semi-desert in soils in Argentina Biodiversity of species of Aspergillus section Fumigati MICROBIOLOGÍA

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R E V I S T A A R G E N T I N A D E

MICROBIOLOGÍA

ORIGINAL ARTICLE

Biodiversity of species of Aspergillus section Fumigati in semi-desert soils in Argentina

Gustavo E. Giusiano

, Eduardo Piontelli

, Mariana S. Fernández

,

Magdalena L. Mangiaterra

, María E. Cattana

, Sándor Kocsubé

, János Varga

aUniversidadNacionaldelNordeste,CONICET,InstitutodeMedicinaRegional,DepartamentodeMicología,Resistencia,Chaco, Argentina

bUniversidaddeValparaíso,EscueladeMedicina,CátedradeMicología,Valparaíso,Chile

cDepartmentofMicrobiology,FacultyofScienceandInformatics,UniversityofSzeged,Szeged,Hungary

Received19February2016;accepted20February2017 Availableonline25May2017

KEYWORDS Aspergillusfelis;

Aspergillus udagawae;

Aspergillus fumigatiaffinis;

Aspergillus fumigatus;

Soil

Abstract ThedistributionofAspergillusspeciesinsoilhasbeenwidelystudiedalloverthe world. Theaim ofthisstudy was thephenotypicand genotypiccharacterization ofspecies Aspergillusbelonging tosectionFumigati presentinsoils fromtwo Argentiniansemi-desert areas havingdifferentgeologicalconditions.Altogether,23 isolatesbelongingtoAspergillus section Fumigati were recoveredandidentified usingapolyphasic approach includingphe- notypic and molecular identifications. Aspergillus fumigatus sensu stricto and Aspergillus fumigatiaffinis had the highest frequency, of occurrence while isolates closely related to AspergillusudagawaeandAspergillusfeliswererarely observed.A. fumigatiaffinisandiso- latesclosertoA.udagawaewereisolatedforthefirsttimefromArgentiniansoilsandthisis thefirstreportontheoccurrenceofspeciesbelongingtotheA.feliscladeinSouthAmerica.

Recentscientificinterestsinbiodiversity,aswellastheincreasingimportanceofaspergillias causativeagentsofhumanandanimaldiseasesincreasetheneedtounderstandthediversity andoccurrenceofthesefungiinnature.

©2017Asociaci´onArgentinadeMicrobiolog´ıa.PublishedbyElsevierEspa˜na,S.L.U.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by- nc-nd/4.0/).

∗Correspondingauthor.

E-mailaddress:gustavogiusiano@yahoo.com.ar(G.E.Giusiano).

† †

http://dx.doi.org/10.1016/j.ram.2017.02.002

0325-7541/©2017Asociaci´onArgentinadeMicrobiolog´ıa.PublishedbyElsevierEspa˜na,S.L.U.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

PALABRASCLAVE Aspergillusfelis;

Aspergillus udagawae;

Aspergillus fumigatiaffinis;

Aspergillus fumigatus;

Suelo

BiodiversidaddeespeciesdeAspergillusdelasecciónFumigatiensuelos semidesérticosdeArgentina

Resumen LadistribucióndeespeciesdeAspergillusenelsuelosehaestudiadoampliamente entodoelmundo.Elobjetivodeestetrabajofuecaracterizarfenotípicaygenotípicamentelas especiespertenecientesalasecciónFumigatipresentesenlossuelosdedoszonassemidesérti- casdeArgentinacondiferentesgeologías.Entotal,23AspergillusdelasecciónFumigatifueron aisladoseidentificadosutilizandounenfoquepolifásicoincluyendoidentificacionesfenotípicas y moleculares.Aspergillusfumigatus sensustrictoy Aspergillusfumigatiaffinisaparecieron conmayorfrecuencia,mientrasquelosaislamientosrelacionadosaAspergillusudagawaeya Aspergillusfelisseobservaronraramente. Esteeselprimerinforme deA.fumigatiaffinisy deaislamientosestrechamenterelacionadosaA.udagawaeensuelosargentinos;tambiénel primerosobrelaocurrenciadeespeciespertenecientesalcladoA.felisenSudamérica.Elemer- genteinteréscientíficoenlabiodiversidad,asícomolacrecienteimportanciadeAspergillus comoagentescausalesdeenfermedadeshumanasyanimales,aumentanlanecesidaddecono- cerladiversidadylaocurrenciadeestoshongosenlanaturaleza.

©2017Asociaci´onArgentinadeMicrobiolog´ıa.PublicadoporElsevierEspa˜na,S.L.U.Esteesun art´ıculoOpenAccessbajolalicenciaCCBY-NC-ND(http://creativecommons.org/licenses/by- nc-nd/4.0/).

Introduction

In the21st century, Aspergillus andits teleomorphs have beeninvestigatedwithpolyphasicmethodstoexaminevari- abilityamongspecies.Currently,accordingtothepolyphasic taxonomy,Houbrakenetal.²⁴ andHubka etal.²⁵ proposed that the genus Aspergillus is classified into four subgen- era (Aspergillus, Circumdati, Fumigati and Nidulantes) and 20 sections and each includes a number of related species.

SectionFumigatiisoneofthemostspecies-richsections in the genus Aspergillus and includes species with over- allsignificanceformedicine,pharmacology,biotechnology, foodandsoil mycology.Atpresent,thesectionconsistsof 51taxa:21strictly anamorphicAspergillus speciesand30 Neosartoryaspecies⁴³.

The distributionof Aspergillus speciesin soil has been widelystudiedallovertheworld³⁰.Informationonthediver- sityof Aspergillus specieswasreportedin more than270 studiesof microfungi fromsoilbetween 0 and46degrees Northor South(N/S)andconcludedthattherelativeper- centageofAspergillusspeciesisgreatestin25---35degrees N/S.ManyrarespeciesandmostnewspeciesofAspergillus have been reported only from tropical and subtropical soils^19,22,29.

ThedistributionofAspergillusspeciesinArgentiniansoils has been treated in several studies, but there are only limiteddataavailableregardingsectionFumigati^14,31,32.The aimofthisstudywasthephenotypicandgenotypiccharac- terizationofspeciesbelongingtosectionFumigatipresent insoilsfromtwoArgentiniansemi-desertareashavingdif- ferentgeologicalconditions.

Materials and methods

Inwinter(July)2011soilsamplesfromTalampayaNational ParkandPampadeAchalawerecollected.

TalampayaNationalParkisinthesouthwestofLaRioja province(29^◦46Sand67^◦54O).Theparkcoversanareaof 2150squarekilometers,atanaltitudeof1300metersabove mean sealevel. This National Park is in asemiarid conti- nentalzone with150---170mmannualrainfall.Theclimate is hotinsummer,withtemperaturesexceeding50^◦C,and goingdownto−7to−9^◦Conwinternights.Onthecharac- teristicsandyandstonysoilsoftheplace,thevegetationis representedbyxerophilousbushesandcactuses^2,3.

PampadeAchalaisahydrologicnaturalreservelocated inthenorthwestofCórdobaprovince(31^◦41Sand64^◦50O).

Itisapampa(plain)of146000squarekilometerswithgen- tleslope andsteep gorge locatedat an altitude between 1500and2790mabovemeansealevel.Theclimateofthe regionistemperate-cold;maximumtemperaturesaregen- erally30^◦Cinthesummer,fallingbelow−20^◦Cduringthe winter. Rainfall takes place from October to April and is about 800mmannually.The Pampa’svegetationischarac- terizedbyscrubandgrasslands^1,3.

Sampling

SoilsampleswerecollectedinTalampayaNationalParkina 55 km-sectionof nationalroad 76.Soil samples werecol- lected in Pampa de Achala in a 30km-section of the old

provincialroute14.Inbothcases,samplingwascarriedout every5km,movingaway200or300mfromthemainroad.

Fivesampleswerecollectedrandomlyina20m²radius. A poolofabout250gwasmade.Thesamplesweregathered withasterilizedspoonfromthesuperficiallayer(2---3cm).In casesinwhichthesoilwashard,aknifetipsterilizedinsitu usingiodizedalcoholwasused.Thesoilsampleswerestored insterilizedpaperbagsat4^◦Cuntiltheywereanalyzed.

Soilprocessing

Dilutiontechnique:Onegramofeachsamplewasdilutedin 10mlsterilizedwater.Fromthis1:10dilutiona1:100dilu- tion wasmade. Of each dilution (1:10and 1:100), 0.2ml weretransferredtoPetridishescontainingpotato-dextrose- agar (PDA) with chloramphenicol (0.25g/l). Each dilution wasculturedtwice.Cultureplateswereincubatedfor7days at37^◦Cinordertoinhibitthegrowthofmesophilousmolds.

The plateswerekept for 20dayswaiting for thepossible growthofassociatedteleomorphs.

ThepHofeachsoilsamplecollected(10g)wasmeasured immediatelyusingapH meter(Jenco6230),afterdilution in125mlsteriledistilledwaterwith5minofagitation.

Isolationandmorphologicaldiagnosis

Primarycultureplateswereexaminedunderastereoscopic microscopeandeveryAspergillusanditsteleomorphswere countedand sub-culturedonspecific media.Each species wascountedonceineachsample,evenifitappearedtwice inthesameplateorintheduplicate.Speciesfrequencywas calculatedbyitspresenceintotalsamples.

Isolates were subcultured on Czapek yeast autolysate agar(CYA),oatmealagar(OA)andmaltextractagar(MEA) andincubatedforaweekat25^◦C.Identificationwasdoneby macroscopicandmicroscopicmorphologycriteriaaccording togeneraltaxonomickeys.Todistinguishgrowingmaximum temperature range, isolates were inoculated on CYA and incubatedfor7daysat10-37-42-45-48and50^◦C30,37,38,40.

Molecularidentification

Culturesweregrownon2mlmaltpeptonebroth[10%(v/v) maltextract(Brix10)and0.1%(w/v)bactopeptone(Difco)], in15mltubes.Thecultureswereincubatedat25^◦Cfor 7 days³⁷.

DNAwasextractedfrommyceliausingtheMasterpure^TM yeastDNApurificationkit(EpicenterBiotechnologies,Madi- son,WI,USA)accordingtothemanufacturer’sinstructions.

Random amplified polymorphic DNA (RAPD)-PCRs under the conditions described by Hong¹⁸ was per- formed toidentifyAspergillus fumigatussensustricto (A.

fumigatus s.s.) and to detect other different band pat- terns.PrimersPELF(5-ATATCATCGAAGCCGC-3)andURP1F (5-ATCCAAGGTCCGAGACAACC-3) were used and the A.

fumigatustypestrainCBS133.61wasusedaspattern.

Isolates genetically different from type strain A.

fumigatus were sent to the Department of Microbiol- ogy, Faculty of Science and Informatics, University of Szeged, Hungary for molecular identification. DNA was

extractedasmentionedabove.Amplificationofthepartial calmodulin gene (calM) and ␤-tubulin(benA) wascarried out using primers cmd5 (5-CCGAGTACAAGGAGGCCTTC- 3), cmd6 (5-CCGATAGAGGTCATAACGTGG-3)¹⁸, and Bt2a (5-GGTAACCAAATCGGTGCTGCTTTC-3), Bt2b (5- ACCCTCAGTGTAGTGACCCTTGGC-3)¹⁵, respectively. The amplified DNA fragments were purified by the QIAquick PCRpurificationkit(Qiagene,Hilden,Germany).Sequence analyses were performed withthe BigDye Terminator 3.1 CycleSequencingReadyReactionKit (ABI 0401041, Foster City,California)forbothstrands.Sequenceswereanalyzed ontheABIPRISM310GeneticAnalyzer(AppliedBiosystems, Carlsbad, CA, USA). Alignment of partial calM and benA sequenceswasdone usingMAFFTv7.149b withtheL-INS-i option²⁸.Phylogeneticreconstructionwasconductedusing MaximumLikelihoodanalysesinraxmlGUIv1.3.1underthe GTR+ model⁴¹. The analysis was run in 1000 bootstrap replicates.ThecalMandbenAsequencesoftypeisolatesof sectionFumigatispecieswereobtainedfromthedatabase oftheNationalCenterforBiotechnologyInformation(NCBI) (Table1).

Nucleotidesequenceaccessionnumbers

Sequencesof calM of the 14Aspergillus section Fumigati isolatesweresubmittedtotheEuropeanNucleotideArchive (ENA)andassignedaccessionnumbersKP824727---KP824740.

Sequences of benA of the isolates related to A. udagawae and to A. felis were submitted to the EuropeanNucleotideArchive(ENA)andassignedaccession numbersLT674551---LT674557.

Results

Atotalof 17 soil sampleswascollected,11 in Talampaya NationalParkand6in PampadeAchala.ThepHvaluesof TalampayaNationalParksoilsampleswerealkaline,ranging from8.00to9.83.Incontrast,thepHvaluesofPampade Achalasoil sampleswere acid, ranging from 4.91 to5.65 (Table2).

From these 17 samples, 39 Aspergillus isolates were obtained. According to their phenotypic features, 23/39 belongtosubgenusFumigati,8/39tosubgenusNidulantes and8/39tosubgenusCircumdati.

WithinsubgenusFumigationlyisolatesbelongingtosec- tionFumigatiwerefound,whichwerepresentinallthe17 samplesanalyzed.

The RAPD-PCR for 23 isolates of Aspergillus section Fumigatiwasconducted.Nine(9/17,52.94%)isolateswere genetically similar to type strain A. fumigatus s.s. (CBS 133.61)(datanot shown)and the14 geneticallydifferent isolateswereidentifiedbysequencingpartsofthecalMgene (Fig.1)andthebenAgene(Fig.2).Nine(9/17,52.94%)iso- lateswereclearlyidentifiedasA.fumigatiaffinisusingthe calMgene.Two(2/17,11.76%)isolateswerecloselyrelated toA. udagawae and 3/17 (17.65%) isolates belong tothe A.feliscladesequencingthecalMandbenAgenes.Table2 shows the Aspergillus section Fumigati identifiedand the areaswheretheywerefound.

A.fumigatuss.s.wereidentifiedby morphologicaland growthcharacteristics:darkblue-greencolorandvelutinous

IMR-MF 702 = KS 702

62

100 71

100

100 61

59

100 54

72 70 93

81

100 100 90 56 90 86

99

99

IMR-MF 705 = KS 705 IMR-MF 704 = KS 704 IMR-MF 701 = KS 701 IMR-MF 700 = KS 700 IMR-MF 695 = KS 695 IMR-MF 696 = KS 696 IMR-MF 698 = KS 698 IMR-MF 699 = KS 699

IMR-MF 707 = KS 707 IMR-MF 697 = KS 697

IMR-MF 703 = KS 703

IMR-MF 708 = KS 708 IMR-MF 706 = KS 706 A. fumigatiaffinis IBT 12703^T

A. novofumigatus IBT 16806^T A. lentulus NRRL 35552^T

A. fumisynnematus IFM 42277^T A. ffischeri NRRL 181^T

A. fumigatus NRRL 5587^T A. takakii CBM-FA-884^T

A. paulistensis CBM FA–0690^T A. laciniosus UAMH 11627 A. spinosus NRL 5034^T

A. parafelis CM 3147^T

A. pseudoviridinutans NIH AV1^T

A. udagawae CBM FA–0702^T A. aureolus NRRL 2244^T

A. siamensis KUFC 6349^T A. wyomingensis CCF 4417^T

A. viridinutans IMI 367415^T A. acroverdensis IFM 61334

A. fennelliae NRRL 5534^T

0 009

N. coreana KACC 41659^T A. felis CBS 130245^T A. pseudofelis CM 6087^T

Figure1 Taxonomic position ofthe strains ofAspergillus section Fumigati isolated from Argentinian soils based on partial Calmodulingenephylogeny.Phylogenetictreeinferredfrommaximumlikelihoodanalysis.Onlybootstrapvalues≥50%areshown.

colony,fastandabundantsporulationonMEAandCYA.Most isolateshadsubclavatevesicles(13-26␮m)andtheconidio- phorestipediameterrangedfrom5to9␮m.AllA.fumigatus s.s.didnotgrowat10^◦Candgrewat50^◦ConCYA,aswas expectedaccordingtotheliterature18,20,38,40.

A. fumigatiaffinis, and the isolates closely related to A. felis and A. udagawae showed less sporulation on MEA and CYA than A. fumigatus s.s. and the colonies were white, with a dull green center. Most isolates of A.fumigatiaffinisshowed(sub)globosevesicles(15---23␮m),

IMR–MF 697 = KS 697

IMR–MF 703 = KS 703

IMR–MF 707 = KS 707

IMR–MF 708 = KS 708

IMR–MF 706 = KS 706

A. pseudofelis CM 6087^T

A. pseudofelis CM 4518

A. felis CBS 130245^T

A. parafelis CM 3147^T

A. pseudoviridinutans NIH AV1^T

A. udagawae CBM FA–0702^T . A. fennelliae NRRL 5534^T

0 008 100

100

100 85

99 99

100

Figure2 TaxonomicpositionoftheisolatesrelatedtoAspergillusudagawaeandAspergillusfelisbasedonpartial␤-tubulingene phylogeny.PhylogenetictreeinferredfromMaximumLikelihoodanalysis.Onlybootstrapvalues≥50%areshown.

while vesicles of isolates belonging to the A. felis clade were subclavate (15---17␮m) and isolates closely related to A. udagawae exhibited hemispherical to flask-shaped vesicles (11---16␮m). The width of conidiophore stipes in A.fumigatiaffinisrangedfrom6to8␮m,whileinisolates belongingtothe A.felis cladefrom5 to9␮m andin the isolates closely related to A. udagawae from 4 to 6␮m.

Alltheseisolateswereabletogrowat10^◦Candunableto growat50^◦C.

All isolates were deposited at Szeged Microbiological CollectionoftheDepartmentofMicrobiology,FacultyofSci- enceandInformatics,UniversityofSzeged,Hungaryunder theassignednumbersKS695---KS708.

Discussion

InavastcompilationofstudiesonAspergillusspeciesinsoil itwasfoundthatAspergillusspeciesmostfrequentlyoccur insubtropicalzones,between25and35degreesN/S^22,23,29. Consideringthattheareastudiedisincludedinthoselati- tudes,theAspergillusspp.frequenciesfoundprobablyratify thatassertion.

Section Fumigati was represented in both eco-regions studied, although with different species distributions. A.

fumigatus s.s. and A. fumigatiaffinis were the most fre- quentspecies,followedbyisolatescloselyrelatedtoA.felis

and A. udagawae. However, unlike the other species, A.

fumigatuss.s.occurredinbothareas,confirmingitscapa- bility of adaptation and ubiquity. A. fumigatiaffinis and isolatescloselyrelatedtoA.udagawaeandbelongingtothe A.felis cladewereisolatedfor thefirsttimefromArgen- tiniansoils.

A.fumigatiaffiniswasonlyisolatedinTalampayaPark.

Theclimaticcharacteristicsandheightabovemeansealevel ofTalampayaParkaresimilartothoseofSocorrocity(USA), whereA.fumigatiaffiniswasreportedforthefirsttime¹⁸.

Thepresence oftwoisolatesclosely relatedtoA.uda- gawaeonlyin PampadeAchala providesmore dataabout theplasticityof this speciesbecause,eventhough it was describedinBrazilinahumidsubtropicalarea,ithasalso beenidentifiedasinfectinghumansanddomesticanimalsin areaswithextremeenvironments23,26,27,42.

A. felis is an important species in Aspergillus section Fumigatidescribedrecently¹⁰.A.felis(neosartorya-morph) isphenotypicallysimilartoA.viridinutans;however,itdif- fersby itsabilitytogrow at 45^◦Cand isphylogenetically relatedtoA.aureolusandA.udagawae²⁷.Inourstudy,the isolatesbelonging totheA.felis cladewereonlyisolated inPampadeAchala.AlthoughA.felis wasisolatedinsoils of Wyoming, USA, this is the first report onthe ambient occurrenceofaspeciesofthiscladeintheSouthAmerican continent³⁴.

G.E.Giusianoetal.

Table1 calMandbenAsequencesoftypeisolatesofsectionFumigatiusedforphylogeneticanalysis

Species^a Referencestrain calM^b benA^b Species Referencestrain calM^b benA^b

A.udagawae CBMFA-070^T AB748566.1 AF132226.1 A.parafelis CM3147^T KJ914702.1 KJ914692.1

A.aureolus NRRL224^T EF669877.1 --- A.pseudofelis CM4518 --- KJ914696.1

A.wyomingensis CCF441^T HF933397.1 --- A.pseudofelis CM6087^T KJ914705.1 KJ914697.1

A.felis CBS13024^T JX021715.1 JX021700.1 A.siamensis KUFC634^T AB776704.1 ---

A.viridinutans IMI36741^T DQ534162.1 --- A.novofumigatus IBT1680^T DQ094893.1 ---

A.arcoverdensis IFM6133^T AB818856.1 --- A.fumigatiaffinis IBT1270^T DQ094891.1 ---

A.takakii CBMFA-88^T AB787566.1 --- A.fumisynnematus IFM4227^T AB259968.1 ---

N.paulistensis CBMFA-069^T AB488766.1 --- A.lentulus NRRL3555^T EF669895.1 ---

A.laciniosus UAMH11627^T JX845619.1 --- A.fischeri NRRL18^T EF669865.1 ---

N.coreana KACC4165^T AY870718.1 --- A.fumigatus NRRL5587^T EF669922.1 ---

A.spinosus NRRL503^T EF669914.1 --- A.fennelliae^* NRRL553^T EF669920.1 EU014108.1

A.pseudoviridinutans NIHAV1^T KJ914708.1 KJ914690.1

a ObtainedfromthedatabaseNationalCenterforBiotechnologyInformation.

b GenBankaccessionnumberforcalmodulin(calM)and␤-tubulin(benA)gene.

* Selectedspeciesasoutgroup.A.:Aspergillus;N.:Neosartorya.

Table2 FrequencyofAspergillussectionFumigatiisolatedandpHofsoilsamples

Soilsamples T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 t P1 P2 P3 P4 P5 P6 t

pH 9.64 9.06 9.02 8.78 8.94 8.30 8.00 9.27 9.52 9.52 9.83 5.65 5.61 5.36 5.04 4.91 5.30

SectionFumigati

Aspergillusfelisclade 0 X X X 3

Aspergillusfumigatiaffinis X X X X X X X X X 9 0

Aspergillusfumigatuss.s. X X X X 4 X X X X X 5

Aspergillusudagawae 0 X X 2

Totalspecies 13 10

T:TalampayaNationalPark;P:PampadeAchala;t:total.

Aspergillusisalargegenusofubiquitousandcosmopoli- tanfungi.Theirhighadaptabilityallowsthemtosurviveat different temperatures,low water activity and variations ofpH andO2concentrationin soil^29,39,45.Thiscouldbean explanationoftheisolationofthesestrainsintheextreme environmentsstudied.

FungicantolerateawidepHrange³⁹.Theavailableevi- denceforthefungalgrowth---pHrelationshipthusindicates a significantly weaker direct connection with pH than in the case of bacteria, although pure culture studies have shown preferencefor certainpH valuesfor differenttaxa ofsoilfungi^12,39,45.Inourstudy,A.fumigatiaffiniswasiso- latedonlyfromalkalinesoilsofTalampayaPark.Incontrast, theisolatescloselyrelatedtoA.felisandA.udagawaewere isolatedonlyfromPampadeAchalawherethesoilpHwas slightlyacid.Furtherinvestigationsareessentialforcorre- latingthepreferenceforcertainpHvaluesforthesetaxain ordertoallowdefinitiveconclusions.

EventhoughA.fumigatusisthemostprevalentagentof aspergillosis,severalotherspeciesofsectionFumigatihave alsobeenreportedfromclinicalsamplesascausativeagents of disease: A. lentulus⁷, A. udagawae^16,26,42,A. felis^9,10,44 and A. fumigatiaffinis⁴. In Argentina, a recent work with clinical isolates shows the circulating Aspergillus section Fumigatispecies. A.fumigatuss.s.wasthemostfrequent followedbyA.udagawae¹⁷.

Furthermore, these species show high in vitro MICsof azole drugs and amphotericin B, therefore they are fre- quentlyrefractorytostandardantifungaltherapy^4,8.

In this work, morphological and physiological charac- teristics were useful for differentiating isolates belonging to section Fumigati from those belonging to other sec- tions. However, sequence-based methods are needed to assign isolates of section Fumigati at the species level.

In thisstudy,the speciesidentification of isolatesrelated to A. udagawae (IMR-MF 706 and IMR-MF 708) and to A.

felis (IMR-MF697,IMR-MF703, IMR-MF707)wasnotclear using only the partial calMsequences. The results of the analysisof partialbenAsequenceswere necessarytosug- gest that IMR-MF 706 and 708 isolates were closer to A.

udagawae and to the others isolate belonging to A. felis clade.

Identification of environmental and clinical isolatesby molecular techniques is common practice in European countries and in the USA4,6,18,20---22,34,40,42,43,46 but there is scantinformationinSouthAmerica5,11,13,17,33,35,36.

Inmedicalmycology,asmolecularmethodsbecomemore available,theywillallowtheaccurateidentificationoffun- galinfectiousagents.The correctidentification ofspecies withinsectionFumigaticouldhelptopredicttheseverityof thediseaseandguideantifungaltherapy.

Thepresentworkwascarriedoutasacontributiontothe knowledgeoftheecologyofsectionFumigati,tounderstand where this sectionoccurs in naturedue tothe increasing importanceoftheseopportunisticpathogens.

Ethical responsibilities

Protection of human and animal subjects.The authors declarethatnoexperimentswereperformedonhumansor animalsforthisstudy.

Confidentialityofdata.Theauthorsdeclarethatnopatient dataappearinthisarticle.

Right to privacy and informed consent.The authors declarethatnopatientdataappearinthisarticle.

Conflict of interest

Theauthorshavenoconflictofinteresttodeclare.

Acknowledgements

WewouldliketothanktothechemistrytechnicianClaudio Szarfsztejnfor theirassistanceinthesampling anddeter- minationofthepHofthesamplesobtained

References

1.Administraciónde ParquesNacionales, Argentina.[Internet].

[updated10March2014,cited4March2015].Availablefrom:

http://www.parquesnacionales.gob.ar/areas-protegidas/

region-centro/pn-quebrada-del-condorito/

2.Administración de Parques Nacionales, Argentina. Parque NacionalTalampaya.[Internet].[updated10March2014,cited 4March2015].Availablefromhttp://www.parquesnacionales.

gob.ar/areas-protegidas/region-centro/pn-talampaya/

3.AdministracióndeParquesNacionales.SistemadeInformación deBiodiversidad.[Internet].[updated10March2014,cited4 March2015].Availablefromhttps://www.sib.gov.ar/

4.Alcazar-Fuoli L, Mellado E, Alastruey-Izquierdo A, Cuenca- EstrellaM,Rodriguez-TudelaJL.AspergillussectionFumigati:

antifungalsusceptibilitypatternsandsequence-basedidentifi- cation.AntimicroblAgentsandChemother.2008;52:1244---51.

5.Alonso V, Aminahuel C, Díaz Vergara L, Pereyra C, Poloni V, Dalcero A, Cavaglieri L. Ecophysiology of environmental Aspergillusfumigatusand comparisonwithclinicalstrainson gliotoxinproductionandelastaseactivity.LettAppMicrobiol.

2016;62:160---8.

6.Araujo R, Amorim A, Gusmão L. Diversity and specificity of microsatelliteswithinAspergillussectionFumigati.BMCMicro- biol.2012;12:154.

7.BalajeeSA,GribskovJL,HanleyE,NickleD,MarrKA.Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus.

EukaryotCell.2005;4:625---32.

8.BalajeeSA,KanoR,BaddleyJW,MoserSA,MarrKA,Alexander BD, KontoyiannisDP, PerroneG, PetersonS, BrandtME, Pap- pasPG,ChillerT.MolecularidentificationofAspergillusspecies collectedfor thetransplant-associated infectionsurveillance network.JClinMicrobiol.2009;47:3138---41.

9.Barrs VR, Halliday C, Martin P, Wilson B, Krockenberger M, GunewM,BennettS,KoehlmeyerE,ThompsonA,FliegnerR, HockingA,SleimanS,O’BrienC,BeattyJA.Sinonasalandsino- orbital aspergillosisin 23cats: aetiology, clinicopathological featuresandtreatmentoutcomes.VetJ.2012;191:58---64.

10.BarrsVR,vanDoornTM,HoubrakenJ,KiddSE,MartinP,Pin- heiroMD,RichardsonM,VargaJ,SamsonRA.Aspergillusfelis sp.nov.,anemergingagentofinvasiveaspergillosisinhumans, cats,anddogs.PLoSONE.2013;8.

11.DogiC,AlonsoV,FochesatoA,PoloniV,CavaglieriL.Comparison oftoxicogenicandimmunosuppressivecapacityofAspergillus fumigatusstrainsisolatedfromclinicalandcornsilagesamples.

JAppMicrobiol.2015;118:175---81.

12.DomschKH,GamsW,AndersonT-H.Compendiumofsoilfungi.

2ndedEching:IHWVerlag;2007.

13.Duarte-EscalanteE,ZunigaG,RamirezON,CordobaS,Refojo N,ArenasR,DelhaesL,Reyes-MontesMD.Populationstructure and diversityofthepathogenicfungusAspergillusfumigatus isolatedfromdifferentsourcesandgeographicorigins.MemInst OswaldoCruz.2009;104:427---33.

14.GiusianoG,PiontelliE,MangiaterraM,SosaMA.Keratinophilic altitudinaldistributionoffungi,epiphytesandendophytesin semiaridsoilsofnorthwesternArgentina(Prov.deJujuy,23^oL.S y66^oL.W).BolMicol.2002;17:51---62.

15.GlassNL,DonaldsonGC.Developmentofprimersetsdesigned forusewiththePCRtoamplifyconservedgenesfromfilamen- tousAscomycetes.AppEnvironMicrobiol.1995;61:1323---30.

16.GyotokuH,IzumikawaK,IkedaH,TakazonoT,MorinagaY,Naka- muraS,ImamuraY,NishinoT,MiyazakiT,KakeyaH,Yamamoto Y,YanagiharaK,YasuokaA,YaguchiT,OhnoH,MiyzakiY,Kamei K,KandaT,KohnoS.Acaseofbronchialaspergillosiscausedby Aspergillusudagawaeanditsmycologicalfeatures.MedMycol.

2012;50:631---6.

17.Hevia A, Abrantes RA, Fernández J, Davel G, Refojo N.

Identificación molecular de Aspergillus Sección Fumigati no fumigatus en Argentina. Libro de Resúmenes-INFOCUS.

2015;7. Available from http://infocus2015.circulomedicocba.

org/docs/LibroderesumenesInfocus2015.pdf.

18.HongSB,GoSJ,ShinHD,FrisvadJC,SamsonRA.Polyphasictax- onomyofAspergillusfumigatusandrelatedspecies.Mycologia.

2005;97:1316---29.

19.Hong SB, Cho HS, Shin HD, Frisvad JC, Samson RA. Novel NeosartoryaspeciesisolatedfromsoilinKorea.IntJSystEvol Microbiol.2006;56:477---86.

20.HongSB,ShinHD,HongJ,FrisvadJC,NielsenPV,VargaJ,Sam- sonRA.NewtaxaofNeosartoryaandAspergillusinAspergillus sectionFumigati.AntonLeeuwIntJG.2008;93:87---98.

21.Hong SB, Kim DH, Park IC, Choi YJ, Shin HD, Sam- son R. Re-identification of Aspergillus fumigatus sensu lato based on a new concept of species delimitation. Microbiol.

2010;48:607---15.

22.Hong SB, Kim DH, Park IC, Samson RA, Shin HD. Isolation andidentificationofAspergillussectionFumigatistrainsfrom arablesoilinKorea.Mycobiol.2010;38:1---6.

23.HorieY,MiyajiM,NishimuraK,FrancoMF,CoelhoK,labukiR.

NewandinterestingspeciesofNeosartoryafromBraziliansoil.

Mycoscience.1995;36:199---204.

24.Houbraken J, de Vries RP, Samson RA. Modern taxonomy of biotechnologically important Aspergillus and Penicillium species.AdvApplMicrobiol.2014;86:199-249.

25.Hubka V, Nováková A, Kolaˇrík M, Jurjevi´c ˇZ, Peterson SW. Revision of Aspergillus section Flavipedes: seven new species and proposal of section Jani sect. nov. Mycologia.

2015;107:169---208.

26.KanoR,ItamotoK,OkudaM,InokumaH,HasegawaA,Balajee SA.IsolationofAspergillusudagawaefromafatalcaseoffeline orbitalaspergillosis.Mycoses.2008;51:360---1.

27.KanoR,ShibahashiA,FujinoY,SakaiH,MoriT,TsujimotoH, YanaiT,HasegawaA. Twocasesoffelineorbitalaspergillosis duetoAspergillusudagawaeandA.viridinutans.JVetMedSci.

2013;75:7---10.

28.KatohK,StandleyDM.MAFFTmultiplesequencealignmentsoft- wareversion7:improvementsinperformanceandusability.Mol BiolEvol.2013;30:772---80.

29.KlichMA.BiogeographyofAspergillusspeciesinsoilandlitter.

Mycologia.2002;94:21---7.

30.Klich MA, Pitt JI. A laboratory guide to the common Aspergillusspeciesandtheirteleomorphs.NorthRyde,N.S.W:

CommonwealthScientificandIndustrialResearchOrganisation, DivisionofFoodProcessing;1988.

31.MangiaterraM,GiusianoG,GonzálezI.Somegeophilicmicro- fungiinsemiaridplainsnorthwestoftheProvinceofSanLuis (Argentina).BolMicol.2006;21:43---8.

32.MangiaterraM,GiusianoG, GonzálezI.Geophilic mushrooms inthewesternpartoftheChacoregion(Argentina).BolMicol.

2007;22:21---9.

33.MontenegroG,SánchezPuchS,JewtuchowiczVM,PinoniMV, RellosoS,TemporittiE,IovannittiCA,MujicaMT.Phenotypic and genotypic characterization of Aspergillus lentulus and Aspergillusfumigatusisolatesinapatientwithprobableinva- siveaspergillosis.JMedMicrobiol.2009;58:391---5.

34.Nováková A, Hubka V, Dudová Z, Matsuzawa T, Kubátová A, Yaguchi T, Kolaˇrík M. New species in Aspergillus section FumigatifromreclamationsitesinWyoming(U.S.A.)andrevi- sion of A. viridinutans complex. Fungal Diversity. 2014;64:

253---74.

35.PenaGA,CoelhoI,ReynosoMM,SoleiroC,CavaglieriLR.Char- acterizationandgeneticvariabilityoffeed-borneandclinical animal/human Aspergillus fumigatus strains using molecular markers.MedMycol.2015;53:699---708.

36.Pena GA, Pereyra CM, Armando MR, Chiacchiera SM, Mag- noli CE, Orlando JL, Dalcero AM, Rosa CA, Cavaglieri LR.

Aspergillusfumigatustoxicityandgliotoxinlevelsinfeedstuff fordomesticanimalsandpetsinArgentina.LettAppMicrobiol.

2010;50:77---81.

37.PetersonS,VargaJ,FrisvadJ,SamsonR.Phylogenyandsub- generictaxonomyofAspergillus.In:PetersonS,VargaJ,Frisvad J,SamsonR,editors.Aspergillusinthegenomicsera.Wagenin- gen:WageningenAcademicPublishers;2008.p.33---56.

38.RaperK,FennellDI.ThegenusAspergillus(R.K&F.D,Eds.).

Baltimore,U.S:TheWilliams&WilkinsCo.;1965.

39.RouskJ,BååthE,BrookesPC,LauberCL,LozuponeC,Caporaso JG, Knight R, Fierer N. Soil bacterial and fungal communi- ties across a pH gradient inan arable soil. ISME J. 2010;4:

1340---51.

40.SamsonRA,HongS,PetersonSW,FrisvadJC,VargaJ.Polyphasic taxonomyofAspergillussectionFumigati anditsteleomorph Neosartorya.StudMycol.2007;59:147---203.

41.Silvestro D, Michalak I. raxmlGUI: a graphical front-end for RAxML.OrgDiversEvol.2012;12:335---7.

42.SuguiJA,VinhDC,NardoneG,SheaYR,ChangYC,ZelaznyAM, MarrKA,HollandSM,Kwon-ChungKJ.Neosartoryaudagawae (Aspergillusudagawae),anemergingagentofaspergillosis:how different is it from Aspergillus fumigatus? J Clin Microbiol.

2010;48:220---8.

43.SuguiJA,PetersonSW,FigatA,HansenB,SamsonRA,Mellado E,Cuenca-EstrellaM,Kwon-ChungKJ.Geneticrelatednessver- susbiologicalcompatibilitybetweenAspergillusfumigatusand relatedspecies.JClinMicrobiol.2014;52:3707---21.

44.Varga J, Szigeti G, Baranyi N, Kocsube S, Samson RA.

18th International Society for Human and Animal Mycology (ISHAM).In:ClinicallyrelevanttaxaofthegenusAspergillus.

2012.Available fromhttp://www.aspergillus.org.uk/content/

clinically-relevant-taxa-genus-aspergillus

45.WheelerKA,HurdmanBF,PittJI.InfluenceofpHonthegrowth ofsometoxigenicspeciesofAspergillus,PenicilliumandFusa- rium.IntJFoodMicrobiol.1991;12:141---9.

46.YaguchiT,HorieY,TanakaR,MatsuzawaT,ItoJ,NishimuraK.

MolecularphylogeneticsofmultiplegenesonAspergillussec- tionFumigatiisolatedfromclinicalspecimensinJapan.JpnJ MedMycol.2007;48:37---46.