Effects of ferulic acid combined with light irradiation on deoxynivalenol and its production in Fusarium graminearum.

This study aimed to investigate the effects of ferulic acid (FA), a natural phenolic phytochemical, in combination with light irradiation at three wavelengths (365, 385 and 405 nm) on the concentration and toxicity of deoxynivalenol (DON), a mycotoxin produced by Fusarium graminearum. Moreover, this study examined the influence of the combination treatment on DON production in the cultured fungus. FA activated by light at a peak wavelength of 365 nm exhibited the most effective decrease in DON concentration of the tested wavelengths; a residual DON ratio of 0.23 at 24 h exposure was observed, compared with the initial concentration. The reduction in DON using 365-nm light was dependent on the concentration of FA, with a good correlation (r2 = 0.979) between the rate constants of DON decrease and FA concentration, which was confirmed by a pseudo-first-order kinetics analysis of the photoreaction with different FA concentrations (50-400 mg/L) for 3 h. The viability of HepG2 cells increased by 56.7% following in vitro treatment with a mixture containing the photoproducts obtained after treatment with 20 mg/L DON and 200 mg/L FA under 365-nm irradiation for 6 h. These results suggested that the photoreaction of FA under 365-nm irradiation induces the detoxification of DON through degradation or modification of DON. The antifungal effects of the combination (FA and 365-nm light) on F. graminearum were investigated. Conidia treated with the combination did not show additive or synergistic inhibition of fungal biomass and DON production in 7-day cultivated fungal samples compared with samples after single treatment. However, successive treatment, composed of 90 min irradiation at 365 nm and then treatment with 200 mg/L FA for 90 min in the dark, suppressed fungal growth and DON yield to 70% and 25% of the untreated sample level, respectively. This photo-technology involving the two treatment methods of 365-nm irradiation and FA addition as a food-grade phenolic acid in combination or successively, can aid in developing alternative approaches to eliminate fungal contaminants in the fields of environmental water and agriculture. However, further research is required to explore the underlying mechanisms of DON decontamination and its biosynthesis in F. graminearum.

The potential of soil microbial communities to transform deoxynivalenol in agricultural soils-a soil microcosm study.

Infestation of cereal fields with toxigenic Fusarium species is identified as an environmental source for the mycotoxin deoxynivalenol (DON). During rain events, DON may be washed off from infested plants and enter the soil, where microbial transformation may occur. Although some studies showed DON transformation potential of soil microbial communities in liquid soil extracts, these findings can not be transferred to environmental conditions. Accordingly, microbial transformation of DON in soil has to be investigated under realistic conditions, e.g., microcosms mimicking field situations. In this study, we investigated the potential of soil microbial communities to transform DON in six different agricultural soils at two levels (0.5 and 5 µg g-1). The dissipation and the formation of transformation products were investigated in a period of 35 days and compared to a sterilized control. In addition, we measured soil respiration and applied the phospholipid-derived fatty acid (PLFA) analysis to assess whether soil microbial community characteristics are related to the microbial transformation potential. Dissipation of DON in non-sterilized soils was fast (50% dissipation within 0.6-3.7 days) compared to the sterile control where almost no dissipation was observed. Thus, dissipation was mainly attributed to microbial transformation. We verified that small amounts of DON are transformed to 3-keto-deoxynivalenol (3-keto-DON) and 3-epi-deoxynivalenol (3-epi-DON), which were not detectable after 16-day incubation, indicating further transformation processes. There was a trend towards faster transformation in soils with active and large microbial communities and low fungi-to-bacteria ratio.

Baicalin ameliorates the gut barrier function and intestinal microbiota of broiler chickens.

The deoxynivalenol (DON)-contaminated feeds can impair chicken gut barrier function, disturb the balance of the intestinal microbiota, decrease chicken growth performance and cause major economic loss. With the aim of investigating the ameliorating effects of baicalin on broiler intestinal barrier damage and gut microbiota dysbiosis induced by DON, a total of 150 Arbor Acres broilers are used in the present study. The morphological damage to the duodenum, jejunum, and ileum caused by DON is reversed by treatment with different doses of baicalin, and the expression of tight junction proteins (ZO-1, claudin-1, and occludin) is also significantly increased in the baicalin-treated groups. Moreover, the disturbance of the intestinal microbiota caused by DON-contaminated feed is altered by baicalin treatment. In particular, compared with those in the DON group, the relative abundances of Lactobacillus, Lachnoclostridium, Ruminiclostridium and other beneficial microbes in the baicalin-treated groups are significantly greater. However, the percentage of unclassified_f__Lachnospiraceae in the baicalin-treated groups is significantly decreased in the DON group. Overall, the current results demonstrate that different doses of baicalin can improve broiler intestinal barrier function and the ameliorating effects on broiler intestinal barrier damage may be related to modulations of the intestinal microbiota.

Trichothecenes and Fumonisins: Key Players in Fusarium-Cereal Ecosystem Interactions.

Fusarium fungi produce a diverse array of mycotoxic metabolites during the pathogenesis of cereals. Some, such as the trichothecenes and fumonisins, are phytotoxic, acting as non-proteinaceous effectors that facilitate disease development in cereals. Over the last few decades, we have gained some depth of understanding as to how trichothecenes and fumonisins interact with plant cells and how plants deploy mycotoxin detoxification and resistance strategies to defend themselves against the producer fungi. The cereal-mycotoxin interaction is part of a co-evolutionary dance between Fusarium and cereals, as evidenced by a trichothecene-responsive, taxonomically restricted, cereal gene competing with a fungal effector protein and enhancing tolerance to the trichothecene and resistance to DON-producing F. graminearum. But the binary fungal-plant interaction is part of a bigger ecosystem wherein other microbes and insects have been shown to interact with fungal mycotoxins, directly or indirectly through host plants. We are only beginning to unravel the extent to which trichothecenes, fumonisins and other mycotoxins play a role in fungal-ecosystem interactions. We now have tools to determine how, when and where mycotoxins impact and are impacted by the microbiome and microfauna. As more mycotoxins are described, research into their individual and synergistic toxicity and their interactions with the crop ecosystem will give insights into how we can holistically breed for and cultivate healthy crops.

Investigating Metabolic Plant Response toward Deoxynivalenol Accumulation in Four Winter Cereals.

Deoxynivalenol (DON) is a phytotoxic agent supporting the spread of fungal diseases in cereals worldwide, i.e., fusarium head blight. It is known that DON accumulation may elicit changes in plant secondary metabolites in response to pathogen attack. This study maps the changes in selected secondary metabolite classes upon DON contamination occurring in fifteen Triticum spp. genotypes, among them emmer, spelt, and soft wheat, and 2 tritordeum varieties, cultivated in two different sites and over two harvest years. The main phenolic classes (i.e., alkylresorcinols, soluble, and cell-wall bound phenolic acids) were targeted analyzed, while changes in the lipidome signature were collected through untargeted HRMS experiments. The results, obtained across multiple Triticum species and in open fields, confirmed the modulation of first-line biological pathways already described in previous studies involving single cereal species or a limited germplasm, thus reinforcing the involvement of nonspecific chemical defenses in the plant response to pathogen attack.

Regulation of TRI5 expression and deoxynivalenol biosynthesis by a long non-coding RNA in Fusarium graminearum.

Deoxynivalenol (DON) is the most frequently detected mycotoxin in cereal grains and processed food or feed. Two transcription factors, Tri6 and Tri10, are essential for DON biosynthesis in Fusarium graminearum. In this study we conduct stranded RNA-seq analysis with tri6 and tri10 mutants and show that Tri10 acts as a master regulator controlling the expression of sense and antisense transcripts of TRI6 and over 450 genes with diverse functions. TRI6 is more specific for regulating TRI genes although it negatively regulates TRI10. Two other TRI genes, including TRI5 that encodes a key enzyme for DON biosynthesis, also have antisense transcripts. Both Tri6 and Tri10 are essential for TRI5 expression and for suppression of antisense-TRI5. Furthermore, we identify a long non-coding RNA (named RNA5P) that is transcribed from the TRI5 promoter region and is also regulated by Tri6 and Tri10. Deletion of RNA5P by replacing the promoter region of TRI5 with that of TRI12 increases TRI5 expression and DON biosynthesis, indicating that RNA5P suppresses TRI5 expression. However, ectopic constitutive overexpression of RNA5P has no effect on DON biosynthesis and TRI5 expression. Nevertheless, elevated expression of RNA5P in situ reduces TRI5 expression and DON production. Our results indicate that TRI10 and TRI6 regulate each other's expression, and both are important for suppressing the expression of RNA5P, a long non-coding RNA with cis-acting inhibitory effects on TRI5 expression and DON biosynthesis in F. graminearum.

Overview of T-2 Toxin Enterotoxicity: From Toxic Mechanisms and Detoxification to Future Perspectives.

Fusarium species produce a secondary metabolite known as T-2 toxin, which is the primary and most harmful toxin found in type A trichothecenes. T-2 toxin is widely found in food and grain-based animal feed and endangers the health of both humans and animals. T-2 toxin exposure in humans and animals occurs primarily through food administration; therefore, the first organ that T-2 toxin targets is the gut. In this overview, the research progress, toxicity mechanism, and detoxification of the toxin T-2 were reviewed, and future research directions were proposed. T-2 toxin damages the intestinal mucosa and destroys intestinal structure and intestinal barrier function; furthermore, T-2 toxin disrupts the intestinal microbiota, causes intestinal flora disorders, affects normal intestinal metabolic function, and kills intestinal epidermal cells by inducing oxidative stress, inflammatory responses, and apoptosis. The primary harmful mechanism of T-2 toxin in the intestine is oxidative stress. Currently, selenium and plant extracts are mainly used to exert antioxidant effects to alleviate the enterotoxicity of T-2 toxin. In future studies, the use of genomic techniques to find upstream signaling molecules associated with T-2 enterotoxin toxicity will provide new ideas for the prevention of this toxicity. The purpose of this paper is to review the progress of research on the intestinal toxicity of T-2 toxin and propose new research directions for the prevention and treatment of T-2 toxin toxicity.

Self-cascade deoxynivalenol detoxification by an artificial enzyme with bifunctions of dehydrogenase and aldo/keto reductase from genome mining.

Due to the severe health risks for human and animal caused by the intake of toxic deoxynivalenol (DON) derived from Fusarium species, elimination DON in food and feed has been initiated as a critical issue. Enzymatic cascade catalysis by dehydrogenase and aldo-keto reductase represents a fascinating strategy for DON detoxification. Here, one quinone-dpendent alcohol dehydrogenase DADH oxidized DON into less-toxic 3-keto-DON and NADPH-dependent aldo-keto reductase AKR13B3 reduced 3-keto-DON into relatively non-toxic 3-epi-DON were identified from Devosia strain A6-243, indicating that degradation of DON on C3 are two-step sequential cascade processes. To establish the bifunctions, fusion enzyme linking DADH and AKR13B3 was successfully assembled to promote one-step DON degradations with accelerated specific activity and efficiency, resulting 93.29 % of DON removal rate in wheat sample. Three-dimensional simulation analysis revealed that the bifunctional enzyme forms an artificial intramolecular channel to minimize the distance of intermediate from DADH to AKR13B3 for two-step enzymatic reactions, and thereby accelerates this enzymatic process. As the first report of directing single step DON detoxification by an interesting bifunctional artificial enzyme, this work revealed a facile and eco-friendly approach to detoxify DON with application potential and gave valuable insights into execute other mycotoxin detoxification for ensuring food safety.

Fhb7-GST catalyzed glutathionylation effectively detoxifies the trichothecene family.

Trichothecene (TCN) contamination in food and feed is a serious challenge due to the negative health and economic impacts. Here, we confirmed that the glutathione S-transferase (GST) Fhb7-GST could broadly catalyze type A, type B and type D TCNs into glutathione epoxide adducts (TCN-13-GSHs). To evaluate the toxicity of TCN-13-GSH adducts, we performed cell proliferation assays in vitro, which demonstrated decreased cytotoxicity of the adducts. Moreover, in vivo assays (repeated-dose treatment in mice) confirmed that TCN-13-GSH adducts were dramatically less toxic than the corresponding TCNs. To establish whether TCN-13-GSH was metabolized back to free toxin during digestion, single-dose metabolic tests were performed in rats; DON-13-GSH was not hydrolyzed in vivo, but rather was quickly metabolized to another low-toxicity compound, DON-13-N-acetylcysteine. These results demonstrate the promise of Fhb7-GST as a candidate of detoxification enzyme potentially applied in TCN-contaminated agricultural samples, minimizing the detrimental effects of the mycotoxin.

Structure-Guided Steric Hindrance Engineering of Devosia Strain A6-243 Quinone-Dependent Dehydrogenase to Enhance Its Catalytic Efficiency.

Deoxynivalenol (DON), the most widely distributed mycotoxin worldwide, causes severe health risks for humans and animals. Quinone-dependent dehydrogenase derived from Devosia strain A6-243 (DADH) can degrade DON into less toxic 3-keto-DON and then aldo-keto reductase AKR13B3 can reduce 3-keto-DON into relatively nontoxic 3-epi-DON. However, the poor catalytic efficiency of DADH made it unsuitable for practical applications, and it has become the rate-limiting step of the two-step enzymatic cascade catalysis. Here, structure-guided steric hindrance engineering was employed to enhance the catalytic efficiency of DADH. After the steric hindrance engineering, the best mutant, V429G/N431V/T432V/L434V/F537A (M5-1), showed an 18.17-fold increase in specific activity and an 11.04-fold increase in catalytic efficiency (kcat/Km) compared with that of wild-type DADH. Structure-based computational analysis provided information on the increased catalytic efficiency in the directions that attenuated steric hindrance, which was attributed to the reshaped substrate-binding pocket with an expanded catalytic binding cavity and a favorable attack distance. Tunnel analysis suggested that reshaping the active cavity by mutation might alter the shape and size of the enzyme tunnels or form one new enzyme tunnel, which might contribute to the improved catalytic efficiency of M5-1. These findings provide a promising strategy to enhance the catalytic efficiency by steric hindrance engineering.

Evaluation of Immunomodulatory Effects of Fusarium Mycotoxins Using Bacterial Endotoxin-Stimulated Bovine Epithelial Cells and Macrophages in Co-Culture.

Mycotoxins are secondary metabolites produced by a variety of fungi that contaminate animal food and feeds and are capable of inducing a wide range of toxicities. Predictive in vitro models represent valuable substitutes for animal experiments to assess the toxicity of mycotoxins. The complexities of the interactions between epithelial and innate immune cells, vital for upholding barrier integrity and averting infections, remain inadequately understood. In the current study, a co-culture model of bovine epithelial cells (MAC-T) and macrophages (BoMac) was used to investigate the impact of exposure to Fusarium mycotoxins, namely deoxynivalenol (DON), zearalenone (ZEN), enniatin B (ENB), and beauvericin (BEA), on the inflammatory response elicited by the bacterial lipopolysaccharide (LPS) endotoxin. The MAC-T cells and BoMac were seeded on the apical side of a Transwell membrane and in the lower chamber, respectively, and mycotoxin exposure on the apical side of the membrane was carried out with the different mycotoxins (LC20; concentrations that elicited 20% cytotoxicity) for 48 h followed by an LPS immunity challenge for 24 h. The culture supernatants were collected from the basolateral compartment and these samples were submitted for cytokine/chemokine multiplex analysis. RNA-Seq analysis was performed using total RNA extracted from the MAC-T cells to acquire a more detailed insight into their cellular functions. The multiplex analysis indicated that IFN-γ, IL-1α, IL-8, and MCP-1 were significantly induced post-DON treatment when compared to control cells, and levels of IL-1α and IL-8 were enhanced significantly in all mycotoxin-treated groups post-LPS challenge. Analysis of the sequencing results showed that there were 341, 357, and 318 differentially expressed MAC-T cell genes that were up-regulated in the DON, ENB, and BEA groups, respectively. Gene ontology and pathway analysis revealed that these DEGs were significantly enriched in various biological processes and pathways related to inflammation, apoptosis signaling, and Wnt signaling. These results provide a comprehensive analysis of the co-culture cytokine/chemokine production and MAC-T cells' gene expression profiles elicited by Fusarium mycotoxins, which further contributes to the understanding of early endotoxemia post-mycotoxin exposure.

Effective protective agents against organ toxicity of deoxynivalenol and their detoxification mechanisms: A review.

Deoxynivalenol (DON) is one of the most prevalent mycotoxins in feed, which causes organ toxicity in animals. Therefore, reducing DON-induced organ toxicity can now be accomplished effectively using protective agents. This review provides an overview of multiple studies on a wide range of protective agents and their molecular mechanisms against DON organ toxicity. Protective agents include plant extracts, yeast products, bacteria, peptides, enzymes, H2, oligosaccharides, amino acids, adsorbents, vitamins and selenium. Among these, biological detoxification of DON using microorganisms to reduce the toxicity of DON without affecting the growth performance of pigs may be the most promising detoxification strategy. This paper also evaluates future developments related to DON detoxification and discusses the detoxification role and application potential of protective agents. This paper provides new perspectives for future research and development of safe and effective feed additives.

Loop Evolutionary Patterns Shape Catalytic Efficiency of TRI101/201 for Trichothecenes: Insights into Protein-Substrate Interactions.

Trichothecenes are highly toxic mycotoxins produced by Fusarium fungi, while TRI101/201 family enzymes play a crucial role in detoxification through acetylation. Studies on the substrate specificity and catalytic kinetics of TRI101/201 have revealed distinct kinetic characteristics, with significant differences observed in catalytic efficiency toward deoxynivalenol, while the catalytic efficiency for T-2 toxin remains relatively consistent. In this study, we used structural bioinformatics analysis and a molecular dynamics simulation workflow to investigate the mechanism underlying the differential catalytic activity of TRI101/201. The findings revealed that the binding stability between trichothecenes and TRI101/201 hinges primarily on a hydrophobic cage structure within the binding site. An intrinsic disordered loop, termed loop cover, defined the evolutionary patterns of the TRI101/201 protein family that are categorized into four subfamilies (V1/V2/V3/M). Furthermore, the unique loop displayed different conformations among these subfamilies' structures, which served to disrupt (V1/V2/V3) or reinforce (M) the hydrophobic cages. The disrupted cages enhanced the water exposure of the hydrophilic moieties of substrates like deoxynivalenol and thereby hindered their binding to the catalytic sites of V-type enzymes. In contrast, this water exposure does not affect substrates like T-2 toxin, which have more hydrophobic substituents, resulting in a comparable catalytic efficiency of both V- and M-type enzymes. Overall, our studies provide theoretical support for understanding the catalytic mechanism of TRI101/201, which shows how an intrinsic disordered loop could impact the protein-ligand binding and suggests a direction for rational protein design in the future.

Phenamacril and carbendazim regulate trichothecene mycotoxin synthesis by affecting ROS levels in F. asiaticum.

Fusarium head blight caused by Fusarium asiaticum is an important cereal crop disease, and the trichothecene mycotoxins produced by F. asiaticum can contaminate wheat grain, which is very harmful to humans and animals. To effectively control FHB in large areas, the application of fungicides is the major strategy; however, the application of different types of fungicides has varying influences on the accumulation of trichothecene mycotoxins in F. asiaticum. In this study, phenamacril inhibited trichothecene mycotoxin accumulation in F. asiaticum; however, carbendazim (N-1H-benzimidazol-2-yl-carbamic acid, methyl ester) induced trichothecene mycotoxin accumulation. Additionally, phenamacril led to a lower level of reactive oxygen species (ROS) by inducing gene expression of the catalase and superoxide dismutase (SOD) pathways in F. asiaticum, whereas carbendazim stimulated ROS accumulation by inhibiting gene expression of the catalase and SOD pathways. Based on these results, we conclude that phenamacril and carbendazim regulate trichothecene mycotoxin synthesis by affecting ROS levels in F. asiaticum.

Toxicokinetics of Deoxynivalenol in Dezhou Male Donkeys after Oral Administration.

Deoxynivalenol (DON) is detected in different types of foods and feeds, inducing toxicity in humans and animals. After entering the organism, DON first appears in the plasma; then, it is rapidly absorbed and distributed in various organs and tends to accumulate in the body to exert its toxic effects. This study was performed to investigate the toxicokinetics of DON on Dezhou male donkeys after a single oral dose of 500 µg/kg·BW (body weight). The plasma of donkeys was collected at 0, 5, 10, 15, 20, 30, 45 min, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 6, 9, 12, 24, 48, 72, 96 and 120 h after administration, and the feces and urine were collected at 0 h and at 6 h intervals up to 24 h, followed by 4 h intervals up to 120 h. The concentrations of DON in plasma, urine and feces were determined by HPLC. The peak concentration of DON in plasma was 174.30 µg/L, which occurred at 1.07 h after oral gavage. The recovery of unchanged DON in urine and feces amounted to 19.98% and 6.74%, respectively. Overall, DON was rapidly absorbed and slowly eliminated in donkeys within 120 h following a single oral dose, which can lead to DON accumulation in the body if ingested for a long time.

Deoxynivalenol: Emerging Toxic Mechanisms and Control Strategies, Current and Future Perspectives.

Deoxynivalenol (DON) is the most frequently present mycotoxin contaminant in food and feed, causing a variety of toxic effects in humans and animals. Currently, a series of mechanisms involved in DON toxicity have been identified. In addition to the activation of oxidative stress and the MAPK signaling pathway, DON can activate hypoxia-inducible factor-1α, which further regulates reactive oxygen species production and cancer cell apoptosis. Noncoding RNA and signaling pathways including Wnt/ß-catenin, FOXO, and TLR4/NF-κB also participate in DON toxicity. The intestinal microbiota and the brain-gut axis play a crucial role in DON-induced growth inhibition. In view of the synergistic toxic effect of DON and other mycotoxins, strategies to detect DON and control it biologically and the development of enzymes for the biodegradation of various mycotoxins and their introduction in the market are the current and future research hotspots.

Global distribution, toxicity to humans and animals, biodegradation, and nutritional mitigation of deoxynivalenol: A review.

Deoxynivalenol (DON) is one of the main types of B trichothecenes, and it causes health-related issues in humans and animals and imposes considerable challenges to food and feed safety globally each year. This review investigates the global hazards of DON, describes the occurrence of DON in food and feed in different countries, and systematically uncovers the mechanisms of the various toxic effects of DON. For DON pollution, many treatments have been reported on the degradation of DON, and each of the treatments has different degradation efficacies and degrades DON by a distinct mechanism. These treatments include physical, chemical, and biological methods and mitigation strategies. Biodegradation methods include microorganisms, enzymes, and biological antifungal agents, which are of great research significance in food processing because of their high efficiency, low environmental hazards, and drug resistance. And we also reviewed the mechanisms of biodegradation methods of DON, the adsorption and antagonism effects of microorganisms, and the different chemical transformation mechanisms of enzymes. Moreover, nutritional mitigation including common nutrients (amino acids, fatty acids, vitamins, and microelements) and plant extracts was discussed in this review, and the mitigation mechanism of DON toxicity was elaborated from the biochemical point of view. These findings help explore various approaches to achieve the best efficiency and applicability, overcome DON pollution worldwide, ensure the sustainability and safety of food processing, and explore potential therapeutic options with the ability to reduce the deleterious effects of DON in humans and animals.

The Fusarium graminearum Transporters Abc1 and Abc6 Are Important for Xenobiotic Resistance, Trichothecene Accumulation, and Virulence to Wheat.

The plant pathogenic fungus Fusarium graminearum is the causal agent of Fusarium head blight (FHB) disease on small-grain cereals. F. graminearum produces trichothecene mycotoxins such as deoxynivalenol (DON) that are required for full virulence. DON must be exported outside the cell to cause FHB disease, a process that may require the involvement of membrane-bound transporters. In this study, we show that the deletion of membrane-bound transporters results in reduced DON accumulation as well as reduced FHB symptoms on wheat. Deletion of the ATP-binding cassette (ABC) transporter gene Abc1 results in the greatest reduction in DON accumulation and virulence. Deletion of another ABC transporter gene, Abc6, also reduces FHB symptoms to a lesser degree. Combining deletions fails to reduce DON accumulation or virulence in an additive fashion, even when a ∆abc1 deletion is included. Heterologous expression of F. graminearum transporters in a DON-sensitive strain of yeast confirms Abc1 as a major DON resistance mechanism; furthermore, it suggests that Abc1 is directly participating in DON transport rather than facilitating DON transport though other means. Yeast expression further indicates that multiple transporters, including Abc1, play an important role in resistance to the wheat phytoalexin 2-benzoxazolinone (BOA) and other xenobiotics. Thus, Abc1 may contribute to virulence on wheat both by facilitating export of DON and by providing resistance to the wheat phytoalexin BOA. This research provides useful information that may aid in designing novel management techniques of FHB or other destructive plant diseases.

A Chemically Defined Medium That Supports Mycotoxin Production by Stachybotrys chartarum Enabled Analysis of the Impact of Nitrogen and Carbon Sources on the Biosynthesis of Macrocyclic Trichothecenes and Stachybotrylactam.

Stachybotrys chartarum (Hypocreales, Ascomycota) is a toxigenic fungus that is frequently isolated from water-damaged buildings or improperly stored feed. The secondary metabolites formed by this mold have been associated with health problems in humans and animals. Several authors have studied the influence of environmental conditions on the production of mycotoxins, but these studies focused on undefined or complex substrates, such as building materials and media that impeded investigations of the influence of specific nutrients. In this study, a chemically defined cultivation medium was used to investigate the impact of several nitrogen and carbon sources on growth of S. chartarum and its production of macrocyclic trichothecenes (MTs) and stachybotrylactam (STLAC). Increasing concentrations of sodium nitrate were found to positively affect mycelial growth, the level of sporulation, and MT production, while ammonium nitrate and ammonium chloride had an inhibitory effect. Potato starch was the superior and most reliable carbon source tested. Additionally, we observed that the level of sporulation was correlated with the production of MTs but not with that of STLAC. In this study, we provide a chemically well-defined cultivation medium suitable for standardized in vitro testing of the capacity of S. chartarum isolates to produce macrocyclic trichothecenes. IMPORTANCE Macrocyclic trichothecenes (MTs) are highly toxic secondary metabolites that are produced by certain Stachybotrys chartarum strains, which consequently pose a risk for animals and humans. To identify hazardous, toxin-producing strains by analytical means, it is important to grow them under conditions that support MT production. Nutrients determine growth and development and thus the synthesis of secondary metabolites. Complex rich media are commonly used for diagnostics, but batch differences of supplements pose a risk for inconsistent data. We have established a chemically defined medium for S. chartarum and used it to analyze the impact of nitrogen and carbon sources. A key finding is that nitrate stimulates MT production, whereas ammonium suppresses it. Defining nutrients that support MT production will enable a more reliable identification of hazardous S. chartarum isolates. The new medium will also be instrumental in analyzing the biosynthetic pathways and regulatory mechanisms that control mycotoxin production in S. chartarum.

Genome-Wide Characterization of the RNA Exosome Complex in Relation to Growth, Development, and Pathogenicity of Fusarium graminearum.

The RNA exosome complex is a conserved, multisubunit RNase complex that contributes to the processing and degradation of RNAs in mammalian cells. However, the roles of the RNA exosome in phytopathogenic fungi and how it relates to fungal development and pathogenicity remain unclear. Herein, we identified 12 components of the RNA exosome in the wheat fungal pathogen Fusarium graminearum. Live-cell imaging showed that all the components of the RNA exosome complex are localized in the nucleus. FgEXOSC1 and FgEXOSCA were successfully knocked out; they are both involved in the vegetative growth, sexual reproduction, and pathogenicity of F. graminearum. Moreover, deletion of FgEXOSC1 resulted in abnormal toxisomes, decreased deoxynivalenol (DON) production, and downregulation of the expression levels of DON biosynthesis genes. The RNA-binding domain and N-terminal region of FgExosc1 are required for its normal localization and functions. Transcriptome sequencing (RNA-seq) showed that the disruption of FgEXOSC1 resulted in differential expression of 3,439 genes. Genes involved in processing of noncoding RNA (ncRNA), rRNA and ncRNA metabolism, ribosome biogenesis, and ribonucleoprotein complex biogenesis were significantly upregulated. Furthermore, subcellular localization, green fluorescent protein (GFP) pulldown, and coimmunoprecipitation (co-IP) assays demonstrated that FgExosc1 associates with the other components of the RNA exosome to form the RNA exosome complex in F. graminearum. Deletion of FgEXOSC1 and FgEXOSCA reduced the relative expression of some of the other subunits of the RNA exosome. Deletion of FgEXOSC1 affected the localization of FgExosc4, FgExosc6, and FgExosc7. In summary, our study reveals that the RNA exosome is involved in vegetative growth, sexual reproduction, DON production, and pathogenicity of F. graminearum. IMPORTANCE The RNA exosome complex is the most versatile RNA degradation machinery in eukaryotes. However, little is known about how this complex regulates the development and pathogenicity of plant-pathogenic fungi. In this study, we systematically identified 12 components of the RNA exosome complex in Fusarium head blight fungus Fusarium graminearum and first unveiled their subcellular localizations and established their biological functions in relation to the fungal development and pathogenesis. All the RNA exosome components are localized in the nucleus. FgExosc1 and FgExoscA are both required for the vegetative growth, sexual reproduction, DON production and pathogenicity in F. graminearum. FgExosc1 is involved in ncRNA processing, rRNA and ncRNA metabolism process, ribosome biogenesis and ribonucleoprotein complex biogenesis. FgExosc1 associates with the other components of RNA exosome complex and form the exosome complex in F. graminearum. Our study provides new insights into the role of the RNA exosome in regulating RNA metabolism, which is associated with fungal development and pathogenicity.