Role of the osaA Gene in Aspergillus fumigatus Development, Secondary Metabolism and Virulence.

Aspergillus fumigatus is the leading cause of aspergillosis, associated with high mortality rates, particularly in immunocompromised individuals. In search of novel genetic targets against aspergillosis, we studied the WOPR transcription factor OsaA. The deletion of the osaA gene resulted in colony growth reduction. Conidiation is also influenced by osaA; both osaA deletion and overexpression resulted in a decrease in spore production. Wild-type expression levels of osaA are necessary for the expression of the conidiation regulatory genes brlA, abaA, and wetA. In addition, osaA is necessary for normal cell wall integrity. Furthermore, the deletion of osaA resulted in a reduction in the ability of A. fumigatus to adhere to surfaces, decreased thermotolerance, as well as increased sensitivity to oxidative stress. Metabolomics analysis indicated that osaA deletion or overexpression led to alterations in the production of multiple secondary metabolites, including gliotoxin. This was accompanied by changes in the expression of genes in the corresponding secondary metabolite gene clusters. These effects could be, at least in part, due to the observed reduction in the expression levels of the veA and laeA global regulators when the osaA locus was altered. Importantly, our study shows that osaA is indispensable for virulence in both neutropenic and corticosteroid-immunosuppressed mouse models.

Homeobox transcription factor HbxA influences expression of over one thousand genes in the model fungus Aspergillus nidulans.

In fungi, conserved homeobox-domain proteins are transcriptional regulators governing development. In Aspergillus species, several homeobox-domain transcription factor genes have been identified, among them, hbxA/hbx1. For instance, in the opportunistic human pathogen Aspergillus fumigatus, hbxA is involved in conidial production and germination, as well as virulence and secondary metabolism, including production of fumigaclavines, fumiquinazolines, and chaetominine. In the agriculturally important fungus Aspergillus flavus, disruption of hbx1 results in fluffy aconidial colonies unable to produce sclerotia. hbx1 also regulates production of aflatoxins, cyclopiazonic acid and aflatrem. Furthermore, transcriptome studies revealed that hbx1 has a broad effect on the A. flavus genome, including numerous genes involved in secondary metabolism. These studies underline the importance of the HbxA/Hbx1 regulator, not only in developmental processes but also in the biosynthesis of a broad number of fungal natural products, including potential medical drugs and mycotoxins. To gain further insight into the regulatory scope of HbxA in Aspergilli, we studied its role in the model fungus Aspergillus nidulans. Our present study of the A. nidulans hbxA-dependent transcriptome revealed that more than one thousand genes are differentially expressed when this regulator was not transcribed at wild-type levels, among them numerous transcription factors, including those involved in development as well as in secondary metabolism regulation. Furthermore, our metabolomics analyses revealed that production of several secondary metabolites, some of them associated with A. nidulans hbxA-dependent gene clusters, was also altered in deletion and overexpression hbxA strains compared to the wild type, including synthesis of nidulanins A, B and D, versicolorin A, sterigmatocystin, austinol, dehydroaustinol, and three unknown novel compounds.

Mutagenicity and genotoxicity assessment of the emerging mycotoxin Versicolorin A, an Aflatoxin B1 precursor.

Aflatoxin B1 (AFB1) is the most potent natural carcinogen among mycotoxins. Versicolorin A (VerA) is a precursor of AFB1 biosynthesis and is structurally related to the latter. Although VerA has already been identified as a genotoxin, data on the toxicity of VerA are still scarce, especially at low concentrations. The SOS/umu and miniaturised version of the Ames test in Salmonella Typhimurium strains used in the present study shows that VerA induces point mutations. This effect, like AFB1, depends primarily on metabolic activation of VerA. VerA also induced chromosomal damage in metabolically competent intestinal cells (IPEC-1) detected by the micronucleus assay. Furthermore, results from the standard and enzyme-modified comet assay confirmed the VerA-mediated DNA damage, and we observed that DNA repair pathways were activated upon exposure to VerA, as indicated by the phosphorylation and/or relocation of relevant DNA-repair biomarkers (γH2AX and 53BP1/FANCD2, respectively). In conclusion, VerA induces DNA damage without affecting cell viability at concentrations as low as 0.03 µM, highlighting the danger associated with VerA exposure and calling for more research on the carcinogenicity of this emerging food contaminant.

Effect of Streptomyces roseolus Cell-Free Supernatants on the Fungal Development, Transcriptome, and Aflatoxin B1 Production of Aspergillus flavus.

Crop contamination by aflatoxin B1 (AFB1), an Aspergillus-flavus-produced toxin, is frequently observed in tropical and subtropical regions. This phenomenon is emerging in Europe, most likely as a result of climate change. Alternative methods, such as biocontrol agents (BCAs), are currently being developed to reduce the use of chemicals in the prevention of mycotoxin contamination. Actinobacteria are known to produce many bioactive compounds, and some of them can reduce in vitro AFB1 concentration. In this context, the present study aims to analyze the effect of a cell-free supernatant (CFS) from Streptomyces roseolus culture on the development of A. flavus, as well as on its transcriptome profile using microarray assay and its impact on AFB1 concentration. Results demonstrated that in vitro, the S. roseolus CFS reduced the dry weight and conidiation of A. flavus from 77% and 43%, respectively, and was therefore associated with a reduction in AFB1 concentration reduction to levels under the limit of quantification. The transcriptomic data analysis revealed that 5198 genes were differentially expressed in response to the CFS exposure and among them 5169 were downregulated including most of the genes involved in biosynthetic gene clusters. The aflatoxins' gene cluster was the most downregulated. Other gene clusters, such as the aspergillic acid, aspirochlorine, and ustiloxin B gene clusters, were also downregulated and associated with a variation in their concentration, confirmed by LC-HRMS.

Inhibition of Aflatoxin B1 Synthesis in Aspergillus flavus by Mate (Ilex paraguariensis), Rosemary (Rosmarinus officinalis) and Green Tea (Camellia sinensis) Extracts: Relation with Extract Antioxidant Capacity and Fungal Oxidative Stress Response Modulation.

Plant extracts may represent an ecofriendly alternative to chemical fungicides to limit aflatoxin B1 (AFB1) contamination of foods and feeds. Mate (Ilex paraguariensis), rosemary (Romarinus officinalis) and green tea (Camellia sinensis) are well known for their beneficial properties, which are mainly related to their richness in bioactive phenolic compounds. AFB1 production is inhibited, with varying efficiency, by acetone/water extracts from these three plants. At 0.45 µg dry matter (DM)/mL of culture medium, mate and green tea extracts were able to completely inhibit AFB1 production in Aspergillus flavus, and rosemary extract completely blocked AFB1 biosynthesis at 3.6 µg DM/mL of culture medium. The anti-AFB1 capacity of the extracts correlated strongly with their phenolic content, but, surprisingly, no such correlation was evident with their antioxidative ability, which is consistent with the ineffectiveness of these extracts against fungal catalase activity. Anti-AFB1 activity correlated more strongly with the radical scavenging capacity of the extracts. This is consistent with the modulation of SOD induced by mate and green tea in Aspergillus flavus. Finally, rutin, a phenolic compound present in the three plants tested in this work, was shown to inhibit AFB1 synthesis and may be responsible for the anti-mycotoxin effect reported herein.

Metabolism of versicolorin A, a genotoxic precursor of aflatoxin B1: Characterization of metabolites using in vitro production of standards.

The toxicity of mycotoxins containing bisfuranoid structures such as aflatoxin B1 (AFB1) depends largely on biotransformation processes. While the genotoxicity and mutagenicity of several bisfuranoid mycotoxins including AFB1 and sterigmatocystin have been linked to in vivo bioactivation of these molecules into reactive epoxide forms, the metabolites of genotoxic and mutagenic AFB1 precursor versicolorin A (VerA) have not yet been characterized. Because this molecule is not available commercially, our strategy was to produce a library of metabolites derived from the biotransformation of in-house purified VerA, following incubation with human liver S9 fractions, in presence of appropriate cofactors. The resulting chromatographic and mass-spectrometric data were used to identify VerA metabolites produced by intestinal cell lines as well as intestinal and liver tissues exposed ex vivo. In this way, we obtained a panel of metabolites suggesting the involvement of phase I (M + O) and phase II (glucuronide and sulfate metabolites) enzymes, the latter of which is implicated in the detoxification process. This first qualitative description of the metabolization products of VerA suggests bioactivation of the molecule into an epoxide form and provides qualitative analytic data to further conduct a precise metabolism study of VerA required for the risk assessment of this emerging mycotoxin.

Tissular Genomic Responses to Oral FB1 Exposure in Pigs.

Fumonisin B1 (FB1) is a widespread mycotoxin produced by fungal Fusarium species-mainly in maize, one of the plants most commonly used for food and feed. Pigs and horses are the animal species most susceptible to this mycotoxin. FB1 exposure can cause highly diverse clinical symptoms, including hepatotoxicity, immunotoxicity, and intestinal barrier function disturbance. Inhibition of ceramide synthetase is a well-understood ubiquitous molecular mechanism of FB1 toxicity, but other more tissue-specific effects remain to be elucidated. To investigate the effects of FB1 in different exposed tissues, we cross-analyzed the transcriptomes of fours organs: liver, jejunum, jejunal Peyer's patches, and spleen. During a four-week study period, pigs were fed a control diet or a FB1-contaminated diet (10 mg/kg feed). In response to oral FB1 exposure, we observed common biological processes in the four organs, including predominant and recurrent processes (extracellular matrix organization, integrin activation, granulocyte chemotaxis, neutrophil migration, and lipid and sterol homeostasis), as well as more tissue-specific processes that appeared to be related to lipid outcomes (cell cycle regulation in jejunum, and gluconeogenesis in liver).

The Solvent Dimethyl Sulfoxide Affects Physiology, Transcriptome and Secondary Metabolism of Aspergillus flavus.

Dimethyl sulfoxide (DSMO) is a simple molecule widely used because of its great solvating ability, but this solvent also has little-known biological effects, especially on fungi. Aspergillus flavus is a notorious pathogenic fungus which may contaminate a large variety of crops worldwide by producing aflatoxins, endangering at the same time food safety and international trade. The aim of this study was to characterize the effect of DMSO on A. flavus including developmental parameters such as germination and sporulation, as well as its transcriptome profile using high-throughput RNA-sequencing assay and its impact on secondary metabolism (SM). After DMSO exposure, A. flavus displayed depigmented conidia in a dose-dependent manner. The four-day exposition of cultures to two doses of DMSO, chosen on the basis of depigmentation intensity (35 mM "low" and 282 mM "high"), led to no significant impact on fungal growth, germination or sporulation. However, transcriptomic data analysis showed that 4891 genes were differentially regulated in response to DMSO (46% of studied transcripts). A total of 4650 genes were specifically regulated in response to the highest dose of DMSO, while only 19 genes were modulated upon exposure to the lowest dose. Secondary metabolites clusters genes were widely affected by the DMSO, with 91% of clusters impacted at the highest dose. Among these, aflatoxins, cyclopiazonic acid and ustiloxin B clusters were totally under-expressed. The genes belonging to the AFB1 cluster were the most negatively modulated ones, the two doses leading to 63% and 100% inhibition of the AFB1 production, respectively. The SM analysis also showed the disappearance of ustiloxin B and a 10-fold reduction of cyclopiazonic acid level when A. flavus was treated by the higher DMSO dose. In conclusion, the present study showed that DMSO impacted widely A. flavus' transcriptome, including secondary metabolism gene clusters with the aflatoxins at the head of down-regulated ones. The solvent also inhibits conidial pigmentation, which could illustrate common regulatory mechanisms between aflatoxins and fungal pigment pathways. Because of its effect on major metabolites synthesis, DMSO should not be used as solvent especially in studies testing anti-aflatoxinogenic compounds.

Regulation of Secondary Metabolism in the Penicillium Genus.

Penicillium, one of the most common fungi occurring in a diverse range of habitats, has a worldwide distribution and a large economic impact on human health. Hundreds of the species belonging to this genus cause disastrous decay in food crops and are able to produce a varied range of secondary metabolites, from which we can distinguish harmful mycotoxins. Some Penicillium species are considered to be important producers of patulin and ochratoxin A, two well-known mycotoxins. The production of these mycotoxins and other secondary metabolites is controlled and regulated by different mechanisms. The aim of this review is to highlight the different levels of regulation of secondary metabolites in the Penicillium genus.

Chemical repertoire and biosynthetic machinery of the Aspergillus flavus secondary metabolome: A review.

Filamentous fungi represent a rich source of extrolites, including secondary metabolites (SMs) comprising a great variety of astonishing structures and interesting bioactivities. State-of-the-art techniques in genome mining, genetic manipulation, and secondary metabolomics have enabled the scientific community to better elucidate and more deeply appreciate the genetic and biosynthetic chemical arsenal of these microorganisms. Aspergillus flavus is best known as a contaminant of food and feed commodities and a producer of the carcinogenic family of SMs, aflatoxins. This fungus produces many SMs including polyketides, ribosomal and nonribosomal peptides, terpenoids, and other hybrid molecules. This review will discuss the chemical diversity, biosynthetic pathways, and biological/ecological role of A. flavus SMs, as well as their significance concerning food safety and security.

Diversity within Aspergillus niger Clade and Description of a New Species: Aspergillus vinaceus sp. nov.

Diversity of species within Aspergillus niger clade, currently represented by A. niger sensu stricto and A. welwitshiae, was investigated combining three-locus gene sequences, Random Amplified Polymorphic DNA, secondary metabolites profile and morphology. Firstly, approximately 700 accessions belonging to this clade were investigated using calmodulin gene sequences. Based on these sequences, eight haplotypes were clearly identified as A. niger (n = 247) and 17 as A. welwitschiae (n = 403). However, calmodulin sequences did not provide definitive species identities for six haplotypes. To elucidate the taxonomic position of these haplotypes, two other loci, part of the beta-tubulin gene and part of the RNA polymerase II gene, were sequenced and used to perform an analysis of Genealogical Concordance Phylogenetic Species Recognition. This analysis enabled the recognition of two new phylogenetic species. One of the new phylogenetic species showed morphological and chemical distinguishable features in comparison to the known species A. welwitschiae and A. niger. This species is illustrated and described as Aspergillus vinaceus sp. nov. In contrast to A. niger and A. welwitschiae, A. vinaceus strains produced asperazine, but none of them were found to produce ochratoxin A and/or fumonisins. Sclerotium production on laboratory media, which does not occur in strains of A. niger and A. welwitschiae, and strictly sclerotium-associated secondary metabolites (14-Epi-hydroxy-10,23-dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydro-21-oxo-aflavinine) were found in A. vinaceus. The strain type of A. vinaceus sp. nov. is ITAL 47,456 (T) (=IBT 35556).

The brlA Gene Deletion Reveals That Patulin Biosynthesis Is Not Related to Conidiation in Penicillium expansum.

Dissemination and survival of ascomycetes is through asexual spores. The brlA gene encodes a C2H2-type zinc-finger transcription factor, which is essential for asexual development. Penicillium expansum causes blue mold disease and is the main source of patulin, a mycotoxin that contaminates apple-based food. A P. expansum PeΔbrlA deficient strain was generated by homologous recombination. In vivo, suppression of brlA completely blocked the development of conidiophores that takes place after the formation of coremia/synnemata, a required step for the perforation of the apple epicarp. Metabolome analysis displayed that patulin production was enhanced by brlA suppression, explaining a higher in vivo aggressiveness compared to the wild type (WT) strain. No patulin was detected in the synnemata, suggesting that patulin biosynthesis stopped when the fungus exited the apple. In vitro transcriptome analysis of PeΔbrlA unveiled an up-regulated biosynthetic gene cluster (PEXP_073960-PEXP_074060) that shares high similarity with the chaetoglobosin gene cluster of Chaetomium globosum. Metabolome analysis of PeΔbrlA confirmed these observations by unveiling a greater diversity of chaetoglobosin derivatives. We observed that chaetoglobosins A and C were found only in the synnemata, located outside of the apple, whereas other chaetoglobosins were detected in apple flesh, suggesting a spatial-temporal organization of the chaetoglobosin biosynthesis pathway.

Aflatoxin Biosynthesis and Genetic Regulation: A Review.

The study of fungal species evolved radically with the development of molecular techniques and produced new evidence to understand specific fungal mechanisms such as the production of toxic secondary metabolites. Taking advantage of these technologies to improve food safety, the molecular study of toxinogenic species can help elucidate the mechanisms underlying toxin production and enable the development of new effective strategies to control fungal toxicity. Numerous studies have been made on genes involved in aflatoxin B1 (AFB1) production, one of the most hazardous carcinogenic toxins for humans and animals. The current review presents the roles of these different genes and their possible impact on AFB1 production. We focus on the toxinogenic strains Aspergillus flavus and A. parasiticus, primary contaminants and major producers of AFB1 in crops. However, genetic reports on A. nidulans are also included because of the capacity of this fungus to produce sterigmatocystin, the penultimate stable metabolite during AFB1 production. The aim of this review is to provide a general overview of the AFB1 enzymatic biosynthesis pathway and its link with the genes belonging to the AFB1 cluster. It also aims to illustrate the role of global environmental factors on aflatoxin production and the recent data that demonstrate an interconnection between genes regulated by these environmental signals and aflatoxin biosynthetic pathway.

Versicolorin A, a precursor in aflatoxins biosynthesis, is a food contaminant toxic for human intestinal cells.

Aflatoxin B1 (AFB1) is the most potent carcinogen among mycotoxins. Its biosynthesis involves the formation of versicolorin A (VerA), whose chemical structure shares many features with AFB1. Our data revealed significant levels of VerA in foodstuff from Central Asia and Africa. Given this emerging food risk, it was of prime interest to compare the toxic effects of the two mycotoxins against cells originating from the intestinal tract. We used human colon cell lines (Caco-2, HCT116) to investigate the cytotoxic process induced by the two mycotoxins. Contrary to AFB1, a low dose of VerA (1 µM) disturbed the expression level of thousands of genes (18 002 genes). We show that the cytotoxic effects of low doses of VerA (1-20 µM) were stronger than the same low doses of AFB1 in both Caco-2 and HCT116 cell lines. In Caco-2 cells, VerA induced DNA strand breaks that led to apoptosis and reduced DNA replication of dividing cells, consequently inhibiting cell proliferation. Although VerA was able to induce the p53 signaling pathway in p53 wild-type HCT116 cells, its toxicity process did not mainly rely on p53 expression since similar cytotoxic effects were also observed in HCT116 cells that do not express p53. In conclusion, this study provides evidence of the risk of food contamination by VerA and shed light on its toxicological effect on human colon cells.

The Transcriptional Regulator HbxA Governs Development, Secondary Metabolism, and Virulence in Aspergillus fumigatus.

Aspergillus fumigatus is the leading cause of invasive aspergillosis, which in immunocompromised patients results in a mortality rate as high as 90%. Earlier studies showed that HbxA is a global regulator in Aspergillus flavus affecting morphological development and secondary metabolism. Here, we determined its role in A. fumigatus, examining whether HbxA influences the regulation of asexual development, natural product biosynthesis, and virulence of this fungus. Our analysis demonstrated that removal of the hbxA gene caused a near-complete loss of conidial production in the mutant strain, as well as a slight reduction in colony growth. Other aspects of asexual development are affected, such as size and germination of conidia. Furthermore, we showed that in A. fumigatus, the loss of hbxA decreased the expression of the brlA central regulatory pathway involved in asexual development, as well as the expression of the "fluffy" genes flbB, flbD, and fluG HbxA was also found to regulate secondary metabolism, affecting the biosynthesis of multiple natural products, including fumigaclavines, fumiquinazolines, and chaetominine. In addition, using a neutropenic mouse infection model, hbxA was found to negatively impact the virulence of A. fumigatusIMPORTANCE The number of immunodepressed individuals is increasing, mainly due to the greater life expectancy in immunodepressed patients due to improvements in modern medical treatments. However, this population group is highly susceptible to invasive aspergillosis. This devastating illness, mainly caused by the fungus Aspergillus fumigatus, is associated with mortality rates reaching 90%. Treatment options for this disease are currently limited, and a better understanding of A. fumigatus genetic regulatory mechanisms is paramount for the design of new strategies to prevent or combat this infection. Our work provides new insight into the regulation of the development, metabolism, and virulence of this important opportunistic pathogen. The transcriptional regulatory gene hbxA has a profound effect on A. fumigatus biology, governing multiple aspects of conidial development. This is relevant since conidia are the main source of inoculum in Aspergillus infections. Importantly, hbxA also regulates the biosynthesis of secondary metabolites and the pathogenicity of this fungus.

Application of MALDI-TOF MS to species complex differentiation and strain typing of food related fungi: Case studies with Aspergillus section Flavi species and Penicillium roqueforti isolates.

Filamentous fungi are one of the main causes of food losses worldwide and their ability to produce mycotoxins represents a hazard for human health. Their correct and rapid identification is thus crucial to manage food safety. In recent years, MALDI-TOF emerged as a rapid and reliable tool for fungi identification and was applied to typing of bacteria and yeasts, but few studies focused on filamentous fungal species complex differentiation and typing. Therefore, the aim of this study was to evaluate the use of MALDI-TOF to identify species of the Aspergillus section Flavi, and to differentiate Penicillium roqueforti isolates from three distinct genetic populations. Spectra were acquired from 23 Aspergillus species and integrated into a database for which cross-validation led to more than 99% of correctly attributed spectra. For P. roqueforti, spectra were acquired from 63 strains and a two-step calibration procedure was applied before database construction. Cross-validation and external validation respectively led to 94% and 95% of spectra attributed to the right population. Results obtained here suggested very good agreement between spectral and genetic data analysis for both Aspergillus species and P. roqueforti, demonstrating MALDI-TOF applicability as a fast and easy alternative to molecular techniques for species complex differentiation and strain typing of filamentous fungi.

Combination of Isotope Labeling and Molecular Networking of Tandem Mass Spectrometry Data To Reveal 69 Unknown Metabolites Produced by Penicillium nordicum.

The secondary metabolome of Penicillium nordicum is poorly documented despite its frequent detection on contaminated food and its capacity to produce toxic metabolites such as ochratoxin A. To characterize metabolites produced by this fungi, we combined a full stable isotopes labeling with the dereplication of tandem mass spectrometry (MS/MS) data by molecular networking. First, the untargeted metabolomic analysis by high-resolution mass spectrometry of a double stable isotope labeling of P. nordicum enabled the specific detection of its metabolites and the unambiguous determination of their elemental composition. Analyses showed that infection of substrate by P. nordicum lead to the production of at least 92 metabolites and that 69 of them were completely unknown. Then, curated molecular networks of MS/MS data were generated with GNPS and MetGem, specifically on the features of interest, which allowed highlighting 13 fungisporin-related metabolites that had not previously been identified in this fungus and 8 that had never been observed in any fungus. The structures of the unknown compounds, namely, a native fungisporin and seven linear peptides, were characterized by tandem mass spectrometry experiments. The analysis of P. nordicum growing on its natural substrates, i.e. pork ham, turkey ham, and cheese, demonstrated that 10 of the known fungisporin-related metabolites and three of the new metabolites were also synthesized. Thus, the curation of data for molecular networking using a specific detection of metabolites of interest with stable isotopes labeling allowed the discovery of new metabolites produced by the food contaminant P. nordicum.

Occurrence and diversity of Aspergillus in commercial yerba mate elaborated for the Brazilian beverage 'chimarrão'.

Dried leaves and stems of Ilex paraguariensis St. Hil. (yerba mate) are used to make a popular beverage in some countries of South America, commonly known as "chimarrão". The present study was designed to evaluate the occurrence of toxigenic Aspergillus in yerba mate in order to define the mycotoxin risk associated with this foodstuff. All samples tested were positive for fungal contamination, and the fungal load per sample ranged from 2.0 × 102 to 1.6 × 104 CFU/g. Aspergillus section Nigri was found in all samples and represented 76.5% of the total fungi isolated. Aspergillus section Circumdati, Aspergillus section Flavi and Aspergillus section Cremei were found at low frequencies. Thirteen different Aspergillus species were identified. The most common species found was A. luchuensis, which does not produce any harmful toxin for humans. A. niger, A. welwitschiae, A. flavus and A. novoparasiticus, all potentially toxigenic species, were found only in small quantities. The A. niger and A. welwitschiae strains were cultured to test for ochratoxin A and fumonisin B2 production. Only one strain producing ochratoxin A was found, but approximately 29% of the strains were positive for fumonisin B2. The A. flavus and A. novoparasiticus strains were tested for aflatoxins production, and 63% were positive. A. pallidofulvus, recently assigned to A. section Circumdati, was reported for the first time in herbs. All A. pallidofulvus strains analyzed in this study were negative for ochratoxin A production. In conclusion, A. section Nigri occurs with high frequency in yerba mate, and A. luchuensis is the predominant species. Although toxigenic species were found in this herb, the incidence was low.