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Identification of pathogenic dematiaceous fungi and related taxa based on large subunit ribosomal DNA D1/D2 domain sequence analysis

Paride Abliz, Kazutaka Fukushima, Kayoko Takizawa, Kazuko Nishimura
DOI: http://dx.doi.org/10.1016/S0928-8244(03)00275-X 41-49 First published online: 1 January 2004


The nucleotide sequences of the D1/D2 domains of large subunit (26S) ribosomal DNA for 76 strains of 46 species of pathogenic dematiaceous fungi and related taxa were determined. Intra-species sequence diversity of medically important dematiaceous fungi including Phialophora verrucosa, Fonsecaea pedrosoi, Fonsecaea compacta, Cladophialophora carrionii, Cladophialophora bantiana, Exophiala dermatitidis, Exophiala jeanselmei, Exophiala spinifera, Exophiala moniliae, and Hortaea werneckii were extremely small; as few as 0 changes were detected in C. bantiana, Fonsecaea and Exophiala species, 1 bp in C. carrionii and H. werneckii, and 2 bp in P. verrucosa. Inter-species nucleotide diversity between most species was higher. These data suggested that the D1/D2 domain is sufficiently variable for identification of pathogenic dematiaceous fungi and relevant species. The phylogenetic trees constructed from the sequence data revealed that most human pathogenic species formed a single cluster and that Cladosporium and Phialophora species were distributed polyphyletically into several clusters.

  • Identification
  • Pathogenic dematiaceous fungus
  • Large subunit ribosomal DNA
  • D1/D2 domains

1 Introduction

Dematiaceous fungi are usually defined as having melanin or melanin-like pigment in the wall of their hyphae and/or spores [1]. They are widely distributed in nature. The major infections caused by dematiaceous human pathogens are classified into two groups of disease: chromoblastomycosis and pheohyphomycosis [2]. Chromoblastomycosis is a chronic infection of cutaneous and subcutaneous tissues. Verrucose lesions and round, brown, and thick-walled muriform cells (sclerotic bodies) in tissues are characteristics of this infection [2,3]. The principal etiologic agents of chromoblastomycosis are Fonsecaea pedrosoi, Fonsecaea compacta, Cladophialophora carrionii, Phialophora verrucosa, and Rhinocladiella aquaspersa[1]. Pheohyphomycosis is a primary or opportunistic infection that ranges from the superficial tissue to deep organs. The etiologic agents are present in host tissues with melanized yeast-like cells, pseudohyphae-like elements, hyphae or any combination of these forms [2]. In recent literature, 59 species of 28 genera and three classes were described as the etiologic agents of pheohyphomycosis [1]. The numbers of case reports of infections with dematiaceous fungi have increased [412]. Outcomes of antifungal therapies for these infections have remained poor. A high mortality rate (79%) was reported in disseminated pheohyphomycosis patients even with antifungal therapy [13]. Identification of pathogenic dematiaceous fungi is typically done by morphological and physiological procedures [1417] however, these procedures are time-consuming, require technical expertise, and are ineffective for identification of species with poor conidia production and a wide diversity in anamorphic life cycles [18].

Genetic methods have high sensitivity and specificity for identifying microorganisms. The D1/D2 domains of the large subunit ribosomal DNA (LSUrDNA) have been reported to be useful for identification of most ascomycetous yeasts [19,20] and medically important zygomycetes [21]. Thus, the sequences of the D1/D2 domains could serve as reliable and practical criteria for identification of most known yeasts. However, no research on the D1/D2 domains of dematiaceous fungi has been done. The objective of the present study was to investigate the efficacy of these domains for identification of medically important dematiaceous fungi. We analyzed D1/D2 domain sequences for 76 strains of 46 species of fungi and related species. The sequence data were then used to study the phylogenetic relation among these organisms, and between the genus Phialophora and pathogenic Chaetothyriales.

2 Materials and methods

2.1 Fungi

The 76 strains of 46 species of pathogenic dematiaceous fungi and related taxa analyzed in the present study are listed in Table 1.

View this table:
Table 1

Strains examined

SpeciesStrainSourceNumber of intra-species nucleotide differencesSequence length (bp)Accession number
Alternaria alternataIFM 41348TIMM 1289614AB100676
Aureobasidium pullulansIFM 4802ATCC 15233614AB104687
Capronia hanlinianaIFM 52023CBS 588.93603AB100681
Cladophialophora carrioniiIFM 4808TATCC 16264613AB100642
IFM 4805ATCC 445350613AB100640
IFM 4810DCU 3001613AB100643
IFM 4812DCU 3020613AB100641
IFM 41446DCU 6061613AB100644
IFM 41641BMU 2371613AB100645
Cladophialopora bantianaIFM 46164CBS 364.80613AB100616
IFM 41433DCU 6070613AB104686
Cladophialophora devriesiiIFM 51369TCBS 147.84613AB100646
Cladophialophora minouraeIFM 4818DCU 428613AB100647
Cladophialophora arxiiIFM 52022TCBS 306.94613AB100683
Cladophialophora boppiiIFM 52024TCBS 126.86612AB100684
Cladophialophora emmonsiiIFM 52025TCBS 979.96613AB100682
Cladosporium cladosporioidesIFM 41447IFO 6368608AB100650
Cladosporium colocasiaeIFM 51371CBS 386.64608AB100649
Cladosporium coralloidesIFM 41451IFO 6536608AB100658
Cladosporium elatumIFM 41452IFO 6372614AB100652
Cladosporium fulvumIFM 40703IAM 5006608AB100653
Cladosporium herbarumIFM 41454TMI614AB100651
Cladosporium minusculumIFM 51370URM 721608AB100648
Cladosporium sphaerospermumIFM 41453IFO 4458608AB100654
Cladosporium variabileIFM 41458IFO 6378608AB100655
Exophiala alcalophilaIFM 4823TIAM 12519616AB100672
Exophiala dermatitidisIFM 41479TCBS 207.35616AB100659
IFM 41818Venezuela0616AB100660
IFM 41828soil, Brazil0616AB100661
IFM 45986tap water0616AB100662
Exophiala jeanselmeiIFM 4852TNCMH 123618AB100664
IFM 41691BMU 27560618AB100663
FM 45989patient0618AB100665
IFM 4974bathroom drainpipe0618AB100666
Exophiala moniliaeIFM 41500TCBS 520.76618AB100667
IFM 41832Venezuela0618AB100668
Exophiala spiniferaIFM 4883TATCC 182180618AB100673
IFM 41504CBS 670.760618AB100679
IFM 41505patient0618AB100680
Fonsecaea pedrosoiIFM 4887TCBS 271.37613AB100632
IFM 4856DCU 6770613AB100631
IFM 4889ATCC 443560613AB100633
IFM 4914Venezuela0613AB100634
IFM 41705bark, China0613AB100635
IFM 46410soil, Brazil0613AB100636
Fonsecaea compactaIFM 4886KUM 9110613AB100637
IFM 41704BMU 48450613AB100638
IFM 41931MTU0613AB100639
Hormoconis resinaeIFM 51372IFO 8588615AB100657
Hortaea werneckiiIFM 4885TCBS 107.67602AB079584
IFM 51373URM 7041602AB100674
IFM 41538patient0602AB079586
IFM 41541patient1602AB079588
Lecythophora hoffmanniiIFM 4922CBS 245.38602AB100627
Lecythophora mutabilisIFM 4923ATCC 26223602AB100628
Phialophora verrucosaIFM 4928ATCC 38561613AB100610
IFM 41710corn, China0613AB100611
IFM 41871soil, Colombia0613AB100612
IFM 41873Venezuela2613AB100613
IFM 41879soil, Colombia0613AB100614
IFM 41898soil, Brazil0613AB100615
Phialophora albaIFM 51363IFO 31973615AB100618
Phialophora americanaIFM 51361CBS 273.37613AB100616
Phialophora atrovirensIFM 51364IFO 6793615AB100617
Phialophora bubakiiIFM 51365IFO 6794615AB100620
Phialophora cinerescensIFM 51366IFO 6849615AB100621
Phialophora fastigiataIFM 41577IFO 6856616AB100625
Phialophora heteromorphaIFM 41578IFO 6878615AB100626
Phialophora lagerbergiiIFM 51367IFO 8576615AB100622
Phialophora meliniiIFM 51362CBS 268.33615AB100617
Phialophora oxysporaIFM 51368URM 2904616AB100630
Phialophora repensIFM 4925CBS 423.73602AB100623
Phialophora richardsiaeIFM 4926KUM 1681602AB100624
Phaeoacremonium parasiticumIFM 4924KUM 1827602AB100629
Rhinocladiella aquaspersaIFM 4930CBS 313.73617AB100677
Rhinocladiella atrovirensIFM 4931TCBS 317.33617AB100678
  • TType strain.

  • Used for matrix study.

  • Used for phylogenetic study.

  • ATCC, American Type Culture Collection, Rockville, MD, USA; BMU, Department of Dermatology, Beijing Medical University, Beijing, China; CBS, Central bureau voor Schimmelcultures, Baarn, The Netherlands; CUH, Department of Laboratory Medicine, School of Medicine, Chiba University, Chiba, Japan; DCU, Department of Dermatology, School of Medicine, Chiba University, Chiba, Japan; IAM, Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan; IFM, Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba, Japan; IFO, Institute for Fermentation, Osaka, Japan; KUM, Department of Dermatology, School of Medicine, Kanazawa University, Kanazawa, Ishikawa, Japan; MTU, Department of Bacteriology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; NCMH, The North Carolina Memorial Hospital, University of North Carolina, Chapel Hill, NC, USA; TIMM, Research Center for Medical Mycology, Teikyo University, Tokyo, Japan; TMI, Tottori Mycological Institute, Tottori, Japan; UNEFM, Universidade Nacional Experimental Francisco de Miranda, Coro, Falcon, Venezuela; URM; Department of Mycology, Federal University of Pernambuco, Recife, PE, Brazil.

2.2 DNA extraction

DNAs were prepared as described previously [22]. Briefly, approximately 50 mg of fungal elements were suspended in 600 µl extraction buffer (200 mM Tris–HCl, pH 7.5, 25 mM EDTA, 0.5% w/v sodium dodecyl sulfate, 250 mM NaCl). The mixture was vortexed for 15 s, incubated at 100°C for 15 min, kept on ice for 60 min, and then centrifuged at 14,000×g for 15 min. Supernatants were transferred to new tubes and extracted with phenol–chloroform–isoamyl alcohol (25:24:1 v/v). Each sample DNA was precipitated with cold isopropanol (−20°C), dried, and resuspended in 100 µl distilled water.

2.3 Amplification and sequencing of D1/D2 domains

The D1/D2 domains of the LSUrDNA were amplified with primers NL-1, 5′-GCATATCAATAAGCGGAGGAAAAG-3′ and NL-4m, 5′-GGTCCGTGTTTCAAGACG-3′[23]. Polymerase chain reaction (PCR) was carried out in 50 µl reactions containing 5 µl of template DNA, 5 µl (2 pmol) each primer, 4 µl (2.5 mM) dNTP mixture (Nippon Gene, Tokyo, Japan), 0.25 µl (5 U µl−1) Taq polymerase (Nippon Gene), and 5 µl 10×reaction buffer (Nippon Gene). Amplification was performed with a PCR Thermal Cycler MP (TaKaRa Shuzo, Tokyo, Japan) under the following conditions: 1 cycle of 95°C for 4 min followed by 30 cycles of 94°C for 1 min, 55°C for 2.5 min, and 72°C for 2.5 min, with a final extension at 72°C for 10 min. The amplified products were purified with SUPREC™-02 (TaKaRa) and subjected to direct sequencing with an ABI Prism 3100 sequencer after labeling with BigDye™ Terminator Cycle Sequencing Ready Reaction (Applied Biosystems, Foster City, CA, USA). The external primers, NL-1 and NL-4m, and the internal primers, NL-2m, 5′-CTTGTGCGCTATCGGTCTC-3′ and NL-3m, 5′-GAGACCGATAGCGCACAAG-3′, were used to sequence each DNA sample.

The sequence data were aligned with CLUSTAL W (version 1.6) [24]. Phylogenetic trees were constructed with the neighbor-joining (NJ) method. The nucleotide sequences for all strains examined were registered in the DNA Data Bank of Japan (DDBJ) under the accession numbers shown in Table 1.

3 Results

The length of the nucleotide sequence for each strain is summarized in Table 1. They ranged from 602 bp to 618 bp. For 10 medically important species, C. carrionii, C. bantiana, E. dermatitidis, E. jeanselmei, E. moniliae, E. spinifera, F. pedrosoi, F. compacta, H. werneckii, and P. verrucosa, multiple strains were analyzed to investigate differences in sequence length and intra-species nucleotide substitutions. Variations in sequence length were not observed in any of the 10 species. These data are summarized in Table 1. To detect nucleotide differences, type strains were selected as the reference species for seven species. For the three remaining species, P. verrucosa, F. compacta, and C. bantiana, the type strains were unavailable or difficult to obtain; therefore, strains IFM 4928, IFM 4886, and IFM 46164 were used as reference strains, respectively. Intra-species nucleotide variation was not detected in C. bantiana, E. dermatitidis, E. jeanselmei, E. moniliae, E. spinifera, F. pedrosoi, and F. compacta. Single nucleotide substitutions were found in C. carrionii and H. werneckii, and changes at two nucleotides were found in P. verrucosa. Therefore, the sequences of D1/D2 domains of the medically important species examined in this study were highly conserved.

A matrix of the nucleotide differences between Cladophialophora (C.) and Cladosporium (Cl.) species is shown in Table 2. The number of nucleotide differences among members of Cladophialophora species ranged from five between C. devriesii, C. minourae, and C. arxii to 26 between C. devriesii, C. minourae and C. bantiana. These data supported the sequences of D1/D2 domains is useful for identification of Cladophialophora species. Cladosporium species showed differences ranging from zero to 134 nucleotides. Cl. coralloides and Cl. cladosporioides had identical sequences, and Cl. fulvum had two nucleotide differences from each of Cl. coralloides, Cl. cladosporioides, and Cl. colocasiae. Cl. colocasiae showed three nucleotide differences from each of Cl. coralloides and Cl. cladosporioides. For the species in which the number of nucleotide differences was less than three, the sequences of D1/D2 domains are considered to be not sufficient criteria to identify confidently each species. However, the other species can be identified by the sequence of the domains.

View this table:
Table 2

Matrix of nucleotide differences in D1/D2 domains of LSUrDNA between Cladophialophora (C.) and Cladosporium (Cl.) species

SpeciesC. carrioniiC. bantianaC. devriesiiC. minouraeC. arxiiC. boppiiC. emmonsiiCl. herbarumCl. fulvumCl. coralloidesCl. cladosporioidesCl. colocasiaeCl. variabileCl. sphaerospermumCl. minusculum
C. bantiana8
C. devriesii2126
C. minourae22268
C. arxii212355
C. boppii1212182219
C. emmonsii252112121124
Cl. herbarum102102100100989895
Cl. fulvum130132125120119117121118
Cl. coralloides1291311211201201171211182
Cl. cladosporioides12812911711911911812111120
Cl. colocasiae128129122119115119100118233
Cl. variabile127128121120118120119114113131315
Cl. sphaerospermum1261271181191171151181151121011135
Cl. minusculum12612711811911711611511411110111311010
Cl. elatum13413313010712612712512810811311114113112111

Nucleotide differences among Phialophora species are shown as a matrix in Table 3. P. verrucosa is the type species of the genus Phialophora and is a human pathogen. P. verrucosa is morphologically and physiologically similar to P. americana, and these species are not clearly separated in the medical literature. The two species differed at five nucleotide positions, whereas they showed a large number of differences from 11 saprophytic and rare pathogenic species of this genus. In the genus Phialophora, P. lagerbergii, P. bubakii, P. atrovirens, and P. heteromorpha had identical sequences, and the sequence of P. melinii differed at only one position. P. cinerescens had four or five differences from the five species listed above. Other species, such as P. richardsiae, P. repens, P. fastigiata, and P. oxyspora, were found to have characteristic sequences that differed from each other with high numbers of nucleotide substitutions, averaging 100. With the exception of the five species with identical sequences or single nucleotide variations, the sequence of the D1/D2 domains is a useful tool for identification of both pathogenic and saprophytic species of genus Phialophora.

View this table:
Table 3

Matrix of nucleotide differences in D1/D2 domains of LSUrDNA of Phialophora species

SpeciesP. verrucosaP. americanaP. albaP. cinerescensP. meliniiP. lagerbergiiP. bubakiiP. atrovirensP. heteromorphaP. richardsiaeP. repensP. fastigiata
P. americana5
P. alba9594
P. cinerescens969542
P. melinii9594414
P. lagerbergii96954051
P. bubakii969840510
P. atrovirens9793414100
P. heteromorpha97974051000
P. richardsiae121115110110114114114114114
P. repens12212311311311711711711711747
P. fastigiata3136102939495959495131122
P. oxyspora63629795949393929313012548

A matrix of the nucleotide differences for 10 medically important species was generated to evaluate the usefulness of these sequences for identification (Table 4). Data were obtained by comparison of D1/D2 domain sequences for the type strains of seven pathogenic species. As described earlier, IFM 4886, IFM 46164, and IFM 4928 were used as reference strains for F. compacta, C. bantiana and P. verrucosa, respectively. F. pedrosoi and its dysplastic variant, F. compacta, were found to have identical sequences as predicted. The smallest difference, three nucleotides, was found between C. bantiana and P. verrucosa, and small differences of six nucleotides were observed both between C. bantiana and C. carrionii and between E. spinifera and E. jeanselmei. The largest difference, 130 nucleotides, was observed between C. bantiana and H. werneckii. In general, the number of differences was distributed in the range of 20–30 nucleotides. These results suggest that the sequences of the D1/D2 domains might be useful for identification of these pathogenic dematiaceous species except for discrimination between C. bantiana and P. verrucosa. We also compared the data for H. werneckii, the causative agent of tinea nigra, and Exophiala species because H. werneckii was classified into the genus Exophiala prior to 1984. As shown in Table 4, there were more than 120 differences between H. werneckii and all five species of Exophiala. This strongly supports the validity of establishing Hortaea as an independent genus.

View this table:
Table 4

Matrix of nucleotide differences in D1/D2 domains of LSUrDNA among medically important dematiaceous fungi

SpeciesC. bantianaC. carrioniiE. dermatitidisE. jeanselmeiE. moniliaeE. spiniferaF. pedrosoiH. werneckii
C. carrionii6
E. dermatitidis3538
E. jeanselmei323113
E. moniliae32321512
E. spinifera303115611
F. pedrosoi251925172621
F. compacta2519251726210
H. werneckii130128123128129127122
P. verrucosa383732343124126

The phylogenetic trees constructed by the NJ method for Chaetothyriales and for all examined species are shown in Figs. 1 and 2, respectively. Aspergillus fumigatus was used as an outgroup. The tree of Fig. 1 shows that almost all human pathogens, including F. pedrosoi, F. compacta, P. verrucosa, P. americana, all species of Cladophialophora and Exophiala form one cluster, and also that Exophiala species are located as a monophyletic cluster separated from human pathogens described above. Polyphyletic characteristics in Phialophora species were inferred from their cluster formations; P. verrucosa and P. americana were closely related to species of the genera Fonsecaea and Cladophialophora, P. fastigiata clustered with the genus Exophiala, and P. oxyspora formed a single-membered cluster independently. Of the 13 species examined, the nine species except the four mentioned above formed a subcluster with comparatively remote distance from other species. The tree in Fig. 2 was constructed for all species examined to study phylogenetic relationships among them. The human pathogenic species analyzed in the present study are classified into the following four orders and five families of the class Euascomycetes: Chaetothyriales Herpotrichiellaceae [A], Dothideales Dothioraceae [B-1], Dothideales Mycosphaerellaceae [B-2], Sordariales Coniochaetaceae [C], and Pleosporales Pleosporaceae [D]. In Fig. 2, Chaetothyriales [A] show their phylogenetically distant relationship from the other orders Dothidiales [B-1, 2], Sordariales [C], and Pleosporales [D]. The results for Chaetothyriales in Fig. 1 were principally not affected by adding other examined species. P. richardsiae and P. repens were closely related to Phaeoacremonium and Lecythophora species of order Sordariales. The species in the order Dothideales, including Aureobasidium pullulans, H. werneckii and Cladosporium species, formed one cluster with Alternaria alternata of the order Pleosporales. Cl. herbarum was more distantly related to other Cladosporium species, with lower sequence homology.

Figure 1

NJ tree for D1/D2 domains of the genus Phialophora and pathogenic Chaetothyriales. Bootstrap values derived from 10,000 replicates are shown as percentages. The scale bar represents a difference corresponding to 0.02 (2%). For full genus and species names see Table 1.

Figure 2

NJ tree for D1/D2 domains of pathogenic dematiaceous fungi and related taxa. Bootstrap values derived from 10,000 replicates are shown as percentages. The scale bar represents a difference corresponding to 0.02 (2%). For full genus and species names see Table 1. A: Chaetothyriales Herpotrichiellaceae, B-1: Dothideales Dothioraceae, B-2: Dothideales Mycosphaerellaceae, C: Sordariales Coniochaetaceae, D: Pleosporales Pleosporaceae.

4 Discussion

Dematiaceous fungi, including medically important species, have been typically identified by morphological and physiological characteristics. Such methods are laborious and sometimes cannot distinguish such species with polymorphic conidiogeneses or lacking conidia formation. Genetic methods such as RAPD (random amplified polymorphic DNA) and RFLP (restriction fragment length polymorphism) have been used to identify medically important dematiaceous species [2529]; however, these methods are considered to be more appropriate for taxonomy, typing, and epidemiological investigation of fungi than identification. Recently, sequences of the internal transcribed spacer region and D1/D2 domains of rDNA have been used for identification purposes due to the higher accuracy and objectivity of such methods. Since sequence data for the D1/D2 domains of dematiaceous fungal taxa have not been reported, the present study aimed to collect the data and then to evaluate them as a criterion for identification of dematiaceous fungi, especially medical pathogens.

We examined multiple strains for each medically important species. High intra-species conservation of nucleotide sequences of the D1/D2 domains was observed in 10 pathogenic species. No intra-species nucleotide substitutions were detected in seven species described previously, and the largest number of substitutions was only two in a strain of P. verrucosa. On the other hand, high nucleotide substitutions were shown between species. F. pedrosoi and F. compacta have identical D1/D2 sequences. The two species are morphologically and physiologically similar [30,31]. RAPD and RFLP methods have been used to investigate genetic variations between these species [26,32]; however, variations were not found. It is possible that F. compacta is not an independent species but a variant of F. pedrosoi. P. verrucosa and C. bantiana have only three nucleotide substitutions and the nucleotide diversity is less than 0.5%. However, the two species have large diversity in their morphology and pathogenicity. The former is a causative agent of chromoblastomycosis, whereas the latter one is associated with cerebral pheohyphomycosis [1,15]. This is an example that morphological characteristics may have a higher contribution to discriminating two species than any genetic evidence including the D1/D2 domain sequence.

Of some saprophytic and pathogenic species of the genera Phialophora and Cladosporium, several species were found to have identical or highly homologous sequences with substitutions at only one or two positions. For such species, the nucleotide sequences are incapable of discriminating each species, and it is expected to find any genetic region with greater nucleotide variation. Four Phialophora species, P. lagerbergii, P. bubakii, P. atrovirens, and P. heteromorpha, have identical sequences in D1/D2. These data may become new evidence to discuss the necessity of their re-identification. Except for the species mentioned above, other species could be discriminated from each other by the sequence of D1/D2 domains. Especially for medically important species, except P. verrucosa and C. bantiana, the domain is applicable for identification of all species.

The tree (Fig. 1) of the species of genus Phialophora and pathogenic Chaetothyriales demonstrated the phylogenetically characteristic relationships among them: the close phylogenetic distances of human pathogens of Fonsecaea species, all Cladophialophora species, and the two species of P. verrucosa and P. americana cluster formation in all Exophiala species examined and the phylogenetic diversity of the genus Phialophora.

In the tree (Fig. 2) for all examined species, the phylogenetic results for the genus Phialophora and Chaetothyriales discussed above are principally not changed by adding other taxa. As a new finding, Rhinocladiella species are demonstrated to have a close interrelation to the genus Exophiala. The tree also includes some interesting information on phylogenetic distances among dematiaceous fungal taxa; the genus Phialophora is a polyphyletic taxon; the genus Cladosporium has a completely remote distance from the genus Cladophialophora, and should be polyphyletic; although much higher numbers of tested fungi need to be analyzed, the genera Lecythophora [C], Phaeoacremonium [C] and Alternaria [D] are supported to be accompanied in different taxa from [A] and [B]; Au. pullulans and H. werneckii, weak pathogens, might form an independent cluster in taxon [B-1]. The phylogenetic relationship of pathogenic dematiaceous fungi was defined from the trees constructed using the D1/D2 domain sequences.

In conclusion, the sequences of the D1/D2 domains were evaluated to be applicable for identification of many taxa of dematiaceous fungi, especially of human pathogenic species. On the other hand, for some pathogenic or saprophytic Phialophora and Cladosporium species which have identical sequences or an inter-species sequence diversity of less than 0.5%, the domain is not useful for their identification. For these species, morphological characteristics may be used as the prevailing criteria, because no other genetic region having a higher ability to discriminate species than D1/D2 domain has been found to date.


This study was performed as part of the program ‘Frontier Studies and International Networking of Genetic Resources in Pathogenic Fungi and Actinomycetes (FN-GRPF)’ through Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government 2003.


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View Abstract