Polyphasic taxonomy of Aspergillus section Aspergillus (formerly Eurotium), and its occurrence in indoor environments and food

Abstract

Aspergillus section Aspergillus (formerly the genus Eurotium) includes xerophilic species with uniseriate conidiophores, globose to subglobose vesicles, green conidia and yellow, thin walled eurotium-like ascomata with hyaline, lenticular ascospores. In the present study, a polyphasic approach using morphological characters, extrolites, physiological characters and phylogeny was applied to investigate the taxonomy of this section. Over 500 strains from various culture collections and new isolates obtained from indoor environments and a wide range of substrates all over the world were identified using calmodulin gene sequencing. Of these, 163 isolates were subjected to molecular phylogenetic analyses using sequences of ITS rDNA, partial β-tubulin (BenA), calmodulin (CaM) and RNA polymerase II second largest subunit (RPB2) genes. Colony characteristics were documented on eight cultivation media, growth parameters at three incubation temperatures were recorded and micromorphology was examined using light microscopy as well as scanning electron microscopy to illustrate and characterize each species. Many specific extrolites were extracted and identified from cultures, including echinulins, epiheveadrides, auroglaucins and anthraquinone bisanthrons, and to be consistent in strains of nearly all species. Other extrolites are species-specific, and thus valuable for identification. Several extrolites show antioxidant effects, which may be nutritionally beneficial in food and beverages. Important mycotoxins in the strict sense, such as sterigmatocystin, aflatoxins, ochratoxins, citrinin were not detected despite previous reports on their production in this section. Adopting a polyphasic approach, 31 species are recognized, including nine new species. ITS is highly conserved in this section and does not distinguish species. All species can be differentiated using CaM or RPB2 sequences. For BenA, Aspergillus brunneus and A. niveoglaucus share identical sequences. Ascospores and conidia morphology, growth rates at different temperatures are most useful characters for phenotypic species identification.

Analysis of the fungal population involved in Katsuobushi production

“…This was consistent with an observation that A. chevarieri produces yellow ascomata and en masse grayish-green conidia (Chen et al, 2017).…”

“…The benA, caM, and rpb2 genes, which encode β-tubulin, calmodulin, and the second largest subunit of RNA polymerase II, respectively, were then sequenced to further identify strains M1, M2, M3, and M4. The benA, caM, and rpb2 genes have been used for species-level identification of Aspergillus (Samson et al, 2014;Chen et al, 2017). Colony PCR analysis was performed using the following primer sets: Bt 2 a and Bt 2 b for benA (Glass and Donaldson, 1995); CMD5 and CMD6 for caM (Hong et al, 2005); 5F and 7CR for rpb2 (Liu et al, 1999).…”

“…Nucleotide BLAST analysis of benA, caM, and rpb2 of strain M4 also showed best hits to those of A. sydowii (data not shown), indicating that strain M4 is classified into A. sydowii. Because comprehensive DNA sequence information of benA, caM, and rpb2 were available for the section Aspergillus (Chen et al, 2017), we performed phylogenetic analysis with those from strains M1, M2, and M3 (Fig. 2).…”

“…However, the recent classification method based on DNA sequence (Chen et al, 2017;Samson et al, 2014) has not been applied for the classification of the fungal population in Katsuobushi. In this study, we characterized the fugal population in Katsuobushi production using DNA sequencing.…”

Complete mitochondrial genome sequence of a xerophilic fungus, Aspergillus pseudoglaucus

“…Aspergillus pseudoglaucus (Chen et al 2017), identified in 1929, is a xerophilic filamentous fungus belonging to Aspergillus section Aspergillus. It has been isolated from seafoods and soil (Smetanina et al 2007;S eguin et al 2014).…”