Aspergillus is monophyletic: Evidence from multiple gene phylogenies and extrolites profiles

Abstract Aspergillus is one of the economically most important fungal genera. Recently, the ICN adopted the single name nomenclature which has forced mycologists to choose one name for fungi (e.g. Aspergillus, Fusarium, Penicillium, etc.). Previously two proposals for the single name nomenclature in Aspergillus were presented: one attributes the name “Aspergillus” to clades comprising seven different teleomorphic names, by supporting the monophyly of this genus; the other proposes that Aspergillus is a non-monophyletic genus, by preserving the Aspergillus name only to species belonging to subgenus Circumdati and maintaining the sexual names in the other clades. The aim of our study was to test the monophyly of Aspergilli by two independent phylogenetic analyses using a multilocus phylogenetic approach. One test was run on the publicly available coding regions of six genes (RPB1, RPB2, Tsr1, Cct8, BenA, CaM), using 96 species of Penicillium, Aspergillus and related taxa. Bayesian (MrBayes) and Ultrafast Maximum Likelihood (IQ-Tree) and Rapid Maximum Likelihood (RaxML) analyses gave the same conclusion highly supporting the monophyly of Aspergillus. The other analyses were also performed by using publicly available data of the coding sequences of nine loci (18S rRNA, 5,8S rRNA, 28S rRNA (D1-D2), RPB1, RPB2, CaM, BenA, Tsr1, Cct8) of 204 different species. Both Bayesian (MrBayes) and Maximum Likelihood (RAxML) trees obtained by this second round of independent analyses strongly supported the monophyly of the genus Aspergillus. The stability test also confirmed the robustness of the results obtained. In conclusion, statistical analyses have rejected the hypothesis that the Aspergilli are non-monophyletic, and provided robust arguments that the genus is monophyletic and clearly separated from the monophyletic genus Penicillium. There is no phylogenetic evidence to split Aspergillus into several genera and the name Aspergillus can be used for all the species belonging to Aspergillus i.e. the clade comprising the subgenera Aspergillus, Circumdati, Fumigati, Nidulantes, section Cremei and certain species which were formerly part of the genera Phialosimplex and Polypaecilum. Section Cremei and the clade containing Polypaecilum and Phialosimplex are proposed as new subgenera of Aspergillus. The phylogenetic analysis also clearly shows that Aspergillus clavatoflavus and A. zonatus do not belong to the genus Aspergillus. Aspergillus clavatoflavus is therefore transferred to a new genus Aspergillago as Aspergillago clavatoflavus and A. zonatus was transferred to Penicilliopsis as P. zonata. The subgenera of Aspergillus share similar extrolite profiles indicating that the genus is one large genus from a chemotaxonomical point of view. Morphological and ecophysiological characteristics of the species also strongly indicate that Aspergillus is a polythetic class in phenotypic characters

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

AbstractAspergillus 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

Phylogeny, identification and nomenclature of the genus Aspergillus

AbstractAspergillus 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

Mycobiota and ochratoxin A in raisins purchased in Hungary

Ochratoxin A is a mycotoxin produced by Aspergillus and Penicillium species. This mycotoxin is a common contaminant of various foods including cereal products, spices, dried fruits, coffee, beer and wine. Besides cereal products, goods of grape origin contribute significantly to ochratoxin exposure of humans. The ochratoxin content and mycobiota of raisins purchased in Hungarian outlets were examined in this study. Ochratoxin A content was examined by an immunochemical technique, and the results were confirmed by HPLC analysis using fluorescent detection. Altogether 20 raisin samples were analyzed. Ochratoxin A was detected in all but two samples with ochratoxin concentrations ranging from 0 to 6.2 mg kg-1. The most heavily contaminated raisin sample came from Iran. However, none of the raisins contained ochratoxin A above 10 mg kg-1, the European Community maximum allowable limit in raisins. The mycobiota of raisin samples was also examined to clarify which species could be responsible for ochratoxin A contamination. All except three raisin samples were contaminated with black aspergilli, some of which produced ochratoxin A. Besides A. carbonarius, ochratoxin producing A. tubingensis isolates dominated in the samples

Nonribosomal peptide synthetase genes in the genome of Fusarium graminearum, causative agent of wheat head blight

Fungal nonribosomal peptide synthetases (NRPSs) are responsible for the biosynthesis of numerous metabolites which serve as virulence factors in several plant-pathogen interactions. The aim of our work was to investigate the diversity of these genes in a Fusarium graminearum sequence database using bioinformatic techniques. Our search identified 15 NRPS sequences, among which two were found to be closely related to peptide synthetases of various fungi taking part in ferrichrome biosynthesis. Another peptide synthetase gene was similar to that identified in Aspergillus oryzae which is possibly responsible for the biosynthesis of fusarinine, an extracellular iron-chelating siderophore. To our knowledge, this is the first report on the identification of a putative NRPS gene possibly responsible for the biosynthesis of fusarinine-type siderophores. The other NRPSs were found to be related to peptide synthetases taking part in the biosynthesis of various peptides in other fungi. Transcription factors carrying ankyrin repeats were observed in the vicinity of four of the identified peptide synthetase genes. Additionally, NRPS related genes similar to putative long-chain fatty acid CoA ligases, acyl CoA ligases, ABC transport proteins, a highly conserved putative transmembrane protein of Aspergillus nidulans, and a-aminoadipate reductases have also been identified. Further studies are in progress to clarify the role of some of the identified NRPS genes in plant pathogenesis

Strain-specific SCAR markers for the detection of Trichoderma harzianum AS12-2, a biological control agent against Rhizocto nia solani, the causal agent of rice sheath blight

In order to identify a specific marker for T. harzianum AS12-2, a strain capable of controlling rice sheath blight caused by Rhizoctonia solani, UP-PCR was performed using five universal primers (UP) both separately and in pairwise combinations. The application of two UP primers resulted in the amplification of unique fragments from the genomic DNA of T. harzianum AS12-2, clearly distinguishing it from other Trichoderma strains. The unique fragments had no significant sequence homology with any other known sequence available in databases. Based on the sequences of the unique fragments, 14 oligonucleotide primers were designed. Two primer sets amplified a fragment of expected size from the DNA of strain T. harzianum AS12-2 but not from any other examined strains belonging to T. harzianum, to other Trichoderma species assayed, or to other common fungi present in paddy fields of Mazandaran province, Iran. In conclusion, SCAR (sequence characterized amplified regions) markers were successfully identified and rapid, reliable tools were provided for the detection of an effective biocontrol Trichoderma strain, which can facilitate studies of its population dynamics and establishment after release into the natural environment

Aspergillus section Nidulantes (formerly Emericella): polyphasic taxonomy, chemistry and biology

Aspergillus section Nidulantes includes species with striking morphological characters, such as biseriate conidiophores with brown-pigmented stipes, and if present, the production of ascomata embedded in masses of Hülle cells with often reddish brown ascospores. The majority of species in this section have a sexual state, which were named Emericella in the dual name nomenclature system. In the present study, strains belonging to subgenus Nidulantes were subjected to multilocus molecular phylogenetic analyses using internal transcribed spacer region (ITS), partial β-tubulin (BenA), calmodulin (CaM) and RNA polymerase II second largest subunit (RPB2) sequences. Nine sections are accepted in subgenus Nidulantes including the new section Cavernicolus. A polyphasic approach using morphological characters, extrolites, physiological characters and phylogeny was applied to investigate the taxonomy of section Nidulantes. Based on this approach, section Nidulantes is subdivided in seven clades and 65 species, and 10 species are described here as new. Morphological characters including colour, shape, size, and ornamentation of ascospores, shape and size of conidia and vesicles, growth temperatures are important for identifying species. Many species of section Nidulantes produce the carcinogenic mycotoxin sterigmatocystin. The most important mycotoxins in Aspergillus section Nidulantes are aflatoxins, sterigmatocystin, emestrin, fumitremorgins, asteltoxins, and paxillin while other extrolites are useful drugs or drug lead candidates such as echinocandins, mulundocandins, calbistrins, varitriols, variecolins and terrain. Aflatoxin B1 is produced by four species: A. astellatus, A. miraensis, A. olivicola, and A. venezuelensis