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Many studies aim at maximizing fungal secondary metabolite production but the influence of light during cultivation has often been neglected. Here, we combined an untargeted isotope-assisted liquid chromatography–high-resolution mass spectrometry-based metabolomics approach with standardized cultivation of Trichoderma atroviride under three defined light regimes (darkness (PD), reduced light (RL) exposure, and 12/12 h light/dark cycle (LD)) to systematically determine the effect of light on secondary metabolite production. Comparative analyses revealed a similar metabolite profile upon cultivation in PD and RL, whereas LD treatment had an inhibiting effect on both the number and abundance of metabolites. Additionally, the spatial distribution of the detected metabolites for PD and RL was analyzed. From the more than 500 detected metabolites, only 25 were exclusively produced upon fungal growth in darkness and 85 were significantly more abundant in darkness. The majority were detected under both cultivation conditions and annotation revealed a cluster of substances whose production followed the pattern observed for the well-known T. atroviride metabolite 6-pentyl-alpha-pyrone. We conclude that cultivation of T. atroviride under RL can be used to maximize secondary metabolite production.

ABSTRACT 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.

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.

Abstract Background Stable isotopically labelled organisms have found wide application in life science research including plant physiology, plant stress and defense as well as metabolism related sciences. Therefore, the reproducible production of plant material enriched with stable isotopes such as 13C and 15N is of considerable interest. A high degree of enrichment (> 96 atom %) with a uniformly distributed isotope (global labelling) is accomplished by a continuous substrate supply during plant growth/cultivation. In the case of plants, 13C-labelling can be achieved by growth in 13CO2(g) atmosphere while global 15N-labelling needs 15N- containing salts in the watering/nutrient solution. Here, we present a method for the preparation of 13C and 15N-labelled plants by the use of closed growth chambers and hydroponic nutrient supply. The method is exemplified with durum wheat. Results In total, 330 g of globally 13C- and 295 g of 15N-labelled Triticum durum wheat was produced during 87 cultivation days. For this, a total of 3.88 mol of 13CO2(g) and 58 mmol of 15N were consumed. The degree of enrichment was determined by LC-HRMS and ranged between 96 and 98 atom % for 13C and 95–99 atom % for 15N, respectively. Additionally, the isotopically labelled plant extracts were successfully used for metabolome-wide internal standardisation of native T.durum plants. Application of an isotope-assisted LC-HRMS workflow enabled the detection of 652 truly wheat-derived metabolites out of which 143 contain N. Conclusion A reproducible cultivation which makes use of climate chambers and hydroponics was successfully adapted to produce highly enriched, uniformly 13C- and 15N-labelled wheat. The obtained plant material is suitable to be used in all kinds of isotope-assisted research. The described technical equipment and protocol can easily be applied to other plants to produce 13C-enriched biological samples when the necessary specific adaptations e.g. temperature and light regime, as well as nutrient supply are considered. Additionally, the 15N-labelling method can also be carried out under regular glasshouse conditions without the need for customised atmosphere.

Environmental exposure to xenoestrogens, i.e., chemicals that imitate the hormone 17β-estradiol, has the potential to influence hormone homeostasis and action. Detailed knowledge of xenobiotic biotransformation processes in cell models is key when transferring knowledge learned from in vitro models to in vivo relevance. This study elucidated the metabolism of two naturally-occurring phyto- and mycoestrogens; namely genistein and zearalenone, in an estrogen receptor positive breast cancer cell line (MCF-7) with the aid of stable isotope-assisted metabolomics and the bioinformatic tool MetExtract II. Metabolism was studied in a time course experiment after 2 h, 6 h and 24 h incubation. Twelve and six biotransformation products of zearalenone and genistein were detected, respectively, clearly demonstrating the abundant xenobiotic biotransformation capability of the cells. Zearalenone underwent extensive phase-I metabolism resulting in α-zearalenol (α-ZEL), a molecule known to possess a significantly higher estrogenicity, and several phase-II metabolites (sulfo- and glycoconjugates) of the native compound and the major phase I metabolite α-ZEL. Moreover, potential adducts of zearalenone with a vitamin and several hydroxylated metabolites were annotated. Genistein metabolism resulted in sulfation, combined sulfation and hydroxylation, acetylation, glucuronidation and unexpectedly adduct formation with pentose- and hexose sugars. Kinetics of metabolite formation and subsequent excretion into the extracellular medium revealed a time-dependent increase in most biotransformation products. The untargeted elucidation of biotransformation products formed during cell culture experiments enables an improved and more meaningful interpretation of toxicological assays and has the potential to identify unexpected or unknown metabolites.

The plant pathogen Fusarium graminearum is a proficient producer of mycotoxins and other in part still unknown secondary metabolites, some of which might act as virulence factors on wheat. The PKS15 gene is expressed only in planta, so far hampering the identification of an associated metabolite. Here we combined the activation of silent gene clusters by chromatin manipulation (kmt6) with blocking the metabolic flow into the competing biosynthesis of the two major mycotoxins deoxynivalenol and zearalenone. Using an untargeted metabolomics approach, two closely related metabolites were found in triple mutants (kmt6 tri5 pks4,13) deficient in production of the major mycotoxins deoxynivalenol and zearalenone, but not in strains with an additional deletion in PKS15 (kmt6 tri5 pks4,13 pks15). Characterization of the metabolites, by LC-HRMS/MS in combination with a stable isotope-assisted tracer approach, revealed that they are likely hybrid polyketides comprising a polyketide part consisting of malonate-derived acetate units and a structurally deviating part. We propose the names gramiketide A and B for the two metabolites. In a biological experiment, both gramiketides were formed during infection of wheat ears with wild-type but not with pks15 mutants. The formation of the two gramiketides during infection correlated with that of the well-known virulence factor deoxynivalenol, suggesting that they might play a role in virulence.

Abstract Background Filamentous fungi have evolved to succeed in nature by efficient growth and degradation of substrates, but also due to the production of secondary metabolites including mycotoxins. For Trichoderma reesei, as a biotechnological workhorse for homologous and heterologous protein production, secondary metabolite secretion is of particular importance for industrial application. Recent studies revealed an interconnected regulation of enzyme gene expression and carbon metabolism with secondary metabolism. Results Here, we investigated gene regulation by YPR2, one out of two transcription factors located within the SOR cluster of T. reesei, which is involved in biosynthesis of sorbicillinoids. Transcriptome analysis showed that YPR2 exerts its major function in constant darkness upon growth on cellulose. Targets (direct and indirect) of YPR2 overlap with induction specific genes as well as with targets of the carbon catabolite repressor CRE1 and a considerable proportion is regulated by photoreceptors as well. Functional category analysis revealed both effects on carbon metabolism and secondary metabolism. Further, we found indications for an involvement of YPR2 in regulation of siderophores. In agreement with transcriptome data, mass spectrometric analyses revealed a broad alteration in metabolite patterns in ∆ypr2. Additionally, YPR2 positively influenced alamethicin levels along with transcript levels of the alamethicin synthase tex1 and is essential for production of orsellinic acid in darkness. Conclusions YPR2 is an important regulator balancing secondary metabolism with carbon metabolism in darkness and depending on the carbon source. The function of YPR2 reaches beyond the SOR cluster in which ypr2 is located and happens downstream of carbon catabolite repression mediated by CRE1.

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 Δtsc1, 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.

The genus Lysobacter includes several bacterial species which show potential for being used in biological control of plant diseases. It was shown recently that several Lysobacter type strains produce volatile organic compounds (VOCs) which controlled the growth of Phytophthora infestans in vitro when the bacteria were grown on a protein rich medium. In the present study, Lysobacter capsici AZ78 (AZ78) has been tested for its potential to produce VOCs that may contribute to the bioactivity against soilborne plant pathogens. To this end, split Petri dish assays of bacterial cultures have been combined with GC-MS measurements with the aim to reveal the identity of the VOCs which inhibit the growth of Pythium ultimum Rhizoctonia solani, and Sclerotinia minor. While AZ78 completely suppressed the growth of P. ultimum and S. minor, the growth of R. solani was still reduced significantly. The GC-MS analysis revealed 22 VOCs to be produced by AZ78, the majority of which were (putatively) identified as mono- and dialkylated methoxypyrazines. Based on additional cultivation and GC-MS experiments, 2,5-dimethylpyrazine, 2-ethyl-3-methoxypyrazine and 2-isopropyl-3-methoxypyrazine were selected as presumable bioactive compounds. Further bioassays employing indirect exposure to standard solutions (1–10 mg per Petri dish) of the synthetic compounds via the gas phase, revealed that each of these pyrazines was able to suppress the growth of the pathogens under investigation. The results of this study highlight the possible future implementation of pyrazine derivatives in the control of soilborne plant diseases and further support the biocontrol potential of L. capsici AZ78.

The necrotrophic mycoparasite Trichoderma atroviride is a biological pest control agent frequently applied in agriculture for the protection of plants against fungal phytopathogens. One of the main secondary metabolites produced by this fungus is 6-pentyl-α-pyrone (6-PP). 6-PP is an organic compound with antifungal and plant growth-promoting activities, whose biosynthesis was previously proposed to involve a lipoxygenase (Lox). In this study, we investigated the role of the single lipoxygenase-encoding gene lox1 encoded in the T. atroviride genome by targeted gene deletion. We found that light inhibits 6-PP biosynthesis but lox1 is dispensable for 6-PP production as well as for the ability of T. atroviride to parasitize and antagonize host fungi. However, we found Lox1 to be involved in T. atroviride conidiation in darkness, in injury-response, in the production of several metabolites, including oxylipins and volatile organic compounds, as well as in the induction of systemic resistance against the plant-pathogenic fungus Botrytis cinerea in Arabidopsis thaliana plants. Our findings give novel insights into the roles of a fungal Ile-group lipoxygenase and expand the understanding of a light-dependent role of these enzymes.

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).

The emerging mycotoxin fusaproliferin is produced by Fusarium proliferatum and other related Fusarium species. Several fungi from other taxonomic groups were also reported to produce fusaproliferin or the deacetylated derivative, known as siccanol or terpestacin. Here, we describe the identification and functional characterization of the Fusarium proliferatum genes encoding the fusaproliferin biosynthetic enzymes: a terpenoid synthase, two cytochrome P450s, a FAD-oxidase and an acetyltransferase. With the exception of one gene encoding a CYP450 (FUP2, FPRN_05484), knock-out mutants of the candidate genes could be generated, and the production of fusaproliferin and intermediates was tested by LC-MS/MS. Inactivation of the FUP1 (FPRN_05485) terpenoid synthase gene led to complete loss of fusaproliferin production. Disruption of a putative FAD-oxidase (FUP4, FPRN_05486) did not only affect oxidation of preterpestacin III to terpestacin, but also of new side products (11-oxo-preterpstacin and terpestacin aldehyde). In the knock-out strains lacking the predicted acetyltransferase (FUP5, FPRN_05487) fusaproliferin was no longer formed, but terpestacin was found at elevated levels. A model for the biosynthesis of fusaproliferin and of novel derivatives found in mutants is presented.

Determining the mode of action of microbial biocontrol agents plays a key role in their development and registration as commercial biopesticides. The biocontrol rhizobacterium Lysobacter capsici AZ78 (AZ78) is able to inhibit a vast array of plant pathogenic oomycetes and Gram-positive bacteria due to the release of antimicrobial secondary metabolites. A combination of MALDI-qTOF-MSI and UHPLC-HRMS/M was applied to finely dissect the AZ78 metabolome and identify the main secondary metabolites involved in the inhibition of plant pathogenic microorganisms. Under nutritionally limited conditions, MALDI-qTOF-MSI revealed that AZ78 is able to release a relevant number of antimicrobial secondary metabolites belonging to the families of 2,5-diketopiperazines, cyclic lipodepsipeptides, macrolactones and macrolides. In vitro tests confirmed the presence of secondary metabolites toxic against Pythium ultimum and Rhodococcus fascians in AZ78 cell-free extracts. Subsequently, UHPLC-HRMS/MS was used to confirm the results achieved with MALDI-qTOF-MSI and investigate for further putative antimicrobial secondary metabolites known to be produced by Lysobacter spp. This technique confirmed the presence of several 2,5-diketopiperazines in AZ78 cell-free extracts and provided the first evidence of the production of the cyclic depsipeptide WAP-8294A2 in a member of L. capsici species. Moreover, UHPLC-HRMS/MS confirmed the presence of dihydromaltophilin/Heat Stable Antifungal Factor (HSAF) in AZ78 cell-free extracts. Due to the production of HSAF by AZ78, cell-free supernatants were effective in controlling Plasmopara viticola on grapevine leaf disks after exposure to high temperatures. Overall, our work determined the main secondary metabolites involved in the biocontrol activity of AZ78 against plant pathogenic oomycetes and Gram-positive bacteria. These results might be useful for the future development of this bacterial strain as the active ingredient of a microbial biopesticide that might contribute to a reduction in the chemical input in agriculture.

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.

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.

Fusarium graminearum is a plant pathogenic fungus which is able to infect wheat and other economically important cereal crop species. The role of ethylene in the interaction with host plants is unclear and controversial. We have analyzed the inventory of genes with a putative function in ethylene production or degradation of the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC). F. graminearum, in contrast to other species, does not contain a candidate gene encoding ethylene-forming enzyme. Three genes with similarity to ACC synthases exist; heterologous expression of these did not reveal enzymatic activity. The F. graminearum genome contains in addition two ACC deaminase candidate genes. We have expressed both genes in E. coli and characterized the enzymatic properties of the affinity-purified products. One of the proteins had indeed ACC deaminase activity, with kinetic properties similar to ethylene-stress reducing enzymes of plant growth promoting bacteria. The other candidate was inactive with ACC but turned out to be a d-cysteine desulfhydrase. Since it had been reported that ethylene insensitivity in transgenic wheat increased Fusarium resistance and reduced the content of the mycotoxin deoxynivalenol (DON) in infected wheat, we generated single and double knockout mutants of both genes in the F. graminearum strain PH-1. No statistically significant effect of the gene disruptions on fungal spread or mycotoxin content was detected, indicating that the ability of the fungus to manipulate the production of the gaseous plant hormones ethylene and H2S is dispensable for full virulence.

Volatile organic compounds (VOCs) play an essential role in microbe–microbe and plant–microbe interactions. We investigated the interaction between two plant growth-promoting rhizobacteria, and their interaction with tomato plants. VOCs produced by Pantoea agglomerans MVC 21 modulates the release of siderophores, the solubilisation of phosphate and potassium by Pseudomonas (Ps.) putida MVC 17. Moreover, VOCs produced by P. agglomerans MVC 21 increased lateral root density (LRD), root and shoot dry weight of tomato seedlings. Among the VOCs released by P. agglomerans MVC 21, only dimethyl disulfide (DMDS) showed effects similar to P. agglomerans MVC 21 VOCs. Because of the effects on plants and bacterial cells, we investigated how P. agglomerans MVC 21 VOCs might influence bacteria–plant interaction. Noteworthy, VOCs produced by P. agglomerans MVC 21 boosted the ability of Ps. putida MVC 17 to increase LRD and root dry weight of tomato seedlings. These results could be explained by the positive effect of DMDS and P. agglomerans MVC 21 VOCs on acid 3-indoleacetic production in Ps. putida MVC 17. Overall, our results clearly indicated that P. agglomerans MVC 21 is able to establish a beneficial interaction with Ps. putida MVC 17 and tomato plants through the emission of DMDS.

Plant beneficial rhizobacteria may antagonize soilborne plant pathogens by producing a vast array of volatile organic compounds (VOCs). The production of these compounds depends on the medium composition used for bacterial cell growth. Accordingly, Lysobacter capsici AZ78 (AZ78) grown on a protein-rich medium was previously found to emit volatile pyrazines with toxic activity against soilborne phypathogenic fungi and oomycetes. However, the discrepancy between the quantity of pyrazines in the gaseous phase and the minimum quantity needed to achieve inhibition of plant pathogens observed, lead us to further investigate the volatile-mediated inhibitory activity of AZ78. Here, we show that, besides VOCs, AZ78 cells produce ammonia in increased amounts when a protein-rich medium is used for bacterial growth. The production of this volatile compound caused the alkalinization of the physically separated culture medium where Rhizoctonia solani was inoculated subsequently. Results achieved in this work clearly demonstrate that VOC, ammonia and the growth medium alkalinization contribute to the overall inhibitory activity of AZ78 against R. solani. Thus, our findings suggest that the volatile-mediated inhibitory activity of rhizobacteria in protein-rich substrates can be regarded as a result of multiple factors interaction, rather than exclusively VOCs production.

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.

The ascomycete Trichoderma atroviride is well known for its mycoparasitic lifestyle. Similar to other organisms, light is an important cue for T. atroviride. However, besides triggering of conidiation, little is known on the physiological responses of T. atroviride to light. In this study, we analyzed how cultivation under different light wavelengths and regimes impacted the behavior of two T. atroviride wild-type strains: IMI206040 and P1. While colony extension of both strains was slightly affected by light, massive differences in their photoconidation responses became evident. T. atroviride P1 colonies conidiated under all conditions tested including growth in complete darkness, while IMI206040 required white, blue or green light to trigger asexual reproduction. Interestingly, deletion of the stress-activated MAP kinase-encoding gene tmk3 abolished the ability of strain P1 to conidiate in red and yellow light as well as in darkness. Furthermore, light-dependent differences in the mycoparasitic activity and in the biosynthesis of the secondary metabolite 6-pentyl-α-pyrone (6-PP) became evident. 6-PP production was highest upon dark incubation, while light, especially exposure to white light as light/dark cycles, had an inhibitory effect on its biosynthesis. We conclude that the response of T. atroviride to light is strain-dependent and impacts differentiation, mycoparasitism, and 6-PP production; hence, this should be considered in experiments testing the mycoparasitic activity of these fungi.

Deoxynivalenol (DON) is a protein synthesis inhibitor produced by the Fusarium species, which frequently contaminates grains used for human or animal consumption. We treated a wheat suspension culture with DON or one of its acetylated derivatives, 3-acetyl-DON (3-ADON), 15-acetyl-DON (15-ADON) and 3,15-diacetyl-DON (3,15-diADON), and monitored the metabolization over a course of 96 h. Supernatant and cell extract samples were analyzed using a tailored LC-MS/MS method for the quantification of DON metabolites. We report the formation of tentatively identified DON-15-O-β-D-glucoside (D15G) and of 15-acetyl-DON-3-sulfate (15-ADON3S) as novel deoxynivalenol metabolites in wheat. Furthermore, we found that the recently identified 15-acetyl-DON-3-O-β-D-glucoside (15-ADON3G) is the major metabolite produced after 15-ADON challenge. 3-ADON treatment led to a higher intracellular content of toxic metabolites after six hours compared to all other treatments. 3-ADON was exclusively metabolized into DON before phase II reactions occurred. In contrast, we found that 15-ADON was directly converted into 15-ADON3G and 15-ADON3S in addition to metabolization into deoxynivalenol-3-O-β-D-glucoside (D3G). This study highlights significant differences in the metabolization of DON and its acetylated derivatives.

Forty-five volatile organic compounds (VOCs) were identified or annotated in the mandibular gland reservoir content (MGRC) of the Southeast Asian ant Colobopsis explodens Laciny and Zettel, 2018 (Hymenoptera: Formicidae), using headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS) and liquid extraction combined with GC-MS. In extension of previous reports on VOCs of C. explodens, members of different compound classes, such as alkanes, aliphatic and aromatic carboxylic acids, and phenolics, were detected. The ketone 2-heptanone and the biochemically related phenolics benzene-1,3,5-triol (phloroglucinol, PG), 1-(2,4,6-trihydroxyphenyl)ethanone (monoacetylphloroglucinol, MAPG), 5,7-dihydroxy-2-methylchromen-4-one (noreugenin), and 1-(3-Acetyl-2,4,6-trihydroxyphenyl)ethanone (2,4-diacetylphloroglucinol, DAPG) dominated the GC-MS chromatograms. The identities of the main phenolics MAPG and noreugenin were further verified by liquid chromatography-high resolution-tandem mass spectrometry (LC-HRMS/MS). A comparative study of MGRC samples originating from three distinct field expeditions revealed differences in the VOC profiles, but the presence and relative abundances of the dominating constituents were largely consistent in all samples. Our study considerably extends the knowledge about the number and type of VOCs occurring in the MGRC of C. explodens. Based on the type of the detected compounds, we propose that the likely irritant and antibiotic phenolic constituents play a role in defense against arthropod opponents or in protection against microbial pathogens.

Fusarium graminearum is an opportunistic pathogen of cereals where it causes severe yield losses and concomitant mycotoxin contamination of the grains. The pathogen has mixed biotrophic and necrotrophic (saprophytic) growth phases during infection and the regulatory networks associated with these phases have so far always been analyzed together. In this study we compared the transcriptomes of fungal cells infecting a living, actively defending plant representing the mixed live style (pathogenic growth on living flowering wheat heads) to the response of the fungus infecting identical, but dead plant tissues (cold-killed flowering wheat heads) representing strictly saprophytic conditions. We found that the living plant actively suppressed fungal growth and promoted much higher toxin production in comparison to the identical plant tissue without metabolism suggesting that molecules signaling secondary metabolite induction are not pre-existing or not stable in the plant in sufficient amounts before infection. Differential gene expression analysis was used to define gene sets responding to the active or the passive plant as main impact factor and driver for gene expression. We correlated our results to the published F. graminearum transcriptomes, proteomes and secretomes and found that only a limited number of in planta- expressed genes require the living plant for induction but the majority uses simply the plant tissue as signal. Many secondary metabolite (SM) gene clusters show a heterogeneous expression pattern within the cluster indicating that different genetic or epigenetic signals govern the expression of individual genes within a physically linked cluster. Our bioinformatic approach also identified fungal genes which were actively repressed by signals derived from the active plant and may thus represent direct targets of the plant defense against the invading pathogen.

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.

In this study, a total of nine different biotransformation products of the Fusarium mycotoxin deoxynivalenol (DON) formed in wheat during detoxification of the toxin are characterized by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The detected metabolites suggest that DON is conjugated to endogenous metabolites via two major metabolism routes, namely 1) glucosylation (DON-3-glucoside, DON-di-hexoside, 15-acetyl-DON-3-glucoside, DON-malonylglucoside) and 2) glutathione conjugation (DON-S-glutathione, "DON-2H"-S-glutathione, DON-S-cysteinyl-glycine and DON-S-cysteine). Furthermore, conjugation of DON to a putative sugar alcohol (hexitol) was found. A molar mass balance for the cultivar 'Remus' treated with 1 mg DON revealed that under the test conditions approximately 15% of the added DON were transformed into DON-3-glucoside and another 19% were transformed to the remaining eight biotransformation products or irreversibly bound to the plant matrix. Additionally, metabolite abundance was monitored as a function of time for each DON derivative and was established for six DON treated wheat lines (1 mg/ear) differing in resistance quantitative trait loci (QTL) Fhb1 and/or Qfhs.ifa-5A. All cultivars carrying QTL Fhb1 showed similar metabolism kinetics: Formation of DON-Glc was faster, while DON-GSH production was less efficient compared to cultivars which lacked the resistance QTL Fhb1. Moreover, all wheat lines harboring Fhb1 showed significantly elevated D3G/DON abundance ratios.

We investigated the hypothesis that resistance to deoxynivalenol (DON) is a major resistance factor in the Fusarium head blight (FHB) resistance complex of wheat. Ninety-six double haploid lines from a cross between ‘CM-82036’ and ‘Remus’ were examined. The lines were tested for DON resistance after application of the toxin in the ear, and for resistances to initial infection and spread of FHB after artificial inoculation with Fusarium spp. Toxin application to flowering ears induced typical FHB symptoms. Quantitative trait locus (QTL) analyses detected one locus with a major effect on DON resistance (logarithm of odds = 53.1, R2 = 92.6). The DON resistance phenotype was closely associated with an important FHB resistance QTL, Qfhs.ndsu-3BS, which previously was identified as governing resistance to spread of symptoms in the ear. Resistance to the toxin was correlated with resistance to spread of FHB (r = 0.74, P < 0.001). In resistant wheat lines, the applied toxin was converted to DON-3-O-glucoside as the detoxification product. There was a close relation between the DON-3-glucoside/DON ratio and DON resistance in the toxintreated ears (R2 = 0.84). We conclude that resistance to DON is important in the FHB resistance complex and hypothesize that Qfhs.ndsu-3BS either encodes a DON-glucosyltransferase or regulates the expression of such an enzyme.

The Fusarium mycotoxins HT-2 toxin (HT2) and T-2 toxin (T2) are frequent contaminants in oats. These toxins, but also their plant metabolites, may contribute to toxicological effects. This work describes the use of 13C-assisted liquid chromatography–high-resolution mass spectrometry for the first comprehensive study on the biotransformation of HT2 and T2 in oats. Using this approach, 16 HT2 and 17 T2 metabolites were annotated including novel glycosylated and hydroxylated forms of the toxins, hydrolysis products, and conjugates with acetic acid, putative malic acid, malonic acid, and ferulic acid. Further targeted quantitative analysis was performed to study toxin metabolism over time, as well as toxin and conjugate mobility within non-treated plant tissues. As a result, HT2-3-O-β-d-glucoside was identified as the major detoxification product of both parent toxins, which was rapidly formed (to an extent of 74% in HT2-treated and 48% in T2-treated oats within one day after treatment) and further metabolised. Mobility of the parent toxins appeared to be negligible, while HT2-3-O-β-d-glucoside was partly transported (up to approximately 4%) through panicle side branches and stem. Our findings demonstrate that the presented combination of untargeted and targeted analysis is well suited for the comprehensive elucidation of mycotoxin metabolism in plants.

The objective of this study was to quantitatively evaluate mycotoxins in samples of maize and poultry feed produced in Brazil. A multimycotoxin method based on HPLC-MS/MS was applied to investigate the occurrence of toxical fungal metabolites in 119 samples collected from poultry feed factory integrated poultry farms: maize grain (74), poultry feed (36), and feed factory residue (9). Twenty of 101 fungal metabolites investigated were detected and quantified in the samples: aflatoxins B1, B2, G1, and G2, fumonisins B1, B2, and B3, hydrolyzed fumonisin B1, zearalenone, agroclavine, chanoclavine, deoxynivalenol, and nivalenol, and enniatin A, A1, B, B1, beauvericin, kojic acid, and moniliformin. Most samples were contaminated with more than one mycotoxin. All samples were contaminated with fumonisins, with medians values of 1,840 μg/kg, 239 μg/kg, and 23,676 μg/kg for maize, feed, and factory residue samples, respectively. Surprisingly, beauvericin was detected in more than 90% of samples. The median contaminations of aflatoxin and trichothecenes were low, near LOD values. The factory residue presented highest contamination levels for all mycotoxins. This is the first study dealing with agroclavine, chanoclavine, enniatin A, A1, B, B1, beauvericin, and kojic acid contamination of maize and poultry feeds from Brazil.

Due to its unsurpassed sensitivity and selectivity, LC-HRMS is one of the major analytical techniques in metabolomics research. However, limited stability of experimental and instrument parameters may cause shifts and drifts of retention time and mass accuracy or the formation of different ion species, thus complicating conclusive interpretation of the raw data, especially when generated in different analytical batches. Here, a novel software tool for the semi-automated alignment of different measurement sequences is presented. The tool is implemented in the Java programming language, it features an intuitive user interface and its main goal is to facilitate the comparison of data obtained from different metabolomics experiments. Based on a feature list (i.e., processed LC-HRMS chromatograms with mass-to-charge ratio (m/z) values and retention times) that serves as a reference, the tool recognizes both m/z and retention time shifts of single or multiple analytical datafiles/batches of interest. MetMatch is also designed to account for differently formed ion species of detected metabolites. Corresponding ions and metabolites are matched and chromatographic peak areas, m/z values and retention times are combined into a single data matrix. The convenient user interface allows for easy manipulation of processing results and graphical illustration of the raw data as well as the automatically matched ions and metabolites. The software tool is exemplified with LC-HRMS data from untargeted metabolomics experiments investigating phenylalanine-derived metabolites in wheat and T-2 toxin/HT-2 toxin detoxification products in barley.

A glutathione (GSH) adduct of the mycotoxin 4-deoxynivalenol (DON), together with a range of related conjugates, has recently been tentatively identified by LC-MS of DON-treated wheat spikelets. In this study, we prepared samples of DON conjugated at the 10- and 13-positions with GSH, Cys, CysGly, γ-GluCys and N-acetylcysteine (NAC). The mixtures of conjugates were used as standards for LC-HRMS analysis of one of the DON-treated wheat spikelet samples, as well as 19 Norwegian grain samples of spring wheat and 16 grain samples of oats that were naturally-contaminated with DON at concentrations higher than 1 mg/kg. The artificially-contaminated wheat spikelets contained conjugates of GSH, CysGly and Cys coupled at the olefinic 10-position of DON, whereas the naturally-contaminated harvest-ripe grain samples contained GSH, CysGly, Cys, and NAC coupled mainly at the 13-position on the epoxy group. The identities of the conjugates were confirmed by LC-HRMS comparison with authentic standards, oxidation to the sulfoxides with hydrogen peroxide, and examination of product-ion spectra from LC-HRMS/MS analysis. No γ-GluCys adducts of DON were detected in any of the samples. The presence of 15-O-acetyl-DON was demonstrated for the first time in Norwegian grain. The results indicate that a small but significant proportion of DON is metabolized via the GSH-conjugation pathway in plants. To our knowledge, this is the first report of in vivo conjugation of trichothecenes via their epoxy group, which has generally been viewed as unreactive. Because conjugation at the 13-position of DON and other trichothecenes has been shown to be irreversible, this type of conjugate may prove useful as a biomarker of exposure to DON and other 12,13-epoxytrichothecenes.

The evaluation of extraction protocols for untargeted metabolomics approaches is still difficult. We have applied a novel stable isotope-assisted workflow for untargeted LC-HRMS-based plant metabolomics , which allows for the first time every detected feature to be considered for method evaluation. The efficiency and complementarity of commonly used extraction solvents, namely 1 + 3 (v/v) mixtures of water and selected organic solvents (methanol, acetonitrile or methanol/acetonitrile 1 + 1 (v/v)), with and without the addition of 0.1% (v/v) formic acid were compared. Four different wheat organs were sampled, extracted and analysed by LC-HRMS. Data evaluation was performed with the in-house-developed MetExtract II software and R. With all tested solvents a total of 871 metabolites were extracted in ear, 785 in stem, 733 in leaf and 517 in root samples, respectively. Between 48% (stem) and 57% (ear) of the metabolites detected in a particular organ were found with all extraction mixtures, and 127 of 996 metabolites were consistently shared between all extraction agent/organ combinations. In aqueous methanol, acidification with formic acid led to pronounced pH dependency regarding the precision of metabolite abundance and the number of detectable metabolites, whereas extracts of acetonitrile-containing mixtures were less affected. Moreover, methanol and acetonitrile have been found to be complementary with respect to extraction efficiency. Interestingly, the beneficial properties of both solvents can be combined by the use of a water-methanol-acetonitrile mixture for global metabolite extraction instead of aqueous methanol or aqueous acetonitrile alone.

Increasing frequencies of 3-acetyl-deoxynivalenol (3-ADON)-producing strains of Fusarium graminearum (3-ADON chemotype) have been reported in North America and Asia. 3-ADON is nearly nontoxic at the level of the ribosomal target and has to be deacetylated to cause inhibition of protein biosynthesis. Plant cells can efficiently remove the acetyl groups of 3-ADON, but the underlying genes are yet unknown. We therefore performed a study of the family of candidate carboxylesterases (CXE) genes of the monocot model plant Brachypodium distachyon. We report the identification and characterization of the first plant enzymes responsible for deacetylation of trichothecene toxins. The product of the BdCXE29 gene efficiently deacetylates T-2 toxin to HT-2 toxin, NX-2 to NX-3, both 3-ADON and 15-acetyl-deoxynivalenol (15-ADON) into deoxynivalenol and, to a lesser degree, also fusarenon X into nivalenol. The BdCXE52 esterase showed lower activity than BdCXE29 when expressed in yeast and accepts 3-ADON, NX-2, 15-ADON and, to a limited extent, fusarenon X as substrates. Expression of these Brachypodium genes in yeast increases the toxicity of 3-ADON, suggesting that highly similar genes existing in crop plants may act as susceptibility factors in Fusarium head blight disease.