Morphological and Molecular Characterization of Aspergillus Section Usti

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 121601^T	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 588.65^T	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 107.25^T	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 756.74^T	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 495.65^T	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 261.67^T	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 489.65^T	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 (link)),
with minor modifications as described by Smedsgaard
(1997 (link)). 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 (link)). Amplifications
of the partial calmodulin and actin genes were set up as described previously
(Hong et al. 2005 (link)).
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 (link)), which
were then used to construct the NJ tree with MEGA v. 3.1
(Kumar et al. 2004 (link)).
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.

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Houbraken J., Due M., Varga J., Meijer M., Frisvad J.C, & Samson R.A. (2007). Polyphasic taxonomy of Aspergillus section Usti. Studies in Mycology, 59, 107-128.

Publication 2007

Actin Agar Asperazine Aspergillosis Aspergillus Aspergillus versicolor Austamide Austdiol Autopsy Bacto peptone Biopsy Bronchial Bronchoalveolar lavage Bronchoalveolar lavage fluid Bull Calmodulin Cataract surgery Characters Creatine Dung Endophthalmitis Forearm Gamma Gel filtration Gene Gene amplification Hplc Infection Inoculations Isolation Kotanin Light microscopy Lung Mouth wash Nucleic acids Ochratoxin a Palma Patient Peptone Primers Prism Reconstruction Replications Retention Rubber Secretion Sephadex g 50 Sequence alignments Skin Sputum Strains Sucrose Sugar Tissue Tongue Trees Tubulin Tumor of brain Woman Yeast

Corresponding Organization :

Other organizations : Westerdijk Fungal Biodiversity Institute

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Variable analysis

independent variables

Growth conditions (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)

dependent variables

Morphological examination of strains

control variables

Medium compositions (according to Samson et al. 2004)
Light microscopy (Olympus BH2 and Zeiss Axioskop 2 Plus)

controls

No positive or negative controls explicitly mentioned.

Annotations

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