Interactions among the composition changes in fungal communities and the main mycotoxins in simulated stored wheat grains.

BACKGROUND: There are significant food safety risks associated with wheat spoilage due to fungal growth and mycotoxin contamination. Nevertheless, a few studies have examined how stored wheat grain microbial communities and mycotoxins vary in different storage conditions. In this study, changes in deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) content were measured with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), and an amplicon sequence analysis of fungi was performed on stored wheat grains from different storage conditions using high-throughput sequencing. The detailed interactions among the composition changes in the fungal community and the DON content of simulated stored wheat grains were also analyzed. RESULTS: Alternaria, Fusarium, Mrakia, and Aspergillus were the core fungal taxa, and the fungal communities of samples stored under different conditions were observed to be different. Aspergillus relative abundances increased, whereas Fusarium decreased. This led to an increase in the content of DON. The content of DON increased about 67% with 12% moisture and at 25 °C after 2 months of storage, which was influenced by the stress response of Fusarium. Correlations in fungal and mycotoxins changes were observed. There may be potential value in these findings for developing control strategies to prevent mildew infestations and mycotoxins contamination during grain storage. CONCLUSION: In storage, the more the fungal community composition and the relative abundance of Fusarium change, the more mycotoxins will be produced. We should therefore reduce competition between fungal communities through pre-storage treatment and through measures during storage. © 2023 Society of Chemical Industry.

A Hydrolase Produced by Rhodococcus erythropolis HQ Is Responsible for the Detoxification of Zearalenone.

Zearalenone (ZEN), an estrogenic mycotoxin, is one of the prevalent contaminants found in food and feed, posing risks to human and animal health. In this study, we isolated a ZEN-degrading strain from soil and identified it as Rhodococcus erythropolis HQ. Analysis of degradation products clarified the mechanism by which R. erythropolis HQ degrades ZEN. The gene zenR responsible for degrading ZEN was identified from strain HQ, in which zenR is the key gene for R. erythropolis HQ to degrade ZEN, and its expression product is a hydrolase named ZenR. ZenR shared 58% sequence identity with the hydrolase ZenH from Aeromicrobium sp. HA, but their enzymatic properties were significantly different. ZenR exhibited maximal enzymatic activity at pH 8.0-9.0 and 55 °C, with a Michaelis constant of 21.14 µM, and its enzymatic activity is 2.8 times that of ZenH. The catalytic triad was identified as S132-D157-H307 via molecular docking and site-directed mutagenesis. Furthermore, the fermentation broth of recombinant Bacillus containing ZenR can be effectively applied to liquefied corn samples, with the residual amount of ZEN decreased to 0.21 µg/g, resulting in a remarkable ZEN removal rate of 93%. Thus, ZenR may serve as a new template for the modification of ZEN hydrolases and a new resource for the industrial application of biological detoxification. Consequently, ZenR could potentially be regarded as a novel blueprint for modifying ZEN hydrolases and as a fresh resource for the industrial implementation of biological detoxification.

Paralogous FgIDO genes with differential roles in tryptophan catabolism, fungal development and virulence in Fusarium graminearum.

Indoleamine 2,3-dioxygenase (Ido) is a tryptophan-degrading enzyme that is widely distributed across species. Ido catalyzes the first step of tryptophan (TRP) degradation and drives the de novo synthesis of nicotinamide adenine dinucleotide (NAD+) coenzymes via the kynurenine (KYN) pathway. The budding yeast Saccharomyces cerevisiae possesses a single IDO gene (BNA2) that is responsible for NAD+ synthesis, whereas a number of fungal species contain multiple IDO genes. However, the biological roles of IDO paralogs in plant pathogens remain unclear. In the current study, we identified three FgIDOs from the wheat head blight fungus Fusarium graminearum. FgIDOA/B/C expression was significantly induced upon TRP treatment. Targeted disruption of FgIDOA and/or FgIDOB caused different levels of NAD+ auxotrophy, thus resulting in pleotropic phenotypic defects. Loss of FgIDOA resulted in abnormal conidial morphology, reduced mycelial growth, decreased virulence in wheat heads and reduced deoxynivalenol accumulation. Exogenous addition of KYN or various intermediates involved in the KYN pathway rescued auxotrophy of the mutants. Metabolomics analysis revealed shifts toward alternative TRP degradation pathways to melatonin and indole derivatives in mutants lacking FgIDOB. Upregulation of partner genes in auxotrophic mutants and the capacity to rescue the auxotroph by overexpressing a partner gene indicated functional complementation among FgIDOA/B/C. Taken together, the results of this study provide insights into differential roles in paralogous FgIDOs and how fungal TRP catabolism modulates fungal development and virulence.

Antifungal activities of a novel triazole fungicide, mefentrifluconazole, against the major maize pathogen Fusarium verticillioides.

Fusarium ear rot (FER) is a serious fungal disease occurring the late growth stage of maize. FER not only reduces the yield of maize but also causes mycotoxin contamination, which affects the quality of maize and threatens human and animal health. Fusarium verticillioides is the predominant causative pathogen of FER worldwide. At present, there is no registered fungicide for use against maize FER in China. The novel isopropyl alcohol-triazole fungicide mefentrifluconazole (MFZ) has been shown to be effective against several Fusarium spp., but little is known about its specific activity against F. verticillioides. MFZ exhibited strong antifungal activities against 50 strains of F. verticillioides collected from the major maize-growing areas in China. MFZ inhibited mycelial growth, conidium production, germination and germ tube elongation of F. verticillioides. MFZ treatment significantly reduced fumonisin production and the expression levels of fumonisin biosynthetic genes. Genome-wide transcriptional profiling of F. verticillioides in response to MFZ indicated that the expression of genes involved in ergosterol biosynthesis, including fungicide target genes (cyp51 genes), was significantly downregulated by MFZ. MFZ treatment resulted in reduced ergosterol production and increased glycerol and malonaldehyde production as well as relative conductivity in F. verticillioides. A 2-year field experiment showed a significant reduction in FER severity in maize after spraying with MFZ at the tasseling stage. This study evaluated the potential of MFZ to control FER in maize and provides insights into its antifungal activities and mechanism of action against F. verticillioides.

Antifungal activities of metconazole against the emerging wheat pathogen Fusarium pseudograminearum.

Fusarium crown rot of wheat is a serious fungal disease that occurs worldwide. The disease has been emerging in the major wheat-growing areas in China since 2010. Fusarium pseudogramineaum is the predominant causative pathogen of crown rot of wheat in China. The 14α-demethylation inhibitor (DMI) fungicide metconazole has been shown to be effective against Fusarium spp., but little is known about its specific activity against F. pseudogramineaum. Metconazole exhibited strong antifungal activities against all thirty-nine F. pseudogramineaum strains collected from the major wheat-growing areas in China. Metconazole inhibited mycelial growth and conidial germ tube elongation of F. pseudograminearum. Metconazole treatment significantly reduced the production of major toxins and the expression levels of toxin biosynthesis genes. Genome-wide transcriptional profiling of F. pseudograminearum in response to metconazole indicated that the expression of genes involved in ergosterol biosynthesis, including fungicide target genes (cyp51 genes), was significantly induced by metconazole. Nine ATP-binding cassette (ABC) transporter-encoding genes were significantly expressed in response to metconazole treatment. Reduced ergosterol production and antioxidant enzyme activities were observed after metconazole treatment. Greenhouse experiments indicated a significant reduction in crown rot occurrence in wheat after seed treatment with metconazole. This study evaluated the potential of metconazole to manage wheat crown rot and provides information to understand its antifungal activities and mechanism of action against F. pseudograminearum.

New Hydrolase from Aeromicrobium sp. HA for the Biodegradation of Zearalenone: Identification, Mechanism, and Application.

Zearalenone (ZEN) is an estrogenic mycotoxin most frequently found in cereals that can cause reproductive disorders in livestock and pose a severe threat to animal husbandry. In this study, we isolated a ZEN-degrading Aeromicrobium strain from soil and found that ZenH, a hydrolase, is responsible for the hydrolysis of ZEN through comparative proteomics and biochemical studies. ZenH exhibited the highest similarity with lactone hydrolase ZHD607 from Phialophora americana at 21.52%. ZenH displayed maximal enzymatic activity at pH 7.0 and 55 °C with a Michaelis constant of 12.64 µM. The catalytic triad of ZenH was identified as S117-D142-H292 by molecular docking and site-directed mutagenesis. ZenH catalyzed the hydrolysis of ZEN to a novel metabolite, (S,E)-4-hydroxy-2-(10-hydroxy-6-oxoundec-1-en-1-yl)-7-oxabicyclo[4.2.0]octa-1,3,5-trien-8-one, which exhibited significantly lower estrogenic toxicity than ZEN. This study illustrates a novel ZEN-degrading enzyme and reveals a new degradation product. Furthermore, the enzyme showed good potential for detoxifying ZEN during food processing.

Isolation, Characterization, and Application of Clostridium sporogenes F39 to Degrade Zearalenone under Anaerobic Conditions.

Zearalenone (ZEN) is produced by Fusarium spp. and is widely found in moldy wheat, corn, and other grains. ZEN has a strong toxicity and causes reproductive and immune disorders and estrogenic syndrome in animals and humans. Biodegradation has been demonstrated as an efficient way to control the hazardous effect of ZEN. A promising way to apply biodegradation in feed is to introduce anaerobic ZEN-degrading microorganisms, which can function during the digestion process in animal intestines. The aim of this study was to isolate anaerobic ZEN-degrading bacteria from anaerobic environments. A strain named F39 was isolated from animal intestinal contents and had a ZEN-degradation rate of 87.35% in 48 h to form trace amount of α- and ß-zearalenol. Based on the morphological and physiological properties and phylogenetic analysis of 16S rRNA and rpoB gene sequences, F39 was identified as Clostridium sporogenes. The optimum temperature for the growth of F39 was 37 °C, the optimum pH was 7.0, and the most suitable carbon source was beef extract, while the optimal conditions for the degradation of ZEN were as follows: 35 °C, pH 7.0, and GAM medium. ZEN was degraded by F39 with a high efficiency in the concentration range of 1-15 mg/L. The bioactive factors responsible for ZEN degradation were mainly distributed intracellularly. F39 can degrade most of the ZEN present, but a small amount is broken down into two secondary metabolites, α- and ß-zearalenol, and the toxicity of the degradation products is reduced. With an efficiency of 49%, F39 can more effectively degrade ZEN in wheat-based feedstuffs than in other feedstuff, and the degradation efficiency was pH related. To the best of our knowledge, this is the first report of Clostridium sporogenes F39's ability to maintain the biodegradation potentials.

The autophagy-related proteins FvAtg4 and FvAtg8 are involved in virulence and fumonisin biosynthesis in Fusarium verticillioides.

Autophagy is the main intracellular degradation system by which cytoplasmic materials are transported to and degraded in the vacuole/lysosome of eukaryotic cells, and it also controls cellular differentiation and virulence in a variety of filamentous fungi. However, the contribution of the autophagic pathway to fungal development and pathogenicity in the important maize pathogen and mycotoxigenic fungus Fusarium verticillioides is still unknown. In this study, we characterized two autophagy-related proteins, FvAtg4 and FvAtg8. The F. verticillioides deletion mutants ΔFvAtg4 and ΔFvAtg8 were impaired in autophagosome formation, aerial hyphal formation, sexual growth, lipid turnover, pigmentation and fungal virulence. Interestingly, ΔFvAtg4 and ΔFvAtg8 were defective in fumonisin B1 (FB1) synthesis, which may have resulted from decreased intracellular levels of alanine in the mutants. Our results indicate that FvAtg4 and FvAtg8 contribute to F. verticillioides pathogenicity by regulating the autophagic pathway to control lipid turnover, fumonisin biosynthesis, and pigmentation during its infectious cycle.

Rapid detection of fumonisin B1 and B2 in ground corn samples using smartphone-controlled portable near-infrared spectrometry and chemometrics.

A portable near-infrared (NIR) spectrometer coupled with chemometrics for the detection of fumonisin B1 and B2 (FBs) in ground corn samples was proposed in the present work. A total of 173 corn samples were collected, and their FB contents were determined by HPLC-MS/MS. Partial least squares (PLS), support vector machine (SVM) and local PLS based on global PLS score (LPLS-S) algorithms were employed to construct quantitative models. The performance of the SVM and LPLS-S was better than that of PLS, and the LPLS-S presented the lowest RMSEP (12.08 mg/kg) and the highest RPD (3.44). Partial least squares-discriminant analysis (PLS-DA) and support vector machine-discriminant analysis (SVM-DA) were used to classify corn samples according to the maximum residue limit (MRL) of FBs, and the discriminant accuracy of both the PLS-DA and SVM-DA algorithms was above 86.0%. Thus, the present study provided a rapid method for monitoring FB contamination in corn samples.

Improved Whole-Genome Sequence of Fusarium meridionale, the Fungal Pathogen Causing Fusarium Head Blight in Rice.

Members of the Fusarium graminearum species complex (FGSC) cause extensive yield losses in cereal production worldwide, and food safety concerns due to the accumulation of Fusarium toxins in infected grains. Among these pathogens, F. meridionale is responsible for Fusarium head blight of wheat and rice, ear and stalk rot of maize, and pod blight of soybean. Here, we present an improved genome assembly of F. meridionale strain SR5 isolated from rice in China based on PacBio long-read sequencing and Illumina short-read sequencing technology. The assembled genome of SR5 has a total size of 36.82 Mb, an N50 scaffold length of 7.82 Mb, nine scaffolds, and encodes 12,409 predicted genes. These high-quality data expand FGSC genomic resources and provide a valuable resource for better understanding their genetic diversity and the molecular basis of pathogenesis, which will facilitate the development of an effective control strategy.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Antioxidant Guaiacol Exerts Fungicidal Activity Against Fungal Growth and Deoxynivalenol Production in Fusarium graminearum.

The main component of creosote obtained from dry wood distillation-guaiacol-is a natural antioxidant that has been widely used in pharmaceutical and food preservation applications. However, the antifungal mechanism of guaiacol against phytopathogens remains unclear. In this study, we found that guaiacol exerts inhibitory effects against mycelial growth, conidial formation and germination, and deoxynivalenol (DON) biosynthesis in Fusarium graminearum in a dose-dependent manner. The median effective concentration (EC50) value of guaiacol for the standard F. graminearum strain PH-1 was 1.838 mM. Guaiacol strongly inhibited conidial production and germination. The antifungal effects of guaiacol may be attributed to its capability to cause damage to the cell membrane by disrupting Ca2+ transport channels. In addition, the decreased malondialdehyde (MDA) levels and catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activity by guaiacol treatment indicate that guaiacol displays activity against DON production by modulating the oxidative response in F. graminearum. Taken together, this study revealed the potentials of antioxidant in inhibiting mycotoxins in F. graminearum.

A direct competitive nanozyme-linked immunosorbent assay based on MnO2 nanosheets as a catalytic label for the determination of fumonisin B1.

A direct competitive nanozyme-linked immunosorbent assay (dcNLISA) based on MnO2 nanosheets (MnO2 NSs) as a nanozyme label was developed for the highly sensitive determination of fumonisin B1 (FB1). MnO2 NS-labeled fumonisin B1-bovine serum albumin was easily synthesized as a competing antigen for the dcNLISA. And color changes derived from the MnO2-3,3',5,5'-tetramethylbenzidine (TMB) system were exploited as the output signals of the dcNLISA. Several experimental parameters including the concentrations of the coating antibody, pH values, ionic strength and methanol concentration were optimized. Under the optimal conditions, the proposed method demonstrated a linear range (1.17-20.74 ng mL-1) with a reliable correlation coefficient (R2 = 0.9989), a satisfactory limit of detection (0.63 ng mL-1) and high selectivity for the detection of FB1. The recoveries of FB1 in spiked corn and wheat samples were in the range of 85.31-108.16% with coefficients of variation (CVs) ranging from 6.14% to 9.23%. Meanwhile, the testing results showed good consistency (R2 = 0.9892) between the developed dcNLISA and the reference method, liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) method. The proposed method was proven to be simple, sensitive, cost-effective and reliable for the screening of FB1 in cereals.