Polyphasic taxonomy of Aspergillus section Usti

Abstract

Aspergillus ustus is a very common species in foods, soil and indoor environments. Based on chemical, molecular and morphological data, A. insuetus is separated from A. ustus and revived. A. insuetus differs from A. ustus in producing drimans and ophiobolin G and H and not producing ustic acid and austocystins. The molecular, physiological and morphological data also indicated that another species, A. keveii sp. nov. is closely related but distinct from A. insuetus. Aspergillus section Usti sensu stricto includes 8 species: A. ustus, A. puniceus, A. granulosus, A. pseudodeflectus, A. calidoustus, A. insuetus and A. keveii together with Emericella heterothallica.

INTRODUCTION

Aspergillus ustus is a very common filamentous fungus found in foods, soil and indoor air environments (Samson et al. 2002). This species is considered as a rare human pathogen that can cause invasive infection in immunocompromised hosts. However, A. ustus has been noted increasingly as causes of invasive aspergillosis in tertiary care centres in the US (Malani & Kaufman 2007). Up to date, 22 invasive aspergillosis cases have been reported to be caused by A. ustus (Verweij et al. 1999; Pavie et al. 2005; Panackal et al. 2006; Yildiran et al. 2006). Several studies indicate that A. ustus isolates are resistant to amphotericin B, echinocandins and azole derivatives (Verweij et al. 1999; Pavie et al. 2005; Gene et al. 2001; Garcia-Martos et al. 2005). Other species related to A. ustus can also cause human or animal infections. Aspergillus granulosus was found to cause disseminated infection in a cardiac transplant patient (Fakih et al. 1995), while A. deflectus has been reported to cause disseminated mycosis in dogs (Robinson et al. 2000; Kahler et al. 1990; Jang et al. 1986).

A. ustus is a variable species. Raper & Fennell (1965) stated that “not a single strain can be cited as wholly representative of the species as described”. Indeed, A. ustus isolates may vary in their colony colour from mud brown to slate grey, with colony reverse colours from uncoloured through yellow to dark brown (Raper & Fennell 1965; Kozakiewicz 1989). Molecular data also indicate that this species is highly variable; RAPD analysis carried out in various laboratories could be used to detect clustering of the isolates (Rath et al. 2002; Panackal et al. 2006), and sequence analysis of parts of the ribosomal RNA gene cluster also detected variability within this species (Henry et al. 2000; Peterson 2000; Hinrikson et al. 2005).

We examined a large set of A. ustus isolates and related species originating from environmental and clinical sources to clarify the taxonomic status of the species, and to clarify the taxonomy of Aspergillus section Usti. The methods used include sequence analysis of the ITS region (intergenic spacer region and the 5.8 S rRNA gene of the rRNA gene cluster), and parts of the β-tubulin, calmodulin and actin genes, analysis of extrolite profiles, and macro- and micromorphological analysis of the isolates.

MATERIALS AND METHODS

Morphological examination. The strains examined are listed in Table 1. Both clinical and environmental strains were grown as 3-point inoculations on Czapek yeast agar (CYA), malt extract agar (MEA), creatine agar (CREA) and yeast extract sucrose agar (YES) at 25 °C, and on CYA at 37 °C for 7 d (medium compositions according to Samson et al. 2004). For micro morphological examination light microscopy (Olympus BH2 and Zeiss Axioskop 2 Plus) was employed.

Table 1.

Isolates in Aspergillus section Usti and related species examined in this study.

Species	Strain No.	Source
A. calidoustus	CBS 112452	Indoor air, Germany
A. calidoustus	CBS 113228	ATCC 38849; IBT 13091
A. calidoustus	CBS 114380	Wooden construction material, Finland
A. calidoustus	CBS 121601T	Bronchoalveolar lavage fluid, proven invasive aspergillosis, Nijmegen, The Netherlands†
A. calidoustus	CBS 121602	Bronchial secretion, proven invasive aspergillosis, Nijmegen, The Netherlands†
A. calidoustus	CBS 121589	Autopsy lung tissue sample, proven invasive aspergillosis, Nijmegen, The Netherlands†
A. calidoustus	CBS 121603	Elevator shaft in hospital, Nijmegen, The Netherlands
A. calidoustus	CBS 121604	Patient room, Nijmegen, The Netherlands
A. calidoustus	CBS 121605	Laboratory, Nijmegen, The Netherlands
A. calidoustus	CBS 121606	Sputum, Nijmegen, The Netherlands
A. calidoustus	CBS 121607	Feces, Nijmegen, The Netherlands
A. calidoustus	CBS 121608	Bronchoalveolar lavage, Nijmegen, The Netherlands
A. calidoustus	7843	Pasteur Institute, Paris, France
A. calidoustus	8623	Oslo, Norway
A. calidoustus	9331	Mouth wash, Nijmegen, The Netherlands
A. calidoustus	9371	Mouth wash, Nijmegen, The Netherlands
A. calidoustus	9420	Bronchial secretion, Nijmegen, The Netherlands
A. calidoustus	9692	Hospital ward, Nijmegen, The Netherlands
A. calidoustus	V02-46	Tongue swab, Nijmegen, The Netherlands
A. calidoustus	V07-21	Bronchial secretion, Nijmegen, The Netherlands
A. calidoustus	V17-43	Bronchial secretion, Nijmegen, The Netherlands
A. calidoustus	V22-60	Skin biopsy, Nijmegen, The Netherlands
A. calidoustus	CBS 121609	Post-cataract surgery endophthalmitis, Turkey
A. calidoustus	907	Post-cataract surgery endophthalmitis, Turkey
A. calidoustus	908	Post-cataract surgery endophthalmitis, Turkey
A. calidoustus	64	Post-cataract surgery endophthalmitis, Turkey
A. calidoustus	67	Post-cataract surgery endophthalmitis, Turkey
A. calidoustus	CBS 121610	Post-cataract surgery endophthalmitis, Turkey
A. calidoustus	351	Osteorickets
A. calidoustus	482	Post-cataract surgery endophthalmitis
A. calidoustus	CBS 121611	Patient 4, Washington, U.S.A.
A. calidoustus	CBS 121616	Environmental, Washington, U.S.A.
A. calidoustus	FH 165	Patient 5b, Washington, U.S.A.
A. calidoustus	CBS 121614	Patient 5a, Washington, U.S.A.
A. calidoustus	CBS 121615	Patient 6, Washington, U.S.A.
A. calidoustus	CBS 121613	Patient 2, Washington, U.S.A.
A. calidoustus	CBS 121612	Patient 1, Washington, U.S.A.
A. calidoustus	FH 91	Patient 1a, Washington, U.S.A.
A. calidoustus	NRRL 26162	Culture contaminant, Peoria, U.S.A.
A. calidoustus	NRRL 281	Thom 5634
A. calidoustus	NRRL 277	Thom 5698.754, Green rubber
A. granulosus	CBS 588.65T	Soil, Fayetteville, Arkansas, U.S.A.
A. granulosus	CBS 119.58	Soil, Texas, U.S.A.
A. granulosus	IBT 23478 = WB 1932 = IMI 017278iii = CBS 588.65	Soil, Fayetteville, Arkansas, U.S.A.
A. insuetus	CBS 107.25T	South Africa
A. insuetus	CBS 119.27	Unknown
A. insuetus	CBS 102278	Subcutaneous infection left forearm and hand of 77-year-old woman
A. keveii	CBS 209.92	Soil, La Palma, Spain
A. keveii	CBS 561.65	Soil, Panama
A. keveii	IBT 10524 = CBS 113227 = NRRL 1254	Soil, Panama
A. keveii	IBT 16751 = DMG 153	Galápagos Islands, Ecuador, D.P. Mahoney
A. pseudodeflectus	CBS 596.65	Sugar, U.S.A., Louisiana
A. pseudodeflectus	CBS 756.74T	Desert soil, Egypt, Western Desert
A. puniceus	CBS 122.33	Unknown
A. puniceus	9377	Mouth wash, Nijmegen, Netherlands
A. puniceus	V41-02	Faeces, Nijmegen, Netherlands
A. puniceus	NRRL 29173	Indoor air, Saskatoon, Canada
A. puniceus	CBS 495.65T	Soil, Zarcero Costa Rica
A. puniceus	CBS 128.62	Soil, Louisiana, U.S.A.
A. ustus	CBS 116057	Antique tapestries, Krakow, Poland
A. ustus	CBS 114901	Carpet, The Netherlands
A. ustus	CBS 261.67T	Culture contaminant, U.S.A.
A. ustus	CBS 133.55	Textile buried in soil, Netherlands
A. ustus	CBS 239.90	Man, biopsy of brain tumor, Netherlands
A. ustus	CBS 113233	IBT 14495
A. ustus	CBS 113232	IBT 14932
A. ustus	NRRL 285	Soil, Iowa, U.S.A.
A. ustus	NRRL 280	Bat dung, Cuba
A. ustus	NRRL 1609	Bat dung, Cuba
A. ustus	NRRL 29172	Indoor air, Edmonton, Canada
E. heterothallica	CBS 489.65T	soil, Costa Rica
E. heterothallica	CBS 488.65	soil, Costa Rica

^†These samples were taken from the same patient (Verweij et al. 1999)

Extrolite analysis. Extrolites were analysed by HPLC using alkylphenone retention indices and diode array UV-VIS detection as described by Frisvad & Thrane (1987), with minor modifications as described by Smedsgaard (1997). Standards of ochratoxin A and B, aflavinine, asperazine, austamide, austdiol, kotanin and other extrolites from the collection at Biocentrum-DTU were used to compare with the extrolites from the species under study.

Isolation and analysis of nucleic acids. The cultures used for the molecular studies were grown on malt peptone (MP) broth using 10 % (v/v) of malt extract (Brix 10) and 0.1 % (w/v) bacto peptone (Difco), 2 mL of medium in 15 mL tubes. The cultures were incubated at 25 °C for 7 d. DNA was extracted from the cells using the Masterpure™ yeast DNA purification kit (Epicentre Biotechnol.) according to the instructions of the manufacturer. Fragments containing the ITS region were amplified using primers ITS1 and ITS4 as described previously (White et al. 1990). Amplification of part of the β-tubulin gene was performed using the primers Bt2a and Bt2b (Glass 1995). Amplifications of the partial calmodulin and actin genes were set up as described previously (Hong et al. 2005). Sequence analysis was performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit for both strands, and the sequences were aligned with the MT Navigator software (Applied Biosystems). All the sequencing reactions were purified by gel filtration through Sephadex G-50 (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated in double-distilled water and analyzed on the ABI PRISM 310 Genetic Analyzer (Applied Biosystems).

Data analysis. The sequence data was optimised using the software package Seqman from DNAStar Inc. Sequence alignments were performed by using CLUSTAL-X (Thompson et al. 1997) and improved manually. The neighbour-joining (NJ) method was used for the phylogenetic analysis. For NJ analysis, the data were first analysed using the Tamura-Nei parameter distance calculation model with gamma-distributed substitution rates (Tamura & Nei 1993), which were then used to construct the NJ tree with MEGA v. 3.1 (Kumar et al. 2004). To determine the support for each clade, a bootstrap analysis was performed with 1000 replications.

For parsimony analysis, the PAUP v. 4.0 software was used (Swofford 2000). Alignment gaps were treated as a fifth character state and all characters were unordered and of equal weight. Maximum parsimony analysis was performed for all data sets using the heuristic search option with 100 random taxa additions and tree bisection and reconstruction (TBR) as the branch-swapping algorithm. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The robustness of the trees obtained was evaluated by 1000 bootstrap replications (Hillis & Bull 1993). An Aspergillus versicolor isolate was used as outgroup in these experiments. Unique sequences of the ITS, actin, calmodulin and β-tubulin gene sequences have been deposited in the GenBank under accession numbers EU076344-EU76377.

RESULTS

Phylogenetic analyses

For the molecular analysis, four genomic regions, the ITS region, and parts of the actin, calmodulin and β-tubulin genes were amplified and sequenced. Phylogenetic analysis of the data was carried out using the neighbour-joining technique and parsimony analysis. The trees obtained by the different approaches were identical, neighbour-joining trees based on the different data sets are shown in Figs 1, 2, 3 and 4. During analysis of part of the β-tubulin gene, 487 characters were analyzed, 111 of which were found to be parsimony informative. The topology of the tree is the same as that of one of the more than 10⁴ maximum parsimony trees constructed by the PAUP program (length: 216 steps, consistency index: 0.8148, retention index: 0.9679). The calmodulin data set included 474 characters, with 172 parsimony informative characters (1 MP tree, tree length: 360, consistency index: 0.8083, retention index: 0.9550). The actin data set included 406 characters, with 161 parsimony informative characters (3 MP trees, tree length: 292, consistency index: 0.8870, retention index: 0.9633). The ITS data set included 482 characters, 26 of which were parsimony informative (>10⁴ MP trees, tree length: 71, consistency index: 0.9155, retention index: 0.9781).

Fig. 1.

Neighbour-joining tree based on β-tubulin sequence data of Aspergillus section Usti. Numbers above branches are bootstrap values. Only values above 70 % are indicated.

Fig. 2.

Neighbour-joining tree based on calmodulin sequence data of Aspergillus section Usti. Numbers above branches are bootstrap values. Only values above 70 % are indicated.

Fig. 3.

Neighbour-joining tree based on actin sequence data of Aspergillus section Usti. Numbers above branches are bootstrap values. Only values above 70 % are indicated.

Fig. 4.

Neighbour-joining tree based on ITS sequence data of Aspergillus section Usti. Numbers above branches are bootstrap values. Only values above 70 % are indicated.

Molecular data revealed that Aspergillus section Usti consists of eight species: A. ustus, A. puniceus, A. granulosus, A. pseudodeflectus, A. calidoustus, A. insuetus and a new species including CBS 209.92 and some other isolates. We propose the name A. keveii sp. nov. for this set of isolates. The trees based on ITS, calmodulin and β-tubulin sequence data indicated that also E. heterothallica belongs to this section, although actin sequence data did not support this finding.

Morphological and physiological studies

Phenotypic comparison of the different members of the section Usti showed that eight taxa could be distinguished. Various characters showed to be valuable for differentiation (see also Table 2). One of the main criteria is the growth rate on CYA at 37 °C. A. calidoustus, A. pseudodeflectus and A. granulosus had high growth rates at this temperature, while E. heterothallica only grew restrictedly. The other members of this section were unable to grow at 37 °C, which reduces the potential of these species to become opportunistic human pathogens. The growth rate and the mycelium colour on creatin agar (CREA) also proved to be a good tool to differentiate between the species examined. Some species, like A. ustus, A. puniceus, A. insuetus and A. keveii have a good growth on this medium. Since sporulation on this medium is often inhibited, this medium was also useful to determine the colour of the mycelium. The colours varied from bright yellow by A. puniceus and E. heterothallica to faint yellow in A. ustus to colourless in the other species. Another useful character was the use of the Ehrlich test to detect the presence of indol metabolites. This feature gave, with the exception of A. keveii, very clear-cut results. Besides these features, the colony diam on YES was also suitable to differentiate between A. insuetus and the other species.

Table 2.

Overview of morphological criteria to differentiate between the members of Aspergillus section Usti.

Species	CYA37 (mm)	YES (mm)	Ehrlich reaction	Reaction on CREA	Conidial colour on MEA**
A. ustus	No growth	43-49	None	Good growth, faint yellow mycelium	Hair brown
A. puniceus	No growth	48-53	None	Moderate to good growth, yellow mycelium	Olive brown
A. calidoustus	20-35	36-41	Violet	Weak to moderate growth, hyaline mycelium	Brownish grey
A. insuetus	No growth	23-30	Violet	Good growth, hyaline mycelium	(Brownish) grey to light grey
A. keveii	No growth	40-46	Violet*	Good growth, hyaline mycelium	Brownish grey / pinkish brown
A. pseudodeflectus	15-20	20-30	None	Weak to moderate growth, hyaline mycelium	No sporulation
A. granulosus	30-35	35-40	Violet	Weak growth, hyaline mycelium	Buff to greyish brown
E. heterothallica	5-10	38-42	None	Weak growth, bright yellow mycelium	No sporulation

^*All have violet reaction, except CBS 113227

^**Colour according Methuen handbook of colours

Extrolite profiles

Aspergillus ustus has been claimed to produce a range of extrolites including austdiol (Vleggaar et al. 1974), Austin (Chexal et al. 1976), austocystins (Steyn & Vleggaar 1974; Kfir et al. 1986), brevianamide A (Steyn 1973), sterigmatocystin (Rabie et al. 1977), austalides (de Jesus et al. 1987), austamide (Steyn 1971), dehydroaustin (Scott et al. 1986), pergillin (Cutler et al. 1980), dehydropergillin (Cutler et al. 1981), phenylahistin (Kanoh et al. 1997), ophiobolins G & H (Cutler et al. 1984), drimans (Hayes et al. 1996), diacetoxyscirpenol (Tuomi et al. 2000) and ustic acid (Raistrick & Strickings 1951).

The mycotoxins and other extrolites found to be produced by the examined species in this study are listed in Table 3. Species assigned to section Usti could clearly be divided in three chemical groups based on the extrolites produced by them. A. ustus, A. granulosus and A. puniceus produced ustic acids in common. A. ustus and A. puniceus also produced austocystins and versicolorins. In the second chemical group, A. pseudodeflectus produced drimans (Hayes et al. 1996) in common with the other species in this group, and also several unique unknown compounds. A. calidoustus isolates produced drimans and ophiobolins in common with A. insuetus and A. keveii, but also produced austins not identified in other species of section Usti. A. insuetus isolates also produced pergillin, while A. keveii together with some other isolates produced nidulol. In the third chemical group, E. heterothallica has been reported to produce emethallicins A-F (Kawahara et al. 1989, 1990a, 1990b), 5”-hydroxyaveranthin (Yabe et al. 1991), emeheterone (Kawahara et al. 1988), emesterones A & B (Hosoe et al. 1998), 5”-hydroxyaveranthin (Yabe et al. 1991), Mer-NF8054X (Mizuno et al. 1995). This latter compound is an 18,22-cyclosterol derivative, and was also identified in an A. ustus isolate (Mizuno et al. 1995). Apart from this chemical similarity Emericella heterothallica appear to be quite different from the anamorphic species in section Usti, in agreement with actin sequencing data. Austamide, deoxybrevianamide E and austdiol could not be detected in any of the strains examined here and the strain producing these mycotoxins should be reexamined.

Table 3.

Extrolites produced by species assigned to Aspergillus section Usti.

Species	Extrolites produced
Chemical group I
A. ustus	Ustic acids, austocystins (and versicolorins), austalides, a compound related to sterigmatocystin, nidulol
A. granulosus	Ustic acids, a compound resembling sterigmatocystin, nidulol, drimans
A. puniceus	Ustic acids, austocystins (and versicolorins), phenylahistin, a compound related to sterigmatocystin, nidulol
Chemical group II
A. pseudodeflectus	Drimans, unknown compounds
A. calidoustus	Drimans, ophiobolins G and H, austins
A. insuetus	Drimans, ophiobolins G and H, pergillin-like
A. keveii	Drimans, ophiobolins G and H, nidulol
Chemical group III
E. heterothallica	Emethallicins A, B, C, D, E & F, emeheterone, emesterones A & B, 5″-hydroxyaveranthin, Mer-NF8054X, sterigmatocystin, versicolorins

Comparing the extrolites profiles of section Usti with other sections within subgenus Nidulantes, nidulol and versicolorins are also produced by members of sections Versicolores and Nidulantes (Cole & Schweikert 2003). Interestingly, versicolorins and 5”-hydroxyaveranthin are intermediates of the aflatoxin biosynthetic pathway and also produced by species assigned to Aspergillus section Flavi and Ochraceorosei (Yabe et al. 1991; Frisvad et al. 2005). However, while the versicolorins are precursors of sterigmatocystin in section Ochraceorosei, Versicolores and Nidulantes, they are precursors of austocystins in section Usti.

Section Usti contains the only Aspergillus species known to produce pergillins, ophiobolins, austins, austocystins, ustic acids, drimans, Mer-NF8054X, austalides, deoxybrevianamides and austamide and thus this section is chemically unique. We have not examined the species for production of emethallicins, emesterones and emeheterones, as standards of these compounds were not available.

DISCUSSION

Raper and Fennell (1965) classified A. ustus in the Aspergillus ustus group together with four other species: A. panamensis, A. puniceus, A. conjunctus and A. deflectus. Later, Kozakiewicz (1989) revised the taxonomy of the group, and included A. ustus, A. pseudodeflectus, A. conjunctus, A. puniceus, A. panamensis and A. granulosus into the A. ustus species group, and established the A. deflectus group including A. deflectus, A pulvinus and A. silvaticus based on morphological studies. Klich (1993) treated A. granulosus as member of section Versicolores, and found that A. pseudodeflectus is only weakly related to this section based on morphological treatment of section Versicolores. Peterson (2000) transferred most species of section Usti to section Nidulantes based on sequence analysis of part of the 28 S rRNA gene. On his cladogram, A. ustus, A. pseudodeflectus, A. granulosus and A. puniceus form a well-supported branch closely related to A. versicolor and its allies, while A. deflectus is on another branch related to A. elongatus and A. lucknowensis. Peterson (2000) transferred A. conjunctus, A. funiculosus, A. silvaticus, A. panamensis and A. anthodesmis to section Sparsi. Recently Varga et al. (submitted) studied large numbers of isolates from clinical and other sources using molecular, morphological and physiological approaches. Phylogenetic analysis of partial β-tubulin, calmodulin, actin and ITS sequences indicated that none of the clinical isolates recognised previously as A. ustus belong to the A. ustus species. All but two of these isolates formed a well-defined clade related to A. pseudodeflectus based on sequence analysis of protein coding regions. Morphological and physiological examination of the isolates indicated that they are able to grow above 37 °C, in contrast with A. ustus isolates, and give a positive Ehrlich reaction, in contrast with related species including A. granulosus, A. ustus, and A. pseudodeflectus. These isolates were described as A. calidoustus.

Aspergillus ustus (Bainier) Thom & Church was redescribed by Thom & Church (1926) based on Sterigmatocystis usta Bainier. In this manual, A. insuetus (Bainier) Thom & Church was also accepted based on S. insueta Bainier (Thom & Chuch, 1926), but later A. insuetus was abandoned (Thom and Raper, 1945) and included in the broad description of A. ustus in Raper and Fennell (1965). Our studies clarified that A. insuetus is a valid species which can be distinguished from A. ustus and other species assigned to Aspergillus section Usti. A. insuetus could be separated from the other members of the section Usti by various phenotypic characters. The most important one is the slower growth rate on YES agar and clear differences in extrolite profiles (Table 2). This finding was supported by all the different data sets used to characterise section Usti. The molecular data showed that this species is more related to A. calidoustus and A. pseudodeflectus than A. ustus. Also different extrolite patterns were observed. There were many differences between A. ustus and A. insuetus, and, like the molecular data, this species was mostly related to A. calidoustus and A. pseudodeflectus. The main difference between the latter species was the production of a pergillin-like compound by A. insuetus (Table 3).

Our polyphasic taxonomic approach revealed that Aspergillus section Usti includes eight species: A. ustus, A. puniceus, A. granulosus, A. pseudodeflectus, A. calidoustus, A. insuetus and A. keveii sp. nov. The phylogenetic trees based on ITS, calmodulin and β-tubulin sequence data indicated that E. heterothallica also belongs to this section. This species has similar morphology of the conidiophores and Hülle cells. In our study we were not able to observe ascospores by crossing the two mating strains but these are described by Raper and Fennell (1965: 502-503).

Aspergillus calidoustus Varga et al. Eukaryotic Cell submitted. Fig. 5.

Fig. 5.

Aspergillus calidoustus. A-B. Colonies at 25 °C after 7 d. A. CYA. B. MEA. C-E, G-H Conidiophores. F. Hülle cells. I. Conidia. Scale bars = 10 μm, except F = 30 μm

Type: CBS 121604 from human, Netherlands

Other no. of the type: strain 677

Description strain

Colony diam, 7 d, in mm: CYA25 27-32; CYA37 20-35; MEA25 35-48; YES 36-41

Colony colour on CYA: blond/greyish yellow, brownish grey or greyish brown

Conidiation on CYA: abundant

Reverse colour (CYA): yellow with beige or olive brown centre

Colony texture: floccose

Conidial heads: loosely columnar

Stipe: 150-300 × 4-7 μm, smooth, brown

Vesicle diam/shape: 9-15 μm, pyriform to broadly spathulate

Conidium size/shape/surface texture: 2.7-3.5 × μm, globose, very rough ornamentation (0.5-0.8 μm high), inner and outer wall visible

Hülle cells: sparsely produced, irregularly elongated, in scattered groups

Ehrlich reaction: violet

Growth on creatine: weak to moderate growth with hyaline mycelium, no acid production

Diagnostic features: good growth at 37 °C, violet Ehrlich reaction, coarsely roughened to echinulate conidia

Cultures examined: CBS 121589, 121601-121616

Similar species: A. pseudodeflectus

Distribution: U.S.A., Turkey, Finland, Germany, Netherlands

Ecology and habitats: indoor air, rubber, construction material, human

Extrolites: Drimans, ophiobolins G and H, austins

Pathogenicity: pathogenic to humans (Verweij et al. 1999; Weiss & Thiemke 1983; Pavie et al. 2005; Panackal et al. 2006; Yildiran et al. 2006; Iwen et al. 1998)

Aspergillus granulosus Raper & Thom, Mycologia 36: 565. 1944. Fig. 6.

Fig. 6.

Aspergillus granulosus. A-B. Colonies at 25 °C after 7 d. A. CYA. B. MEA. C-H Conidiophores. I. Conidia. Scale bars = 10 μm, except C = 30 μm.

Type: CBS 588.65, from soil, Fayetteville, Arkansas, U.S.A.

Other no. of the type: ATCC 16837, NRRL 1932, WB 1932, CBS 452.93

Description

Colony diam, 7 d, in mm: CYA25 30-48; CYA37 30-51; MEA25 25-37; YES25 35-45; CZA25 17-25

Colony colour: buff to dull brown

Conidiation: moderate

Reverse colour (CYA): dull yellow to red brown

Colony texture: floccose, plane or irregularly furrowed

Conidial head: hemispherical to radiate

Stipe: 100-600 × 5.5-8 μm, thin-walled, smooth, straight, tan to light brown

Vesicle diam/shape: 15-25 × 12-18 μm, ovoid to elliptical

Conidium size/shape/surface texture: (3.3-)4-4.5(-5.5) μm, globose, delicately echinulate

Hülle cells: irregularly globose, ovoid to elongate, 12-30 μm, in colourless clusters at colony margins

Ehrlich reaction: violet

Growth on creatine: poor growth with inconspicuous mycelium, no acid production

Cultures examined: CBS 119.58, CBS 588.65, IBT 23478

Diagnostic features: small colourless clusters of irregularly globose Hülle cells, giving the colony a characteristic granular appearance, good growth at 37 °C and violet Ehrlich reaction

Similar species: -

Distribution: U.S.A.

Ecology and habitats: soil

Extrolites: Ustic acids, a compound resembling sterigmatocystin, nidulol, drimans

Pathogenicity: pathogenic to humans (Fakih et al. 1995)

Aspergillus insuetus (Bainier) Thom & Church, Manual of the aspergilli: 153. 1929. Fig. 7.

Fig. 7.

Aspergillus insuetus. A-B. Colonies at 25 °C after 7 d. A. CYA. B. MEA. C-H Conidiophores. I. Conidia. Scale bars = 10 μm, except C = 30 μm.

• = Sterigmatocystis insueta Bainier (1908)

Type: CBS 107.25, from South Africa, Sartory

Other no. of the type: ATCC 1033; IFO 4128; NRRL 279; NRRL 1726; Thom No. 4658.245

Description

Colony diam, 7 d, in mm: CYA 28-32; CYA37 no growth; MEA25 36-41; YES 23-30

Colony colour: almost black in center, shading through gray to white sterile floccose marginal areas

Conidiation on CYA: moderate to good

Reverse colour (CYA): yellow olive to blackish brown with age

Colony texture: floccose

Conidial head: radiate to hemispherical

Stipe: 300 × 4-8 μm, smooth, brown

Vesicle diam/shape: 11-16 μm, hemispherical to subglobose

Conidium size/shape/surface texture: 3.2-4 μm, globose, distinct roughened and inner and outer wall visible, fuligeneous, the colour mostly aggregated into echinulations of the cell-wall, and even forming bars and tubercules at times

Hülle cells: variously coiled or curved, in scattered groups

Ehrlich reaction: violet

Growth on creatine: good growth with hyaline mycelium, no acid production

Cultures examined: CBS 107.25, CBS 119.27, CBS 102278

Similar species: A. keveii

Distribution: South Africa, Spain

Diagnostic features: no growth at 37 °C, violet Ehrlich reaction, restricted growth on YES, coarsely roughened to echinulate conidia

Ecology and habitats: soil (?), human

Extrolites: Drimans, ophiobolins G and H, pergillin-like

Pathogenicity: caused subcutaneous infection (Gené et al. 2001)

Aspergillus keveii sp. nov. Varga, Frisvad & Samson - MycoBank MB505570. Fig. 8.

Fig. 8.

Aspergillus kerveii. A-B. Colonies at 25 °C after 7 d. A. CYA. B. MEA. C-H Conidiophores. I. Conidia. Scale bars = 10 μm.

Holotype of Aspergillus keveii, here designated as CBS 209.92^T (dried culture) isolated from soil, Las Palmas, Spain.

Coloniae in 7 dieibus et 25 °C in agaro MEA 36-41 mm, in CYA 30-39 mm, in YES 40-46 mm, in CREA 25-32 mm diam; auctus in 7 dieibus et 37 °C in agaro CYA nullus. Sporulatio in CYA abundans; colonia brunneogrisea vel subroseobrunnea; textura coloniae floccosa; colonia reversa flavide olivaceobrunnea vel atrobrunnea. Capitula conidialia laxe columnaria; stipites 150-300 × 4-6 μm, pariete laevi, brunneo; vesciculae pyriformes, 9-13 μm in lat., biseriatae; metulae 4.7-6.7 × 2.8-3.6 μm; phialides 5.7-7 × 2-3 μm; conidia globosa, 2.4-2.8 μm diam., ornamento exasperato vel echinulato. Cellulae “hülle” irregulariter elongatae, (10-) 25-40(-65) μm in long., in cumulis dispersis.

Colonies on MEA 36-41 mm, on CYA 30-39 mm, on YES 40-46 mm, on CREA 25-32 mm in diam. after 7 d at 25 °C, no growth on CYA after 7 d at 37 °C. Conidial heads abundant on CYA, colony colour brownish grey to pinkish brown, colony texture floccose, reverse yellow olive brown to dark brown. Conidial heads loosely columnar; stipes 150-300 × 4-6 μm, smooth walled, brown in colour; vesicles 9-13 μm wide, pyriform, biseriate; metulae covering the upper half to three-fourths of the vesicle, measuring 4.7-6.7 × 2.8-3.6 μm μm; phialides 5.7-7 × 2-3 μm; conidia globose 2.4-2.8 μm, coarsely roughened to echinulate. Hülle cells (10-)25-40(-65) μm, irregularly elongated, produced in scattered groups.

Etymology: named after Prof. Ferenc Kevei, eminent mycologist devoting his life to Aspergillus research.

Type: CBS 209.92

Ehrlich reaction: violet, with exception of CBS 113227

Growth on creatine: good growth with hyaline mycelium, no or weak acid production

Diagnostic features: no growth at 37 °C, good growth on CREA and YES, coarsely roughened to echinulate conidia; Hülle cells in scattered groups, violet Ehrlich reaction

Cultures examined: CBS 561.65, CBS 209.92 and CBS 113227

Similar species: A. insuetus

Distribution: U.S.A., Turkey, Finland, Germany, Netherlands

Ecology and habitats: indoor air, rubber, construction material, human

Extrolites: Drimans, ophiobolins G and H, nidulol

Pathogenicity: not reported

Notes: CBS 113227 is deviating in having larger conidial heads and small (2.6 μm), finely roughened pinkish brown coloured conidia

Aspergillus pseudodeflectus Samson & Mouchacca, Antonie van Leeuwenhoek 41(3): 325. 1975. Fig. 9.

Fig. 9.

Aspergillus pseudodeflectus. A-C. Colonies at 25 °C after 7 d. A. MEA + 40 % sucrose. B. CYA + 20 % sucrose. C. MEA. D-I. Conidiophores. H. Conidia. Scale bars = 10 μm.

Type: CBS 756.74, from desert soil, Western Desert, Egypt

Other no. of the type: IMI 278381

Description

Colony diam, 7 d, in mm: CYA25 43-49; CYA37 15-20; MEA25 35-45; YES 20-30; CZA25 25-26

Colony colour: white mycelial felt intermixed with brown conidiogenous structures

Conidiation: sparse

Reverse colour (CZA): yellow

Colony texture: velvety appearance, no sporulation

Conidial head: radiate, brown

Stipe: 35-200 × 2.5-3.5 μm, rough-walled with warty protuberances, brown

Vesicle diam/shape: 4-12 μm, globose to clavate

Conidium size/shape/surface texture: 3.5-5 μm, globose to ellipsoidal, brown, ornamented with small warts and colour bars

Hülle cells: absent

Ehrlich reaction: none

Growth on creatine: weak to moderate growth with hyaline mycelium, no acid production

Diagnostic features: Growth at 37 °C, curved brown conidiophores and the ornamented conidia, absence of Hülle cells

Cultures examined: CBS 756.74, CBS 596.65

Similar species: A. calidoustus

Distribution: Egypt, U.S.A.

Ecology and habitats: soil

Extrolites: Drimans (Hayes et al. 1996), unknown compounds

Pathogenicity: not reported

Aspergillus puniceus Kwon and Fennell, The genus Aspergillus: 547. 1965. Fig. 10.

Fig. 10.

Aspergillus puniceus. A-B. Colonies at 25 °C after 7 d. A. CYA. B. MEA. C-H Conidiophores. I. Sclerotia. J. Conidia. Scale bars = 10 μm, except D = 30 μm.

• = A. ustus var. laevis Blochwitz (1945)

Type: CBS 495.65, from soil, Zarcero, Costa Rica

Other no. of the type: ATCC 16800; IMI 126692; WB 5077

Description

Colony diam, 7 d, in mm: CYA 40-50; CYA37 no growth; MEA25 40-45; YES 48-53; CZA25: 40-50 mm

Colony colour: pinkish orange near vinaceous pink, with wine red exudate droplets

Conidiation: moderate

Reverse colour (CYA): dark yellow brown or crème brown

Colony texture: floccose

Conidial head: radiate to short columnar, dull green becoming light drab with age

Stipe: 150-250(-300) × 5.5-6(-8) μm, aerially borne stipes up to 135 × 3-4 μm, straight, smooth

Vesicle diam/shape: 8-16 μm (subglobose), 15-18 × 13-15 μm (elliptical)

Conidium size/shape/surface texture: 2.5-3.3 μm, globose, roughened

Hülle cells: elongate, crescent shaped or irregularly twisted, often aggregated into yellowish masses

Ehrlich reaction: no reaction

Growth on creatine: moderate to good growth with bright yellow mycelium, no acid production (in some isolates weak acid production under colony)

Cultures examined: CBS 495.65, CBS 122.33, CBS 128.62, 9377, V41-02, NRRL 29173

Diagnostic features: No growth at 37 °C, good growth on creatine with brightly pigmented yellow mycelium, Hülle cells aggregated into yellowish masses

Similar species: A. ustus

Distribution: Costa Rica, U.S.A., Canada, Netherlands

Ecology and habitats: soil, indoor air, human

Extrolites: ustic acids, austocystins, nidulol, versicolorins, phenylahistin, sterigmatocystin-related compound (in CBS 128.62)

Pathogenicity: isolated from mouth wash and faeces

Aspergillus ustus (Bainier) Thom & Church, The aspergilli: 152. 1924. Fig. 11.

Fig 11.

Aspergillus ustus. A-B. Colonies at 25 °C after 7 d. A. CYA. B. MEA. C-E. G-H Conidiophores. F. Hülle cells. I. Conidia. Scale bars = 10 μm, except F = 30μm.

• = Sterigmatocystis usta Bainier (1881)

• = Aspergillus humus Abbott (1926)

Type: CBS 261.67, culture contaminant, U.S.A.

Other no. of the type: ATCC 1041; ATCC 16818; IMI 211805; NRRL 275; QM 7477; WB 275; Thom 3556

Description

Colony diam, 7 d, in mm: CYA 36-43; CYA37 no growth; MEA25 39-46; YES 42-50

Colony colour: greyish brown to dark brown

Conidiation on CYA: moderate

Reverse colour (CZA): yellow-olive edge with olive brown centre

Colony texture: floccose, plane, sulcate or umbonate

Conidial head: radiate to hemispherical

Stipe: 400 × 3-6 μm, aerially borne stipes up to 125 × 2-5 μm, smooth, brownish

Vesicle diam/shape: 7-15 μm, hemispherical to subglobose

Conidium size/shape/surface texture: 3.2-4.5 μm, globose, roughened, greenish to dark yellow brown

Hülle cells: irregularly ovoid or elongate, usually scattered

Ehrlich reaction: no reaction

Growth on creatine: good growth with faint yellow mycelium, no acid production

Cultures examined: CBS 116057, CBS 114901, CBS 261.67, CBS 133.55, CBS 239.90, CBS 113233, CBS 113232, NRRL 285, NRRL 280, NRRL 1609, NRRL 29172

Diagnostic features: No growth at 37 °C; good growth on creatine with faint yellow pigmented mycelium; Hülle cells typically scattered or form irregular masses and not associated with pigmented mycelium

Similar species: A. puniceus

Distribution: U.S.A., Poland, Netherlands, Canada

Ecology and habitats: soil, indoor air, bat dung

Extrolites: Ustic acids, austocystins, versicolorins, austalides, a compound related to sterigmatocystin, nidulol

Pathogenicity: isolated from biopsy of man with brain tumour (CBS 239.90). However, this isolate does not grow at 37 °C on normal agar media and might therefore be a culture contamination.

Emericella heterothallica (Kwon-Chung, Fennell & Raper) Malloch & Cain [anamorph: A. compatibilis Samson & Gams], Can. J. Bot. 50: 62. 1972. Fig. 12.

Fig. 12.

Emericella heterothallica. A-C. Colonies at 25 °C after 7 d. A. CYA. B. MEA. C. Crossing of mating strains. D-E, F-H. Conidiophores. I. Conidia. Scale bars = 10 μm.

Type: CBS 489.65, from soil, Costa Rica

Other no. of the type: ATCC 16824; IHEM 2064; IMI 139278; RV 34434; WB 5097; IBT 22604

Description

Colony diam, 7 d, in mm: CYA25 35-39; CYA37 5-8; MEA25 40-42; YES25 38-42

Colony colour: cream to yellow to orange

Conidiation: limited

Reverse colour (CYA): yellow to orange to pink becoming dark reddish brown

Colony texture: floccose

Conidial head: hemispherical to short columnar

Stipe: 185-410 × 5-11 μm, generally sinuous, brownish with age, smooth

Vesicle diam/shape: 13-20 μm

Conidium size/shape/surface texture: 2.5-4 μm, globose, echinulate, yellow green

Hülle cells: 600-700(-1000) μm, pyriform to oval to elongate to twisted, in globose to subglobose masses

Cleistothecia: produced in a heterothallic manner, 270-510 μm, cinnamon to dark purple, surrounded by Hülle cells

Ascospores: 4-4.5 × 3.5-4 μm, lenticular, orange brown in colour, with two pleated equatorial crests (1.5-2 μm), with convex smooth

Ehrlich reaction: none

Growth on creatine: weak growth with yellow coloured mycelium, no acid production

Diagnostic features: heterothallic species, weak growth at 37 °C

Cultures examined: CBS 489.65, CBS 488.65 = IBT 22607

Similar species: -

Distribution: Costa Rica

Ecology and habitats: soil

Extrolites: Found in this study: Sterigmatocystin, versicolorins, Mer-NF8054X. Literature data: emethallicins A-F (Kawahara et al. 1989, 1990a), 5”-hydroxyaveranthin (Yabe et al. 1991), emeheterone (Kawahara et al. 1988), emesterones A & B (Hosoe et al. 1998), 5”-hydroxyaveranthin (Yabe et al. 1991), Mer-NF8054X (Mizuno et al. 1995).

Pathogenicity: not reported