New and revisited species in Aspergillus section Nigri.

Four new species, Aspergillus eucalypticola, A. neoniger, A. fijiensis and A. indologenus are described and illustrated. Aspergillus eucalypticola was isolated from Eucalyptus leaf from Australia, and is related to A. tubingensis and A. costaricaensis, but could clearly be distinguished from them based on either β-tubulin or calmodulin sequence data. Aspergillus eucalypticola produced pyranonigrin A, funalenone, aurasperone B and other naphtho-γ-pyrones. Aspergillus neoniger is also a biseriate species isolated from desert sand in Namibia, and mangrove water in Venezuela, which produces aurasperone B and pyranonigrin A. Aspergillus fijiensis is a uniseriate species related to A. aculeatinus, and was isolated from soil in Fiji, and from Lactuca sativa in Indonesia. This species is able to grow at 37 °C, and produces asperparalines and okaramins. Aspergillus indologenus was isolated from soil, India. This species also belongs to the uniseriate group of black aspergilli, and was found to be related to, but clearly distinguishable from A. uvarum based on β-tubulin, calmodulin and ITS sequence data. Aspergillus indologenus produced the insecticidal compounds okaramins A, B, H, and two types of indol-alkaloids which have not been structure elucidated. Two other species, A. violaceofuscus and A. acidus, are revalidated based on molecular and extrolite data. Aspergillus violaceofuscus was found to be related to A. japonicus, and produced some of the same interesting indol-alkaloids as A. indologenus, and also produced several families of partially characterised extrolites that were also found in A. heteromorphus. Aspergillus acidus (previously known as A. foetidus var. pallidus and A. foetidus var. acidus) is also a valid species, while A. foetidus is a synonym of A. niger based on molecular and physiological data. Two other species described previously, A. coreanus and A. lacticoffeatus, were found to be colour mutants of A. acidus and A. niger, respectively. Methods which could be used to distinguish the two closely related and economically important species A. niger and A. awamori are also detailed. Although these species differ in their occurrence and several physiological means (elastase activities, abilities to utilise 2-deoxy-D-glucose as sole carbon source), our data indicate that only molecular approaches including sequence analysis of calmodulin or β-tubulin genes, AFLP analysis, UP-PCR analysis or mtDNA RFLP analysis can be used reliably to distinguish these sibling species. Aspergillus section Nigri now includes 26 taxa.

INTRODUCTION

The black aspergilli (Aspergillus section Nigri; Gams ) are an important group of species in food mycology, medical mycology and biotechnology. Many species cause food spoilage, but on the other hand are also used in the fermentation industry to produce hydrolytic enzymes, such as amylases or lipases, and organic acids, such as citric acid and gluconic acid (Varga ). They are also candidates for genetic manipulation in the biotechnology industries since A. niger used under certain industrial conditions has been granted the GRAS (Generally Regarded As Safe) status by the Food and Drug Administration of the US government. Although the main source of black aspergilli is soil, members of this section have been isolated from various other sources (Kozakiewicz 1989, Abarca , Samson , Ferracin ). Black aspergilli are one of the more difficult groups concerning classification and identification, and several taxonomic schemes have been proposed. New molecular approaches have shown that there is a high biodiversity, but that species are occasionally difficult to recognise based solely on their phenotypic characters (Samson ).

During a study of the genetic relationships among black aspergilli collected worldwide, four isolates have been identified which did not fit to any of the currently accepted 19 species of Aspergillus section Nigri (Samson , Noonim , Perrone ). We used a polyphasic approach including sequence analysis of parts of the β-tubulin and calmodulin genes and the ITS region, macro- and micromorpholocigal analyses and examination of extrolite profiles of the isolates to describe four new species in this section. Besides, the applicability of various approaches for distinguishing the two closely related species A. niger and A. awamori has also been examined. The methods tested include morphological, physiological, ecological and molecular approaches.

MATERIALS AND METHODS

Isolates

The strains used in this study are listed in Table 1.

Table 1.

Isolates examined in this study.

Accession No.	Species	Origin and information (abbreviation)	β-tubulin	calmodulin	ITS
CBS 564.65T	A. acidus	Unknown substratum, Japan	AY585533	AY585533	AJ280009
CBS 121060T	A. aculeatinus	Arabica green coffee bean, Thailand	EU159220	EU159241	EU159211
CBS 172.66T	A. aculeatus	Tropical soil, unknown origin	AY585540	AJ964877	AJ279988
CBS 557.65T	A. awamori	NRRL 4948 = WB 4948	AY820001	AJ964874	AM087614
CBS 101740T	A. brasiliensis	Soil, Sao Paulo, Pedreira, Brazil	AY820006	AM295175	AJ280010
CBS 111.26T	A. carbonarius	Paper, origin unknown	AY585532	AJ964873	DQ900605
CBS 119384T	A. coreanus	Nuruk, Boun-up, Bounkun, Chungbuk Prov., Korea	FJ491693	FJ4916702	FJ491684
CBS 115574T	A. costaricaensis	Soil, Taboga Island, Gauguin garden, Costa Rica	AY820014	EU163268	DQ900602
CBS 707.79T	A. ellipticus	Soil, Costa Rica	AY585530	AM117809	AJ280014
CBS 122712T	A. eucalypticola	Eucalyptus leaves, Australia	EU482435	EU482433	EU482439
CBS 119.49	A. fijiensis	Lactuca sativa, Palembang, Indonesia	FJ491689	FJ491701	FJ491679
CBS 313.89T	A. fijiensis	Soil, Fiji	FJ491688	FJ491695	FJ491680
CBS 121.28NT	A. foetidus	Awamori-koji alcoholic beverage, Ryuku island, Japan	FJ491690	FJ491694	FJ491683
CBS 114.49T	A. foetidus	NRRL 341, Thom 5135.17; K. Yakoyama, Japan	EF661090	EF661155	EF661187
CBS 117.55T	A. heteromorphus	Culture contaminant, Brazil	AY585529	AM421461	AJ280013
CBS 101889T	A. homomorphus	Soil, near Dead Sea, Israel	AY820015	AM887865	EF166063
CBS 121593T	A. ibericus	Grapes, Portugal	AM419748	AJ971805	AY656625
CBS 114.80T	A. indologenus	Soil, India	AY585539	AM419750	AJ280005
CBS 114.51T	A. japonicus	Saito 5087, origin unknown	AY585542	AJ964875	AJ279985
CBS 101883T	A. lacticoffeatus	Coffee bean, South Sumatra, Indonesia	AY819998	EU163270	DQ900604
CBS 115657	A. neoniger	Desert sand, Namibia	FJ491692	FJ491699	FJ491681
CBS 115656T	A. neoniger	Mangrove water, Mochima Bay, Venezuela	FJ491691	FJ491700	FJ491682
CBS 554.65T	A. niger	Connecticut, USA	AY585536	AJ964872	AJ223852
CBS 112811T	A. piperis	Black pepper, Denmark	AY820013	EU163267	DQ900603
CBS 121057T	A. sclerotiicarbonarius	Robusta coffee bean, Thailand	EU159229	EU159235	EU159216
CBS 127449T	A. saccharolyticus	under a toilet seat made of treated oak wood, Gentofte, Denmark	HM853553	HM853554	HM853552
CBS 115572T	A. sclerotioniger	Coffee bean, Karnataka, India	AY819996	EU163271	DQ900606
CBS 134.48T	A. tubingensis	Origin unknown	AY820007	AJ964876	AJ223853
CBS 121591T	A. uvarum Healthy	Cisternino grape, Italy	AM745751	AM745755	AM745751
CBS 113365T	A. vadensis	Origin unknown	AY585531	EU163269	AY585549
CBS 115571	A. violaceofuscus	Marine environment, Bahamas	EU482434	EU482432	EU482440
CBS 123.27NT	A. violaceofuscus	Soil, Puerto Rico; Thom 3522.30	FJ491685	FJ491698	FJ491678
CBS 122.35	A. violaceofuscus mut. grisea	WB 4880	FJ491687	FJ491696	FJ491676
CBS 102.23T	A. violaceofuscus	Received from D. Borrel, Strassbourg, France	FJ491686	FJ491697	FJ491677

T = Type, NT = Neotype

Morphological analysis

For macromorphological observations, Czapek Yeast Autolysate (CYA) agar, Malt Extract Autolysate (MEA) agar, Yeast Extract Sucrose Agar (YES), Creatine Agar (CREA), and Oatmeal Agar (OA) were used (Samson ). The isolates were inoculated at three points on each plate of each medium and incubated at 25 °C and 37 °C in the dark for 7 d. For micromorphological observations, microscopic mounts were made in lactic acid from MEA colonies and a drop of alcohol was added to remove air bubbles and excess conidia.

Extrolite analysis

The isolates were grown on CYA and YES at 25 °C for 7 d. Extrolites were extracted after incubation. Five plugs of each agar medium were taken and pooled together into same vial for extraction with 0.75 mL of a mixture of ethyl acetate/dichloromethane/methanol (3:2:1) (v/v/v) with 1 % (v/v) formic acid. The extracts were filtered and analysed by HPLC using alkylphenone retention indices and diode array UV-VIS detection as described by Frisvad & Thrane (1987, 1993), with minor modifications as described by Smedsgaard (1997). The column used was a 50 x 2 mm Luna C-18 (II) reversed phase column (Phenomenex, Torrance, CA, USA) fitted with a 2 x 2 mm guard column. Standards of ochratoxin A and B, secalonic acid D, neoxaline and other extrolites from the collection at Department of Systems Biology-DTU were used to confirm the identity of the detected extrolites.

Physiological analysis

Aspergillus niger and A. awamori isolates were analysed using various methods. Elastase activity was studied on Czapek Dox minimal medium without NaNO3, with 0.05 % elastin (Sigma, St Louis, MO, USA) and 0.05 % Rose Bengal (Sigma, St Louis, MO, USA), buffered to pH 7.6 with NaOH (modified after Blanco ). Conidial suspensions of A. niger and A. awamori strains were prepared from cultures grown on YPD agar slants. The suspensions were diluted in 1 mL bidistilled water. Plates were inoculated in a central spot with 20 μL of the conidium suspensions and were incubated for 7 d at 37 °C. The diameters of the halo of elastin lysis were measured. The experiment was repeated three times, and the average diameters were calculated for each strain. Statistical analysis was made using the R software package (http://www.r-project.org/). The assumptions of ANOVA were tested using the diagnostic plots in R. According to Quantile-Quantile (QQ) Plot the data were not normally distributed, thus the Kruskal-Wallis test was applied to compare the average diameters between the two species.

Carbon source assimilation tests were performed on minimal medium (MM: 0.5 % (NH4)2SO4, 0.1 % KH2PO4, 0.05 % MgSO4, 2 % agar) with 0.2 % single carbon source. Conidial suspensions of eight A. niger and eight A. awamori strains were prepared from 5-d-old cultures grown on YPD agar slants. The suspensions were diluted in bidistilled water and conidia were filtered. An YPD plate was inoculated in 16 points with 15 μL of the conidium suspensions and was incubated for 3 d at 25 °C. Strains were replicated to the MM plates, which contained single carbon sources using a 16-pronged replicator. Plates were incubated for 7 d at 25 °C. The experiment was repeated twice and control series was made on MM plates without carbon source and YPD plates.

Thirty carbon sources were tested, which were selected based on previous carbon source utilisation experiments (Varga ): glucose, D-xylose, galactose, D-lyxose, L-sorbose, L-rhamnose, lactose, eritrit, galactit, L-valine, L-β-phenilalanine, L-triptophane, L-treonine, L-serine, L-cysteine, L-asparagine acid, L-tyrosine, L-lysine, L-histidine, L-citrulline, cis-aconitic acid, vanillin, vanillin acid, L-ascorbic acid, D-glucoseamine, glycylglycine, salicin, pectin, melezitose, α-ketoglutaric acid. Different growth patterns of the strains belonging to the two species were observed simply in the case of L-sorbose, so the test was extended to 2-deoxy-D-glucose because of the structural similarity of these two compounds.

Genotypic analysis

The cultures used for the molecular studies were grown on malt peptone (MP) broth using 10 % (v/v) of malt extract (Oxoid) 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. The ITS region and parts of the β-tubulin and calmodulin genes were amplified and sequenced as described previously (Varga et al. 2007a, 2007b, 2007c).

Part of the FUM8 gene was amplified using primers vnF1 and vnR3 as described by Susca et al. (2010). Primer sets were also designed to target part of the chloroperoxidase gene of black aspergilli presumably taking part in ochratoxin biosynthesis. Construction of the primers was carried out by using the homologous sequences identified in the genomic sequences of Aspegillus niger CBS 513.88 and Aspergillus carbonarius ITEM 5010 isolates. The designed chloroperoxidase specific PCR primers were BCPOF (5'-CTGGGCGACTGCATCCAC – 3') and BCPOR (5'-TTCATCGTACGGCAGACGCT - 3') which generated specific amplicons of about 250 base pairs. Amplifications were performed on a PTC-0148 Mini48 thermocycler (BioRad, USA), using the following amplification steps: 4 min of initial denaturation at 94 °C followed by 35 amplification cycles of 20 s at 94 °C, 15 s at 62 °C and 30 s at 2 °C and a final extension step for 1 min at 72 °C.

DNA sequences were edited with the DNASTAR computer package and an alignment of the sequences and neighbour joining analyses were performed using the MEGA v. 4 software (Tamura ). To determine the support for each clade, a bootstrap analysis was performed with 1 000 replications. Aspergillus flavus CBS 100927T was used as outgroup in these analyses.

Phylogenetic analysis of sequence data was also performed using PAUP* v. 4.0b10 (Swofford 2000). Alignment gaps were treated as fifth character state, parsimony uninformative characters were excluded and all characters were unordered and equal weight. Maximum parsimony analysis was performed for all data sets using the heuristic search option. To assess the robustness of the topology, 1 000 bootstrap replicates were run by maximum parsimony (Hillis & Bull 1993). Other measures including tree length, consistency index and retention index were also calculated. Sequences were deposited at GenBank under accession numbers listed in Table 1.

UP-PCR analyses were carried out according to Bulat et al. (2000). DNA was isolated as described in the literature (Leach ). The primers used were L45, AS15inv, L15/AS19, AA2M2, L21, 3-2, AS4, AS15 (Lübeck , Bulat ). The amplification process consisted of a predenaturation step for 1 min at 94 °C, followed by 35 cycles (30 s at 94 °C, 45 s at 55 °C, and 1 min at 72 °C), plus a final extension of 2 min at 72 °C. The amplification products were separated by electrophoresis in 1 % agarose gels, stained with ethidium bromide, and visualised under UV light. All amplifications were repeated at least two times. The faint bands which did not appear in all repeated experiments were not counted during cluster analysis.

Altogether 88 fragments were noted and a binomial matrix was created so that presence and absence of DNA fragments were scored as 1 or 0, respectively. Cluster analysis was carried out by using PHYLIP v. 3.67 software package (Felsenstein 2007). Phylogenetical tree was created by using neighbor-joining method (Saitou et al. 1987) with the program NEIGHBOR from the PHYLIP program package.

RESULTS AND DISCUSSION

Phylogenetic analysis of sequence data

The calmodulin data set consisted of 478 characters including 218 parsimony informative sites; MP analysis resulted in 33 most parsimonious trees (length = 621, consistency index = 0.620787, retention index = 0.873655), one of which is presented in Fig. 1. Of the aligned β-tubulin sequences, a portion of 468 positions including 221 parsimony informative characters was selected for the analysis; MP analysis of the sequence data resulted in 29 similar, equally most parsimonious trees (tree length = 464 steps, consistency index = 0.622172, retention index = 0.882394), one of which is shown in Fig. 2. The ITS data set consisted of 478 characters including 81 parsimony informative sites; MP analysis resulted in 211 equally most parsimonious trees (length = 143, consistency index = 0.837989, retention index = 0.957910), one of which is presented in Fig. 3.

Fig. 1.

One of the MP trees obtained based on phylogenetic analysis of calmodulin sequence data of Aspergillus section Nigri. Numbers above branches are bootstrap values. Only values above 70 % are indicated.

Fig. 2.

The single MP tree obtained based on phylogenetic analysis of β-tubulin sequence data of Aspergillus section Nigri. Numbers above branches are bootstrap values. Only values above 70 % are indicated.

Fig. 3.

One of the MP trees obtained based on phylogenetic analysis of ITS sequence data of Aspergillus section Nigri. Numbers above branches are bootstrap values. Only values above 70 % are indicated.

All four species could be distinguished from the currently accepted species of Aspergillus section Nigri (Samson ) based on either calmodulin or β-tubulin sequence data, and isolate CBS 114.80 also exhibited unique ITS sequences (Fig. 3). Aspergillus eucalypticola was found to be related to A. costaricaensis, however, their ITS sequences were different (Fig. 3). Aspergillus violaceofuscus and A. indologenus belonged to a clade including A. uvarum and A. aculeatinus on the tree based on β-tubulin sequence data (Fig. 1), and to a clade including A. uvarum and A. japonicus on the tree based on calmodulin sequence data (Fig. 2).

Sequence data also indicate that both the A. foetidus ex-type strain (CBS 114.49 = NRRL 341) and the neotype strain (CBS 121.28), and A. lacticoffeatus are synonyms of A. niger, while A. coreanus Yu et al. (2004) is a synonym of A. foetidus var. acidus (validly named A. acidus by Kozakiewicz 1989). Aspergillus. lacticoffeatus and A. coreanus differ from other black aspergilli in producing brownish conidia, and not producing naphtho-γ-pyrones. There appears to be a link between the black pigment and the naphtho-γ-pyrones via the gene PksA (Jørgensen et al. 2011, Chiang et al. 2011) Besides, A. coreanus also does not produce antafumicins, produced by some A. acidus isolates. Aspergillus lacticoffeatus also carries the fumonisin biosynthetic genes, similarly to A. niger (Meijer et al., unpubl. data). Based on these observations, we consider A. foetidus and A. lacticoffeatus as synonyms of A. niger, and A. coreanus of A. acidus, respectively.

Our data indicate that Aspergillus section Nigri comprises 26 species including the new species A. saccharolyticus Sørensen et al. (2011). These taxa can be divided into five main clades (called series in Frisvad ; Fig. 1). The A. niger clade includes 10 biseriate species, and was divided into three subclades based on β-tubulin and calmodulin sequence data: the A. tubingensis, A. niger and A. brasiliensis subclades. Only A. niger is known to be able to produce ochratoxin A and fumonisins from this clade (Samson , Frisvad ). Another main clade includes relatives of A. carbonarius (A. ibericus, A. sclerotioniger and A. sclerotiicarbonarius). These species series are characterised by relatively large conidia and two of them, A. carbonarius and A. sclerotioniger are able to produce ochratoxin A. Aspergillus ellipticus and A. heteromorphus form another clade, while the biseriate A. homomorphus forms a distinct clade. All uniseriate species belong to the A. aculeatus clade, involving seven species (Fig. 1).

Extrolites

The extrolites produce by black Aspergillus species have been reviewed by Samson et al. (2007) and Nielsen et al. (2009). The new species described here produce a series of bioactive extrolites, especially the uniseriate species A. indologenus and A. fijiensis.

Aspergillus eucalypticola CBS 122712 produced pyranonigrin A, funalenone, aurasperone B and other naphtho-γ-pyrones, and an unknown extrolite (“MYC”) in common with A. neoniger (CBS 115656 = IBT 20973 and CBS 115657 = IBT 23434). Aspergillus neoniger produced funalenone, naphtho-γ-pyrones, pyranonigrin A and “MYC”, and it is chemically very closely related to A. eucalypticola.

Aspergillus indologenus CBS 114.80 = IBT 3679 produced the insecticidal compounds okaramins A, B, H earlier also reported from an A. aculeatus isolate (Hayashi ), partially characterised polar alkaloids, a series of very apolar sclerotial indol-alkaloids (related to aflavinins) and unique indol-alkaloids with similar UV spectra as the fumitremorgins. Aspergillus violaceofuscus produced some indol-alkaloids also found in A. indologenus, but it also produced several families of partially characterised extrolites that have also been found in A. heteromorphus [“SMIF”, “PON”, “SENGLAB” (a pyranonigrin-related compound), and yellow compounds with characteristic UV spectra]. Aspergillus fijiensis CBS 313.89 and CBS 119.49 produced asperparalins, secalonic acid D, F and the partially characterised “BAM”, “PON” = “FIB1” & “FIB2”, “GLABRINOL”, “SENGLAB”, and “YE1”. CBS 313.89 in addition produced “DERH” and “YE2” and CBS 119.49 additionally produced neoxaline, and “TRU”. Asperparaline A (= aspergillimide = VM55598), asperparaline B and C have earlier been reported from Aspergillus japonicus ATCC 204480 (Hayashi et al. 1997, 2000) and asperparaline A, 16-keto aspergillimide, VM54159, SB203105 and SB 200437 have been isolated from “a black Aspergillus with pink sclerotia” IMI 337664 (Banks ) and neoxaline has been isolated from A. japonicus (Hirano et al. 1979). Based on the extrolite data, ATCC 204480 and IMI 337664 may indeed belong to A. fijiensis, but we have not examined these cultures yet.

Species related to A. niger, such as A. eucalypticola and A. neoniger and the well known species A. carbonarius and A. tubingensis, produce different combinations of pyranonigrins, tensidols, kotanins, fumonisins, funalenones, naphtho-γ-pyrones, ochratoxins, asperazines, and pyrophen (Samson et al. 2004b, 2007), while species related to A. aculeatus, A. aculeatinus, A. japonicus, A. uvarum, and the new species described here, A. indologenus, A. fijiensis and the revived A. violaceofuscus produce different combinations of asperparalins, okaramins, neoxaline, sclerotial indol-alkaloids, and secalonic acids (Parenicova , Samson , Samson , Noonim ).

Approaches to distinguish between isolates of the sibling species A. niger and A. awamori

Aspergillus awamori has recently been revalidated as a cryptic species within the A. niger species (Perrone ). These species cannot be reliably separated from each other using either morphological or extrolite data. However, molecular data including sequence-based approaches using either β-tubulin, calmodulin or translation elongation factor a sequences and AFLP analysis were found to be useful for distinguishing these species (Perrone ). Aspergillus niger and A. awamori are economically important as isolates of both species are able to produce fumonisins and/or ochratoxins (Varga , Perrone ). In view of the importance of these species in mycotoxin contamination of various agricultural products (see below), we examined other possibilities which could be used for the easy identification of these species.

Molecular approaches

UP-PCR analysis (Bulat ) was found to be also useful for species delineation (Fig. 4). This technique is similar to RAPD, but is more reliable as it uses higher annealing temperatures and longer primers (Bulat ). Besides, UP-PCR analysis is easier to perform than AFLP analysis. Similarly to AFLP analysis (Perrone ), this technique could also be used successfully to separate the examined A. niger and A. awamori isolates into two clusters (Fig. 4).

Fig. 4.

Cluster analysis of A. niger and A. awamori isolates based on UP-PCR profiles.

Another possibility is the application of mitochondrial DNA RFLP analyses. This technique was previously used to assign isolates of the A. niger species aggregate to different haplotypes (Varga et al. 1993, 1994). Our study revealed that one of these types previously called mtDNA type 1c actually corresponds to A. awamori (data not shown).

Attempts have also been made to use sequences of mycotoxin biosynthetic genes for distinguishing A. niger from A. awamori. Susca et al. (2010) examined the presence of FUM8 encoding an a-oxoamine synthase in black aspergilli came from grapes. They found no strict correlation between the phylogenetic trees based on sequences of partial calmodulin gene and FUM8 (Fig. 5). Similar results were found in our laboratory using sequences of either FUM8, or another fumonisin biosynthetic gene, FUM1, encoding for a polyketide synthase taking part in fumonisin biosynthesis (Varga et al., unpubl. data). It was suggested that, similarly to that observed in the trichothecene biosynthesis gene cluster of the Fusarium graminearum species complex, balancing selection could be responsible for maintaining sequence polymorphisms within the fumonisin gene cluster (Ward , Susca ).

Fig. 5.

One of the 73 MP trees obtained based on phylogenetic analysis of FUM8 sequence data of A. niger and A. awamori isolates (tree length: 411, consistency index: 0.961240, retention index: 0.980843). Numbers above branches are bootstrap values. Only values above 70 % are indicated.

The applicability of another mycotoxin biosynthetic gene, a chloroperoxidase gene presumably taking part in ochratoxin biosynthesis was also examined for distinguishing A. niger and A. awamori. This gene has been found to take part in ochratoxin biosynthesis in Penicillium verrucosum and P. nordicum (Geisen 2007). Homologues of these genes were identified in the full genome sequences of A. niger and A. carbonarius, and primers were designed to amplify orthologues in species assigned to the A. niger species complex. Phylogenetic analysis of the sequence data indicate that sequences of a chloroperoxidase gene are useful for species delineation in the A. niger species aggregate (Fig. 6). Aspergillus niger and A. awamori could also be distinguished based on their chloroperoxidase sequences.

Fig. 6.

One of the 153 MP trees obtained based on phylogenetic analysis of chloroperoxidase sequence data of A. niger and A. awamori isolates (tree length: 113, consistency index: 0.767677, retention index: 0.886700). Numbers above branches are bootstrap values. Only values above 70 % are indicated.

Morphological and physiological approaches

Molecular methods are commonly used today for species identification among fungi. However, in accordance with the polyphasic species concept, other criteria have also been searched for. Aspergillus niger and A. awamori cannot be distinguished based on morphology alone. Regarding extrolite production, isolates of both species produce several metabolites in common including the mycotoxins ochratoxin A and fumonisin B2, and they also share the production of pyranonigrin A, tensidol B, funalenone, malformins and naphtho-γ-pyrones. The growth rates of the isolates of these species are also similar at different temperatures (Varga et al., unpubl. data).

Carbon source utilisation tests revealed that A. niger and A. awamori has very similar utilisation spectra (data not shown). Different growth of the strains belonging to the two species was observed only in the case of L-sorbose: A. awamori strains grew less intensively on this sugar than A. niger strains. Consequently, the test was extended to 2-deoxy-D-glucose because of the structural similarity of these two compounds. Of the 30 isolates examined, 13 of the examined A. niger isolates grew well, while 13 of the 15 examined A. awamori isolates failed to grow on 2-deoxy-D-glucose as sole carbon source (data not shown). Microscopical analysis of the colonies indicated that conidial germination was inhibited in the case of A. awamori isolates (data not shown). Furthermore 2-deoxy-D-glucose was earlier found to inhibit conidium germination in Penicillium expansum (Kazi ).

Antifungal susceptibilities of the isolates has also been examined using five antifungal drugs including amphotericin B, fluconazole, itraconazole, ketoconazole and terbinafine (Szigeti ). Species-specific differences were not observed between A. niger and A. awamori isolates. All isolates were highly susceptible to terbinafine, while exhibited moderate susceptibilities against amphotericin B, fluconazole and ketoconazole. However, in general, A. niger and A. awamori were found to have higher MICs for azoles than A. tubingensis (Szigeti ).

Elastase production is treated as a virulence factor in Aspergillus fumigatus, contributing to the invasiveness of the fungus during infection (Denning , Kolattukudy , Blanco , Garcia ). Elastase activities of the isolates were tested according to the method of Blanco et al. (2002). The data revealed that A. awamori isolates in general exhibit higher elastase activities in a much narrower range than A. niger (Fig. 7). We found significant difference between the elastase activities of the isolates belonging to the two species (χ2 = 8.017; p=0.0046; Kruskal-Wallis test). In general A. awamori isolates showed more intensive elastin lysis than A. niger isolates, although an outlier was found in the case of an A. niger strain, which exhibited very high elastase activities (Fig. 7). The high elastase activity of this isolate could be due to simple mutations as has been proposed for A. fumigatus recently (Alvarez-Perez ).

Fig. 7.

The difference of elastin lysis between the isolates belonging to A. niger and A. awamori.

Ecology

Several differences have been found recently regarding the distribution of A. niger and A. awamori is various habitats. Both species have been found in various ratios in indoor air in various buildings in Southern Hungary and Serbia, although A. awamori was more frequently encountered in Serbia than in Hungary (Varga J., unpubl. data). Similarly, both species were present on dried vine fruits collected in various countries (Varga ). However, neither A. niger nor A. awamori could be isolated from pistachio nuts from Iran (Sedaghati ), nor from dates from Iran and Tunesia (Varga J., unpubl. data). Aspergillus awamori was found to be the predominant black Aspergillus species on onions cultivated in Hungary, and is presumably the causative agent of black mold rot in this country (Varga et al., unpubl. data), and was also found to be the causative agent of seed rot of Welwitschia mirabilis in Namibia (Varga et al., unpubl. data). However, A. awamori was not detected on figs from Tunesia, Turkey and Iran (Varga J., unpubl. data). Instead, A. tubingensis and A. niger were found to contaminate these fig samples.

Regarding clinical significance, both species have been identified as causative agents of otomycosis in Iran and in Hungary, although at different frequencies, with A. niger being the dominant species in Iran, while A. awamori was most frequently identified in Hungary (Szigeti , unpubl. data).

In conclusion, A. niger and A. awamori are two very closely related species which seem to be in the course of speciation, similarly to the recently described species of Fusarium graminearum sensu lato (Ward , Starkey ). Although these species differ in their occurrence on various substrates and several physiological characteristics (elastase activities, abilities to utilise 2-deoxy-D-glucose as sole carbon source), our data indicate that only molecular approaches including sequence analysis of calmodulin or β-tubulin genes, AFLP analysis, UP-PCR analysis or mtDNA RFLP analysis can be used reliably to distinguish these sibling species.

Species descriptions

Kozak. Mycol. Pap. 161: 110 (1989) Fig. 8.

Fig. 8.

Aspergillus acidus A–C. Colonies incubated at 25 °C for 7 d, A. CYA, B. MEA, C. CREA, D–I. Conidiophores and conidia. Scale bars = 10 μm.

Culture ex-type: IMI 104688 = CBS 564.65, Japan, Nakazawa, 1936.

CYA, 7 d, 25 °C: 37–80 mm; MEA, 7 d, 25 °C: 43–68 mm; YES, 7 d, 25 °C: 38–80 mm; OA, 7 d, 25 °C: 38–55 mm; CYA, 7 d, 37 °C: 30–67 mm; CREA: poor growth but good acid production; CYAS: 16–69 mm (strong sporulation on all media, except CREA). Colony reverse colour on CYA: cream yellow, reverse colour on YES: yellow to cream yellow.

Conidiophores biseriate with globose vesicles 55–80 μm, stipe smooth–walled to finely roughened, hyaline, 17–22 μm. Conidia globose, 3–4 μm, brown, smooth-walled to roughened. Sclerotia not observed.

Kozakiewics (1989) proposed the name A. acidus for the variety acidus of A. foetidus on the basis on the verrucose conidium ornamentation as seen by scanning electron microscopy. This variety was recognised by Raper & Fennell (1965) as a variety of A. citricus. Al-Musallan (1980) however, could not distinguish this variety from the A. niger aggregate.

Aspergillus acidus seems to be the dominant black Aspergillus species on tea leaves (Mogensen ), and has also been identified in human aspergillosis cases (Alcazar-Fuoli ). Aspergillus coreanus, isolated from Korean fermented nuruk, was invalidly described because a Latin diagnosis was lacking. The ex-type strain of this species is morphologically different of A. acidus, because it was described with yellow green colonies. We observed that the colonies were light yellow brown.

Varga, Frisvad & Samson, sp. nov. MycoBank MB560387. Fig. 9.

Fig. 9.

Aspergillus eucalypticola sp. nov. A–C. Colonies incubated at 25 °C for 7 d, A. CYA, B. MEA, C. CREA, D–I. Conidiophores and conidia. Scale bars = 10 μm.

Conidiophoris biseriatis, vesiculis globosis, 30–55 μm diam, stipitibus levibus vel subtiliter exasperatis. hyalinis, 8–14 μm latis. Conidiis globosis, 2.5–3.5 μm, brunneis, levibus vel grosse exasperatis. Sclerotia nulla.

Typus: ex leaves of Eucalyptus sp., New South Wales, Australia, isolated by P.W. Crous, 2007. (CBS H-20627 -- holotypus, culture ex-type CBS 122712 = IBT 29274).

CYA, 7 d, 25 °C: 68–72 mm; MEA, 7 d, 25 °C: 46–51 mm; YES, 7 d, 25 °C: 70–80 mm; OA, 7 d, 25 °C: 45–50 mm; CYA, 7 d, 37 °C: 30–50 mm; CREA: poor growth but good acid production; CYAS: 50–54 mm (strong sporulation on all media, except CREA). Colony reverse colour on CYA: beige to cream yellow, reverse colour on YES: yellow.

Conidiophores biseriate with globose vesicles 30–55 μm, stipe smooth-walled to finely roughened, hyaline, 8–14 μm. Conidia globose, 2.5–3.5 μm, brown, smooth-walled to coarsely roughened. Sclerotia not observed.

Aspergillus eucalypticola was isolated from an Eucalyptus leaf from Australia, and resembles morphologically A. tubingensis and A. costaricaensis. It can be distinguished from these two taxa by the β-tubulin or calmodulin sequence data. Aspergillus eucalypticola produces pyranonigrin A, funalenone, aurasperone B and other naphtho-γ-pyrones.

Varga, Frisvad & Samson, sp. nov. MycoBank MB560388. Fig. 10.

Fig. 10.

Aspergillus fijiensis sp. nov. A–C. Colonies incubated at 25 °C for 7 d, A. CYA, B. MEA, C. CREA, D–I. Conidiophores and conidia. Scale bars = 10 μm.

Conidiophoris uniseriatis, vesiculis globosis vel ellipsoideis, 35–70 μm diam, stipitibus levibus vel subtiliter exasperatis, hyalinis, 8–12 μm latis. Conidiis ellipsoideis vel leniter fusiformibus, 3–3.5 × 3.4–4 μm, brunneis, grosse exasperatis vel echinulatis. Sclerotia nulla.

Typus: ex soil, Fiji Islands, K. Bundgaard. (CBS H-20628 -- holotypus, culture ex-type CBS 313.89 = IBT 13989).

Additional isolate: CBS 119.49 = IBT 4580, ex Lactuca sativa, Palembang, Indonesia).

CYA, 7 d, 25 °C: 71–78 mm; MEA, 7 d, 25 °C: 46–57 mm; YES, 7 d, 25 °C: 74–80 mm; OA, 7 d, 25 °C: 46–56 mm; CYA, 7 d, 37 °C: 12–25 mm; CREA: poor growth but moderate acid production; CYAS: 52–57 mm (strong sporulation on all media, except CREA). Colony reverse colour on CYA: beige to yellow, reverse colour on YES: yellow.

Conidiophores uniseriate with globose to ellipsoidal vesicles 35–70 μm wide, stipe smooth-walled to finely roughened, hyaline, 8–12 μm. Conidia ellipsoidal to slightly fusiform, 3–3.5 × 3.4–4 μm, brown, coarsely roughened to echinulate. Sclerotia not observed.

Aspergillus fijiensis is characterised by uniseriate conidial heads and is related to A. aculeatinus. It was isolated from soil in Fiji, and from Lactuca sativa in Indonesia. This species is able to grow at 37 °C, and produces asperparalines and okaramins.

Frisvad, Varga & Samson, sp. nov. MycoBank MB560389. Fig. 11.

Fig. 11.

Aspergillus indologenus sp. nov. A–C. Colonies incubated at 25 °C for 7 d, A. CYA, B. MEA, C. CREA, D–I. Conidiophores and conidia. Scale bars = 10 μm.

Conidiophoris uniseriatis, vesiculis ellipsoideis, 20–45 μm diam, stipitibus levibus vel subtiliter exasperatis, hyalinis, 5–11 μm latis. Conidiis globosis, 3–4 μm diam, brunneis, grosse exasperatis vel echinulatis. Sclerotia nulla.

Typus: ex soil India (CBS H-20629 -- holotypus, culture ex-type CBS 114.80 = IBT 3679).

CYA, 7 d, 25 °C: 63–70 mm; MEA, 7 d, 25 °C: 57–70 mm; YES, 7 d, 25 °C: 76–80 mm; OA, 7 d, 25 °C: 50–58 mm; CYA, 7 d, 37 °C: 20–33 mm; CREA: poor growth and no acid production; CYAS: 1–2 mm (strong sporulation on all media, except CYAS and CREA). Colony reverse colour on CYA: dark brown, reverse colour on YES: brown.

Conidiophores uniseriate with ellipsoidal vesicles 20–45 μm wide, stipe smooth-walled to finely roughened, hyaline, 5–11 μm. Conidia globose, 3–4 μm, brown, coarsely roughened to echinulate. Sclerotia not observed.

Aspergillus indologenus has uniseriate conidial heads and is related to, but clearly distinguishable from A. uvarum based on β-tubulin, calmodulin and ITS sequence data. Aspergillus uvarum has typical echinulate conida, while A. indologenus has conidia which are coarsely roughenmed to echinulate. Aspergillus indologenus produced the insecticidal compounds okaramins A, B, H, and two types of indol-alkaloids which have not been structure elucidated.

Varga, Frisvad & Samson, sp. nov. MycoBank MB560390. Fig. 12.

Fig. 12.

Aspergillus neoniger sp. nov. A–C. Colonies incubated at 25 °C for 7 d, A. CYA, B. MEA, C. CREA, D–I. Conidiophores and conidia. Scale bars = 10 μm.

Conidiophoris biseriatis, vesiculis globosis, 30–50 μm diam, stipitibus levibus vel subtiliter exasperatis, hyalinis, 8–12 μm. Conidiis globosis, 3.5–5 μm diam, brunneis, grosse exasperatis vel echinulatis. Sclerotia nulla.

Typus: ex Verongia species (sulphur sponge, Porifera), Morro of Garrapatá, Mochima Bay, Venezuela, isolated by E.K. Lyhne, 1997. (CBS H-20630 -- holotypus, culture ex-type CBS 115656 = IBT 20973 = Ven97 M64).

Other isolate: CBS 115657, ex desert soil, Namibia.

CYA, 7 d, 25 °C: 72–80 mm; MEA, 7 d, 25 °C: 54–61 mm; YES, 7 d, 25 °C: 74–80 mm; OA, 7 d, 25 °C: 50–72 mm; CYA, 7 d, 37 °C: 37–67 mm; CREA: poor growth but good acid production; CYAS: 50–54 mm (strong sporulation on all media, except CREA). Colony reverse colour on CYA: beige to cream yellow, reverse colour on YES: yellow.

Conidiophores biseriate with globose vesicles 30–50 μm, stipe smooth-walled to finely roughened, hyaline, 8–12 μm. Conidia globose, 3.5–5 μm, brown, coarsely roughened to echinulate. Sclerotia not observed.

Aspergillus neoniger is also a biseriate species isolated from desert sand in Namibia, and mangrove water in Venezuela. Morphologicaly it resembles A. niger and A. tubingensis and produces aurasperone B and pyranonigrin A. This species has also been identified in desert soil close to Welwitschia mirabilis, Namibia (Varga et al., unpubl. data).

Gasperini, Atti della Societa Toscana di Scienze Naturali 2: 326 (1887). Fig. 13.

Fig. 13.

Aspergillus violaceofuscus A–C. Colonies incubated at 25 °C for 7 d, A. CYA, B. MEA, C. CREA, D–I. Conidiophores and conidia. Scale bars = 10 μm.

Culture ex-neotype: CBS 123.27 = Thom 3522.30, ex soil, Puerto Rico, deposited by M.B. Church.

CYA, 7 d, 25 °C: 50–77 mm; MEA, 7 d, 25 °C: 64–74 mm; YES, 7 d, 25 °C: 74–80 mm; OA, 7 d, 25 °C: 36–74 mm; CYA, 7 d, 37 °C: 0 mm (but 28–32 mm in CBS 122.35); CREA: poor growth and no acid production; CYAS: 0 mm (but 46–50 mm in CBS 115571) (strong sporulation on all media, except CYAS and CREA). Colony reverse colour on CYA: brown to cream yellow, reverse colour on YES: cream yellow.

Conidiophores uniseriate with globose vesicles 10–18 μm wide; stipe sometimes bent, smooth-walled to finely roughened, hyaline, 2.5–5 μm. Conidia ellipsoidal to slightly fusiform, 3.5–4 × 4–5.5 μm, brown, coarsely roughened to echinulate. Sclerotia not observed.

Raper & Fennell (1965) considered A. violaceofuscus as a possible synonym of A. aculeatus. In our study we have observed that the neotype strain is phylogenetically different from A. aculeatus. Aspergilus violaceofuscus has typical ellipsoidal to fusiform conidia, which are coarsely roughened to echinulate.