are the most alkalihalotolerant isolates in thisstudy

The tolerance of the strains to extreme 45∘C was testedand results indicated that Aspergillus fumigatus JA10 Alter-naria alternata JA23 Ulocladium sp JA17 and Aspergillusfumigatiaffinis JA11 were able to grow Of particular interestthe latter two strains retained 100 of the growth rate

BioMed Research International 9

and biomass production as estimated by colony diameterMoreover their ability to grow at low temperature may allowthem to better adapt to the big temperature fluctuation indesert environments Additionally exposure to substratesgenerating oxidative stress such as H

2O2at 10mM and

paraquat at 500 120583M did not alter significantly the growth ofalmost tested strains demonstrating their ability to tolerateoxidative stress These findings may explain their presence indesert regions that are considered amongst the most stressfulenvironments on Earth because of the high UV radiationdesiccation increased salinity low nutrient availability sea-sonal and daily temperature variation and solar irradiation[6 10]

It has been postulated that microorganisms sharing arich and particular extracellular enzymatic activities arecommon in harsh conditions characterizing their ecologicalhabitat including high level of aridity temperature ionicstrength and particularly the low nutrient availability [31]This implies the need by microorganisms for an effectiveutilization of each possible available organic compound [45]Moreover fungal isolates from hot desert were revealed toplay an important role in seeds germination by breakingdormancy and increasing water uptake [46] In the presentstudy the capacity of fungal isolates to produce extracellularenzymes was assayed in the presence of 10 of NaClEnzymes tested were the following amylase for degradationof starch abundant carbohydrate polymer in many planttissues protease for degradation of plant and animal proteinscellulase which hydrolyses the cellulose themain componentof wood ubiquitous substrate for fungi and finally the laccaseinvolved in plant material delignification and in the synthesisof the melanin and related compounds to protect fungiagainst radiationThirteen strains displayed high productionsat least for one of the five-screened activities while noclear correlation of enzyme production profile with fungalsystematic groups was noted The abundance of proteaseactivity is in line with previous data on fungal isolatesfrom extreme environments showing high caseinase activitieswith little effect of salinity and temperature on enzymeproduction [36] The relative limited number of isolatesdisplaying cellulase amylase lipase and laccase activitiessuggests that high concentration of salt may have an adverseeffect on enzymeproduction andor activityTheir energywasprobably oriented to avoid salt stress due to 10 NaCl ratherthan the production of bioactive extrolites [47] Howevernot detecting the enzyme is not absolute confirmation ofan isolate inability to produce it It could also mean thatthe enzyme was not released from the mycelium or thatthe medium is inadequate for its detection [48] Laccaseproduction in the presence of 10 of salt by theCladosporiumgroup may be of biotechnological interest for example inmycoremediation of high salty environments contaminatedby organic pollutants

In conclusion fungal community described in this studywas similar to those reported in inhospitable habitats characterized by limitation of nutrients moisture deficit and

exposure to high solar radiation Further studies are neededin order to elucidate their biogeochemical roles in such anextreme environment and to exploit their promising potentialto produce new biomolecules such as enzymes and protectiveagents against oxidative stress

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support from theEuropean Union in the ambit of the Project BIODESERT(EU FP7-CSA-SA REGPOT-2008-2 Grant agreement no245746) and the Tunisian Ministry of Higher Education andScientific Research in the ambit of the laboratory Project LRMBA20 Atef Jaouani wants to thank the Tunisian Society forMicrobial Ecology (ATEM) for supporting publication fees ofthis work

References

[1] J KWarren ldquoSabkhas saline mudflats and pansrdquo in EvaporitesSediments Resources and Hydrocarbons pp 139ndash220 SpringerBerlin Germany 2006

[2] A A Gorbushina and W E Krumbein ldquoRock dwelling fungalcommunities diversity of life styles and colony structurerdquo inJourney to Diverse Microbial Worlds J Seckbach Ed pp 317ndash334 Kluwer Academic Publishers Amsterdam The Nether-lands 2000

[3] L J Rothschild and R L Mancinelli ldquoLife in extreme environ-mentsrdquo Nature vol 409 no 6823 pp 1092ndash1101 2001

[4] S BHumayounN Bano and J THollibaugh ldquoDepth distribu-tion ofmicrobial diversity inmono lake ameromictic soda lakein Californiardquo Applied and Environmental Microbiology vol 69no 2 pp 1030ndash1042 2003

[5] C Demergasso E O Casamayor G Chong P GalleguillosL Escudero and C Pedros-Alio ldquoDistribution of prokaryoticgenetic diversity in athalassohaline lakes of the AtacamaDesertNorthern Chilerdquo FEMS Microbiology Ecology vol 48 no 1 pp57ndash69 2004

[6] N Gunde-Cimerman A Oren and A Plemenitas ldquoAdaptationto life in high salt concentrationsrdquo in Archaea Bacteria andEukarya p 576 Springer Dordrecht The Netherlands 2005

[7] L Maturrano F Santos R Rossello-Mora and J AntonldquoMicrobial diversity in Maras salterns a hypersaline envi-ronment in the Peruvian Andesrdquo Applied and EnvironmentalMicrobiology vol 72 no 6 pp 3887ndash3895 2006

[8] Q Wu G Zwart M Schauer M Kamst-van Agterveld andM Hahn ldquoBacterioplankton community composition alonga salinity gradient of sixteen high-mountain lakes locatedon the Tibetan Plateau Chinardquo Applied and EnvironmentalMicrobiology vol 72 no 8 pp 5478ndash5485 2006

[9] C Gostincar M Grube S De Hoog P Zalar and N Gunde-Cimerman ldquoExtremotolerance in fungi evolution on the edgerdquoFEMS Microbiology Ecology vol 71 no 1 pp 2ndash11 2010

10 BioMed Research International

[10] K Sterflinger D Tesei and K Zakharova ldquoFungi in hot andcold deserts with particular reference to microcolonial fungirdquoFungal Ecology vol 5 no 4 pp 453ndash462 2012

[11] M J Liszka M E Clark E Schneider and D S ClarkldquoNature versus nurture developing enzymes that functionunder extreme conditionsrdquo Annual Review of Chemical andBiomolecular Engineering vol 3 pp 77ndash102 2012

[12] J P Perthuisot La Sebkha ElMelah de Zarzis genese et evolutiondrsquoun bassin salin paralique Les gisements neolithiques des abordsde la Sebkha el Melah vol 9 of de Travaux du Laboratoire degeologieEcole normale superieure Ecole Normale Superieure1975

[13] I cLellan A Varela M Blahgen et al ldquoHarmonisation ofphysical and chemical methods for soil management in CorkOak forestsmdashlessons from collaborative investigationsrdquoAfricanJournal of Environmental Science and Technology vol 7 no 6pp 386ndash401 2013

[14] J HWarcup ldquoThe soil-plate method for isolation of fungi fromsoilrdquo Nature vol 166 no 4211 pp 117ndash118 1950

[15] K H Domsch W Gams and T H Anderson Compendium ofSoil Fungi Academic Press London UK 1980

[16] J A von ArxThe Genera of Fungi Sporulating in Pure CultureJ Cramer Vaduz Germany 1981

[17] E Kiffer and M Morelet Les deuteromycetes Classification etcles didentification generique INRA Editions Paris France1997

[18] D Liu S Coloe R Baird and J Pedersen ldquoRapid mini-preparation of fungal DNA for PCRrdquo Journal of Clinical Micro-biology vol 38 no 1 article 471 2000

[19] T J White T Bruns S Lee and J Talor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Innes D H Gelfand J J Sninsky and T JWhite Eds pp 315ndash322 Academic Press New York NY USA1990

[20] G Luo and T G Mitchell ldquoRapid identification of pathogenicfungi directly from cultures by using multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 8 pp 2860ndash2865 2002

[21] P Rodrigues C Santos A Venancio and N Lima ldquoSpeciesidentification of Aspergillus section Flavi isolates from Por-tuguese almonds using phenotypic including MALDI-TOFICMS and molecular approachesrdquo Journal of Applied Microbi-ology vol 111 no 4 pp 877ndash892 2011

[22] K Bensch U Braun J Z Groenewald and P W Crous ldquoThegenus cladosporiumrdquo Studies in Mycology vol 72 pp 1ndash4012012

[23] M L Berbee M Pirseyedi and S Hubbard ldquoCochliobo-lus phylogenetics and the origin of known highly virulentpathogens inferred from ITS and glyceraldehyde-3-phosphatedehydrogenase gene sequencesrdquo Mycologia vol 91 no 6 pp964ndash977 1999

[24] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous Ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995

[25] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[26] H M Hassan and I Fridovich ldquoParaquat and Escherichia coliMechanism of production of extracellular superoxide radicalrdquoJournal of Biological Chemistry vol 254 no 21 pp 10846ndash108521979

[27] M B Angelova S B Pashova B K Spasova S V Vassilevand L S Slokoska ldquoOxidative stress response of filamentousfungi induced by hydrogen peroxide and paraquatrdquoMycologicalResearch vol 109 no 2 pp 150ndash158 2005

[28] A de Groot V Chapon P Servant et al ldquoDeinococcus desertisp nov a gamma-radiation-tolerant bacterium isolated fromthe Sahara Desertrdquo International Journal of Systematic andEvolutionary Microbiology vol 55 no 6 pp 2441ndash2446 2005

[29] A Chanal V Chapon K Benzerara et al ldquoThe desert ofTataouine an extreme environment that hosts a wide diversityof microorganisms and radiotolerant bacteriardquo EnvironmentalMicrobiology vol 8 no 3 pp 514ndash525 2006

[30] M Gtari D Daffonchio and A Boudabous ldquoAssessment ofthe genetic diversity of Frankia microsymbionts of Elaeagnusangustifolia L plants growing in a Tunisian date-palm oasis byanalysis of PCR amplified nifD-K intergenic spacerrdquo CanadianJournal of Microbiology vol 53 no 3 pp 440ndash445 2007

[31] I Essoussi F Ghodhbane-Gtari H Amairi et al ldquoEsterase asan enzymatic signature of Geodermatophilaceae adaptabilityto Sahara desert stones and monumentsrdquo Journal of AppliedMicrobiology vol 108 no 5 pp 1723ndash1732 2010

[32] I Sbissi M Neffati A Boudabous C Murat and M GtarildquoPhylogenetic affiliation of the desert truffles Picoa juniperi andPicoa lefebvreirdquo Antonie van Leeuwenhoek vol 98 no 4 pp429ndash436 2010

[33] I Sbissi F Ghodhbane-Gtari M Neffati H Ouzari A Boud-abous and M Gtari ldquoDiversity of the desert truffle Terfeziaboudieri chatin in southern Tunisiardquo Canadian Journal ofMicrobiology vol 57 no 7 pp 599ndash605 2011

[34] C A Conley G Ishkhanova C P McKay and K Cullings ldquoApreliminary survey of non-lichenized fungi cultured from thehyperarid Atacama Desert of Chilerdquo Astrobiology vol 6 no 4pp 521ndash526 2006

[35] E V Smolyanyuk and E N Bilanenko ldquoCommunities ofhalotolerant micromycetes from the areas of natural salinityrdquoMicrobiology vol 80 no 6 pp 877ndash883 2011

[36] H PMolitoris A S Buchalo I Kurchenko et al ldquoPhysiologicaldiversity of the fIrst fIlamentous fungi isolated from the hyper-saline Dead Seardquo inAquatic Mycology Across theMillennium KD Hyde W H Ho and S B Pointing Eds vol 5 pp 55ndash70Fungal Diversity Press 2000

[37] S I I Abdel-Hafez ldquoSurvey of the mycoflora of desert soils inSaudi ArabiardquoMycopathologia vol 80 no 1 pp 3ndash8 1982

[38] A H M El-Said and A Saleem ldquoEcological and physiologicalstudies on soil fungi at Western Region Libyardquo Mycobiologyvol 36 pp 1ndash9 2008

[39] P Zalar G S de Hoog H-J Schroers P W Crous J Z Groe-newald and N Gunde-Cimerman ldquoPhylogeny and ecology ofthe ubiquitous saprobe Cladosporium sphaerospermum withdescriptions of seven new species from hypersaline environ-mentsrdquo Studies in Mycology vol 58 pp 157ndash183 2007

[40] J KMisra ldquoOccurrence distribution and seasonality of aquaticfungi as affected by chemical factors in six alkaline ponds ofIndiardquo Hydrobiologia vol 97 no 2 pp 185ndash191 1982

[41] F T El-Hissy S A El-Zayat A M Khallil and M S MassoudldquoAquatic fungi from the submerged mud of Aswan High DamLakerdquoMicrobiological Research vol 152 no 1 pp 27ndash32 1997

[42] AM Khallil F T El-Hissy and EH Ali ldquoSeasonal fluctuationsof aquatic fungi recovered from Egyptian soil (Delta Region)rdquoJournal of Basic Microbiology vol 35 no 2 pp 93ndash102 1995

BioMed Research International 11

[43] P B Matheny J A Gossmann P Zalar T K A Kumarand D S Hibbett ldquoResolving the phylogenetic position of theWallemiomycetes an enigmatic major lineage of Basidiomy-cotardquoCanadian Journal of Botany vol 84 no 12 pp 1794ndash18052006

[44] A Blomberg and L Adler ldquoTolerence of fungi to NaClrdquo in StressTolerance of Fungi D Jennings Ed pp 209ndash231 1993

[45] A Oren ldquoBioenergetic aspects of halophilismrdquo MicrobiologyandMolecular Biology Reviews vol 63 no 2 pp 334ndash348 1999

[46] P Delgado-Sanchez M A Ortega-Amaro J F Jimenez-Bremont and J Flores ldquoAre fungi important for breaking seeddormancy in desert species Experimental evidence in Opuntiastreptacantha (Cactaceae)rdquo Plant Biology vol 13 no 1 pp 154ndash159 2011

[47] J C Frisvad ldquoHalotolerant and halophilic fungi and theirextrolite productionrdquo in Adaptation to Life at High Salt Con-centrations in Archaea Bacteria and Eukarya N Gunde-Cimerman A Oren and A Plemenitas Eds pp 426ndash440Springer Amsterdam The Netherlands 2005

[48] A Abdel-Raheem and C A Shearer ldquoExtracellular enzymeproduction by freshwater ascomycetesrdquo Fungal Diversity vol 11pp 1ndash19 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 BioMed Research International

continental sebkha More recently around 8000 years BPthe lagoon evolved into an evaporite basin The sebkhasediments are composed of several saliferous layers of rocksalt and gypsum (calcium sulfate) andor polyhalite (sulfateof potassium calcium and magnesium) [12] Here we reportthe isolation ofmoderately halotolerant fungi from Sebkha ElMelah Strains have been identified based on morphologicaland molecular markers and their resistance to salt thermalalkaline and oxidative stresses was assessed Their ability toproduce different hydrolytic and oxidative enzymes undersalt stress was also evaluated

2 Material and Methods

21 Sampling Site Description and Fungal Isolation Threelocations from the Sebkha El Melah margins (L133∘23101584001110158401015840N 10∘54101584056810158401015840E L2 33∘21101584042110158401015840N 10∘55101584005510158401015840Eand L3 33∘23101584037710158401015840N 10∘53101584040210158401015840E) were chosen for sampling(Figure 1) From each location a composite sample wasprepared aseptically from five subsamples (1ndash10 cm deep)and collected from the arms and center of an X (each armwas 1m in length) [13] One cm soil from the ground surfacewas firstly removed to avoid contamination during samplingprocedure Samples were then transported to the laboratoryin a cool box and stored at 4∘C prior to processing

Fungi were isolated on potato dextrose agar (PDA)containing 10 of NaCl and 005 of chloramphenicol usingthe soil plate method where few milligrams of sample weredirectly spread on the agar mediumThis method has a slightedge over the dilution plate method since it allows highertotal number of isolates and limits invasion by species whichsporulate heavily [14]

22 Morphological and Molecular Identification Isolatedfungi were identified conventionally according to theirmacroscopic and microscopic features After determinationof their genera [15ndash17] they were transferred to the mediarecommended of selected genus monographs for speciesidentification

DNA extraction was achieved as described by Liu etal [18] the amplification of the internal transcribed spacerregions (nuclear-encoded 18S rRNA-ITS1-58S rRNA-ITS2-28S rRNA) was performed using the couple of universalprimers ITS1 (51015840-TCC GTA GGT GAA CCT GCG G-31015840) andITS4 (51015840-TCC TCC GCT TAT TGA TAT GC-31015840) [19] andthe thermal cycler conditions according to Luo and Mitchell[20] PCRwas carried out in 25 120583L volumes containing 25 120583Lof 1X PCR reaction buffer (100mM Tris-HCl 500mM KClpH 83) 15 120583L MgCl

2 02 120583molL (each) primer 02 120583molL

(each) dNTP and 25 units of Taq polymerase (Dream TaqFermentas) and 1 120583L of DNA template Depending on thefungus genus different gene sequences were amplified Forthe Aspergillus flavus group the calmodulin gene was ampli-fied using the primers CL1 (51015840-GARTWCAAGGAGGCC-TTCTC-31015840) andCL2A (51015840-TTTTGCATCATGAGTTGGAC-31015840) according to Rodrigues et al [21] for the Cladosporium

L1L2

L3

Alg

eria Tunisia

Med

iterr

anea

n Se

aLi

bya

Figure 1 Map of Sebkha El Melah (Google Earth) L1 L2 and L3indicate locations of sampling

genus the actin gene was amplified using the primers ACT-512F (51015840-ATGTGCAAGGCCGGTTTCGC-31015840) and ACT-783R (51015840-TACGAGTCCTTCTGGCCCAT-31015840) according toBensch et al [22] for Alternaria genus the glyceraldehyde-3-phosphate dehydrogenase gene was amplified using theprimers GPD1 (51015840-CAACGGCTTCGGTCGCATTG-31015840) andGPD2 (51015840-GCCAAGCAGTTGGTTGTGC-31015840) according toBerbee et al [23] for Penicillium and Aspergillus generathe 120573-tubulin gene was amplified using the primers Bt2a(51015840-GGTAACCAAATCGGTGCTGCTTTC-31015840) and Bt2B(51015840-ACCCTCAGTGTAGTGACCCTTGGC-31015840) according toGlass and Donaldson [24]

The PCR products were purified with QIAquick WizardPCR purification Kit (Promega) according to the manu-facturerrsquos instructions and the sequences were determinedby cycle sequencing using the Taq Dye Deoxy Termi-nator Cycle Sequencing kit (Applied Biosystems HTDSTunisia) and fragment separation in an ABI PrismTM 3130DNA sequencer (Applied Biosystems HTDS Tunisia) Thesequences obtained were compared reference sequences inthe NCBI GenBank database using the BLASTN searchoption [25]

23 Effect of pH Salinity Temperature and Oxidative StressPDAmediumwas used to study the effect of different stresseson solid media For oxidative stresses H

2O2or paraquat was

filter sterilized and added separately to melted PDA mediumpreviously autoclaved Paraquat is a redox-cycling agentwidely used to generate reactive oxygen species and induceoxidative stress in bacteria [26] and fungi [27] For pH stressPDA medium was buffered with 100mM Glycine-NaOH topH 10 before autoclaving Salt stress in solid media wasstudied in PDA medium containing different concentrationsof salts The inoculated plates with 3mm cylindrical mycelialplugs were then incubated at 30∘C for oxidative salt andpH stresses and at 4∘C and 45∘C for thermal stresses andradial growth was measured daily Results were expressedas relative growth of fungal strains under different stressesas follows (Colony diameter under stresscolony diameterwithout stress after 7 days incubation) times 100

The effect of salinity in liquid medium was carried out inBiolog system a commercially redox based test (Biolog IncHayward CA) Malt extracts (2) containing 0 5 1015 and 20of salt were inoculated by a suspension of spores

BioMed Research International 3

and fragmentedmycelium according to the supplierrsquos instruc-tions in 96-well microtiter plates After 15 days incubationat 30∘C the numeric results were extracted using PM DataAnalysis 13 software The fungal growth was assimilated tothe reduction of the redox indicatorThe ability of the fungusto grow in the presence of salt was expressed as the ratio ofkinetic curve surface under stress versus without stress

24 Extracellular Enzymes Production Profiling The capacityof fungal isolates to produce extracellular enzymes namelyamylase cellulase protease laccase and lipase was assayedin the presence of 10 of NaCl Inoculation was madeby transferring 3mm of cylindrical mycelial plugs on thecorresponding media Amylase production was assayed onPDA containing 1 soluble starch Enzyme production isshown by the presence of clear halo when iodine waspoured onto the plates Cellulase production was testedon PDA medium containing 1 of carboxymethylcelluloseThe presence of activity is reflected by a clear halo on redbackground after flooding the plates with 02 Congo redfor 30min Protease production was revealed on skim milkagar by the appearance of a clear zone corresponding tocasein hydrolysissolubilization surrounding the microbialcolonyThe laccase productionwas detected on PDAmediumcontaining 5mM of 26 dimethoxyphenol (DMP) Oxidationof the substrate is indicated by the appearance of brown colorLipase production was tested on PDA medium containing10mLL of Tween 20 and 01 gL of CaCl

2 Positive reaction

is accompanied by the presence of precipitates around thefungal colony The enzymes production was expressed asactivity ratio (PR) which corresponds to the activity diameter(halo of enzymatic reaction) divided by the colony diameterafter 7 days incubation at 30∘C

25 Statistical Analysis The data presented are the average ofthe results of at least three replicates with a standard error ofless than 10

3 Results

31 Isolation and Identification of Halotolerant FungiTwenty-one fungal isolates were obtained on halophilicmedium containing 10 NaCl and subjected to morpho-logical and molecular identification Seventeen strains wereidentified at genus level based on 28S rRNA gene sequenceswhile four were identified based on ITS regions Finalassignment was based on combination of morphologicaland 120573-tubulin actin calmodulin and glyceraldehyde-3-phosphate dehydrogenase genes sequencing (Table 1) The 21strains have been identified as Cladosporium cladosporioides(119899 = 2) Cladosporium halotolerans (119899 = 1) Cladosporiumsphaerospermum (119899 = 2) Alternaria tenuissima (119899 = 1)Aspergillus flavus (119899 = 1) Aspergillus fumigatiaffinis (119899 = 1)Aspergillus fumigatus (119899 = 1) Penicillium canescens (119899 = 1)Penicillium chrysogenum (119899 = 3) Penicillium sp (119899 = 1)Alternaria alternata (119899 = 3) Ulocladium consortiale (119899 = 1)Ulocladium sp (119899 = 1) Engyodontium album (119899 = 1) andEmbellisia phragmospora (119899 = 1) species All the strains have

been deposited at the Mycotheca Universitatis Taurinensis(MUT) in the University of Turin

32 Salt Tolerance of Fungal Isolates Salt tolerance of thefungal isolates was assessed on solid and liquid media forNaCl content ranging from 5 to 20 In solid media salttolerance was estimated as relative growth represented bythe ratio of colony diameter under salt stress to that withoutsalt stress As illustrated in Table 2 all the isolated strainssucceeded to grow in the presence of 10 of salt While 19isolates remain able to grow under 15 NaCl only 7 isolatestolerated 20 NaCl Penicillium chrysogenum JA1 and JA22Cladosporium halotolerans JA8 Cladosporium sphaerosper-mum JA2 Cladosporium cladosporioides JA18 Aspergillusflavus JA4 and Engyodontium album JA7

When liquid cultures were used fungal isolates seemedto become more sensitive to salt stress Indeed none of thestrains was able to grow in the presence of 20NaCl whereasonly 8 strains and 19 strains tolerated 15 and 10 NaClrespectively (Table 2)

33 Alkaline Temperature and Oxidative Stress ExceptingCladosporium cladosporioides JA18 all tested strains wereable to grow at pH 10 All isolates were able to grow at 4∘Cwhile only five strains Aspergillus fumigatus JA10 Aspergillusfumigatiaffinis JA11 Alternaria alternata JA23 Ulocladiumconsortiale JA12 andUlocladium sp JA17 showed a significantgrowth at 45∘C All 21 strains tolerated oxidative stressgenerated by 10mMH

2O2and 500 120583M paraquat (Table 3)

34 Enzymatic Profiling of Isolates Among the 21 strainstested 13 strains displayed at least one of the five-screenedactivities protease amylase cellulase lipase and laccase inthe presence of 10 NaCl (Table 4) Protease and amylasewere the most abundant activities shown by 9 and 6 strainsrespectively Four strains belonging to Cladosporium andPenicillium genera produced laccase while Cladosporiumsphaerospermum JA2 Aspergillus flavus JA4 and Engyodon-tium album JA7 were able to produce lipase Cellulase activitywas detected only in Penicillium sp JA15

4 Discussion

With regard to bacteria that have been well explored insouthern desert region of Tunisia [28ndash31] data related tofungi are scarce and are limited to truffle and mycorrhiza sofar considered as real specialists of desert environments [3233] To the best of our knowledge this is the first report on theisolation and characterization of fungi from Tunisian desertand particularly from salt flat A collection of 21 fungi isolateshave been established from samples ashes collected fromSebkha El Melah These alkalihalotolerant fungi have beenassigned to 15 taxa belonging to 6 genera of AscomycetesSeveral studies showed that fungi belonging toCladosporiumAlternaria and Ulocladium genera were clearly predominantunder desert and salty environments [34 35] These fungihave in common thick-walled and strongly melanized sporeswhich are important for UV radiation and desiccation

4 BioMed Research International

Table1Identifi

catio

nof

fung

aliso

lates

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA1

Penicilliu

m

Penicilliu

mflavigenu

mJX997105

(100)

Pconfertum

JX997081

(100)

PdipodomyisJX9

97080(100)

Pcommun

eKC3

33882(100)

Pchrysogenu

mKC

009827

(100)

Pgrise

ofulvum

JQ781833

(100)

Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417559

ITSKF

417577

JA2

Cladosporiu

m

Cladosporiu

mspG

U017498

(100)

Hyalodend

ronspA

M176721

(100)

Csphaerosperm

umAB5

72902(99

)C

cladosporioidesEF

568045

(99

)

Cladosporiu

msphaerosperm

um(A

ctin)

nd

Cladosporiu

msphaerosperm

umPenzig

28SKF

417560

ITSKF

417578

JA3

Penicilliu

mchrysogenu

mPenicilliu

mcanescensH

Q607858(99

)Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417561

ITSKF

417579

JA4

Aspergillus

Aspergillus

aureofulgens

EF669617

(100)

Aspergillus

flavus

(calmodulin)

ndAspergillus

flavusL

ink

28SKF

417562

ITSKF

417580

JA5

ndPenicilliu

mdesertorum

JX997039

(100)

Pchrysogenu

mKC

009826

(99

)Penicilliu

mcanescensgroup

(120573-tu

bulin

andcalm

odulin)

ndPenicilliu

mcanescens

Sopp

ITSKF

417581

JA6

Alternaria

Alternariatriticim

aculan

sJN867470

(100)

Atenu

issim

aJN

867469

(100)

AmaliJN867468

(100)

AalternataJQ

690087

(100)

Alternariatenu

issim

a(G

PD)

Alternariaalternata

Alternariatenu

issim

a(N

ees)

Wiltshire

28SKF

417563

ITSKF

417582

JA7

ndEn

gyodontiu

malbum

HM214540

(100)

Engyodontiu

malbum

Engyodontiu

malbum

(Lim

ber)de

Hoo

gITSKF

417583

JA8

Cladosporiu

m

Cladosporiu

mcla

dosporioidesEF

568045

(100)

Csphaerosperm

umAM176719

(100)

Chalotoleran

sJX5

35318(99

)

Cladosporiu

mhalotoleran

s(Actin)

Cladosporiu

mcla

dosporioideshalotoleran

s

Cladosporiu

mhalotoleran

sZa

larde

Hoo

gand

Gun

de-C

imerman

28SKF

417564

ITSKF

417584

JA9

Embellisia

Chalasto

sporaEm

bellisia

phragm

ospora

JN383493

(100)

Alternariatenu

issim

asim

ilarity

Alternariaarborescens

Alternariaalternata(G

PD)

Embellisia

phragm

ospora

Embellisia

phragm

ospora

(Emden)

EG

28SKF

417565

ITSKF

417585

BioMed Research International 5

Table1Con

tinued

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA10

Aspergillus

Aspergillus

lentulusJN943567

(99

)A

afffum

igatus

JN2460

66(99

)A

fumigatiaffinisH

F545316(99

)A

novofumigatus

FR733874

(99

)

Aspergillus

fumiga

tus

(120573-tu

bulin

)Aspergillus

fumiga

tiaffinis

Aspergillus

fumiga

tus

Fresenius

28SKF

417566

ITSKF

417586

JA11

ndAspergillus

afffum

igatus

JN2460

66(100)

AfumigatiaffinisK

C253955(99

)

Aspergillus

fumiga

tiaffinis

(120573-tu

bulin

)nd

Aspergillus

fumiga

tiaffinis

Hon

gFrisv

adand

Samson

ITSKF

417587

JA12

Ulocla

dium

Ulocla

dium

consortia

leJQ

585682

(100)

Alternariaradicin

aHM2044

57(99

)Ulocla

dium

consortia

le(G

PD)

Ulocla

dium

tuberculatum

consortiale

Ulocla

dium

consortia

le(Th

umen)E

GSim

mon

s28SKF

417567

ITSKF

417588

JA13

Cladosporiu

mCladosporiu

mcla

dosporioidesJX868638

(99

)C

sphaerosperm

umHM999943

(99

)

Cladosporiu

msphaerosperm

umgrou

p(A

ctin)

Cladosporiu

msphaerosperm

um

Cladosporiu

msphaerosperm

umPenzig

28SKF

417568

ITSKF

417589

JA14

Cladosporiu

mD

avidiella

Cladosporiu

mcla

dosporioidesKC

009539

(99

)DavidiellatassianaGU248332

(98

)nd

nd

Cladosporiu

mcla

dosporioides

(Fresenius)G

AdeV

ries

28SKF

417569

ITSKF

417590

JA15

Penicilliu

mPenicilliu

mspinulosum

KC167852

(100)

Pgla

brum

KC00

9784

(100)

ndPenicilliu

mgla

brum

Penicilliu

msp

28SKF

417570

ITSKF

417591

JA17

Ulocla

dium

Ulocla

dium

consortia

leJQ

585682

(100)

Uchartarum

JN942881

(99

)nd

Ulocla

dium

sp

Ulocla

dium

sp

28SKF

417572

ITSKF

417593

JA18

Cladosporiu

mCladosporiu

mcla

dosporioidesHQ380770

(100)

ndCladosporiu

mcla

dosporioides

Cladosporiu

mcla

dosporioides

(Fresenius)G

AdeV

ries

28SKF

417573

ITSKF

417594

JA19

Alternaria

AlternariaspK

C139473(100)

AarborescensJQ781762

(100)

AalternataJN

107734

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

28SKF

417574

ITSKF

417595

6 BioMed Research International

Table1Con

tinued

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA20

Alternaria

Alternariabrassicae

JX290150

(100)

AporriH

Q821479

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

28SKF

417575

ITSKF

417596

JA22

Penicilliu

m

Penicilliu

mchrysogenu

mKC

341721

(99

)PdipodomyicolaJX232278

(99

)Prubens

JX003126

(99

)Pcommun

eJN676122

(99

)

Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417576

ITSKF

417597

JA23

nd

AlternariaalternataJQ

809324

(100)

AquercusK

C329620(100)

Atenu

issim

aKC

329619

(100)

Aatrans

KC329618

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

ITSKF

417598

BioMed Research International 7

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 0JA3 Penicillium chrysogenum 100 72 37 0 96 46 11 0JA22 Penicillium chrysogenum 100 82 41 25 90 46 0 0AJ5 Penicillium canescens 70 30 20 0 79 44 0 0JA15 Penicillium sp 83 70 34 0 53 18 0 0JA8 Cladosporium halotolerans 80 68 32 18 30 0 0 0JA2 Cladosporium sphaerospermum 76 64 34 22 47 19 11 0JA13 Cladosporium sphaerospermum 100 49 25 0 81 79 12 0JA14 Cladosporium cladosporioides 40 30 10 0 0 0 0 0JA18 Cladosporium cladosporioides 58 40 24 8 63 61 4 0JA4 Aspergillus flavus 90 80 48 26 56 16 0 0JA10 Aspergillus fumigatus 100 76 35 0 100 57 11 0JA11 Aspergillus fumigatiaffinis 100 46 25 0 52 30 12 0JA19 Alternaria alternata 52 38 24 0 57 9 0 0JA20 Alternaria alternata 60 40 0 0 37 24 0 0JA23 Alternaria alternata 100 68 20 0 65 12 0 0JA6 Alternaria tenuissima 100 60 22 0 80 55 17 0JA9 Embellisia phragmospora 94 50 10 0 78 26 0 0JA12 Ulocladium consortiale 72 28 0 0 32 10 0 0JA17 Ulocladium sp 100 70 28 0 67 20 0 0JA7 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) times 100 (2) Relative growth in liquid mediaafter 7 days incubation = (kinetic curve surface under salt stresskinetic curve surface without salt stress) times 100

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 [10mM] Paraquat [500120583M]

JA1 Penicillium chrysogenum 43 39 mdash 66 74JA3 Penicillium chrysogenum 42 50 mdash 84 71JA22 Penicillium chrysogenum 47 45 mdash 68 53JA5 Penicillium canescens 26 28 mdash 59 63JA15 Penicillium sp 43 100 mdash 100 100JA8 Cladosporium halotolerans 34 26 mdash 44 40JA2 Cladosporium sphaerospermum 21 24 mdash 52 48JA13 Cladosporium sphaerospermum 21 43 mdash 55 44JA14 Cladosporium cladosporioides 34 38 mdash 20 31JA18 Cladosporium cladosporioides mdash 41 mdash 18 16JA4 Aspergillus flavus 46 22 mdash 47 39JA10 Aspergillus fumigatus 89 41 61 100 100JA11 Aspergillus fumigatiaffinis 94 26 100 100 100JA19 Alternaria alternata 49 35 mdash 69 89JA20 Alternaria alternata 58 48 mdash 100 100JA23 Alternaria alternata 100 83 40 57 52JA6 Alternaria tenuissima 57 30 mdash 81 100JA9 Embellisia phragmospora 58 67 mdash 100 100JA12 Ulocladium consortiale 44 37 36 56 100JA17 Ulocladium sp 93 28 100 81 100JA7 Engyodontium album 34 18 mdash 66 53Relative growth of fungal strains under different stresses after 7 days incubation was expressed as follows (1) ( colony at pH 10 colony at pH 5) times 100 (2)( colony at 45∘C or 4∘C colony at 30∘C) times 100 (3) ( colony with H2O2 or paraquat colony without stress) times 100 mdash not significant growth

8 BioMed Research International

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

Strain code Strain Protease Amylase Cellulase Lipase LaccaseJA1 Penicillium chrysogenum ++ + minus minus minusJA3 Penicillium chrysogenum ++ minus minus minus minusJA22 Penicillium chrysogenum + + minus minus minusAJ5 Penicillium canescens minus minus minus minus +JA15 Penicillium sp minus minus + minus minusJA8 Cladosporium halotolerans + minus minus minus +JA2 Cladosporium sphaerospermum +++ minus minus + +JA13 Cladosporium sphaerospermum minus + minus minus +JA14 Cladosporium cladosporioides + + minus minus minus

JA18 Cladosporium cladosporioides minus minus minus minus minus

JA4 Aspergillus flavus + minus minus + minus

JA10 Aspergillus fumigatus minus minus minus minus minus

JA11 Aspergillus fumigatiaffinis minus minus minus minus minus

JA19 Alternaria alternata minus minus minus minus minus

JA20 Alternaria alternata minus + minus minus minus

JA23 Alternaria alternata minus minus minus minus minus

JA6 Alternaria tenuissima + minus minus minus minus

JA9 Embellisia phragmospora minus minus minus minus minus

JA12 Ulocladium consortiale minus minus minus minus minus

JA17 Ulocladium sp minus minus minus minus minus

JA7 Engyodontium album + + minus ++ minus

AR activity ratio = ( activity colony) minus no activity + AR lt 1 ++ 1 lt AR lt 2 +++ 2 lt AR lt 3

tolerance [10] On the other hand Molitoris et al [36]reported that other halotolerant and halophilic fungi suchas Aspergillus and Cladosporium spp are predominant insaline desert soil of Dead Sea Many Aspergillus species havebeen also reported to constitute dominant fungi in desert ofSaudi Arabia and Libya [37 38] and halotolerant speciesincludingAspergillus sppPenicillium spp andCladosporiumsphaerospermum have been consistently isolated fromhyper-saline environments around the globe [39] In this studycontrary to many reports on hypersaline environments nospecies belonging to the genera EurotiumThrimmatostromaEmericella and Phaeotheca [9] have been obtained probablybecause of the initial alkaline pH of the Sebkha El Melahsalt lake Actually the effect of pH on the fungal diversity iscontroversialMisra [40] observed that fungal diversity varieswith the pH while other investigators found no significanteffect of pH values of water and soil habitats on fungaloccurrence [41] It is more likely that the number of theisolated fungi is directly correlated to the organic mattercontent of water mud and soil samples [42]

Beside the identification of the recovered fungal isolatesfrom Sebkha El Melah the second goal of the current studywas the detection of some of their physiological and biochem-ical featuresThis allows understanding ecological adaptationto extreme environment and predicts some biotechnologicalusage The 21 strains have been screened for tolerance toextreme NaCl concentrations basic pH temperature andoxidative stress and for the production of important enzy-matic activities in presence of 10 NaCl

Excepting Cladosporium cladosporioides JA18 all isolatesobtained in this study can be considered as moderatelyhaloalkaliphilic fungi as deduced from their ability to grow

at pH 10 and 10 of NaCl However the isolates were ableto grow when salt was not added to their growing mediaExcepting some Wallemia ichthyophaga the most strictlyhalophilic fungus [43] all other fungal strains known todate are able to grow without salt a fact confirmed inour study However gradual decrease in fungal growth wasobserved with the increasing of salt concentration in theculture medium Nineteen strains remain able to grow under15 of NaCl whereas 7 strains were able to tolerate 20 ofNaCl This result was confirmed by salt tolerance assay inliquid media as estimated by Biolog system It is noteworthythat fungi were more sensitive to salt stress in liquid mediathan in solid mediaThis could be explained by the alterationof the osmotic gradient forcing the fungi to expend moreenergy in the osmoregulatory processes resulting in slowergrowth [44] Moreover at higher salt concentration deathoccurs

Regarding the stress of pH the capacity of the majority ofisolates to growth at pH 10 implies firstly that some habitats inthe salt lake may have a varying pH and secondly that fungican tolerate a wide pH range Prima facie the overall resultsin solid and liquid media showed that Penicillium chryso-genum JA1 and JA3 Cladosporium sphaerospermum JA2 andJA13 Cladosporium cladosporioides JA18 Aspergillus fumi-gatus JA10 Aspergillus fumigatiaffinis JA11 and Alternariatenuissima JA6 are the most alkalihalotolerant isolates in thisstudy

The tolerance of the strains to extreme 45∘C was testedand results indicated that Aspergillus fumigatus JA10 Alter-naria alternata JA23 Ulocladium sp JA17 and Aspergillusfumigatiaffinis JA11 were able to grow Of particular interestthe latter two strains retained 100 of the growth rate

BioMed Research International 9

and biomass production as estimated by colony diameterMoreover their ability to grow at low temperature may allowthem to better adapt to the big temperature fluctuation indesert environments Additionally exposure to substratesgenerating oxidative stress such as H

2O2at 10mM and

paraquat at 500 120583M did not alter significantly the growth ofalmost tested strains demonstrating their ability to tolerateoxidative stress These findings may explain their presence indesert regions that are considered amongst the most stressfulenvironments on Earth because of the high UV radiationdesiccation increased salinity low nutrient availability sea-sonal and daily temperature variation and solar irradiation[6 10]

It has been postulated that microorganisms sharing arich and particular extracellular enzymatic activities arecommon in harsh conditions characterizing their ecologicalhabitat including high level of aridity temperature ionicstrength and particularly the low nutrient availability [31]This implies the need by microorganisms for an effectiveutilization of each possible available organic compound [45]Moreover fungal isolates from hot desert were revealed toplay an important role in seeds germination by breakingdormancy and increasing water uptake [46] In the presentstudy the capacity of fungal isolates to produce extracellularenzymes was assayed in the presence of 10 of NaClEnzymes tested were the following amylase for degradationof starch abundant carbohydrate polymer in many planttissues protease for degradation of plant and animal proteinscellulase which hydrolyses the cellulose themain componentof wood ubiquitous substrate for fungi and finally the laccaseinvolved in plant material delignification and in the synthesisof the melanin and related compounds to protect fungiagainst radiationThirteen strains displayed high productionsat least for one of the five-screened activities while noclear correlation of enzyme production profile with fungalsystematic groups was noted The abundance of proteaseactivity is in line with previous data on fungal isolatesfrom extreme environments showing high caseinase activitieswith little effect of salinity and temperature on enzymeproduction [36] The relative limited number of isolatesdisplaying cellulase amylase lipase and laccase activitiessuggests that high concentration of salt may have an adverseeffect on enzymeproduction andor activityTheir energywasprobably oriented to avoid salt stress due to 10 NaCl ratherthan the production of bioactive extrolites [47] Howevernot detecting the enzyme is not absolute confirmation ofan isolate inability to produce it It could also mean thatthe enzyme was not released from the mycelium or thatthe medium is inadequate for its detection [48] Laccaseproduction in the presence of 10 of salt by theCladosporiumgroup may be of biotechnological interest for example inmycoremediation of high salty environments contaminatedby organic pollutants

In conclusion fungal community described in this studywas similar to those reported in inhospitable habitats characterized by limitation of nutrients moisture deficit and

exposure to high solar radiation Further studies are neededin order to elucidate their biogeochemical roles in such anextreme environment and to exploit their promising potentialto produce new biomolecules such as enzymes and protectiveagents against oxidative stress

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support from theEuropean Union in the ambit of the Project BIODESERT(EU FP7-CSA-SA REGPOT-2008-2 Grant agreement no245746) and the Tunisian Ministry of Higher Education andScientific Research in the ambit of the laboratory Project LRMBA20 Atef Jaouani wants to thank the Tunisian Society forMicrobial Ecology (ATEM) for supporting publication fees ofthis work

References

[1] J KWarren ldquoSabkhas saline mudflats and pansrdquo in EvaporitesSediments Resources and Hydrocarbons pp 139ndash220 SpringerBerlin Germany 2006

[2] A A Gorbushina and W E Krumbein ldquoRock dwelling fungalcommunities diversity of life styles and colony structurerdquo inJourney to Diverse Microbial Worlds J Seckbach Ed pp 317ndash334 Kluwer Academic Publishers Amsterdam The Nether-lands 2000

[3] L J Rothschild and R L Mancinelli ldquoLife in extreme environ-mentsrdquo Nature vol 409 no 6823 pp 1092ndash1101 2001

[4] S BHumayounN Bano and J THollibaugh ldquoDepth distribu-tion ofmicrobial diversity inmono lake ameromictic soda lakein Californiardquo Applied and Environmental Microbiology vol 69no 2 pp 1030ndash1042 2003

[5] C Demergasso E O Casamayor G Chong P GalleguillosL Escudero and C Pedros-Alio ldquoDistribution of prokaryoticgenetic diversity in athalassohaline lakes of the AtacamaDesertNorthern Chilerdquo FEMS Microbiology Ecology vol 48 no 1 pp57ndash69 2004

[6] N Gunde-Cimerman A Oren and A Plemenitas ldquoAdaptationto life in high salt concentrationsrdquo in Archaea Bacteria andEukarya p 576 Springer Dordrecht The Netherlands 2005

[7] L Maturrano F Santos R Rossello-Mora and J AntonldquoMicrobial diversity in Maras salterns a hypersaline envi-ronment in the Peruvian Andesrdquo Applied and EnvironmentalMicrobiology vol 72 no 6 pp 3887ndash3895 2006

[8] Q Wu G Zwart M Schauer M Kamst-van Agterveld andM Hahn ldquoBacterioplankton community composition alonga salinity gradient of sixteen high-mountain lakes locatedon the Tibetan Plateau Chinardquo Applied and EnvironmentalMicrobiology vol 72 no 8 pp 5478ndash5485 2006

[9] C Gostincar M Grube S De Hoog P Zalar and N Gunde-Cimerman ldquoExtremotolerance in fungi evolution on the edgerdquoFEMS Microbiology Ecology vol 71 no 1 pp 2ndash11 2010

10 BioMed Research International

[10] K Sterflinger D Tesei and K Zakharova ldquoFungi in hot andcold deserts with particular reference to microcolonial fungirdquoFungal Ecology vol 5 no 4 pp 453ndash462 2012

[11] M J Liszka M E Clark E Schneider and D S ClarkldquoNature versus nurture developing enzymes that functionunder extreme conditionsrdquo Annual Review of Chemical andBiomolecular Engineering vol 3 pp 77ndash102 2012

[12] J P Perthuisot La Sebkha ElMelah de Zarzis genese et evolutiondrsquoun bassin salin paralique Les gisements neolithiques des abordsde la Sebkha el Melah vol 9 of de Travaux du Laboratoire degeologieEcole normale superieure Ecole Normale Superieure1975

[13] I cLellan A Varela M Blahgen et al ldquoHarmonisation ofphysical and chemical methods for soil management in CorkOak forestsmdashlessons from collaborative investigationsrdquoAfricanJournal of Environmental Science and Technology vol 7 no 6pp 386ndash401 2013

[14] J HWarcup ldquoThe soil-plate method for isolation of fungi fromsoilrdquo Nature vol 166 no 4211 pp 117ndash118 1950

[15] K H Domsch W Gams and T H Anderson Compendium ofSoil Fungi Academic Press London UK 1980

[16] J A von ArxThe Genera of Fungi Sporulating in Pure CultureJ Cramer Vaduz Germany 1981

[17] E Kiffer and M Morelet Les deuteromycetes Classification etcles didentification generique INRA Editions Paris France1997

[18] D Liu S Coloe R Baird and J Pedersen ldquoRapid mini-preparation of fungal DNA for PCRrdquo Journal of Clinical Micro-biology vol 38 no 1 article 471 2000

[19] T J White T Bruns S Lee and J Talor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Innes D H Gelfand J J Sninsky and T JWhite Eds pp 315ndash322 Academic Press New York NY USA1990

[20] G Luo and T G Mitchell ldquoRapid identification of pathogenicfungi directly from cultures by using multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 8 pp 2860ndash2865 2002

[21] P Rodrigues C Santos A Venancio and N Lima ldquoSpeciesidentification of Aspergillus section Flavi isolates from Por-tuguese almonds using phenotypic including MALDI-TOFICMS and molecular approachesrdquo Journal of Applied Microbi-ology vol 111 no 4 pp 877ndash892 2011

[22] K Bensch U Braun J Z Groenewald and P W Crous ldquoThegenus cladosporiumrdquo Studies in Mycology vol 72 pp 1ndash4012012

[23] M L Berbee M Pirseyedi and S Hubbard ldquoCochliobo-lus phylogenetics and the origin of known highly virulentpathogens inferred from ITS and glyceraldehyde-3-phosphatedehydrogenase gene sequencesrdquo Mycologia vol 91 no 6 pp964ndash977 1999

[24] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous Ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995

[25] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[26] H M Hassan and I Fridovich ldquoParaquat and Escherichia coliMechanism of production of extracellular superoxide radicalrdquoJournal of Biological Chemistry vol 254 no 21 pp 10846ndash108521979

[27] M B Angelova S B Pashova B K Spasova S V Vassilevand L S Slokoska ldquoOxidative stress response of filamentousfungi induced by hydrogen peroxide and paraquatrdquoMycologicalResearch vol 109 no 2 pp 150ndash158 2005

[28] A de Groot V Chapon P Servant et al ldquoDeinococcus desertisp nov a gamma-radiation-tolerant bacterium isolated fromthe Sahara Desertrdquo International Journal of Systematic andEvolutionary Microbiology vol 55 no 6 pp 2441ndash2446 2005

[29] A Chanal V Chapon K Benzerara et al ldquoThe desert ofTataouine an extreme environment that hosts a wide diversityof microorganisms and radiotolerant bacteriardquo EnvironmentalMicrobiology vol 8 no 3 pp 514ndash525 2006

[30] M Gtari D Daffonchio and A Boudabous ldquoAssessment ofthe genetic diversity of Frankia microsymbionts of Elaeagnusangustifolia L plants growing in a Tunisian date-palm oasis byanalysis of PCR amplified nifD-K intergenic spacerrdquo CanadianJournal of Microbiology vol 53 no 3 pp 440ndash445 2007

[31] I Essoussi F Ghodhbane-Gtari H Amairi et al ldquoEsterase asan enzymatic signature of Geodermatophilaceae adaptabilityto Sahara desert stones and monumentsrdquo Journal of AppliedMicrobiology vol 108 no 5 pp 1723ndash1732 2010

[32] I Sbissi M Neffati A Boudabous C Murat and M GtarildquoPhylogenetic affiliation of the desert truffles Picoa juniperi andPicoa lefebvreirdquo Antonie van Leeuwenhoek vol 98 no 4 pp429ndash436 2010

[33] I Sbissi F Ghodhbane-Gtari M Neffati H Ouzari A Boud-abous and M Gtari ldquoDiversity of the desert truffle Terfeziaboudieri chatin in southern Tunisiardquo Canadian Journal ofMicrobiology vol 57 no 7 pp 599ndash605 2011

[34] C A Conley G Ishkhanova C P McKay and K Cullings ldquoApreliminary survey of non-lichenized fungi cultured from thehyperarid Atacama Desert of Chilerdquo Astrobiology vol 6 no 4pp 521ndash526 2006

[35] E V Smolyanyuk and E N Bilanenko ldquoCommunities ofhalotolerant micromycetes from the areas of natural salinityrdquoMicrobiology vol 80 no 6 pp 877ndash883 2011

[36] H PMolitoris A S Buchalo I Kurchenko et al ldquoPhysiologicaldiversity of the fIrst fIlamentous fungi isolated from the hyper-saline Dead Seardquo inAquatic Mycology Across theMillennium KD Hyde W H Ho and S B Pointing Eds vol 5 pp 55ndash70Fungal Diversity Press 2000

[37] S I I Abdel-Hafez ldquoSurvey of the mycoflora of desert soils inSaudi ArabiardquoMycopathologia vol 80 no 1 pp 3ndash8 1982

[38] A H M El-Said and A Saleem ldquoEcological and physiologicalstudies on soil fungi at Western Region Libyardquo Mycobiologyvol 36 pp 1ndash9 2008

[39] P Zalar G S de Hoog H-J Schroers P W Crous J Z Groe-newald and N Gunde-Cimerman ldquoPhylogeny and ecology ofthe ubiquitous saprobe Cladosporium sphaerospermum withdescriptions of seven new species from hypersaline environ-mentsrdquo Studies in Mycology vol 58 pp 157ndash183 2007

[40] J KMisra ldquoOccurrence distribution and seasonality of aquaticfungi as affected by chemical factors in six alkaline ponds ofIndiardquo Hydrobiologia vol 97 no 2 pp 185ndash191 1982

[41] F T El-Hissy S A El-Zayat A M Khallil and M S MassoudldquoAquatic fungi from the submerged mud of Aswan High DamLakerdquoMicrobiological Research vol 152 no 1 pp 27ndash32 1997

[42] AM Khallil F T El-Hissy and EH Ali ldquoSeasonal fluctuationsof aquatic fungi recovered from Egyptian soil (Delta Region)rdquoJournal of Basic Microbiology vol 35 no 2 pp 93ndash102 1995

BioMed Research International 11

[43] P B Matheny J A Gossmann P Zalar T K A Kumarand D S Hibbett ldquoResolving the phylogenetic position of theWallemiomycetes an enigmatic major lineage of Basidiomy-cotardquoCanadian Journal of Botany vol 84 no 12 pp 1794ndash18052006

[44] A Blomberg and L Adler ldquoTolerence of fungi to NaClrdquo in StressTolerance of Fungi D Jennings Ed pp 209ndash231 1993

[45] A Oren ldquoBioenergetic aspects of halophilismrdquo MicrobiologyandMolecular Biology Reviews vol 63 no 2 pp 334ndash348 1999

[46] P Delgado-Sanchez M A Ortega-Amaro J F Jimenez-Bremont and J Flores ldquoAre fungi important for breaking seeddormancy in desert species Experimental evidence in Opuntiastreptacantha (Cactaceae)rdquo Plant Biology vol 13 no 1 pp 154ndash159 2011

[47] J C Frisvad ldquoHalotolerant and halophilic fungi and theirextrolite productionrdquo in Adaptation to Life at High Salt Con-centrations in Archaea Bacteria and Eukarya N Gunde-Cimerman A Oren and A Plemenitas Eds pp 426ndash440Springer Amsterdam The Netherlands 2005

[48] A Abdel-Raheem and C A Shearer ldquoExtracellular enzymeproduction by freshwater ascomycetesrdquo Fungal Diversity vol 11pp 1ndash19 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

and fragmentedmycelium according to the supplierrsquos instruc-tions in 96-well microtiter plates After 15 days incubationat 30∘C the numeric results were extracted using PM DataAnalysis 13 software The fungal growth was assimilated tothe reduction of the redox indicatorThe ability of the fungusto grow in the presence of salt was expressed as the ratio ofkinetic curve surface under stress versus without stress

24 Extracellular Enzymes Production Profiling The capacityof fungal isolates to produce extracellular enzymes namelyamylase cellulase protease laccase and lipase was assayedin the presence of 10 of NaCl Inoculation was madeby transferring 3mm of cylindrical mycelial plugs on thecorresponding media Amylase production was assayed onPDA containing 1 soluble starch Enzyme production isshown by the presence of clear halo when iodine waspoured onto the plates Cellulase production was testedon PDA medium containing 1 of carboxymethylcelluloseThe presence of activity is reflected by a clear halo on redbackground after flooding the plates with 02 Congo redfor 30min Protease production was revealed on skim milkagar by the appearance of a clear zone corresponding tocasein hydrolysissolubilization surrounding the microbialcolonyThe laccase productionwas detected on PDAmediumcontaining 5mM of 26 dimethoxyphenol (DMP) Oxidationof the substrate is indicated by the appearance of brown colorLipase production was tested on PDA medium containing10mLL of Tween 20 and 01 gL of CaCl

2 Positive reaction

is accompanied by the presence of precipitates around thefungal colony The enzymes production was expressed asactivity ratio (PR) which corresponds to the activity diameter(halo of enzymatic reaction) divided by the colony diameterafter 7 days incubation at 30∘C

25 Statistical Analysis The data presented are the average ofthe results of at least three replicates with a standard error ofless than 10

3 Results

31 Isolation and Identification of Halotolerant FungiTwenty-one fungal isolates were obtained on halophilicmedium containing 10 NaCl and subjected to morpho-logical and molecular identification Seventeen strains wereidentified at genus level based on 28S rRNA gene sequenceswhile four were identified based on ITS regions Finalassignment was based on combination of morphologicaland 120573-tubulin actin calmodulin and glyceraldehyde-3-phosphate dehydrogenase genes sequencing (Table 1) The 21strains have been identified as Cladosporium cladosporioides(119899 = 2) Cladosporium halotolerans (119899 = 1) Cladosporiumsphaerospermum (119899 = 2) Alternaria tenuissima (119899 = 1)Aspergillus flavus (119899 = 1) Aspergillus fumigatiaffinis (119899 = 1)Aspergillus fumigatus (119899 = 1) Penicillium canescens (119899 = 1)Penicillium chrysogenum (119899 = 3) Penicillium sp (119899 = 1)Alternaria alternata (119899 = 3) Ulocladium consortiale (119899 = 1)Ulocladium sp (119899 = 1) Engyodontium album (119899 = 1) andEmbellisia phragmospora (119899 = 1) species All the strains have

been deposited at the Mycotheca Universitatis Taurinensis(MUT) in the University of Turin

32 Salt Tolerance of Fungal Isolates Salt tolerance of thefungal isolates was assessed on solid and liquid media forNaCl content ranging from 5 to 20 In solid media salttolerance was estimated as relative growth represented bythe ratio of colony diameter under salt stress to that withoutsalt stress As illustrated in Table 2 all the isolated strainssucceeded to grow in the presence of 10 of salt While 19isolates remain able to grow under 15 NaCl only 7 isolatestolerated 20 NaCl Penicillium chrysogenum JA1 and JA22Cladosporium halotolerans JA8 Cladosporium sphaerosper-mum JA2 Cladosporium cladosporioides JA18 Aspergillusflavus JA4 and Engyodontium album JA7

When liquid cultures were used fungal isolates seemedto become more sensitive to salt stress Indeed none of thestrains was able to grow in the presence of 20NaCl whereasonly 8 strains and 19 strains tolerated 15 and 10 NaClrespectively (Table 2)

33 Alkaline Temperature and Oxidative Stress ExceptingCladosporium cladosporioides JA18 all tested strains wereable to grow at pH 10 All isolates were able to grow at 4∘Cwhile only five strains Aspergillus fumigatus JA10 Aspergillusfumigatiaffinis JA11 Alternaria alternata JA23 Ulocladiumconsortiale JA12 andUlocladium sp JA17 showed a significantgrowth at 45∘C All 21 strains tolerated oxidative stressgenerated by 10mMH

2O2and 500 120583M paraquat (Table 3)

34 Enzymatic Profiling of Isolates Among the 21 strainstested 13 strains displayed at least one of the five-screenedactivities protease amylase cellulase lipase and laccase inthe presence of 10 NaCl (Table 4) Protease and amylasewere the most abundant activities shown by 9 and 6 strainsrespectively Four strains belonging to Cladosporium andPenicillium genera produced laccase while Cladosporiumsphaerospermum JA2 Aspergillus flavus JA4 and Engyodon-tium album JA7 were able to produce lipase Cellulase activitywas detected only in Penicillium sp JA15

4 Discussion

With regard to bacteria that have been well explored insouthern desert region of Tunisia [28ndash31] data related tofungi are scarce and are limited to truffle and mycorrhiza sofar considered as real specialists of desert environments [3233] To the best of our knowledge this is the first report on theisolation and characterization of fungi from Tunisian desertand particularly from salt flat A collection of 21 fungi isolateshave been established from samples ashes collected fromSebkha El Melah These alkalihalotolerant fungi have beenassigned to 15 taxa belonging to 6 genera of AscomycetesSeveral studies showed that fungi belonging toCladosporiumAlternaria and Ulocladium genera were clearly predominantunder desert and salty environments [34 35] These fungihave in common thick-walled and strongly melanized sporeswhich are important for UV radiation and desiccation

4 BioMed Research International

Table1Identifi

catio

nof

fung

aliso

lates

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA1

Penicilliu

m

Penicilliu

mflavigenu

mJX997105

(100)

Pconfertum

JX997081

(100)

PdipodomyisJX9

97080(100)

Pcommun

eKC3

33882(100)

Pchrysogenu

mKC

009827

(100)

Pgrise

ofulvum

JQ781833

(100)

Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417559

ITSKF

417577

JA2

Cladosporiu

m

Cladosporiu

mspG

U017498

(100)

Hyalodend

ronspA

M176721

(100)

Csphaerosperm

umAB5

72902(99

)C

cladosporioidesEF

568045

(99

)

Cladosporiu

msphaerosperm

um(A

ctin)

nd

Cladosporiu

msphaerosperm

umPenzig

28SKF

417560

ITSKF

417578

JA3

Penicilliu

mchrysogenu

mPenicilliu

mcanescensH

Q607858(99

)Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417561

ITSKF

417579

JA4

Aspergillus

Aspergillus

aureofulgens

EF669617

(100)

Aspergillus

flavus

(calmodulin)

ndAspergillus

flavusL

ink

28SKF

417562

ITSKF

417580

JA5

ndPenicilliu

mdesertorum

JX997039

(100)

Pchrysogenu

mKC

009826

(99

)Penicilliu

mcanescensgroup

(120573-tu

bulin

andcalm

odulin)

ndPenicilliu

mcanescens

Sopp

ITSKF

417581

JA6

Alternaria

Alternariatriticim

aculan

sJN867470

(100)

Atenu

issim

aJN

867469

(100)

AmaliJN867468

(100)

AalternataJQ

690087

(100)

Alternariatenu

issim

a(G

PD)

Alternariaalternata

Alternariatenu

issim

a(N

ees)

Wiltshire

28SKF

417563

ITSKF

417582

JA7

ndEn

gyodontiu

malbum

HM214540

(100)

Engyodontiu

malbum

Engyodontiu

malbum

(Lim

ber)de

Hoo

gITSKF

417583

JA8

Cladosporiu

m

Cladosporiu

mcla

dosporioidesEF

568045

(100)

Csphaerosperm

umAM176719

(100)

Chalotoleran

sJX5

35318(99

)

Cladosporiu

mhalotoleran

s(Actin)

Cladosporiu

mcla

dosporioideshalotoleran

s

Cladosporiu

mhalotoleran

sZa

larde

Hoo

gand

Gun

de-C

imerman

28SKF

417564

ITSKF

417584

JA9

Embellisia

Chalasto

sporaEm

bellisia

phragm

ospora

JN383493

(100)

Alternariatenu

issim

asim

ilarity

Alternariaarborescens

Alternariaalternata(G

PD)

Embellisia

phragm

ospora

Embellisia

phragm

ospora

(Emden)

EG

28SKF

417565

ITSKF

417585

BioMed Research International 5

Table1Con

tinued

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA10

Aspergillus

Aspergillus

lentulusJN943567

(99

)A

afffum

igatus

JN2460

66(99

)A

fumigatiaffinisH

F545316(99

)A

novofumigatus

FR733874

(99

)

Aspergillus

fumiga

tus

(120573-tu

bulin

)Aspergillus

fumiga

tiaffinis

Aspergillus

fumiga

tus

Fresenius

28SKF

417566

ITSKF

417586

JA11

ndAspergillus

afffum

igatus

JN2460

66(100)

AfumigatiaffinisK

C253955(99

)

Aspergillus

fumiga

tiaffinis

(120573-tu

bulin

)nd

Aspergillus

fumiga

tiaffinis

Hon

gFrisv

adand

Samson

ITSKF

417587

JA12

Ulocla

dium

Ulocla

dium

consortia

leJQ

585682

(100)

Alternariaradicin

aHM2044

57(99

)Ulocla

dium

consortia

le(G

PD)

Ulocla

dium

tuberculatum

consortiale

Ulocla

dium

consortia

le(Th

umen)E

GSim

mon

s28SKF

417567

ITSKF

417588

JA13

Cladosporiu

mCladosporiu

mcla

dosporioidesJX868638

(99

)C

sphaerosperm

umHM999943

(99

)

Cladosporiu

msphaerosperm

umgrou

p(A

ctin)

Cladosporiu

msphaerosperm

um

Cladosporiu

msphaerosperm

umPenzig

28SKF

417568

ITSKF

417589

JA14

Cladosporiu

mD

avidiella

Cladosporiu

mcla

dosporioidesKC

009539

(99

)DavidiellatassianaGU248332

(98

)nd

nd

Cladosporiu

mcla

dosporioides

(Fresenius)G

AdeV

ries

28SKF

417569

ITSKF

417590

JA15

Penicilliu

mPenicilliu

mspinulosum

KC167852

(100)

Pgla

brum

KC00

9784

(100)

ndPenicilliu

mgla

brum

Penicilliu

msp

28SKF

417570

ITSKF

417591

JA17

Ulocla

dium

Ulocla

dium

consortia

leJQ

585682

(100)

Uchartarum

JN942881

(99

)nd

Ulocla

dium

sp

Ulocla

dium

sp

28SKF

417572

ITSKF

417593

JA18

Cladosporiu

mCladosporiu

mcla

dosporioidesHQ380770

(100)

ndCladosporiu

mcla

dosporioides

Cladosporiu

mcla

dosporioides

(Fresenius)G

AdeV

ries

28SKF

417573

ITSKF

417594

JA19

Alternaria

AlternariaspK

C139473(100)

AarborescensJQ781762

(100)

AalternataJN

107734

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

28SKF

417574

ITSKF

417595

6 BioMed Research International

Table1Con

tinued

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA20

Alternaria

Alternariabrassicae

JX290150

(100)

AporriH

Q821479

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

28SKF

417575

ITSKF

417596

JA22

Penicilliu

m

Penicilliu

mchrysogenu

mKC

341721

(99

)PdipodomyicolaJX232278

(99

)Prubens

JX003126

(99

)Pcommun

eJN676122

(99

)

Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417576

ITSKF

417597

JA23

nd

AlternariaalternataJQ

809324

(100)

AquercusK

C329620(100)

Atenu

issim

aKC

329619

(100)

Aatrans

KC329618

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

ITSKF

417598

BioMed Research International 7

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 0JA3 Penicillium chrysogenum 100 72 37 0 96 46 11 0JA22 Penicillium chrysogenum 100 82 41 25 90 46 0 0AJ5 Penicillium canescens 70 30 20 0 79 44 0 0JA15 Penicillium sp 83 70 34 0 53 18 0 0JA8 Cladosporium halotolerans 80 68 32 18 30 0 0 0JA2 Cladosporium sphaerospermum 76 64 34 22 47 19 11 0JA13 Cladosporium sphaerospermum 100 49 25 0 81 79 12 0JA14 Cladosporium cladosporioides 40 30 10 0 0 0 0 0JA18 Cladosporium cladosporioides 58 40 24 8 63 61 4 0JA4 Aspergillus flavus 90 80 48 26 56 16 0 0JA10 Aspergillus fumigatus 100 76 35 0 100 57 11 0JA11 Aspergillus fumigatiaffinis 100 46 25 0 52 30 12 0JA19 Alternaria alternata 52 38 24 0 57 9 0 0JA20 Alternaria alternata 60 40 0 0 37 24 0 0JA23 Alternaria alternata 100 68 20 0 65 12 0 0JA6 Alternaria tenuissima 100 60 22 0 80 55 17 0JA9 Embellisia phragmospora 94 50 10 0 78 26 0 0JA12 Ulocladium consortiale 72 28 0 0 32 10 0 0JA17 Ulocladium sp 100 70 28 0 67 20 0 0JA7 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) times 100 (2) Relative growth in liquid mediaafter 7 days incubation = (kinetic curve surface under salt stresskinetic curve surface without salt stress) times 100

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 [10mM] Paraquat [500120583M]

JA1 Penicillium chrysogenum 43 39 mdash 66 74JA3 Penicillium chrysogenum 42 50 mdash 84 71JA22 Penicillium chrysogenum 47 45 mdash 68 53JA5 Penicillium canescens 26 28 mdash 59 63JA15 Penicillium sp 43 100 mdash 100 100JA8 Cladosporium halotolerans 34 26 mdash 44 40JA2 Cladosporium sphaerospermum 21 24 mdash 52 48JA13 Cladosporium sphaerospermum 21 43 mdash 55 44JA14 Cladosporium cladosporioides 34 38 mdash 20 31JA18 Cladosporium cladosporioides mdash 41 mdash 18 16JA4 Aspergillus flavus 46 22 mdash 47 39JA10 Aspergillus fumigatus 89 41 61 100 100JA11 Aspergillus fumigatiaffinis 94 26 100 100 100JA19 Alternaria alternata 49 35 mdash 69 89JA20 Alternaria alternata 58 48 mdash 100 100JA23 Alternaria alternata 100 83 40 57 52JA6 Alternaria tenuissima 57 30 mdash 81 100JA9 Embellisia phragmospora 58 67 mdash 100 100JA12 Ulocladium consortiale 44 37 36 56 100JA17 Ulocladium sp 93 28 100 81 100JA7 Engyodontium album 34 18 mdash 66 53Relative growth of fungal strains under different stresses after 7 days incubation was expressed as follows (1) ( colony at pH 10 colony at pH 5) times 100 (2)( colony at 45∘C or 4∘C colony at 30∘C) times 100 (3) ( colony with H2O2 or paraquat colony without stress) times 100 mdash not significant growth

8 BioMed Research International

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

Strain code Strain Protease Amylase Cellulase Lipase LaccaseJA1 Penicillium chrysogenum ++ + minus minus minusJA3 Penicillium chrysogenum ++ minus minus minus minusJA22 Penicillium chrysogenum + + minus minus minusAJ5 Penicillium canescens minus minus minus minus +JA15 Penicillium sp minus minus + minus minusJA8 Cladosporium halotolerans + minus minus minus +JA2 Cladosporium sphaerospermum +++ minus minus + +JA13 Cladosporium sphaerospermum minus + minus minus +JA14 Cladosporium cladosporioides + + minus minus minus

JA18 Cladosporium cladosporioides minus minus minus minus minus

JA4 Aspergillus flavus + minus minus + minus

JA10 Aspergillus fumigatus minus minus minus minus minus

JA11 Aspergillus fumigatiaffinis minus minus minus minus minus

JA19 Alternaria alternata minus minus minus minus minus

JA20 Alternaria alternata minus + minus minus minus

JA23 Alternaria alternata minus minus minus minus minus

JA6 Alternaria tenuissima + minus minus minus minus

JA9 Embellisia phragmospora minus minus minus minus minus

JA12 Ulocladium consortiale minus minus minus minus minus

JA17 Ulocladium sp minus minus minus minus minus

JA7 Engyodontium album + + minus ++ minus

AR activity ratio = ( activity colony) minus no activity + AR lt 1 ++ 1 lt AR lt 2 +++ 2 lt AR lt 3

tolerance [10] On the other hand Molitoris et al [36]reported that other halotolerant and halophilic fungi suchas Aspergillus and Cladosporium spp are predominant insaline desert soil of Dead Sea Many Aspergillus species havebeen also reported to constitute dominant fungi in desert ofSaudi Arabia and Libya [37 38] and halotolerant speciesincludingAspergillus sppPenicillium spp andCladosporiumsphaerospermum have been consistently isolated fromhyper-saline environments around the globe [39] In this studycontrary to many reports on hypersaline environments nospecies belonging to the genera EurotiumThrimmatostromaEmericella and Phaeotheca [9] have been obtained probablybecause of the initial alkaline pH of the Sebkha El Melahsalt lake Actually the effect of pH on the fungal diversity iscontroversialMisra [40] observed that fungal diversity varieswith the pH while other investigators found no significanteffect of pH values of water and soil habitats on fungaloccurrence [41] It is more likely that the number of theisolated fungi is directly correlated to the organic mattercontent of water mud and soil samples [42]

Beside the identification of the recovered fungal isolatesfrom Sebkha El Melah the second goal of the current studywas the detection of some of their physiological and biochem-ical featuresThis allows understanding ecological adaptationto extreme environment and predicts some biotechnologicalusage The 21 strains have been screened for tolerance toextreme NaCl concentrations basic pH temperature andoxidative stress and for the production of important enzy-matic activities in presence of 10 NaCl

Excepting Cladosporium cladosporioides JA18 all isolatesobtained in this study can be considered as moderatelyhaloalkaliphilic fungi as deduced from their ability to grow

at pH 10 and 10 of NaCl However the isolates were ableto grow when salt was not added to their growing mediaExcepting some Wallemia ichthyophaga the most strictlyhalophilic fungus [43] all other fungal strains known todate are able to grow without salt a fact confirmed inour study However gradual decrease in fungal growth wasobserved with the increasing of salt concentration in theculture medium Nineteen strains remain able to grow under15 of NaCl whereas 7 strains were able to tolerate 20 ofNaCl This result was confirmed by salt tolerance assay inliquid media as estimated by Biolog system It is noteworthythat fungi were more sensitive to salt stress in liquid mediathan in solid mediaThis could be explained by the alterationof the osmotic gradient forcing the fungi to expend moreenergy in the osmoregulatory processes resulting in slowergrowth [44] Moreover at higher salt concentration deathoccurs

Regarding the stress of pH the capacity of the majority ofisolates to growth at pH 10 implies firstly that some habitats inthe salt lake may have a varying pH and secondly that fungican tolerate a wide pH range Prima facie the overall resultsin solid and liquid media showed that Penicillium chryso-genum JA1 and JA3 Cladosporium sphaerospermum JA2 andJA13 Cladosporium cladosporioides JA18 Aspergillus fumi-gatus JA10 Aspergillus fumigatiaffinis JA11 and Alternariatenuissima JA6 are the most alkalihalotolerant isolates in thisstudy

The tolerance of the strains to extreme 45∘C was testedand results indicated that Aspergillus fumigatus JA10 Alter-naria alternata JA23 Ulocladium sp JA17 and Aspergillusfumigatiaffinis JA11 were able to grow Of particular interestthe latter two strains retained 100 of the growth rate

BioMed Research International 9

and biomass production as estimated by colony diameterMoreover their ability to grow at low temperature may allowthem to better adapt to the big temperature fluctuation indesert environments Additionally exposure to substratesgenerating oxidative stress such as H

2O2at 10mM and

paraquat at 500 120583M did not alter significantly the growth ofalmost tested strains demonstrating their ability to tolerateoxidative stress These findings may explain their presence indesert regions that are considered amongst the most stressfulenvironments on Earth because of the high UV radiationdesiccation increased salinity low nutrient availability sea-sonal and daily temperature variation and solar irradiation[6 10]

It has been postulated that microorganisms sharing arich and particular extracellular enzymatic activities arecommon in harsh conditions characterizing their ecologicalhabitat including high level of aridity temperature ionicstrength and particularly the low nutrient availability [31]This implies the need by microorganisms for an effectiveutilization of each possible available organic compound [45]Moreover fungal isolates from hot desert were revealed toplay an important role in seeds germination by breakingdormancy and increasing water uptake [46] In the presentstudy the capacity of fungal isolates to produce extracellularenzymes was assayed in the presence of 10 of NaClEnzymes tested were the following amylase for degradationof starch abundant carbohydrate polymer in many planttissues protease for degradation of plant and animal proteinscellulase which hydrolyses the cellulose themain componentof wood ubiquitous substrate for fungi and finally the laccaseinvolved in plant material delignification and in the synthesisof the melanin and related compounds to protect fungiagainst radiationThirteen strains displayed high productionsat least for one of the five-screened activities while noclear correlation of enzyme production profile with fungalsystematic groups was noted The abundance of proteaseactivity is in line with previous data on fungal isolatesfrom extreme environments showing high caseinase activitieswith little effect of salinity and temperature on enzymeproduction [36] The relative limited number of isolatesdisplaying cellulase amylase lipase and laccase activitiessuggests that high concentration of salt may have an adverseeffect on enzymeproduction andor activityTheir energywasprobably oriented to avoid salt stress due to 10 NaCl ratherthan the production of bioactive extrolites [47] Howevernot detecting the enzyme is not absolute confirmation ofan isolate inability to produce it It could also mean thatthe enzyme was not released from the mycelium or thatthe medium is inadequate for its detection [48] Laccaseproduction in the presence of 10 of salt by theCladosporiumgroup may be of biotechnological interest for example inmycoremediation of high salty environments contaminatedby organic pollutants

In conclusion fungal community described in this studywas similar to those reported in inhospitable habitats characterized by limitation of nutrients moisture deficit and

exposure to high solar radiation Further studies are neededin order to elucidate their biogeochemical roles in such anextreme environment and to exploit their promising potentialto produce new biomolecules such as enzymes and protectiveagents against oxidative stress

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support from theEuropean Union in the ambit of the Project BIODESERT(EU FP7-CSA-SA REGPOT-2008-2 Grant agreement no245746) and the Tunisian Ministry of Higher Education andScientific Research in the ambit of the laboratory Project LRMBA20 Atef Jaouani wants to thank the Tunisian Society forMicrobial Ecology (ATEM) for supporting publication fees ofthis work

References

[1] J KWarren ldquoSabkhas saline mudflats and pansrdquo in EvaporitesSediments Resources and Hydrocarbons pp 139ndash220 SpringerBerlin Germany 2006

[2] A A Gorbushina and W E Krumbein ldquoRock dwelling fungalcommunities diversity of life styles and colony structurerdquo inJourney to Diverse Microbial Worlds J Seckbach Ed pp 317ndash334 Kluwer Academic Publishers Amsterdam The Nether-lands 2000

[3] L J Rothschild and R L Mancinelli ldquoLife in extreme environ-mentsrdquo Nature vol 409 no 6823 pp 1092ndash1101 2001

[4] S BHumayounN Bano and J THollibaugh ldquoDepth distribu-tion ofmicrobial diversity inmono lake ameromictic soda lakein Californiardquo Applied and Environmental Microbiology vol 69no 2 pp 1030ndash1042 2003

[5] C Demergasso E O Casamayor G Chong P GalleguillosL Escudero and C Pedros-Alio ldquoDistribution of prokaryoticgenetic diversity in athalassohaline lakes of the AtacamaDesertNorthern Chilerdquo FEMS Microbiology Ecology vol 48 no 1 pp57ndash69 2004

[6] N Gunde-Cimerman A Oren and A Plemenitas ldquoAdaptationto life in high salt concentrationsrdquo in Archaea Bacteria andEukarya p 576 Springer Dordrecht The Netherlands 2005

[7] L Maturrano F Santos R Rossello-Mora and J AntonldquoMicrobial diversity in Maras salterns a hypersaline envi-ronment in the Peruvian Andesrdquo Applied and EnvironmentalMicrobiology vol 72 no 6 pp 3887ndash3895 2006

[8] Q Wu G Zwart M Schauer M Kamst-van Agterveld andM Hahn ldquoBacterioplankton community composition alonga salinity gradient of sixteen high-mountain lakes locatedon the Tibetan Plateau Chinardquo Applied and EnvironmentalMicrobiology vol 72 no 8 pp 5478ndash5485 2006

[9] C Gostincar M Grube S De Hoog P Zalar and N Gunde-Cimerman ldquoExtremotolerance in fungi evolution on the edgerdquoFEMS Microbiology Ecology vol 71 no 1 pp 2ndash11 2010

10 BioMed Research International

[10] K Sterflinger D Tesei and K Zakharova ldquoFungi in hot andcold deserts with particular reference to microcolonial fungirdquoFungal Ecology vol 5 no 4 pp 453ndash462 2012

[11] M J Liszka M E Clark E Schneider and D S ClarkldquoNature versus nurture developing enzymes that functionunder extreme conditionsrdquo Annual Review of Chemical andBiomolecular Engineering vol 3 pp 77ndash102 2012

[12] J P Perthuisot La Sebkha ElMelah de Zarzis genese et evolutiondrsquoun bassin salin paralique Les gisements neolithiques des abordsde la Sebkha el Melah vol 9 of de Travaux du Laboratoire degeologieEcole normale superieure Ecole Normale Superieure1975

[13] I cLellan A Varela M Blahgen et al ldquoHarmonisation ofphysical and chemical methods for soil management in CorkOak forestsmdashlessons from collaborative investigationsrdquoAfricanJournal of Environmental Science and Technology vol 7 no 6pp 386ndash401 2013

[14] J HWarcup ldquoThe soil-plate method for isolation of fungi fromsoilrdquo Nature vol 166 no 4211 pp 117ndash118 1950

[15] K H Domsch W Gams and T H Anderson Compendium ofSoil Fungi Academic Press London UK 1980

[16] J A von ArxThe Genera of Fungi Sporulating in Pure CultureJ Cramer Vaduz Germany 1981

[17] E Kiffer and M Morelet Les deuteromycetes Classification etcles didentification generique INRA Editions Paris France1997

[18] D Liu S Coloe R Baird and J Pedersen ldquoRapid mini-preparation of fungal DNA for PCRrdquo Journal of Clinical Micro-biology vol 38 no 1 article 471 2000

[19] T J White T Bruns S Lee and J Talor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Innes D H Gelfand J J Sninsky and T JWhite Eds pp 315ndash322 Academic Press New York NY USA1990

[20] G Luo and T G Mitchell ldquoRapid identification of pathogenicfungi directly from cultures by using multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 8 pp 2860ndash2865 2002

[21] P Rodrigues C Santos A Venancio and N Lima ldquoSpeciesidentification of Aspergillus section Flavi isolates from Por-tuguese almonds using phenotypic including MALDI-TOFICMS and molecular approachesrdquo Journal of Applied Microbi-ology vol 111 no 4 pp 877ndash892 2011

[22] K Bensch U Braun J Z Groenewald and P W Crous ldquoThegenus cladosporiumrdquo Studies in Mycology vol 72 pp 1ndash4012012

[23] M L Berbee M Pirseyedi and S Hubbard ldquoCochliobo-lus phylogenetics and the origin of known highly virulentpathogens inferred from ITS and glyceraldehyde-3-phosphatedehydrogenase gene sequencesrdquo Mycologia vol 91 no 6 pp964ndash977 1999

[24] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous Ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995

[25] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[26] H M Hassan and I Fridovich ldquoParaquat and Escherichia coliMechanism of production of extracellular superoxide radicalrdquoJournal of Biological Chemistry vol 254 no 21 pp 10846ndash108521979

[27] M B Angelova S B Pashova B K Spasova S V Vassilevand L S Slokoska ldquoOxidative stress response of filamentousfungi induced by hydrogen peroxide and paraquatrdquoMycologicalResearch vol 109 no 2 pp 150ndash158 2005

[28] A de Groot V Chapon P Servant et al ldquoDeinococcus desertisp nov a gamma-radiation-tolerant bacterium isolated fromthe Sahara Desertrdquo International Journal of Systematic andEvolutionary Microbiology vol 55 no 6 pp 2441ndash2446 2005

[29] A Chanal V Chapon K Benzerara et al ldquoThe desert ofTataouine an extreme environment that hosts a wide diversityof microorganisms and radiotolerant bacteriardquo EnvironmentalMicrobiology vol 8 no 3 pp 514ndash525 2006

[30] M Gtari D Daffonchio and A Boudabous ldquoAssessment ofthe genetic diversity of Frankia microsymbionts of Elaeagnusangustifolia L plants growing in a Tunisian date-palm oasis byanalysis of PCR amplified nifD-K intergenic spacerrdquo CanadianJournal of Microbiology vol 53 no 3 pp 440ndash445 2007

[31] I Essoussi F Ghodhbane-Gtari H Amairi et al ldquoEsterase asan enzymatic signature of Geodermatophilaceae adaptabilityto Sahara desert stones and monumentsrdquo Journal of AppliedMicrobiology vol 108 no 5 pp 1723ndash1732 2010

[32] I Sbissi M Neffati A Boudabous C Murat and M GtarildquoPhylogenetic affiliation of the desert truffles Picoa juniperi andPicoa lefebvreirdquo Antonie van Leeuwenhoek vol 98 no 4 pp429ndash436 2010

[33] I Sbissi F Ghodhbane-Gtari M Neffati H Ouzari A Boud-abous and M Gtari ldquoDiversity of the desert truffle Terfeziaboudieri chatin in southern Tunisiardquo Canadian Journal ofMicrobiology vol 57 no 7 pp 599ndash605 2011

[34] C A Conley G Ishkhanova C P McKay and K Cullings ldquoApreliminary survey of non-lichenized fungi cultured from thehyperarid Atacama Desert of Chilerdquo Astrobiology vol 6 no 4pp 521ndash526 2006

[35] E V Smolyanyuk and E N Bilanenko ldquoCommunities ofhalotolerant micromycetes from the areas of natural salinityrdquoMicrobiology vol 80 no 6 pp 877ndash883 2011

[36] H PMolitoris A S Buchalo I Kurchenko et al ldquoPhysiologicaldiversity of the fIrst fIlamentous fungi isolated from the hyper-saline Dead Seardquo inAquatic Mycology Across theMillennium KD Hyde W H Ho and S B Pointing Eds vol 5 pp 55ndash70Fungal Diversity Press 2000

[37] S I I Abdel-Hafez ldquoSurvey of the mycoflora of desert soils inSaudi ArabiardquoMycopathologia vol 80 no 1 pp 3ndash8 1982

[38] A H M El-Said and A Saleem ldquoEcological and physiologicalstudies on soil fungi at Western Region Libyardquo Mycobiologyvol 36 pp 1ndash9 2008

[39] P Zalar G S de Hoog H-J Schroers P W Crous J Z Groe-newald and N Gunde-Cimerman ldquoPhylogeny and ecology ofthe ubiquitous saprobe Cladosporium sphaerospermum withdescriptions of seven new species from hypersaline environ-mentsrdquo Studies in Mycology vol 58 pp 157ndash183 2007

[40] J KMisra ldquoOccurrence distribution and seasonality of aquaticfungi as affected by chemical factors in six alkaline ponds ofIndiardquo Hydrobiologia vol 97 no 2 pp 185ndash191 1982

[41] F T El-Hissy S A El-Zayat A M Khallil and M S MassoudldquoAquatic fungi from the submerged mud of Aswan High DamLakerdquoMicrobiological Research vol 152 no 1 pp 27ndash32 1997

[42] AM Khallil F T El-Hissy and EH Ali ldquoSeasonal fluctuationsof aquatic fungi recovered from Egyptian soil (Delta Region)rdquoJournal of Basic Microbiology vol 35 no 2 pp 93ndash102 1995

BioMed Research International 11

[43] P B Matheny J A Gossmann P Zalar T K A Kumarand D S Hibbett ldquoResolving the phylogenetic position of theWallemiomycetes an enigmatic major lineage of Basidiomy-cotardquoCanadian Journal of Botany vol 84 no 12 pp 1794ndash18052006

[44] A Blomberg and L Adler ldquoTolerence of fungi to NaClrdquo in StressTolerance of Fungi D Jennings Ed pp 209ndash231 1993

[45] A Oren ldquoBioenergetic aspects of halophilismrdquo MicrobiologyandMolecular Biology Reviews vol 63 no 2 pp 334ndash348 1999

[46] P Delgado-Sanchez M A Ortega-Amaro J F Jimenez-Bremont and J Flores ldquoAre fungi important for breaking seeddormancy in desert species Experimental evidence in Opuntiastreptacantha (Cactaceae)rdquo Plant Biology vol 13 no 1 pp 154ndash159 2011

[47] J C Frisvad ldquoHalotolerant and halophilic fungi and theirextrolite productionrdquo in Adaptation to Life at High Salt Con-centrations in Archaea Bacteria and Eukarya N Gunde-Cimerman A Oren and A Plemenitas Eds pp 426ndash440Springer Amsterdam The Netherlands 2005

[48] A Abdel-Raheem and C A Shearer ldquoExtracellular enzymeproduction by freshwater ascomycetesrdquo Fungal Diversity vol 11pp 1ndash19 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

Table1Identifi

catio

nof

fung

aliso

lates

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA1

Penicilliu

m

Penicilliu

mflavigenu

mJX997105

(100)

Pconfertum

JX997081

(100)

PdipodomyisJX9

97080(100)

Pcommun

eKC3

33882(100)

Pchrysogenu

mKC

009827

(100)

Pgrise

ofulvum

JQ781833

(100)

Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417559

ITSKF

417577

JA2

Cladosporiu

m

Cladosporiu

mspG

U017498

(100)

Hyalodend

ronspA

M176721

(100)

Csphaerosperm

umAB5

72902(99

)C

cladosporioidesEF

568045

(99

)

Cladosporiu

msphaerosperm

um(A

ctin)

nd

Cladosporiu

msphaerosperm

umPenzig

28SKF

417560

ITSKF

417578

JA3

Penicilliu

mchrysogenu

mPenicilliu

mcanescensH

Q607858(99

)Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417561

ITSKF

417579

JA4

Aspergillus

Aspergillus

aureofulgens

EF669617

(100)

Aspergillus

flavus

(calmodulin)

ndAspergillus

flavusL

ink

28SKF

417562

ITSKF

417580

JA5

ndPenicilliu

mdesertorum

JX997039

(100)

Pchrysogenu

mKC

009826

(99

)Penicilliu

mcanescensgroup

(120573-tu

bulin

andcalm

odulin)

ndPenicilliu

mcanescens

Sopp

ITSKF

417581

JA6

Alternaria

Alternariatriticim

aculan

sJN867470

(100)

Atenu

issim

aJN

867469

(100)

AmaliJN867468

(100)

AalternataJQ

690087

(100)

Alternariatenu

issim

a(G

PD)

Alternariaalternata

Alternariatenu

issim

a(N

ees)

Wiltshire

28SKF

417563

ITSKF

417582

JA7

ndEn

gyodontiu

malbum

HM214540

(100)

Engyodontiu

malbum

Engyodontiu

malbum

(Lim

ber)de

Hoo

gITSKF

417583

JA8

Cladosporiu

m

Cladosporiu

mcla

dosporioidesEF

568045

(100)

Csphaerosperm

umAM176719

(100)

Chalotoleran

sJX5

35318(99

)

Cladosporiu

mhalotoleran

s(Actin)

Cladosporiu

mcla

dosporioideshalotoleran

s

Cladosporiu

mhalotoleran

sZa

larde

Hoo

gand

Gun

de-C

imerman

28SKF

417564

ITSKF

417584

JA9

Embellisia

Chalasto

sporaEm

bellisia

phragm

ospora

JN383493

(100)

Alternariatenu

issim

asim

ilarity

Alternariaarborescens

Alternariaalternata(G

PD)

Embellisia

phragm

ospora

Embellisia

phragm

ospora

(Emden)

EG

28SKF

417565

ITSKF

417585

BioMed Research International 5

Table1Con

tinued

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA10

Aspergillus

Aspergillus

lentulusJN943567

(99

)A

afffum

igatus

JN2460

66(99

)A

fumigatiaffinisH

F545316(99

)A

novofumigatus

FR733874

(99

)

Aspergillus

fumiga

tus

(120573-tu

bulin

)Aspergillus

fumiga

tiaffinis

Aspergillus

fumiga

tus

Fresenius

28SKF

417566

ITSKF

417586

JA11

ndAspergillus

afffum

igatus

JN2460

66(100)

AfumigatiaffinisK

C253955(99

)

Aspergillus

fumiga

tiaffinis

(120573-tu

bulin

)nd

Aspergillus

fumiga

tiaffinis

Hon

gFrisv

adand

Samson

ITSKF

417587

JA12

Ulocla

dium

Ulocla

dium

consortia

leJQ

585682

(100)

Alternariaradicin

aHM2044

57(99

)Ulocla

dium

consortia

le(G

PD)

Ulocla

dium

tuberculatum

consortiale

Ulocla

dium

consortia

le(Th

umen)E

GSim

mon

s28SKF

417567

ITSKF

417588

JA13

Cladosporiu

mCladosporiu

mcla

dosporioidesJX868638

(99

)C

sphaerosperm

umHM999943

(99

)

Cladosporiu

msphaerosperm

umgrou

p(A

ctin)

Cladosporiu

msphaerosperm

um

Cladosporiu

msphaerosperm

umPenzig

28SKF

417568

ITSKF

417589

JA14

Cladosporiu

mD

avidiella

Cladosporiu

mcla

dosporioidesKC

009539

(99

)DavidiellatassianaGU248332

(98

)nd

nd

Cladosporiu

mcla

dosporioides

(Fresenius)G

AdeV

ries

28SKF

417569

ITSKF

417590

JA15

Penicilliu

mPenicilliu

mspinulosum

KC167852

(100)

Pgla

brum

KC00

9784

(100)

ndPenicilliu

mgla

brum

Penicilliu

msp

28SKF

417570

ITSKF

417591

JA17

Ulocla

dium

Ulocla

dium

consortia

leJQ

585682

(100)

Uchartarum

JN942881

(99

)nd

Ulocla

dium

sp

Ulocla

dium

sp

28SKF

417572

ITSKF

417593

JA18

Cladosporiu

mCladosporiu

mcla

dosporioidesHQ380770

(100)

ndCladosporiu

mcla

dosporioides

Cladosporiu

mcla

dosporioides

(Fresenius)G

AdeV

ries

28SKF

417573

ITSKF

417594

JA19

Alternaria

AlternariaspK

C139473(100)

AarborescensJQ781762

(100)

AalternataJN

107734

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

28SKF

417574

ITSKF

417595

6 BioMed Research International

Table1Con

tinued

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA20

Alternaria

Alternariabrassicae

JX290150

(100)

AporriH

Q821479

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

28SKF

417575

ITSKF

417596

JA22

Penicilliu

m

Penicilliu

mchrysogenu

mKC

341721

(99

)PdipodomyicolaJX232278

(99

)Prubens

JX003126

(99

)Pcommun

eJN676122

(99

)

Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417576

ITSKF

417597

JA23

nd

AlternariaalternataJQ

809324

(100)

AquercusK

C329620(100)

Atenu

issim

aKC

329619

(100)

Aatrans

KC329618

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

ITSKF

417598

BioMed Research International 7

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 0JA3 Penicillium chrysogenum 100 72 37 0 96 46 11 0JA22 Penicillium chrysogenum 100 82 41 25 90 46 0 0AJ5 Penicillium canescens 70 30 20 0 79 44 0 0JA15 Penicillium sp 83 70 34 0 53 18 0 0JA8 Cladosporium halotolerans 80 68 32 18 30 0 0 0JA2 Cladosporium sphaerospermum 76 64 34 22 47 19 11 0JA13 Cladosporium sphaerospermum 100 49 25 0 81 79 12 0JA14 Cladosporium cladosporioides 40 30 10 0 0 0 0 0JA18 Cladosporium cladosporioides 58 40 24 8 63 61 4 0JA4 Aspergillus flavus 90 80 48 26 56 16 0 0JA10 Aspergillus fumigatus 100 76 35 0 100 57 11 0JA11 Aspergillus fumigatiaffinis 100 46 25 0 52 30 12 0JA19 Alternaria alternata 52 38 24 0 57 9 0 0JA20 Alternaria alternata 60 40 0 0 37 24 0 0JA23 Alternaria alternata 100 68 20 0 65 12 0 0JA6 Alternaria tenuissima 100 60 22 0 80 55 17 0JA9 Embellisia phragmospora 94 50 10 0 78 26 0 0JA12 Ulocladium consortiale 72 28 0 0 32 10 0 0JA17 Ulocladium sp 100 70 28 0 67 20 0 0JA7 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) times 100 (2) Relative growth in liquid mediaafter 7 days incubation = (kinetic curve surface under salt stresskinetic curve surface without salt stress) times 100

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 [10mM] Paraquat [500120583M]

JA1 Penicillium chrysogenum 43 39 mdash 66 74JA3 Penicillium chrysogenum 42 50 mdash 84 71JA22 Penicillium chrysogenum 47 45 mdash 68 53JA5 Penicillium canescens 26 28 mdash 59 63JA15 Penicillium sp 43 100 mdash 100 100JA8 Cladosporium halotolerans 34 26 mdash 44 40JA2 Cladosporium sphaerospermum 21 24 mdash 52 48JA13 Cladosporium sphaerospermum 21 43 mdash 55 44JA14 Cladosporium cladosporioides 34 38 mdash 20 31JA18 Cladosporium cladosporioides mdash 41 mdash 18 16JA4 Aspergillus flavus 46 22 mdash 47 39JA10 Aspergillus fumigatus 89 41 61 100 100JA11 Aspergillus fumigatiaffinis 94 26 100 100 100JA19 Alternaria alternata 49 35 mdash 69 89JA20 Alternaria alternata 58 48 mdash 100 100JA23 Alternaria alternata 100 83 40 57 52JA6 Alternaria tenuissima 57 30 mdash 81 100JA9 Embellisia phragmospora 58 67 mdash 100 100JA12 Ulocladium consortiale 44 37 36 56 100JA17 Ulocladium sp 93 28 100 81 100JA7 Engyodontium album 34 18 mdash 66 53Relative growth of fungal strains under different stresses after 7 days incubation was expressed as follows (1) ( colony at pH 10 colony at pH 5) times 100 (2)( colony at 45∘C or 4∘C colony at 30∘C) times 100 (3) ( colony with H2O2 or paraquat colony without stress) times 100 mdash not significant growth

8 BioMed Research International

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

Strain code Strain Protease Amylase Cellulase Lipase LaccaseJA1 Penicillium chrysogenum ++ + minus minus minusJA3 Penicillium chrysogenum ++ minus minus minus minusJA22 Penicillium chrysogenum + + minus minus minusAJ5 Penicillium canescens minus minus minus minus +JA15 Penicillium sp minus minus + minus minusJA8 Cladosporium halotolerans + minus minus minus +JA2 Cladosporium sphaerospermum +++ minus minus + +JA13 Cladosporium sphaerospermum minus + minus minus +JA14 Cladosporium cladosporioides + + minus minus minus

JA18 Cladosporium cladosporioides minus minus minus minus minus

JA4 Aspergillus flavus + minus minus + minus

JA10 Aspergillus fumigatus minus minus minus minus minus

JA11 Aspergillus fumigatiaffinis minus minus minus minus minus

JA19 Alternaria alternata minus minus minus minus minus

JA20 Alternaria alternata minus + minus minus minus

JA23 Alternaria alternata minus minus minus minus minus

JA6 Alternaria tenuissima + minus minus minus minus

JA9 Embellisia phragmospora minus minus minus minus minus

JA12 Ulocladium consortiale minus minus minus minus minus

JA17 Ulocladium sp minus minus minus minus minus

JA7 Engyodontium album + + minus ++ minus

AR activity ratio = ( activity colony) minus no activity + AR lt 1 ++ 1 lt AR lt 2 +++ 2 lt AR lt 3

tolerance [10] On the other hand Molitoris et al [36]reported that other halotolerant and halophilic fungi suchas Aspergillus and Cladosporium spp are predominant insaline desert soil of Dead Sea Many Aspergillus species havebeen also reported to constitute dominant fungi in desert ofSaudi Arabia and Libya [37 38] and halotolerant speciesincludingAspergillus sppPenicillium spp andCladosporiumsphaerospermum have been consistently isolated fromhyper-saline environments around the globe [39] In this studycontrary to many reports on hypersaline environments nospecies belonging to the genera EurotiumThrimmatostromaEmericella and Phaeotheca [9] have been obtained probablybecause of the initial alkaline pH of the Sebkha El Melahsalt lake Actually the effect of pH on the fungal diversity iscontroversialMisra [40] observed that fungal diversity varieswith the pH while other investigators found no significanteffect of pH values of water and soil habitats on fungaloccurrence [41] It is more likely that the number of theisolated fungi is directly correlated to the organic mattercontent of water mud and soil samples [42]

Beside the identification of the recovered fungal isolatesfrom Sebkha El Melah the second goal of the current studywas the detection of some of their physiological and biochem-ical featuresThis allows understanding ecological adaptationto extreme environment and predicts some biotechnologicalusage The 21 strains have been screened for tolerance toextreme NaCl concentrations basic pH temperature andoxidative stress and for the production of important enzy-matic activities in presence of 10 NaCl

Excepting Cladosporium cladosporioides JA18 all isolatesobtained in this study can be considered as moderatelyhaloalkaliphilic fungi as deduced from their ability to grow

at pH 10 and 10 of NaCl However the isolates were ableto grow when salt was not added to their growing mediaExcepting some Wallemia ichthyophaga the most strictlyhalophilic fungus [43] all other fungal strains known todate are able to grow without salt a fact confirmed inour study However gradual decrease in fungal growth wasobserved with the increasing of salt concentration in theculture medium Nineteen strains remain able to grow under15 of NaCl whereas 7 strains were able to tolerate 20 ofNaCl This result was confirmed by salt tolerance assay inliquid media as estimated by Biolog system It is noteworthythat fungi were more sensitive to salt stress in liquid mediathan in solid mediaThis could be explained by the alterationof the osmotic gradient forcing the fungi to expend moreenergy in the osmoregulatory processes resulting in slowergrowth [44] Moreover at higher salt concentration deathoccurs

Regarding the stress of pH the capacity of the majority ofisolates to growth at pH 10 implies firstly that some habitats inthe salt lake may have a varying pH and secondly that fungican tolerate a wide pH range Prima facie the overall resultsin solid and liquid media showed that Penicillium chryso-genum JA1 and JA3 Cladosporium sphaerospermum JA2 andJA13 Cladosporium cladosporioides JA18 Aspergillus fumi-gatus JA10 Aspergillus fumigatiaffinis JA11 and Alternariatenuissima JA6 are the most alkalihalotolerant isolates in thisstudy

The tolerance of the strains to extreme 45∘C was testedand results indicated that Aspergillus fumigatus JA10 Alter-naria alternata JA23 Ulocladium sp JA17 and Aspergillusfumigatiaffinis JA11 were able to grow Of particular interestthe latter two strains retained 100 of the growth rate

BioMed Research International 9

and biomass production as estimated by colony diameterMoreover their ability to grow at low temperature may allowthem to better adapt to the big temperature fluctuation indesert environments Additionally exposure to substratesgenerating oxidative stress such as H

2O2at 10mM and

paraquat at 500 120583M did not alter significantly the growth ofalmost tested strains demonstrating their ability to tolerateoxidative stress These findings may explain their presence indesert regions that are considered amongst the most stressfulenvironments on Earth because of the high UV radiationdesiccation increased salinity low nutrient availability sea-sonal and daily temperature variation and solar irradiation[6 10]

It has been postulated that microorganisms sharing arich and particular extracellular enzymatic activities arecommon in harsh conditions characterizing their ecologicalhabitat including high level of aridity temperature ionicstrength and particularly the low nutrient availability [31]This implies the need by microorganisms for an effectiveutilization of each possible available organic compound [45]Moreover fungal isolates from hot desert were revealed toplay an important role in seeds germination by breakingdormancy and increasing water uptake [46] In the presentstudy the capacity of fungal isolates to produce extracellularenzymes was assayed in the presence of 10 of NaClEnzymes tested were the following amylase for degradationof starch abundant carbohydrate polymer in many planttissues protease for degradation of plant and animal proteinscellulase which hydrolyses the cellulose themain componentof wood ubiquitous substrate for fungi and finally the laccaseinvolved in plant material delignification and in the synthesisof the melanin and related compounds to protect fungiagainst radiationThirteen strains displayed high productionsat least for one of the five-screened activities while noclear correlation of enzyme production profile with fungalsystematic groups was noted The abundance of proteaseactivity is in line with previous data on fungal isolatesfrom extreme environments showing high caseinase activitieswith little effect of salinity and temperature on enzymeproduction [36] The relative limited number of isolatesdisplaying cellulase amylase lipase and laccase activitiessuggests that high concentration of salt may have an adverseeffect on enzymeproduction andor activityTheir energywasprobably oriented to avoid salt stress due to 10 NaCl ratherthan the production of bioactive extrolites [47] Howevernot detecting the enzyme is not absolute confirmation ofan isolate inability to produce it It could also mean thatthe enzyme was not released from the mycelium or thatthe medium is inadequate for its detection [48] Laccaseproduction in the presence of 10 of salt by theCladosporiumgroup may be of biotechnological interest for example inmycoremediation of high salty environments contaminatedby organic pollutants

In conclusion fungal community described in this studywas similar to those reported in inhospitable habitats characterized by limitation of nutrients moisture deficit and

exposure to high solar radiation Further studies are neededin order to elucidate their biogeochemical roles in such anextreme environment and to exploit their promising potentialto produce new biomolecules such as enzymes and protectiveagents against oxidative stress

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support from theEuropean Union in the ambit of the Project BIODESERT(EU FP7-CSA-SA REGPOT-2008-2 Grant agreement no245746) and the Tunisian Ministry of Higher Education andScientific Research in the ambit of the laboratory Project LRMBA20 Atef Jaouani wants to thank the Tunisian Society forMicrobial Ecology (ATEM) for supporting publication fees ofthis work

References

[1] J KWarren ldquoSabkhas saline mudflats and pansrdquo in EvaporitesSediments Resources and Hydrocarbons pp 139ndash220 SpringerBerlin Germany 2006

[2] A A Gorbushina and W E Krumbein ldquoRock dwelling fungalcommunities diversity of life styles and colony structurerdquo inJourney to Diverse Microbial Worlds J Seckbach Ed pp 317ndash334 Kluwer Academic Publishers Amsterdam The Nether-lands 2000

[3] L J Rothschild and R L Mancinelli ldquoLife in extreme environ-mentsrdquo Nature vol 409 no 6823 pp 1092ndash1101 2001

[4] S BHumayounN Bano and J THollibaugh ldquoDepth distribu-tion ofmicrobial diversity inmono lake ameromictic soda lakein Californiardquo Applied and Environmental Microbiology vol 69no 2 pp 1030ndash1042 2003

[5] C Demergasso E O Casamayor G Chong P GalleguillosL Escudero and C Pedros-Alio ldquoDistribution of prokaryoticgenetic diversity in athalassohaline lakes of the AtacamaDesertNorthern Chilerdquo FEMS Microbiology Ecology vol 48 no 1 pp57ndash69 2004

[6] N Gunde-Cimerman A Oren and A Plemenitas ldquoAdaptationto life in high salt concentrationsrdquo in Archaea Bacteria andEukarya p 576 Springer Dordrecht The Netherlands 2005

[7] L Maturrano F Santos R Rossello-Mora and J AntonldquoMicrobial diversity in Maras salterns a hypersaline envi-ronment in the Peruvian Andesrdquo Applied and EnvironmentalMicrobiology vol 72 no 6 pp 3887ndash3895 2006

[8] Q Wu G Zwart M Schauer M Kamst-van Agterveld andM Hahn ldquoBacterioplankton community composition alonga salinity gradient of sixteen high-mountain lakes locatedon the Tibetan Plateau Chinardquo Applied and EnvironmentalMicrobiology vol 72 no 8 pp 5478ndash5485 2006

[9] C Gostincar M Grube S De Hoog P Zalar and N Gunde-Cimerman ldquoExtremotolerance in fungi evolution on the edgerdquoFEMS Microbiology Ecology vol 71 no 1 pp 2ndash11 2010

10 BioMed Research International

[10] K Sterflinger D Tesei and K Zakharova ldquoFungi in hot andcold deserts with particular reference to microcolonial fungirdquoFungal Ecology vol 5 no 4 pp 453ndash462 2012

[11] M J Liszka M E Clark E Schneider and D S ClarkldquoNature versus nurture developing enzymes that functionunder extreme conditionsrdquo Annual Review of Chemical andBiomolecular Engineering vol 3 pp 77ndash102 2012

[12] J P Perthuisot La Sebkha ElMelah de Zarzis genese et evolutiondrsquoun bassin salin paralique Les gisements neolithiques des abordsde la Sebkha el Melah vol 9 of de Travaux du Laboratoire degeologieEcole normale superieure Ecole Normale Superieure1975

[13] I cLellan A Varela M Blahgen et al ldquoHarmonisation ofphysical and chemical methods for soil management in CorkOak forestsmdashlessons from collaborative investigationsrdquoAfricanJournal of Environmental Science and Technology vol 7 no 6pp 386ndash401 2013

[14] J HWarcup ldquoThe soil-plate method for isolation of fungi fromsoilrdquo Nature vol 166 no 4211 pp 117ndash118 1950

[15] K H Domsch W Gams and T H Anderson Compendium ofSoil Fungi Academic Press London UK 1980

[16] J A von ArxThe Genera of Fungi Sporulating in Pure CultureJ Cramer Vaduz Germany 1981

[17] E Kiffer and M Morelet Les deuteromycetes Classification etcles didentification generique INRA Editions Paris France1997

[18] D Liu S Coloe R Baird and J Pedersen ldquoRapid mini-preparation of fungal DNA for PCRrdquo Journal of Clinical Micro-biology vol 38 no 1 article 471 2000

[19] T J White T Bruns S Lee and J Talor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Innes D H Gelfand J J Sninsky and T JWhite Eds pp 315ndash322 Academic Press New York NY USA1990

[20] G Luo and T G Mitchell ldquoRapid identification of pathogenicfungi directly from cultures by using multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 8 pp 2860ndash2865 2002

[21] P Rodrigues C Santos A Venancio and N Lima ldquoSpeciesidentification of Aspergillus section Flavi isolates from Por-tuguese almonds using phenotypic including MALDI-TOFICMS and molecular approachesrdquo Journal of Applied Microbi-ology vol 111 no 4 pp 877ndash892 2011

[22] K Bensch U Braun J Z Groenewald and P W Crous ldquoThegenus cladosporiumrdquo Studies in Mycology vol 72 pp 1ndash4012012

[23] M L Berbee M Pirseyedi and S Hubbard ldquoCochliobo-lus phylogenetics and the origin of known highly virulentpathogens inferred from ITS and glyceraldehyde-3-phosphatedehydrogenase gene sequencesrdquo Mycologia vol 91 no 6 pp964ndash977 1999

[24] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous Ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995

[25] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[26] H M Hassan and I Fridovich ldquoParaquat and Escherichia coliMechanism of production of extracellular superoxide radicalrdquoJournal of Biological Chemistry vol 254 no 21 pp 10846ndash108521979

[27] M B Angelova S B Pashova B K Spasova S V Vassilevand L S Slokoska ldquoOxidative stress response of filamentousfungi induced by hydrogen peroxide and paraquatrdquoMycologicalResearch vol 109 no 2 pp 150ndash158 2005

[28] A de Groot V Chapon P Servant et al ldquoDeinococcus desertisp nov a gamma-radiation-tolerant bacterium isolated fromthe Sahara Desertrdquo International Journal of Systematic andEvolutionary Microbiology vol 55 no 6 pp 2441ndash2446 2005

[29] A Chanal V Chapon K Benzerara et al ldquoThe desert ofTataouine an extreme environment that hosts a wide diversityof microorganisms and radiotolerant bacteriardquo EnvironmentalMicrobiology vol 8 no 3 pp 514ndash525 2006

[30] M Gtari D Daffonchio and A Boudabous ldquoAssessment ofthe genetic diversity of Frankia microsymbionts of Elaeagnusangustifolia L plants growing in a Tunisian date-palm oasis byanalysis of PCR amplified nifD-K intergenic spacerrdquo CanadianJournal of Microbiology vol 53 no 3 pp 440ndash445 2007

[31] I Essoussi F Ghodhbane-Gtari H Amairi et al ldquoEsterase asan enzymatic signature of Geodermatophilaceae adaptabilityto Sahara desert stones and monumentsrdquo Journal of AppliedMicrobiology vol 108 no 5 pp 1723ndash1732 2010

[32] I Sbissi M Neffati A Boudabous C Murat and M GtarildquoPhylogenetic affiliation of the desert truffles Picoa juniperi andPicoa lefebvreirdquo Antonie van Leeuwenhoek vol 98 no 4 pp429ndash436 2010

[33] I Sbissi F Ghodhbane-Gtari M Neffati H Ouzari A Boud-abous and M Gtari ldquoDiversity of the desert truffle Terfeziaboudieri chatin in southern Tunisiardquo Canadian Journal ofMicrobiology vol 57 no 7 pp 599ndash605 2011

[34] C A Conley G Ishkhanova C P McKay and K Cullings ldquoApreliminary survey of non-lichenized fungi cultured from thehyperarid Atacama Desert of Chilerdquo Astrobiology vol 6 no 4pp 521ndash526 2006

[35] E V Smolyanyuk and E N Bilanenko ldquoCommunities ofhalotolerant micromycetes from the areas of natural salinityrdquoMicrobiology vol 80 no 6 pp 877ndash883 2011

[36] H PMolitoris A S Buchalo I Kurchenko et al ldquoPhysiologicaldiversity of the fIrst fIlamentous fungi isolated from the hyper-saline Dead Seardquo inAquatic Mycology Across theMillennium KD Hyde W H Ho and S B Pointing Eds vol 5 pp 55ndash70Fungal Diversity Press 2000

[37] S I I Abdel-Hafez ldquoSurvey of the mycoflora of desert soils inSaudi ArabiardquoMycopathologia vol 80 no 1 pp 3ndash8 1982

[38] A H M El-Said and A Saleem ldquoEcological and physiologicalstudies on soil fungi at Western Region Libyardquo Mycobiologyvol 36 pp 1ndash9 2008

[39] P Zalar G S de Hoog H-J Schroers P W Crous J Z Groe-newald and N Gunde-Cimerman ldquoPhylogeny and ecology ofthe ubiquitous saprobe Cladosporium sphaerospermum withdescriptions of seven new species from hypersaline environ-mentsrdquo Studies in Mycology vol 58 pp 157ndash183 2007

[40] J KMisra ldquoOccurrence distribution and seasonality of aquaticfungi as affected by chemical factors in six alkaline ponds ofIndiardquo Hydrobiologia vol 97 no 2 pp 185ndash191 1982

[41] F T El-Hissy S A El-Zayat A M Khallil and M S MassoudldquoAquatic fungi from the submerged mud of Aswan High DamLakerdquoMicrobiological Research vol 152 no 1 pp 27ndash32 1997

[42] AM Khallil F T El-Hissy and EH Ali ldquoSeasonal fluctuationsof aquatic fungi recovered from Egyptian soil (Delta Region)rdquoJournal of Basic Microbiology vol 35 no 2 pp 93ndash102 1995

BioMed Research International 11

[43] P B Matheny J A Gossmann P Zalar T K A Kumarand D S Hibbett ldquoResolving the phylogenetic position of theWallemiomycetes an enigmatic major lineage of Basidiomy-cotardquoCanadian Journal of Botany vol 84 no 12 pp 1794ndash18052006

[44] A Blomberg and L Adler ldquoTolerence of fungi to NaClrdquo in StressTolerance of Fungi D Jennings Ed pp 209ndash231 1993

[45] A Oren ldquoBioenergetic aspects of halophilismrdquo MicrobiologyandMolecular Biology Reviews vol 63 no 2 pp 334ndash348 1999

[46] P Delgado-Sanchez M A Ortega-Amaro J F Jimenez-Bremont and J Flores ldquoAre fungi important for breaking seeddormancy in desert species Experimental evidence in Opuntiastreptacantha (Cactaceae)rdquo Plant Biology vol 13 no 1 pp 154ndash159 2011

[47] J C Frisvad ldquoHalotolerant and halophilic fungi and theirextrolite productionrdquo in Adaptation to Life at High Salt Con-centrations in Archaea Bacteria and Eukarya N Gunde-Cimerman A Oren and A Plemenitas Eds pp 426ndash440Springer Amsterdam The Netherlands 2005

[48] A Abdel-Raheem and C A Shearer ldquoExtracellular enzymeproduction by freshwater ascomycetesrdquo Fungal Diversity vol 11pp 1ndash19 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

Table1Con

tinued

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA10

Aspergillus

Aspergillus

lentulusJN943567

(99

)A

afffum

igatus

JN2460

66(99

)A

fumigatiaffinisH

F545316(99

)A

novofumigatus

FR733874

(99

)

Aspergillus

fumiga

tus

(120573-tu

bulin

)Aspergillus

fumiga

tiaffinis

Aspergillus

fumiga

tus

Fresenius

28SKF

417566

ITSKF

417586

JA11

ndAspergillus

afffum

igatus

JN2460

66(100)

AfumigatiaffinisK

C253955(99

)

Aspergillus

fumiga

tiaffinis

(120573-tu

bulin

)nd

Aspergillus

fumiga

tiaffinis

Hon

gFrisv

adand

Samson

ITSKF

417587

JA12

Ulocla

dium

Ulocla

dium

consortia

leJQ

585682

(100)

Alternariaradicin

aHM2044

57(99

)Ulocla

dium

consortia

le(G

PD)

Ulocla

dium

tuberculatum

consortiale

Ulocla

dium

consortia

le(Th

umen)E

GSim

mon

s28SKF

417567

ITSKF

417588

JA13

Cladosporiu

mCladosporiu

mcla

dosporioidesJX868638

(99

)C

sphaerosperm

umHM999943

(99

)

Cladosporiu

msphaerosperm

umgrou

p(A

ctin)

Cladosporiu

msphaerosperm

um

Cladosporiu

msphaerosperm

umPenzig

28SKF

417568

ITSKF

417589

JA14

Cladosporiu

mD

avidiella

Cladosporiu

mcla

dosporioidesKC

009539

(99

)DavidiellatassianaGU248332

(98

)nd

nd

Cladosporiu

mcla

dosporioides

(Fresenius)G

AdeV

ries

28SKF

417569

ITSKF

417590

JA15

Penicilliu

mPenicilliu

mspinulosum

KC167852

(100)

Pgla

brum

KC00

9784

(100)

ndPenicilliu

mgla

brum

Penicilliu

msp

28SKF

417570

ITSKF

417591

JA17

Ulocla

dium

Ulocla

dium

consortia

leJQ

585682

(100)

Uchartarum

JN942881

(99

)nd

Ulocla

dium

sp

Ulocla

dium

sp

28SKF

417572

ITSKF

417593

JA18

Cladosporiu

mCladosporiu

mcla

dosporioidesHQ380770

(100)

ndCladosporiu

mcla

dosporioides

Cladosporiu

mcla

dosporioides

(Fresenius)G

AdeV

ries

28SKF

417573

ITSKF

417594

JA19

Alternaria

AlternariaspK

C139473(100)

AarborescensJQ781762

(100)

AalternataJN

107734

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

28SKF

417574

ITSKF

417595

6 BioMed Research International

Table1Con

tinued

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA20

Alternaria

Alternariabrassicae

JX290150

(100)

AporriH

Q821479

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

28SKF

417575

ITSKF

417596

JA22

Penicilliu

m

Penicilliu

mchrysogenu

mKC

341721

(99

)PdipodomyicolaJX232278

(99

)Prubens

JX003126

(99

)Pcommun

eJN676122

(99

)

Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417576

ITSKF

417597

JA23

nd

AlternariaalternataJQ

809324

(100)

AquercusK

C329620(100)

Atenu

issim

aKC

329619

(100)

Aatrans

KC329618

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

ITSKF

417598

BioMed Research International 7

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 0JA3 Penicillium chrysogenum 100 72 37 0 96 46 11 0JA22 Penicillium chrysogenum 100 82 41 25 90 46 0 0AJ5 Penicillium canescens 70 30 20 0 79 44 0 0JA15 Penicillium sp 83 70 34 0 53 18 0 0JA8 Cladosporium halotolerans 80 68 32 18 30 0 0 0JA2 Cladosporium sphaerospermum 76 64 34 22 47 19 11 0JA13 Cladosporium sphaerospermum 100 49 25 0 81 79 12 0JA14 Cladosporium cladosporioides 40 30 10 0 0 0 0 0JA18 Cladosporium cladosporioides 58 40 24 8 63 61 4 0JA4 Aspergillus flavus 90 80 48 26 56 16 0 0JA10 Aspergillus fumigatus 100 76 35 0 100 57 11 0JA11 Aspergillus fumigatiaffinis 100 46 25 0 52 30 12 0JA19 Alternaria alternata 52 38 24 0 57 9 0 0JA20 Alternaria alternata 60 40 0 0 37 24 0 0JA23 Alternaria alternata 100 68 20 0 65 12 0 0JA6 Alternaria tenuissima 100 60 22 0 80 55 17 0JA9 Embellisia phragmospora 94 50 10 0 78 26 0 0JA12 Ulocladium consortiale 72 28 0 0 32 10 0 0JA17 Ulocladium sp 100 70 28 0 67 20 0 0JA7 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) times 100 (2) Relative growth in liquid mediaafter 7 days incubation = (kinetic curve surface under salt stresskinetic curve surface without salt stress) times 100

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 [10mM] Paraquat [500120583M]

JA1 Penicillium chrysogenum 43 39 mdash 66 74JA3 Penicillium chrysogenum 42 50 mdash 84 71JA22 Penicillium chrysogenum 47 45 mdash 68 53JA5 Penicillium canescens 26 28 mdash 59 63JA15 Penicillium sp 43 100 mdash 100 100JA8 Cladosporium halotolerans 34 26 mdash 44 40JA2 Cladosporium sphaerospermum 21 24 mdash 52 48JA13 Cladosporium sphaerospermum 21 43 mdash 55 44JA14 Cladosporium cladosporioides 34 38 mdash 20 31JA18 Cladosporium cladosporioides mdash 41 mdash 18 16JA4 Aspergillus flavus 46 22 mdash 47 39JA10 Aspergillus fumigatus 89 41 61 100 100JA11 Aspergillus fumigatiaffinis 94 26 100 100 100JA19 Alternaria alternata 49 35 mdash 69 89JA20 Alternaria alternata 58 48 mdash 100 100JA23 Alternaria alternata 100 83 40 57 52JA6 Alternaria tenuissima 57 30 mdash 81 100JA9 Embellisia phragmospora 58 67 mdash 100 100JA12 Ulocladium consortiale 44 37 36 56 100JA17 Ulocladium sp 93 28 100 81 100JA7 Engyodontium album 34 18 mdash 66 53Relative growth of fungal strains under different stresses after 7 days incubation was expressed as follows (1) ( colony at pH 10 colony at pH 5) times 100 (2)( colony at 45∘C or 4∘C colony at 30∘C) times 100 (3) ( colony with H2O2 or paraquat colony without stress) times 100 mdash not significant growth

8 BioMed Research International

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

Strain code Strain Protease Amylase Cellulase Lipase LaccaseJA1 Penicillium chrysogenum ++ + minus minus minusJA3 Penicillium chrysogenum ++ minus minus minus minusJA22 Penicillium chrysogenum + + minus minus minusAJ5 Penicillium canescens minus minus minus minus +JA15 Penicillium sp minus minus + minus minusJA8 Cladosporium halotolerans + minus minus minus +JA2 Cladosporium sphaerospermum +++ minus minus + +JA13 Cladosporium sphaerospermum minus + minus minus +JA14 Cladosporium cladosporioides + + minus minus minus

JA18 Cladosporium cladosporioides minus minus minus minus minus

JA4 Aspergillus flavus + minus minus + minus

JA10 Aspergillus fumigatus minus minus minus minus minus

JA11 Aspergillus fumigatiaffinis minus minus minus minus minus

JA19 Alternaria alternata minus minus minus minus minus

JA20 Alternaria alternata minus + minus minus minus

JA23 Alternaria alternata minus minus minus minus minus

JA6 Alternaria tenuissima + minus minus minus minus

JA9 Embellisia phragmospora minus minus minus minus minus

JA12 Ulocladium consortiale minus minus minus minus minus

JA17 Ulocladium sp minus minus minus minus minus

JA7 Engyodontium album + + minus ++ minus

AR activity ratio = ( activity colony) minus no activity + AR lt 1 ++ 1 lt AR lt 2 +++ 2 lt AR lt 3

tolerance [10] On the other hand Molitoris et al [36]reported that other halotolerant and halophilic fungi suchas Aspergillus and Cladosporium spp are predominant insaline desert soil of Dead Sea Many Aspergillus species havebeen also reported to constitute dominant fungi in desert ofSaudi Arabia and Libya [37 38] and halotolerant speciesincludingAspergillus sppPenicillium spp andCladosporiumsphaerospermum have been consistently isolated fromhyper-saline environments around the globe [39] In this studycontrary to many reports on hypersaline environments nospecies belonging to the genera EurotiumThrimmatostromaEmericella and Phaeotheca [9] have been obtained probablybecause of the initial alkaline pH of the Sebkha El Melahsalt lake Actually the effect of pH on the fungal diversity iscontroversialMisra [40] observed that fungal diversity varieswith the pH while other investigators found no significanteffect of pH values of water and soil habitats on fungaloccurrence [41] It is more likely that the number of theisolated fungi is directly correlated to the organic mattercontent of water mud and soil samples [42]

Beside the identification of the recovered fungal isolatesfrom Sebkha El Melah the second goal of the current studywas the detection of some of their physiological and biochem-ical featuresThis allows understanding ecological adaptationto extreme environment and predicts some biotechnologicalusage The 21 strains have been screened for tolerance toextreme NaCl concentrations basic pH temperature andoxidative stress and for the production of important enzy-matic activities in presence of 10 NaCl

Excepting Cladosporium cladosporioides JA18 all isolatesobtained in this study can be considered as moderatelyhaloalkaliphilic fungi as deduced from their ability to grow

at pH 10 and 10 of NaCl However the isolates were ableto grow when salt was not added to their growing mediaExcepting some Wallemia ichthyophaga the most strictlyhalophilic fungus [43] all other fungal strains known todate are able to grow without salt a fact confirmed inour study However gradual decrease in fungal growth wasobserved with the increasing of salt concentration in theculture medium Nineteen strains remain able to grow under15 of NaCl whereas 7 strains were able to tolerate 20 ofNaCl This result was confirmed by salt tolerance assay inliquid media as estimated by Biolog system It is noteworthythat fungi were more sensitive to salt stress in liquid mediathan in solid mediaThis could be explained by the alterationof the osmotic gradient forcing the fungi to expend moreenergy in the osmoregulatory processes resulting in slowergrowth [44] Moreover at higher salt concentration deathoccurs

Regarding the stress of pH the capacity of the majority ofisolates to growth at pH 10 implies firstly that some habitats inthe salt lake may have a varying pH and secondly that fungican tolerate a wide pH range Prima facie the overall resultsin solid and liquid media showed that Penicillium chryso-genum JA1 and JA3 Cladosporium sphaerospermum JA2 andJA13 Cladosporium cladosporioides JA18 Aspergillus fumi-gatus JA10 Aspergillus fumigatiaffinis JA11 and Alternariatenuissima JA6 are the most alkalihalotolerant isolates in thisstudy

The tolerance of the strains to extreme 45∘C was testedand results indicated that Aspergillus fumigatus JA10 Alter-naria alternata JA23 Ulocladium sp JA17 and Aspergillusfumigatiaffinis JA11 were able to grow Of particular interestthe latter two strains retained 100 of the growth rate

BioMed Research International 9

and biomass production as estimated by colony diameterMoreover their ability to grow at low temperature may allowthem to better adapt to the big temperature fluctuation indesert environments Additionally exposure to substratesgenerating oxidative stress such as H

2O2at 10mM and

paraquat at 500 120583M did not alter significantly the growth ofalmost tested strains demonstrating their ability to tolerateoxidative stress These findings may explain their presence indesert regions that are considered amongst the most stressfulenvironments on Earth because of the high UV radiationdesiccation increased salinity low nutrient availability sea-sonal and daily temperature variation and solar irradiation[6 10]

It has been postulated that microorganisms sharing arich and particular extracellular enzymatic activities arecommon in harsh conditions characterizing their ecologicalhabitat including high level of aridity temperature ionicstrength and particularly the low nutrient availability [31]This implies the need by microorganisms for an effectiveutilization of each possible available organic compound [45]Moreover fungal isolates from hot desert were revealed toplay an important role in seeds germination by breakingdormancy and increasing water uptake [46] In the presentstudy the capacity of fungal isolates to produce extracellularenzymes was assayed in the presence of 10 of NaClEnzymes tested were the following amylase for degradationof starch abundant carbohydrate polymer in many planttissues protease for degradation of plant and animal proteinscellulase which hydrolyses the cellulose themain componentof wood ubiquitous substrate for fungi and finally the laccaseinvolved in plant material delignification and in the synthesisof the melanin and related compounds to protect fungiagainst radiationThirteen strains displayed high productionsat least for one of the five-screened activities while noclear correlation of enzyme production profile with fungalsystematic groups was noted The abundance of proteaseactivity is in line with previous data on fungal isolatesfrom extreme environments showing high caseinase activitieswith little effect of salinity and temperature on enzymeproduction [36] The relative limited number of isolatesdisplaying cellulase amylase lipase and laccase activitiessuggests that high concentration of salt may have an adverseeffect on enzymeproduction andor activityTheir energywasprobably oriented to avoid salt stress due to 10 NaCl ratherthan the production of bioactive extrolites [47] Howevernot detecting the enzyme is not absolute confirmation ofan isolate inability to produce it It could also mean thatthe enzyme was not released from the mycelium or thatthe medium is inadequate for its detection [48] Laccaseproduction in the presence of 10 of salt by theCladosporiumgroup may be of biotechnological interest for example inmycoremediation of high salty environments contaminatedby organic pollutants

In conclusion fungal community described in this studywas similar to those reported in inhospitable habitats characterized by limitation of nutrients moisture deficit and

exposure to high solar radiation Further studies are neededin order to elucidate their biogeochemical roles in such anextreme environment and to exploit their promising potentialto produce new biomolecules such as enzymes and protectiveagents against oxidative stress

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support from theEuropean Union in the ambit of the Project BIODESERT(EU FP7-CSA-SA REGPOT-2008-2 Grant agreement no245746) and the Tunisian Ministry of Higher Education andScientific Research in the ambit of the laboratory Project LRMBA20 Atef Jaouani wants to thank the Tunisian Society forMicrobial Ecology (ATEM) for supporting publication fees ofthis work

References

[1] J KWarren ldquoSabkhas saline mudflats and pansrdquo in EvaporitesSediments Resources and Hydrocarbons pp 139ndash220 SpringerBerlin Germany 2006

[2] A A Gorbushina and W E Krumbein ldquoRock dwelling fungalcommunities diversity of life styles and colony structurerdquo inJourney to Diverse Microbial Worlds J Seckbach Ed pp 317ndash334 Kluwer Academic Publishers Amsterdam The Nether-lands 2000

[3] L J Rothschild and R L Mancinelli ldquoLife in extreme environ-mentsrdquo Nature vol 409 no 6823 pp 1092ndash1101 2001

[4] S BHumayounN Bano and J THollibaugh ldquoDepth distribu-tion ofmicrobial diversity inmono lake ameromictic soda lakein Californiardquo Applied and Environmental Microbiology vol 69no 2 pp 1030ndash1042 2003

[5] C Demergasso E O Casamayor G Chong P GalleguillosL Escudero and C Pedros-Alio ldquoDistribution of prokaryoticgenetic diversity in athalassohaline lakes of the AtacamaDesertNorthern Chilerdquo FEMS Microbiology Ecology vol 48 no 1 pp57ndash69 2004

[6] N Gunde-Cimerman A Oren and A Plemenitas ldquoAdaptationto life in high salt concentrationsrdquo in Archaea Bacteria andEukarya p 576 Springer Dordrecht The Netherlands 2005

[7] L Maturrano F Santos R Rossello-Mora and J AntonldquoMicrobial diversity in Maras salterns a hypersaline envi-ronment in the Peruvian Andesrdquo Applied and EnvironmentalMicrobiology vol 72 no 6 pp 3887ndash3895 2006

[8] Q Wu G Zwart M Schauer M Kamst-van Agterveld andM Hahn ldquoBacterioplankton community composition alonga salinity gradient of sixteen high-mountain lakes locatedon the Tibetan Plateau Chinardquo Applied and EnvironmentalMicrobiology vol 72 no 8 pp 5478ndash5485 2006

[9] C Gostincar M Grube S De Hoog P Zalar and N Gunde-Cimerman ldquoExtremotolerance in fungi evolution on the edgerdquoFEMS Microbiology Ecology vol 71 no 1 pp 2ndash11 2010

10 BioMed Research International

[10] K Sterflinger D Tesei and K Zakharova ldquoFungi in hot andcold deserts with particular reference to microcolonial fungirdquoFungal Ecology vol 5 no 4 pp 453ndash462 2012

[11] M J Liszka M E Clark E Schneider and D S ClarkldquoNature versus nurture developing enzymes that functionunder extreme conditionsrdquo Annual Review of Chemical andBiomolecular Engineering vol 3 pp 77ndash102 2012

[12] J P Perthuisot La Sebkha ElMelah de Zarzis genese et evolutiondrsquoun bassin salin paralique Les gisements neolithiques des abordsde la Sebkha el Melah vol 9 of de Travaux du Laboratoire degeologieEcole normale superieure Ecole Normale Superieure1975

[13] I cLellan A Varela M Blahgen et al ldquoHarmonisation ofphysical and chemical methods for soil management in CorkOak forestsmdashlessons from collaborative investigationsrdquoAfricanJournal of Environmental Science and Technology vol 7 no 6pp 386ndash401 2013

[14] J HWarcup ldquoThe soil-plate method for isolation of fungi fromsoilrdquo Nature vol 166 no 4211 pp 117ndash118 1950

[15] K H Domsch W Gams and T H Anderson Compendium ofSoil Fungi Academic Press London UK 1980

[16] J A von ArxThe Genera of Fungi Sporulating in Pure CultureJ Cramer Vaduz Germany 1981

[17] E Kiffer and M Morelet Les deuteromycetes Classification etcles didentification generique INRA Editions Paris France1997

[18] D Liu S Coloe R Baird and J Pedersen ldquoRapid mini-preparation of fungal DNA for PCRrdquo Journal of Clinical Micro-biology vol 38 no 1 article 471 2000

[19] T J White T Bruns S Lee and J Talor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Innes D H Gelfand J J Sninsky and T JWhite Eds pp 315ndash322 Academic Press New York NY USA1990

[20] G Luo and T G Mitchell ldquoRapid identification of pathogenicfungi directly from cultures by using multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 8 pp 2860ndash2865 2002

[21] P Rodrigues C Santos A Venancio and N Lima ldquoSpeciesidentification of Aspergillus section Flavi isolates from Por-tuguese almonds using phenotypic including MALDI-TOFICMS and molecular approachesrdquo Journal of Applied Microbi-ology vol 111 no 4 pp 877ndash892 2011

[22] K Bensch U Braun J Z Groenewald and P W Crous ldquoThegenus cladosporiumrdquo Studies in Mycology vol 72 pp 1ndash4012012

[23] M L Berbee M Pirseyedi and S Hubbard ldquoCochliobo-lus phylogenetics and the origin of known highly virulentpathogens inferred from ITS and glyceraldehyde-3-phosphatedehydrogenase gene sequencesrdquo Mycologia vol 91 no 6 pp964ndash977 1999

[24] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous Ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995

[25] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[26] H M Hassan and I Fridovich ldquoParaquat and Escherichia coliMechanism of production of extracellular superoxide radicalrdquoJournal of Biological Chemistry vol 254 no 21 pp 10846ndash108521979

[27] M B Angelova S B Pashova B K Spasova S V Vassilevand L S Slokoska ldquoOxidative stress response of filamentousfungi induced by hydrogen peroxide and paraquatrdquoMycologicalResearch vol 109 no 2 pp 150ndash158 2005

[28] A de Groot V Chapon P Servant et al ldquoDeinococcus desertisp nov a gamma-radiation-tolerant bacterium isolated fromthe Sahara Desertrdquo International Journal of Systematic andEvolutionary Microbiology vol 55 no 6 pp 2441ndash2446 2005

[29] A Chanal V Chapon K Benzerara et al ldquoThe desert ofTataouine an extreme environment that hosts a wide diversityof microorganisms and radiotolerant bacteriardquo EnvironmentalMicrobiology vol 8 no 3 pp 514ndash525 2006

[30] M Gtari D Daffonchio and A Boudabous ldquoAssessment ofthe genetic diversity of Frankia microsymbionts of Elaeagnusangustifolia L plants growing in a Tunisian date-palm oasis byanalysis of PCR amplified nifD-K intergenic spacerrdquo CanadianJournal of Microbiology vol 53 no 3 pp 440ndash445 2007

[31] I Essoussi F Ghodhbane-Gtari H Amairi et al ldquoEsterase asan enzymatic signature of Geodermatophilaceae adaptabilityto Sahara desert stones and monumentsrdquo Journal of AppliedMicrobiology vol 108 no 5 pp 1723ndash1732 2010

[32] I Sbissi M Neffati A Boudabous C Murat and M GtarildquoPhylogenetic affiliation of the desert truffles Picoa juniperi andPicoa lefebvreirdquo Antonie van Leeuwenhoek vol 98 no 4 pp429ndash436 2010

[33] I Sbissi F Ghodhbane-Gtari M Neffati H Ouzari A Boud-abous and M Gtari ldquoDiversity of the desert truffle Terfeziaboudieri chatin in southern Tunisiardquo Canadian Journal ofMicrobiology vol 57 no 7 pp 599ndash605 2011

[34] C A Conley G Ishkhanova C P McKay and K Cullings ldquoApreliminary survey of non-lichenized fungi cultured from thehyperarid Atacama Desert of Chilerdquo Astrobiology vol 6 no 4pp 521ndash526 2006

[35] E V Smolyanyuk and E N Bilanenko ldquoCommunities ofhalotolerant micromycetes from the areas of natural salinityrdquoMicrobiology vol 80 no 6 pp 877ndash883 2011

[36] H PMolitoris A S Buchalo I Kurchenko et al ldquoPhysiologicaldiversity of the fIrst fIlamentous fungi isolated from the hyper-saline Dead Seardquo inAquatic Mycology Across theMillennium KD Hyde W H Ho and S B Pointing Eds vol 5 pp 55ndash70Fungal Diversity Press 2000

[37] S I I Abdel-Hafez ldquoSurvey of the mycoflora of desert soils inSaudi ArabiardquoMycopathologia vol 80 no 1 pp 3ndash8 1982

[38] A H M El-Said and A Saleem ldquoEcological and physiologicalstudies on soil fungi at Western Region Libyardquo Mycobiologyvol 36 pp 1ndash9 2008

[39] P Zalar G S de Hoog H-J Schroers P W Crous J Z Groe-newald and N Gunde-Cimerman ldquoPhylogeny and ecology ofthe ubiquitous saprobe Cladosporium sphaerospermum withdescriptions of seven new species from hypersaline environ-mentsrdquo Studies in Mycology vol 58 pp 157ndash183 2007

[40] J KMisra ldquoOccurrence distribution and seasonality of aquaticfungi as affected by chemical factors in six alkaline ponds ofIndiardquo Hydrobiologia vol 97 no 2 pp 185ndash191 1982

[41] F T El-Hissy S A El-Zayat A M Khallil and M S MassoudldquoAquatic fungi from the submerged mud of Aswan High DamLakerdquoMicrobiological Research vol 152 no 1 pp 27ndash32 1997

[42] AM Khallil F T El-Hissy and EH Ali ldquoSeasonal fluctuationsof aquatic fungi recovered from Egyptian soil (Delta Region)rdquoJournal of Basic Microbiology vol 35 no 2 pp 93ndash102 1995

BioMed Research International 11

[43] P B Matheny J A Gossmann P Zalar T K A Kumarand D S Hibbett ldquoResolving the phylogenetic position of theWallemiomycetes an enigmatic major lineage of Basidiomy-cotardquoCanadian Journal of Botany vol 84 no 12 pp 1794ndash18052006

[44] A Blomberg and L Adler ldquoTolerence of fungi to NaClrdquo in StressTolerance of Fungi D Jennings Ed pp 209ndash231 1993

[45] A Oren ldquoBioenergetic aspects of halophilismrdquo MicrobiologyandMolecular Biology Reviews vol 63 no 2 pp 334ndash348 1999

[46] P Delgado-Sanchez M A Ortega-Amaro J F Jimenez-Bremont and J Flores ldquoAre fungi important for breaking seeddormancy in desert species Experimental evidence in Opuntiastreptacantha (Cactaceae)rdquo Plant Biology vol 13 no 1 pp 154ndash159 2011

[47] J C Frisvad ldquoHalotolerant and halophilic fungi and theirextrolite productionrdquo in Adaptation to Life at High Salt Con-centrations in Archaea Bacteria and Eukarya N Gunde-Cimerman A Oren and A Plemenitas Eds pp 426ndash440Springer Amsterdam The Netherlands 2005

[48] A Abdel-Raheem and C A Shearer ldquoExtracellular enzymeproduction by freshwater ascomycetesrdquo Fungal Diversity vol 11pp 1ndash19 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

Table1Con

tinued

Strain

code

28Sidentifi

catio

nITSidentifi

catio

nIdentifi

catio

nbasedon

specific

prim

ers

Morph

ologicalidentifi

catio

nFinalidentificatio

nand

accessionnu

mberin

NCB

I

JA20

Alternaria

Alternariabrassicae

JX290150

(100)

AporriH

Q821479

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

28SKF

417575

ITSKF

417596

JA22

Penicilliu

m

Penicilliu

mchrysogenu

mKC

341721

(99

)PdipodomyicolaJX232278

(99

)Prubens

JX003126

(99

)Pcommun

eJN676122

(99

)

Penicilliu

mchrysogenu

m(120573-tu

bulin

)Penicilliu

mchrysogenu

m

Penicilliu

mchrysogenu

mTh

om28SKF

417576

ITSKF

417597

JA23

nd

AlternariaalternataJQ

809324

(100)

AquercusK

C329620(100)

Atenu

issim

aKC

329619

(100)

Aatrans

KC329618

(100)

Alternariatenu

issim

aAlternariaarborescens

Alternariaalternata

(GPD

)

Alternariaalternata

Alternariaalternata

Keissler

ITSKF

417598

BioMed Research International 7

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 0JA3 Penicillium chrysogenum 100 72 37 0 96 46 11 0JA22 Penicillium chrysogenum 100 82 41 25 90 46 0 0AJ5 Penicillium canescens 70 30 20 0 79 44 0 0JA15 Penicillium sp 83 70 34 0 53 18 0 0JA8 Cladosporium halotolerans 80 68 32 18 30 0 0 0JA2 Cladosporium sphaerospermum 76 64 34 22 47 19 11 0JA13 Cladosporium sphaerospermum 100 49 25 0 81 79 12 0JA14 Cladosporium cladosporioides 40 30 10 0 0 0 0 0JA18 Cladosporium cladosporioides 58 40 24 8 63 61 4 0JA4 Aspergillus flavus 90 80 48 26 56 16 0 0JA10 Aspergillus fumigatus 100 76 35 0 100 57 11 0JA11 Aspergillus fumigatiaffinis 100 46 25 0 52 30 12 0JA19 Alternaria alternata 52 38 24 0 57 9 0 0JA20 Alternaria alternata 60 40 0 0 37 24 0 0JA23 Alternaria alternata 100 68 20 0 65 12 0 0JA6 Alternaria tenuissima 100 60 22 0 80 55 17 0JA9 Embellisia phragmospora 94 50 10 0 78 26 0 0JA12 Ulocladium consortiale 72 28 0 0 32 10 0 0JA17 Ulocladium sp 100 70 28 0 67 20 0 0JA7 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) times 100 (2) Relative growth in liquid mediaafter 7 days incubation = (kinetic curve surface under salt stresskinetic curve surface without salt stress) times 100

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 [10mM] Paraquat [500120583M]

JA1 Penicillium chrysogenum 43 39 mdash 66 74JA3 Penicillium chrysogenum 42 50 mdash 84 71JA22 Penicillium chrysogenum 47 45 mdash 68 53JA5 Penicillium canescens 26 28 mdash 59 63JA15 Penicillium sp 43 100 mdash 100 100JA8 Cladosporium halotolerans 34 26 mdash 44 40JA2 Cladosporium sphaerospermum 21 24 mdash 52 48JA13 Cladosporium sphaerospermum 21 43 mdash 55 44JA14 Cladosporium cladosporioides 34 38 mdash 20 31JA18 Cladosporium cladosporioides mdash 41 mdash 18 16JA4 Aspergillus flavus 46 22 mdash 47 39JA10 Aspergillus fumigatus 89 41 61 100 100JA11 Aspergillus fumigatiaffinis 94 26 100 100 100JA19 Alternaria alternata 49 35 mdash 69 89JA20 Alternaria alternata 58 48 mdash 100 100JA23 Alternaria alternata 100 83 40 57 52JA6 Alternaria tenuissima 57 30 mdash 81 100JA9 Embellisia phragmospora 58 67 mdash 100 100JA12 Ulocladium consortiale 44 37 36 56 100JA17 Ulocladium sp 93 28 100 81 100JA7 Engyodontium album 34 18 mdash 66 53Relative growth of fungal strains under different stresses after 7 days incubation was expressed as follows (1) ( colony at pH 10 colony at pH 5) times 100 (2)( colony at 45∘C or 4∘C colony at 30∘C) times 100 (3) ( colony with H2O2 or paraquat colony without stress) times 100 mdash not significant growth

8 BioMed Research International

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

Strain code Strain Protease Amylase Cellulase Lipase LaccaseJA1 Penicillium chrysogenum ++ + minus minus minusJA3 Penicillium chrysogenum ++ minus minus minus minusJA22 Penicillium chrysogenum + + minus minus minusAJ5 Penicillium canescens minus minus minus minus +JA15 Penicillium sp minus minus + minus minusJA8 Cladosporium halotolerans + minus minus minus +JA2 Cladosporium sphaerospermum +++ minus minus + +JA13 Cladosporium sphaerospermum minus + minus minus +JA14 Cladosporium cladosporioides + + minus minus minus

JA18 Cladosporium cladosporioides minus minus minus minus minus

JA4 Aspergillus flavus + minus minus + minus

JA10 Aspergillus fumigatus minus minus minus minus minus

JA11 Aspergillus fumigatiaffinis minus minus minus minus minus

JA19 Alternaria alternata minus minus minus minus minus

JA20 Alternaria alternata minus + minus minus minus

JA23 Alternaria alternata minus minus minus minus minus

JA6 Alternaria tenuissima + minus minus minus minus

JA9 Embellisia phragmospora minus minus minus minus minus

JA12 Ulocladium consortiale minus minus minus minus minus

JA17 Ulocladium sp minus minus minus minus minus

JA7 Engyodontium album + + minus ++ minus

AR activity ratio = ( activity colony) minus no activity + AR lt 1 ++ 1 lt AR lt 2 +++ 2 lt AR lt 3

tolerance [10] On the other hand Molitoris et al [36]reported that other halotolerant and halophilic fungi suchas Aspergillus and Cladosporium spp are predominant insaline desert soil of Dead Sea Many Aspergillus species havebeen also reported to constitute dominant fungi in desert ofSaudi Arabia and Libya [37 38] and halotolerant speciesincludingAspergillus sppPenicillium spp andCladosporiumsphaerospermum have been consistently isolated fromhyper-saline environments around the globe [39] In this studycontrary to many reports on hypersaline environments nospecies belonging to the genera EurotiumThrimmatostromaEmericella and Phaeotheca [9] have been obtained probablybecause of the initial alkaline pH of the Sebkha El Melahsalt lake Actually the effect of pH on the fungal diversity iscontroversialMisra [40] observed that fungal diversity varieswith the pH while other investigators found no significanteffect of pH values of water and soil habitats on fungaloccurrence [41] It is more likely that the number of theisolated fungi is directly correlated to the organic mattercontent of water mud and soil samples [42]

Beside the identification of the recovered fungal isolatesfrom Sebkha El Melah the second goal of the current studywas the detection of some of their physiological and biochem-ical featuresThis allows understanding ecological adaptationto extreme environment and predicts some biotechnologicalusage The 21 strains have been screened for tolerance toextreme NaCl concentrations basic pH temperature andoxidative stress and for the production of important enzy-matic activities in presence of 10 NaCl

Excepting Cladosporium cladosporioides JA18 all isolatesobtained in this study can be considered as moderatelyhaloalkaliphilic fungi as deduced from their ability to grow

at pH 10 and 10 of NaCl However the isolates were ableto grow when salt was not added to their growing mediaExcepting some Wallemia ichthyophaga the most strictlyhalophilic fungus [43] all other fungal strains known todate are able to grow without salt a fact confirmed inour study However gradual decrease in fungal growth wasobserved with the increasing of salt concentration in theculture medium Nineteen strains remain able to grow under15 of NaCl whereas 7 strains were able to tolerate 20 ofNaCl This result was confirmed by salt tolerance assay inliquid media as estimated by Biolog system It is noteworthythat fungi were more sensitive to salt stress in liquid mediathan in solid mediaThis could be explained by the alterationof the osmotic gradient forcing the fungi to expend moreenergy in the osmoregulatory processes resulting in slowergrowth [44] Moreover at higher salt concentration deathoccurs

Regarding the stress of pH the capacity of the majority ofisolates to growth at pH 10 implies firstly that some habitats inthe salt lake may have a varying pH and secondly that fungican tolerate a wide pH range Prima facie the overall resultsin solid and liquid media showed that Penicillium chryso-genum JA1 and JA3 Cladosporium sphaerospermum JA2 andJA13 Cladosporium cladosporioides JA18 Aspergillus fumi-gatus JA10 Aspergillus fumigatiaffinis JA11 and Alternariatenuissima JA6 are the most alkalihalotolerant isolates in thisstudy

The tolerance of the strains to extreme 45∘C was testedand results indicated that Aspergillus fumigatus JA10 Alter-naria alternata JA23 Ulocladium sp JA17 and Aspergillusfumigatiaffinis JA11 were able to grow Of particular interestthe latter two strains retained 100 of the growth rate

BioMed Research International 9

and biomass production as estimated by colony diameterMoreover their ability to grow at low temperature may allowthem to better adapt to the big temperature fluctuation indesert environments Additionally exposure to substratesgenerating oxidative stress such as H

2O2at 10mM and

paraquat at 500 120583M did not alter significantly the growth ofalmost tested strains demonstrating their ability to tolerateoxidative stress These findings may explain their presence indesert regions that are considered amongst the most stressfulenvironments on Earth because of the high UV radiationdesiccation increased salinity low nutrient availability sea-sonal and daily temperature variation and solar irradiation[6 10]

It has been postulated that microorganisms sharing arich and particular extracellular enzymatic activities arecommon in harsh conditions characterizing their ecologicalhabitat including high level of aridity temperature ionicstrength and particularly the low nutrient availability [31]This implies the need by microorganisms for an effectiveutilization of each possible available organic compound [45]Moreover fungal isolates from hot desert were revealed toplay an important role in seeds germination by breakingdormancy and increasing water uptake [46] In the presentstudy the capacity of fungal isolates to produce extracellularenzymes was assayed in the presence of 10 of NaClEnzymes tested were the following amylase for degradationof starch abundant carbohydrate polymer in many planttissues protease for degradation of plant and animal proteinscellulase which hydrolyses the cellulose themain componentof wood ubiquitous substrate for fungi and finally the laccaseinvolved in plant material delignification and in the synthesisof the melanin and related compounds to protect fungiagainst radiationThirteen strains displayed high productionsat least for one of the five-screened activities while noclear correlation of enzyme production profile with fungalsystematic groups was noted The abundance of proteaseactivity is in line with previous data on fungal isolatesfrom extreme environments showing high caseinase activitieswith little effect of salinity and temperature on enzymeproduction [36] The relative limited number of isolatesdisplaying cellulase amylase lipase and laccase activitiessuggests that high concentration of salt may have an adverseeffect on enzymeproduction andor activityTheir energywasprobably oriented to avoid salt stress due to 10 NaCl ratherthan the production of bioactive extrolites [47] Howevernot detecting the enzyme is not absolute confirmation ofan isolate inability to produce it It could also mean thatthe enzyme was not released from the mycelium or thatthe medium is inadequate for its detection [48] Laccaseproduction in the presence of 10 of salt by theCladosporiumgroup may be of biotechnological interest for example inmycoremediation of high salty environments contaminatedby organic pollutants

In conclusion fungal community described in this studywas similar to those reported in inhospitable habitats characterized by limitation of nutrients moisture deficit and

exposure to high solar radiation Further studies are neededin order to elucidate their biogeochemical roles in such anextreme environment and to exploit their promising potentialto produce new biomolecules such as enzymes and protectiveagents against oxidative stress

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support from theEuropean Union in the ambit of the Project BIODESERT(EU FP7-CSA-SA REGPOT-2008-2 Grant agreement no245746) and the Tunisian Ministry of Higher Education andScientific Research in the ambit of the laboratory Project LRMBA20 Atef Jaouani wants to thank the Tunisian Society forMicrobial Ecology (ATEM) for supporting publication fees ofthis work

References

[1] J KWarren ldquoSabkhas saline mudflats and pansrdquo in EvaporitesSediments Resources and Hydrocarbons pp 139ndash220 SpringerBerlin Germany 2006

[2] A A Gorbushina and W E Krumbein ldquoRock dwelling fungalcommunities diversity of life styles and colony structurerdquo inJourney to Diverse Microbial Worlds J Seckbach Ed pp 317ndash334 Kluwer Academic Publishers Amsterdam The Nether-lands 2000

[3] L J Rothschild and R L Mancinelli ldquoLife in extreme environ-mentsrdquo Nature vol 409 no 6823 pp 1092ndash1101 2001

[4] S BHumayounN Bano and J THollibaugh ldquoDepth distribu-tion ofmicrobial diversity inmono lake ameromictic soda lakein Californiardquo Applied and Environmental Microbiology vol 69no 2 pp 1030ndash1042 2003

[5] C Demergasso E O Casamayor G Chong P GalleguillosL Escudero and C Pedros-Alio ldquoDistribution of prokaryoticgenetic diversity in athalassohaline lakes of the AtacamaDesertNorthern Chilerdquo FEMS Microbiology Ecology vol 48 no 1 pp57ndash69 2004

[6] N Gunde-Cimerman A Oren and A Plemenitas ldquoAdaptationto life in high salt concentrationsrdquo in Archaea Bacteria andEukarya p 576 Springer Dordrecht The Netherlands 2005

[7] L Maturrano F Santos R Rossello-Mora and J AntonldquoMicrobial diversity in Maras salterns a hypersaline envi-ronment in the Peruvian Andesrdquo Applied and EnvironmentalMicrobiology vol 72 no 6 pp 3887ndash3895 2006

[8] Q Wu G Zwart M Schauer M Kamst-van Agterveld andM Hahn ldquoBacterioplankton community composition alonga salinity gradient of sixteen high-mountain lakes locatedon the Tibetan Plateau Chinardquo Applied and EnvironmentalMicrobiology vol 72 no 8 pp 5478ndash5485 2006

[9] C Gostincar M Grube S De Hoog P Zalar and N Gunde-Cimerman ldquoExtremotolerance in fungi evolution on the edgerdquoFEMS Microbiology Ecology vol 71 no 1 pp 2ndash11 2010

10 BioMed Research International

[10] K Sterflinger D Tesei and K Zakharova ldquoFungi in hot andcold deserts with particular reference to microcolonial fungirdquoFungal Ecology vol 5 no 4 pp 453ndash462 2012

[11] M J Liszka M E Clark E Schneider and D S ClarkldquoNature versus nurture developing enzymes that functionunder extreme conditionsrdquo Annual Review of Chemical andBiomolecular Engineering vol 3 pp 77ndash102 2012

[12] J P Perthuisot La Sebkha ElMelah de Zarzis genese et evolutiondrsquoun bassin salin paralique Les gisements neolithiques des abordsde la Sebkha el Melah vol 9 of de Travaux du Laboratoire degeologieEcole normale superieure Ecole Normale Superieure1975

[13] I cLellan A Varela M Blahgen et al ldquoHarmonisation ofphysical and chemical methods for soil management in CorkOak forestsmdashlessons from collaborative investigationsrdquoAfricanJournal of Environmental Science and Technology vol 7 no 6pp 386ndash401 2013

[14] J HWarcup ldquoThe soil-plate method for isolation of fungi fromsoilrdquo Nature vol 166 no 4211 pp 117ndash118 1950

[15] K H Domsch W Gams and T H Anderson Compendium ofSoil Fungi Academic Press London UK 1980

[16] J A von ArxThe Genera of Fungi Sporulating in Pure CultureJ Cramer Vaduz Germany 1981

[17] E Kiffer and M Morelet Les deuteromycetes Classification etcles didentification generique INRA Editions Paris France1997

[18] D Liu S Coloe R Baird and J Pedersen ldquoRapid mini-preparation of fungal DNA for PCRrdquo Journal of Clinical Micro-biology vol 38 no 1 article 471 2000

[19] T J White T Bruns S Lee and J Talor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Innes D H Gelfand J J Sninsky and T JWhite Eds pp 315ndash322 Academic Press New York NY USA1990

[20] G Luo and T G Mitchell ldquoRapid identification of pathogenicfungi directly from cultures by using multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 8 pp 2860ndash2865 2002

[21] P Rodrigues C Santos A Venancio and N Lima ldquoSpeciesidentification of Aspergillus section Flavi isolates from Por-tuguese almonds using phenotypic including MALDI-TOFICMS and molecular approachesrdquo Journal of Applied Microbi-ology vol 111 no 4 pp 877ndash892 2011

[22] K Bensch U Braun J Z Groenewald and P W Crous ldquoThegenus cladosporiumrdquo Studies in Mycology vol 72 pp 1ndash4012012

[23] M L Berbee M Pirseyedi and S Hubbard ldquoCochliobo-lus phylogenetics and the origin of known highly virulentpathogens inferred from ITS and glyceraldehyde-3-phosphatedehydrogenase gene sequencesrdquo Mycologia vol 91 no 6 pp964ndash977 1999

[24] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous Ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995

[25] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[26] H M Hassan and I Fridovich ldquoParaquat and Escherichia coliMechanism of production of extracellular superoxide radicalrdquoJournal of Biological Chemistry vol 254 no 21 pp 10846ndash108521979

[27] M B Angelova S B Pashova B K Spasova S V Vassilevand L S Slokoska ldquoOxidative stress response of filamentousfungi induced by hydrogen peroxide and paraquatrdquoMycologicalResearch vol 109 no 2 pp 150ndash158 2005

[28] A de Groot V Chapon P Servant et al ldquoDeinococcus desertisp nov a gamma-radiation-tolerant bacterium isolated fromthe Sahara Desertrdquo International Journal of Systematic andEvolutionary Microbiology vol 55 no 6 pp 2441ndash2446 2005

[29] A Chanal V Chapon K Benzerara et al ldquoThe desert ofTataouine an extreme environment that hosts a wide diversityof microorganisms and radiotolerant bacteriardquo EnvironmentalMicrobiology vol 8 no 3 pp 514ndash525 2006

[30] M Gtari D Daffonchio and A Boudabous ldquoAssessment ofthe genetic diversity of Frankia microsymbionts of Elaeagnusangustifolia L plants growing in a Tunisian date-palm oasis byanalysis of PCR amplified nifD-K intergenic spacerrdquo CanadianJournal of Microbiology vol 53 no 3 pp 440ndash445 2007

[31] I Essoussi F Ghodhbane-Gtari H Amairi et al ldquoEsterase asan enzymatic signature of Geodermatophilaceae adaptabilityto Sahara desert stones and monumentsrdquo Journal of AppliedMicrobiology vol 108 no 5 pp 1723ndash1732 2010

[32] I Sbissi M Neffati A Boudabous C Murat and M GtarildquoPhylogenetic affiliation of the desert truffles Picoa juniperi andPicoa lefebvreirdquo Antonie van Leeuwenhoek vol 98 no 4 pp429ndash436 2010

[33] I Sbissi F Ghodhbane-Gtari M Neffati H Ouzari A Boud-abous and M Gtari ldquoDiversity of the desert truffle Terfeziaboudieri chatin in southern Tunisiardquo Canadian Journal ofMicrobiology vol 57 no 7 pp 599ndash605 2011

[34] C A Conley G Ishkhanova C P McKay and K Cullings ldquoApreliminary survey of non-lichenized fungi cultured from thehyperarid Atacama Desert of Chilerdquo Astrobiology vol 6 no 4pp 521ndash526 2006

[35] E V Smolyanyuk and E N Bilanenko ldquoCommunities ofhalotolerant micromycetes from the areas of natural salinityrdquoMicrobiology vol 80 no 6 pp 877ndash883 2011

[36] H PMolitoris A S Buchalo I Kurchenko et al ldquoPhysiologicaldiversity of the fIrst fIlamentous fungi isolated from the hyper-saline Dead Seardquo inAquatic Mycology Across theMillennium KD Hyde W H Ho and S B Pointing Eds vol 5 pp 55ndash70Fungal Diversity Press 2000

[37] S I I Abdel-Hafez ldquoSurvey of the mycoflora of desert soils inSaudi ArabiardquoMycopathologia vol 80 no 1 pp 3ndash8 1982

[38] A H M El-Said and A Saleem ldquoEcological and physiologicalstudies on soil fungi at Western Region Libyardquo Mycobiologyvol 36 pp 1ndash9 2008

[39] P Zalar G S de Hoog H-J Schroers P W Crous J Z Groe-newald and N Gunde-Cimerman ldquoPhylogeny and ecology ofthe ubiquitous saprobe Cladosporium sphaerospermum withdescriptions of seven new species from hypersaline environ-mentsrdquo Studies in Mycology vol 58 pp 157ndash183 2007

[40] J KMisra ldquoOccurrence distribution and seasonality of aquaticfungi as affected by chemical factors in six alkaline ponds ofIndiardquo Hydrobiologia vol 97 no 2 pp 185ndash191 1982

[41] F T El-Hissy S A El-Zayat A M Khallil and M S MassoudldquoAquatic fungi from the submerged mud of Aswan High DamLakerdquoMicrobiological Research vol 152 no 1 pp 27ndash32 1997

[42] AM Khallil F T El-Hissy and EH Ali ldquoSeasonal fluctuationsof aquatic fungi recovered from Egyptian soil (Delta Region)rdquoJournal of Basic Microbiology vol 35 no 2 pp 93ndash102 1995

BioMed Research International 11

[43] P B Matheny J A Gossmann P Zalar T K A Kumarand D S Hibbett ldquoResolving the phylogenetic position of theWallemiomycetes an enigmatic major lineage of Basidiomy-cotardquoCanadian Journal of Botany vol 84 no 12 pp 1794ndash18052006

[44] A Blomberg and L Adler ldquoTolerence of fungi to NaClrdquo in StressTolerance of Fungi D Jennings Ed pp 209ndash231 1993

[45] A Oren ldquoBioenergetic aspects of halophilismrdquo MicrobiologyandMolecular Biology Reviews vol 63 no 2 pp 334ndash348 1999

[46] P Delgado-Sanchez M A Ortega-Amaro J F Jimenez-Bremont and J Flores ldquoAre fungi important for breaking seeddormancy in desert species Experimental evidence in Opuntiastreptacantha (Cactaceae)rdquo Plant Biology vol 13 no 1 pp 154ndash159 2011

[47] J C Frisvad ldquoHalotolerant and halophilic fungi and theirextrolite productionrdquo in Adaptation to Life at High Salt Con-centrations in Archaea Bacteria and Eukarya N Gunde-Cimerman A Oren and A Plemenitas Eds pp 426ndash440Springer Amsterdam The Netherlands 2005

[48] A Abdel-Raheem and C A Shearer ldquoExtracellular enzymeproduction by freshwater ascomycetesrdquo Fungal Diversity vol 11pp 1ndash19 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

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 0JA3 Penicillium chrysogenum 100 72 37 0 96 46 11 0JA22 Penicillium chrysogenum 100 82 41 25 90 46 0 0AJ5 Penicillium canescens 70 30 20 0 79 44 0 0JA15 Penicillium sp 83 70 34 0 53 18 0 0JA8 Cladosporium halotolerans 80 68 32 18 30 0 0 0JA2 Cladosporium sphaerospermum 76 64 34 22 47 19 11 0JA13 Cladosporium sphaerospermum 100 49 25 0 81 79 12 0JA14 Cladosporium cladosporioides 40 30 10 0 0 0 0 0JA18 Cladosporium cladosporioides 58 40 24 8 63 61 4 0JA4 Aspergillus flavus 90 80 48 26 56 16 0 0JA10 Aspergillus fumigatus 100 76 35 0 100 57 11 0JA11 Aspergillus fumigatiaffinis 100 46 25 0 52 30 12 0JA19 Alternaria alternata 52 38 24 0 57 9 0 0JA20 Alternaria alternata 60 40 0 0 37 24 0 0JA23 Alternaria alternata 100 68 20 0 65 12 0 0JA6 Alternaria tenuissima 100 60 22 0 80 55 17 0JA9 Embellisia phragmospora 94 50 10 0 78 26 0 0JA12 Ulocladium consortiale 72 28 0 0 32 10 0 0JA17 Ulocladium sp 100 70 28 0 67 20 0 0JA7 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) times 100 (2) Relative growth in liquid mediaafter 7 days incubation = (kinetic curve surface under salt stresskinetic curve surface without salt stress) times 100

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 [10mM] Paraquat [500120583M]

JA1 Penicillium chrysogenum 43 39 mdash 66 74JA3 Penicillium chrysogenum 42 50 mdash 84 71JA22 Penicillium chrysogenum 47 45 mdash 68 53JA5 Penicillium canescens 26 28 mdash 59 63JA15 Penicillium sp 43 100 mdash 100 100JA8 Cladosporium halotolerans 34 26 mdash 44 40JA2 Cladosporium sphaerospermum 21 24 mdash 52 48JA13 Cladosporium sphaerospermum 21 43 mdash 55 44JA14 Cladosporium cladosporioides 34 38 mdash 20 31JA18 Cladosporium cladosporioides mdash 41 mdash 18 16JA4 Aspergillus flavus 46 22 mdash 47 39JA10 Aspergillus fumigatus 89 41 61 100 100JA11 Aspergillus fumigatiaffinis 94 26 100 100 100JA19 Alternaria alternata 49 35 mdash 69 89JA20 Alternaria alternata 58 48 mdash 100 100JA23 Alternaria alternata 100 83 40 57 52JA6 Alternaria tenuissima 57 30 mdash 81 100JA9 Embellisia phragmospora 58 67 mdash 100 100JA12 Ulocladium consortiale 44 37 36 56 100JA17 Ulocladium sp 93 28 100 81 100JA7 Engyodontium album 34 18 mdash 66 53Relative growth of fungal strains under different stresses after 7 days incubation was expressed as follows (1) ( colony at pH 10 colony at pH 5) times 100 (2)( colony at 45∘C or 4∘C colony at 30∘C) times 100 (3) ( colony with H2O2 or paraquat colony without stress) times 100 mdash not significant growth

8 BioMed Research International

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

Strain code Strain Protease Amylase Cellulase Lipase LaccaseJA1 Penicillium chrysogenum ++ + minus minus minusJA3 Penicillium chrysogenum ++ minus minus minus minusJA22 Penicillium chrysogenum + + minus minus minusAJ5 Penicillium canescens minus minus minus minus +JA15 Penicillium sp minus minus + minus minusJA8 Cladosporium halotolerans + minus minus minus +JA2 Cladosporium sphaerospermum +++ minus minus + +JA13 Cladosporium sphaerospermum minus + minus minus +JA14 Cladosporium cladosporioides + + minus minus minus

JA18 Cladosporium cladosporioides minus minus minus minus minus

JA4 Aspergillus flavus + minus minus + minus

JA10 Aspergillus fumigatus minus minus minus minus minus

JA11 Aspergillus fumigatiaffinis minus minus minus minus minus

JA19 Alternaria alternata minus minus minus minus minus

JA20 Alternaria alternata minus + minus minus minus

JA23 Alternaria alternata minus minus minus minus minus

JA6 Alternaria tenuissima + minus minus minus minus

JA9 Embellisia phragmospora minus minus minus minus minus

JA12 Ulocladium consortiale minus minus minus minus minus

JA17 Ulocladium sp minus minus minus minus minus

JA7 Engyodontium album + + minus ++ minus

AR activity ratio = ( activity colony) minus no activity + AR lt 1 ++ 1 lt AR lt 2 +++ 2 lt AR lt 3

tolerance [10] On the other hand Molitoris et al [36]reported that other halotolerant and halophilic fungi suchas Aspergillus and Cladosporium spp are predominant insaline desert soil of Dead Sea Many Aspergillus species havebeen also reported to constitute dominant fungi in desert ofSaudi Arabia and Libya [37 38] and halotolerant speciesincludingAspergillus sppPenicillium spp andCladosporiumsphaerospermum have been consistently isolated fromhyper-saline environments around the globe [39] In this studycontrary to many reports on hypersaline environments nospecies belonging to the genera EurotiumThrimmatostromaEmericella and Phaeotheca [9] have been obtained probablybecause of the initial alkaline pH of the Sebkha El Melahsalt lake Actually the effect of pH on the fungal diversity iscontroversialMisra [40] observed that fungal diversity varieswith the pH while other investigators found no significanteffect of pH values of water and soil habitats on fungaloccurrence [41] It is more likely that the number of theisolated fungi is directly correlated to the organic mattercontent of water mud and soil samples [42]

Beside the identification of the recovered fungal isolatesfrom Sebkha El Melah the second goal of the current studywas the detection of some of their physiological and biochem-ical featuresThis allows understanding ecological adaptationto extreme environment and predicts some biotechnologicalusage The 21 strains have been screened for tolerance toextreme NaCl concentrations basic pH temperature andoxidative stress and for the production of important enzy-matic activities in presence of 10 NaCl

Excepting Cladosporium cladosporioides JA18 all isolatesobtained in this study can be considered as moderatelyhaloalkaliphilic fungi as deduced from their ability to grow

at pH 10 and 10 of NaCl However the isolates were ableto grow when salt was not added to their growing mediaExcepting some Wallemia ichthyophaga the most strictlyhalophilic fungus [43] all other fungal strains known todate are able to grow without salt a fact confirmed inour study However gradual decrease in fungal growth wasobserved with the increasing of salt concentration in theculture medium Nineteen strains remain able to grow under15 of NaCl whereas 7 strains were able to tolerate 20 ofNaCl This result was confirmed by salt tolerance assay inliquid media as estimated by Biolog system It is noteworthythat fungi were more sensitive to salt stress in liquid mediathan in solid mediaThis could be explained by the alterationof the osmotic gradient forcing the fungi to expend moreenergy in the osmoregulatory processes resulting in slowergrowth [44] Moreover at higher salt concentration deathoccurs

Regarding the stress of pH the capacity of the majority ofisolates to growth at pH 10 implies firstly that some habitats inthe salt lake may have a varying pH and secondly that fungican tolerate a wide pH range Prima facie the overall resultsin solid and liquid media showed that Penicillium chryso-genum JA1 and JA3 Cladosporium sphaerospermum JA2 andJA13 Cladosporium cladosporioides JA18 Aspergillus fumi-gatus JA10 Aspergillus fumigatiaffinis JA11 and Alternariatenuissima JA6 are the most alkalihalotolerant isolates in thisstudy

The tolerance of the strains to extreme 45∘C was testedand results indicated that Aspergillus fumigatus JA10 Alter-naria alternata JA23 Ulocladium sp JA17 and Aspergillusfumigatiaffinis JA11 were able to grow Of particular interestthe latter two strains retained 100 of the growth rate

BioMed Research International 9

and biomass production as estimated by colony diameterMoreover their ability to grow at low temperature may allowthem to better adapt to the big temperature fluctuation indesert environments Additionally exposure to substratesgenerating oxidative stress such as H

2O2at 10mM and

paraquat at 500 120583M did not alter significantly the growth ofalmost tested strains demonstrating their ability to tolerateoxidative stress These findings may explain their presence indesert regions that are considered amongst the most stressfulenvironments on Earth because of the high UV radiationdesiccation increased salinity low nutrient availability sea-sonal and daily temperature variation and solar irradiation[6 10]

It has been postulated that microorganisms sharing arich and particular extracellular enzymatic activities arecommon in harsh conditions characterizing their ecologicalhabitat including high level of aridity temperature ionicstrength and particularly the low nutrient availability [31]This implies the need by microorganisms for an effectiveutilization of each possible available organic compound [45]Moreover fungal isolates from hot desert were revealed toplay an important role in seeds germination by breakingdormancy and increasing water uptake [46] In the presentstudy the capacity of fungal isolates to produce extracellularenzymes was assayed in the presence of 10 of NaClEnzymes tested were the following amylase for degradationof starch abundant carbohydrate polymer in many planttissues protease for degradation of plant and animal proteinscellulase which hydrolyses the cellulose themain componentof wood ubiquitous substrate for fungi and finally the laccaseinvolved in plant material delignification and in the synthesisof the melanin and related compounds to protect fungiagainst radiationThirteen strains displayed high productionsat least for one of the five-screened activities while noclear correlation of enzyme production profile with fungalsystematic groups was noted The abundance of proteaseactivity is in line with previous data on fungal isolatesfrom extreme environments showing high caseinase activitieswith little effect of salinity and temperature on enzymeproduction [36] The relative limited number of isolatesdisplaying cellulase amylase lipase and laccase activitiessuggests that high concentration of salt may have an adverseeffect on enzymeproduction andor activityTheir energywasprobably oriented to avoid salt stress due to 10 NaCl ratherthan the production of bioactive extrolites [47] Howevernot detecting the enzyme is not absolute confirmation ofan isolate inability to produce it It could also mean thatthe enzyme was not released from the mycelium or thatthe medium is inadequate for its detection [48] Laccaseproduction in the presence of 10 of salt by theCladosporiumgroup may be of biotechnological interest for example inmycoremediation of high salty environments contaminatedby organic pollutants

In conclusion fungal community described in this studywas similar to those reported in inhospitable habitats characterized by limitation of nutrients moisture deficit and

exposure to high solar radiation Further studies are neededin order to elucidate their biogeochemical roles in such anextreme environment and to exploit their promising potentialto produce new biomolecules such as enzymes and protectiveagents against oxidative stress

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support from theEuropean Union in the ambit of the Project BIODESERT(EU FP7-CSA-SA REGPOT-2008-2 Grant agreement no245746) and the Tunisian Ministry of Higher Education andScientific Research in the ambit of the laboratory Project LRMBA20 Atef Jaouani wants to thank the Tunisian Society forMicrobial Ecology (ATEM) for supporting publication fees ofthis work

References

[1] J KWarren ldquoSabkhas saline mudflats and pansrdquo in EvaporitesSediments Resources and Hydrocarbons pp 139ndash220 SpringerBerlin Germany 2006

[2] A A Gorbushina and W E Krumbein ldquoRock dwelling fungalcommunities diversity of life styles and colony structurerdquo inJourney to Diverse Microbial Worlds J Seckbach Ed pp 317ndash334 Kluwer Academic Publishers Amsterdam The Nether-lands 2000

[3] L J Rothschild and R L Mancinelli ldquoLife in extreme environ-mentsrdquo Nature vol 409 no 6823 pp 1092ndash1101 2001

[4] S BHumayounN Bano and J THollibaugh ldquoDepth distribu-tion ofmicrobial diversity inmono lake ameromictic soda lakein Californiardquo Applied and Environmental Microbiology vol 69no 2 pp 1030ndash1042 2003

[5] C Demergasso E O Casamayor G Chong P GalleguillosL Escudero and C Pedros-Alio ldquoDistribution of prokaryoticgenetic diversity in athalassohaline lakes of the AtacamaDesertNorthern Chilerdquo FEMS Microbiology Ecology vol 48 no 1 pp57ndash69 2004

[6] N Gunde-Cimerman A Oren and A Plemenitas ldquoAdaptationto life in high salt concentrationsrdquo in Archaea Bacteria andEukarya p 576 Springer Dordrecht The Netherlands 2005

[7] L Maturrano F Santos R Rossello-Mora and J AntonldquoMicrobial diversity in Maras salterns a hypersaline envi-ronment in the Peruvian Andesrdquo Applied and EnvironmentalMicrobiology vol 72 no 6 pp 3887ndash3895 2006

[8] Q Wu G Zwart M Schauer M Kamst-van Agterveld andM Hahn ldquoBacterioplankton community composition alonga salinity gradient of sixteen high-mountain lakes locatedon the Tibetan Plateau Chinardquo Applied and EnvironmentalMicrobiology vol 72 no 8 pp 5478ndash5485 2006

[9] C Gostincar M Grube S De Hoog P Zalar and N Gunde-Cimerman ldquoExtremotolerance in fungi evolution on the edgerdquoFEMS Microbiology Ecology vol 71 no 1 pp 2ndash11 2010

10 BioMed Research International

[10] K Sterflinger D Tesei and K Zakharova ldquoFungi in hot andcold deserts with particular reference to microcolonial fungirdquoFungal Ecology vol 5 no 4 pp 453ndash462 2012

[11] M J Liszka M E Clark E Schneider and D S ClarkldquoNature versus nurture developing enzymes that functionunder extreme conditionsrdquo Annual Review of Chemical andBiomolecular Engineering vol 3 pp 77ndash102 2012

[12] J P Perthuisot La Sebkha ElMelah de Zarzis genese et evolutiondrsquoun bassin salin paralique Les gisements neolithiques des abordsde la Sebkha el Melah vol 9 of de Travaux du Laboratoire degeologieEcole normale superieure Ecole Normale Superieure1975

[13] I cLellan A Varela M Blahgen et al ldquoHarmonisation ofphysical and chemical methods for soil management in CorkOak forestsmdashlessons from collaborative investigationsrdquoAfricanJournal of Environmental Science and Technology vol 7 no 6pp 386ndash401 2013

[14] J HWarcup ldquoThe soil-plate method for isolation of fungi fromsoilrdquo Nature vol 166 no 4211 pp 117ndash118 1950

[15] K H Domsch W Gams and T H Anderson Compendium ofSoil Fungi Academic Press London UK 1980

[16] J A von ArxThe Genera of Fungi Sporulating in Pure CultureJ Cramer Vaduz Germany 1981

[17] E Kiffer and M Morelet Les deuteromycetes Classification etcles didentification generique INRA Editions Paris France1997

[18] D Liu S Coloe R Baird and J Pedersen ldquoRapid mini-preparation of fungal DNA for PCRrdquo Journal of Clinical Micro-biology vol 38 no 1 article 471 2000

[19] T J White T Bruns S Lee and J Talor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Innes D H Gelfand J J Sninsky and T JWhite Eds pp 315ndash322 Academic Press New York NY USA1990

[20] G Luo and T G Mitchell ldquoRapid identification of pathogenicfungi directly from cultures by using multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 8 pp 2860ndash2865 2002

[21] P Rodrigues C Santos A Venancio and N Lima ldquoSpeciesidentification of Aspergillus section Flavi isolates from Por-tuguese almonds using phenotypic including MALDI-TOFICMS and molecular approachesrdquo Journal of Applied Microbi-ology vol 111 no 4 pp 877ndash892 2011

[22] K Bensch U Braun J Z Groenewald and P W Crous ldquoThegenus cladosporiumrdquo Studies in Mycology vol 72 pp 1ndash4012012

[23] M L Berbee M Pirseyedi and S Hubbard ldquoCochliobo-lus phylogenetics and the origin of known highly virulentpathogens inferred from ITS and glyceraldehyde-3-phosphatedehydrogenase gene sequencesrdquo Mycologia vol 91 no 6 pp964ndash977 1999

[24] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous Ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995

[25] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[26] H M Hassan and I Fridovich ldquoParaquat and Escherichia coliMechanism of production of extracellular superoxide radicalrdquoJournal of Biological Chemistry vol 254 no 21 pp 10846ndash108521979

[27] M B Angelova S B Pashova B K Spasova S V Vassilevand L S Slokoska ldquoOxidative stress response of filamentousfungi induced by hydrogen peroxide and paraquatrdquoMycologicalResearch vol 109 no 2 pp 150ndash158 2005

[28] A de Groot V Chapon P Servant et al ldquoDeinococcus desertisp nov a gamma-radiation-tolerant bacterium isolated fromthe Sahara Desertrdquo International Journal of Systematic andEvolutionary Microbiology vol 55 no 6 pp 2441ndash2446 2005

[29] A Chanal V Chapon K Benzerara et al ldquoThe desert ofTataouine an extreme environment that hosts a wide diversityof microorganisms and radiotolerant bacteriardquo EnvironmentalMicrobiology vol 8 no 3 pp 514ndash525 2006

[30] M Gtari D Daffonchio and A Boudabous ldquoAssessment ofthe genetic diversity of Frankia microsymbionts of Elaeagnusangustifolia L plants growing in a Tunisian date-palm oasis byanalysis of PCR amplified nifD-K intergenic spacerrdquo CanadianJournal of Microbiology vol 53 no 3 pp 440ndash445 2007

[31] I Essoussi F Ghodhbane-Gtari H Amairi et al ldquoEsterase asan enzymatic signature of Geodermatophilaceae adaptabilityto Sahara desert stones and monumentsrdquo Journal of AppliedMicrobiology vol 108 no 5 pp 1723ndash1732 2010

[32] I Sbissi M Neffati A Boudabous C Murat and M GtarildquoPhylogenetic affiliation of the desert truffles Picoa juniperi andPicoa lefebvreirdquo Antonie van Leeuwenhoek vol 98 no 4 pp429ndash436 2010

[33] I Sbissi F Ghodhbane-Gtari M Neffati H Ouzari A Boud-abous and M Gtari ldquoDiversity of the desert truffle Terfeziaboudieri chatin in southern Tunisiardquo Canadian Journal ofMicrobiology vol 57 no 7 pp 599ndash605 2011

[34] C A Conley G Ishkhanova C P McKay and K Cullings ldquoApreliminary survey of non-lichenized fungi cultured from thehyperarid Atacama Desert of Chilerdquo Astrobiology vol 6 no 4pp 521ndash526 2006

[35] E V Smolyanyuk and E N Bilanenko ldquoCommunities ofhalotolerant micromycetes from the areas of natural salinityrdquoMicrobiology vol 80 no 6 pp 877ndash883 2011

[36] H PMolitoris A S Buchalo I Kurchenko et al ldquoPhysiologicaldiversity of the fIrst fIlamentous fungi isolated from the hyper-saline Dead Seardquo inAquatic Mycology Across theMillennium KD Hyde W H Ho and S B Pointing Eds vol 5 pp 55ndash70Fungal Diversity Press 2000

[37] S I I Abdel-Hafez ldquoSurvey of the mycoflora of desert soils inSaudi ArabiardquoMycopathologia vol 80 no 1 pp 3ndash8 1982

[38] A H M El-Said and A Saleem ldquoEcological and physiologicalstudies on soil fungi at Western Region Libyardquo Mycobiologyvol 36 pp 1ndash9 2008

[39] P Zalar G S de Hoog H-J Schroers P W Crous J Z Groe-newald and N Gunde-Cimerman ldquoPhylogeny and ecology ofthe ubiquitous saprobe Cladosporium sphaerospermum withdescriptions of seven new species from hypersaline environ-mentsrdquo Studies in Mycology vol 58 pp 157ndash183 2007

[40] J KMisra ldquoOccurrence distribution and seasonality of aquaticfungi as affected by chemical factors in six alkaline ponds ofIndiardquo Hydrobiologia vol 97 no 2 pp 185ndash191 1982

[41] F T El-Hissy S A El-Zayat A M Khallil and M S MassoudldquoAquatic fungi from the submerged mud of Aswan High DamLakerdquoMicrobiological Research vol 152 no 1 pp 27ndash32 1997

[42] AM Khallil F T El-Hissy and EH Ali ldquoSeasonal fluctuationsof aquatic fungi recovered from Egyptian soil (Delta Region)rdquoJournal of Basic Microbiology vol 35 no 2 pp 93ndash102 1995

BioMed Research International 11

[43] P B Matheny J A Gossmann P Zalar T K A Kumarand D S Hibbett ldquoResolving the phylogenetic position of theWallemiomycetes an enigmatic major lineage of Basidiomy-cotardquoCanadian Journal of Botany vol 84 no 12 pp 1794ndash18052006

[44] A Blomberg and L Adler ldquoTolerence of fungi to NaClrdquo in StressTolerance of Fungi D Jennings Ed pp 209ndash231 1993

[45] A Oren ldquoBioenergetic aspects of halophilismrdquo MicrobiologyandMolecular Biology Reviews vol 63 no 2 pp 334ndash348 1999

[46] P Delgado-Sanchez M A Ortega-Amaro J F Jimenez-Bremont and J Flores ldquoAre fungi important for breaking seeddormancy in desert species Experimental evidence in Opuntiastreptacantha (Cactaceae)rdquo Plant Biology vol 13 no 1 pp 154ndash159 2011

[47] J C Frisvad ldquoHalotolerant and halophilic fungi and theirextrolite productionrdquo in Adaptation to Life at High Salt Con-centrations in Archaea Bacteria and Eukarya N Gunde-Cimerman A Oren and A Plemenitas Eds pp 426ndash440Springer Amsterdam The Netherlands 2005

[48] A Abdel-Raheem and C A Shearer ldquoExtracellular enzymeproduction by freshwater ascomycetesrdquo Fungal Diversity vol 11pp 1ndash19 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 BioMed Research International

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

Strain code Strain Protease Amylase Cellulase Lipase LaccaseJA1 Penicillium chrysogenum ++ + minus minus minusJA3 Penicillium chrysogenum ++ minus minus minus minusJA22 Penicillium chrysogenum + + minus minus minusAJ5 Penicillium canescens minus minus minus minus +JA15 Penicillium sp minus minus + minus minusJA8 Cladosporium halotolerans + minus minus minus +JA2 Cladosporium sphaerospermum +++ minus minus + +JA13 Cladosporium sphaerospermum minus + minus minus +JA14 Cladosporium cladosporioides + + minus minus minus

JA18 Cladosporium cladosporioides minus minus minus minus minus

JA4 Aspergillus flavus + minus minus + minus

JA10 Aspergillus fumigatus minus minus minus minus minus

JA11 Aspergillus fumigatiaffinis minus minus minus minus minus

JA19 Alternaria alternata minus minus minus minus minus

JA20 Alternaria alternata minus + minus minus minus

JA23 Alternaria alternata minus minus minus minus minus

JA6 Alternaria tenuissima + minus minus minus minus

JA9 Embellisia phragmospora minus minus minus minus minus

JA12 Ulocladium consortiale minus minus minus minus minus

JA17 Ulocladium sp minus minus minus minus minus

JA7 Engyodontium album + + minus ++ minus

AR activity ratio = ( activity colony) minus no activity + AR lt 1 ++ 1 lt AR lt 2 +++ 2 lt AR lt 3

tolerance [10] On the other hand Molitoris et al [36]reported that other halotolerant and halophilic fungi suchas Aspergillus and Cladosporium spp are predominant insaline desert soil of Dead Sea Many Aspergillus species havebeen also reported to constitute dominant fungi in desert ofSaudi Arabia and Libya [37 38] and halotolerant speciesincludingAspergillus sppPenicillium spp andCladosporiumsphaerospermum have been consistently isolated fromhyper-saline environments around the globe [39] In this studycontrary to many reports on hypersaline environments nospecies belonging to the genera EurotiumThrimmatostromaEmericella and Phaeotheca [9] have been obtained probablybecause of the initial alkaline pH of the Sebkha El Melahsalt lake Actually the effect of pH on the fungal diversity iscontroversialMisra [40] observed that fungal diversity varieswith the pH while other investigators found no significanteffect of pH values of water and soil habitats on fungaloccurrence [41] It is more likely that the number of theisolated fungi is directly correlated to the organic mattercontent of water mud and soil samples [42]

Beside the identification of the recovered fungal isolatesfrom Sebkha El Melah the second goal of the current studywas the detection of some of their physiological and biochem-ical featuresThis allows understanding ecological adaptationto extreme environment and predicts some biotechnologicalusage The 21 strains have been screened for tolerance toextreme NaCl concentrations basic pH temperature andoxidative stress and for the production of important enzy-matic activities in presence of 10 NaCl

Excepting Cladosporium cladosporioides JA18 all isolatesobtained in this study can be considered as moderatelyhaloalkaliphilic fungi as deduced from their ability to grow

at pH 10 and 10 of NaCl However the isolates were ableto grow when salt was not added to their growing mediaExcepting some Wallemia ichthyophaga the most strictlyhalophilic fungus [43] all other fungal strains known todate are able to grow without salt a fact confirmed inour study However gradual decrease in fungal growth wasobserved with the increasing of salt concentration in theculture medium Nineteen strains remain able to grow under15 of NaCl whereas 7 strains were able to tolerate 20 ofNaCl This result was confirmed by salt tolerance assay inliquid media as estimated by Biolog system It is noteworthythat fungi were more sensitive to salt stress in liquid mediathan in solid mediaThis could be explained by the alterationof the osmotic gradient forcing the fungi to expend moreenergy in the osmoregulatory processes resulting in slowergrowth [44] Moreover at higher salt concentration deathoccurs

Regarding the stress of pH the capacity of the majority ofisolates to growth at pH 10 implies firstly that some habitats inthe salt lake may have a varying pH and secondly that fungican tolerate a wide pH range Prima facie the overall resultsin solid and liquid media showed that Penicillium chryso-genum JA1 and JA3 Cladosporium sphaerospermum JA2 andJA13 Cladosporium cladosporioides JA18 Aspergillus fumi-gatus JA10 Aspergillus fumigatiaffinis JA11 and Alternariatenuissima JA6 are the most alkalihalotolerant isolates in thisstudy

The tolerance of the strains to extreme 45∘C was testedand results indicated that Aspergillus fumigatus JA10 Alter-naria alternata JA23 Ulocladium sp JA17 and Aspergillusfumigatiaffinis JA11 were able to grow Of particular interestthe latter two strains retained 100 of the growth rate

BioMed Research International 9

and biomass production as estimated by colony diameterMoreover their ability to grow at low temperature may allowthem to better adapt to the big temperature fluctuation indesert environments Additionally exposure to substratesgenerating oxidative stress such as H

2O2at 10mM and

paraquat at 500 120583M did not alter significantly the growth ofalmost tested strains demonstrating their ability to tolerateoxidative stress These findings may explain their presence indesert regions that are considered amongst the most stressfulenvironments on Earth because of the high UV radiationdesiccation increased salinity low nutrient availability sea-sonal and daily temperature variation and solar irradiation[6 10]

It has been postulated that microorganisms sharing arich and particular extracellular enzymatic activities arecommon in harsh conditions characterizing their ecologicalhabitat including high level of aridity temperature ionicstrength and particularly the low nutrient availability [31]This implies the need by microorganisms for an effectiveutilization of each possible available organic compound [45]Moreover fungal isolates from hot desert were revealed toplay an important role in seeds germination by breakingdormancy and increasing water uptake [46] In the presentstudy the capacity of fungal isolates to produce extracellularenzymes was assayed in the presence of 10 of NaClEnzymes tested were the following amylase for degradationof starch abundant carbohydrate polymer in many planttissues protease for degradation of plant and animal proteinscellulase which hydrolyses the cellulose themain componentof wood ubiquitous substrate for fungi and finally the laccaseinvolved in plant material delignification and in the synthesisof the melanin and related compounds to protect fungiagainst radiationThirteen strains displayed high productionsat least for one of the five-screened activities while noclear correlation of enzyme production profile with fungalsystematic groups was noted The abundance of proteaseactivity is in line with previous data on fungal isolatesfrom extreme environments showing high caseinase activitieswith little effect of salinity and temperature on enzymeproduction [36] The relative limited number of isolatesdisplaying cellulase amylase lipase and laccase activitiessuggests that high concentration of salt may have an adverseeffect on enzymeproduction andor activityTheir energywasprobably oriented to avoid salt stress due to 10 NaCl ratherthan the production of bioactive extrolites [47] Howevernot detecting the enzyme is not absolute confirmation ofan isolate inability to produce it It could also mean thatthe enzyme was not released from the mycelium or thatthe medium is inadequate for its detection [48] Laccaseproduction in the presence of 10 of salt by theCladosporiumgroup may be of biotechnological interest for example inmycoremediation of high salty environments contaminatedby organic pollutants

In conclusion fungal community described in this studywas similar to those reported in inhospitable habitats characterized by limitation of nutrients moisture deficit and

exposure to high solar radiation Further studies are neededin order to elucidate their biogeochemical roles in such anextreme environment and to exploit their promising potentialto produce new biomolecules such as enzymes and protectiveagents against oxidative stress

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support from theEuropean Union in the ambit of the Project BIODESERT(EU FP7-CSA-SA REGPOT-2008-2 Grant agreement no245746) and the Tunisian Ministry of Higher Education andScientific Research in the ambit of the laboratory Project LRMBA20 Atef Jaouani wants to thank the Tunisian Society forMicrobial Ecology (ATEM) for supporting publication fees ofthis work

References

[1] J KWarren ldquoSabkhas saline mudflats and pansrdquo in EvaporitesSediments Resources and Hydrocarbons pp 139ndash220 SpringerBerlin Germany 2006

[2] A A Gorbushina and W E Krumbein ldquoRock dwelling fungalcommunities diversity of life styles and colony structurerdquo inJourney to Diverse Microbial Worlds J Seckbach Ed pp 317ndash334 Kluwer Academic Publishers Amsterdam The Nether-lands 2000

[3] L J Rothschild and R L Mancinelli ldquoLife in extreme environ-mentsrdquo Nature vol 409 no 6823 pp 1092ndash1101 2001

[4] S BHumayounN Bano and J THollibaugh ldquoDepth distribu-tion ofmicrobial diversity inmono lake ameromictic soda lakein Californiardquo Applied and Environmental Microbiology vol 69no 2 pp 1030ndash1042 2003

[5] C Demergasso E O Casamayor G Chong P GalleguillosL Escudero and C Pedros-Alio ldquoDistribution of prokaryoticgenetic diversity in athalassohaline lakes of the AtacamaDesertNorthern Chilerdquo FEMS Microbiology Ecology vol 48 no 1 pp57ndash69 2004

[6] N Gunde-Cimerman A Oren and A Plemenitas ldquoAdaptationto life in high salt concentrationsrdquo in Archaea Bacteria andEukarya p 576 Springer Dordrecht The Netherlands 2005

[7] L Maturrano F Santos R Rossello-Mora and J AntonldquoMicrobial diversity in Maras salterns a hypersaline envi-ronment in the Peruvian Andesrdquo Applied and EnvironmentalMicrobiology vol 72 no 6 pp 3887ndash3895 2006

[8] Q Wu G Zwart M Schauer M Kamst-van Agterveld andM Hahn ldquoBacterioplankton community composition alonga salinity gradient of sixteen high-mountain lakes locatedon the Tibetan Plateau Chinardquo Applied and EnvironmentalMicrobiology vol 72 no 8 pp 5478ndash5485 2006

[9] C Gostincar M Grube S De Hoog P Zalar and N Gunde-Cimerman ldquoExtremotolerance in fungi evolution on the edgerdquoFEMS Microbiology Ecology vol 71 no 1 pp 2ndash11 2010

10 BioMed Research International

[10] K Sterflinger D Tesei and K Zakharova ldquoFungi in hot andcold deserts with particular reference to microcolonial fungirdquoFungal Ecology vol 5 no 4 pp 453ndash462 2012

[11] M J Liszka M E Clark E Schneider and D S ClarkldquoNature versus nurture developing enzymes that functionunder extreme conditionsrdquo Annual Review of Chemical andBiomolecular Engineering vol 3 pp 77ndash102 2012

[12] J P Perthuisot La Sebkha ElMelah de Zarzis genese et evolutiondrsquoun bassin salin paralique Les gisements neolithiques des abordsde la Sebkha el Melah vol 9 of de Travaux du Laboratoire degeologieEcole normale superieure Ecole Normale Superieure1975

[13] I cLellan A Varela M Blahgen et al ldquoHarmonisation ofphysical and chemical methods for soil management in CorkOak forestsmdashlessons from collaborative investigationsrdquoAfricanJournal of Environmental Science and Technology vol 7 no 6pp 386ndash401 2013

[14] J HWarcup ldquoThe soil-plate method for isolation of fungi fromsoilrdquo Nature vol 166 no 4211 pp 117ndash118 1950

[15] K H Domsch W Gams and T H Anderson Compendium ofSoil Fungi Academic Press London UK 1980

[16] J A von ArxThe Genera of Fungi Sporulating in Pure CultureJ Cramer Vaduz Germany 1981

[17] E Kiffer and M Morelet Les deuteromycetes Classification etcles didentification generique INRA Editions Paris France1997

[18] D Liu S Coloe R Baird and J Pedersen ldquoRapid mini-preparation of fungal DNA for PCRrdquo Journal of Clinical Micro-biology vol 38 no 1 article 471 2000

[19] T J White T Bruns S Lee and J Talor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Innes D H Gelfand J J Sninsky and T JWhite Eds pp 315ndash322 Academic Press New York NY USA1990

[20] G Luo and T G Mitchell ldquoRapid identification of pathogenicfungi directly from cultures by using multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 8 pp 2860ndash2865 2002

[21] P Rodrigues C Santos A Venancio and N Lima ldquoSpeciesidentification of Aspergillus section Flavi isolates from Por-tuguese almonds using phenotypic including MALDI-TOFICMS and molecular approachesrdquo Journal of Applied Microbi-ology vol 111 no 4 pp 877ndash892 2011

[22] K Bensch U Braun J Z Groenewald and P W Crous ldquoThegenus cladosporiumrdquo Studies in Mycology vol 72 pp 1ndash4012012

[23] M L Berbee M Pirseyedi and S Hubbard ldquoCochliobo-lus phylogenetics and the origin of known highly virulentpathogens inferred from ITS and glyceraldehyde-3-phosphatedehydrogenase gene sequencesrdquo Mycologia vol 91 no 6 pp964ndash977 1999

[24] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous Ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995

[25] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[26] H M Hassan and I Fridovich ldquoParaquat and Escherichia coliMechanism of production of extracellular superoxide radicalrdquoJournal of Biological Chemistry vol 254 no 21 pp 10846ndash108521979

[27] M B Angelova S B Pashova B K Spasova S V Vassilevand L S Slokoska ldquoOxidative stress response of filamentousfungi induced by hydrogen peroxide and paraquatrdquoMycologicalResearch vol 109 no 2 pp 150ndash158 2005

[28] A de Groot V Chapon P Servant et al ldquoDeinococcus desertisp nov a gamma-radiation-tolerant bacterium isolated fromthe Sahara Desertrdquo International Journal of Systematic andEvolutionary Microbiology vol 55 no 6 pp 2441ndash2446 2005

[29] A Chanal V Chapon K Benzerara et al ldquoThe desert ofTataouine an extreme environment that hosts a wide diversityof microorganisms and radiotolerant bacteriardquo EnvironmentalMicrobiology vol 8 no 3 pp 514ndash525 2006

[30] M Gtari D Daffonchio and A Boudabous ldquoAssessment ofthe genetic diversity of Frankia microsymbionts of Elaeagnusangustifolia L plants growing in a Tunisian date-palm oasis byanalysis of PCR amplified nifD-K intergenic spacerrdquo CanadianJournal of Microbiology vol 53 no 3 pp 440ndash445 2007

[31] I Essoussi F Ghodhbane-Gtari H Amairi et al ldquoEsterase asan enzymatic signature of Geodermatophilaceae adaptabilityto Sahara desert stones and monumentsrdquo Journal of AppliedMicrobiology vol 108 no 5 pp 1723ndash1732 2010

[32] I Sbissi M Neffati A Boudabous C Murat and M GtarildquoPhylogenetic affiliation of the desert truffles Picoa juniperi andPicoa lefebvreirdquo Antonie van Leeuwenhoek vol 98 no 4 pp429ndash436 2010

[33] I Sbissi F Ghodhbane-Gtari M Neffati H Ouzari A Boud-abous and M Gtari ldquoDiversity of the desert truffle Terfeziaboudieri chatin in southern Tunisiardquo Canadian Journal ofMicrobiology vol 57 no 7 pp 599ndash605 2011

[34] C A Conley G Ishkhanova C P McKay and K Cullings ldquoApreliminary survey of non-lichenized fungi cultured from thehyperarid Atacama Desert of Chilerdquo Astrobiology vol 6 no 4pp 521ndash526 2006

[35] E V Smolyanyuk and E N Bilanenko ldquoCommunities ofhalotolerant micromycetes from the areas of natural salinityrdquoMicrobiology vol 80 no 6 pp 877ndash883 2011

[36] H PMolitoris A S Buchalo I Kurchenko et al ldquoPhysiologicaldiversity of the fIrst fIlamentous fungi isolated from the hyper-saline Dead Seardquo inAquatic Mycology Across theMillennium KD Hyde W H Ho and S B Pointing Eds vol 5 pp 55ndash70Fungal Diversity Press 2000

[37] S I I Abdel-Hafez ldquoSurvey of the mycoflora of desert soils inSaudi ArabiardquoMycopathologia vol 80 no 1 pp 3ndash8 1982

[38] A H M El-Said and A Saleem ldquoEcological and physiologicalstudies on soil fungi at Western Region Libyardquo Mycobiologyvol 36 pp 1ndash9 2008

[39] P Zalar G S de Hoog H-J Schroers P W Crous J Z Groe-newald and N Gunde-Cimerman ldquoPhylogeny and ecology ofthe ubiquitous saprobe Cladosporium sphaerospermum withdescriptions of seven new species from hypersaline environ-mentsrdquo Studies in Mycology vol 58 pp 157ndash183 2007

[40] J KMisra ldquoOccurrence distribution and seasonality of aquaticfungi as affected by chemical factors in six alkaline ponds ofIndiardquo Hydrobiologia vol 97 no 2 pp 185ndash191 1982

[41] F T El-Hissy S A El-Zayat A M Khallil and M S MassoudldquoAquatic fungi from the submerged mud of Aswan High DamLakerdquoMicrobiological Research vol 152 no 1 pp 27ndash32 1997

[42] AM Khallil F T El-Hissy and EH Ali ldquoSeasonal fluctuationsof aquatic fungi recovered from Egyptian soil (Delta Region)rdquoJournal of Basic Microbiology vol 35 no 2 pp 93ndash102 1995

BioMed Research International 11

[43] P B Matheny J A Gossmann P Zalar T K A Kumarand D S Hibbett ldquoResolving the phylogenetic position of theWallemiomycetes an enigmatic major lineage of Basidiomy-cotardquoCanadian Journal of Botany vol 84 no 12 pp 1794ndash18052006

[44] A Blomberg and L Adler ldquoTolerence of fungi to NaClrdquo in StressTolerance of Fungi D Jennings Ed pp 209ndash231 1993

[45] A Oren ldquoBioenergetic aspects of halophilismrdquo MicrobiologyandMolecular Biology Reviews vol 63 no 2 pp 334ndash348 1999

[46] P Delgado-Sanchez M A Ortega-Amaro J F Jimenez-Bremont and J Flores ldquoAre fungi important for breaking seeddormancy in desert species Experimental evidence in Opuntiastreptacantha (Cactaceae)rdquo Plant Biology vol 13 no 1 pp 154ndash159 2011

[47] J C Frisvad ldquoHalotolerant and halophilic fungi and theirextrolite productionrdquo in Adaptation to Life at High Salt Con-centrations in Archaea Bacteria and Eukarya N Gunde-Cimerman A Oren and A Plemenitas Eds pp 426ndash440Springer Amsterdam The Netherlands 2005

[48] A Abdel-Raheem and C A Shearer ldquoExtracellular enzymeproduction by freshwater ascomycetesrdquo Fungal Diversity vol 11pp 1ndash19 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 9

and biomass production as estimated by colony diameterMoreover their ability to grow at low temperature may allowthem to better adapt to the big temperature fluctuation indesert environments Additionally exposure to substratesgenerating oxidative stress such as H

2O2at 10mM and

paraquat at 500 120583M did not alter significantly the growth ofalmost tested strains demonstrating their ability to tolerateoxidative stress These findings may explain their presence indesert regions that are considered amongst the most stressfulenvironments on Earth because of the high UV radiationdesiccation increased salinity low nutrient availability sea-sonal and daily temperature variation and solar irradiation[6 10]

It has been postulated that microorganisms sharing arich and particular extracellular enzymatic activities arecommon in harsh conditions characterizing their ecologicalhabitat including high level of aridity temperature ionicstrength and particularly the low nutrient availability [31]This implies the need by microorganisms for an effectiveutilization of each possible available organic compound [45]Moreover fungal isolates from hot desert were revealed toplay an important role in seeds germination by breakingdormancy and increasing water uptake [46] In the presentstudy the capacity of fungal isolates to produce extracellularenzymes was assayed in the presence of 10 of NaClEnzymes tested were the following amylase for degradationof starch abundant carbohydrate polymer in many planttissues protease for degradation of plant and animal proteinscellulase which hydrolyses the cellulose themain componentof wood ubiquitous substrate for fungi and finally the laccaseinvolved in plant material delignification and in the synthesisof the melanin and related compounds to protect fungiagainst radiationThirteen strains displayed high productionsat least for one of the five-screened activities while noclear correlation of enzyme production profile with fungalsystematic groups was noted The abundance of proteaseactivity is in line with previous data on fungal isolatesfrom extreme environments showing high caseinase activitieswith little effect of salinity and temperature on enzymeproduction [36] The relative limited number of isolatesdisplaying cellulase amylase lipase and laccase activitiessuggests that high concentration of salt may have an adverseeffect on enzymeproduction andor activityTheir energywasprobably oriented to avoid salt stress due to 10 NaCl ratherthan the production of bioactive extrolites [47] Howevernot detecting the enzyme is not absolute confirmation ofan isolate inability to produce it It could also mean thatthe enzyme was not released from the mycelium or thatthe medium is inadequate for its detection [48] Laccaseproduction in the presence of 10 of salt by theCladosporiumgroup may be of biotechnological interest for example inmycoremediation of high salty environments contaminatedby organic pollutants

In conclusion fungal community described in this studywas similar to those reported in inhospitable habitats characterized by limitation of nutrients moisture deficit and

exposure to high solar radiation Further studies are neededin order to elucidate their biogeochemical roles in such anextreme environment and to exploit their promising potentialto produce new biomolecules such as enzymes and protectiveagents against oxidative stress

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support from theEuropean Union in the ambit of the Project BIODESERT(EU FP7-CSA-SA REGPOT-2008-2 Grant agreement no245746) and the Tunisian Ministry of Higher Education andScientific Research in the ambit of the laboratory Project LRMBA20 Atef Jaouani wants to thank the Tunisian Society forMicrobial Ecology (ATEM) for supporting publication fees ofthis work

References

[1] J KWarren ldquoSabkhas saline mudflats and pansrdquo in EvaporitesSediments Resources and Hydrocarbons pp 139ndash220 SpringerBerlin Germany 2006

[2] A A Gorbushina and W E Krumbein ldquoRock dwelling fungalcommunities diversity of life styles and colony structurerdquo inJourney to Diverse Microbial Worlds J Seckbach Ed pp 317ndash334 Kluwer Academic Publishers Amsterdam The Nether-lands 2000

[3] L J Rothschild and R L Mancinelli ldquoLife in extreme environ-mentsrdquo Nature vol 409 no 6823 pp 1092ndash1101 2001

[4] S BHumayounN Bano and J THollibaugh ldquoDepth distribu-tion ofmicrobial diversity inmono lake ameromictic soda lakein Californiardquo Applied and Environmental Microbiology vol 69no 2 pp 1030ndash1042 2003

[5] C Demergasso E O Casamayor G Chong P GalleguillosL Escudero and C Pedros-Alio ldquoDistribution of prokaryoticgenetic diversity in athalassohaline lakes of the AtacamaDesertNorthern Chilerdquo FEMS Microbiology Ecology vol 48 no 1 pp57ndash69 2004

[6] N Gunde-Cimerman A Oren and A Plemenitas ldquoAdaptationto life in high salt concentrationsrdquo in Archaea Bacteria andEukarya p 576 Springer Dordrecht The Netherlands 2005

[7] L Maturrano F Santos R Rossello-Mora and J AntonldquoMicrobial diversity in Maras salterns a hypersaline envi-ronment in the Peruvian Andesrdquo Applied and EnvironmentalMicrobiology vol 72 no 6 pp 3887ndash3895 2006

[8] Q Wu G Zwart M Schauer M Kamst-van Agterveld andM Hahn ldquoBacterioplankton community composition alonga salinity gradient of sixteen high-mountain lakes locatedon the Tibetan Plateau Chinardquo Applied and EnvironmentalMicrobiology vol 72 no 8 pp 5478ndash5485 2006

[9] C Gostincar M Grube S De Hoog P Zalar and N Gunde-Cimerman ldquoExtremotolerance in fungi evolution on the edgerdquoFEMS Microbiology Ecology vol 71 no 1 pp 2ndash11 2010

10 BioMed Research International

[10] K Sterflinger D Tesei and K Zakharova ldquoFungi in hot andcold deserts with particular reference to microcolonial fungirdquoFungal Ecology vol 5 no 4 pp 453ndash462 2012

[11] M J Liszka M E Clark E Schneider and D S ClarkldquoNature versus nurture developing enzymes that functionunder extreme conditionsrdquo Annual Review of Chemical andBiomolecular Engineering vol 3 pp 77ndash102 2012

[12] J P Perthuisot La Sebkha ElMelah de Zarzis genese et evolutiondrsquoun bassin salin paralique Les gisements neolithiques des abordsde la Sebkha el Melah vol 9 of de Travaux du Laboratoire degeologieEcole normale superieure Ecole Normale Superieure1975

[13] I cLellan A Varela M Blahgen et al ldquoHarmonisation ofphysical and chemical methods for soil management in CorkOak forestsmdashlessons from collaborative investigationsrdquoAfricanJournal of Environmental Science and Technology vol 7 no 6pp 386ndash401 2013

[14] J HWarcup ldquoThe soil-plate method for isolation of fungi fromsoilrdquo Nature vol 166 no 4211 pp 117ndash118 1950

[15] K H Domsch W Gams and T H Anderson Compendium ofSoil Fungi Academic Press London UK 1980

[16] J A von ArxThe Genera of Fungi Sporulating in Pure CultureJ Cramer Vaduz Germany 1981

[17] E Kiffer and M Morelet Les deuteromycetes Classification etcles didentification generique INRA Editions Paris France1997

[18] D Liu S Coloe R Baird and J Pedersen ldquoRapid mini-preparation of fungal DNA for PCRrdquo Journal of Clinical Micro-biology vol 38 no 1 article 471 2000

[19] T J White T Bruns S Lee and J Talor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Innes D H Gelfand J J Sninsky and T JWhite Eds pp 315ndash322 Academic Press New York NY USA1990

[20] G Luo and T G Mitchell ldquoRapid identification of pathogenicfungi directly from cultures by using multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 8 pp 2860ndash2865 2002

[21] P Rodrigues C Santos A Venancio and N Lima ldquoSpeciesidentification of Aspergillus section Flavi isolates from Por-tuguese almonds using phenotypic including MALDI-TOFICMS and molecular approachesrdquo Journal of Applied Microbi-ology vol 111 no 4 pp 877ndash892 2011

[22] K Bensch U Braun J Z Groenewald and P W Crous ldquoThegenus cladosporiumrdquo Studies in Mycology vol 72 pp 1ndash4012012

[23] M L Berbee M Pirseyedi and S Hubbard ldquoCochliobo-lus phylogenetics and the origin of known highly virulentpathogens inferred from ITS and glyceraldehyde-3-phosphatedehydrogenase gene sequencesrdquo Mycologia vol 91 no 6 pp964ndash977 1999

[24] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous Ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995

[25] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[26] H M Hassan and I Fridovich ldquoParaquat and Escherichia coliMechanism of production of extracellular superoxide radicalrdquoJournal of Biological Chemistry vol 254 no 21 pp 10846ndash108521979

[27] M B Angelova S B Pashova B K Spasova S V Vassilevand L S Slokoska ldquoOxidative stress response of filamentousfungi induced by hydrogen peroxide and paraquatrdquoMycologicalResearch vol 109 no 2 pp 150ndash158 2005

[28] A de Groot V Chapon P Servant et al ldquoDeinococcus desertisp nov a gamma-radiation-tolerant bacterium isolated fromthe Sahara Desertrdquo International Journal of Systematic andEvolutionary Microbiology vol 55 no 6 pp 2441ndash2446 2005

[29] A Chanal V Chapon K Benzerara et al ldquoThe desert ofTataouine an extreme environment that hosts a wide diversityof microorganisms and radiotolerant bacteriardquo EnvironmentalMicrobiology vol 8 no 3 pp 514ndash525 2006

[30] M Gtari D Daffonchio and A Boudabous ldquoAssessment ofthe genetic diversity of Frankia microsymbionts of Elaeagnusangustifolia L plants growing in a Tunisian date-palm oasis byanalysis of PCR amplified nifD-K intergenic spacerrdquo CanadianJournal of Microbiology vol 53 no 3 pp 440ndash445 2007

[31] I Essoussi F Ghodhbane-Gtari H Amairi et al ldquoEsterase asan enzymatic signature of Geodermatophilaceae adaptabilityto Sahara desert stones and monumentsrdquo Journal of AppliedMicrobiology vol 108 no 5 pp 1723ndash1732 2010

[32] I Sbissi M Neffati A Boudabous C Murat and M GtarildquoPhylogenetic affiliation of the desert truffles Picoa juniperi andPicoa lefebvreirdquo Antonie van Leeuwenhoek vol 98 no 4 pp429ndash436 2010

[33] I Sbissi F Ghodhbane-Gtari M Neffati H Ouzari A Boud-abous and M Gtari ldquoDiversity of the desert truffle Terfeziaboudieri chatin in southern Tunisiardquo Canadian Journal ofMicrobiology vol 57 no 7 pp 599ndash605 2011

[34] C A Conley G Ishkhanova C P McKay and K Cullings ldquoApreliminary survey of non-lichenized fungi cultured from thehyperarid Atacama Desert of Chilerdquo Astrobiology vol 6 no 4pp 521ndash526 2006

[35] E V Smolyanyuk and E N Bilanenko ldquoCommunities ofhalotolerant micromycetes from the areas of natural salinityrdquoMicrobiology vol 80 no 6 pp 877ndash883 2011

[36] H PMolitoris A S Buchalo I Kurchenko et al ldquoPhysiologicaldiversity of the fIrst fIlamentous fungi isolated from the hyper-saline Dead Seardquo inAquatic Mycology Across theMillennium KD Hyde W H Ho and S B Pointing Eds vol 5 pp 55ndash70Fungal Diversity Press 2000

[37] S I I Abdel-Hafez ldquoSurvey of the mycoflora of desert soils inSaudi ArabiardquoMycopathologia vol 80 no 1 pp 3ndash8 1982

[38] A H M El-Said and A Saleem ldquoEcological and physiologicalstudies on soil fungi at Western Region Libyardquo Mycobiologyvol 36 pp 1ndash9 2008

[39] P Zalar G S de Hoog H-J Schroers P W Crous J Z Groe-newald and N Gunde-Cimerman ldquoPhylogeny and ecology ofthe ubiquitous saprobe Cladosporium sphaerospermum withdescriptions of seven new species from hypersaline environ-mentsrdquo Studies in Mycology vol 58 pp 157ndash183 2007

[40] J KMisra ldquoOccurrence distribution and seasonality of aquaticfungi as affected by chemical factors in six alkaline ponds ofIndiardquo Hydrobiologia vol 97 no 2 pp 185ndash191 1982

[41] F T El-Hissy S A El-Zayat A M Khallil and M S MassoudldquoAquatic fungi from the submerged mud of Aswan High DamLakerdquoMicrobiological Research vol 152 no 1 pp 27ndash32 1997

[42] AM Khallil F T El-Hissy and EH Ali ldquoSeasonal fluctuationsof aquatic fungi recovered from Egyptian soil (Delta Region)rdquoJournal of Basic Microbiology vol 35 no 2 pp 93ndash102 1995

BioMed Research International 11

[43] P B Matheny J A Gossmann P Zalar T K A Kumarand D S Hibbett ldquoResolving the phylogenetic position of theWallemiomycetes an enigmatic major lineage of Basidiomy-cotardquoCanadian Journal of Botany vol 84 no 12 pp 1794ndash18052006

[44] A Blomberg and L Adler ldquoTolerence of fungi to NaClrdquo in StressTolerance of Fungi D Jennings Ed pp 209ndash231 1993

[45] A Oren ldquoBioenergetic aspects of halophilismrdquo MicrobiologyandMolecular Biology Reviews vol 63 no 2 pp 334ndash348 1999

[46] P Delgado-Sanchez M A Ortega-Amaro J F Jimenez-Bremont and J Flores ldquoAre fungi important for breaking seeddormancy in desert species Experimental evidence in Opuntiastreptacantha (Cactaceae)rdquo Plant Biology vol 13 no 1 pp 154ndash159 2011

[47] J C Frisvad ldquoHalotolerant and halophilic fungi and theirextrolite productionrdquo in Adaptation to Life at High Salt Con-centrations in Archaea Bacteria and Eukarya N Gunde-Cimerman A Oren and A Plemenitas Eds pp 426ndash440Springer Amsterdam The Netherlands 2005

[48] A Abdel-Raheem and C A Shearer ldquoExtracellular enzymeproduction by freshwater ascomycetesrdquo Fungal Diversity vol 11pp 1ndash19 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 BioMed Research International

[10] K Sterflinger D Tesei and K Zakharova ldquoFungi in hot andcold deserts with particular reference to microcolonial fungirdquoFungal Ecology vol 5 no 4 pp 453ndash462 2012

[11] M J Liszka M E Clark E Schneider and D S ClarkldquoNature versus nurture developing enzymes that functionunder extreme conditionsrdquo Annual Review of Chemical andBiomolecular Engineering vol 3 pp 77ndash102 2012

[12] J P Perthuisot La Sebkha ElMelah de Zarzis genese et evolutiondrsquoun bassin salin paralique Les gisements neolithiques des abordsde la Sebkha el Melah vol 9 of de Travaux du Laboratoire degeologieEcole normale superieure Ecole Normale Superieure1975

[13] I cLellan A Varela M Blahgen et al ldquoHarmonisation ofphysical and chemical methods for soil management in CorkOak forestsmdashlessons from collaborative investigationsrdquoAfricanJournal of Environmental Science and Technology vol 7 no 6pp 386ndash401 2013

[14] J HWarcup ldquoThe soil-plate method for isolation of fungi fromsoilrdquo Nature vol 166 no 4211 pp 117ndash118 1950

[15] K H Domsch W Gams and T H Anderson Compendium ofSoil Fungi Academic Press London UK 1980

[16] J A von ArxThe Genera of Fungi Sporulating in Pure CultureJ Cramer Vaduz Germany 1981

[17] E Kiffer and M Morelet Les deuteromycetes Classification etcles didentification generique INRA Editions Paris France1997

[18] D Liu S Coloe R Baird and J Pedersen ldquoRapid mini-preparation of fungal DNA for PCRrdquo Journal of Clinical Micro-biology vol 38 no 1 article 471 2000

[19] T J White T Bruns S Lee and J Talor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Innes D H Gelfand J J Sninsky and T JWhite Eds pp 315ndash322 Academic Press New York NY USA1990

[20] G Luo and T G Mitchell ldquoRapid identification of pathogenicfungi directly from cultures by using multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 8 pp 2860ndash2865 2002

[21] P Rodrigues C Santos A Venancio and N Lima ldquoSpeciesidentification of Aspergillus section Flavi isolates from Por-tuguese almonds using phenotypic including MALDI-TOFICMS and molecular approachesrdquo Journal of Applied Microbi-ology vol 111 no 4 pp 877ndash892 2011

[22] K Bensch U Braun J Z Groenewald and P W Crous ldquoThegenus cladosporiumrdquo Studies in Mycology vol 72 pp 1ndash4012012

[23] M L Berbee M Pirseyedi and S Hubbard ldquoCochliobo-lus phylogenetics and the origin of known highly virulentpathogens inferred from ITS and glyceraldehyde-3-phosphatedehydrogenase gene sequencesrdquo Mycologia vol 91 no 6 pp964ndash977 1999

[24] N L Glass and G C Donaldson ldquoDevelopment of primersets designed for use with the PCR to amplify conserved genesfrom filamentous Ascomycetesrdquo Applied and EnvironmentalMicrobiology vol 61 no 4 pp 1323ndash1330 1995

[25] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[26] H M Hassan and I Fridovich ldquoParaquat and Escherichia coliMechanism of production of extracellular superoxide radicalrdquoJournal of Biological Chemistry vol 254 no 21 pp 10846ndash108521979

[27] M B Angelova S B Pashova B K Spasova S V Vassilevand L S Slokoska ldquoOxidative stress response of filamentousfungi induced by hydrogen peroxide and paraquatrdquoMycologicalResearch vol 109 no 2 pp 150ndash158 2005

[28] A de Groot V Chapon P Servant et al ldquoDeinococcus desertisp nov a gamma-radiation-tolerant bacterium isolated fromthe Sahara Desertrdquo International Journal of Systematic andEvolutionary Microbiology vol 55 no 6 pp 2441ndash2446 2005

[29] A Chanal V Chapon K Benzerara et al ldquoThe desert ofTataouine an extreme environment that hosts a wide diversityof microorganisms and radiotolerant bacteriardquo EnvironmentalMicrobiology vol 8 no 3 pp 514ndash525 2006

[30] M Gtari D Daffonchio and A Boudabous ldquoAssessment ofthe genetic diversity of Frankia microsymbionts of Elaeagnusangustifolia L plants growing in a Tunisian date-palm oasis byanalysis of PCR amplified nifD-K intergenic spacerrdquo CanadianJournal of Microbiology vol 53 no 3 pp 440ndash445 2007

[31] I Essoussi F Ghodhbane-Gtari H Amairi et al ldquoEsterase asan enzymatic signature of Geodermatophilaceae adaptabilityto Sahara desert stones and monumentsrdquo Journal of AppliedMicrobiology vol 108 no 5 pp 1723ndash1732 2010

[32] I Sbissi M Neffati A Boudabous C Murat and M GtarildquoPhylogenetic affiliation of the desert truffles Picoa juniperi andPicoa lefebvreirdquo Antonie van Leeuwenhoek vol 98 no 4 pp429ndash436 2010

[33] I Sbissi F Ghodhbane-Gtari M Neffati H Ouzari A Boud-abous and M Gtari ldquoDiversity of the desert truffle Terfeziaboudieri chatin in southern Tunisiardquo Canadian Journal ofMicrobiology vol 57 no 7 pp 599ndash605 2011

[34] C A Conley G Ishkhanova C P McKay and K Cullings ldquoApreliminary survey of non-lichenized fungi cultured from thehyperarid Atacama Desert of Chilerdquo Astrobiology vol 6 no 4pp 521ndash526 2006

[35] E V Smolyanyuk and E N Bilanenko ldquoCommunities ofhalotolerant micromycetes from the areas of natural salinityrdquoMicrobiology vol 80 no 6 pp 877ndash883 2011

[36] H PMolitoris A S Buchalo I Kurchenko et al ldquoPhysiologicaldiversity of the fIrst fIlamentous fungi isolated from the hyper-saline Dead Seardquo inAquatic Mycology Across theMillennium KD Hyde W H Ho and S B Pointing Eds vol 5 pp 55ndash70Fungal Diversity Press 2000

[37] S I I Abdel-Hafez ldquoSurvey of the mycoflora of desert soils inSaudi ArabiardquoMycopathologia vol 80 no 1 pp 3ndash8 1982

[38] A H M El-Said and A Saleem ldquoEcological and physiologicalstudies on soil fungi at Western Region Libyardquo Mycobiologyvol 36 pp 1ndash9 2008

[39] P Zalar G S de Hoog H-J Schroers P W Crous J Z Groe-newald and N Gunde-Cimerman ldquoPhylogeny and ecology ofthe ubiquitous saprobe Cladosporium sphaerospermum withdescriptions of seven new species from hypersaline environ-mentsrdquo Studies in Mycology vol 58 pp 157ndash183 2007

[40] J KMisra ldquoOccurrence distribution and seasonality of aquaticfungi as affected by chemical factors in six alkaline ponds ofIndiardquo Hydrobiologia vol 97 no 2 pp 185ndash191 1982

[41] F T El-Hissy S A El-Zayat A M Khallil and M S MassoudldquoAquatic fungi from the submerged mud of Aswan High DamLakerdquoMicrobiological Research vol 152 no 1 pp 27ndash32 1997

[42] AM Khallil F T El-Hissy and EH Ali ldquoSeasonal fluctuationsof aquatic fungi recovered from Egyptian soil (Delta Region)rdquoJournal of Basic Microbiology vol 35 no 2 pp 93ndash102 1995

BioMed Research International 11

[43] P B Matheny J A Gossmann P Zalar T K A Kumarand D S Hibbett ldquoResolving the phylogenetic position of theWallemiomycetes an enigmatic major lineage of Basidiomy-cotardquoCanadian Journal of Botany vol 84 no 12 pp 1794ndash18052006

[44] A Blomberg and L Adler ldquoTolerence of fungi to NaClrdquo in StressTolerance of Fungi D Jennings Ed pp 209ndash231 1993

[45] A Oren ldquoBioenergetic aspects of halophilismrdquo MicrobiologyandMolecular Biology Reviews vol 63 no 2 pp 334ndash348 1999

[46] P Delgado-Sanchez M A Ortega-Amaro J F Jimenez-Bremont and J Flores ldquoAre fungi important for breaking seeddormancy in desert species Experimental evidence in Opuntiastreptacantha (Cactaceae)rdquo Plant Biology vol 13 no 1 pp 154ndash159 2011

[47] J C Frisvad ldquoHalotolerant and halophilic fungi and theirextrolite productionrdquo in Adaptation to Life at High Salt Con-centrations in Archaea Bacteria and Eukarya N Gunde-Cimerman A Oren and A Plemenitas Eds pp 426ndash440Springer Amsterdam The Netherlands 2005

[48] A Abdel-Raheem and C A Shearer ldquoExtracellular enzymeproduction by freshwater ascomycetesrdquo Fungal Diversity vol 11pp 1ndash19 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 11

[43] P B Matheny J A Gossmann P Zalar T K A Kumarand D S Hibbett ldquoResolving the phylogenetic position of theWallemiomycetes an enigmatic major lineage of Basidiomy-cotardquoCanadian Journal of Botany vol 84 no 12 pp 1794ndash18052006

[44] A Blomberg and L Adler ldquoTolerence of fungi to NaClrdquo in StressTolerance of Fungi D Jennings Ed pp 209ndash231 1993

[45] A Oren ldquoBioenergetic aspects of halophilismrdquo MicrobiologyandMolecular Biology Reviews vol 63 no 2 pp 334ndash348 1999

[46] P Delgado-Sanchez M A Ortega-Amaro J F Jimenez-Bremont and J Flores ldquoAre fungi important for breaking seeddormancy in desert species Experimental evidence in Opuntiastreptacantha (Cactaceae)rdquo Plant Biology vol 13 no 1 pp 154ndash159 2011

[47] J C Frisvad ldquoHalotolerant and halophilic fungi and theirextrolite productionrdquo in Adaptation to Life at High Salt Con-centrations in Archaea Bacteria and Eukarya N Gunde-Cimerman A Oren and A Plemenitas Eds pp 426ndash440Springer Amsterdam The Netherlands 2005

[48] A Abdel-Raheem and C A Shearer ldquoExtracellular enzymeproduction by freshwater ascomycetesrdquo Fungal Diversity vol 11pp 1ndash19 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology