Jens Christian Frisvad

Identification and nomenclature of the genus Penicillium.

Penicillium is a diverse genus occurring worldwide and its species play important roles as decomposers of organic materials and cause destructive rots in the food industry where they produce a wide range of mycotoxins. Other species are considered enzyme factories or are common indoor air allergens. Although DNA sequences are essential for robust identification of Penicillium species, there is currently no comprehensive, verified reference database for the genus. To coincide with the move to one fungus one name in the International Code of Nomenclature for algae, fungi and plants, the generic concept of Penicillium was re-defined to accommodate species from other genera, such as Chromocleista, Eladia, Eupenicillium, Torulomyces and Thysanophora, which together comprise a large monophyletic clade. As a result of this, and the many new species described in recent years, it was necessary to update the list of accepted species in Penicillium. The genus currently contains 354 accepted species, including new combinations for Aspergillus crystallinus, A. malodoratus and A. paradoxus, which belong to Penicillium section Paradoxa. To add to the taxonomic value of the list, we also provide information on each accepted species MycoBank number, living ex-type strains and provide GenBank accession numbers to ITS, β-tubulin, calmodulin and RPB2 sequences, thereby supplying a verified set of sequences for each species of the genus. In addition to the nomenclatural list, we recommend a standard working method for species descriptions and identifications to be adopted by laboratories working on this genus.

Phylogenetic analysis of nucleotide sequences from the ITS region of terverticillate Penicillium species

The genetic variability within 600 bp of DNA sequence from the ribosomal internal transcribed spacers (ITS 1 and ITS 2) and the 5.8 S rRNA gene was examined in 52 strains belonging to 29 terverticillate taxa in Penicillium subg. Penicillium from diverse sources and locations. The sequenced region is extremely conserved within the terverticillate penicillia, with only 29 positions differing in one or more taxa. The highest degree of ITS variability was seen among the species close to Penicillium roqueforti ( P. roqueforti, P. carneum and P. paneum ) whereas the ITS variability was very low between closely related taxa, e.g. among the taxa near P. aurantiogriseum. The relationships among the terverticillate penicillia, and related teleomorphs in Eupenicillium , were analysed from bootstrapped ITS sequence data sets using the neighbour-joining method. The terverticillate penicillia form a well supported clade with Eupenicillium crustaceum. Sequence analysis generally confirmed the overall taxonomy in Penicillium subg. Penicillium but relationships between all the terverticillate taxa could not be clearly established due to the low degree of ITS variability. Nevertheless, clades of Penicillium species sharing environmental characteristics did emerge, e.g. species growing on protein and lipid rich substrates (e.g. P. crustosum and P. commune ), species prevalent in dry habitats (e.g. P. chrysogenum and P. nalgiovense ), and those prevalent on carbohydrate rich substrates (e.g. P. aurantiogriseum and P. freii ).

A critical review of producers of small lactone mycotoxins: patulin, penicillic acid and moniliformin

A very large number of filamentous fungi has been reported to produce the small lactone mycotoxins patulin, penicillic acid and moniliformin. Among the 167 reported fungal producers of patulin, only production by 29 species could be confirmed. Patulin is produced by 3 Aspergillus species, 3 Paecilomyces species, 22 Penicillium species from 7 sections of Penicillium, and one Xylaria species. Among 101 reported producers of penicillic acid, 48 species could produce this mycotoxin. Penicillic acid is produced by 23 species in section Aspergillus subgenus Circumdati section Circumdati, by Malbranchea aurantiaca and by 24 Penicillium species from 9 sections in Penicillium and one species that does not actually belong to Penicillium (P. megasporum). Among 40 reported producers of moniliformin, five species have been regarded as doubtful producers of this mycotoxin or are now regarded as taxonomic synonyms. Moniliformin is produced by 34 Fusarium species and one Penicillium species. All the accepted producers of...

On the safety of Aspergillus niger - a review

Abstract.Aspergillus niger is one of the most important microorganisms used in biotechnology. It has been in use already for many decades to produce extracellular (food) enzymes and citric acid. In fact, citric acid and many A. niger enzymes are considered GRAS by the United States Food and Drug Administration. In addition, A. niger is used for biotransformations and waste treatment. In the last two decades, A. niger has been developed as an important transformation host to over-express food enzymes. Being pre-dated by older names, the name A. niger has been conserved for economical and information retrieval reasons and there is a taxonomical consensus based on molecular data that the only other common species closely related to A. niger in the Aspergillus series Nigri is A. tubingensis. A. niger, like other filamentous fungi, should be treated carefully to avoid the formation of spore dust. However, compared with other filamentous fungi, it does not stand out as a particular problem concerning allergy or mycopathology. A few medical cases, e.g. lung infections, have been reported, but always in severely immunocompromised patients. In tropical areas, ear infections (otomycosis) do occur due to A. niger invasion of the outer ear canal but this may be caused by mechanical damage of the skin barrier. A. niger strains produce a series of secondary metabolites, but it is only ochratoxin A that can be regarded as a mycotoxin in the strict sense of the word. Only 3–10% of the strains examined for ochratoxin A production have tested positive under favourable conditions. New and unknown isolates should be checked for ochratoxin A production before they are developed as production organisms. It is concluded, with these restrictions, that A. niger is a safe production organism.

Standardized high-performance liquid chromatography of 182 mycotoxins and other fungal metabolites based on alkylphenone retention indices and UV—VIS spectra (diodearray detection)

Abstract A general standardized method for the analysis of mycotoxins and other fungal secondary metabolites has been developed, based on high-performance liquid chromatography (HPLC) with an alkulphenone retention index and photodiode-array detection combined with thin-layer chromatography (TLC) in two different eluents. Each fungal secondary metabolite is characterized by its bracketed alkylphenone retention time index, its UV—VIS absorption maxima and its retardation factors relative to griseofulvin in two TLC eluents. This system is effective for the comparison of chemotaxonomic data in different laboratories and for a precise identification of fungi based on organic solvent extracts of fungal cultures. All important groups of mycotoxins and other fungal secondary metabolites could be detected in the HPLC system described and data are listed for 182 metabolites. The fungal secondary metabolites separated and characterized include aflatoxin B 1 , B 2 , G 1 and G 2 , ochratoxin A, citrinin, penicillin acid, viomellein, penitrem A, patulin, sterigmatocystin, alternariol, tenuazonic acid, trichothecenes, requefortines, fusarin C, zearalenone, PR-toxin, citreoviridin, viridicatumtoxin, verruculogen, rugulosin, cyclopiazonic acid, penicillin G and many other alkaloids, polyketides and terpenes.

TERVERTICILLATE PENICILLIA: CHEMOTAXONOMY AND MYCOTOXIN PRODUCTION

A total of 4346 isolates of terverticillate Penicillium species was examined for production of my? cotoxins and other important secondary metabolites. Nearly all taxa and chemotypes (38 in all) produced one or more known mycotoxins consistently. Profiles of secondary metabolites were specific for each taxon, but several mycotoxins were produced by more than one species, suggesting a high degree of relatedness in the terverticillate penicillia. A high number of misidentified Penicillium isolates were reidentified, clarifying connections between taxa and profiles of mycotoxins and other secondary me? tabolites. Confirmed production of several mycotoxins in different taxa is reported for the first time. Penicillium chrysogenum, P. atramentosum, P. coprophilum, P. crustosum, P. expansum, P. glandicola var. glandicola and var. glaucovenetum, P. griseofulvum, P. hirsutum var. hirsutum, var. albocoremium, var. allii and var. hordei, and P. vulpinum are reported for the first time to be consistent producers of roquefortine C. Griseofulvin is reported from P. coprophilum. Tremorgen penitrem A was consistently produced by P. crustosum, P. glandicola var. glandicola and P. clavigerum. Terrestric acid is reported from P. hirsutum and its varieties, and P. crustosum. P. hirsutum var. hirsutum and P. solitum produce compactin. Penicillium mononematosum produces cyclopaldic acid and isochromantoxin. P. expansum was found to produce the mycotoxin chaetoglobosin C and P. atramentosum produces rugulovasine A. Several new varieties, chemotypes and combinations are proposed, based on profiles of secondary metabolites, morphology and physiological characters. Living ex type and authentic cultures of taxa believed to be synonyms of important species were examined and revised lists of synonyms are pre? sented.

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.

LaeA, a Regulator of Morphogenetic Fungal Virulence Factors

ABSTRACT Opportunistic animal and plant pathogens, well represented by the genus Aspergillus, have evolved unique mechanisms to adapt to and avoid host defenses. Aspergillus fumigatus, an increasingly serious pathogen owing to expanding numbers of immunocompromised patients, causes the majority of human infections; however, an inability to identify bona fide virulence factors has impeded therapeutic advances. We show that an A. fumigatus mutation in a developmentally expressed transcriptional regulator (ΔlaeA) coordinating morphological and chemical differentiation reduces virulence in a murine model; impaired virulence is associated with decreased levels of pulmonary gliotoxin and multiple changes in conidial and hyphal susceptibility to host phagocytes ex vivo. LaeA, a conserved protein in filamentous fungi, is a developmental regulator of virulence genes and, possibly, the first antimicrobial target specific to filamentous fungi that are pathogenic to plants and animals.

Diagnostic tools to identify black aspergilli

The present taxonomy of the black aspergilli reveals that there are 19 accepted taxa. However the identification of species of Aspergillus section Nigri is often problematic in spite of the existence of numerous methods proposed. An overview is provided of phenotypic and molecular methods to identify the accepted species of the black aspergilli. Colony morphology, conidial size and ornamentation of the ex type cultures is presented in a pictorial overview. The temperature range of all species is given and their growth characteristics on creatine agar and boscalid agar, a medium which was developed as a selective medium for the isolation of A. carbonarius are also shown. The extrolites produced by each species are listed while the response of the Ehrlich reaction is described. The literature on the various molecular methods to be used for species identification is reviewed and a critical evaluation of the usefulness of various techniques and genomic loci for species identification of black aspergilli is presented.

Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compel additional exploration. We therefore undertook whole-genome sequencing of the acidogenic A. niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence, and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was used to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 Mb of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis supported up-regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases, and protein transporters in the protein producing CBS 513.88 strain. Our results and data sets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi.