Molecular and aflatoxigenicity analyses of Aspergillus flavus isolates indigenous to grain corn in Malaysia; potentials for biological control.

AIMS: The present work aimed to distinguish the indigenous Aspergillus flavus isolates obtained from the first (pioneer) grain corn farms in Terengganu, Malaysia, into aflatoxigenic and non-aflatoxigenic by molecular and aflatoxigenicity analyses, and determine the antagonistic capability of the non-aflatoxigenic isolates against aflatoxigenic counterparts and their aflatoxin production in vitro. METHODS AND RESULTS: Seven A. flavus isolates previously obtained from the farms were characterized molecularly and chemically. All isolates were examined for the presence of seven aflatoxin biosynthesis genes, and their aflatoxigenicity was confirmed using high performance liquid chromatography with fluorescence detector. Phylogenetic relationships of all isolates were tested using ITS and ß-tubulin genes. Of the seven isolates, two were non-aflatoxigenic, while the remaining were aflatoxigenic based on the presence of all aflatoxin biosynthesis genes tested and the productions of aflatoxins B1 and B2. All isolates were also confirmed as A. flavus following phylogenetic analysis. The indigenous non-aflatoxigenic isolates were further examined for their antagonistic potential against aflatoxigenic isolates on 3% grain corn agar. Both non-aflatoxigenic isolates significantly reduced AFB1 production of the aflatoxigenic isolates. CONCLUSION: The indigenous non-aflatoxigenic A. flavus strains identified in the present work were effective in controlling the aflatoxin production by the aflatoxigenic A. flavus isolates in vitro and can be utilized for in situ testing.

Fusion Expression of Peptides with AflR Binuclear Zinc Finger Motif and Their Enhanced Inhibition of Aspergillus flavus: A Study of Engineered Antimicrobial Peptides.

This study reports a peptide design model for engineering fusion-expressed antimicrobial peptides (AMPs) with the AflR dinuclear zinc finger motif to improve the defense against aflatoxins and Aspergillus flavus. The study identified AflR, a Zn2Cys6-type sequence-specific DNA-binding protein, as a key player in the regulation of aflatoxin biosynthesis. By integrating the AflR motif into AMPs, we demonstrate that these novel fusion peptides significantly lower the minimum inhibitory concentrations (MICs) and reduce aflatoxin B1 and B2 levels, outperforming traditional AMPs. Comprehensive analysis, including bioinformatics and structural determination, elucidates the enhanced structure-function relationship underlying their efficacy. Furthermore, the study reveals the possibility that the fusion peptides have the potential to bind to the DNA binding sites of transcriptional regulators, binding DNA sites of key transcriptional regulators, thereby inhibiting genes critical for aflatoxin production. This research not only deepens our understanding of aflatoxin inhibition mechanisms but also presents a promising avenue for developing advanced antifungal agents, which are essential for global food safety and crop protection.

Bioprospecting endophytic fungi for bioactive metabolites with seed germination promoting potentials.

There is an urgent need for new bioactive molecules with unique mechanisms of action and chemistry to address the issue of incorrect use of chemical fertilizers and pesticides, which hurts both the environment and the health of humans. In light of this, research was done for this work to isolate, identify, and evaluate the germination-promoting potential of various plant species' fungal endophytes. Zea mays L. (maize) seed germination was examined using spore suspension of 75 different endophytic strains that were identified. Three promising strains were identified through screening to possess the ability mentioned above. These strains Alternaria alternate, Aspergilus flavus, and Aspergillus terreus were isolated from the stem of Tecoma stans, Delonix regia, and Ricinus communis, respectively. The ability of the three endophytic fungal strains to produce siderophore and indole acetic acid (IAA) was also examined. Compared to both Aspergillus flavus as well as Aspergillus terreus, Alternaria alternata recorded the greatest rates of IAA, according to the data that was gathered. On CAS agar versus blue media, all three strains failed to produce siderophores. Moreover, the antioxidant and antifungal potentials of extracts from these fungi were tested against different plant pathogens. The obtained results indicated the antioxidant and antifungal activities of the three fungal strains. GC-Mass studies were carried out to determine the principal components in extracts of all three strains of fungi. The three strains' fungus extracts included both well-known and previously unidentified bioactive compounds. These results may aid in the development of novel plant growth promoters by suggesting three different fungal strains as sources of compounds that may improve seed germination. According to the study that has been given, as unexplored sources of bioactive compounds, fungal endophytes have great potential.

Divergent Aspergillus flavus corn population is composed of prolific conidium producers: Implications for saprophytic disease cycle.

The ascomycete fungus Aspergillus flavus infects and contaminates corn, peanuts, cottonseed, and tree nuts with toxic and carcinogenic aflatoxins. Subdivision between soil and host plant populations suggests that certain A. flavus strains are specialized to infect peanut, cotton, and corn despite having a broad host range. In this study, the ability of strains isolated from corn and/or soil in 11 Louisiana fields to produce conidia (field inoculum and male gamete) and sclerotia (resting bodies and female gamete) was assessed and compared with genotypic single-nucleotide polymorphism (SNP) differences between whole genomes. Corn strains produced upward of 47× more conidia than strains restricted to soil. Conversely, corn strains produced as much as 3000× fewer sclerotia than soil strains. Aspergillus flavus strains, typified by sclerotium diameter (small S-strains, <400 µm; large L-strains, >400 µm), belonged to separate clades. Several strains produced a mixture (M) of S and L sclerotia, and an intermediate number of conidia and sclerotia, compared with typical S-strains (minimal conidia, copious sclerotia) and L-strains (copious conidia, minimal sclerotia). They also belonged to a unique phylogenetic mixed (M) clade. Migration from soil to corn positively correlated with conidium production and negatively correlated with sclerotium production. Genetic differences correlated with differences in conidium and sclerotium production. Opposite skews in female (sclerotia) or male (conidia) gametic production by soil or corn strains, respectively, resulted in reduced effective breeding population sizes when comparing male:female gamete ratio with mating type distribution. Combining both soil and corn populations increased the effective breeding population, presumably due to contribution of male gametes from corn, which fertilize sclerotia on the soil surface. Incongruencies between aflatoxin clusters, strain morphotype designation, and whole genome phylogenies suggest a history of sexual reproduction within this Louisiana population, demonstrating the importance of conidium production, as infectious propagules and as fertilizers of the A. flavus soil population.

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.

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.

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.

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.

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.

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.

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.

Magnetization and ZIF-67 modification of Aspergillus flavus biomass for tetracycline removal from aqueous solutions: A stable and efficient composite.

In the present work, the biomass of Aspergillus flavus (AF) was modified using magnetic nanoparticles MnFe2O4 and metal-organic framework of ZIF-67, and its ability to remove tetracycline antibiotic (TCH) was investigated. With the help of physicochemical tests, AF biomass modification with ZIF-67 and MnFe2O4 magnetic nanoparticles was confirmed. Based on the BET value, AF-MnFe2O4-ZIF-67 (139.83 m2/g) has a higher surface value than AF (0.786 m2/g) and AF/MnFe2O4 (17.504 m2/g). Also, the magnetic saturation value revealed that the modified biomass can be isolated from the treated solution using a simple magnetic field. Maximum TCH elimination (99.04%) using AF-MnFe2O4-ZIF-67 was obtained at pH 7, adsorber mass of 1 g/L, adsorption time of 40 min, and TCH content of 10 mg/L. The thermodynamic study indicated that the TCH abatement using the desired composite is spontaneous and exothermic. The experimental results showed that the adsorption process is compatible with the pseudo-second-order kinetic and Freundlich model. The maximum adsorption capacity for AF, AF-MnFe2O4, and AF-MnFe2O4-ZIF-67 was quantified to be 9.75 mg/g, 25.59 mg/g, and 43.87 mg/g, respectively. The reusability of the desired adsorbers was examined in up to 8 steps. The outcomes showed that the adsorbers can be used several times in TCH elimination. The provided composite can remove TCH from hospital wastewater, so it can be suggested for use in water and wastewater treatment works.

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.

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.

Nanofabrication of citronellal with chitosan biopolymer to boost its efficacy against aflatoxin B1 and Aspergillus flavus mediated biodeterioration of active ingredient of Piper longum.

The study reports the efficacy of nanofabricated citronellal inside the chitosan biopolymer (NeCn) against Aspergillus flavus growth, aflatoxin B1 (AFB1) production, and active ingredient biodeterioration (Piperine) in Piper longum L. The prepared NeCn was characterized by Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), and Fourier Transform Infrared Spectroscopy (FTIR). The results revealed that the NeCn exhibited distantly improved antifungal (1.25 µL/mL) and AFB1 inhibition (1.0 µL/mL) compared to free Cn. The perturbances in membrane function, mitochondrial membrane potential, antioxidant defense system, and regulatory genes (Ver-1 and Nor-1) of AFB1 biosynthesis were reported as probable modes of action of NeCn. The NeCn (1.25 µL/mL) effectively protects the P. longum from A. flavus (78.8%), AFB1 contamination (100%), and deterioration of Piperine (62.39%), thus demonstrating its potential as a promising novel antifungal agent for food preservation.

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.

Production of kojic acid by Aspergillus flavus OL314748 using box-Behnken statistical design and its antibacterial and anticancer applications using molecular docking technique.

Kojic acid is a wonderful fungal secondary metabolite that has several applications in the food, medical, and agriculture sectors. Many human diseases become resistant to normal antibiotics and normal treatments. We need to search for alternative treatment sources and understand their mode of action. Aspergillus flavus ASU45 (OL314748) was isolated from the caraway rhizosphere as a non-aflatoxin producer and identified genetically using 18S rRNA gene sequencing. After applying the Box-Behnken statistical design to maximize KA production, the production raised from 39.96 to 81.59 g/l utilizing (g/l) glucose 150, yeast extract 5, KH2PO4 1, MgSO4.7H2O 2, and medium pH 3 with a coefficient (R2) of 98.45%. Extracted KA was characterized using FTIR, XRD, and a scanning electron microscope. Crystalized KA was an effective antibacterial agent against six human pathogenic bacteria (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Serratia marcescens, and Serratia plymuthica). KA achieves high inhibition activity against Bacillus cereus, K. pneumonia, and S. plymuthica at 100 µg/ml concentration by 2.75, 2.85, and 2.85 compared with chloramphenicol which gives inhibition zones 1, 1.1, and 1.6, respectively. Crystalized KA had anticancer activity versus three types of cancer cell lines (Mcf-7, HepG2, and Huh7) and demonstrated high cytotoxic capabilities on HepG-2 cells that propose strong antitumor potent of KA versus hepatocellular carcinoma. The antibacterial and anticancer modes of action were illustrated using the molecular docking technique. Crystalized kojic acid from a biological source represented a promising microbial metabolite that could be utilized as an alternative antibacterial and anticancer agent effectively.

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.