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.

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.

Antifungal mechanisms of binary combinations of volatile organic compounds produced by lactic acid bacteria strains against Aspergillusflavus.

Aspergillus flavus（A. flavus）, a common humic fungus known for its ability to infect agricultural products, served as the subject of investigation in this study. The primary objective was to assess the antifungal efficacy and underlying mechanisms of binary combinations of five volatile organic compounds (VOCs) produced by lactic acid bacteria, specifically in their inhibition of A. flavus. This assessment was conducted through a comprehensive analysis, involving biochemical characterization and transcriptomic scrutiny. The results showed that VOCs induce notable morphological abnormalities in A. flavus conidia and hyphae. Furthermore, they disrupt the integrity of the fungal cell membrane and cell wall, resulting in the leakage of intracellular contents and an increase in extracellular electrical conductivity. In terms of cellular components, VOC exposure led to an elevation in malondialdehyde content while concurrently inhibiting the levels of total lipids, ergosterol, soluble proteins, and reducing sugars. Additionally, the impact of VOCs on A. flavus energy metabolism was evident, with significant inhibition observed in the activities of key enzymes, such as Na+/K+-ATPase, malate dehydrogenase, succinate dehydrogenase, and chitinase. And they were able to inhibit aflatoxin B1 synthesis. The transcriptomic analysis offered further insights, highlighting that differentially expressed genes (DEGs) were predominantly associated with membrane functionality and enriched in pathways about carbohydrate and amino acid metabolism. Notably, DEGs linked to cellular components and energy-related mechanisms exhibited down-regulation, thereby corroborating the findings from the biochemical analyses. In summary, these results elucidate the principal antifungal mechanisms of VOCs, which encompass the disruption of cell membrane integrity and interference with carbohydrate and amino acid metabolism in A. flavus.

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.

Glass bead system to study mycotoxin production of Aspergillus spp. on corn and rice starches.

Mycotoxin production by aflatoxin B1 (AFB1) -producing Aspergillus flavus Zt41 and sterigmatocystin (ST) -hyperproducer Aspergillus creber 2663 mold strains on corn and rice starch, both of high purity and nearly identical amylose-amylopectin composition, as the only source of carbon, was studied. Scanning electron microscopy revealed average starch particle sizes of 4.54 ± 0.635 µm and 10.9 ± 2.78 µm, corresponding to surface area to volume ratios of 127 1/µm for rice starch and 0.49 1/µm for corn starch. Thus, a 2.5-fold difference in particle size correlated to a larger, 259-fold difference in surface area. To allow starch, a water-absorbing powder, to be used as a sole food source for Aspergillus strains, a special glass bead system was applied. AFB1 production of A. flavus Zt41 was determined to be 437.6 ± 128.4 ng/g and 90.0 ± 44.8 ng/g on rice and corn starch, respectively, while corresponding ST production levels by A. creber 2663 were 72.8 ± 10.0 µg/g and 26.8 ± 11.6 µg/g, indicating 3-fivefold higher mycotoxin levels on rice starch than on corn starch as sole carbon and energy sources. KEY POINTS: • A glass bead system ensuring the flow of air when studying powders was developed. • AFB1 and ST production of A. flavus and A. creber on rice and corn starches were studied. • 3-fivefold higher mycotoxin levels on rice starch than on corn starch were detected.

AFB1 Toxicity in Human Food and Animal Feed Consumption: A Review of Experimental Treatments and Preventive Measures.

AIMS: The current review aims to outline and summarize the latest research on aflatoxin, with research studies describing natural, herbal and chemical compound applications in animal (pig) models and in vitro cellular studies. Aflatoxin, a carcinogenic toxin metabolite, is produced by Aspergillus flavus in humid environments, posing a threat to human health and crop production. The current treatment involves the prevention of exposure to aflatoxin and counteracting its harmful toxic effects, enabling survival and research studies on an antidote for aflatoxin. OBJECTIVES: To summarize current research prospects and to outline the influence of aflatoxin on animal forage in farm production, food and crop processing. The research application of remedies to treat aflatoxin is undergoing development to pinpoint biochemical pathways responsible for aflatoxin effects transmission and actions of treatment. SIGNIFICANCE: To underline the environmental stress of aflatoxin on meat and dairy products; to describe clinical syndromes associated with aflatoxicosis on human health that are counteracted with proposed treatment and preventive interventions. To understand how to improve the health of farm animals with feed conditions.

Bio-stimulating effect of endophytic Aspergillus flavus AUMC 16068 and its respective ex-polysaccharides in lead stress tolerance of Triticum aestivum plant.

Heavy metal accumulation is one of the major agronomic challenges that has seriously threatened food safety. As a result, metal-induced phytotoxicity concerns require quick and urgent action to retain and maintain the physiological activities of microorganisms, the nitrogen pool of soils, and the continuous yields of wheat in a constantly worsening environment. The current study was conducted to evaluate the plant growth-promoting endophytic Aspergillus flavus AUMC 16,068 and its EPS for improvement of plant growth, phytoremediation capacity, and physiological consequences on wheat plants (Triticum aestivum) under lead stress. After 60 days of planting, the heading stage of wheat plants, data on growth metrics, physiological properties, minerals content, and lead content in wheat root, shoot, and grains were recorded. Results evoked that lead pollution reduced wheat plants' physiological traits as well as growth at all lead stress concentrations; however, inoculation with lead tolerant endophytic A. flavus AUMC 16,068 and its respective EPS alleviated the detrimental impact of lead on the plants and promoted the growth and physiological characteristics of wheat in lead-contaminated conditions and also lowering oxidative stress through decreasing (CAT, POD, and MDA), in contrast to plants growing in the un-inoculated lead polluted dealings. In conclusion, endophytic A. flavus AUMC 16,068 spores and its EPS are regarded as eco-friendly, safe, and powerful inducers of wheat plants versus contamination with heavy metals, with a view of protecting plant, soil, and human health.

Terpinen-4-ol from tea tree oil prevents Aspergillus flavus growth in postharvest wheat grain.

Plant volatile organic compounds (PVOCs) have gained increasing attention for their role in preventing fungal spoilage and insect contamination in postharvest agro-products owing to their effectiveness and sustainability. In this study, the essential oil was extracted from fresh M. alternifolia (tea tree) leaves, and the fumigation vapor of tea tree oil (TTO) completely inhibited the growth of Aspergillus flavus on agar plates at a concentration of 1.714 µL/mL. Terpinen-4-ol was identified as the major component (40.76 %) of TTO volatiles analyzed using headspace gas chromatography-mass spectrometry. Terpinen-4-ol vapor completely inhibited the A. flavus growth on agar plates and 20 % moisture wheat grain at 0.556 and 1.579 µL/mL, respectively, indicating that terpinen-4-ol serves as the main antifungal constituent in TTO volatiles. The minimum inhibitory concentration of terpinen-4-ol in liquid-contact culture was 1.6 µL/mL. Terpinen-4-ol treatment caused depressed, wrinkled, and punctured mycelial morphology and destroyed the plasma membrane integrity of A. flavus. Metabolomics analysis identified significant alterations in 93 metabolites, with 79 upregulated and 14 downregulated in A. flavus mycelia exposed to 1.6 µL/mL terpinen-4-ol for 6 h, involved in multiple cellular processes including cell membrane permeability and integrity, the ABC transport system, pentose phosphate pathway, and the tricarboxylic acid cycle. Biochemical analysis and 2,7-dichlorofluorescein diacetate staining showed that terpinen-4-ol induced oxidative stress and mitochondrial dysfunction in A. flavus mycelia. This study provides new insights into the antifungal effects of the main TTO volatile compounds terpinen-4-ol on the growth of 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.

Biogenic carbon quantum dots from marine endophytic fungi (Aspergillus flavus) to enhance the curcumin production and growth in Curcuma longa L.

In this study, we have investigated the effect of carbon quantum dots (FM-CQDs) synthesized from marine fungal extract on Curcuma longa to improve the plant growth and curcumin production. The isolated fungus, Aspergillus flavus has produced a high amount of indole-3-acetic acid (IAA) (0.025 mg g-1), when treated with tryptophan. CQDs were synthesized from the A. flavus extract and it was characterized using ultraviolet visible spectrophotometer (UV-Vis) and high-resolution transmission electron microscopy (HR-TEM). The synthesized CQDs were excited at 365 nm in an UV-Vis and the HR-TEM analysis showed approximately 7.4 nm in size with a spherical shape. Both fungal crude extract (FCE) at 0-100 mg L-1 and FM-CQDs 0-5 mg L-1 concentrations were tested on C. longa. About 80 mg L-1 concentration FCE treated plants has shown a maximum height of 21 cm and FM-CQDs at 4 mg L-1 exhibited a maximum height of 25 cm compared to control. The FM-CQDs significantly increased the photosynthetic pigments such as total chlorophyll (1.08 mg g-1 FW) and carotenoids (17.32 mg g-1 FW) in C. longa. Further, antioxidant enzyme analysis confirmed that the optimum concentrations of both extracts did not have any toxic effects on the plants. FM-CQDs treated plants increased the curcumin content up to 0.060 mg g-1 by HPLC analysis. Semi quantitative analysis revealed that FCE and FM-CQDs significantly upregulated ClCURS1 gene expression in curcumin production.

Chitosan nanoemulsion incorporated with Carum carvi essential oil as ecofriendly alternative for mitigation of aflatoxin B1 contamination in stored herbal raw materials.

The present investigation entails the first report on entrapment of Carum carvi essential oil (CCEO) into chitosan polymer matrix for protection of stored herbal raw materials against fungal inhabitation and aflatoxin B1 (AFB1) production. Physico-chemical characterization of nanoencapsulated CCEO was performed through Fourier transform infrared spectroscopy, dynamic light scattering, X-ray diffractometry, and scanning electron microscopy. The nanoencapsulated CCEO displayed improved antifungal and AFB1 suppressing potentiality along with controlled delivery over unencapsulated CCEO. The encapsulated CCEO nanoemulsion obstructed the ergosterol production and escalated the efflux of cellular ions, thereby suggesting plasma membrane as prime target of antifungal action in Aspergillus flavus cells. The impairment in methyglyoxal production and modeling based carvone interaction with Afl-R protein validated the antiaflatoxigenic mechanism of action. In addition, CCEO displayed augmentation in antioxidant potentiality after encapsulation into chitosan nanomatrix. Moreover, the in-situ study demonstrated the effective protection of Withania somnifera root samples (model herbal raw material) against fungal infestation and AFB1 contamination along with prevention of lipid peroxidation. The acceptable organoleptic qualities of W. somnifera root samples and favorable safety profile in mice (animal model) strengthen the application of nanoencapsulated CCEO emulsion as nano-fungitoxicant for preservation of herbal raw materials against fungi and AFB1 mediated biodeterioration.

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.

Melanin depletion affects Aspergillus flavus conidial surface proteins, architecture, and virulence.

Melanin is an Aspergillus flavus cell wall component that provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile, and virulence of melanized conidia (MC) and non-melanized conidia (NMC) of A. flavus. Kojic acid treatment inhibited melanin synthesis in A. flavus, and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathways were found only in the formic acid extracts of NMC. Scanning electron microscopy (SEM) analysis showed the conidial surface morphology difference between the NMC and MC, indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor white staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining between MC and NMC. Evaluation of the virulence of MC and NMC in the Galleria mellonella model showed NMC was less virulent compared to MC. Our findings showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer, and hence, melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. The G. mellonella virulence assay also confirmed that the NMC were susceptible to host defense as in other Aspergillus pathogens. KEY POINTS: • l-DOPA melanin production was inhibited in A. flavus isolates by kojic acid, and for the first time, scanning electron microscopy (SEM) analysis revealed morphological differences between MC and NMC of A. flavus strains • Proteome profile of non-melanized conidia showed more conidial surface proteins and these proteins were mainly involved in the virulence, oxidative stress, and metabolism pathways • Non-melanized conidia of A. flavus strains were shown to be less virulent than melanised conidia in an in vivo virulence experiment with the G. melonella model.

m6A methyltransferase AflIme4 orchestrates mycelial growth, development and aflatoxin B1 biosynthesis in Aspergillus flavus.

Aflatoxin B1 (AFB1), a highly toxic secondary metabolite produced by Aspergillus flavus, poses a severe threat to agricultural production, food safety and human health. The methylation of mRNA m6A has been identified as a regulator of both the growth and AFB1 production of A. flavus. However, its intracellular occurrence and function needs to be elucidated. Here, we identified and characterized a m6A methyltransferase, AflIme4, in A. flavus. The enzyme was localized in the cytoplasm, and knockout of AflIme4 significantly reduced the methylation modification level of mRNA. Compared with the control strains, ΔAflIme4 exhibited diminished growth, conidial formation, mycelial hydrophobicity, sclerotium yield, pathogenicity and increased sensitivity to CR, SDS, NaCl and H2O2. Notably, AFB1 production was markedly inhibited in the A. flavus ΔAflIme4 strain. RNA-Seq coupled with RT-qPCR validation showed that the transcriptional levels of genes involved in the AFB1 biosynthesis pathway including aflA, aflG, aflH, aflK, aflL, aflO, aflS, aflV and aflY were significantly upregulated. Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) analysis demonstrated a significant increase in m6A methylation modification levels of these pathway-specific genes, concomitant with a decrease in mRNA stability. These results suggest that AflIme4 attenuates the mRNA stability of genes in AFB1 biosynthesis by enhancing their mRNA m6A methylation modification, leading to impaired AFB1 biosynthesis. Our study identifies a novel m6A methyltransferase AflIme4 and highlights it as a potential target to control A. flavus growth, development and aflatoxin pollution.

Synergistic antifungal mechanism of cinnamaldehyde and nonanal against Aspergillus flavus and its application in food preservation.

Aspergillus flavus colonization on agricultural products during preharvest and postharvest results in tremendous economic losses. Inspired by the synergistic antifungal effects of essential oils, the aims of this study were to explore the mechanism of combined cinnamaldehyde and nonanal (SCAN) against A. flavus and to evaluate the antifungal activity of SCAN loading into diatomite (DM). Shriveled mycelia were observed by scanning electron microscopy, especially in the SCAN treatment group. Calcofluor white staining, transmission electron microscopy, dichloro-dihydro-fluorescein diacetate staining and the inhibition of key enzymes in tricarboxylic acid cycle indicated that the antifungal mechanism of SCAN against A. flavus was related to the cell wall damage, reactive oxygen species accumulation and energy metabolism interruption. RNA sequencing revealed that some genes involved in antioxidation were upregulated, whereas genes responsible for cell wall biosynthesis, oxidative stress, cell cycle and spore development were significantly downregulated, supporting the occurrence of cellular apoptosis. In addition, compared with the control group, conidia production in 1.5 mg/mL DM/cinnamaldehyde, DM/nonanal and DM/SCAN groups were decreased by 27.16%, 48.22% and 76.66%, respectively, and the aflatoxin B1 (AFB1) contents decreased by 2.00%, 73.02% and 84.15%, respectively. These finding suggest that DM/SCAN complex has potential uses in food preservation.

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.

Nitric oxide-mediated regulation of Aspergillus flavus asexual development by targeting TCA cycle and mitochondrial function.

Nitric oxide (NO) is a signaling molecule with diverse roles in various organisms. However, its role in the opportunistic pathogen Aspergillus flavus remains unclear. This study investigates the potential of NO, mediated by metabolites from A. oryzae (AO), as an antifungal strategy against A. flavus. We demonstrated that AO metabolites effectively suppressed A. flavus asexual development, a critical stage in its lifecycle. Transcriptomic analysis revealed that AO metabolites induced NO synthesis genes, leading to increased intracellular NO levels. Reducing intracellular NO content rescued A. flavus spores from germination inhibition caused by AO metabolites. Furthermore, exogenous NO treatment and dysfunction of flavohemoglobin Fhb1, a key NO detoxification enzyme, significantly impaired A. flavus asexual development. RNA-sequencing and metabolomic analyses revealed significant metabolic disruptions within tricarboxylic acid (TCA) cycle upon AO treatment. NO treatment significantly reduced mitochondrial membrane potential (Δψm) and ATP generation. Additionally, aberrant metabolic flux within the TCA cycle was observed upon NO treatment. Further analysis revealed that NO induced S-nitrosylation of five key TCA cycle enzymes. Genetic analysis demonstrated that the S-nitrosylated Aconitase Acon and one subunit of succinate dehydrogenase Sdh2 played crucial roles in A. flavus development by regulating ATP production. This study highlights the potential of NO as a novel antifungal strategy to control A. flavus by compromising its mitochondrial function and energy metabolism.

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.

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.