Eco-friendly one-step egg white gel preparation for sensitive detection of 13 trichothecenes in oats using UHPLC-MS/MS.

Oat products have gained widespread recognition as a health food due to their rich and balanced nutritional profile and convenience. However, the unique matrix composition of oats, which differs significantly from other cereals, presents specific challenges for mycotoxin analysis. This study presents an ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method enhanced with an innovative egg white gel pretreatment for the simultaneous analysis of 13 regulated and unregulated trichothecenes in oats. The method demonstrated excellent performance with high accuracy (> 87.5%), repeatability (< 5.7%), and reproducibility (< 8.1%). Analysis of 100 commercial oat products revealed a concerning detection rate (78%) for at least one of the 11 trichothecenes investigated. Notably, deoxynivalenol, exceeding the standard limit in 2% of samples, exhibited the highest detection rate (62%). Additionally, concerning co-occurrence patterns and positive correlations were observed, highlighting potential synergistic effects. The first-time detection of unregulated mycotoxins (T-2 triol, 4,15-diacetoxyscirpenol, 15-acetoxyscirpenol, and neosolaniol) underscores the need for comprehensive monitoring. This method, while developed for oats, shows potential for broader application to other cereals, though further investigation and confirmation are necessary. These findings suggest a potentially underestimated risk of trichothecenes in oats, necessitating continuous monitoring to ensure consumer safety.

The identification of a key gene highlights macrocyclic ring's role in trichothecene toxicity.

Trichothecenes are toxins produced by certain species from several fungal genera, including Aspergillus, Fusarium, Isaria, Paramyrothecium, Stachybotrys, Trichoderma, and Trichothecium. These toxins are of interest because they contribute to the toxigenicity, plant pathogenicity, and/or biological control activities of some fungi. All trichothecenes have the same core (12,13-epoxytrichothec-9-ene or EPT) structure but can differ from one another by the presence or absence of a macrocyclic ring formed from polyketide and isoprenoid substituents esterified to carbon atoms 4 and 15 of EPT, respectively. Genes required for formation and some modifications of EPT have been elucidated, but almost nothing is known about genes specific to the formation of the macrocyclic ring. Therefore, we used genomic, transcriptomic, metabolomic, and gene deletion analyses to identify genes that are required specifically for the formation of the macrocyclic ring. These analyses identified one gene, TRI24, that is predicted to encode an acyltransferase and that is required for macrocyclic ring formation during biosynthesis of macrocyclic trichothecenes by the fungus Paramyrothecium roridum. In addition, a TRI24 deletion mutant of P. roridum caused less severe disease symptoms on common bean and had less antifungal activity than its wild-type progenitor strain. We propose that the reduced aggressiveness and antifungal activity of the mutant resulted from its inability to produce trichothecenes with a macrocyclic ring. To our knowledge, this is the first report of a gene required specifically for the formation of the macrocyclic ring of trichothecenes and that loss of the macrocyclic ring of trichothecenes can alter the biological activities of a fungus. KEY POINTS: • TRI24 gene is found in all known macrocyclic trichothecene-producing fungi. • A tri24-deletion mutant exhibits a reduction in antifungal and plant disease activities. • TRI24 is the first described gene specific to macrocyclic trichothecene biosynthesis.

Equine Mycotoxins.

The main mycotoxins involved in adverse equine health issues are aflatoxins, fumonisins, trichothecenes, and probably ergovaline (fescue grass endophyte toxicosis). Most exposures are through contaminated grains and grain byproducts, although grasses and hays can contain mycotoxins. Clinical signs are often nonspecific and include feed refusal, colic, diarrhea, and liver damage but can be dramatic with neurologic signs associated with equine leukoencephalomalacia and tremorgens. Specific antidotes for mycotoxicosis are rare, and treatment involves stopping the use of contaminated feed, switching to a "clean" feed source, and providing supportive care.

NX Trichothecenes Are Required for Fusarium graminearum Infection of Wheat.

Fusarium graminearum causes Fusarium head blight (FHB) on wheat and barley and contaminates grains with various mycotoxins that are toxic to humans and animals. Deoxynivalenol (DON), a type B trichothecene, is an essential virulence factor that is required for F. graminearum to spread within a wheat head. Recently, novel type A trichothecenes NX-2 and NX-3 (NX) have been found in F. graminearum. NX trichothecenes lack a keto group at the C8 position. To determine if NX trichothecenes play a role similar to that of DON during F. graminearum infection, deletion mutants of TRI5, the first gene for trichothecene biosynthesis, were generated from strains PH-1, NRRL46422, and NRRL44211 (hereafter 44211) representing the 15-acetyl-DON, 3-acetyl-DON, and NX chemotypes. No trichothecene production was detected in any of the Δtri5 mutants in cultures or inoculated wheat heads. FHB symptoms were restricted to the inoculated wheat spikelets when point-inoculated with the Δtri5 mutants, confirming the necessity of NX and DON for FHB spread. Furthermore, whole-head dip inoculations revealed significant reductions in disease and fungal biomass in wheat heads inoculated with 44211Δtri5 compared with 44211. Introduction of the native 44211 TRI5 and a Trichoderma arundinaceum TRI5 ortholog in the 44211Δtri5 mutant complemented trichothecene production in vitro; however, introducing both TRI5 partially restored wild-type levels of NX in infected heads. Our results demonstrate that NX trichothecenes play an important role in Fusarium graminearum initial infection as well as FHB spread. Thus, TRI5 may serve as an ideal target to control plant infection, FHB spread, and mycotoxin production simultaneously. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

De novo genome assembly and functional annotation for Fusarium langsethiae.

BACKGROUND: Fusarium langsethiae is a T-2 and HT-2 mycotoxins producing species firstly characterised in 2004. It is commonly isolated from oats in Northern Europe. T-2 and HT-2 mycotoxins exhibit immunological and haemotological effects in animal health mainly through inhibition of protein, RNA and DNA synthesis. The development of a high-quality and comprehensively annotated assembly for this species is therefore essential in providing the molecular understanding and the mechanism of T-2 and HT-2 biosynthesis in F. langsethiae to help develop effective control strategies. RESULTS: The F. langsethiae assembly was produced using PacBio long reads, which were then assembled independently using Canu, SMARTdenovo and Flye. A total of 19,336 coding genes were identified using RNA-Seq informed ab-initio gene prediction. Finally, predicting genes were annotated using the basic local alignment search tool (BLAST) against the NCBI non-redundant (NR) genome database and protein hits were annotated using InterProScan. Genes with blast hits were functionally annotated with Gene Ontology. CONCLUSIONS: We developed a high-quality genome assembly of a total length of 59 Mb and N50 of 3.51 Mb. Raw sequence reads and assembled genome is publicly available and can be downloaded from: GenBank under the accession JAFFKB000000000. All commands used to generate this assembly are accessible via GitHub: https://github.com/FadyMohareb/fusarium_langsethiae .

Identification of polyketide synthase genes required for aspinolide biosynthesis in Trichoderma arundinaceum.

The fungus Trichoderma arundinaceum exhibits biological control activity against crop diseases caused by other fungi. Two mechanisms that likely contribute to this activity are upregulation of plant defenses and production of two types of antifungal secondary metabolites: the sesquiterpenoid harzianum A (HA) and the polyketide-derived aspinolides. The goal of the current study was to identify aspinolide biosynthetic genes as part of an effort to understand how these metabolites contribute to the biological control activity of T. arundinaceum. Comparative genomics identified two polyketide synthase genes (asp1 and asp2) that occur in T. arundinaceum and Aspergillus ochraceus, which also produces aspinolides. Gene deletion and biochemical analyses in T. arundinaceum indicated that both genes are required for aspinolide production: asp2 for formation of a 10-member lactone ring and asp1 for formation of a butenoyl subsituent at position 8 of the lactone ring. Gene expression and comparative genomics analyses indicated that asp1 and asp2 are located within a gene cluster that occurs in both T. arundinaceum and A. ochraceus. A survey of genome sequences representing 35 phylogenetically diverse Trichoderma species revealed that intact homologs of the cluster occurred in only two other species, which also produced aspinolides. An asp2 mutant inhibited fungal growth more than the wild type, but an asp1 mutant did not, and the greater inhibition by the asp2 mutant coincided with increased HA production. These findings indicate that asp1 and asp2 are aspinolide biosynthetic genes and that loss of either aspinolide or HA production in T. arundinaceum can be accompanied by increased production of the other metabolite(s). KEY POINTS: • Two polyketide synthase genes are required for aspinolide biosynthesis. • Blocking aspinolide production increases production of the terpenoid harzianum A. • Aspinolides and harzianum A act redundantly in antibiosis of T. arundinaceum.

New Trichothecenes Isolated from the Marine Algicolous Fungus Trichoderma brevicompactum.

Eight trichothecenes, including four new compounds 1-4 and four known entities 5-8, together with one known cyclonerane (9) were isolated from the solid-state fermentation of Trichoderma brevicompactum NTU439 isolated from the marine alga Mastophora rosea. The structures of 1-9 were determined by 1D/2D NMR (nuclear magnetic resonance), MS (mass spectrometry), and IR (infrared spectroscopy) spectroscopic data. All of the compounds were evaluated for cytotoxic activity against HCT-116, PC-3, and SK-Hep-1 cancer cells by the SRB assay, and compound 8 showed promising cytotoxic activity against all three cancer cell lines with the IC50 values of 3.3 ± 0.3, 5.3 ± 0.3, and 1.8 ± 0.8 µM, respectively. Compounds 1-2, 4-6, and 7-8 potently inhibited LPS-induced NO production, and compounds 5 and 8 showed markedly inhibited gelatinolysis of MMP-9 in S1 protein-stimulated THP-1 monocytes.

Computational Strategy for Minimizing Mycotoxins in Cereal Crops: Assessment of the Biological Activity of Compounds Resulting from Virtual Screening.

Cereal crops are frequently affected by toxigenic Fusarium species, among which the most common and worrying in Europe are Fusarium graminearum and Fusarium culmorum. These species are the causal agents of grain contamination with type B trichothecene (TCTB) mycotoxins. To help reduce the use of synthetic fungicides while guaranteeing low mycotoxin levels, there is an urgent need to develop new, efficient and environmentally-friendly plant protection solutions. Previously, F. graminearum proteins that could serve as putative targets to block the fungal spread and toxin production were identified and a virtual screening undertaken. Here, two selected compounds, M1 and M2, predicted, respectively, as the top compounds acting on the trichodiene synthase, a key enzyme of TCTB biosynthesis, and the 24-sterol-C-methyltransferase, a protein involved in ergosterol biosynthesis, were submitted for biological tests. Corroborating in silico predictions, M1 was shown to significantly inhibit TCTB yield by a panel of strains. Results were less obvious with M2 that induced only a slight reduction in fungal biomass. To go further, seven M1 analogs were assessed, which allowed evidencing of the physicochemical properties crucial for the anti-mycotoxin activity. Altogether, our results provide the first evidence of the promising potential of computational approaches to discover new anti-mycotoxin solutions.

Genome-wide association study for deoxynivalenol production and aggressiveness in wheat and rye head blight by resequencing 92 isolates of Fusarium culmorum.

BACKGROUND: Fusarium culmorum is an important pathogen causing head blight of cereals in Europe. This disease is of worldwide importance leading to reduced yield, grain quality, and contamination by mycotoxins. These mycotoxins are harmful for livestock and humans; therefore, many countries have strict regulatory limits for raw materials and processed food. Extensive genetic diversity is described among field populations of F. culmorum isolates for aggressiveness and production of the trichothecene mycotoxin deoxynivalenol (DON). However, the causes for this quantitative variation are not clear, yet. We analyzed 92 isolates sampled from different field populations in Germany, Russia, and Syria together with an international collection for aggressiveness and DON production in replicated field experiments at two locations in two years with two hosts, wheat and rye. The 30x coverage whole-genome resequencing of all isolates resulted in the identification of 130,389 high quality single nucleotide polymorphisms (SNPs) that were used for the first genome-wide association study in this phytopathogenic fungus. RESULTS: In wheat, 20 and 27 SNPs were detected for aggressiveness and DON content, respectively, of which 10 overlapped. Additionally, two different SNPs were significantly associated with aggressiveness in rye that were among those SNPs being associated with DON production in wheat. Most of the SNPs explained only a small proportion of genotypic variance (pG), however, four SNPs were associated with major quantitative trait loci (QTLs) with pG ranging from 12 to 48%. The QTL with the highest pG was involved in DON production and associated with a SNP most probably located within the Tri4 gene. CONCLUSIONS: The diversity of 92 isolates of F. culmorum were captured using a heuristic approach. Key phenotypic traits, SNPs, and candidate genes underlying aggressiveness and DON production were identified. Clearly, many QTLs are responsible for aggressiveness and DON content in wheat, both traits following a quantitative inheritance. Several SNPs involved in DON metabolism, among them the Tri4 gene of the trichothecene pathway, were inferred as important source of variation in fungal aggressiveness. Using this information underlying the phenotypic variation will be of paramount importance in evaluating strategies for successful resistance breeding.

Fusarium graminearum species complex occurrence on soybean and F. graminearum sensu stricto inoculum maintenance on residues in soybean-wheat rotation under field conditions.

AIMS: The aims of this study were to determine the occurrence of Fusarium graminearum species complex (FGSC) on soybean pods, seeds and roots, including rhizoplane, during the period of soybean crop in rotation with wheat and to evaluate the FGSC dynamics on wheat and soybean residues during two soybean growing seasons in rotation with wheat, particularly F. graminearum sensu stricto (FGss). METHODS AND RESULTS: Soybean roots, pods and seeds were analysed during 2012/13 and 2013/14 seasons. The morphological identification of FGSC and mycotoxin analysis was done. Crop residues were taken in both soybean season in wheat rotation and FGss were quantificated by real-time PCR. The results showed that Fusarium species, mainly FGSC, survive in a soybean crop in rotation with wheat. Isolation frequency of these species was higher on soybean pods than on seeds at R6 stage. Deoxynivalenol contamination on soybean seeds was higher in the 2013/14 season in comparison with the 2012/13 season. Low isolation levels of Fusarium species and species that did not belong to FGSC were observed in soybean root, whereas in rhizoplane a higher level was observed. Fusarium species inoculum on residues remained stable during crop succession and the FGSC were recovered from both wheat and soybean residues. Real time PCR data showed a higher DNA concentration of FGss in wheat residues in the first developmental stages of soybean plants, being the levels more significant during 2012/13 season. With regard to soybean residues collected during the wheat growing stages, an increase in DNA from anthesis until wheat harvest was observed. CONCLUSIONS: In a no-till production system, the populations of FGSC can colonize wheat and soybean residues to become an inoculum source. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new data on the occurrence of FGSC populations in soybean plant and FGss on residues in soybean-wheat rotation, a cultural practice commonly used in in Argentina.

Accumulation of 4-Deoxy-7-hydroxytrichothecenes, but Not 4,7-Dihydroxytrichothecenes, in Axenic Culture of a Transgenic Nivalenol Chemotype Expressing the NX-Type FgTri1 Gene.

Fusarium graminearum species complex produces type B trichothecenes oxygenated at C-7. In axenic liquid culture, F. graminearum mainly accumulates one of the three types of trichothecenes, namely 3-acetyldeoxyinvalenol, 15-acetyldeoxyinvalenol, or mixtures of 4,15-diacetylnivalenol/4-acetylnivalenol, depending on each strain's genetic background. The acetyl groups of these trichothecenes are slowly deacetylated to give deoxynivalenol (DON) or nivalenol (NIV) on solid medium culture. Due to the evolution of F. graminearum FgTri1, encoding a cytochrome P450 monooxygenase responsible for hydroxylation at both C-7 and C-8, new derivatives of DON, designated as NX-type trichothecenes, have recently emerged. To assess the risks of emergence of new NX-type trichothecenes, we examined the effects of replacing FgTri1 in the three chemotypes with FgTri1_NX chemotype, which encodes a cytochrome P450 monooxygenase that can only hydroxylate C-7 of trichothecenes. Similar to the transgenic DON chemotypes, the transgenic NIV chemotype strain accumulated NX-type 4-deoxytrichothecenes in axenic liquid culture. C-4 oxygenated trichothecenes were marginal, despite the presence of a functional FgTri13 encoding a C-4 hydroxylase. At present, outcrossing of the currently occurring NX chemotype with NIV chemotype strains of F. graminearum in the natural environment likely will not yield a new strain that produces a C-4 oxygenated NX-type trichothecene.

T-2 Toxin-The Most Toxic Trichothecene Mycotoxin: Metabolism, Toxicity, and Decontamination Strategies.

Among trichothecenes, T-2 toxin is the most toxic fungal secondary metabolite produced by different Fusarium species. Moreover, T-2 is the most common cause of poisoning that results from the consumption of contaminated cereal-based food and feed reported among humans and animals. The food and feed most contaminated with T-2 toxin is made from wheat, barley, rye, oats, and maize. After exposition or ingestion, T-2 is immediately absorbed from the alimentary tract or through the respiratory mucosal membranes and transported to the liver as a primary organ responsible for toxin's metabolism. Depending on the age, way of exposure, and dosage, intoxication manifests by vomiting, feed refusal, stomach necrosis, and skin irritation, which is rarely observed in case of mycotoxins intoxication. In order to eliminate T-2 toxin, various decontamination techniques have been found to mitigate the concentration of T-2 toxin in agricultural commodities. However, it is believed that 100% degradation of this toxin could be not possible. In this review, T-2 toxin toxicity, metabolism, and decontamination strategies are presented and discussed.

Screening of Wood/Forest and Vine By-Products as Sources of New Drugs for Sustainable Strategies to Control Fusarium graminearum and the Production of Mycotoxins.

Fusarium graminearum is a fungal pathogen that can colonize small-grain cereals and maize and secrete type B trichothecene (TCTB) mycotoxins. The development of environmental-friendly strategies guaranteeing the safety of food and feed is a key challenge facing agriculture today. One of these strategies lies on the promising capacity of products issued from natural sources to counteract crop pests. In this work, the in vitro efficiency of sixteen extracts obtained from eight natural sources using subcritical water extraction at two temperatures was assessed against fungal growth and TCTB production by F. graminearum. Maritime pine sawdust extract was shown to be extremely efficient, leading to a significant inhibition of up to 89% of the fungal growth and up to 65% reduction of the mycotoxin production by F. graminearum. Liquid chromatography/mass spectrometry analysis of this active extract revealed the presence of three families of phenolics with a predominance of methylated compounds and suggested that the abundance of methylated structures, and therefore of hydrophobic compounds, could be a primary factor underpinning the activity of the maritime pine sawdust extract. Altogether, our data support that wood/forest by-products could be promising sources of bioactive compounds for controlling F. graminearum and its production of mycotoxins.

Trichothecenes in Food and Feed, Relevance to Human and Animal Health and Methods of Detection: A Systematic Review.

Trichothecene mycotoxins are sesquiterpenoid compounds primarily produced by fungi in taxonomical genera such as Fusarium, Myrothecium, Stachybotrys, Trichothecium, and others, under specific climatic conditions on a worldwide basis. Fusarium mold is a major plant pathogen and produces a number of trichothecene mycotoxins including deoxynivalenol (or vomitoxin), nivalenol, diacetoxyscirpenol, and T-2 toxin, HT-2 toxin. Monogastrics are sensitive to vomitoxin, while poultry and ruminants appear to be less sensitive to some trichothecenes through microbial metabolism of trichothecenes in the gastrointestinal tract. Trichothecene mycotoxins occur worldwide however both total concentrations and the particular mix of toxins present vary with environmental conditions. Proper agricultural practices such as avoiding late harvests, removing overwintered stubble from fields, and avoiding a corn/wheat rotation that favors Fusarium growth in residue can reduce trichothecene contamination of grains. Due to the vague nature of toxic effects attributed to low concentrations of trichothecenes, a solid link between low level exposure and a specific trichothecene is difficult to establish. Multiple factors, such as nutrition, management, and environmental conditions impact animal health and need to be evaluated with the knowledge of the mycotoxin and concentrations known to cause adverse health effects. Future research evaluating the impact of low-level exposure on livestock may clarify the potential impact on immunity. Trichothecenes are rapidly excreted from animals, and residues in edible tissues, milk, or eggs are likely negligible. In chronic exposures to trichothecenes, once the contaminated feed is removed and exposure stopped, animals generally have an excellent prognosis for recovery. This review shows the occurrence of trichothecenes in food and feed in 2011-2020 and their toxic effects and provides a summary of the discussions on the potential public health concerns specifically related to trichothecenes residues in foods associated with the exposure of farm animals to mycotoxin-contaminated feeds and impact to human health. Moreover, the article discusses the methods of their detection.

Occurrence of type A, B and D trichothecenes, zearalenone and stachybotrylactam in straw.

Straw is the main by-product of grain production, used as bedding material and animal feed. If produced or stored under adverse hygienic conditions, straw is prone to the growth of filamentous fungi. Some of them, e.g. Aspergillus, Fusarium and Stachybotrys spp. are well-known mycotoxin producers. Since studies on mycotoxins in straw are scarce, 192 straw samples (wheat n = 80; barley n = 79; triticale n = 12; oat n = 11; rye n = 12) were collected across Germany within the German official feed surveillance and screened for the presence of 21 mycotoxins. The following mycotoxins (positive samples for at least one mycotoxin n = 184) were detected: zearalenone (n = 86, 6.0-785 µg/kg), nivalenol (n = 51, 30-2,600 µg/kg), deoxynivalenol (n = 156, 20-24,000 µg/kg), 15-acetyl-deoxynivalenol (n = 34, 20-2,400 µg/kg), 3-acetyl-deoxynivalenol (n = 16, 40-340 µg/kg), scirpentriol (n = 14, 40-680 µg/kg), T-2 toxin (n = 67, 10-250 µg/kg), HT-2 toxin (n = 92, 20-800 µg/kg), T-2 tetraol (n = 13, 70-480 µg/kg). 15-monoacetoxyscirpenol (30 µg/kg) and T-2 triol (60 µg/kg) were only detected in one barley sample. Macrocyclic trichothecenes (satratoxin G, F, roridin E, and verrucarin J) were also found in only one barley sample (quantified as roridin A equivalent: total 183 µg/kg). The occurrence of stachybotrylactam was monitored for the first time in four samples (n = 4, 0.96-7.4 µg/kg). Fusarenon-X, 4,15-diacetoxyscirpenol, neosolaniol, satratoxin H and roridin-L2 were not detectable in the samples. The results indicate a non-negligible contribution of straw to oral and possibly inhalation exposure to mycotoxins of animals or humans handling contaminated straw.

Genetic regulation of aflatoxin, ochratoxin A, trichothecene, and fumonisin biosynthesis: A review.

Mycotoxins are a significant food safety concern. Aflatoxins, trichothecenes, fumonisins, and ochratoxin A are considered the most important mycotoxins due to their frequent occurrence in food products and their well-known toxicity. The regulation of mycotoxin biosynthesis occurs mainly at transcriptional level, and specific regulators have been described in every biosynthetic cluster. Secondary metabolite production, including mycotoxin synthesis, is also regulated by general regulator pathways affected by light, osmotic stress and oxidative stress, among others. This review is focused on this genetic regulation of mycotoxin biosynthesis by specific genes and global regulators.

Novel analytical methods to study the fate of mycotoxins during thermal food processing.

Food processing can lead to a reduction of contaminants, such as mycotoxins. However, for food processing operations where thermal energy is employed, it is often not clear whether a reduction of mycotoxins also results in a mitigation of the toxicological impact. This is often due to the reason that the formed degradation products are not characterized and data on their toxicity is scarce. From the perspective of an analytical chemist, the elucidation of the fate of a contaminant in a complex food matrix is extremely challenging. An overview of the analytical approaches is given here, and the application and limitations are exemplified based on cases that can be found in recent literature. As most studies rely on targeted analysis, it is not clear whether the predetermined set of compounds differs from the degradation products that are actually formed during food processing. Although untargeted analysis allows for the elucidation of the complete spectrum of degradation products, only one such study is available so far. Further pitfalls include insufficient precision, natural contamination with masked forms of mycotoxins and interferences that are caused by the food matrix. One topic that is of paramount importance for both targeted and untargeted approaches is the availability of reference standards to identity and quantity the formed degradation products. Our vision is that more studies need to be published that characterize the formed degradation products, collect data on their toxicity and thereby complete the knowledge about the mycotoxin mitigating effect during food processing.

Differentiation of S. chartarum (Ehrenb.) S. Hughes Chemotypes A and S via FT-IR Spectroscopy.

Stachybotrys (S.) chartarum is a cellulolytic mould with the ability to produce highly cytotoxic macrocyclic trichothecenes. Two chemotypes are defined according to their ability to produce either atranones or satratoxins. S. chartarum has been well known as the causative agent of the lethal disease stachybotryotoxicosis in horses. Further investigations revealed that this disease is strictly correlated with the presence of macrocyclic trichothecenes. Furthermore, their occurrence in water-damaged buildings has been linked to adverse health effects such as the sick building syndrome. As the chemotypes cannot be characterized via phenotypic criteria, different methods such as PCR, MALDI-TOF MS, LC-MS/MS, thin-layer chromatography and cytotoxicity assays have been used so far. Fourier-transform-infrared spectroscopy (FT-IR) is commonly used for the differentiation of bacteria and yeasts, but this technique is also applicable to filamentous fungi. Hence, this study aimed at evaluating to which extent a reliable differentiation of S. chartarum chemotypes A and S is possible. Besides, another objective was to verify if the recently introduced third genotype of S. chartarum can be identified. Therefore, 28 strains including the two chemotypes and the third genotype H were cultivated on malt extract agar (MEA) and potato dextrose agar in three biological replicates. Each sample was applied to FT-IR measurements on day 7, 14 and 21 of cultivation. In this study, we achieved a distinction of the chemotypes A and S via FT-IR spectroscopy after incubation for 7 days on MEA. In terms of genotype differentiation, the PCR detecting satratoxin- and atranone-gene clusters remained the only applicable method.

Role of Trichoderma arundinaceum tri10 in regulation of terpene biosynthetic genes and in control of metabolic flux.

Production of trichothecene toxins occurs in phylogenetically diverse fungi with different lifestyles. In these fungi, most homologs of the trichothecene biosynthetic gene cluster include the transcription factor genes tri6 and tri10. Analyses of phytopathogenic species of Fusarium indicate that the TRI6 and TRI10 proteins positively regulate genes required for synthesis of trichothecenes as well as farnesyl diphosphate (FPP), a precursor of the trichothecene and other terpenoids (e.g., ergosterol). However, the apparent absence of tri6 and tri10 in some trichothecene-producing fungi, and the presence of multiple paralogs of the genes in others suggest considerable variability in genetic regulation of trichothecene biosynthesis. To begin to investigate this variability, we functionally characterized tri10 in the saprotrophic fungus Trichoderma arundinaceum. We found that TRI10 is required for wild-type expression of tri genes and trichothecene production during the first 12 h of growth of T. arundinaceum. Comparison of the effect of tri10 deletion in T. arundinaceum and Fusarium species has provided evidence for similarities in the genetic regulation of trichothecene biosynthesis in these two fungi with different lifestyles. In contrast to trichothecenes, tri10 deletion increased production of ergosterol and the polyketide-derived metabolites aspinolides, which is more likely caused by an increase in the intracellular pool of FPP resulting from loss of trichothecene production. Furthermore, although it is unclear how TRI10 affects polyketide production, one possibility is that it does so by rechanneling terpene precursors.