Identification of SclB, a Zn(II)2Cys6 transcription factor involved in sclerotium formation in Aspergillus niger.

Certain Aspergillus species such as Aspergillus flavus and A. parasiticus are well known for the formation of sclerotia. These developmental structures are thought to act as survival structures during adverse environmental conditions but are also a prerequisite for sexual reproduction. We previously described an A. niger mutant (scl-2) which formed sclerotium-like structures, suggesting a possible first stage of sexual development in this species. Several lines of evidence presented in this study support the previous conclusion that the sclerotium-like structures of scl-2 are indeed sclerotia. These included the observations that: (i) safranin staining of the sclerotia-like structures produced by the scl-2 mutant showed the typical cellular structure of a sclerotium; (ii) metabolite analysis revealed specific production of indoloterpenes, which have previously been connected to sclerotium formation; (iii) formation of the sclerotium-like structures is dependent on a functional NADPH complex, as shown for other fungi forming sclerotia. The mutation in scl-2 responsible for sclerotium formation was identified using parasexual crossing and bulk segregant analysis followed by high throughput sequencing and subsequent complementation analysis. The scl-2 strain contains a mutation that introduces a stop codon in the putative DNA binding domain of a previously uncharacterized Zn(II)2Cys6 type transcription factor (An08g07710). Targeted deletion of this transcription factor (sclB) confirmed its role as a repressor of sclerotial formation and in the promotion of asexual reproduction in A. niger. Finally, a genome-wide transcriptomic comparison of RNA extracted from sclerotia versus mycelia revealed major differences in gene expression. Induction of genes related to indoloterpene synthesis was confirmed and also let to the identification of a gene cluster essential for the production of aurasperones during sclerotium formation. Expression analysis of genes encoding other secondary metabolites, cell wall related genes, transcription factors, and genes related to reproductive processes identified many interesting candidate genes to further understand the regulation and biosynthesis of sclerotia in A. niger. The newly identified SclB transcription factor acts as a repressor of sclerotium formation and manipulation of sclB may represent a first prerequisite step towards engineering A. niger strains capable of sexual reproduction. This will provide exciting opportunities for further strain improvement in relation to protein or metabolite production in A. niger.

Investigation of inter- and intraspecies variation through genome sequencing of Aspergillus section Nigri.

Aspergillus section Nigri comprises filamentous fungi relevant to biomedicine, bioenergy, health, and biotechnology. To learn more about what genetically sets these species apart, as well as about potential applications in biotechnology and biomedicine, we sequenced 23 genomes de novo, forming a full genome compendium for the section (26 species), as well as 6 Aspergillus niger isolates. This allowed us to quantify both inter- and intraspecies genomic variation. We further predicted 17,903 carbohydrate-active enzymes and 2,717 secondary metabolite gene clusters, which we condensed into 455 distinct families corresponding to compound classes, 49% of which are only found in single species. We performed metabolomics and genetic engineering to correlate genotypes to phenotypes, as demonstrated for the metabolite aurasperone, and by heterologous transfer of citrate production to Aspergillus nidulans. Experimental and computational analyses showed that both secondary metabolism and regulation are key factors that are significant in the delineation of Aspergillus species.

Cyclopiamines C and D: Epoxide Spiroindolinone Alkaloids from Penicillium sp. CML 3020.

Cyclopiamines C (1) and D (2) were isolated from the extract of Penicillium sp. CML 3020, a fungus sourced from an Atlantic Forest soil sample. Their structures and relative configuration were determined by 1D and 2D NMR, HRMS, and UV/vis data analysis. Cyclopiamines C and D belong to a small subset of rare spiroindolinone compounds containing an alkyl nitro group and a 4,5-dihydro-1 H-pyrrolo[3,2,1- ij]quinoline-2,6-dione ring system. NMR and MS/HRMS data confirmed the presence of an epoxide unit (C-17-O-C-18) and a hydroxy group at C-5, not observed for their known congeners. Cytotoxic and antimicrobial activities were evaluated.

Biosynthesis of the antimicrobial cyclic lipopeptides nunamycin and nunapeptin by Pseudomonas fluorescens strain In5 is regulated by the LuxR-type transcriptional regulator NunF.

Nunamycin and nunapeptin are two antimicrobial cyclic lipopeptides (CLPs) produced by Pseudomonas fluorescens In5 and synthesized by nonribosomal synthetases (NRPS) located on two gene clusters designated the nun-nup regulon. Organization of the regulon is similar to clusters found in other CLP-producing pseudomonads except for the border regions where putative LuxR-type regulators are located. This study focuses on understanding the regulatory role of the LuxR-type-encoding gene nunF in CLP production of P. fluorescens In5. Functional analysis of nunF coupled with liquid chromatography-high-resolution mass spectrometry (LC-HRMS) showed that CLP biosynthesis is regulated by nunF. Quantitative real-time PCR analysis indicated that transcription of the NRPS genes catalyzing CLP production is strongly reduced when nunF is mutated indicating that nunF is part of the nun-nup regulon. Swarming and biofilm formation was reduced in a nunF knockout mutant suggesting that these CLPs may also play a role in these phenomena as observed in other pseudomonads. Fusion of the nunF promoter region to mCherry showed that nunF is strongly upregulated in response to carbon sources indicating the presence of a fungus suggesting that environmental elicitors may also influence nunF expression which upon activation regulates nunamycin and nunapeptin production required for the growth inhibition of phytopathogens.

A Dereplication and Bioguided Discovery Approach to Reveal New Compounds from a Marine-Derived Fungus Stilbella fimetaria.

A marine-derived Stilbella fimetaria fungal strain was screened for new bioactive compounds based on two different approaches: (i) bio-guided approach using cytotoxicity and antimicrobial bioassays; and (ii) dereplication based approach using liquid chromatography with both diode array detection and high resolution mass spectrometry. This led to the discovery of several bioactive compound families with different biosynthetic origins, including pimarane-type diterpenoids and hybrid polyketide-non ribosomal peptide derived compounds. Prefractionation before bioassay screening proved to be a great aid in the dereplication process, since separate fractions displaying different bioactivities allowed a quick tentative identification of known antimicrobial compounds and of potential new analogues. A new pimarane-type diterpene, myrocin F, was discovered in trace amounts and displayed cytotoxicity towards various cancer cell lines. Further media optimization led to increased production followed by the purification and bioactivity screening of several new and known pimarane-type diterpenoids. A known broad-spectrum antifungal compound, ilicicolin H, was purified along with two new analogues, hydroxyl-ilicicolin H and ilicicolin I, and their antifungal activity was evaluated.

Global occurrence and heterogeneity of the Roseobacter-clade species Ruegeria mobilis.

Tropodithietic acid (TDA)-producing Ruegeria mobilis strains of the Roseobacter clade have primarily been isolated from marine aquaculture and have probiotic potential due to inhibition of fish pathogens. We hypothesized that TDA producers with additional novel features are present in the oceanic environment. We isolated 42 TDA-producing R. mobilis strains during a global marine research cruise. While highly similar on the 16S ribosomal RNA gene level (99-100% identity), the strains separated into four sub-clusters in a multilocus sequence analysis. They were further differentiated to the strain level by average nucleotide identity using pairwise genome comparison. The four sub-clusters could not be associated with a specific environmental niche, however, correlated with the pattern of sub-typing using co-isolated phages, the number of prophages in the genomes and the distribution in ocean provinces. Major genomic differences within the sub-clusters include prophages and toxin-antitoxin systems. In general, the genome of R. mobilis revealed adaptation to a particle-associated life style and querying TARA ocean data confirmed that R. mobilis is more abundant in the particle-associated fraction than in the free-living fraction occurring in 40% and 6% of the samples, respectively. Our data and the TARA data, although lacking sufficient data from the polar regions, demonstrate that R. mobilis is a globally distributed marine bacterial species found primarily in the upper open oceans. It has preserved key phenotypic behaviors such as the production of TDA, but contains diverse sub-clusters, which could provide new capabilities for utilization in aquaculture.

Production of the Bioactive Compounds Violacein and Indolmycin Is Conditional in a maeA Mutant of Pseudoalteromonas luteoviolacea S4054 Lacking the Malic Enzyme.

It has previously been reported that some strains of the marine bacterium Pseudoalteromonas luteoviolacea produce the purple bioactive pigment violacein as well as the antibiotic compound indolmycin, hitherto only found in Streptomyces. The purpose of the present study was to determine the relative role of each of these two compounds as antibacterial compounds in P. luteoviolacea S4054. Using Tn10 transposon mutagenesis, a mutant strain that was significantly reduced in violacein production in mannose-containing substrates was created. Full genome analyses revealed that the vio-biosynthetic gene cluster was not interrupted by the transposon; instead the insertion was located to the maeA gene encoding the malic enzyme. Supernatant of the mutant strain inhibited Vibrio anguillarum and Staphylococcus aureus in well diffusion assays and in MIC assays at the same level as the wild type strain. The mutant strain killed V. anguillarum in co-culture experiments as efficiently as the wild type. Using UHPLC-UV/Vis analyses, we quantified violacein and indolmycin, and the mutant strain only produced 7-10% the amount of violacein compared to the wild type strain. In contrast, the amount of indolmycin produced by the mutant strain was about 300% that of the wild type. Since inhibition of V. anguillarum and S. aureus by the mutant strain was similar to that of the wild type, it is concluded that violacein is not the major antibacterial compound in P. luteoviolacea. We furthermore propose that production of violacein and indolmycin may be metabolically linked and that yet unidentified antibacterial compound(s) may be play a role in the antibacterial activity of P. luteoviolacea.

Diversity in Secondary Metabolites Including Mycotoxins from Strains of Aspergillus Section Nigri Isolated from Raw Cashew Nuts from Benin, West Africa.

In a previous study, raw cashew kernels were assayed for the fungal contamination focusing on strains belonging to the genus Aspergillus and on aflatoxins producers. These samples showed high contamination with Aspergillus section Nigri species and absence of aflatoxins. To investigate the diversity of secondary metabolites, including mycotoxins, the species of A. section Nigri may produce and thus threaten to contaminate the raw cashew kernels, 150 strains were isolated from cashew samples and assayed for their production of secondary metabolites using liquid chromatography high resolution mass spectrometry (LC-HRMS). Seven species of black Aspergilli were isolated based on morphological and chemical identification: A. tubingensis (44%), A. niger (32%), A. brasiliensis (10%), A. carbonarius (8.7%), A. luchuensis (2.7%), A. aculeatus (2%) and A. aculeatinus (0.7%). From these, 45 metabolites and their isomers were identified. Aurasperone and pyranonigrin A, produced by all species excluding A. aculeatus and A. aculeatinus, were most prevalent and were encountered in 146 (97.3%) and 145 (95.7%) isolates, respectively. Three mycotoxins groups were detected: fumonisins (B2 and B4) (2.7%) ochratoxin A (13.3%), and secalonic acids (2%), indicating that these mycotoxins could occur in raw cashew nuts. Thirty strains of black Aspergilli were randomly sampled for verification of species identity based on sequences of ß-tubulin and calmodulin genes. Among them, 27 isolates were positive to the primers used and 11 were identified as A. niger, 7 as A. tubingensis, 6 as A. carbonarius, 2 as A. luchuensis and 1 as A. welwitschiae confirming the species names as based on morphology and chemical features. These strains clustered in 5 clades in A. section Nigri. Chemical profile clustering also showed also 5 groups confirming the species specific metabolites production.

Draft genome sequence and chemical profiling of Fusarium langsethiae, an emerging producer of type A trichothecenes.

Fusarium langsethiae is a widespread pathogen of small grain cereals, causing problems with T-2 and HT-2 toxin contamination in grains every year. In an effort to better understand the biology of this fungus, we present a draft genome sequence of F. langsethiae Fl201059 isolated from oats in Norway. The assembly was fragmented, but reveals a genome of approximately 37.5 Mb, with a GC content around 48%, and 12,232 predicted protein-coding genes. Focusing on secondary metabolism we identified candidate genes for 12 polyketide synthases, 13 non-ribosomal peptide synthetases, and 22 genes for terpene/isoprenoid biosynthesis. Some of these were found to be unique compared to sequence databases. The identified putative Tri5 cluster was highly syntenic to the cluster reported in F. sporotrichioides. Fusarium langsethiae Fl201059 produces a high number of secondary metabolites on Yeast Extract Sucrose (YES) agar medium, dominated by type A trichothecenes. Interestingly we found production of glucosylated HT-2 toxin (Glu-HT-2), previously suggested to be formed by the host plant and not by the fungus itself. In greenhouse inoculations of F. langsethiae Fl201059 on barley and oats, we detected the type A trichothecenes: neosolaniol, HT-2 toxin, T-2 toxin, Glu-HT-2 and numerous derivatives of these.

Influence of Iron on Production of the Antibacterial Compound Tropodithietic Acid and Its Noninhibitory Analog in Phaeobacter inhibens.

Tropodithietic acid (TDA) is an antibacterial compound produced by some Phaeobacter and Ruegeria spp. of the Roseobacter clade. TDA production is studied in marine broth or agar since antibacterial activity in other media is not observed. The purpose of this study was to determine how TDA production is influenced by substrate components. High concentrations of ferric citrate, as present in marine broth, or other iron sources were required for production of antibacterially active TDA. However, when supernatants of noninhibitory, low-iron cultures of Phaeobacter inhibens were acidified, antibacterial activity was detected in a bioassay. The absence of TDA in nonacidified cultures and the presence of TDA in acidified cultures were verified by liquid chromatography-high-resolution mass spectrometry. A noninhibitory TDA analog (pre-TDA) was produced by P. inhibens, Ruegeria mobilis F1926, and Phaeobacter sp. strain 27-4 under low-iron concentrations and was instantaneously converted to TDA when pH was lowered. Production of TDA in the presence of Fe(3+) coincides with formation of a dark brown substance, which could be precipitated by acid addition. From this brown pigment TDA could be liberated slowly with aqueous ammonia, and both direct-infusion mass spectrometry and elemental analysis indicated a [Fe(III)(TDA)2]x complex. The pigment could also be produced by precipitation of pure TDA with FeCl3. Our results raise questions about how biologically active TDA is produced in natural marine settings where iron is typically limited and whether the affinity of TDA to iron points to a physiological or ecological function of TDA other than as an antibacterial compound.

Identification of a Classical Mutant in the Industrial Host Aspergillus niger by Systems Genetics: LaeA Is Required for Citric Acid Production and Regulates the Formation of Some Secondary Metabolites.

The asexual filamentous fungus Aspergillus niger is an important industrial cell factory for citric acid production. In this study, we genetically characterized a UV-generated A. niger mutant that was originally isolated as a nonacidifying mutant, which is a desirable trait for industrial enzyme production. Physiological analysis showed that this mutant did not secrete large amounts of citric acid and oxalic acid, thus explaining the nonacidifying phenotype. As traditional complementation approaches to characterize the mutant genotype were unsuccessful, we used bulk segregant analysis in combination with high-throughput genome sequencing to identify the mutation responsible for the nonacidifying phenotype. Since A. niger has no sexual cycle, parasexual genetics was used to generate haploid segregants derived from diploids by loss of whole chromosomes. We found that the nonacidifying phenotype was caused by a point mutation in the laeA gene. LaeA encodes a putative methyltransferase-domain protein, which we show here to be required for citric acid production in an A. niger lab strain (N402) and in other citric acid production strains. The unexpected link between LaeA and citric acid production could provide new insights into the transcriptional control mechanisms related to citric acid production in A. niger. Interestingly, the secondary metabolite profile of a ΔlaeA strain differed from the wild-type strain, showing both decreased and increased metabolite levels, indicating that LaeA is also involved in regulating the production of secondary metabolites. Finally, we show that our systems genetics approach is a powerful tool to identify trait mutations.

Rumen microbial communities influence metabolic phenotypes in lambs.

The rumen microbiota is an essential part of ruminants shaping their nutrition and health. Despite its importance, it is not fully understood how various groups of rumen microbes affect host-microbe relationships and functions. The aim of the study was to simultaneously explore the rumen microbiota and the metabolic phenotype of lambs for identifying host-microbe associations and potential biomarkers of digestive functions. Twin lambs, separated in two groups after birth were exposed to practices (isolation and gavage with rumen fluid with protozoa or protozoa-depleted) that differentially restricted the acquisition of microbes. Rumen microbiota, fermentation parameters, digestibility and growth were monitored for up to 31 weeks of age. Microbiota assembled in isolation from other ruminants lacked protozoa and had low bacterial and archaeal diversity whereas digestibility was not affected. Exposure to adult sheep microbiota increased bacterial and archaeal diversity independently of protozoa presence. For archaea, Methanomassiliicoccales displaced Methanosphaera. Notwithstanding, protozoa induced differences in functional traits such as digestibility and significantly shaped bacterial community structure, notably Ruminococcaceae and Lachnospiraceae lower up to 6 folds, Prevotellaceae lower by ~40%, and Clostridiaceae and Veillonellaceae higher up to 10 folds compared to microbiota without protozoa. An orthogonal partial least squares-discriminant analysis of urinary metabolome matched differences in microbiota structure. Discriminant metabolites were mainly involved in amino acids and protein metabolic pathways while a negative interaction was observed between methylotrophic methanogens Methanomassiliicoccales and trimethylamine N-oxide. These results stress the influence of gut microbes on animal phenotype and show the potential of metabolomics for monitoring rumen microbial functions.

Review on Mycotoxin Issues in Ruminants: Occurrence in Forages, Effects of Mycotoxin Ingestion on Health Status and Animal Performance and Practical Strategies to Counteract Their Negative Effects.

Ruminant diets include cereals, protein feeds, their by-products as well as hay and grass, grass/legume, whole-crop maize, small grain or sorghum silages. Furthermore, ruminants are annually or seasonally fed with grazed forage in many parts of the World. All these forages could be contaminated by several exometabolites of mycotoxigenic fungi that increase and diversify the risk of mycotoxin exposure in ruminants compared to swine and poultry that have less varied diets. Evidence suggests the greatest exposure for ruminants to some regulated mycotoxins (aflatoxins, trichothecenes, ochratoxin A, fumonisins and zearalenone) and to many other secondary metabolites produced by different species of Alternaria spp. (e.g., AAL toxins, alternariols, tenuazonic acid or 4Z-infectopyrone), Aspergillus flavus (e.g., kojic acid, cyclopiazonic acid or ß-nitropropionic acid), Aspergillus fuminatus (e.g., gliotoxin, agroclavine, festuclavines or fumagillin), Penicillium roqueforti and P. paneum (e.g., mycophenolic acid, roquefortines, PR toxin or marcfortines) or Monascus ruber (citrinin and monacolins) could be mainly related to forage contamination. This review includes the knowledge of mycotoxin occurrence reported in the last 15 years, with special emphasis on mycotoxins detected in forages, and animal toxicological issues due to their ingestion. Strategies for preventing the problem of mycotoxin feed contamination under farm conditions are discussed.

Combining UHPLC-High Resolution MS and Feeding of Stable Isotope Labeled Polyketide Intermediates for Linking Precursors to End Products.

We present the results from stable isotope labeled precursor feeding studies combined with ultrahigh performance liquid chromatography-high resolution mass spectrometry for the identification of labeled polyketide (PK) end-products. Feeding experiments were performed with (13)C8-6-methylsalicylic acid (6-MSA) and (13)C14-YWA1, both produced in-house, as well as commercial (13)C7-benzoic acid and (2)H7-cinnamic acid, in species of Fusarium, Byssochlamys, Aspergillus, and Penicillium. Incorporation of 6-MSA into terreic acid or patulin was not observed in any of six evaluated species covering three genera, because the 6-MSA was shunted into (2Z,4E)-2-methyl-2,4-hexadienedioic acid. This indicates that patulin and terreic acid may be produced in a closed compartment of the cell and that (2Z,4E)-2-methyl-2,4-hexadienedioic acid is a detoxification product toward terreic acid and patulin. In Fusarium spp., YWA1 was shown to be incorporated into aurofusarin, rubrofusarin, and antibiotic Y. In A. niger, benzoic acid was shown to be incorporated into asperrubrol. Incorporation levels of 0.7-20% into the end-products were detected in wild-type strains. Thus, stable isotope labeling is a promising technique for investigation of polyketide biosynthesis and possible compartmentalization of toxic metabolites.

Combining Stable Isotope Labeling and Molecular Networking for Biosynthetic Pathway Characterization.

Filamentous fungi are a rich source of bioactive compounds, ranging from statins over immunosuppressants to antibiotics. The coupling of genes to metabolites is of large commercial interest for production of the bioactives of the future. To this end, we have investigated the use of stable isotope labeled amino acids (SILAAs). SILAAs were added to the cultivation media of the filamentous fungus Aspergillus nidulans for the study of the cyclic tetrapeptide nidulanin A. Analysis by UHPLC-TOFMS confirmed that the SILAAs were incorporated into produced nidulanin A, and the change in observed m/z could be used to determine whether a compound (known or unknown) incorporated any of the added amino acids. Samples were then analyzed using MS/MS and the data used to perform molecular networking. The molecular network revealed several known and unknown compounds that were also labeled. Assisted by the isotope labeling, it was possible to determine the sequence of several of the compounds, one of which was the known metabolite fungisporin, not previously described in A. nidulans. Several novel analogues of nidulanin A and fungisporin were detected and tentatively identified, and it was determined that these metabolites were all produced by the same nonribosomal peptide synthase. The combination of stable isotope labeling and molecular network generation was shown to very effective for the automated detection of structurally related nonribosomal peptides, while the labeling was effective for determination of the peptide sequence, which could be used to provide information on biosynthesis of bioactive compounds.

The importance of mass spectrometric dereplication in fungal secondary metabolite analysis.

Having entered the Genomic Era, it is now evident that the biosynthetic potential of filamentous fungi is much larger than was thought even a decade ago. Fungi harbor many cryptic gene clusters encoding for the biosynthesis of polyketides, non-ribosomal peptides, and terpenoids - which can all undergo extensive modifications by tailoring enzymes - thus potentially providing a large array of products from a single pathway. Elucidating the full chemical profile of a fungal species is a challenging exercise, even with elemental composition provided by high-resolution mass spectrometry (HRMS) used in combination with chemical databases (e.g., AntiBase) to dereplicate known compounds. This has led to a continuous effort to improve chromatographic separation in conjunction with improvement in HRMS detection. Major improvements have also occurred with 2D chromatography, ion-mobility, MS/MS and MS(3), stable isotope labeling feeding experiments, classic UV/Vis, and especially automated data-mining and metabolomics software approaches as the sheer amount of data generated is now the major challenge. This review will focus on the development and implementation of dereplication strategies and will highlight the importance of each stage of the process from sample preparation to chromatographic separation and finally toward both manual and more targeted methods for automated dereplication of fungal natural products using state-of-the art MS instrumentation.

Dereplication-guided isolation of depsides thielavins S-T and lecanorins D-F from the endophytic fungus Setophoma sp.

Dereplication methodology using UHPLC-DAD-QTOFMS was applied during the metabolic profiling investigation of the endophyte Setophoma sp., a fungus isolated from symptomless guava fruits. The approach performed allowed a fast analysis of the microbial secondary metabolites. From this fungus, seven highly C-alkylated depsides were isolated and identified as polyketides thielavins S, T, U and V and lecanorins D, E and F. Their structures were elucidated through spectroscopic methods including NMR, HRMS and especially with assistance of HRMS/MS experiments. The compounds were tested for quorum sensing regulation activity in the virulence gene expression of Staphylococcus aureus, but no inhibitory effect was detected. Nevertheless, moderate antibacterial activity was encountered in three of tested depsides, particularly with thielavin T, whose MIC was 6.25 µg/mL against S. aureus.

Characterization of four new antifungal yanuthones from Aspergillus niger.

Four new yanuthone analogs (1-4) were isolated from the filamentous fungus Aspergillus niger. The structures of the new compounds were elucidated on the basis of UHPLC-DAD-HRMS data and one-dimensional and two-dimensional NMR spectroscopy. Labeling studies with (13)C8-6-methylsalicylic acid identified three class I yanuthones originating from the polyketide 6-methylsalicylic acid (yanuthone K, L and M (1-3)) and a class II yanuthone, which was named yanuthone X2 (4). The four new compounds were tested toward the pathogenic yeast Candida albicans and all displayed antifungal activity. Yanuthone X2 represents the first example of a bioactive class II yanuthone, demonstrating the pharmaceutical potential of this class.

Characterization of Alternaria strains from Argentinean blueberry, tomato, walnut and wheat.

Alternaria species have the ability to produce a variety of secondary metabolite, which plays important roles in food safety. Argentina is the second largest exporter of fresh and processed food products to Europe, however, few studies on Alternaria mycotoxins and other bioactive secondary metabolites have been carried out on Argentinean cereals, fruit and vegetables. Knowing the full chemical potential and the distribution of Alternaria spp. on crops, it is necessary to establish a toxicological risk assessment for food products for human consumption. In the present study, 87 Alternaria strains from different substrates (tomato, wheat, blueberries and walnuts) were characterized according to morphology and metabolite production. Aggressive dereplication (accurate mass, isotopic patterns and lists of all described compounds from Alternaria) was used for high-throughput evaluation of the chemical potential. Four strains belonged to the Alternaria infectoria sp.-grp., 6 to the Alternaria arborescens sp.-grp., 6 showed a sporulation pattern similar to that of "M" according to Simmons, 1 to that of Alternaria vaccinii, and the remaining 70 constituted a diverse group belonging to morphological groups "G" and "H". The cluster analysis yielded 16 almost identical dendrograms and grouped the Alternaria strains into four clusters and 11 singletons and outlier groups. The chemical analysis showed that AOH and AME were the most common metabolites produced, followed by TEN, ALXs and TeA. The A. infectoria sp.-grp. had no metabolites in common with the rest of the strains. Several secondary metabolites isolated from large-spored Alternaria species or other fungal genera were detected, such as dehydrocurvularin, pyrenochaetic acid and alternarienonic acid. The strains isolated from tomato produced lower amounts of metabolites than strains from blueberries, walnut and wheat, although individual strains from tomato produced the highest amount of some metabolites. The A. infectoria sp.-grp. was unique to cereals, whereas strains classified as belonging to the A. arborescens sp.-grp or having sporulation pattern "M" were only isolated from tomatoes. Otherwise, no clear association between substrate and identity could be found. The analyses in the study show that at least 75% of the Argentinean strains are able to produce potential mycotoxins.