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Mycotoxins are a major threat for human health. Food safety control relies on the identification of the toxins or the detection of the expression of the respective genes. Contamination of food and feed with toxin-producing fungi is a major threat in agriculture and for human health. The filamentous fungus Alternaria alternata is one of the most widespread postharvest contaminants and a weak plant pathogen. It produces a large variety of secondary metabolites with alternariol and its derivatives as characteristic mycotoxin. Other important phyto- and mycotoxins are perylene quinones (PQs), some of which have anticancer properties. Here, we discovered that the PQ altertoxin (ATX) biosynthesis shares most enzymes with the 1,8-dihydroxynaphthalene (1,8-DHN) melanin pathway. However, melanin was formed in aerial hyphae and spores, and ATXs were synthesized in substrate hyphae. This spatial separation is achieved through the promiscuity of a polyketide synthase, presumably producing a pentaketide (T4HN), a hexaketide (AT4HN), and a heptaketide (YWA1) as products. T4HN directly enters the altertoxin and DHN melanin pathway, whereas AT4HN and YWA1 can be converted only in aerial hyphae, which probably leads to a higher T4HN concentration, favoring 1,8-DHN melanin formation. Whereas the production of ATXs was strictly dependent on the CmrA transcription factor, melanin could still be produced in the absence of CmrA to some extent. This suggests that different cues regulate melanin and toxin formation. Since DHN melanin is produced by many fungi, PQs or related compounds may be produced in many more fungi than so far assumed. IMPORTANCE Mycotoxins are a major threat for human health. Food safety control relies on the identification of the toxins or the detection of the expression of the respective genes. The latter method, however, relies on the knowledge of the biosynthetic pathway and the key genes. Alternaria alternata is a major food contaminant and produces many different mycotoxins with altertoxins and other perylene quinones as prominent examples. Here, we discovered that the biosynthetic pathway for altertoxins shares most of the enzymes with the dihydroxynaphthalene (DHN) melanin pathway. Because the DHN melanin pathway is widespread among fungi, the production of mycotoxins of the perylene quinone class could be more widespread than so far anticipated.

Trichoderma atroviride (Ascomycota, Sordariomycetes) is a well-known mycoparasite applied for protecting plants against fungal pathogens. Its mycoparasitic activity involves processes shared with plant and human pathogenic fungi such as the production of cell wall degrading enzymes and secondary metabolites and is tightly regulated by environmental cues. In eukaryotes, the conserved Target of Rapamycin (TOR) kinase serves as a central regulator of cellular growth in response to nutrient availability. Here we describe how alteration of the activity of TOR1, the single and essential TOR kinase of T . atroviride , by treatment with chemical TOR inhibitors or by genetic manipulation of selected TOR pathway components affected various cellular functions. Loss of TSC1 and TSC2, that are negative regulators of TOR complex 1 (TORC1) in mammalian cells, resulted in altered nitrogen source-dependent growth of T . atroviride , reduced mycoparasitic overgrowth and, in the case of Δ tsc1 , a diminished production of numerous secondary metabolites. Deletion of the gene encoding the GTPase RHE2, whose mammalian orthologue activates mTORC1, led to rapamycin hypersensitivity and altered secondary metabolism, but had an only minor effect on vegetative growth and mycoparasitic overgrowth. The latter also applied to mutants missing the npr1-1 gene that encodes a fungus-specific kinase known as TOR target in yeast. Genome-wide transcriptome analysis confirmed TOR1 as a regulatory hub that governs T . atroviride metabolism and processes associated to ribosome biogenesis, gene expression and translation. In addition, mycoparasitism-relevant genes encoding terpenoid and polyketide synthases, peptidases, glycoside hydrolases, small secreted cysteine-rich proteins, and G protein coupled receptors emerged as TOR1 targets. Our results provide the first in-depth insights into TOR signaling in a fungal mycoparasite and emphasize its importance in the regulation of processes that critically contribute to the antagonistic activity of T . atroviride .

Many metabolomics studies use mixtures of (acidified) methanol and water for sample extraction. In the present study, we investigated if the extraction with methanol can result in artifacts. To this end, wheat leaves were extracted with mixtures of native and deuterium-labeled methanol and water, with or without 0.1% formic acid. Subsequently, the extracts were analyzed immediately or after storage at 10 °C, −20 °C or −80 °C with an HPLC-HESI-QExactive HF-Orbitrap instrument. Our results showed that 88 (8%) of the >1100 detected compounds were derived from the reaction with methanol and either formed during sample extraction or short-term storage. Artifacts were found for various substance classes such as flavonoids, carotenoids, tetrapyrrols, fatty acids and other carboxylic acids that are typically investigated in metabolomics studies. 58 of 88 artifacts were common between the two tested extraction variants. Remarkably, 34 of 73 (acidified extraction solvent) and 33 of 73 (non-acidified extraction solvent) artifacts were formed de novo as none of these meth(ox)ylated metabolites were found after extraction of native leaf samples with CD3OH/H2O. Moreover, sample extracts stored at 10 °C for several days, as can typically be the case during longer measurement sequences, led to an increase in both the number and abundance of methylated artifacts. In contrast, frozen sample extracts were relatively stable during a storage period of one week. Our study shows that caution has to be exercised if methanol is used as the extraction solvent as the detected metabolites might be artifacts rather than natural constituents of the biological system. In addition, we recommend storing sample extracts in deep freezers immediately after extraction until measurement.

Lyophilization is a common method used for stabilizing biological samples prior to storage or to concentrate extracts. However, it is possible that this process may alter the metabolic composition or lead to the loss of metabolites. In this study, the performance of lyophilization is investigated in the example of wheat roots. To this end, native and 13C-labelled, fresh or already lyophilized root samples, and (diluted) extracts with dilution factors up to 32 and authentic reference standards were investigated. All samples were analyzed using RP-LC-HRMS. Results show that using lyophilization for the stabilization of plant material altered the metabolic sample composition. Overall, 7% of all wheat metabolites detected in non-lyophilized samples were not detected in dried samples anymore, and up to 43% of the remaining metabolites exhibited significantly increased or decreased abundances. With respect to extract concentration, less than 5% of the expected metabolites were completely lost by lyophilization and the recovery rates of the remaining metabolites were slightly reduced with increasing concentration factors to an average of 85% at an enrichment factor of 32. Compound annotation did not indicate specific classes of wheat metabolites to be affected.

Screening for new bioactive microbial metabolites, we found a novel okaramine derivative, for which we propose the trivial name lemmokaramine, as well as two already known okaramine congeners – okaramine H and okaramine J - responsible for antimicrobial activity of the recently described microscopic filamentous fungus, Keratinophyton lemmensii BiMM-F76 (= CCF 6359). In addition, two novel substances, a new cyclohexyl denominated lemmensihexol and a new tetrahydroxypyrane denominated lemmensipyrane, were purified and characterized. The compounds were isolated from the culture extract of the fungus grown on modified yeast extract sucrose medium by means of flash chromatography followed by preparative HPLC. The chemical structures were elucidated by NMR and LC-MS. The new okaramine (lemmokaramine) exerted antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and fungi and anticancer activity against different mammalian cell lines (Caco-2, HCT116, HT29, SW480, MCM G1, and MCM DLN). Furthermore, we found a significant antioxidant effect of lemmokaramine following H 2 O 2 treatment indicated by activation of the Nrf2 pathway. This is the first report describing analysis and structural elucidation of bioactive metabolites for the onygenalean genus Keratinophyton .

The major Fusarium mycotoxin deoxynivalenol (DON) is a virulence factor in wheat and has also been shown to induce defense responses in host plant tissue. In this study, global and tracer labeling with 13C were combined to annotate the overall metabolome of wheat spikes and to evaluate the response of phenylalanine-related pathways upon treatment with DON. At anthesis, spikes of resistant and susceptible cultivars as well as two related near isogenic wheat lines (NILs) differing in the presence/absence of the major resistance QTL Fhb1 were treated with 1 mg DON or water (control), and samples were collected at 0, 12, 24, 48, and 96 h after treatment (hat). A total of 172 Phe-derived wheat constituents were detected with our untargeted approach employing 13C-labeled phenylalanine and subsequently annotated as flavonoids, lignans, coumarins, benzoic acid derivatives, hydroxycinnamic acid amides (HCAAs), as well as peptides. Ninety-six hours after the DON treatment, up to 30% of the metabolites biosynthesized from Phe showed significantly increased levels compared to the control samples. Major metabolic changes included the formation of precursors of compounds implicated in cell wall reinforcement and presumed antifungal compounds. In addition, also dipeptides, which presumably are products of proteolytic degradation of truncated proteins generated in the presence of the toxin, were significantly more abundant upon DON treatment. An in-depth comparison of the two NILs with correlation clustering of time course profiles revealed some 70 DON-responsive Phe derivatives. While several flavonoids had constitutively different abundance levels between the two NILs differing in resistance, other Phe-derived metabolites such as HCAAs and hydroxycinnamoyl quinates were affected differently in the two NILs after treatment with DON. Our results suggest a strong activation of the general phenylpropanoid pathway and that coumaroyl-CoA is mainly diverted towards HCAAs in the presence of Fhb1 , whereas the metabolic route to monolignol(-conjugates), lignans, and lignin seems to be favored in the absence of the Fhb1 resistance quantitative trait loci.

Untargeted approaches and thus biological interpretation of metabolomics results are still hampered by the reliable assignment of the global metabolome as well as classification and (putative) identification of metabolites. In this work we present an liquid chromatography-mass spectrometry (LC-MS)–based stable isotope assisted approach that combines global metabolome and tracer based isotope labeling for improved characterization of (unknown) metabolites and their classification into tracer derived submetabolomes. To this end, wheat plants were cultivated in a customized growth chamber, which was kept at 400 ± 50 ppm 13CO2 to produce highly enriched uniformly 13C-labeled sample material. Additionally, native plants were grown in the greenhouse and treated with either 13C9-labeled phenylalanine (Phe) or 13C11-labeled tryptophan (Trp) to study their metabolism and biochemical pathways. After sample preparation, liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis and automated data evaluation, the results of the global metabolome- and tracer-labeling approaches were combined. A total of 1,729 plant metabolites were detected out of which 122 respective 58 metabolites account for the Phe- and Trp-derived submetabolomes. Besides m/z and retention time, also the total number of carbon atoms as well as those of the incorporated tracer moieties were obtained for the detected metabolite ions. With this information at hand characterization of unknown compounds was improved as the additional knowledge from the tracer approaches considerably reduced the number of plausible sum formulas and structures of the detected metabolites. Finally, the number of putative structure formulas was further reduced by isotope-assisted annotation tandem mass spectrometry (MS/MS) derived product ion spectra of the detected metabolites. A major innovation of this paper is the classification of the metabolites into submetabolomes which turned out to be valuable information for effective filtering of database hits based on characteristic structural subparts. This allows the generation of a final list of true plant metabolites, which can be characterized at different levels of specificity.

Background Members of the fungal genus Trichoderma are well-known for their mycoparasitic and plant protecting activities, rendering them important biocontrol agents. One of the most significant specialized metabolites (SMs) produced by various Trichoderma species is the unsaturated lactone 6-pentyl-alpha-pyrone (6-PP). Although first identified more than 50 years ago and having pronounced antifungal and plant growth-promoting properties, the biosynthetic pathway of 6-PP still remains unresolved. Results Here, we demonstrate that 6-PP is biosynthesized via the polyketide biosynthesis pathway. We identified Pks1, an iterative type I polyketide synthase, as crucial for its biosynthesis in Trichoderma atroviride , a species recognized for its prominent 6-PP production abilities. Phylogenetic and comparative genomic analyses revealed that the pks1 gene is part of a biosynthetic gene cluster conserved in those Trichoderma species that are known to produce 6-PP. Deletion of pks1 caused a complete loss of 6-PP production in T. atroviride and a significant reduction in antifungal activity against Botrytis cinerea and Rhizoctonia solani . Surprisingly, the absence of pks1 led to enhanced lateral root formation in Arabidopsis thaliana during interaction with T. atroviride . Transcriptomic analysis revealed co-regulation of pks1 with adjacent genes, including candidates coding for a C3H1-type zinc finger protein and lytic polysaccharide monooxygenase, suggesting coordination between 6-PP biosynthesis and environmental response mechanisms. Conclusion Our findings establish pks1 as an essential gene for 6-PP biosynthesis in T. atroviride , providing novel insights into the production of one of the most significant compounds of this mycoparasite. These findings may pave the way for the development of improved biocontrol agents and the application of 6-PP as potent biopesticide contributing to an eco-friendly and sustainable way of plant disease management.

In the process of screening for new bioactive microbial metabolites we found a novel ƴ-pyrone derivative for which we propose the trivial name luteapyrone, in a recently described microscopic filamentous fungus, Metapochonia lutea BiMM-F96/DF4. The compound was isolated from the culture extract of the fungus grown on modified yeast extract sucrose medium by means of flash chromatography followed by preparative HPLC. The chemical structure was elucidated by NMR and LC-MS. The new compound was found to be non-cytotoxic against three mammalian cell lines (HEK 263, KB-3.1 and Caco-2). Similarly, no antimicrobial activity was observed in tested microorganisms (gram positive and negative bacteria, yeast and fungi).

Stable isotope-assisted approaches can improve untargeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) metabolomics studies. Here, we demonstrate at the example of chemically stressed wheat that metabolome-wide internal standardization by globally 13C-labeled metabolite extract (GLMe-IS) of experimental-condition-matched biological samples can help to improve the detection of treatment-relevant metabolites and can aid in the post-acquisition assessment of putative matrix effects in samples obtained upon different treatments. For this, native extracts of toxin- and mock-treated (control) wheat ears were standardized by the addition of uniformly 13C-labeled wheat ear extracts that were cultivated under similar experimental conditions (toxin-treatment and control) and measured with LC-HRMS. The results show that 996 wheat-derived metabolites were detected with the non-condition-matched 13C-labeled metabolite extract, while another 68 were only covered by the experimental-condition-matched GLMe-IS. Additional testing is performed with the assumption that GLMe-IS enables compensation for matrix effects. Although on average no severe matrix differences between both experimental conditions were found, individual metabolites may be affected as is demonstrated by wrong decisions with respect to the classification of significantly altered metabolites. When GLMe-IS was applied to compensate for matrix effects, 272 metabolites showed significantly altered levels between treated and control samples, 42 of which would not have been classified as such without GLMe-IS.