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.

AaTAS1 and AaMFS1 Genes for Biosynthesis or Efflux Transport of Tenuazonic Acid and Pathogenicity of Alternaria alternata.

Taking tenuazonic acid (TeA) synthetase 1 (TAS1) in Pyricularia oryzae as a reference, the homolog AaTAS1 was first anchored in Alternaria alternata via de novo sequencing. Subsequently, AaMFS1, as a major facilitator superfamily (MFS) protein-encoding gene in the adjacent upstream region, was followed with interest. As hypothesized, AaTAS1 is required for TeA biosynthesis, while AaMFS1 is an efflux pump for the transmembrane transport of TeA. Comparatively, the TeA yield of ΔAaTAS1 and ΔAaMFS1 dropped significantly compared with that of the wild-type strain. Specifically, the A domain of AaTAS1 catalyzed the start of TeA biosynthesis in vitro. Simultaneously, the pathogenicity of ΔAaTAS1 was also significantly decreased. Transcriptome analysis confirmed the abovementioned consistency between the TeA-producing phenotypes and related gene expression. Moreover, the proteins AaTAS1 and AaMFS1 were found present in the cytoplasm, plasma membrane, and intracellular membrane system, respectively, by fluorescence localization. Namely, AaTAS1 was responsible for the biosynthesis of TeA, and AaMFS1 was responsible for the efflux transport of TeA. Certainly, AaTAS1 indirectly regulated the expression of AaMFS1 through the level of synthetic TeA. Overall, data on the novel AaTAS1 and AaMFS1 genes mainly contribute to theoretical advances in mycotoxin biosynthesis and the pathogenicity of phytopathogens to agricultural foods.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Mycotoxin deoxynivalenol affects myoblast differentiation via downregulating cytoskeleton and ECM-integrin-FAK-RAC-PAK signaling pathway.

As a common mycotoxin, deoxynivalenol (DON) contaminates cereal grains and feed in field or during processing and storage. DON elicits a spectrum of adverse effects in animals including anorexia and growth retardation. Especially, the presence of DON has also been detected in muscle, suggesting that DON may has the potential to affect the development of muscle. However, the relevant research is very rare and the molecular mechanism remains unclear. Myoblasts differentiation into multinucleated myotubes is one of the crucial steps of skeletal muscle development. In the present study, we investigated the effects of DON on differentiation of myoblasts using murine C2C12 cells model. The results indicated that DON dose-dependent inhibited the formation of myotubes in C2C12 cells. After performing omics techniques, a total of 149 differentially expressed genes were identified. The expression of cytoskeleton proteins and extracellular matrix (ECM) proteins were downregulated by DON. Furthermore, DON significantly downregulated the expression of integrin αv and integrin ß5, leading to inhibition of the ECM-integrin receptor interaction. The focal adhesion kinase (FAK) and phosphorylated forms, ras-related C3 botulinum toxin substrate (RAC) and p21-activated kinases 1 (PAK1) were also downregulated by DON. Taken together, our findings suggest that DON has the potent to affect the differentiation of myoblasts via downregulating of cytoskeleton and ECM-integrin-FAK-RAC-PAK signaling pathway.

Evaluation of the Individual and Combined Toxicity of Fumonisin Mycotoxins in Human Gastric Epithelial Cells.

Fumonisin contaminates food and feed extensively throughout the world, causing chronic and acute toxicity in human and animals. Currently, studies on the toxicology of fumonisins mainly focus on fumonisin B1 (FB1). Considering that FB1, fumonisin B2 (FB2) and fumonisin B3 (FB3) could coexist in food and feed, a study regarding a single toxin, FB1, may not completely reflect the toxicity of fumonisin. The gastrointestinal tract is usually exposed to these dietary toxins. In our study, the human gastric epithelial cell line (GES-1) was used as in vitro model to evaluate the toxicity of fumonisin. Firstly, we found that they could cause a decrease in cell viability, and increase in membrane leakage, cell death and the induction of expression of markers for endoplasmic reticulum (ER) stress. Their toxicity potency rank is FB1 > FB2 >> FB3. The results also showed that the synergistic effect appeared in the combinations of FB1 + FB2 and FB1 + FB3. Nevertheless, the combinations of FB2 + FB3 and FB1 + FB2 + FB3 showed a synergistic effect at low concentration and an antagonistic effect at high concentration. We also found that myriocin (ISP-1) could alleviate the cytotoxicity induced by fumonisin in GES-1 cells. Finally, this study may help to determine or optimize the legal limits and risk assessment method of mycotoxins in food and feed and provide a potential method to block the fumonisin toxicity.

PPIM: A Protein-Protein Interaction Database for Maize.

Maize (Zea mays) is one of the most important crops worldwide. To understand the biological processes underlying various traits of the crop (e.g. yield and response to stress), a detailed protein-protein interaction (PPI) network is highly demanded. Unfortunately, there are very few such PPIs available in the literature. Therefore, in this work, we present the Protein-Protein Interaction Database for Maize (PPIM), which covers 2,762,560 interactions among 14,000 proteins. The PPIM contains not only accurately predicted PPIs but also those molecular interactions collected from the literature. The database is freely available at http://comp-sysbio.org/ppim with a user-friendly powerful interface. We believe that the PPIM resource can help biologists better understand the maize crop.

Simultaneous Raising of Rabbit Monoclonal Antibodies to Fluoroquinolones with Diverse Recognition Functionalities via Single Mixture Immunization.

Highly specific monoclonal and polyclonal antibodies are the key components in a diverse set of immunoassay applications, from research work to routine monitoring and analysis. In the current manuscript, combinatorial strategies for a single mixture immunization, screening and rabbit hybridoma cell technology were described. Fluoroquinolones (FQs) drugs were chosen as representative analytes. Six FQs were conjugated with bovine serum albumin and used as immunogens for subsequent immunization, while a mixture of all was injected for coimmunization. The hybridomas obtained against the individual and multiple FQs were used for the production of diverse varieties of rabbit monoclonal antibodies (RabMAbs) against the target analytes. As was proven by indirect competitive ELISA and quantitative lateral flow immunoassay, this approach opens a new way for simultaneously obtaining functional monoclonal antibodies which are capable of recognizing both individual and multiple analytes in a single preparation circle. This addresses various needs of different monitoring regulations as analytical methodology advances.

Multi-mycotoxin analysis of animal feed and animal-derived food using LC-MS/MS system with timed and highly selective reaction monitoring.

Mycotoxins have the potential to enter the human food chain through carry-over of contaminants from feed into animal-derived products. The objective of the study was to develop a reliable and sensitive method for the analysis of 30 mycotoxins in animal feed and animal-derived food (meat, edible animal tissues, and milk) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the study, three extraction procedures, as well as various cleanup procedures, were evaluated to select the most suitable sample preparation procedure for different sample matrices. In addition, timed and highly selective reaction monitoring on LC-MS/MS was used to filter out isobaric matrix interferences. The performance characteristics (linearity, sensitivity, recovery, precision, and specificity) of the method were determined according to Commission Decision 2002/657/EC and 401/2006/EC. The established method was successfully applied to screening of mycotoxins in animal feed and animal-derived food. The results indicated that mycotoxin contamination in feed directly influenced the presence of mycotoxin in animal-derived food. Graphical abstract Multi-mycotoxin analysis of animal feed and animal-derived food using LC-MS/MS.

Multiplex lateral flow immunoassay for mycotoxin determination.

A new lateral flow immunoassay (LFA) is proposed for qualitative and/or semiquantitative determination of aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON), and their analogues (AFs, ZEAs, DONs) in cereal samples. Each of the mycotoxin specific antibody was class specific and there was no cross reactivity to other groups of compounds. The visual limits of detection (vLOD) of the strip were 0.03, 1.6, and 10 µg/kg for AFB1, ZEA and DON, respectively. The calculated limits of detection (cLOD) were 0.05, 1, and 3 µg/kg, respectively. Meanwhile the cutoff values were achieved at 1, 50, and 60 µg/kg for AFB1, ZEA and DON, respectively. Recoveries ranged from 80% to 122% and RSD from 5% to 20%. Both the vLOD and cLOD for the three mycotoxins were lower than the EU maximum levels. Analysis of naturally contaminated maize samples resulted in a good agreement between the multiplex LFA and LC-MS/MS (100% for DONs and AFs, and 81% for ZEAs). Careful analysis of the results further explained the general overestimation of LFA compared to chromatographic methods for quantification of mycotoxins.

A quick, easy, cheap, effective, rugged, and safe sample pretreatment and liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of 33 mycotoxins in Lentinula edodes.

Lentinula edodes, one of the most cultivated edible fungi in the world, are usually neglected for mycotoxins contamination due to the initial thinking of its resistance to mycotoxingenic molds. In the present study, a sensitive and reliable liquid chromatography with tandem mass spectrometry method was developed for the simultaneous quantification of 33 mycotoxins in L. edodes. Targeted mycotoxins were extracted using a quick, easy, cheap, effective, rugged, and safe procedure without any further clean-up step, and analyzed by liquid chromatography with tandem mass spectrometry on an Agilent Poroshell 120 EC-C18 column (100 × 3 mm, 2.7 µm) with a linear gradient elution program using water containing 5 mM ammonium acetate and methanol as the mobile phase. After validation by determining linearity (R(2) > 0.99), sensitivity (LOQ ≤ 20 ng/kg), recovery (73.6-117.9%), and precision (0.8-19.5%), the established method has been successfully applied to reveal the contamination states of various mycotoxins in L. edodes. Among the 30 tested samples, 22 were contaminated by various mycotoxins with the concentration levels ranging from 3.3-28,850.7 µg/kg, predicting that the edible fungus could be infected by the mycotoxins-producing fungi. To the best of our knowledge, this is the first report about real mycotoxins contamination in L. edodes.

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.

Development of a new rabbit monoclonal antibody and its based competitive indirect enzyme-linked immunosorbent assay for rapid detection of sulfonamides.

BACKGROUND: Monoclonal antibodies generally obtained through the classic mouse hybridoma system were requisite for the establishment of various immunoassays. In this study, a new rabbit monoclonal antibody (RabMAb) against sulfonamides (SAs) was first produced via hybridoma technique in rabbit. The related enzyme-linked immunosorbent assay (ELISA) was then developed and applied to real sample analysis. RESULTS: A sensitive competitive indirect ELISA method based on a novel RabMAb for rapid detection of sulfonamides was first established. The obtained half-maximum inhibition concentration (IC(50)) values for four SAs were all below 10 ng mL(-1) , with 0.68 ng mL(-1) sulfathiazole (STZ), 1.11 ng mL(-1) sulfadiazine (SD), 1.15 ng mL(-1) sulfapyridine (SP) and 5.27 ng mL(-1) sulfamethoxazole (SMX). Desirable recoveries when detecting different spiked swine urine and milk samples were achieved ranging from 92.6% to 104.3% and from 61.1% to 81.6%, respectively. CONCLUSION: The proposed immunoassay with the newly developed RabMAb is capable of detection of four SAs (STZ, SD, SP and SMX) with proven satisfactory performance and is applicable for routine large-scale analysis in practical uses.

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.

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.

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.

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.

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.

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.

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.