Sándor Kocsubé

Phylogeny, identification and nomenclature of the genus Aspergillus

Aspergillus comprises a diverse group of species based on morphological, physiological and phylogenetic characters, which significantly impact biotechnology, food production, indoor environments and human health. Aspergillus was traditionally associated with nine teleomorph genera, but phylogenetic data suggest that together with genera such as Polypaecilum, Phialosimplex, Dichotomomyces and Cristaspora, Aspergillus forms a monophyletic clade closely related to Penicillium. Changes in the International Code of Nomenclature for algae, fungi and plants resulted in the move to one name per species, meaning that a decision had to be made whether to keep Aspergillus as one big genus or to split it into several smaller genera. The International Commission of Penicillium and Aspergillus decided to keep Aspergillus instead of using smaller genera. In this paper, we present the arguments for this decision. We introduce new combinations for accepted species presently lacking an Aspergillus name and provide an updated accepted species list for the genus, now containing 339 species. To add to the scientific value of the list, we include information about living ex-type culture collection numbers and GenBank accession numbers for available representative ITS, calmodulin, β-tubulin and RPB2 sequences. In addition, we recommend a standard working technique for Aspergillus and propose calmodulin as a secondary identification marker.

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.

Aspergillus brasiliensis sp. nov., a biseriate black Aspergillus species with world-wide distribution

A novel species, Aspergillus brasiliensis sp. nov., is described within Aspergillus section Nigri. This species can be distinguished from other black aspergilli based on intergenic transcribed region, beta-tubulin and calmodulin gene sequences, by amplified fragment length polymorphism analysis and by extrolite profiles. A. brasiliensis isolates produced naphtho-gamma-pyrones, tensidol A and B and pyrophen in common with Aspergillus niger and Aspergillus tubingensis, but also several unique compounds, justifying their treatment as representing a separate species. None of the isolates were found to produce ochratoxin A, kotanins, funalenone or pyranonigrins. The novel species was most closely related to A. niger, and was isolated from soil from Brazil, Australia, USA and The Netherlands, and from grape berries from Portugal. The type strain of Aspergillus brasiliensis sp. nov. is CBS 101740(T) (=IMI 381727(T)=IBT 21946(T)).

Aspergillus uvarum sp nov., an uniseriate black Aspergillus species isolated from grapes in Europe

A novel species, Aspergillus uvarum sp. nov., is described within Aspergillus section Nigri. This species can be distinguished from other black aspergilli based on internal transcribed spacers (ITS), beta-tubulin and calmodulin gene sequences, by AFLP analysis and by extrolite profiles. Aspergillus uvarum sp. nov. isolates produced secalonic acid, common to other Aspergillus japonicus-related taxa, and geodin, erdin and dihydrogeodin, which are not produced by any other black aspergilli. None of the isolates were found to produce ochratoxin A. The novel species is most closely related to two atypical strains of Aspergillus aculeatus, CBS 114.80 and CBS 620.78, and was isolated from grape berries in Portugal, Italy, France, Israel, Greece and Spain. The type strain of Aspergillus uvarum sp. nov. is IMI 388523T=CBS 127591T=ITEM 4834T=IBT26606T.

Fumonisin contamination and fumonisin producing black Aspergilli in dried vine fruits of different origin.

Aspergillus niger isolates are able to produce fumonisins in high quantities on agar media with a low water activity. Several agricultural products fit this criterion, including dried vine fruits, dates and figs. Data on the occurrence and role of this species in fumonisin contamination of agricultural products with high sugar content are needed to clarify the importance of A. niger in human health. The mycobiota and fumonisin contamination of various dried vine fruit samples collected from different countries were examined to clarify the role of black Aspergilli in fumonisin contamination of such products. All except two of the examined samples were contaminated with black Aspergilli. Species assignment of the isolates was carried out using sequence analysis of part of the calmodulin gene. The range of fumonisin isomers present in the raisins samples, and produced by A. niger isolates collected from dried vine fruits was also examined using reversed-phase high-performance liquid chromatography/electrospray ionization-ion trap mass spectrometry (RP-HPLC/ESI-ITMS). Among the 30 A. niger/A. awamori isolates identified, 20 were found to be able to produce fumonisins (average contamination: 5.16 mg/kg; range: 0.017-19.6 mg/kg). The average fumonisin content of the 7 dried vine fruit samples which were found to be contaminated by potential fumonisin producing black Aspergilli was 7.22 mg/kg (range: 4.55-35.49 mg/kg). The isolates produced several fumonisin isomers also present in the dried vine fruit samples, including fumonisins B(1-4), 3-epi-FB(3), 3-epi-FB(4), iso-FB(1), and two iso-FB(2,3) forms. Fumonisin B(1) was detected for the first time in A. niger cultures. Most of these isomers have previously only been identified in Fusarium species. Our data indicate that A. niger and A. awamori are responsible for fumonisin contamination of dried vine fruits worldwide. The observed levels of contamination are alarming and pose a new threat for food safety.

Diversity of polyketide synthase gene sequences in Aspergillus species.

Fungal polyketide synthases are responsible for the biosynthesis of several mycotoxins and other secondary metabolites. The aim of our work was to investigate the diversity of polyketide synthases in Aspergillus species using two approaches: PCR amplification using oligonucleotide primers, and bioinformatics. Ketosynthase domain probes amplified DNA fragments of about 700 bp in each examined isolate. Sequences of these domains were aligned and analyzed by phylogenetic methods. The ketosynthase domain sequences were highly diverse indicating that they most probably represent polyketide synthases responsible for different functions. A. albertensis and A. niger ketosynthase domain sequences clustered together with sequences of genes required for pigment biosynthesis (wA) in A. nidulans and P. patulum, while the ketosynthase domain sequence of A. muricatus was most closely related to an A. parasiticus wA type domain sequence, and those of the A. ochraceus isolates formed a distinct clade on the tree. These sequences were highly homologous to an A. terreus naphthopyrone synthase gene. An Aspergillus fumigatus genomic database was also searched for ketosynthase domain sequences, which have been included in the phylogenetic analysis. Altogether 14 putative ketosynthase domain sequences were identified. Clustering of the ketosynthase domain sequences correlated well with the type of metabolites produced by the corresponding polyketide synthases. At least 8 clusters with putative ketosynthase domain sequences of unknown function have been identified. Further studies are in progress to clarify the role of some of the identified polyketide synthase genes.

Ochratoxin production and taxonomy of the yellow aspergilli (Aspergillus section Circumdati)

Aspergillus section Circumdati or the Aspergillus ochraceus group, includes species with rough walled stipes, biseriate conidial heads, yellow to ochre conidia and sclerotia that do not turn black. Several species are able to produce mycotoxins including ochratoxins, penicillic acids, and xanthomegnins. Some species also produce drug lead candidates such as the notoamides. A polyphasic approach was applied using morphological characters, extrolite data and partial calmodulin, β-tubulin and ITS sequences to examine the evolutionary relationships within this section. Based on this approach the section Circumdati is revised and 27 species are accepted, introducing seven new species: A. occultus, A. pallidofulvus, A. pulvericola, A. salwaensis, A. sesamicola, A. subramanianii and A. westlandensis. In addition we correctly apply the name A. fresenii (≡ A. sulphureus (nom. illeg.)). A guide for the identification of these 27 species is provided. These new species can be distinguished from others based on morphological characters, sequence data and extrolite profiles. The previously described A. onikii and A. petrakii were found to be conspecific with A. ochraceus, whilst A. flocculosus is tentatively synonymised with A. ochraceopetaliformis, despite extrolite differences between the two species. Based on the extrolite data, 13 species of section Circumdati produce large amounts of ochratoxin A: A. affinis, A. cretensis, A. fresenii, A. muricatus, A. occultus, A. ochraceopetaliformis (A. flocculosus), A. ochraceus, A. pseudoelegans, A. pulvericola, A. roseoglobulosus, A. sclerotiorum, A. steynii and A. westerdijkiae. Seven additional species produce ochratoxin A inconsistently and/or in trace amounts: A. melleus, A. ostianus, A. persii, A. salwaensis, A. sesamicola, A. subramanianii and A. westlandensis. The most important species regarding potential ochratoxin A contamination in agricultural products are A. ochraceus, A. steynii and A. westerdijkiae.

Evolutionary relationships among Aspergillus terreus isolates and their relatives

Aspergillus terreus is a ubiquitous fungus in our environment. It is an opportunistic human pathogen and economically important as the main producer of lovastatin, a cholesterol lowering drug. Our aim was to examine the genetic variability of A. terreus and closely related species using molecular and analytical techniques. Lovastatin production was examined by HPLC. Lovastatin was produced by seven isolates belonging to the species A. terreus. RAPD analyses were carried out using 25 different random primers. Neighbor-joining analysis of RAPD data (120 characters) resulted in clustering of the A. terreus isolates into distinct groups. Some correlation was observed between lovastatin producing abilities of the isolates and their position on the dendrogram based on RAPD profiles. The internal transcribed spacer region and the 5.8S rRNA gene of A. terreus and related isolates was also sequenced. Phylogenetic analysis of sequence data let us classify the isolates into different clades which mostly correspond to the species Aspergillus terreus, Aspergillus flavipes, Aspergillus niveus, Aspergillus carneus and Aspergillus janus/A. janus var. brevis. Aspergillus allahabadii, A. terreus var. aureus and A. niveus var. indicus belonged to the A. niveus clade, while an Aspergillus isolate previously classified as A. niveus was most closely related to A. flavipes isolates. Aspergillus anthodesmis formed a distinct branch on the tree. Although it was previously suggested based on 28S rDNA sequence data that Aspergillus section Terrei should include A. carneus and A. niveus isolates, phylogenetic analysis of ITS sequences indicate that A. flavipes isolates are more closely related to A. terreus than A. carneus isolates. Our data suggest that sections Terrei and Flavipedes should be merged. However, further loci should be analysed to draw more definite conclusions.

Re-mind the gap! Insertion - deletion data reveal neglected phylogenetic potential of the nuclear ribosomal internal transcribed spacer (ITS) of fungi.

Rapidly evolving, indel-rich phylogenetic markers play a pivotal role in our understanding of the relationships at multiple levels of the tree of life. There is extensive evidence that indels provide conserved phylogenetic signal, however, the range of phylogenetic depths for which gaps retain tree signal has not been investigated in detail. Here we address this question using the fungal internal transcribed spacer (ITS), which is central in many phylogenetic studies, molecular ecology, detection and identification of pathogenic and non-pathogenic species. ITS is repeatedly criticized for indel-induced alignment problems and the lack of phylogenetic resolution above species level, although these have not been critically investigated. In this study, we examined whether the inclusion of gap characters in the analyses shifts the phylogenetic utility of ITS alignments towards earlier divergences. By re-analyzing 115 published fungal ITS alignments, we found that indels are slightly more conserved than nucleotide substitutions, and when included in phylogenetic analyses, improved the resolution and branch support of phylogenies across an array of taxonomic ranges and extended the resolving power of ITS towards earlier nodes of phylogenetic trees. Our results reconcile previous contradicting evidence for the effects of data exclusion: in the case of more sophisticated indel placement, the exclusion of indel-rich regions from the analyses results in a loss of tree resolution, whereas in the case of simpler alignment methods, the exclusion of gapped sites improves it. Although the empirical datasets do not provide to measure alignment accuracy objectively, our results for the ITS region are consistent with previous simulations studies alignment algorithms. We suggest that sophisticated alignment algorithms and the inclusion of indels make the ITS region and potentially other rapidly evolving indel-rich loci valuable sources of phylogenetic information, which can be exploited at multiple taxonomic levels.

Chemical, Physical and Biological Approaches to Prevent Ochratoxin Induced Toxicoses in Humans and Animals

Ochratoxins are polyketide derived fungal secondary metabolites with nephrotoxic, immunosuppressive, teratogenic, and carcinogenic properties. Ochratoxin-producing fungi may contaminate agricultural products in the field (preharvest spoilage), during storage (postharvest spoilage), or during processing. Ochratoxin contamination of foods and feeds poses a serious health hazard to animals and humans. Several strategies have been investigated for lowering the ochratoxin content in agricultural products. These strategies can be classified into three main categories: prevention of ochratoxin contamination, decontamination or detoxification of foods contaminated with ochratoxins, and inhibition of the absorption of consumed ochratoxins in the gastrointestinal tract. This paper gives an overview of the strategies that are promising with regard to lowering the ochratoxin burden of animals and humans.