Potential fungicidal and antiaflatoxigenic effects of cinnamon essential oils on Aspergillus flavus inhabiting the stored wheat grains.

Wheat is one of the essential crops for the human and animal nutrition, however, contamination with aflatoxigenic fungi, due to the improper storage conditions and high humidity, was the main global threats. So, preventing the growth of aflatoxigenic fungi in stored wheat grains, by using different essential oils was the main objective of this work. Aspergillus flavus EFBL-MU12 PP087400, EFBL-MU23 PP087401 and EFBL-MU36 PP087403 isolates were the most potent aflatoxins producers inhabiting wheat grains. The effect of storage conditions of wheat grains "humidity, temperature, incubation period, and pH" on growth of A. flavus, was assessed by the response surface methodology using Plackett-Burman design and FCCD. The highest yield of aflatoxins EFBL-MU12 B1 and B2 by A. flavus grown on wheat grains were 145.3 and 7.6 µg/kg, respectively, at incubation temperature 35°C, 16% moisture contents, initial pH 5.0, and incubated for 14 days. The tested oils had a powerful antifungal activity for the growth and aflatoxins production by A. flavus in a concentration-dependent manner. Among these oils, cinnamon oil had the highest fungicidal activity for A. flavus at 0.125%, with about 85-90 % reduction to the aflatoxins B1 and B2, conidial pigmentation and chitin contents on wheat grains. From the SEM analysis, cinnamon oils had the most deleterious effect on A. flavus with morphological aberrations to the conidial heads, vegetative mycelia, alteration in conidiophores identity, hyphae shrank, and winding. To emphasize the effect of the essential oils on the aflatoxins producing potency of A. flavus, the molecular expression of the aflatoxins biosynthetic genes was estimated by RT-qPCR. The molecular expression of nor-1, afLR, pKsA and afLJ genes was suppressed by 94-96%, due to cinnamon oil at 0.062% compared to the control. Conclusively, from the results, cinnamon oils followed by the peppermint oils displayed the most fungicidal activity for the growth and aflatoxins production by A. flavus grown on wheat grains.

Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Non-destructive SPE-UPLC-based Quantification of Aflatoxins and Stilbenoid Phytoalexins in Single Peanut (Arachis spp.) Seeds.

Aflatoxins are highly carcinogenic secondary metabolites of some fungal species, particularly Aspergillus flavus. Aflatoxins often contaminate economically important agricultural commodities, including peanuts, posing a high risk to human and animal health. Due to the narrow genetic base, peanut cultivars demonstrate limited resistance to fungal pathogens. Therefore, numerous wild peanut species with tolerance to Aspergillus have received substantial consideration by scientists as sources of disease resistance. Exploring plant germplasm for resistance to aflatoxins is difficult since aflatoxin accumulation does not follow a normal distribution, which dictates the need for the analyses of thousands of single peanut seeds. Sufficiently hydrated peanut (Arachis spp.) seeds, when infected by Aspergillus species, are capable of producing biologically active stilbenes (stilbenoids) that are considered defensive phytoalexins. Peanut stilbenes inhibit fungal development and aflatoxin production. Therefore, it is crucial to analyze the same seeds for peanut stilbenoids to explain the nature of seed resistance/susceptibility to the Aspergillus invasion. None of the published methods offer single-seed analyses for aflatoxins and/or stilbene phytoalexins. We attempted to fulfill the demand for such a method that is environment-friendly, uses inexpensive consumables, and is sensitive and selective. In addition, the method is non-destructive since it uses only half of the seed and leaves the other half containing the embryonic axis intact. Such a technique allows germination and growth of the peanut plant to full maturity from the same seed used for the aflatoxin and stilbenoid analysis. The integrated part of this method, the manual challenging of the seeds with Aspergillus, is a limiting step that requires more time and labor compared to other steps in the method. The method has been used for the exploration of wild Arachis germplasm to identify species resistant to Aspergillus and to determine and characterize novel sources of genetic resistance to this fungal pathogen.

Chitin Deacetylase Homologous Gene cda Contributes to Development and Aflatoxin Synthesis in Aspergillus flavus.

The fungal cell wall serves as the primary interface between fungi and their external environment, providing protection and facilitating interactions with the surroundings. Chitin is a vital structural element in fungal cell wall. Chitin deacetylase (CDA) can transform chitin into chitosan through deacetylation, providing various biological functions across fungal species. Although this modification is widespread in fungi, the biological functions of CDA enzymes in Aspergillus flavus remain largely unexplored. In this study, we aimed to investigate the biofunctions of the CDA family in A. flavus. The A. flavus genome contains six annotated putative chitin deacetylases. We constructed knockout strains targeting each member of the CDA family, including Δcda1, Δcda2, Δcda3, Δcda4, Δcda5, and Δcda6. Functional analyses revealed that the deletion of CDA family members neither significantly affects the chitin content nor exhibits the expected chitin deacetylation function in A. flavus. However, the Δcda6 strain displayed distinct phenotypic characteristics compared to the wild-type (WT), including an increased conidia count, decreased mycelium production, heightened aflatoxin production, and impaired seed colonization. Subcellular localization experiments indicated the cellular localization of CDA6 protein within the cell wall of A. flavus filaments. Moreover, our findings highlight the significance of the CBD1 and CBD2 structural domains in mediating the functional role of the CDA6 protein. Overall, we analyzed the gene functions of CDA family in A. flavus, which contribute to a deeper understanding of the mechanisms underlying aflatoxin contamination and lay the groundwork for potential biocontrol strategies targeting A. flavus.

The potential of antifungal peptides derived from Lactiplantibacillus plantarum WYH for biocontrol of Aspergillus flavus contamination.

Aspergillus flavus is a notorious fungus that contaminates food crops with toxic aflatoxins, posing a serious threat to human health and the agricultural economy. To overcome the inadequacy of traditional control methods and meet consumer preferences for natural-sources additives, there is an urgent demand for novel biocontrol agents that are safe and efficient. This study aims to investigate the antifungal properties of a novel antifungal agent derived from the biologically safe Lactiplantibacillus plantarum WYH. Firstly, antifungal peptides (AFPs) with a molecular weight of less than 3kD, exhibiting remarkable temperature stability and effectively retarding fungal growth in a dose-dependent manner specifically against A. flavus, were concentrated from the fermentation supernatant of L. plantarum WYH and were named as AFPs-WYH. Further analysis demonstrated that AFPs-WYH might exert antifungal effects through the induction of oxidative stress, disruption of mitochondrial function, alteration of membrane permeability, and cell apoptosis in A. flavus. To further validate our findings, a transcriptomics analysis was conducted on A. flavus treated with 2 and 5 mg/mL of AFPs-WYH, which elucidated the potential effect of AFPs-WYH administration on the regulation of genes involved in impairing fungal development and preventing aflatoxin biosynthesis pathways. Overall, AFPs-WYH reduced the A. flavus proliferation and affected the AFB1 biosynthesis, exhibiting a promising potential for food industry applications as a biopreservative and biocontrol agent.

Antifungal mechanisms investigation of lactic acid bacteria against Aspergillus flavus: through combining microbial metabolomics and co-culture system.

AIMS: To develop antifungal lactic acid bacteria (LAB) and investigate their antifungal mechanisms against Aspergillus flavus in aflatoxin (AF) production. METHODS AND RESULTS: We isolated 179 LABs from cereal-based fermentation starters and investigated their antifungal mechanism against A. flavus through liquid chromatography-mass spectrometry and co-culture analysis techniques. Of the 179 isolates, antifungal activity was identified in Pediococcus pentosaceus, Lactobacillus crustorum, and Weissella paramesenteroides. These LABs reduced AF concentration by (i) inhibiting mycelial growth, (ii) binding AF to the cell wall, and (iii) producing antifungal compounds. Species-specific activities were also observed, with P. pentosaceus inhibiting AF production and W. paramesenteroides showing AF B1 binding activity. In addition, crucial extracellular metabolites for selecting antifungal LAB were involved in the 2',3'-cAMP-adenosine and nucleoside pathways. CONCLUSIONS: This study demonstrates that P. pentosaceus, L. crustorum, and W. paramesenteroides are key LAB strains with distinct antifungal mechanisms against A. flavus, suggesting their potential as biological agents to reduce AF in food materials.

Efficacy of atoxigenic Aspergillus flavus from southern China as biocontrol agents against aflatoxin contamination in corn and peanuts.

Aspergillus flavus is a ubiquitous facultative pathogen that routinely infects important crops leading to formation of aflatoxins during crop development and after harvest. Corn and peanuts in warm and/or drought-prone regions are highly susceptible to aflatoxin contamination. Controlling aflatoxin using atoxigenic A. flavus is a widely adopted strategy. However, no A. flavus genotypes are currently approved for use in China. The current study aimed to select atoxigenic A. flavus endemic to Guangxi Zhuang Autonomous Region with potential as active ingredients of aflatoxin biocontrol products. A total of 204 A. flavus isolates from corn, peanuts, and field soil were evaluated for ability to produce the targeted mycotoxins. Overall, 57.3% could not produce aflatoxins while 17.15% were incapable of producing both aflatoxins and CPA. Atoxigenic germplasm endemic to Guangxi was highly diverse, yielding 8 different gene deletion patterns in the aflatoxin and CPA biosynthesis gene clusters ranging from no deletion to deletion of both clusters. Inoculation of corn and peanuts with both an aflatoxin producer and selected atoxigenic genotypes showed significant reduction (74 to 99%) in aflatoxin B1 (AFB1) formation compared with inoculation with the aflatoxin producer alone. Atoxigenic genotypes also efficiently degraded AFB1 (61%). Furthermore, atoxigenic isolates were also highly efficient at reducing aflatoxin concentrations even when present at lower concentrations than aflatoxin producers. The use of multiple atoxigenics was not always as effective as the use of a single atoxigenic. Effective atoxigenic genotypes of A. flavus with known mechanisms of atoxigenicity are demonstrated to be endemic to Southern China. These A. flavus may be utilized as active ingredients of biocontrol products without concern for detrimental impacts that may result from introduction of exotic fungi. Field efficacy trials in the agroecosystems of Southern China are needed to determine the extent to which such products may allow the production of safer food and feed.

Fus3 Interacts with Gal83, Revealing the MAPK Crosstalk to Snf1/AMPK to Regulate Secondary Metabolic Substrates in Aspergillus flavus.

Aflatoxins (AFs), highly carcinogenic natural products, are produced by the secondary metabolism of fungi such as Aspergillus flavus. Essential for the fungi to respond to environmental changes and aflatoxin synthesis, the pheromone mitogen-activated protein kinase (MAPK) is a potential regulator of aflatoxin biosynthesis. However, the mechanism by which pheromone MAPK regulates aflatoxin biosynthesis is not clear. Here, we showed Gal83, a new target of Fus3, and identified the pheromone Fus3-MAPK signaling pathway as a regulator of the Snf1/AMPK energy-sensing pathway modulating aflatoxins synthesis substrates. The screening for Fus3 target proteins identified the ß subunit of Snf1/AMPK complexes using tandem affinity purification and multiomics. This subunit physically interacted with Fus3 both in vivo and in vitro and received phosphorylation from Fus3. Although the transcript levels of aflatoxin synthesis genes were not noticeably downregulated in both gal83 and fus3 deletion mutant strains, the levels of aflatoxin B1 and its synthesis substrates and gene expression levels of primary metabolizing enzymes were significantly reduced. This suggests that both the Fus3-MAPK and Snf1/AMPK pathways respond to energy signals. In conclusion, all the evidence unlocks a novel pathway of Fus3-MAPK to regulate AFs synthesis substrates by cross-talking with the Snf1/AMPK complexes.

How does active yeast supplementation reduce the deleterious effects of aflatoxins in Wistar rats? A radiolabeled assay and histopathological study.

The aim of this study was to investigate the mechanisms by which yeasts (Saccharomyces cerevisiae) control the toxic effects of aflatoxins, which are not yet fully understood. Radiolabeled aflatoxin B1 (AFB13H) was administered by gavage to Wistar rats fed with aflatoxin (AflDiet) and aflatoxin supplemented with active dehydrated yeast Y904 (AflDiet + Yeast). The distribution of AFB13H and its metabolites were analyzed at 24, 48 and 72 h by tracking back of the radioactivity. No significant differences were observed between the AflDiet and AflDiet + Yeast groups in terms of the distribution of labeled aflatoxin. At 72 h, for the AflDiet group the radiolabeled aflatoxin was distributed as following: feces (79.5%), carcass (10.5%), urine (1.7%), and intestine (7.4%); in the AflDiet + Yeast the following distribution was observed: feces (76%), carcass (15%), urine (2.9%), and intestine (4.9%). These values were below 1% in other organs. These findings indicate that even after 72 h considerable amounts of aflatoxins remains in the intestines, which may play a significant role in the distribution and metabolism of aflatoxins and its metabolites over time. The presence of yeast may not significantly affect this process. Furthermore, histopathological examination of hepatic tissues showed that the presence of active yeast reduced the severity of liver damage caused by aflatoxins, indicating that yeasts control aflatoxin damage through biochemical mechanisms. These findings contribute to a better understanding of the mechanisms underlying the protective effects of yeasts against aflatoxin toxicity.

Meat Starter Culture Reduces Aspergillus parasiticus Production of Aflatoxins on Meat-Based and Salami Model Media.

There is great concern about the risk posed by the consumption of food contaminated with aflatoxins (AF), produced mostly by Aspergillus strains, that can also be found in dry-fermented meat products (DFMPs). The aim of this study was to investigate the inhibitory effect of meat starter culture (SC), frequently used for fermentation in the meat industry, on A. parasiticus growth and the production of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), and sterigmatocystin (STE) on different meat-based (CMA) and salami model (SM-G) media. Incubation was carried out under optimal conditions for fungal growth and under typical conditions for ripening of DFMPs for 21 days. Reversed-phase UPLC-MS/MS analysis was performed to determine mycotoxin production. SC reduced A. parasiticus growth more on CMA than on SM-G media. AFB1 formation was inhibited on both types of SC-containing media, although SC generally had a stronger inhibitory effect on AFB1 production on CMA than on SM-G. AFB1 and AFB2 were produced on CMA, while AFB1 dominated in SM-G, AFG1, and AFG2 were not detected in any media. The results show that SC inhibited AFB1 formation of A. parasiticus on SM-G media after 21 days of incubation under typical conditions for the production of DFMPs. These results indicate the necessity to investigate AF on natural matrices in an environment that is as similar as possible to real conditions in the production of DFMPs.

The Gene vepN Regulated by Global Regulatory Factor veA That Affects Aflatoxin Production, Morphological Development and Pathogenicity in Aspergillus flavus.

Velvet (VeA), a light-regulated protein that shuttles between the cytoplasm and the nucleus, serves as a key global regulator of secondary metabolism in various Aspergillus species and plays a pivotal role in controlling multiple developmental processes. The gene vepN was chosen for further investigation through CHIP-seq analysis due to significant alterations in its interaction with VeA under varying conditions. This gene (AFLA_006970) contains a Septin-type guanine nucleotide-binding (G) domain, which has not been previously reported in Aspergillus flavus (A. flavus). The functional role of vepN in A. flavus was elucidated through the creation of a gene knockout mutant and a gene overexpression strain using a well-established dual-crossover recombinational technique. A comparison between the wild type (WT) and the ΔvepN mutant revealed distinct differences in morphology, reproductive capacity, colonization efficiency, and aflatoxin production. The mutant displayed reduced growth rate; dispersion of conidial heads; impaired cell wall integrity; and decreased sclerotia formation, colonization capacity, and aflatoxin levels. Notably, ΔvepN exhibited complete growth inhibition under specific stress conditions, highlighting the essential role of vepN in A. flavus. This study provides evidence that vepN positively influences aflatoxin production, morphological development, and pathogenicity in A. flavus.

One-pot green synthesis of ZIF-8/IgG composite for the precise orientation and protection of antibody and its application in purification and detection of aflatoxins in peanut oil.

This study presents a novel approach toward the one-pot green synthesis of ZIF-8/IgG composite, focusing on its precise orientation and protection of the anti-aflatoxins antibody. The antibody orientation is achieved through the specific binding of IgG to the Fc region of the antibody, while the antibody protection is accomplished by the structural change restriction of ZIF-8 framework to the antibody. Consequently, the antibody exhibits enhanced target capability and significantly improved tolerance to organic solvents. The ZIF-8/IgG/anti-AFT was employed for the purification and detection of AFTs by coupling with UPLC. Under optimized conditions, the recoveries of spiked AFTs in peanut oils are between 86.1% and 106.4%, with relative standard deviations (RSDs) ranging from 0.8% to 8.8%. The linearity range is 0.5-20.0 ng for AFB1 and AFG1, 0.125-5.0 ng for AFB2 and AFG2, the limit of detection is 0.1 ng for AFB1 and AFG1, 0.03 ng for AFB2 and AFG2.

Synthesis, Characterization, and Antifungal Activity of Benzimidazole-Grafted Chitosan against Aspergillus flavus.

Aspergillus flavus contamination in agriculture and food industries poses threats to human health, leading to a requirement of a safe and effective method to control fungal contamination. Chitosan-based nitrogen-containing derivatives have attracted much attention due to their safety and enhanced antimicrobial applications. Herein, a new benzimidazole-grafted chitosan (BAC) was synthesized by linking the chitosan (CS) with a simple benzimidazole compound, 2-benzimidazolepropionic acid (BA). The characterization of BAC was confirmed by Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance spectroscopy (1H and 13C NMR). Then, the efficiency of BAC against A. flavus ACCC 32656 was investigated in terms of spore germination, mycelial growth, and aflatoxin production. BAC showed a much better antifungal effect than CS and BA. The minimum inhibitory concentration (MIC) value was 1.25 mg/mL for BAC, while the highest solubility of CS (16.0 mg/mL) or BA (4.0 mg/mL) could not completely inhibit the growth of A. flavus. Furthermore, results showed that BAC inhibited spore germination and elongation by affecting ergosterol biosynthesis and the cell membrane integrity, leading to the permeabilization of the plasma membrane and leakage of intracellular content. The production of aflatoxin was also inhibited when treated with BAC. These findings indicate that benzimidazole-derived natural CS has the potential to be used as an ideal antifungal agent for food preservation.

HacA, a key transcription factor for the unfolded protein response, is required for fungal development, aflatoxin biosynthesis and pathogenicity of Aspergillus flavus.

Aspergillus flavus is a fungus notorious for contaminating food and feed with aflatoxins. As a saprophytic fungus, it secretes large amounts of enzymes to access nutrients, making endoplasmic reticulum (ER) homeostasis important for protein folding and secretion. The role of HacA, a key transcription factor in the unfolded protein response pathway, remains poorly understood in A. flavus. In this study, the hacA gene in A. flavus was knockout. Results showed that the absence of hacA led to a decreased pathogenicity of the strain, as it failed to colonize intact maize kernels. This may be due to retarded vegetable growth, especially the abnormal development of swollen tips and shorter hyphal septa. Deletion of hacA also hindered conidiogenesis and sclerotial development. Notably, the mutant strain failed to produce aflatoxin B1. Moreover, compared to the wild type, the mutant strain showed increased sensitivity to ER stress inducer such as Dithiothreitol (DTT), and heat stress. It also displayed heightened sensitivity to other environmental stresses, including cell wall, osmotic, and pH stresses. Further transcriptomic analysis revealed the involvement of the hacA in numerous biological processes, including filamentous growth, asexual reproduction, mycotoxin biosynthetic process, signal transduction, budding cell apical bud growth, invasive filamentous growth, response to stimulus, and so on. Taken together, HacA plays a vital role in fungal development, pathogenicity and aflatoxins biosynthesis. This highlights the potential of targeting hacA as a novel approach for early prevention of A. flavus contamination.

Prevalence and concentration of mycotoxins in bovine feed and feed components: A global systematic review and meta-analysis.

Mycotoxins are secondary metabolites produced by fungi and identified as contaminants in animal feed. They have potentially harmful effects, including carcinogenicity, mutagenicity, and repro-toxicity in animals and humans. As a result of climate change, there is the potential for a change in the prevalence and concentration of mycotoxins in animal feed components. This necessitates an assessment of the present and emerging threats to the food supply chain from mycotoxins. This systematic review and meta-analysis study synthesised studies on mycotoxin contamination and prevalence in cattle feed components. The studies were collected from scientific databases Web of Knowledge, Scopus, and Embase between 2011 and 2022. The meta-analysis synthesised 97 studies on the prevalence and the concentration of aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, fumonisin and T-2/HT-2 toxins in feed components. Aflatoxin was highly prevalent (59 %), with a concentration of 2.58-3.92 µg kg-1 in feed components. Ochratoxin A had a global prevalence of 31 % with a concentration of 5.56-12.41 µg kg-1. Deoxynivalenol had a global concentration of 233.17-327.73 µg kg-1 and a prevalence of 74 %. Zearalenone had a prevalence of 70 % and a concentration of 42.47-66.19 µg kg-1. The concentration and prevalence of fumonisins was 232.19-393.07 µg kg-1 and 65 %, respectively. The prevalence and concentration of T-2/HT-2 toxins were 45 % and 23.54-35.12 µg kg-1, respectively. The synthesised concentration of the mycotoxins in the overall feed components was lower than the regulated and guidance values set by the European Union. However, in a few cases, the 95th percentile exceeded these concentration values due to high levels of uncertainty attributed to lower sample size, and thus, need to be considered while conducting risk assessments. The study highlights climates and regions likely to be conducive to the emergence of mycotoxin risk, especially considering the potential influences of climate change.

Exposure assessment and risk characterisation of aflatoxins in randomly collected rice samples from local markets of Hyderabad, India.

Aflatoxin (AF) poisoning of staple foods, such as rice, is caused by fungal contamination by Aspergillus species. These AFs are genotoxic, carcinogenic and suppress the immune system. Hence, the present study was conducted to elucidate the prevalence of AF contamination in rice samples collected from local markets of Hyderabad, Telangana, India. The rice samples collected were analysed for AF by using HPLC-fluorescence detection (HPLC-FLD). Based on AF contamination levels and dietary intake of rice, the health risk was assessed by the margin of exposure (MOE) and liver cancer risk in adults, adolescence and children. The percentage detected contamination with AFB1 and AFB2 of rice samples was 54% and 34%, with the concentration ranging between 0-20.35 µg/kg and 0-1.54 µg/kg, respectively. Three rice samples exceeded the Food Safety and Standards Authority of India (FSSAI) total AF acceptable limit of 15 µg/kg. The average MOE values were 53.73, 50.58 and 35.69 (all <10,000) for adults, adolescence and children, respectively. The average liver cancer risk associated with rice consumption in the population of Hyderabad was found to be 0.27, 0.28 and 0.40 hepatocellular carcinoma (HCC) cases/year/100,000 individuals in adults, adolescence and children, respectively. This study revealed an adverse health risk to population of Hyderabad due to consumption of AF contaminated rice.

The occurrence of aflatoxins and labelling compliance of locally produced za'atar mix products.

Za'atar mix products are mainly composed of the dried and ground leaves and/or blossoms of wild and cultivated plant species (Origanum, Thymbra, Thymus, and Satureja) with the addition of condiments. The aim of this study was to evaluate the occurrence of aflatoxins, chemical composition (carbohydrates, fibre, fat, protein, moisture, ash, and acid contents), mineral content (Na, Ca, and K), and colour traits (L*a*b*) in relation to food label and food standards compliance. Measured and labelled fat content did not agree for approximately 91% of the samples. There was also no agreement between the measured and labelled fibre contents. The total content of aflatoxins in the tested samples ranged from 2 to 63.7 ng g-1. Eleven (69%) of the 16 analysed products had total aflatoxins higher than the maximum permitted limit of the European Commission. The KAS and LAZ products had significantly lighter colour (the highest L* values), while the ALAQ product had the darkest colour (lowest L* value). The range of sodium content in the tested products was 105.1-1425.3 mg/100 g. In conclusion, za'atar mix products that are available in local markets do not have accurate nutritional labelling information, and the occurrence of aflatoxins was very high. Further studies are needed to evaluate the reasons for these quality defects.

AflaILVB/G/I and AflaILVD are involved in mycelial production, aflatoxin biosynthesis, and fungal virulence in Aspergillus flavus.

Aflatoxins (AFs) are produced by fungi such as Aspergillus flavus and A. parasiticus and are one of the most toxic mycotoxins found in agricultural products and food. Aflatoxin contamination, which requires the control of A. flavus, remains problematic because of the lack of effective strategies and the exploration of new compounds that can inhibit A. flavus growth and mycotoxin production is urgently required to alleviate potential deleterious effects. Acetohydroxy acid synthase (AHAS) and dihydroxy acid dehydratase are important enzymes in the biosynthetic pathways of branched-chain amino acids (BCAAs), including isoleucine, leucine, and valine. Enzymes involved in BCAA biosynthesis are present in bacteria, plants, and fungi, but not in mammals, and are therefore, attractive targets for antimicrobial and herbicide development. In this study, we characterized AflaILVB/G/I and AflaILVD, which encode the catalytic and regulatory subunits of AHAS and dihydroxy acid dehydratase, from the pathogenic fungus Aspergillus flavus. The AflaILVB/G/I and AflaILVD deletion mutant grew slower and produced smaller colonies than the wild-type strain when grown on glucose minimal medium, potato dextrose agar, and yeast extract medium for three days at 28°C, and disruption of AflaILVB/G/I caused a significant reduction in conidia production when grown on all kinds of media. Cellular stress assays determined that all strains were sensitive to H2O2. Importantly, the pathogenicity and aflatoxin production were affected when AflaILVB/G/I and AflaILVD were knocked out, particularly AflaILVB/G/I. A series of genes that encoded enzymes involved in aflatoxin synthesis were downregulated, meaning that the knockout of AflaILVB/G/I influenced aflatoxin synthesis in A. flavus strain WT. Collectively, our results demonstrate the potential value of antifungals targeting AflaILVB/G/I in A. flavus.

Structure of Aspergillus flavus populations associated with maize in Greece, Spain, and Serbia: Implications for aflatoxin biocontrol on a regional scale.

Aspergillus flavus is the most frequently identified producer of aflatoxins. Non-aflatoxigenic members of the A. flavus L strains are used in various continents as active ingredients of bioprotectants directed at preventing aflatoxin contamination by competitive displacement of aflatoxin producers. The current research examined the genetic diversity of A. flavus L strain across southern Europe to gain insights into the population structure and evolution of this species and to evaluate the prevalence of genotypes closely related to MUCL54911, the active ingredient of AF-X1. A total of 2173L strain isolates recovered from maize collected across Greece, Spain, and Serbia in 2020 and 2021 were subjected to simple sequence repeat (SSR) genotyping. The analysis revealed high diversity within and among countries and dozens of haplotypes shared. Linkage disequilibrium analysis indicated asexual reproduction and clonal evolution of A. flavus L strain resident in Europe. Moreover, haplotypes closely related to MUCL54911 were found to belong to the same vegetative compatibility group (VCG) IT006 and were relatively common in all three countries. The results indicate that IT006 is endemic to southern Europe and may be utilized as an aflatoxin mitigation tool for maize across the region without concern for potential adverse impacts associated with the introduction of an exotic microorganism.

Development and validation of a liquid chromatography tandem mass spectrometry method for the determination of 10 mycotoxins in beer of the Chinese market and exposure estimate.

Mycotoxins are important risk factors in beer. In this study, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to determine 10 mycotoxins in beer within 6 min. The method is fast, efficient, and has a simple and quick sample preparation. Validation was conducted based on the performance standards specified in Commission Decision 657/2002/EC, and the results demonstrated excellent linearity (R2 > 0.99), repeatability (RSD < 5 %), quantification limits (0.005-20.246 µg/L), and recovery rates (77 %-118 %). The prevalence of the 10 mycotoxins in 96 beers purchased from the Chinese market was analyzed, and the exposure of the Chinese population to mycotoxins through beer consumption was assessed. Deoxynivalenol (DON) was detected in 93.75 % of the beers, and the incidence of fumonisins (FBs) and zearalenone (ZEN) exceeded 50 %. Beer intake contributed significantly to the exposure of aflatoxins (AFs) and DON, especially in males. Correlation analysis between mycotoxin content in beer, raw materials, and the brewing process revealed that the brewing process significantly affected the content of DON (P < 0.001), while auxiliary materials also had a significant impact on the content of FBs and DON (P < 0.001). This study holds great significance in producing higher quality and safer beer.