black yeasts from the family herpotrichiellaceae ( order chaetothyriales ) are fungi with a remarkable dual ecology . on the one hand , they have a unique ability to adapt to extreme environments ( exposure to toxic chemicals , high temperature , scarcity of nutrients , acidic , and/or dry conditions ) , while on the other hand , they exhibit a significant human pathogenic potential . unlike common opportunistic fungi , herpotrichiellaceous black yeasts frequently cause infections in individuals without known underlying disease   and only occasionally in immunocompromised patients . also , more often than any other fungal group , these organisms have been reported from environments that are rich in aromatic compounds .
the first evidence on such phenomenon arose from the frequent isolation of black yeasts from wood treated with creosote , while their occurrence on untreated wood was comparatively low , indicating that accumulation of aromatic compounds might promote the growth of these fungi .
the concurrence of these two ecological traits in a single species has been observed for exophiala dermatitidis , isolated abundantly not only on tropical creosoted railway ties but also from clinical cases of severe mycoses [ 26 , 27 ] .
it has been hypothesized that wild berries constitute a natural niche of e. dermatitidis , which might then be ingested by birds and humans and sporadically resulting in mycoses .
deposition of feces on creosoted railway ties subsequently leads to the massive enrichment of this fungus .
the related species exophiala bergeri , exophiala heteromorpha , exophiala oligosperma , and exophiala xenobiotica have also been reported as opportunistic fungi causing infections that are generally less serious and from creosoted wood [ 57 , 11 , 27 ] . however , the connection between route of infection and natural occurrence is less evident with these species .
the tendency of herpotrichiellaceous fungi toward aromatic metabolism has been confirmed by their recurrent isolation from biofilters treating vapors of volatile aromatic hydrocarbons . besides hydrocarbon exposure , environmental conditions in biofilters
are characterized by a relative low water activity and acidification of the filter bed , which might lead to biomass inhibition problems . in this
respect , fungal colonization in air biofilters has generally been related to an improved bioreactor performance .
detailed metabolic studies on fungi from gas biofilters have demonstrated their capacity to assimilate alkylbenzene hydrocarbons as the sole source of carbon and energy [ 3 , 20 , 29 ] , which appears to be quite an uncommon metabolic feature in the eukaryotes .
molecular phylogenetic characterization of these  biofilter fungi  has shown a predominant affiliation to the genera exophiala and cladophialophora ( fam .
herpotrichiellaceae ) , particularly to the species exophiala lecanii - corni , e. oligosperma , and the recently described e. xenobiotica , cladophialophora saturnica , and cladophialophora inmunda [ 1 , 6 ] .
despite the medical and environmental relevance of herpotrichiellaceous black yeasts , little is understood on their biodiversity and natural occurrence . in order to address questions on niche shifts and environmental prevalence in relation to virulence factors and routes of transmission ,
the aim of the present work is to apply a specific isolation method based on the enrichment of black yeasts by simulating in batch solid state - like cultures the environmental conditions that are found in gas biofilters for the treatment of volatile aromatic hydrocarbons .
different environmental samples related to the life cycle of e. dermatitidis were used as inocula .
the list of sampled sites that were used as source of inoculum for the subsequent enrichment cultures is presented in table  1 .
these samples were collected around utrecht ( the netherlands ) and concerned wild berries from different plants , guano - rich soil , as well as samples from creosote - treated oak railway ties , which might contain both fecal pollution and contamination with aromatic hydrocarbons .
environmental samples were collected with sterile lab tools , placed in plastic bags and plates , then stored at 4c , and processed in the laboratory within 7 to 14  days .
the same locations had previously been sampled for black yeasts , but without enrichment on volatile aromatic hydrocarbons . 

table  1samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbonscodesamplesampling locationgeographic coordinates ( wgs84)a1oak railway tie , outside railsnear station , hollandsche rading521041.95  n , 51045.96  ea2oak railway tie , between railsnear station , hollandsche rading521041.95  n , 51045.96  ea3oak railway tie , outside railsforest area , hilversum521220.68  n , 5119.66  ea4oak railway tie , between railsforest area , hilversum521220.68  n , 5119.66  ea5concrete railway tie , between railsforest area , hilversum521220.68  n , 5119.66  ec1berry , sorbus aucupariaroadside , de bilt52 717.18  n , 5 948.13  ec2berry , sorbus aucuparialight forest , voordaansepad , groenekan52 749.19  n , 5 931.42  ec3berry , sorbus aucuparialight hedge , oostveensepad , maartensdijk52 833.70  n , 5 951.98  ec4berry , sorbus aucuparialapersveld park , hilversum521254.74  n , 51110.25  ec5berry , viburnum opuluslight hedge , oostveensepad , maartensdijk52 833.70  n , 5 951.98  ec6berry , crataegus monogynahedge , vuursche pad , hollandsche rading521010.52  n , 51051.99  ed1guano - rich soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed2guano - rich soil covered with hedera sp.lapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed3guano - tic soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed4fresh goose feceslapersveld park , hilversum521254.74  n , 51110.25  ed5old goose feceslapersveld park , hilversum521254.74  n , 51110.25  e samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbons the solid state - like batch culture technique on a hydrocarbon atmosphere was employed to select for fungi that are able to grow on volatile aromatic hydrocarbons as the sole carbon and energy source .
serum flasks of 100  ml were filled with approximately 25  ml of perlite granules , saturated with mineral medium . each environmental sample ( 13  g )
was washed with phosphate - buffered saline , and the suspension ( 40  ml ) was used to inoculate four different batches ( 10  ml each batch ) . as a control
, these suspensions were also plated on sabouraud s glucose agar ( sga ) amended with antibiotics in order to prevent bacterial growth .
the inoculated flasks were then closed with a cotton  wool plug covered with aluminum foil and placed inside four desiccators where they were exposed , respectively , to a gaseous phase of benzene , toluene , xylene , or naphthalene .
this gas phase was generated by placing 10  ml of a 5% ( v / v ) solution of the aromatic substrate in dibutyl - phthalate .
a solution of 140  g  l of nacl was also added at the bottom of the desiccators to maintain an internal water activity value of 0.9 , and the whole set was incubated at 30c for at least 3  months . after this time
, the perlite granules in each flask were washed with 5060  ml sterile water .
one milliliter of 1- , 10- , or 100-fold dilutions from each soil suspension were plated in duplicate on 2% malt extract agar containing penicillin and streptomycin and incubated at 30c .
colony growth was observed daily , and black yeast - like colonies were transferred to fresh potato dextrose agar plates for purification and provisional identification upon morphological characters .
a sterile blade was used to scrape off the mycelium from the surface of agar plate cultures of previously isolated fungi .
dna was extracted using an ultra clean microbial dna isolation kit ( mobio , carlsbad , ca 92010 , usa ) according to the manufacturer s instructions .
the internal transcribed spacer ( its ) regions and the small subunit ( its1 - 5.8s - its2 ) of the rrna genes were amplified by using the primer set its1 ( 5-tcc gta ggt gaa cct gcg g-3 ) and its4 ( 5-tcc tcc gct tat tga tat gc-3 ) .
pcr reactions were performed on a gene amp pcr system 9700 ( applied biosystems , foster city , ca , usa ) in 50  l volumes containing 25  ng of template dna , 5  l reaction buffer ( 0.1 m tris  hcl , ph  8.0 , 0.5  m kcl , 15  mm mgcl2 , 0.1% gelatine , 1% triton x-100 ) , 0.2  mm of each dntp and 2.0  u taq dna polymerase ( itk diagnostics , leiden , the netherlands ) .
amplification was performed with cycles of 2  min at 94c for primary denaturation , followed by 35  cycles at 94c ( 45  s ) , 52c ( 30  s ) and 72c ( 120  s ) , with a final 7-min extension step at 72c .
amplicons were purified using gfx pcr dna and gel band purification kit ( ge healthcare , ltd . , buckinghamshire , uk ) .
sequence pcr was performed as follows : 95c for 1  min , followed by 30  cycles consisting of 95c for 10  s , 50c for 5  s , and 60c for 2  min .
reactions were purified with sephadex g-50 fine ( ge healthcare bio - sciences ab , uppsala , sweden ) , and sequencing was done on an abi 3730xl automatic sequencer ( applied biosystems ) .
sequence data obtained in this study were adjusted using the seqman of lasergene software ( dnastar inc . , madison , wisconsin , usa ) .
for the phylogenetic assignment , the obtained dna sequences were compared against reference sequences by using the blastn algorithm on local cbs - knaw and public genbank databases ( ncbi , usa ) .
phylogenetic analyses were conducted using mega version 4 ( center for evolutionary functional genomics , the biodesign institute , usa ) .
the list of sampled sites that were used as source of inoculum for the subsequent enrichment cultures is presented in table  1 .
these samples were collected around utrecht ( the netherlands ) and concerned wild berries from different plants , guano - rich soil , as well as samples from creosote - treated oak railway ties , which might contain both fecal pollution and contamination with aromatic hydrocarbons .
environmental samples were collected with sterile lab tools , placed in plastic bags and plates , then stored at 4c , and processed in the laboratory within 7 to 14  days .
the same locations had previously been sampled for black yeasts , but without enrichment on volatile aromatic hydrocarbons . 

table  1samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbonscodesamplesampling locationgeographic coordinates ( wgs84)a1oak railway tie , outside railsnear station , hollandsche rading521041.95  n , 51045.96  ea2oak railway tie , between railsnear station , hollandsche rading521041.95  n , 51045.96  ea3oak railway tie , outside railsforest area , hilversum521220.68  n , 5119.66  ea4oak railway tie , between railsforest area , hilversum521220.68  n , 5119.66  ea5concrete railway tie , between railsforest area , hilversum521220.68  n , 5119.66  ec1berry , sorbus aucupariaroadside , de bilt52 717.18  n , 5 948.13  ec2berry , sorbus aucuparialight forest , voordaansepad , groenekan52 749.19  n , 5 931.42  ec3berry , sorbus aucuparialight hedge , oostveensepad , maartensdijk52 833.70  n , 5 951.98  ec4berry , sorbus aucuparialapersveld park , hilversum521254.74  n , 51110.25  ec5berry , viburnum opuluslight hedge , oostveensepad , maartensdijk52 833.70  n , 5 951.98  ec6berry , crataegus monogynahedge , vuursche pad , hollandsche rading521010.52  n , 51051.99  ed1guano - rich soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed2guano - rich soil covered with hedera sp.lapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed3guano - tic soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed4fresh goose feceslapersveld park , hilversum521254.74  n , 51110.25  ed5old goose feceslapersveld park , hilversum521254.74  n , 51110.25  e samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbons
the solid state - like batch culture technique on a hydrocarbon atmosphere was employed to select for fungi that are able to grow on volatile aromatic hydrocarbons as the sole carbon and energy source .
serum flasks of 100  ml were filled with approximately 25  ml of perlite granules , saturated with mineral medium . each environmental sample ( 13  g ) was washed with phosphate - buffered saline , and the suspension ( 40  ml ) was used to inoculate four different batches ( 10  ml each batch ) . as a control
, these suspensions were also plated on sabouraud s glucose agar ( sga ) amended with antibiotics in order to prevent bacterial growth .
the inoculated flasks were then closed with a cotton  wool plug covered with aluminum foil and placed inside four desiccators where they were exposed , respectively , to a gaseous phase of benzene , toluene , xylene , or naphthalene .
this gas phase was generated by placing 10  ml of a 5% ( v / v ) solution of the aromatic substrate in dibutyl - phthalate .
a solution of 140  g  l of nacl was also added at the bottom of the desiccators to maintain an internal water activity value of 0.9 , and the whole set was incubated at 30c for at least 3  months . after this time ,
one milliliter of 1- , 10- , or 100-fold dilutions from each soil suspension were plated in duplicate on 2% malt extract agar containing penicillin and streptomycin and incubated at 30c .
colony growth was observed daily , and black yeast - like colonies were transferred to fresh potato dextrose agar plates for purification and provisional identification upon morphological characters .
a sterile blade was used to scrape off the mycelium from the surface of agar plate cultures of previously isolated fungi .
dna was extracted using an ultra clean microbial dna isolation kit ( mobio , carlsbad , ca 92010 , usa ) according to the manufacturer s instructions .
the internal transcribed spacer ( its ) regions and the small subunit ( its1 - 5.8s - its2 ) of the rrna genes were amplified by using the primer set its1 ( 5-tcc gta ggt gaa cct gcg g-3 ) and its4 ( 5-tcc tcc gct tat tga tat gc-3 ) .
pcr reactions were performed on a gene amp pcr system 9700 ( applied biosystems , foster city , ca , usa ) in 50  l volumes containing 25  ng of template dna , 5  l reaction buffer ( 0.1 m tris  hcl , ph  8.0 , 0.5  m kcl , 15  mm mgcl2 , 0.1% gelatine , 1% triton x-100 ) , 0.2  mm of each dntp and 2.0  u taq dna polymerase ( itk diagnostics , leiden , the netherlands ) .
amplification was performed with cycles of 2  min at 94c for primary denaturation , followed by 35  cycles at 94c ( 45  s ) , 52c ( 30  s ) and 72c ( 120  s ) , with a final 7-min extension step at 72c .
amplicons were purified using gfx pcr dna and gel band purification kit ( ge healthcare , ltd . , buckinghamshire , uk ) .
sequence pcr was performed as follows : 95c for 1  min , followed by 30  cycles consisting of 95c for 10  s , 50c for 5  s , and 60c for 2  min .
reactions were purified with sephadex g-50 fine ( ge healthcare bio - sciences ab , uppsala , sweden ) , and sequencing was done on an abi 3730xl automatic sequencer ( applied biosystems ) .
sequence data obtained in this study were adjusted using the seqman of lasergene software ( dnastar inc . , madison , wisconsin , usa ) . for the phylogenetic assignment ,
the obtained dna sequences were compared against reference sequences by using the blastn algorithm on local cbs - knaw and public genbank databases ( ncbi , usa ) .
phylogenetic analyses were conducted using mega version 4 ( center for evolutionary functional genomics , the biodesign institute , usa ) .
a total of 71 fungal strains were isolated upon enrichment of environmental samples on atmospheres of different volatile aromatic hydrocarbons ( table  2 ) . of those ,
a single mold ( aspergillus fumigatus ) other than black yeasts was isolated from berries of sorbus aucuparia ( sample c1 ) .
the highest isolation rates were achieved with samples taken from creosoted wooden railway ties , particularly from those arising from the tie section located outside the rails and using toluene as the incubation substrate ( 39 isolates , samples a1 and a3 ) .
these samples were the only ones that yielded isolates when xylene or benzene was applied as enrichment substrates ( 17 and 10 strains , respectively ) , but not a single fungus was isolated when naphthalene was supplied as the sole carbon source .
though in lower numbers , black yeasts were also isolated from the tie section located between the rails ( 14 strains , samples a2 and a4 ) . besides creosote , these samples were likely to contain fecal and mineral oil contamination as well .
table  2number of fungal isolates obtained from solid state - like enrichment cultures incubated at 30c and a water activity of 0.9 , under an atmosphere rich in specific volatile aromatic hydrocarbonssample codebenzenetoluenexylenenaphthalenetotal strainsa10411015a203003a31086024a40110011c105005c207007d206006total strains104317071one isolated strain within this treatment was identified as aspergillus fumigatus ; this was the only non - black yeast strain from the whole obtained strain collection number of fungal isolates obtained from solid state - like enrichment cultures incubated at 30c and a water activity of 0.9 , under an atmosphere rich in specific volatile aromatic hydrocarbons one isolated strain within this treatment was identified as aspergillus fumigatus ; this was the only non - black yeast strain from the whole obtained strain collection from the natural sampled environments , black yeasts were isolated in two out of four berries from s. aucuparia ( 12 isolates , samples c1 and c2 ) , but no fungi were obtained from berry samples of viburnum opulus and crataegus monogyna ( samples c5 and c6 , respectively ) . soil mixed with bird feces ( guano ) also yielded melanized fungi in one out of five tested samples ( six isolates , sample d2 ) .
no black yeasts were isolated when suspensions of the samples described above ( table  1 ) were directly plated onto sga plates , without the enrichment step on volatile aromatic hydrocarbons , while several common and heavily sporulating fungal species were encountered .
phylogenetic analysis of aligned its1 - 5.8s - its2 rrna sequences from the obtained strain collection showed that the isolates were affiliated to five major groups ( fig .  1 ) .
sequences from reference strains deposited at the cbs collection ( utrecht , the netherlands ) that were related to the isolates in terms of their sequence homology and/or ecophysiology were also included in the analysis .
those encompassed the type strains of e. xenobiotica , e. bergeri , exophiala spinifera , e. oligosperma , e. heteromorpha , e. dermatitidis , e. lecanii - corni , and phialophora sessilis , as well as from those of the cladophialophora species that have been so far related to the metabolism of aromatic hydrocarbons : cladophialophora immunda , c. saturnica , and cladophialophora psammophila sp . nov . , the latter species being in the process of description ( badali et al . , submitted )
. strains of e. xenobiotica , e. bergeri , and e. dermatitidis that have previously been isolated from creosoted railway ties were also used . 

figure  1neighbor joining phylogenetic tree ( kimura 2-parameter model ) on aligned its1 - 5.8s - its2 rrna gene sequences from the fungi isolated in this study , in relation to the isolation sample and enrichment substrate .
sequences from relevant reference type strains ( bold characters ) and from other isolates obtained previously from creosoted wood and related environments ( underlined characters ) were also added [ 26 , 27 ] . for phylogenetically unassigned groups , sequences from close genbank matches
were also included in the analysis ( genbank sequence codes are given between square brackets ) neighbor joining phylogenetic tree ( kimura 2-parameter model ) on aligned its1 - 5.8s - its2 rrna gene sequences from the fungi isolated in this study , in relation to the isolation sample and enrichment substrate .
sequences from relevant reference type strains ( bold characters ) and from other isolates obtained previously from creosoted wood and related environments ( underlined characters ) were also added [ 26 , 27 ] . for phylogenetically unassigned groups , sequences from close genbank matches
were also included in the analysis ( genbank sequence codes are given between square brackets ) sequence comparisons against those from reference strains revealed that most of the isolates were similar to exophiala xenobiotica ( 32 strains ) .
the majority was highly homologous ( 99% ) to the ex - type strain , and some were slightly deviating ( 97% homology ) .
a second group of isolates ( nine strains ) was identified as belonging to e. bergeri on the basis of a 99% sequence homology to the ex - type strain .
the cluster encompassing e. bergeri also included a major group ( 24 strains ) , the sequence of which deviated significantly from the ex - type strain ( 94% sequence homology ) , so that its affiliation to e. bergeri might be put into question .
blast searches on this latter group into the genbank genomic database revealed a close match ( 98% homology ) with an unidentified exophiala strain isolated from a rock surface .
a minor e. bergeri sibling group was composed of two strains for which no known sequence match was found .
a third group ( three strains ) did not match any known reference type strain , but genbank searches revealed a high sequence homology ( 98% ) to the sequence of an uncultivated fungus from a municipal composting plant . except for a single isolate ( dh18150 )
obtained from a s. aucuparia berry ( sample c1 ) , the e. xenobiotica - related strains were isolated exclusively from creosoted oak tie sections located outside the rails ( samples a1 and a3 ) .
within this group , three strains were obtained under benzene enrichment , while the remaining 29 strains were isolated with toluene or xylene as enrichment substrates .
besides e. xenobiotica , sample a3 also yielded five isolates upon benzene enrichment , which were identified as e. bergeri .
the related between - the - rails section from that same tie ( sample a4 ) and the guano - rich soil ( sample d2 ) yielded 1 and 3 strains of e. bergeri , respectively , using toluene as the enrichment substrate .
the occurrence of black yeasts in the human - dominated environment has previously been underestimated due to the application of routine microbial isolation methods . under common laboratory culture conditions ,
numerous protocols for the selective isolation of black yeasts have been developed during the last decades ( an overview is presented in table  3 ) , which have yielded new data on the presence of these fungi in a wide range of artificial environments , sometimes with no obvious counterpart in nature .
several methods are based on osmotolerant and oligotrophic abilities of target fungi . with these regimens ,
the isolated black yeasts are mainly osmotolerant members of the saprophytic order dothideales , although the rock - inhabiting fungi appear to be an area of overlap between the dothideales and the chaetothyriales .
applying enrichment methods based on the inoculation of rodents , exposure to aromatic hydrocarbons , assimilation of rare sugars , and/or incubation at high temperature , most isolated black yeast - like fungi are members of the chaetothyriales .
it should be noted though that neither of these enrichment conditions might represent the natural niche for these fungi , and they are therefore classified with difficulty in any of the known ecological categories . 
 table  3overview of selective methods used for environmental isolation of black yeasts and related melanized fungi , with approximate resultsmethodprevalent genusorderecologyreferenceanimal baitexophiala , fonsecaea , cladophialophorachaetothyrialesopportunists[2 , 8]erythritolexophialachaetothyrialesopportunistsmineral oilexophiala , fonsecaeachaetothyrialesopportunistsraulin 40cexophialachaetothyrialesopportunistsneedleconiosporiumchaetothyrialesrock fungialkylbenzene vaporscladophialophora , exophialachaetothyrialesxenobioticsacidicexophialachaetothyrialesacidophileshortaeadothidealescrushconiosporiumchaetothyrialesrock fungihormonemadothidealeslow strengthexophialachaetothyrialesoligotrophssarcinomycesdothidealesrock fungicadophoraleotialesoligotrophshigh saltaureobasidium , hortaeadothidealeshalophiles[31 , 32]suspendcryomyces , friedmanniomycesdothidealespsychrophilesethanolbaudoiniadothidealesethanophiles overview of selective methods used for environmental isolation of black yeasts and related melanized fungi , with approximate results the present study demonstrates that enrichment on inert solid media incubated under a controlled atmosphere rich on volatile aromatic hydrocarbons compounds displays an extraordinary selectiveness toward chaetothyrialian fungi from the herpotrichiellaceae family .
for example , under hydrocarbon atmospheres , the berry samples were free from aureobasidium pullulans , which is a member of the dothideales , otherwise an extremely common component of honeydew mycobiota and slightly osmotic surfaces of fruits and berries .
this species was encountered abundantly along with many contaminants on sga control plates without alkylbenzene enrichment .
the presence of heavily sporulating airborne fungi , frequently found in soil and litter , was strongly reduced by alkylbenzenes and limited to a single strain of a. fumigatus .
interestingly , the dothidiaceous species amorphoteca resinae , known as  creosote fungus  for its common isolation from creosoted wood , was found neither in this study nor in previous fungal isolation surveys from creosoted wood [ 26 , 27 ] .
consistent and relatively abundant isolation of herpotrichiellaceous black yeasts from unpolluted environmental samples , such as wild berries , only after addition of volatile aromatic hydrocarbons strongly suggests that these substances play an essential role in the ecology and competitive ability of those fungi . in particular , the utilization of aromatic compounds as carbon and energy source might explain the key factor determining their success in anthropized environments .
with advanced analytical techniques , it has been shown that volatile aromatic hydrocarbons that traditionally were associated with environmental pollution are in fact ubiquitously present in nature , though at very low concentrations .
toluene , for example , is produced biologically in different natural environments including maturing berries .
on the other hand , as extremophilic and slow - growing microorganisms , incubation on a support that is relatively dry and poor in nutrients is also a determinant factor for the enrichment of black yeasts , when compared to traditional liquid enrichment cultures . regarding both
the presence of aromatic compounds and exposure to fluctuating and/or extreme environmental conditions , wood treated with creosote can be regarded as a paradigmatic environmental niche for black yeasts .
creosote is a distillate derived entirely from tar , which is rich in a wide variety of polycyclic aromatic hydrocarbons , phenols , and cresols , and has widely been used as wood preservative against microbial decay . due to its carcinogenic character
, the use of creosote has been banned in the european union . yet , creosoted wood utilities are still widely present in the open environment , such as telephone poles , railway cross ties , switch ties , and bridge timbers .
also , creosote is an important soil contaminant in former wood creosoting plants . in a previous study ,
e. dermatitidis was isolated from wild berries and massively from creosoted railway ties in thailand when using the pre - incubation in raulin s solution at low ph and incubated at 40c , this protocol being quite selective for e. dermatitidis .
it was hypothesized that the life cycle of this species involved the occurrence on wild berries , ingestion and passage through the human intestinal tract , and feces deposition on railway ties .
subsequent enrichment on creosoted wood might then be related not only to the oligotrophy , thermotolerance ( particularly under tropical conditions ) , acidotolerance , and moderate osmotolerance of e. dermatitidis but also to the presence of aromatic compounds .
the temperate wild berry sampling locations that were the subject of our study ( table  1 ) had previously been analyzed for the occurrence of black yeasts [ 18 , 26 ] .
collected berries were homogenized , diluted , and used for spread plates or were subjected to the raulin s pre - incubation protocol . in these earlier studies involving several hundreds of samples , only a single strain of e. dermatitidis
the lack of this fungus in temperate climate conditions suggests that e. dermatitidis has an origin in the tropics .
this hypothesis is further supported by the isolation of e. dermatitidis from creosoted railway ties in brazil , using the mineral oil - flotation protocol [ 15 , 24 ] . from the present enrichment program on volatile alkylbenzenes
, it appears that exophiala species , though other than the thermophilic e. dermatitidis , are regularly present on substrates that are characteristic from the latter species ( berries , bird feces , and creosoted railway ties ) .
it is known to be associated with mild cutaneous infections in humans   and is also commonly found in habitats rich in xenobiotics such as hydrocarbon - polluted soil and gas biofilters for the biodegradation of btex compounds [ 6 , 24 , 26 ] .
, it was regarded to be an extremely rare opportunist , although a recent study showed that it was underdiagnosed due to a general lack of application of sequencing for identification . using the selective oil - flotation method ,
e. bergeri was isolated from samples of wood treated or non - treated with creosote preservatives .
a distinct cluster was identified within the e. bergeri group , which might belong to a yet - undescribed species .
strains from this group were predominantly isolated from the section between the rails of sampled wooden ties , which were partly contaminated by machine oil and human feces .
the remaining strains of e. xenobiotica and e. bergeri senso stricto were obtained from the section outside the rails , where this contaminating effect was less marked .
three identical strains were isolated from guano - rich soil covered with hedera by enrichment under a toluene atmosphere .
no close sequence homology against reference strains was found for these fungi , indicating that a yet - undescribed species is concerned .
the phylogenetic affiliation of the undescribed strains will be reported in detail in a subsequent study .
the extremophilic nature of black yeast , in combination with their capacity to metabolize aromatic pollutants , makes them ideal candidates for specific bioremediation purposes .
prospects on the biotechnological application of these fungi until recently have been hampered by potential biohazard , but , as phylogenetic data on these organisms accumulates , it seems that severe pathogens and hydrocarbon - associated species are consistently well - separated siblings ( de hoog , unpublished ) .
indeed , enrichment on volatile aromatic hydrocarbons in gas biofilters and similar systems thus far has never yielded pathogenic species of the higher fungal biohazard risk categories , such as cladophialophora bantiana or e. dermatitidis .
this suggests that a certain degree of speciation between pathogenic and aromatic hydrocarbon metabolizing species has occurred in evolutionary terms . yet
, more studies are needed to emphasize the importance of selective methods to isolate black yeasts and acquire more understanding of their ecological niches .
the application of the enrichment method used in the present paper contributes to the isolation of fungi that have a potential for bioremediation of sites polluted with monoaromatic hydrocarbons .
 black yeast members of the herpotrichiellaceae present a complex ecological behavior : they are often isolated from rather extreme environments polluted with aromatic hydrocarbons , while they are also regularly involved in human opportunistic infections . 
 a selective technique to promote the in vitro growth of herpotrichiellaceous fungi was applied to investigate their ecophysiology . 
 samples from natural ecological niches and man - made environments that might contain black yeasts were enriched on an inert solid support at low humidity and under a controlled atmosphere rich in volatile aromatic hydrocarbons . 
 benzene , toluene , and xylene were provided separately as the sole carbon and energy source via the gas phase . 
 the assayed isolation protocol was highly specific toward mesophilic exophiala species ( 70 strains of this genus out of 71 isolates ) . 
 those were obtained predominantly from creosote - treated railway ties ( 53 strains ) , but isolates were also found on wild berries ( 11 strains ) and in guano - rich soil samples ( six strains ) . 
 most of the isolates were obtained on toluene ( 43 strains ) , but enrichments on xylene and benzene also yielded herpotrichiellaceous fungi ( 17 and 10 isolates , respectively ) . based upon morphological characterizations and dna sequences of the full internal transcriber spacers ( its ) and the 8.5s rrna genes , the majority of the obtained isolates were affiliated to the recently described species exophiala xenobiotica ( 32 strains ) and exophiala bergeri ( nine strains ) . 
 members of two other phylogenetic groups ( 24 and two strains , respectively ) somewhat related to e. bergeri were also found , and a last group ( three strains ) corresponded to an undescribed exophiala species . 
Introduction
Materials and Methods
Sampling
Isolation
Molecular Identification
Results
Discussion