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Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.

Pathogenic filamentous fungi pose a significant threat to global food security and human health. The limitations of available antifungal agents, including resistance and toxicity, highlight the need for developing innovative antifungal strategies. Antifungal proteins (AFPs) are a class of secreted small proteins that exhibit potent antifungal activity against filamentous fungi, yet the underlying mechanism remains partially understood. In this study, we investigate the molecular and cellular effects of two AFPs, PgAFP and AfAFP, on Aspergillus flavus , a representative filamentous fungus. These AFPs affect various fungal phenotypes and exert an intracellular effect by interacting with Ntp1, a fungi exclusive protein modulating diverse fungal traits. We find that Ntp1 amino acids 417–588 are critical for AFP binding and play a role in regulating growth, development, sporulation, sclerotia formation, toxin synthesis, and pathogenicity. Results generated from this study will help to control pathogenic fungi. Antifungal proteins (AFPs) produced by filamentous ascomycetes are effective against fungal infection e.g., Aspergillus flavus that can cause aspergillosis on corn and peanut and aflatoxin production. Here, the author report two AFPs and their interacting protein Ntp1 in mediating fungal growth, toxin synthesis and pathogenicity.

Aspergillus flavus is a clinically and agriculturally important saprotrophic fungus responsible for severe human infections and extensive crop losses. Here, we analyze genomic data from 300 (117 clinical and 183 environmental) A. flavus isolates from 13 countries, including 82 clinical isolates sequenced in this study, to examine population and pan-genome structure and their relationship to pathogenicity. We use single nucleotide polymorphisms to build a phylogeny, analyze admixture, and perform discriminant analysis of principal components. We identify five A. flavus populations, including a new population, D, corresponding to distinct clades in the genome-wide phylogeny. Strikingly, > 75% of clinical isolates were in population D and <5% in population B. We also use orthogroup clustering to identify core and accessory genes within the pan-genome. Accessory genes, including genes within biosynthetic gene clusters, were significantly more common in some populations but rare in others. Our functional annotations show that population D is enriched for genes associated with carbohydrate metabolism, lipid metabolism and certain types of hydrolase activity, whereas a non-clinical population is depleted in genes related to zinc ion binding. In contrast to previous results from the major human pathogen Aspergillus fumigatus , isolation of A. flavus from human specimens is associated with population structure, providing a promising system for future investigations into the contributions of population-specific genetic differences to human infection. Genetic diversity between clinical and environmental fungal isolates of Aspergillus flavus is poorly studied. Here, the authors analysed genomic data from a global set of clinical and environmental A. flavus isolates and report that clinical prevalence is associated with population structure.

Cinnamaldehyde (CIN) is a promising natural preservative and generally recognized as safe for commodities as well as consumers. In this work, the antifungal effects of CIN on Aspergillus flavus were evaluated both in solid and in liquid culture conditions. Our results indicated that CIN effectively inhibited radial growth, spore production, mycelium formation, and aflatoxin B₁ biosynthesis by A. flavus in a dose-dependent manner. At the concentration of 104 mg L⁻¹, CIN exposure was able to completely inhibit fungal growth as well as aflatoxin B₁ production. Furthermore, the inhibitory activities of CIN were closely connected with the treatment period and the tested fungal species. Compared with the control strains, CIN dose dependently changed the morphology and ultrastructure of mycelium in different degree. Especially, the reduction of hydrogen peroxide was considered to follow the destruction of mitochondrial. Meanwhile, CIN significantly cut the levels of lipid peroxidation and reduced glutathione. The activity of total superoxide dismutase was significantly inhibited after CIN treatment at the end of incubation, whereas the activities of catalase and glutathione peroxidase were opposite. These results indicated that the inhibitory effect of CIN could attribute to oxidative stress alleviation possibly induced by modifications of cellular structure as well as redox status.

Aspergillus flavus is a ubiquitous opportunistic pathogen of plants and animals, which produces carcinogenic and toxic secondary metabolite aflatoxin. A. flavus and aflatoxin contamination have emerged as a global food safety concern. Currently, post-translational modification plays crucial modulatory roles in the fungal development and virulence, but the role of benzoylation in fungal pathogenicity remains undetermined, which limits the development of prevention and control technique. Here, we first identified 46 benzoylated proteins in A. flavus , and found that benzoyltransferase GcnE exerted effects on pathogenicity and aflatoxin production by regulating the benzoylation of AdhB. This finding not only provided valuable information for prevention and control of A. flavus contamination, but also offered basic knowledge for investigation of the regulation mechanism of secondary metabolism in other fungi.

Various strains of Aspergillus oryzae , regarded as a domesticated variant of aflatoxigenic Aspergillus flavus , are utilized in the soybean fermentation industry of Korea. This study compared A. oryzae/flavus strains isolated from various environments in Korea including industrial settings, Meju (brick of dried fermented soybeans), and wild conditions with globally reported strains using genomic analysis to determine their taxonomic positions and risk of mycotoxicity. Using population genomics, five distinct groups (A to E) were identified, with all aflatoxigenic Korean strains in Group C and non-aflatoxigenic Korean strains in Groups A, B, and E. Korean strains from Meju and wild conditions are distributed across Groups A and B, and most of the Korean industrial strains form a sub-cluster with Japanese industrial strains in Group A. Comparing secondary metabolite gene cluster mutation pattern, three gene clusters (Aflatoxin, Cyclopiazonic acid and Ditryptophenaline) were revealed as group specific ones. In aflatoxin and cyclopiazonic acid clusters, most of the Group C strains had intact regions compared to strains in other groups. Since most of the Group C strains produce aflatoxin and have intact Aflatoxin and Cyclopiazonic acid gene clusters, we considered that this group represent A. flavus . Profiling using MALDI-TOF MS analysis also distinguished Group C from Groups A, B and E by specific three proteomic peaks. Among the three peaks, those around 12,700 to 12,900 m/z (Da) are expected to correspond to AflF (nor B), an enzyme involved in Aflatoxin metabolism. These results showed taxonomic positions of Korean strains of A. oryzae/flavus from various environments and also showed possibility to differentiate between A. oryzae and A. flavus with genome and MALDI-TOF MS analysis.

Aflatoxin B 1 (AFB 1 ), the predominant and most carcinogenic naturally polyketide, is mainly produced by Aspergillus flavus and Aspergillus parasiticus . Cinnamaldehyde has been reported for inhibiting the growth and aflatoxin biosynthesis in A. flavus . But its molecular mechanism of action still remains largely ambiguous. Here, the anti-aflatoxigenic mechanism of cinnamaldehyde in A. flavus was investigated via a comparative transcriptomic analysis. The results indicated that twenty five of thirty genes in aflatoxin cluster showed down-regulation by cinnamaldehyde although the cluster regulators aflR and aflS were slightly up-regulated. This may be due to the up-regulation of the oxidative stress-related genes srrA , msnA and atfB being caused by the significant down-regulation of the diffusible factor FluG . Cinnamaldehyde also inhibited aflatoxin formation by perturbing GPCRs and oxylipins normal function, cell wall biosynthesis and redox equilibrium. In addition, accumulation of NADPH due to up-regulation of pentose phosphate pathway drove acetyl-CoA to lipids synthesis rather than polyketides. Both GO and KEGG analysis suggested that pyruvate and phenylalanine metabolism, post-transcriptional modification and key enzymes biosynthesis might be involved in the suppression of AFB 1 production by cinnamaldehyde. This study served to decipher the anti-aflatoxigenic properties of cinnamaldehyde in A. flavus and provided powerful evidence for its use in practice.

Forkhead transcription factors regulate several important biological processes in many eukaryotic species including fungi. Bioinformatic analysis of the Aspergillus flavus genome revealed four putative forkhead transcription factor genes. Genetic disruption of ( AFLA_005634 ), a homolog of the Aspergillus nidulans fhpA / fkhA gene ( AN4521 ), revealed that the fhpA gene is a negative regulator of both asexual spore production and aflatoxin B 1 production in A. flavus . Furthermore, disruption of the fhpA gene caused a complete loss of sclerotial formation. Overexpression of the fhpA gene caused A. flavus to become more sensitive to sodium chloride whereas disruption of the fhpA gene did not change the ability of A. flavus to respond to any osmotic stress agent tested. Interestingly, both disruption and overexpression of the fhpA gene led to increases in sensitivity to the oxidative stress agent menadione. Overall, these results suggest that fhpA is an important regulator of morphological and chemical development in addition to stress response in A. flavus .

The efficacy of eleven essential oils (EOs) against Aspergillus flavus NRRL 3357 was investigated. The highest antifungal activity against this aflatoxigenic fungus was exhibited by cinnamon, oregano and lemongrass, which showed low minimum inhibitory concentration (MIC) values under vapor conditions. Interactions of the three EOs were evaluated by the fractional inhibition concentration index (FICI), and the composite essential oils (CEO) showed synergistic inhibitory activities. Chemical analysis of the composite essential oils of cinnamon, oregano, and lemongrass (COL-CEO) revealed that (Z)-citral (33.44%), (E)-citral (32.88%) and carvacrol (19.84%) were the dominant components, followed by limonene (4.29%) and cinnamaldehyde (3.76%). COL-CEO not only inhibited fungal growth but also decreased aflatoxin B1 production by A. flavus. Downregulation of the relative expression of aflatoxin genes in the aflatoxin biosynthetic pathway by COL-CEO revealed its anti-aflatoxigenic mechanism. COL-CEO could also affect the colonization of A. flavus on maize grains. Therefore, COL-CEO may be considered as a potential natural antifungal agent, which could be used for the storage of maize and other grains.

The grain contamination by Aspergillus spp. has been a serious issue. This study exhibited the excellent antifungal effects of the essential oil compounds (EOCs) geraniol and citral against common grain pathogens (A. flavus and A. ochraceus) in vitro and in situ. The inhibitory mechanisms were also evaluated from the perspective of cell membrane permeability, reactive oxygen species (ROS) generation, and Aspergillus spp. growth-related gene expression. Meanwhile, the combined effects of EOCs in the vapor phase and modified atmosphere packaging (MAP) were examined to find an alternative preservation method for controlling Aspergillus spp. The results indicated that citral exhibited the antifungal activity mainly by downregulating the sporulation- and growth-related genes for both pathogens. Geraniol displayed inhibitory effectiveness against A. flavus predominantly by inducing the intracellular ROS accumulation and showed toxicity against A. ochraceus principally by changing cell membrane permeability. Furthermore, the synthetic effects of EOCs and MAP (75% CO2 and 25% N2) induced better grain quality than the current commercial fumigant AlP. These findings reveal that EOCs have potential to be a novel grain preservative for further application.