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.

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.

Potential of Trichoderma spp. for Biocontrol of Aflatoxin-Producing Aspergillus flavus.

The inhibitory action of 20 antagonistic Trichoderma isolates against the aflatoxigenic isolate A. flavus ITEM 9 (Af-9) and their efficacy in reducing aflatoxin formation in vitro were examined. Production of metabolites with inhibitory effect by the Trichoderma isolates was also investigated. Antagonistic effect against Af-9 was assessed by inhibition of radial growth of the colonies and by fungal interactions in dual confrontation tests. A total of 8 out of 20 isolates resulted in a significant growth inhibition of 3-day-old cultures of Af-9, ranging from 13% to 65%. A total of 14 isolates reduced significantly the aflatoxin B1 (AfB1) content of 15-day-old Af-9 cultures; 4 were ineffective, and 2 increased AfB1. Reduction of AfB1 content was up to 84.9% and 71.1% in 7- and 15-day-old cultures, respectively. Since the inhibition of Af-9 growth by metabolites of Trichoderma was not necessarily associated with inhibition of AfB1 production and vice versa, we investigated the mechanism of reduction of AfB1 content at the molecular level by examining two strains: one (T60) that reduced both growth and mycotoxin content; and the other (T44) that reduced mycotoxin content but not Af-9 growth. The expression analyses for the two regulatory genes aflR and aflS, and the structural genes aflA, aflD, aflO and aflQ of the aflatoxin biosynthesis cluster indicated that neither strain was able to downregulate the aflatoxin synthesis, leading to the conclusion that the AfB1 content reduction by these Trichoderma strains was based on other mechanisms, such as enzyme degradation or complexation. Although further studies are envisaged to identify the metabolites involved in the biocontrol of A. flavus and prevention of aflatoxin accumulation, as well as for assessment of the efficacy under controlled and field conditions, Trichoderma spp. qualify as promising agents and possible alternative options to other biocontrol agents already in use.

Fus3, as a Critical Kinase in MAPK Cascade, Regulates Aflatoxin Biosynthesis by Controlling the Substrate Supply in Aspergillus flavus, Rather than the Cluster Genes Modulation.

The Fus3-MAP kinase module is a conserved phosphorylation signal system in eukaryotes that responds to environmental stress and transduction of external signals from the outer membrane to the nucleus. Aspergillus flavus can produce aflatoxins (AF), which seriously threaten human and animal health. In this study, we determined the functions of Fus3, confirmed Ste50-Ste11-Ste7-Fus3 protein interactions and phosphorylation, and explored the possible phosphorylation motifs and potential targets of Fus3. The regulatory mechanism of Fus3 on the biosynthesis of AF was partly revealed in this study. AF production was downregulated in Δfus3, but the transcriptional expression of most AF cluster genes was upregulated. It is notable that the levels of acetyl-CoA and malonyl-CoA, the substrates of AF, were significantly decreased in fus3 defective strains. Genes involved in acetyl-CoA and malonyl-CoA biosynthesis were significantly downregulated at transcriptional or phosphorylation levels. Specifically, AccA might be a direct target of Fus3, which led to acetyl-CoA carboxylase activities were decreased in null-deletion and site mutagenesis strains. The results concluded that Fus3 could regulate the expression of acetyl-CoA and malonyl-CoA biosynthetic genes directly or indirectly, and then affect the AF production that relies on the regulation of AF substrate rather than the modulation of AF cluster genes. IMPORTANCE Aspergillus flavus is an important saprophytic fungus that produces aflatoxins (AF), which threaten food and feed safety. MAP (mitogen-activated protein) kanases are essential for fungal adaptation to diverse environments. Fus3, as the terminal kinase of a MAPK cascade, interacts with other MAPK modules and phosphorylates downstream targets. We provide evidence that Fus3 could affect AF biosynthesis by regulating the production of acetyl-CoA and malonyl-CoA, but this does not depend on the regulation of AF biosynthetic genes. Our results partly reveal the regulatory mechanism of Fus3 on AF biosynthesis and provide a novel AF modulation pattern, which may contribute to the discovery of new strategies in controlling A. flavus and AF contamination.

New Insights of Transcriptional Regulator AflR in Aspergillus flavus Physiology.

Aspergillus flavus aflR, a gene encoding a Zn(II)2Cys6 DNA-binding domain, is an important transcriptional regulator of the aflatoxin biosynthesis gene cluster. Our previous results of Gene ontology (GO) analysis for the binding sites of AflR in A. flavus suggest that AflR may play an integrative regulatory role. In this study the ΔaflR and overexpression (OE) strains based on the well-established double-crossover recombinational technique were constructed to investigate the integrative function of the aflR gene in A. flavus. The disruption of aflR severely affected the aflatoxin biosynthetic pathway, resulting in a significant decrease in aflatoxin production. The aflatoxin B1 (AFB1) of the ΔaflR strain was 180 ng/mL and aflatoxin B2 (AFB2) was 2.95 ng/mL on YES medium for 5 days, which was 1/1,000 of that produced by the wild-type strain (WT). In addition, the ΔaflR strain produced relatively sparse conidia and a very small number of sclerotia. On the seventh day, the sclerotia yield on each plate of the WT and OE strains exceeded 1,000, while the sclerotial formation of the ΔaflR strain was not detected until 14 days. However, the biosynthesis of cyclopiazonic acid (CPA) was not affected by aflR gene disruption. Transcriptomic analysis of the ΔaflR strain grown on potato dextrose agar (PDA) plates at 0 h, 24 h, and 72 h showed that expression of clustering genes involved in the biosynthesis of aflatoxin was significantly downregulated. Meanwhile, the ΔaflR strain compared with the WT strain showed significant expression differences in genes involved in spore germination, sclerotial development, and carbohydrate metabolism compared to the WT. The results demonstrated that the A. flavus aflR gene also played a positive role in the fungal growth and development in addition to aflatoxin biosynthesis. IMPORTANCE Past studies of the A. flavus aflR gene and its orthologues in related Aspergillus species were solely focused on their roles in secondary metabolism. In this study, we used the ΔaflR and OE strains to demonstrate the role of aflR in growth and development of A. flavus. For the first time, we confirmed that the ΔaflR strain also was defective in production of conidia and sclerotia, asexual propagules of A. flavus. Our transcriptomic analysis further showed that genes involved in spore germination, sclerotial development, aflatoxin biosynssssthesis, and carbohydrate metabolism exhibited significant differences in the ΔaflR strain compared with the WT strain. Our study indicates that AflR not only plays an important role in regulating aflatoxin synthesis but also in playing a positive role in the conidial formation and sclerotial development in A. flavus. This study reveals the critical and positive role of the aflR gene in fungal growth and development, and provides a theoretical basis for the genetic studies of other aspergilli.

Histone 2-Hydroxyisobutyryltransferase Encoded by Afngg1 Is Involved in Pathogenicity and Aflatoxin Biosynthesis in Aspergillus flavus.

Aflatoxin, a carcinogenic secondary metabolite produced by Aspergillus flavus, is a significant threat to human health and agricultural production. Histone 2-hydroxyisobutyrylation is a novel post-translational modification that regulates various biological processes, including secondary metabolism. In this study, we identified the novel histone 2-hydroxyisobutyryltransferase Afngg1 in A. flavus, and explored its role in cell growth, development and aflatoxin biosynthesis. Afngg1 gene deletion markedly decreased lysine 2-hydroxyisobutyrylation modification of histones H4K5 and H4K8 compared with the control strain. Additionally, Afngg1 deletion inhibited mycelial growth of A. flavus, and the number of conidia and hydrophobicity were significantly decreased. Notably, aflatoxin B1 biosynthesis and sclerotia production were completely inhibited in the ΔAfngg1 strain. Furthermore, the pathogenicity of the ΔAfngg1 strain infecting peanut and corn grains was also diminished, including reduced spore production and aflatoxin biosynthesis compared with A. flavus control and Afngg1 complementation strains. Transcriptome analysis showed that, compared with control strains, differentially expressed genes in ΔAfngg1 were mainly involved in chromatin remodelling, cell development, secondary metabolism and oxidative stress. These results suggest that Afngg1 is involved in histone 2-hydroxyisobutyrylation and chromatin modification, and thus affects cell development and aflatoxin biosynthesis in A. flavus. Our results lay a foundation for in-depth research on the 2-hydroxyisobutyrylation modification in A. flavus, and may provide a novel target for aflatoxin contamination prevention.

aflN Is Involved in the Biosynthesis of Aflatoxin and Conidiation in Aspergillus flavus.

Aspergillus flavus poses a threat to society economy and public health due to aflatoxin production. aflN is a gene located in the aflatoxin gene cluster, but the function of AflN is undefined in Aspergillus flavus. In this study, aflN is knocked out and overexpressed to study the function of AflN. The results indicated that the loss of AflN leads to the defect of aflatoxin biosynthesis. AflN is also found to play a role in conidiation but not hyphal growth and sclerotia development. Moreover, AlfN is related to the response to environmental oxidative stress and intracellular levels of reactive oxygen species. At last, AflN is involved in the pathogenicity of Aspergillus flavus to host. These results suggested that AflN played important roles in aflatoxin biosynthesis, conidiation and reactive oxygen species generation in Aspergillus flavus, which will be helpful for the understanding of aflN function, and will be beneficial to the prevention and control of Aspergillus flavus and aflatoxins contamination.

Genetic fingerprinting and aflatoxin production of Aspergillus section Flavi associated with groundnut in eastern Ethiopia.

BACKGROUND: Aspergillus species cause aflatoxin contamination in groundnut kernels, being a health threat in agricultural products and leading to commodity rejection by domestic and international markets. Presence of Aspergillus flavus and A. parasiticus colonizing groundnut in eastern Ethiopia, as well as presence of aflatoxins have been reported, though in this region, no genetic studies have been done of these species in relation to their aflatoxin production. RESULTS: In this study, 145 Aspergillus isolates obtained from groundnut kernels in eastern Ethiopia were genetically fingerprinted using 23 Insertion/Deletion (InDel) markers within the aflatoxin-biosynthesis gene cluster (ABC), identifying 133 ABC genotypes. Eighty-four isolates were analyzed by Ultra-Performance Liquid Chromatography (UPLC) for in vitro aflatoxin production. Analysis of genetic distances based on the approximately 85 kb-ABC by Neighbor Joining (NJ), 3D-Principal Coordinate Analysis (3D-PCoA), and Structure software, clustered the isolates into three main groups as a gradient in their aflatoxin production. Group I, contained 98% A. flavus, including L- and non-producers of sclerotia (NPS), producers of B1 and B2 aflatoxins, and most of them collected from the lowland-dry Babile area. Group II was a genetic admixture population of A. flavus (NPS) and A. flavus S morphotype, both low producers of aflatoxins. Group III was primarily represented by A. parasiticus and A. flavus S morphotype isolates both producers of B1, B2 and G1, G2 aflatoxins, and originated from the regions of Darolabu and Gursum. The highest in vitro producer of aflatoxin B1 was A. flavus NPS N1436 (77.98 µg/mL), and the highest producer of aflatoxin G1 was A. parasiticus N1348 (50.33 µg/mL), these isolates were from Gursum and Darolabu, respectively. CONCLUSIONS: To the best of our knowledge, this is the first study that combined the use of InDel fingerprinting of the ABC and corresponding aflatoxin production capability to describe the genetic diversity of Aspergillus isolates from groundnut in eastern Ethiopia. Three InDel markers, AFLC04, AFLC08 and AFLC19, accounted for the main assignment of individuals to the three Groups; their loci corresponded to aflC (pksA), hypC, and aflW (moxY) genes, respectively. Despite InDels within the ABC being often associated to loss of aflatoxin production, the vast InDel polymorphism observed in the Aspergillus isolates did not completely impaired their aflatoxin production in vitro.

Rhamnolipids inhibit aflatoxins production in Aspergillus flavus by causing structural damages in the fungal hyphae and down-regulating the expression of their biosynthetic genes.

Aflatoxins are hepatotoxic and carcinogenic fungal secondary metabolites that usually contaminate crops and represent a serious health hazard for humans and animals worldwide. In this work, the effect of rhamnolipids (RLs) produced by Pseudomonas aeruginosa #112 on the growth and aflatoxins production by Aspergillus flavus MUM 17.14 was studied in vitro. At concentrations between 45 and 1500 mg/L, RLs reduced the mycelial growth of A. flavus by 23-40% and the production of aflatoxins by 93.9-99.5%. Purified mono-RLs and di-RLs exhibited a similar inhibitory activity on fungal growth. However, the RL mixture had a stronger inhibitory effect on aflatoxins production at concentrations up to 190 mg/L, probably due to a synergistic effect resulting from the combination of both congeners. Using transmission electron microscopy, it was demonstrated that RLs damaged the cell wall and the cytoplasmic membrane of the fungus, leading to the loss of intracellular content. This disruptive phenomenon explains the growth inhibition observed. Furthermore, RLs down-regulated the expression of genes aflC, aflE, aflP and aflQ involved in the aflatoxins biosynthetic pathway (6.4, 44.3, 38.1 and 2.0-fold, respectively), which is in agreement with the almost complete inhibition of aflatoxins production. Overall, the results herein gathered demonstrate for the first time that RLs could be used against aflatoxigenic fungi to attenuate the production of aflatoxins, and unraveled some of their mechanisms of action.

Transcriptomic responses of Aspergillus flavus to temperature and oxidative stresses during aflatoxin production.

Aflatoxin is a group of polyketide-derived carcinogenic and mutagenic secondary metabolites produced by Aspergillus flavus that negatively impact global food security and threaten the health of both humans and livestock. Aflatoxin biosynthesis is strongly affected by the fungal developmental stage, cultivation conditions, and environmental stress. In this study, a novel float culture method was used to examine the direct responses of the A. flavus transcriptome to temperature stress, oxidative stress, and their dual effects during the aflatoxin production stage. The transcriptomic response of A. flavus illustrated that the co-regulation of different secondary metabolic pathways likely contributes to maintaining cellular homeostasis and promoting cell survival under stress conditions. In particular, aflatoxin biosynthetic gene expression was downregulated, while genes encoding secondary metabolites with antioxidant properties, such as kojic acid and imizoquins, were upregulated under stress conditions. Multiple mitochondrial function-related genes, including those encoding NADH:ubiquinone oxidoreductase, ubiquinol-cytochrome C reductase, and alternative oxidase, were differentially expressed. These data can provide insights into the important mechanisms through which secondary metabolism in A. flavus is co-regulated and facilitate the deployment of various approaches for the effective control and prevention of aflatoxin contamination in food crops.

Synthetic antimicrobial agents inhibit aflatoxin production.

Antimicrobial peptides (AMPs) are biologically active molecules that can eradicate bacteria by destroying the bacterial membrane structure, causing the bacteria to rupture. However, little is known about the extent and effect of AMPs on filamentous fungi. In this study, we synthesized small molecular polypeptides by an inexpensive heat conjugation approach and examined their effects on the growth of Aspergillus flavus and its secondary metabolism. The antimicrobial agents significantly inhibited aflatoxin production, conidiation, and sclerotia formation in A. flavus. Furthermore, we found that the expression of aflatoxin structural genes was significantly inhibited, and the intracellular reactive oxygen species (ROS) level was reduced. Additionally, the antimicrobial agents can change membrane permeability. Overall, our results demonstrated that antimicrobial agents, safe to mammalian cells, have an obvious impact on aflatoxin production, which indicated that antimicrobial agents may be adopted as a new generation of potential agents for controlling aflatoxin contamination.

A novel phosphoinositide kinase Fab1 regulates biosynthesis of pathogenic aflatoxin in Aspergillus flavus.

Aspergillus flavus (A. flavus) is one of the most important model environmental fungi which can produce a potent toxin and carcinogen known as aflatoxin. Aflatoxin contamination causes massive agricultural economic loss and a critical human health issue each year. Although a functional vacuole has been highlighted for its fundamental importance in fungal virulence, the molecular mechanisms of the vacuole in regulating the virulence of A. flavus remain largely unknown. Here, we identified a novel vacuole-related protein in A. flavus, the ortholog of phosphatidylinositol-3-phosphate-5-kinase (Fab1) in Saccharomyces cerevisiae. This kinase was located at the vacuolar membrane, and loss of fab1 function was found to affect the growth, conidia and sclerotial development, cellular acidification and metal ion homeostasis, aflatoxin production and pathogenicity of A. flavus. Further functional analysis revealed that Fab1 was required to maintain the vacuole size and cell morphology. Additional quantitative proteomic analysis suggested that Fab1 was likely to play an important role in maintaining vacuolar/cellular homeostasis, with vacuolar dysregulation upon fab1 deletion leading to impaired aflatoxin synthesis in this fungus. Together, these results provide insight into the molecular mechanisms by which this pathogen produces aflatoxin and mediates its pathogenicity, and may facilitate dissection of the vacuole-mediated regulatory network in A. flavus.

Chemical repertoire and biosynthetic machinery of the Aspergillus flavus secondary metabolome: A review.

Filamentous fungi represent a rich source of extrolites, including secondary metabolites (SMs) comprising a great variety of astonishing structures and interesting bioactivities. State-of-the-art techniques in genome mining, genetic manipulation, and secondary metabolomics have enabled the scientific community to better elucidate and more deeply appreciate the genetic and biosynthetic chemical arsenal of these microorganisms. Aspergillus flavus is best known as a contaminant of food and feed commodities and a producer of the carcinogenic family of SMs, aflatoxins. This fungus produces many SMs including polyketides, ribosomal and nonribosomal peptides, terpenoids, and other hybrid molecules. This review will discuss the chemical diversity, biosynthetic pathways, and biological/ecological role of A. flavus SMs, as well as their significance concerning food safety and security.

Ssu72 Regulates Fungal Development, Aflatoxin Biosynthesis and Pathogenicity in Aspergillus flavus.

The RNA polymerase II (Pol II) transcription process is coordinated by the reversible phosphorylation of its largest subunit-carboxy terminal domain (CTD). Ssu72 is identified as a CTD phosphatase with specificity for phosphorylation of Ser5 and Ser7 and plays critical roles in regulation of transcription cycle in eukaryotes. However, the biofunction of Ssu72 is still unknown in Aspergillus flavus, which is a plant pathogenic fungus and produces one of the most toxic mycotoxins-aflatoxin. Here, we identified a putative phosphatase Ssu72 and investigated the function of Ssu72 in A. flavus. Deletion of ssu72 resulted in severe defects in vegetative growth, conidiation and sclerotia formation. Additionally, we found that phosphatase Ssu72 positively regulates aflatoxin production through regulating expression of aflatoxin biosynthesis cluster genes. Notably, seeds infection assays indicated that phosphatase Ssu72 is crucial for pathogenicity of A. flavus. Furthermore, the Δssu72 mutant exhibited more sensitivity to osmotic and oxidative stresses. Taken together, our study suggests that the putative phosphatase Ssu72 is involved in fungal development, aflatoxin production and pathogenicity in A. flavus, and may provide a novel strategy to prevent the contamination of this pathogenic fungus.

Modelling the effect of temperature and water activity on the growth rate of Aspergillus flavus and aflatoxin production in peanut meal extract agar.

Aspergillus flavus is the predominant species that produce aflatoxins in stored peanuts under favourable conditions. This study aimed to describe the growth and aflatoxin production by two A. flavus strains isolated from imported raw peanuts and to model the effects of temperature and aw on their colony growth rate as a function of temperature and aw in Peanut Meal Extract Agar (PMEA). A full factorial design with seven aw levels (0.85-0.98 aw) and five temperature levels (20-40 °C) was used to investigate the growth and aflatoxin production. Colony diameter was measured daily for 28 days while AFB1 and total aflatoxin were determined on day 3, 7, 14, and 21. The maximum colony growth rate, µmax (mm/day) was estimated by using the primary model of Baranyi, and the µmax was then fitted to the secondary model; second-order polynomial and linear Arrhenius-Davey to describe the colony growth rate as a function of temperature and aw. The results indicated that both strains failed to grow at temperature of 20 °C with aw <0.94 and aw of 0.85 for all temperatures except 30 °C. The highest growth rate was observed at 30 °C, with 0.98 aw for both strains. The analysis of variance showed a significant effect of strain, temperature, and aw on the fungal growth and aflatoxin production (p < 0.05). Furthermore, both secondary models were in good agreement with the observed µmax. However, the polynomial model was found to be a better predictor of the experimental data. A similar pattern was observed in aflatoxin production but in a narrower range of temperature (25-35 °C) and aw (0.92-0.98 aw). The highest production of aflatoxins was observed on day 21 at 30 °C with the aw level of 0.98 for both strains. Overall, the current findings may help in improving the mycotoxin management and intervention strategies in peanuts, especially during storage.

Sisters in structure but different in character, some benzaldehyde and cinnamaldehyde derivatives differentially tune Aspergillus flavus secondary metabolism.

Great are the expectations for a new generation of antimicrobials, and strenuous are the research efforts towards the exploration of diverse molecular scaffolds-possibly of natural origin - aimed at the synthesis of new compounds against the spread of hazardous fungi. Also high but winding are the paths leading to the definition of biological targets specifically fitting the drug's structural characteristics. The present study is addressed to inspect differential biological behaviours of cinnamaldehyde and benzaldehyde thiosemicarbazone scaffolds, exploiting the secondary metabolism of the mycotoxigenic phytopathogen Aspergillus flavus. Interestingly, owing to modifications on the parent chemical scaffold, some thiosemicarbazones displayed an increased specificity against one or more developmental processes (conidia germination, aflatoxin biosynthesis, sclerotia production) of A. flavus biology. Through the comparative analysis of results, the ligand-based screening strategy here described has allowed us to delineate which modifications are more promising for distinct purposes: from the control of mycotoxins contamination in food and feed commodities, to the environmental management of microbial pathogens, to the investigation of specific structure-activity features for new generation drug discovery.

Transcriptome Sequencing Revealed an Inhibitory Mechanism of Aspergillus flavus Asexual Development and Aflatoxin Metabolism by Soy-Fermenting Non-Aflatoxigenic Aspergillus.

Aflatoxins (AFs) have always been regarded as the most effective carcinogens, posing a great threat to agriculture, food safety, and human health. Aspergillus flavus is the major producer of aflatoxin contamination in crops. The prevention and control of A. flavus and aflatoxin continues to be a global problem. In this study, we demonstrated that the cell-free culture filtrate of Aspergillus oryzae and a non-aflatoxigenic A. flavus can effectively inhibit the production of AFB1 and the growth and reproduction of A. flavus, indicating that both of the non-aflatoxigenic Aspergillus strains secrete inhibitory compounds. Further transcriptome sequencing was performed to analyze the inhibitory mechanism of A. flavus treated with fermenting cultures, and the results revealed that genes involved in the AF biosynthesis pathway and other biosynthetic gene clusters were significantly downregulated, which might be caused by the reduced expression of specific regulators, such as AflS, FarB, and MtfA. The WGCNA results further revealed that genes involved in the TCA cycle and glycolysis were potentially involved in aflatoxin biosynthesis. Our comparative transcriptomics also revealed that two conidia transcriptional factors, brlA and abaA, were found to be significantly downregulated, which might lead to the downregulation of conidiation-specific genes, such as the conidial hydrophobins genes rodA and rodB. In summary, our research provides new insights for the molecular mechanism of controlling AF synthesis to control the proliferation of A. flavus and AF pollution.

Dioctatin Activates ClpP to Degrade Mitochondrial Components and Inhibits Aflatoxin Production.

Aflatoxin contamination of crops is a serious problem worldwide. Utilization of aflatoxin production inhibitors is attractive, as the elucidation of their modes of action contributes to clarifying the mechanism of aflatoxin production. Here, we identified mitochondrial protease ClpP as the target of dioctatin, an inhibitor of aflatoxin production of Aspergillus flavus. Dioctatin conferred uncontrolled caseinolytic capacity on ClpP of A. flavus and Escherichia coli. Dioctatin-bound ClpP selectively degraded mitochondrial energy-related proteins in vitro, including a subunit of respiratory chain complex V, which was also reduced by dioctatin in a ClpP-dependent manner in vivo. Dioctatin enhanced glycolysis and alcohol fermentation while reducing tricarboxylic acid cycle metabolites. These disturbances were accompanied by reduced histone acetylation and reduced expression of aflatoxin biosynthetic genes. Our results suggest that dioctatin inhibits aflatoxin production by inducing ClpP-mediated degradation of mitochondrial energy-related components, and that mitochondrial energy metabolism functions as a key determinant of aflatoxin production.

Genetic polymorphisms associated to SDHI fungicides resistance in selected Aspergillus flavus strains and relation with aflatoxin production.

Aspergillus flavus is a common and ubiquitous fungal species able to colonize several agricultural commodities, in both pre- and post-harvest conditions. This species represents a very harmful plant pathogen for its ability to synthesize aflatoxin B1, responsible for human primary hepatocellular carcinoma and classified as a group I (human carcinogenic) by the International Agency for Research on Cancer. Several approaches have been proposed to control A. flavus development and related aflatoxin production in field and storage conditions. The Succinate Dehydrogenase Inhibitor (SDHI) fungicide boscalid has been shown to control A. flavus growth and aflatoxin contamination both in vitro and in field experiments. However, this compound is classified as medium-high risk fungicide for triggering fungal resistance and, indeed, resistant strains can occur on crops treated with boscalid. In this paper, we selected laboratory A. flavus strains resistant to boscalid grown on agar medium containing 50 mg/L of boscalid. In order to investigate the molecular mechanism responsible for the resistant phenotype, specific primer pairs were designed to amplify the whole SdhB, SdhC and SdhD genes. By amino acid sequence analysis, two point mutations, Tyrosine replacing Histidine at codon 249 of SdhB (H249Y) and Arginine replacing Glycine at codon 91 of SdhC (G91R), were identified. The effect of SDHI boscalid and isopyrazam on mycelial growth and conidial germination was evaluated. Both resistant genotypes showed high resistance (MIC and EC50 > 1000 mg/L) to boscalid. A positive cross-resistance was found between boscalid and isopyrazam. Specific sub-lethal doses of both fungicides (0.5 mg/L of boscalid and 0.01 mg/L of isopyrazam) interfered with the mechanisms associated to pigmentation of colonies. In particular, fungal colonies appeared depigmented lacking the typical A. flavus green colour shown on un-amended fungicide medium. A strict correlation between lack of pigmentation and increasing aflatoxin production was also observed.