Detoxifying bacterial genes for deoxynivalenol epimerization confer durable resistance to Fusarium head blight in wheat.

Fusarium head blight (FHB) and the presence of mycotoxin deoxynivalenol (DON) pose serious threats to wheat production and food safety worldwide. DON, as a virulence factor, is crucial for the spread of FHB pathogens on plants. However, germplasm resources that are naturally resistant to DON and DON-producing FHB pathogens are inadequate in plants. Here, detoxifying bacteria genes responsible for DON epimerization were used to enhance the resistance of wheat to mycotoxin DON and FHB pathogens. We characterized the complete pathway and molecular basis leading to the thorough detoxification of DON via epimerization through two sequential reactions in the detoxifying bacterium Devosia sp. D6-9. Epimerization efficiently eliminates the phytotoxicity of DON and neutralizes the effects of DON as a virulence factor. Notably, co-expressing of the genes encoding quinoprotein dehydrogenase (QDDH) for DON oxidation in the first reaction step, and aldo-keto reductase AKR13B2 for 3-keto-DON reduction in the second reaction step significantly reduced the accumulation of DON as virulence factor in wheat after the infection of pathogenic Fusarium, and accordingly conferred increased disease resistance to FHB by restricting the spread of pathogenic Fusarium in the transgenic plants. Stable and improved resistance was observed in greenhouse and field conditions over multiple generations. This successful approach presents a promising avenue for enhancing FHB resistance in crops and reducing mycotoxin contents in grains through detoxification of the virulence factor DON by exogenous resistance genes from microbes.

Utilization of a Novel Soil-Isolated Strain Devosia insulae FS10-7 for Deoxynivalenol Degradation and Biocontrol of Fusarium Crown Rot in Wheat.

Deoxynivalenol (DON) is the most widespread mycotoxin contaminant hazardous to human and animal health globally. It acts as a crucial virulence factor to stimulate the spread of pathogenic Fusarium within wheat plants. Control of DON and Fusarium disease contributes enormously to food safety, which relies on chemical fungicides. Here, we report the biodegradation of DON using a novel soil bacterium, Devosia insulae FS10-7, and its biocontrol effect against Fusarium crown rot. We demonstrated that strain FS10-7 degraded DON to 3-epi-DON by forming a 3-keto-DON intermediate. Such degradation activity can be maintained at a wide range of pH (4 to 10) and temperature (16 to 42°C) values under aerobic conditions. Notably, strain FS10-7 exhibited practical inhibitory effects on Fusarium crown rot disease caused by F. graminearum and F. pseudograminearum in the in vitro Petri dish test under laboratory conditions and the pot experiment under greenhouse conditions. The mechanisms underlying the biocontrol ability of strain FS10-7 were preliminarily investigated to be associated with its high DON-degrading activity rather than direct antagonism. These results establish the foundation to develop further bioagents capable of biodegrading mycotoxins in cereals and derived products and, accordingly, biocontrol plant diseases caused by DON-producing pathogens.

Crude Lipopeptides Produced by Bacillus amyloliquefaciens Could Control the Growth of Alternaria alternata and Production of Alternaria Toxins in Processing Tomato.

Alternaria spp. and its toxins are the main contaminants in processing tomato. Based on our earlier research, the current study looked into the anti-fungal capacity of crude lipopeptides from B. amyloliquefaciens XJ-BV2007 against A. alternata. We found that the crude lipopeptides significantly inhibited A. alternata growth and reduced tomato black spot disease incidence. SEM analysis found that the crude lipopeptides could change the morphology of mycelium and spores of A. alternata. Four main Alternaria toxins were detected using UPLC-MS/MS, and the findings demonstrated that the crude lipopeptides could lessen the accumulation of Alternaria toxins in vivo and in vitro. Meanwhile, under the stress of crude lipopeptides, the expression of critical biosynthetic genes responsible for TeA, AOH, and AME was substantially down-regulated. The inhibitory mechanism of the crude lipopeptides was demonstrated to be the disruption of the mycelial structure of A. alternata, as well as the integrity and permeability of the membrane of A. alternata sporocytes. Taken together, crude lipopeptides extracted from B. amyloliquefaciens XJ-BV2007 are an effective biological agent for controlling tomato black spot disease and Alternaria toxins contamination.

Fusarium Mycotoxins and OTA in Beer from Shanghai, the Largest Megacity in China: Occurrence and Dietary Risk Assessment.

Beer is susceptible to mycotoxin contamination originating from infected grains. It could be that mycotoxins are not completely removed during the brewing process and remain in the final product. Nevertheless, there have been no surveys of exposure to mycotoxin for Chinese inhabitants through beer consumption. This study aimed to investigate the presence of eight mycotoxins in 158 beer samples purchased in Shanghai, the largest megacity in China. The multiple mycotoxins determination was carried out using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Our findings revealed that 48.1% (76/158) of the beer samples were contaminated with Fusarium toxins. Deoxynivalenol-3-glucoside (D3G) and zearalenone (ZEN) were detected in 34.81% and 16.46% of the total samples, respectively. The significant differences between D3G/ZEN contamination and various beer types were performed. Furthermore, this study performed a health risk assessment for Shanghai residents based on data for Fusarium toxins and ochratoxin A (OTA) present in beer for the first time. The results revealed that the 95th percentile dietary exposures of Shanghai residents did not pose any chronic or acute health risks, either individually or in combination. Dietary exposures to Fusarium toxins revealed different risk levels among residents. The cumulative health risk for women is higher than that for men at the same beer consumption. In addition, the acute risk of DONs exposure for adults deserves concern. The insights obtained from this study may be of assistance for beer manufacturers and governmental regulators to further develop beer monitoring and guarantee public health.

Mycotoxin deoxynivalenol-induced intestinal flora disorders, dysfunction and organ damage in broilers and pigs.

Deoxynivalenol (DON) is a common environmental contaminant that causes food refusal and growth retardation in animals. DON targets the intestine and is hazardous to animal, however, it is not clear whether its effect on animals is consistent. Chickens and pigs are the two main animals affected by DON exposure with different susceptibilities. In this study, we found that DON inhibited animal growth and caused damage to the intestine, liver and kidney. DON caused intestinal flora disorders in both chickens and pigs, such as changes of flora diversity and the relative abundance of dominant phyla. Functional analysis showed that changes in the intestinal flora induced by DON were mainly related to metabolic and digestive functions, indicated that the intestinal flora may be associated with the DON-induced intestinal dysfunction. Comparative analysis of differentially altered bacteria suggested that Prevotella may play an important role in maintaining intestinal health, and the presence of differentially altered bacteria in the two animals suggested that DON may have different toxicity modes in animals. In summary, we confirmed the multi-organ toxicity of DON in two major livestock and poultry animals, and speculated that the intestinal flora may be related to the toxic damage caused by DON through species comparison analysis.

Cytotoxicities of Co-occurring alternariol, alternariol monomethyl ether and tenuazonic acid on human gastric epithelial cells.

Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) are the three major Alternaria toxin contaminants in food. In the present study, we conducted their single and combined toxicity analyses using human gastric epithelial cell line (GES-1) that was first exposed to the toxins when they entered the human body. By comparing the cytotoxicity IC50, we found that compared to several other mycotoxins with limit standards there was cytotoxicity DON > OTA > AME > AOH > ZEN > TeA. Further, we obtained combination index (CI)-isobologram equation by the Chou-Talalay method according to a toxin ratio of 1:1:2 and carried out the combined toxicity analysis of the three binary and ternary compounds, and the results showed that AOH + AME + TeA showed synergistic toxic effects. Based on the co-occurring status, we also carried out the combined toxicity analysis of AME and AOH at different ratios and found antagonistic effects at low cytotoxic concentrations as well as synergistic and additive effects at high concentrations. Also, we found that all three and their combinations caused apoptosis, activation of caspase-3 cleavage, activation of DNA damage pathways ATR-Chk1-P53 and ATM-Chk2-P53. In conclusion, we used GES-1 cells to inform the risk of coaction of AOH, AME, and TeA in dietary exposure.

Deciphering Alternaria metabolic responses in microbial confrontation via an integrated mass spectrometric targeted and non-targeted strategy.

Microbial confrontation is ubiquitously present in nature, such as mycoparasitism, competition and antibiosis between biocontrol agents and microbial pathogens. However, the internal metabolic responses of fungal pathogens under microbial interaction scenario have been scarcely investigated. In this study, we set up an integrated mycotoxin analysis and non-targeted metabolomics workflow to decipher metabolic changes of Alternaria pathogens when confronted with selected Trichoderma strains, as well as mycotoxin metabolization in the Trichoderma spp. Results demonstrated that Trichoderma spp. significantly influenced mycotoxin production and whole metabolome of Alternaria pathogens when in cocultivation, and one Trichoderma strain could metabolize alternariol into its hydroxylated form. These differential metabolites revealed fungal physiological alternations in various confrontation conditions. In all, a MS-based strategy was proposed to investigate microbial metabolic profiles under fungal/fungal and fungal/mycotoxin cocultivation, and this generic methodology would be significant for understanding the occurrence and change of food contaminants in microbial interactions.

Mycotoxins in Wheat Flours Marketed in Shanghai, China: Occurrence and Dietary Risk Assessment.

The risk of exposure to mycotoxins through the consumption of wheat flours has long been a concern. A total of 299 wheat flours marketed in Shanghai Province of China were surveyed and analyzed for the co-occurrence of 13 mycotoxins through an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. The detection rates of mycotoxins in wheat flours ranged from 0.7~74.9% and their average contamination levels in wheat flours (0.2~57.6 µg kg-1) were almost lower than the existing regulations in cereals. However, their co-contamination rate was as high as 98.1%, especially Fusarium and Alternaria mycotoxins. Comparative analysis of different types of wheat flours showed that the average contamination levels in refined wheat flours with low-gluten were lower. Based on these contamination data and the existing consumption data of Shanghai residents, point evaluation and the Monte Carlo assessment model were used to preliminarily evaluate the potential dietary exposure risk. The probable daily intakes of almost all mycotoxins, except for alternariol, were under the health-based guidance values for 90% of different consumer groups. Health risks of dietary exposure to alternariol should be a concern and further studied in conjunction with an internal exposure assessment.

Cyanidin-3-O-glucoside promotes stress tolerance and lifespan extension of Caenorhabditis elegans exposed to polystyrene via DAF-16 pathway.

Microplastic pollution has attracted growing attention due to its prevalent and persistent exposure to general population through the food chain, but few reports have focused on the toxicological prevention of polystyrene (PS). Using the wild-type and mutant strains, this study explored the impacts of PS and cyanidin-3-O-glucoside (C3G) on stress tolerance and lifespan of Caenorhabditis elegans (C. elegans). In N2 nematodes, PS exposure initiated the oxidative stress and subsequent lifespan reduction, while these adverse impacts could be positively improved by C3G treatment. Considering the pivotal role of DAF-16 pathway in stress tolerance and lifespan regulation, the expression of the daf-16 gene and its downstream antioxidant genes (clt-2, hsp-16.1, sod-3, sod-5) were examined, and found to be significantly enhanced by C3G. Since the sod-3 gene was up-regulated the most fold by C3G, the activity of SOD enzyme that encoded by the sod-3 was examined, and could be obviously enhanced upon C3G treatment. This explained the improved oxidative stress and delayed oxidation-associated aging after C3G intervention. Nevertheless, these positive effects of C3G were weakened in daf-16(-) mutant strain (with deleted DAF-16 gene), for which the beneficial effects of C3G in promoting stress resistance and lifespan extension were inhibited. These findings suggested that the DAF-16 gene and its downstream antioxidant genes, have participated in C3G's regulations on redox balance and lifespan that impacted by nano-polystyrene particles. This study highlighted the link between dietary components and environmentally driven disturbance.

Identification of Candidate Genes Associated with Trichothecene Biosynthesis in Fusarium graminearum Species Complex Combined with Transcriptomic and Proteomic Analysis.

The Fusarium graminearum species complex is the main causal agent of wheat head blight worldwide. Trichothecenes produced by the pathogen in infected grains have important food safety implications. Previously reported studies on trichothecene production have all focused on the conditions conducive to mycotoxin production, while the molecular mechanisms of trichothecene biosynthesis in Fusarium strains under normal or non-inducing conditions are still unclear. Here, a global analysis of the fungal gene expression of three strains using the Affymetrix Fusarium GeneChip under non-inducing conditions is reported. Differentially expressed genes were identified among strains with different trichothecene-production ability, and some novel genes associated with trichothecene biosynthesis were found by bioinformatics analysis. To verify the transcriptome results, proteomic analyses of the three strains were conducted under the same culture conditions. In total, 69 unique fungal proteins were identified in 77 protein spots. Combined with transcriptome and proteome analysis, 27 novel genes were predicted to be associated with trichothecene mycotoxin production. A protein, encoded by FGSG_01403, was found to be associated with trichothecene production via proteome analysis. Gene knock-out mutations of FGSG_01403 resulted in mutants with increased production of trichothecenes. Future functional analysis of the candidate genes identified in this study may reveal new insights into the negative regulation of trichothecene production in the Fusarium graminearum species complex.

Mycotoxins in Tea ((Camellia sinensis (L.) Kuntze)): Contamination and Dietary Exposure Profiling in the Chinese Population.

Tea is popular worldwide with multiple health benefits. It may be contaminated by the accidental introduction of toxigenic fungi during production and storage. The present study focuses on potential mycotoxin contamination in tea and the probable dietary exposure assessments associated with consumption. The contamination levels for 16 mycotoxins in 352 Chinese tea samples were determined by ultra-performance liquid chromatography-tandem mass spectrometry. Average concentrations of almost all mycotoxins in tea samples were below the established regulations, except for ochratoxin A in the dark tea samples. A risk assessment was performed for the worst-case scenarios by point evaluation and Monte Carlo assessment model using the obtained mycotoxin levels and the available green, oolong, black, and dark tea consumption data from cities in China. Additionally, we discuss dietary risk through tea consumption as beverages and dietary supplements. In conclusion, there is no dietary risk of exposure to mycotoxins through tea consumption in the Chinese population.

Exploration of Mycotoxin Accumulation and Transcriptomes of Different Wheat Cultivars during Fusarium graminearum Infection.

Fusarium graminearum is one of the most devastating diseases of wheat worldwide, and can cause Fusarium head blight (FHB). F. graminearum infection and mycotoxin production mainly present in wheat and can be influenced by environmental factors and wheat cultivars. The objectives of this study were to examine the effect of wheat cultivars and interacting conditions of temperature and water activity (aw) on mycotoxin production by two strains of F. graminearum and investigate the response mechanisms of different wheat cultivars to F. graminearum infection. In this regard, six cultivars of wheat spikes under field conditions and three cultivars of post-harvest wheat grains under three different temperature conditions combined with five water activity (aw) conditions were used for F. graminearum infection in our studies. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis showed significant differences in the concentration of Fusarium mycotoxins deoxynivalenol (DON) and its derivative deoxynivalenol-3-glucoside (D3G) resulting from wheat cultivars and environmental factors. Transcriptome profiles of wheat infected with F. graminearum revealed the lower expression of disease defense-factor-related genes, such as mitogen-activated protein kinases (MAPK)-encoding genes and hypersensitivity response (HR)-related genes of infected Annong 0711 grains compared with infected Sumai 3 grains. These findings demonstrated the optimal temperature and air humidity resulting in mycotoxin accumulation, which will be beneficial in determining the conditions of the relative level of risk of contamination with FHB and mycotoxins. More importantly, our transcriptome profiling illustrated differences at the molecular level between wheat cultivars with different FHB resistances, which will lay the foundation for further research on mycotoxin biosynthesis of F. graminearum and regulatory mechanisms of wheat to F. graminearum.

Biodegradation of Fumonisins by the Consecutive Action of a Fusion Enzyme.

Fumonisins (FBs) are toxic mycotoxins that commonly exist in food and feed. FBs can induce many aspects of toxicity, leading to adverse effects on human and animal health; therefore, investigating methods to reduce fumonisin contamination is necessary. In our study, we generated a recombinant fusion enzyme called FUMDI by linking the carboxylesterase gene (fumD) and the aminotransferase gene (fumI) by overlapping polymerase chain reaction (PCR). The fusion enzyme FUMDI was successfully, secretively expressed in the host Pichia pastoris (P. pastoris) GS115, and its expression was optimized. Our results demonstrated that the fusion enzyme FUMDI had high biodegradation activity of fumonisin B1 (FB1) and other common FBs, such as fumonisin B2 (FB2) and fumonisin B3 (FB3), and almost completely degraded 5 µg/mL of each toxin within 24 h. We also found that FUMDI enzyme and its reaction products had no negative effect on cell viability and did not induce cell apoptosis, oxidative stress, or endoplasmic reticulum (ER) stress in a human gastric epithelial cell line (GES-1). The results indicated that these FBs degradation products cannot have adverse effects in a cell model. In conclusion, a safe and efficient fumonisin-degrading enzyme was discovered, which could be a new a technical method for hazard control of FBs in the future.

Bacillus velezensis A2 Inhibited the Cecal Inflammation Induced by Zearalenone by Regulating Intestinal Flora and Short-Chain Fatty Acids.

Zearalenone (ZEA) as an estrogen-like mycotoxin can cause the inflammatory injury of the cecum. How to reduce the harm that ZEA causes to humans and animals is a current concern for researchers. In this study, we aimed to ascertain whether Bacillus velezensis A2 (A2) could alleviate injury caused by ZEA by regulating the intestinal flora and the content of short chain fatty acids in the cecum among mice. Our results showed that Bacillus velezensis A2 improved the fold height, myometrial thickness, and crypt depth of the cecum induced by ZEA. Enzyme-linked immunosorbent assay and Western blotting results showed that A2 could decrease the ZEA-induced increase in expression levels of IL-2, IL-6, IFN-γ, TNF-α, and FC. Studies also showed that A2 increased the content of SCFA in the cecum which was decreased by ZEA. The microbial communities in the cecum were changed when given ZEA or A2. A2 was found to greatly reduce the ZEN-induced increase in the relative abundance of p_Actinobacteria, p_Protebacteria, o_Coriobacteriales, g_Anaerotruncus, g_Pseudoflavonifractor, g_Lachnoclostridium, g_Enterorhabdus, and f_Oscillospiraceae, and increase the ZEN-induced decrease in the relative abundance of f_Coriobacteriales. Results indicated that Bacillus velezensis A2 can largely ameliorate the intestinal inflammatory injury induced by ZEA in mice by regulating the microflora and short chain fatty acids content.

Cellobiose inhibits the release of deoxynivalenol from transformed deoxynivalenol-3-glucoside from Lactiplantibacillus plantarum.

The masked mycotoxin deoxynivalenol-3-glucoside (D3G) has been reported to be a detoxification product in plants, but can be hydrolyzed into its toxic precursor, deoxynivalenol (DON). Herein, we reported that Lactiplantibacillus plantarum (L. plantarum) NMM.1, isolated from Inner Mongolia raw milk, can efficiently transform D3G to DON in a short time. The global transcriptome microarray profiling of L. plantarum NMM.1 revealed differential expression of genes related to the phosphotransferase system (PTS) when D3G was used as the sole carbohydrate source. By adding an exogenous carbon source, we found that cellobiose efficiently inhibited the conversion of D3G into its precursor toxin by L. plantarum NMM.1. Overall, substrate depletion studies, transcriptome analysis, and carbohydrate intervention studies of L. plantarum NMM.1 suggested that cellobiose could be used to prevent the transformation of D3G into its free native DON by L. plantarum, thereby preventing harm to the human body.

Effects of Fumonisin B and Hydrolyzed Fumonisin B on Growth and Intestinal Microbiota in Broilers.

Fumonisins are mainly produced by Fusarium verticillioides and proliferatum, which causes a variety of toxicities in humans and animals, including fumonisin Bs (FBs) as the main form. After they are metabolized by plants or microorganisms, modified fumonisins are difficult to detect by conventional methods, which result in an underestimation of their contamination level. Fumonisins widely contaminate maize and maize products, especially in broiler feed. As an economically important food, broilers are often adversely affected by mycotoxins, leading to food safety hazards and high economic losses. However, there are few studies regarding the adverse effects of FBs on broiler growth and health, especially modified FBs. Our data shows that after exposure to FBs or hydrolyzed fumonisin Bs (HFBs), the body weight and tissue weight of broilers decreased significantly, especially the testes. Moreover, they significantly affect the intestinal microbiota and the relative abundance of bacteria from phylum-to-species levels, with the differentially affected bacteria mainly belonging to Firmicutes and Proteobacteria. Our findings suggest that both the parent and hydrolyzed FBs could induce growth retardation, tissue damage and the imbalance of intestinal microbiota in broilers. This indicated that the harmful effects of HFBs cannot be ignored during food safety risk assessment.

Development of a Novel UPLC-MS/MS Method for the Simultaneous Determination of 16 Mycotoxins in Different Tea Categories.

The contamination of potential mycotoxins in tea production and consumption has always been a concern. However, the risk monitoring on multiple mycotoxins remains a challenge by existing methods due to the high cost and complex operation in tea matrices. This research has developed a simple ultra-performance liquid chromatography-tandem mass spectrometry strategy based on our homemade purification column, which can be applied in the detections of mycotoxins in complex tea matrices with high-effectively purifying and removing pigment capacity for 16 mycotoxins. The limits of detection and the limits of quantification were in the ranges of 0.015~15.00 and 0.03~30.00 µg·kg-1 for 16 mycotoxins, respectively. Recoveries from mycotoxin-fortified tea samples (0.13~1200 µg·kg-1) in different tea matrices ranged from 61.27 to 118.46%, with their relative standard deviations below 20%. Moreover, this method has been successfully applied to the analysis and investigation of the levels of 16 mycotoxins in major categories of tea and the monitoring of multiple mycotoxins in processed samples of ripened Pu-erh. In conclusion, the proposed strategy is simple, effective, time-saving, and low-cost for the determination of a large number of tea samples.

MicroRNA regulates the toxicological mechanism of four mycotoxins in vivo and in vitro.

Mycotoxins can cause body poisoning and induce carcinogenesis, often with a high mortality rate. Therefore, it is of great significance to seek new targets that indicate mycotoxin activity and to diagnose and intervene in mycotoxin-induced diseases in their early stages. MicroRNAs (miRNAs) are physiological regulators whose dysregulation is closely related to the development of diseases. They are thus important markers for the occurrence and development of diseases. In this review, consideration is given to the toxicological mechanisms associated with four major mycotoxins (ochratoxin A, aflatoxin B1, deoxynivalenol, and zearalenone). The roles that miRNAs play in these mechanisms and the interactions between them and their target genes are explained, and summarize the important role of histone modifications in their toxicity. As a result, the ways that miRNAs are regulated in the pathogenicity signaling pathways are revealed which highlights the roles played by miRNAs in preventing and controlling the harmful effects of the mycotoxins. It is hoped that this review will provide a theoretical basis for the prevention and control of the damage caused by these mycotoxins.

LC-MS/MS Analysis of Fumonisin B1, B2, B3, and Their Hydrolyzed Metabolites in Broiler Chicken Feed and Excreta.

An accurate, reliable, and specific method was developed for the quantitative determination of fumonisins B1, B2, B3, and their hydrolyzed metabolites, HFB1, HFB2, and HFB3, in broiler chicken feed and excreta using ultra-performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS). The samples were extracted and diluted for the determination of parent fumonisins. Another portion of the extracted samples was alkaline-hydrolyzed and cleaned using a strong anionic exchange adsorbent (MAX) for the determination of hydrolyzed fumonisins. Chromatographic separation was performed on a CORTECS C18 column (2.1 mm × 100 mm, 1.6 µm) using 0.2% formic acid aqueous solution and methanol with 0.2% formic acid as the mobile phase under gradient elution. The six fumonisins, FB1, FB2, FB3, HFB1, HFB2, and HFB3, were analyzed by tandem mass spectrometry using multiple-reaction monitoring (MRM) mode. The six fumonisins showed good linearity, with relative coefficients of r > 0.99. The limits of quantitation (LOQs) were 160 µg/kg. At the low, medium, and high spiked levels, the recovery of fumonisins in chicken feed and excreta ranged from 82.6 to 115.8%, with a precision (RSD) of 3.9-18.9%. This method was successfully applied to investigate the migration and transformation of fumonisins in broiler chickens.