Keywords: actin, Aspergillus, β-tubulin, calmodulin, extrolite profiles, ITS, phylogenetics, polyphasic taxonomy.
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Copyright © Copyright 2007 CBS Fungal Biodiversity Centre P.O. Box
85167, 3508 AD Utrecht, The Netherlands. Polyphasic taxonomy of Aspergillus section Usti *Correspondence: J. Houbraken,
j.houbraken/at/cbs.knaw.nl You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author's moral rights. | |||||||||||||||||
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. Keywords: actin, Aspergillus, β-tubulin, calmodulin, extrolite profiles, ITS, phylogenetics, polyphasic taxonomy. | |||||||||||||||||
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.
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).
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.
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.
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.
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
36-41Colony 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.
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 17-25Colony 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.
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
23-30Colony 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.
Holotype of Aspergillus keveii, here designated as CBS 209.92T (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.
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 25-26Colony 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.
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 mmColony 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.
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
42-50Colony 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.
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
38-42Colony 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 | |||||||||||||||||
Acknowledgments We would like to thank the Danish Technical Research Council for support to Center for Microbial Biotechnology and Ellen Kirstine Lyhne for technical support. Dr Emory Simmons kindly provided a Latin diagnosis for the new species. | |||||||||||||||||
Notes Taxonomic novelties: Aspergillus insuetus revived,
Aspergillus keveii sp. nov. | |||||||||||||||||
References
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