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Peanut is a legume widespread in the world, but its high susceptibility to Aspergillus flavus infection poses a significant challenge due to the risk of aflatoxin contamination. It is predicted that changing climatic conditions will result in warmer, drier periods with elevated CO₂ levels, which promote the growth of A. flavus. The most effective pre-harvest mitigation strategy is the use of non-aflatoxigenic strains of biocontrol; however, future climatic conditions may influence the effectiveness of this practice. Thus, the objective of this study was to assess the impact of simulated climate change conditions on the efficacy of a non-aflatoxigenic A. flavus strain, the active agent of a biocontrol product, in reducing fungal growth and mycotoxin production. A range of temperature conditions (T = 25, 30, 35 °C), water activity (a w  = 0.85, 0.90, 0.95) and CO2 concentration (400, 1000 ppm) were selected for investigation. The assay was conducted using three ratios of A. flavus spore suspensions (100 % aflatoxigenic, 100 % non-aflatoxigenic, 50/50 % aflatoxigenic:non-aflatoxigenic; 105 spores mL -1 ) inoculated in vitro on a peanut-based medium (PBM), and in situ on peanut seeds, and incubated for 10 days. Results of in vitro studies showed a significant influence of T and a w on fungal growth rates (μ), with a reduction when the a w decreased and T shifted from the fungus's optimum of 30 °C. The highest mycotoxin concentration was detected on PBM, with an aflatoxin B1 (AFB1) production by the aflatoxigenic strain in situ 50 % lower than in vitro. However, for all the treatments, the application of the biocontrol agent inhibited AFB1 production with a general reduction of 55 % in vitro and 71 % in situ, even though a significant increase in kojic acid production was observed. The effectiveness of the non-aflatoxigenic strain increased when T was raised up to 35 °C with higher AFB1 reductions both in vitro and in situ, of respectively 58 and 76 %. These observations provided the first evidence that climate change will not negatively influence the ability of the Italian A. flavus non-aflatoxigenic strain, which represents the biocontrol agent of the commercial product AF-X1, to reduce AFB1 contamination in peanuts., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study aimed to optimize a novel green CEO nanoemulsions (CEO NEs) and explore its physicochemical properties and the effect on the shelf-life of strawberries during storage at environmental temperature (20-25 °C). We used CEO as oil phase and tea saponin (TS) as a natural surfactant to formulate the novel green CEO NEs, and its potential as an antimicrobial agent was also investigated. The results showed that CEO NEs had a droplet size about 170 nm with uniform distribution and regularly spherical. These CEO NEs exhibited excellent storage stability, thermal stability, pH stability and centrifugal stability. The antimicrobial test indicated that the minimal inhibitory concentration and the minimal bactericidal (fungicidal) concentration of CEO NEs against Escherichia coli, Botrytis cinerea and Aspergillus flavus were 17.81 μg/mL and 35.62 μg/mL, 35.62 μg/mL and 71.25 μg/mL, 2.23 μg/mL and 4.45 μg/mL, respectively, which were significantly lower than those of pure CEO (333.75 μg/mL and 667.5 μg/mL, 667.5 μg/mL and 1335 μg/mL, 41.72 μg/mL and 83.44 μg/mL). More interestingly, after soaking strawberries in CEO NEs for 2 min, the shelf-life of strawberries can be extended to 7 days at environmental temperature, and a lower rate of weight loss and mildew were showed in the group of CEO NEs than other control groups, especially the strawberries in ultrapure water group went bad first, obviously shranked, and contaminated by molds after 3 days. The above results indicate that CEO NEs prepared in this study has great potential as a new green antimicrobial agent in fruit preservation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nitrogen is important for fungal growth and development, and the GATA transcription factor AreA has been widely studied as a key regulator of nitrogen catabolite repression (NCR) in many fungi. However, AreB, another GATA transcription factor in the NCR pathway, remains less studied, and its role in Aspergillus flavus is still unclear. In this study, we characterized areB in A. flavus and investigated its role in regulating nitrogen utilization, fungal growth, and aflatoxin production. The areB gene produces three transcripts, with areB-α being the most abundantly expressed, particularly under nitrogen-limited conditions. Gene expression analysis via qPCR confirmed that areB acts as a negative regulator of NCR, as its deletion led to the upregulation of NCR-related genes under nitrogen-limiting conditions. Gene function analysis of areB revealed that its deletion impaired hyphal growth, reduced conidia production, and delayed conidial germination. Additionally, deletion of areB led to increased aflatoxin production, particularly under less favorable nitrogen sources, while overexpression of areB reduced aflatoxin levels. Furthermore, areB influenced sclerotia formation in a nitrogen-source-dependent manner. These findings reveal the multifaceted role of areB in nitrogen regulation, fungal development, and secondary metabolism, offering insights for controlling aflatoxin contamination and fungal growth., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The worldwide textile industry extensively uses azo dyes, which pose serious health and environmental risks. Effective cleanup is necessary but challenging. Developing bioremediation methods for textile effluents will improve color removal efficiency. The recent attention to effectively utilizing microbes to convert toxic industrial azo dyes into non-hazardous compounds has garnered significant attention. In the present study, four fungal strains-Aspergillus flavus, Aspergillus terreus, Aspergillus niger, and Fusarium oxysporium-were employed to screen for the degradation and detoxification of azo dyes including congo red, crystal violet, bromophenol blue, and malachite green. After eight days, A. flavus had degraded azo dyes at the maximum proportion. The maximum decolorization (%) was achieved at 50 mg/L of dye concentration, 8 days of incubation, pH 6, 30 °C temperature, sucrose as a carbon source, NaNO 3 as a nitrogen source, Ca +2 as minerals, and using static culture. The efficient production of laccases, lignin peroxidase, and manganese peroxidase enzymes by A. flavus proved that the enzyme played a crucial role in decolorizing the harmful azo dyes. The Fourier Transform Infrared spectrometer (FT-IR) data validated the decolorization and degradation process brought on by absorption and biodegradation. Compared to control plants, the results of the phytotoxicity assay showed that the degraded product was less harmful to maize and common bean plant's growth and germination rates. As a result, the findings indicate that A. flavus is a viable option for remediating azo dyes. This aids in the biodegradation of azo dyes found in wastewater., Competing Interests: Declarations. Ethics approval and consent to participate: This study has complied with relevant institutional, national, and international guidelines and legislation. This study does not contain any studies with human participants or animals performed by any of the authors. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Lysine benzoylation (Kbz) is a newly identified post-translational modification associated with active transcription and metabolism in eukaryotes. However, whether Kbz exists in pathogenic fungi and its function remains unknown. Here, we demonstrated for the first time that Kbz is present in Aspergillus flavus and identified 60 benzoylated sites on 46 benzoylated proteins by global benzoylome analysis. Our data demonstrated that alcohol dehydrogenase B (AdhB) is regulated by benzoylation on lysine 321 (K321), and mutations of Kbz site in AdhB significantly reduced the alcohol dehydrogenase activity in vivo and in vitro . Both adhB deletion mutant and benzoylated site mutants (K321R and K321A) exhibited similar phenotype, including decreased conidiation and seed colonization, increased sclerotia formation and aflatoxin production, and more sensitive to cell wall damage stress. We also found that GcnE has benzoyltransferase activity in vitro and in vivo , and its repression leads to decreased Kbz level and enzymatic activity of AdhB. The catalytic site E139 is important for the benzoyltransferase function of GcnE. Our study uncovers a previously unknown mechanism by which benzoylation regulates AdhB activity to affect the development, secondary metabolism, pathogenicity, and stress response of A. flavus . Meanwhile, it points out the important role of Kbz in the pathogenicity of pathogenic fungi.IMPORTANCE 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., Competing Interests: The authors declare no conflict of interest.

Peanuts and corn are susceptible to various soil-borne fungi, leading to significant economic losses. Atoxigenic Aspergillus flavus have been widely used as biocontrol agents for managing aflatoxin contamination because of their minimal environmental impact, strong competitive ability, and sustained inhibition effect. After multiple identifications and cluster amplification pattern (CAP) analysis, three atoxigenic A. flavus PA04, PA10 and PA67 were isolated from peanut samples in Shandong Province, which can reduce aflatoxin levels by up to 90 %. Our study revealed that atoxigenic A. flavus also competed vigorously with Sclerotium rolfsii and Fusarium proliferatum for nutrition and space, achieving notable inhibition rates of up to 90.4 % and 90.6 %, respectively. The supernatants of atoxigenic A. flavus also inhibited the growth of S. rolfsii and F. proliferatum, with PA67 demonstrating the most significant effect. Whole genome sequencing revealed that PA67 contains multiple glycoside hydrolases and metabolites with antifungal activity. The kojic acid production of PA67 was higher than that of PA04 and PA10, reaching 17.48 g/L, which has a significant inhibition on sclerotia germination. PA67 supernatant significantly inhibited the hyphae growth of S. rolfsii and F. proliferatum, and down-regulated genes related to sclerotia and fumonisin formation. This study demonstrates the biocontrol potential of PA67 against three soil-borne fungi and is the first investigation of atoxigenic A. flavus to inhibit S. rolfsii and F. proliferatum., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aflatoxins are a group of high toxic mycotoxins in food chain. Recent studies showed that aflatoxins might contaminate post-fermented tea, but the result remains controversial. Here, Aspgergillus flavus growth and aflatoxin production were characterized in Puerh tea, peanut and polished rice at different initial water activity (a w ) values for long-term storage. As a result, food initial a w value was the critical factor for A. flavus growth and aflatoxin production, and A. flavus almost not grew on foods at a w value lower than 0.8. A. flavus grew best in peanut, followed by rice, but growth on Puerh tea was limited. A. flavus growth was inhibited significantly by adding tea to Potato Dextrose Agar (PDA). Accordingly, aflatoxins produced dramatically in peanut, followed by rice at the first 90 days storage. However, aflatoxin neither produced in Puerh tea nor on tea modified PDA, indicating tea components inhibited A. flavus growth and aflatoxins synthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the study in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aspergillus flavus occurs as a contaminant of various foods and animal feeds and can produce the mycotoxin aflatoxin B 1 that is a danger to human and animal health. Here, we develop models to predict the behaviour of A. flavus in maize extract agar and maize grains. Growth and aflatoxin B 1 production were recorded on maize extract agar at 20-35 °C and water activities from 0.84 to 0.90. We then obtained probability models-using temperature, water activity, and time as explanatory variables-based on data of growth and aflatoxin B 1 production. Additional data were generated under two dynamically changing temperature regimes. Initial water activity, and relative humidity during incubation, were recorded. Predicted probability of growth under dynamic conditions based on models built under static conditions depended on the temperature regime and substrate, concordance ranging from 66 to 100%, with lower concordances obtained for aflatoxin B 1 production prediction. Interestingly, aflatoxin B 1 production was higher on maize grains than on maize extract agar. Moreover, this work suggests that the safe water activity for a cereal may depend on the previous water activity and temperatures which may have allowed fungal growth and so trigger later toxin production under water stress., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Heat-resistant fungal conidia are a common source of contamination and can cause significant difficulties in producing spawns. Through the use of PCR method, Aspergillus tubingensis and Aspergillus flavus as common microbial contaminants found in wheat grain spawn were identified that had been sterilized at 120 ºc for 2 h. Since these conidia are highly resistant to standard sterilization techniques, alternative methods were used to treat them with NaOCl and cold plasma and evaluate their effectiveness in reducing contamination. Optical emission spectroscopy (OES) analysis of the plasma showed dominant emissions from the N 2 second positive system and N 2 + first negative system, while reactive oxygen species (ROS) spectral lines were undetected due to collision-induced quenching effects. Field Emission Scanning Electron Microscopy (FESEM) and Energy Dispersive X-ray Spectroscopy (EDXS) analyses revealed notable alterations in the elemental makeup of conidia surfaces, as evidenced by a marked rise in levels of Na, O, Cl (in the case of NaOCl treatment) and N (in the case of plasma treatment). The conidia size was reduced at lower levels of NaOCl, but with increased concentrations and plasma treatment, the conidia underwent rupture and, in some cases, pulverization. The research suggests that utilizing a combined approach can be highly effective in eliminating heat-resistant fungal conidia and drastically cutting down the sterilization time for producing wheat spawn to only 30 s., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Research involving plants: All authors comply with the IUCN Policy Statement on Research Involving Species at Risk of Extinction., (© 2024. The Author(s).)

Almonds are susceptible to infestation by Aspergillus flavus , an aflatoxin-producing fungus. The objective of this study was to investigate the effects of kernel type (inshell, shelled and split almonds) on the ability of A. flavus to grow and produce aflatoxins at different combinations of temperature (20, 27 and 35 °C), water activity (0.85, 0.92, 0.95 and 0.98 a w ) and incubation period (10, 20 and 30 days). There was no fungal growth at 0.85 a w on any of the kernel types. At 0.92 a w , only the split kernels supported growth and aflatoxin synthesis. The fungus was able to grow and produce aflatoxins on all three kernels at 0.95-0.98 a w and 20-35 °C. At 0.98 a w , high total aflatoxin concentrations (>300 µg/kg) were found on the shelled and split kernels at all temperatures. On the inshell nuts, the fungus produced up to 372 µg/kg of total aflatoxins at 0.98 a w and 27 °C. Regression analysis showed that significantly higher levels of aflatoxins were produced at 27 °C (as compared to at 20 and 35 °C) on shelled and split almonds. Incubation time was also a significant predictor of aflatoxin accumulation. The results of this study indicated that shipping almonds below 0.85 a w and reducing storage time would significantly decrease the risk of infestation and aflatoxin production by A. flavus .

The epigenetic reader SntB was identified as an important transcriptional regulator of growth, development, and secondary metabolite synthesis in Aspergillus flavus . However, the underlying molecular mechanism is still unclear. In this study, by gene deletion and complementation, we found SntB is essential for mycelia growth, conidial production, sclerotia formation, aflatoxin synthesis, and host colonization. Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) analysis revealed that SntB played key roles in oxidative stress response of A. flavus , influencing related gene activity, especially catC encoding catalase. SntB regulated the expression activity of catC with or without oxidative stress, and was related to the expression level of the secretory lipase (G4B84_008359). The deletion of catC showed that CatC participated in the regulation of fungal morphogenesis, reactive oxygen species (ROS) level, and aflatoxin production, and that CatC significantly regulated fungal sensitive reaction and AFB1 yield under oxidative stress. Our study revealed the potential machinery that SntB regulated fungal morphogenesis, mycotoxin anabolism, and fungal virulence through the axle of from H3K36me3 modification to fungal virulence and mycotoxin biosynthesis. The results of this study shed light into the SntB-mediated transcript regulation pathways of fungal mycotoxin anabolism and virulence, which provided potential strategy to control the contamination of A. flavus and its aflatoxins., Competing Interests: DW, CY, YY, DM, HL, LH, WX, KY, MS, YH, YY, ZZ No competing interests declared, (© 2024, Wu, Yang et al.)

The mycotoxin-producing fungi contamination of foodstuffs and agricultural commodities is a serious problem. In this study, the efficacy of acetic acid (AcA), chitosan (CS), and CS with extra AcA (CS/extra AcA) against Aspergillus flavus (A. flavus) in vitro and in vivo were investigated. Results showed that CS/extra AcA strongly inhibited the mycelial growth and aflatoxins production of A. flavus at lower concentrations than CS (MIC > 16.0 mg/mL) or AcA (MIC: 0.31 %). Significant inhibition was achieved in the presence of 4.0/0.12 and 8.0/0.08 CS (mg/mL)/extra AcA (%). CS/extra AcA exhibited remarkable antifungal activities based on the results of spore germination inhibition, abnormalities of spore morphology, disruption of cell membrane, mitochondrial dysfunction, and induction of apoptosis of hyphae. Moreover, pathogenicity test in peanut kernels showed that 8.0/0.16 CS (mg/mL)/extra AcA (%) could effectively inhibit the fungal infection and aflatoxin B 1 production. The efficacy of CS/extra AcA is possibly due to the synergy of the antifungal property of CS, and undissociated AcA. The findings referred that CS/extra AcA is safe, efficient, and economical, and it could be used as a promising way to control A. flavus contamination in agro-foods preservation., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this study, garlic-oil-combined cellulose fibers were prepared by using Borassus flabellifer (Asian Palmyra palm) to enhance the post-harvest shelf life of tomatoes. The physicochemical properties of the prepared cellulose fibers were characterized by using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and field emission scanning electron microscopy (FE-SEM). The B. flabellifer cellulose fibers combined with garlic oil (BCF/GO) coatings exhibited significant antifungal properties against Aspergillus flavus. In addition, the BCF/GO coating resulted in a notable extension in the shelf life of tomatoes regarding parameters such as weight loss, firmness, pH, ascorbic acid content, lycopene level, moisture content, and titratable acidity after 25 days of storage at 25-29 °C with 85 % relative humidity. The synergistic combination of BCF and GO presents a natural and sustainable solution for extending the shelf life of tomatoes with the potential to significantly reduce post-harvest losses in the food industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

To improve the application value of peanuts, the fungicidal effect and physicochemical properties of the protein in peanuts were investigated by combining high voltage atmospheric cold plasma (HVCP) and ultraviolet-cold plasma (UVCP) in this study. Compared to the single HVCP or UVCP treatment, the combined treatments exhibited a higher fungicidal efficiency of A. flavus spores in peanuts, decreasing by 0.79-2.97 log 10 cfu/g after 8-min treatment. The A. flavus growth and aflatoxin production in peanuts during storage were also lower than the single plasma groups. Moreover, cold plasma treatments could modify the molecular structures of protein in peanuts by changing secondary and tertiary structures, decreasing particle size and increasing zeta potential, which contributed to improve the solubility and emulsification of protein. Overall, this research provides a unique strategy for the combined application of cold plasma in grain decontamination and protein modification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The peanut seed coat acts as a physical and biochemical barrier against Aspergillus flavus infection; however, the nature of the inhibitory chemicals in the peanut seed coat in general is not known. This study identified and characterized peanut seed coat metabolites that inhibit A. flavus growth and aflatoxin contamination. Selected peanut accessions grown under well-watered and water-deficit conditions were assayed for A. flavus resistance, and seed coats were metabolically profiled using liquid chromatography mass spectrometry. Kyoto Encyclopedia of Genes and Genome phenylpropanoid pathway reference analysis resulted in the identification of several seed coat metabolic compounds, and ten selected metabolites were tested for inhibition of A. flavus growth and aflatoxin contamination. Radial growth bioassay demonstrated that 2,5-dihydroxybenzaldehyde inhibited A. flavus growth (98.7%) and reduced the aflatoxin contamination estimate from 994 to 1 μg/kg. Scanning electron micrographs showed distorted hyphae and conidiophores in cultures of 2,5-dihydroxybenzaldehyde-treated A. flavus , indicating its potential use for field application as well as seed coat metabolic engineering.

Mycotoxins are representative contaminants causing food losses and food safety problems worldwide. Thymol can effectively inhibit pathogen infestation and aflatoxin accumulation during grain storage, but high volatility limits its application. Here, a thymol-betaine co-crystal system was synthesized through grinding-induced self-assembly. The THY-TMG co-crystal exhibited excellent thermal stability with melting point of 91.2 °C owing to abundant intermolecular interactions. Remarkably, after 15 days at 30 °C, the release rate of thymol from co-crystal was only 55%, far surpassing that of pure thymol. Notably, the co-crystal demonstrated the ability to bind H 2 O in the environment while controlling the release of thymol, essentially acting as a desiccant. Moreover, the co-crystals effectively inhibited the growth of Aspergillus flavus and the biosynthesis of aflatoxin B 1 . In practical terms, the THY-TMG co-crystal was successful in preventing AFB 1 contamination and nutrients loss in peanuts, thereby prolonging their shelf-life under conditions of 28 °C and 70% RH., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The toxicity of the contaminated powder contributed to toxic aflatoxins has been well-known in the literature. However, before this study, the specific fungal strain behind aflatoxin production remained unidentified. Our research aimed to isolate and identify fungi from the tainted sandwiches while also assessing the preservation of sandwiches in ambient conditions. The study pinpointed Aspergillus flavus as the fungus responsible for aflatoxin production. Analysis revealed that the sandwich samples contaminated with pure A. flavus exhibited a significant Aflatoxin B1 (AFB1) concentration of 55.2 ± 0.21 ng/g, accompanied by a spore count of 2 × 10 6 Colony-Forming Unit (CFU)/g after ten days. In contrast, sandwich samples contaminated with the unspecified fungi displayed a lower AFB1 content of 16.21 ± 0.42 ng/g, with a spore count of 2.2 × 10 2  CFU/g after the same duration. In the prevention study, the efficacy of the ethanol spray method for inhibiting aflatoxin from A. flavus was investigated. Results demonstrated that a 70 % ethanol concentration at a ratio of 2.0 % total weight of the sandwich proved highly effective, significantly impeding fungal growth. This method extended the preservation time by sevenfold compared to the control. Importantly, tests at 2.0 % ethanol of the sandwich weight did not detect aflatoxin presence., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aspergillus flavus infects important crops and produces carcinogenic aflatoxins, posing a serious threat to food safety and human health. Biochemical analysis and RNA-seq were performed to investigate the effects and mechanisms of piperitone on A. flavus growth and aflatoxin B1 biosynthesis. Piperitone significantly inhibited the growth of A. flavus, AFB1 production, and its pathogenicity on peanuts and corn flour. Differentially expressed genes (DEGs) associated with the synthesis of chitin, glucan, and ergosterol were markedly down-regulated, and the ergosterol content was reduced, resulting in a disruption in the integrity of the cell wall and cell membrane. Moreover, antioxidant genes were down-regulated, the correspondingly activities of antioxidant enzymes such as catalase, peroxidase, and superoxide dismutase were reduced, and levels of superoxide anion and hydrogen peroxide were increased, leading to a burst of reactive oxygen species (ROS). Accompanied by ROS accumulation, DNA fragmentation and cell autophagy were observed, and 16 aflatoxin cluster genes were down-regulated. Overall, piperitone disrupts the integrity of the cell wall and cell membrane, triggers the accumulation of ROS, causes DNA fragmentation and cell autophagy, ultimately leading to defective growth and impaired AFB1 biosynthesis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aspergillus flavus conidia are widespread in air; they attach to food and feed crops and secrete aflatoxins, which results in serious contamination. Germination of A. flavus conidia is the most critical step in contamination of food by A. flavus . This study aims to gain an insight into A. flavus conidia through dormancy to germination to provide a theoretical basis for inhibition of A. flavus conidia germination. The morphological changes and regulation mechanism of A. flavus conidia germination at 0, 4, 8, and 12 hours were observed. Transcriptomic and metabolomic analyses showed that conidia became active from dormancy (0 hour) to the initial stage of germination (4 hours), cellular respiration and energy metabolism increased, and amino acids and lipids were synthesized rapidly. The number of differentially expressed genes and differential metabolites was highest at this stage. Besides, we found that conidia germination had selectivity for different carbon and nitrogen sources. Compared with monosaccharides, disaccharides, as the only carbon source, significantly promoted the germination of conidia. Moreover, MepA , one of genes in the ammonium transporter family was studied. The gene deletion mutant ΔMepA had a significant growth defect, and the expression of MeaA was significantly upregulated in ΔMepA compared with the wild-type, indicating that both MepA and MeaA played an important role in transporting ammonium ions.IMPORTANCEThis is the first study to use combined transcriptomic and metabolomics analyses to explore the biological changes during germination of Aspergillus flavus conidia. The biological process with the highest changes occurred in 0-4 hours at the initial stage of germination. Compared with polysaccharides, monosaccharides significantly increased the size of conidia, while significantly decreasing the germination rate of conidia. Both MeaA and MepA were involved in ammonia transport and metabolism during conidia germination., Competing Interests: The authors declare no conflict of interest.

Background: The recurrent contaminations of feed materials with mycotoxigenic fungi can endanger both farmed animals and humans. Biosynthesized nanomaterials are assumingly the ideal agents to overcome fungal invasion in feed/foodstuffs, especially when utilizing sustainable sources for synthesis. Herein, the phycosynthesis of selenium nanoparticles (SeNPs) was targeted using Cystoseira myrica algal extract (CE), and the conjugation of CE/SeNPs with chitosan nanoparticles (NCt) to produce potential antifungal nanocomposites for controlling Aspergillus flavus isolates in fish feed., Results: The phycosynthesis of SeNPs with CE was effectually carried out and validated using visible/UV analysis, X-ray diffraction and transmission microscopy; CE/SeNPs had diameters of 8.7 nm and spherical shapes. NCt/CE/SeNPs nanocomposite (173.3 nm mean diameter) was achieved and the component interactions were validated via infrared spectroscopic analysis. The antifungal assessment of screened nanomaterials against three Aspergillus flavus strains indicated that NCt/CE/SeNPs exceeded the fluconazole action using qualitative/quantitative assays. Severe alteration/distortions in A. flavus mycelial structure and morphology were microscopically observed within 48 h of NCt/CE/SeNPs treatment. The treatment of feed ingredients (crushed corn and feed powder) by blending with nanomaterials (NCt, CE/SeNPs and NCt/CE/SeNPs) led to significant reduction in A. flavus count/growth after storage for 7 days; NCt/CE/SeNPs could completely inhibit any fungal growth in feed material., Conclusion: The pioneering phycosynthesis of CE/SeNPs and their nanoconjugation with NCt generated bioactive antifungal agents to control A. flavus strains. The innovatively constructed NCt/CE/SeNPs nanocomposite is advised for application as an effectual, biosafe and natural fungicidal conjugate for the protection of fish feed from mycotoxigenic fungi. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Aspergillus flavus , the primary mold that causes food spoilage, poses significant health and economic problems worldwide. Eliminating A. flavus growth is essential to ensure the safety of agricultural products, and extracellular compounds (ECCs) produced by Bacillus spp. have been demonstrated to inhibit the growth of this pathogen. In this study, we aimed to identify microorganisms efficient at inhibiting A. flavus growth and degrading aflatoxin B 1 . We isolated microorganisms from soil samples using a culture medium containing coumarin (CM medium) as the sole carbon source. Of the 498 isolates grown on CM medium, only 132 bacterial strains were capable of inhibiting A. flavus growth. Isolate 3BS12-4, identified as Bacillus siamensis , exhibited the highest antifungal activity with an inhibition ratio of 43.10%, and was therefore selected for further studies. The inhibition of A. flavus by isolate 3BS12-4 was predominantly attributed to ECCs, with a minimum inhibitory concentration and minimum fungicidal concentration of 0.512 g/ml. SEM analysis revealed that the ECCs disrupted the mycelium of A. flavus . The hydrolytic enzyme activity of the ECCs was assessed by protease, β-1,3-glucanase, and chitinase activity. Our results demonstrate a remarkable 96.11% aflatoxin B 1 degradation mediated by ECCs produced by isolate 3BS12-4. Furthermore, treatment with these compounds resulted in a significant 97.93% inhibition of A. flavus growth on peanut seeds. These findings collectively present B. siamensis 3BS12-4 as a promising tool for developing environmentally friendly products to manage aflatoxin-producing fungi and contribute to the enhancement of agricultural product safety and food security.

Application of actinobacteria has grown exponentially in recent years in sustainable agricultural. Most actinobacterial inoculants are tailored to function as either biocontrol agents or biofertilizers. Hence, there is the need to obtain and include multifunctional actinobacterial strains in inocula formulations. In this research, 90 actinobacterial isolates were isolated from rhizospheric and non-rhizospheric soils of Algerian Saharan arid regions and were screened for their activity against the phytopathogenic fungi Alternaria alternata, Aspergillus flavus, Botrytis cinerea, Fusarium oxysporum, and Fusarium solani. Five isolates that inhibited at least three of these fungi were characterized according to morphological, environmental and biochemical parameters, and were preliminarily identified as Streptomyces enissocaesilis A1, Streptomyces olivoverticillatus A5, Streptomyces erumpens A6, Streptomyces cavourensis A8, and Streptomyces microflavus A20. These strains were then screened for plant growth promoting activities. All strains produced siderophores, hydrocyanic acid, ammonia and the auxin indole-3-acetic acid (IAA) and were capable of solubilizing phosphate. The highest producer of siderophores (69.19 percent siderophore units), ammonia (70.56 μg mL -1 ) and IAA (148.76 μg mL -1 ) was strain A8, A20, and A5, respectively. These findings showed that the five actinobacteria are multipurpose strains with simultaneous antifungal and plant growth promoting activities and have the potential to be used for sustainable agricultural practices, particularly in arid regions., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The climate-change-coupled fungal burden in crop management and the need to reduce chemical pesticide usage highlight the importance of finding sustainable ways to control Aspergillus flavus . This study examines the effectiveness of 50 Pseudomonas isolates obtained from corn rhizospheres against A. flavus in both solid and liquid co-cultures. The presence and quantity of aflatoxin B1 (AFB1) and AFB1-related compounds were determined using high-performance liquid chromatography-high resolution mass spectrometry analysis. Various enzymatic- or non-enzymatic mechanisms are proposed to interpret the decrease in AFB1 production, accompanied by the accumulation of biosynthetic intermediates (11-hydroxy-O-methylsterigmatocystin, aspertoxin, 11-hydroxyaspertoxin) or degradation products (the compounds C 16 H 10 O 6 , C 16 H 14 O 5 , C 18 H 16 O 7 , and C 19 H 16 O 8 ). Our finding implies the upregulation or enhanced activity of fungal oxidoreductases and laccases in response to bacterial bioactive compound(s). Furthermore, non-enzymatic reactions resulted in the formation of additional degradation products due to acid accumulation in the fermented broth. Three isolates completely inhibited AFB1 or any AFB1-related compounds without significantly affecting fungal growth. These bacterial isolates supposedly block the entire pathway for AFB1 production in the fungus during interaction. Apart from identifying effective Pseudomonas isolates as potential biocontrol agents, this work lays the foundation for exploring new bacterial bioactive compounds.

The study reports the efficacy of nanofabricated citronellal inside the chitosan biopolymer (NeCn) against Aspergillus flavus growth, aflatoxin B 1 (AFB 1 ) 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 AFB 1 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 AFB 1 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%), AFB 1 contamination (100%), and deterioration of Piperine (62.39%), thus demonstrating its potential as a promising novel antifungal agent for food preservation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Contamination of crop seeds and feed with Aspergillus flavus and its associated aflatoxins presents a significant threat to human and animal health due to their hepatotoxic and carcinogenic properties. To address this challenge, researchers have screened for potential biological control agents in peanut soil and pods. This study identified a promising candidate, a strain of the nonpigmented bacterium, Achromobacter xylosoxidans ZJS2-1, isolated from the peanut rhizosphere in Zhejiang Province, China, exhibiting notable antifungal and antiaflatoxin activities. Further investigations demonstrated that ZJS2-1 active substances (ZAS) effectively inhibited growth at a MIC of 60 μL/mL and nearly suppressed AFB1 production by 99%. Metabolomic analysis revealed that ZAS significantly affected metabolites involved in cell wall and membrane biosynthesis, leading to compromised cellular integrity and induced apoptosis in A. flavus through the release of cytochrome c . Notably, ZAS targeted SrbA, a key transcription factor involved in ergosterol biosynthesis and cell membrane integrity, highlighting its crucial role in ZJS2-1's biocontrol mechanism. Moreover, infection of crop seeds and plant wilt caused by A. flavus can be efficiently alleviated by ZAS. Additionally, ZJS2-1 and ZAS demonstrated significant inhibitory effects on various Aspergillus species, with inhibition rates ranging from 80 to 99%. These findings highlight the potential of ZJS2-1 as a biocontrol agent against Aspergillus species, offering a promising solution to enhance food safety and protect human health.

Fungal contaminations of cereal grains are a profound food-safety and food-security concern worldwide, threatening consumers' and animals' health and causing enormous economic burdens. Because far-ultraviolet C (far-UVC) light at 222 nm has recently been shown to be human-safe, we investigated its efficacy as an alternative to thermal, chemical, and conventional 254 nm UVC anti-fungal treatments. Our microplasma-based far-UVC lamp system achieved a 5.21-log reduction in the conidia of Aspergillus flavus suspended in buffer with a dose of 1032.0 mJ/cm 2 , and a 5.11-log reduction of Fusarium graminearum conidia in suspension with a dose of 619.2 mJ/cm 2 . We further observed that far-UVC treatments could induce fungal-cell apoptosis, alter mitochondrial membrane potential, lead to the accumulation of intracellular reactive oxygen species, cause lipid peroxidation, and result in cell-membrane damage. The lamp system also exhibited a potent ability to inhibit the mycelial growth of both A. flavus and F. graminearum. On potato dextrose agar plates, such growth was completely inhibited after doses of 576.0 mJ/cm 2 and 460.8 mJ/cm 2 , respectively. To test our approach's efficacy at decontaminating actual cereal grains, we designed a cubical 3D treatment chamber fitted with six lamps. At a dose of 780.0 mJ/cm 2 on each side, the chamber achieved a 1.88-log reduction of A. flavus on dried yellow corn kernels and a 1.11-log reduction of F. graminearum on wheat grains, without significant moisture loss to either cereal type (p > 0.05). The treatment did not cause significant changes in the propensity of wheat grains to germinate in the week following treatment (p > 0.05). However, it increased the germination propensity of corn kernels by more than 71% in the same timeframe (p < 0.05). Collectively, our results demonstrate that 222 nm far-UVC radiation can effectively inactivate fungal growth in liquid, on solid surfaces, and on cereal grains. If scalable, its emergence as a safe, cost-effective alternative tool for reducing fungi-related post-harvest cereal losses could have important positive implications for the fight against world hunger and food insecurity., Competing Interests: Declaration of competing interest The authors declare the following competing financial interest(s): Professor Yi-Cheng Wang is concurrently collaborating with the co-founder and chief technology officer of Eden Park Illumination, Inc., Dr. Sung-Jin Park, on a Small Business Innovation Research project sponsored by the U.S. Department of Energy, which is unconnected to the present work. Dr. Park provided technical support with the far-UVC lamps used in the present work, but had no direct involvement in it, and placed no restrictions on any part of its study design; its data collection, analysis, or interpretation; or its publication., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Phellinus caribaeo-quercicola is a basidiomycetous fungus, isolated as an endophyte in this study from the healthy and symptomless leaves of Inula racemosa Hook. f., an important medicinal herb growing in Kashmir Himalaya. This study combines morphological, molecular and phylogenetic techniques to identify the fungal endophyte, using the ITS sequence of nrDNA. A detached leaf assay was conducted to assess the pathogenicity of the fungal endophyte suggesting its mutually symbiotic relationship with the host. The authors also investigated the antifungal potential of the isolated endophytic strain to ascertain its use as a biocontrol agent. The study shows that P. caribaeo-quercicola INL3-2 strain exhibits biocontrol activity against four key fungal phytopathogens that cause significant agronomic and economic losses: Aspergillus flavus, Aspergillus niger, Fusarium solani, and Fusarium oxysporum. Notably, P. caribaeo-quercicola INL3-2 strain is highly effective against A. flavus, with an inhibition percentage of 57.63%. In addition, this study investigates the antioxidant activity of P. caribaeo-quercicola INL3-2 strain crude extracts using ethyl acetate and methanol as solvents. The results showed that the methanolic fraction of P. caribaeo-quercicola exhibits potential as an antioxidant agent, with an IC 50 value of 171.90 ± 1.15 µg/mL. This investigation is first of its kind and marks the initial report of this fungal basidiomycete, P. caribaeo-quercicola, as an endophyte associated with a medicinal plant. The findings of this study highlight the potential of P. caribaeo-quercicola INL3-2 strain as a dual-action agent with both biocontrol and antioxidant properties consistent with the medicinal properties of Inula racemosa. This endophytic fungus could be a promising source of natural compounds for use in agriculture, medicine, and beyond., (© 2024 Wiley‐VCH GmbH.)

Food crops around the world are commonly contaminated with Aspergillus flavus , which can produce the carcinogenic mycotoxin aflatoxin B1 (AFB1). The objective of this study is to test an X-ray irradiation sterilization method for studying AFB1 in contaminated maize samples in the laboratory. Maize that had been naturally contaminated with 300 ppb AFB1 by the growth of aflatoxigenic A. flavus was ground and then irradiated at 0.0, 1.0, 1.5, 2.0, 2.5, and 3.0 kGy. A. flavus was quantified by dilution plating on potato dextrose agar (PDA) and modified Rose Bengal media (MDRB) for viability and qPCR for gene presence. AFB1 was quantified by HPLC and ELISA. A. flavus viability, but not gene copies, significantly decreased with increasing doses of radiation (PDA: p < 0.001; MDRB: p < 0.001; qPCR: p = 0.026). AFB1 concentration did not significantly change with increasing doses of radiation (HPLC: p = 0.153; ELISA: p = 0.567). Our results imply that X-ray irradiation is an effective means of reducing viable A. flavus without affecting AFB1 concentrations. Reducing the hazard of fungal spores and halting AFB1 production at the targeted dose are important steps to safely and reproducibly move forward research on the global mycotoxin challenge.

Chemical pesticides help reduce crop loss during production and storage. However, the carbon footprints and ecological costs associated with this strategy are unsustainable. Here, we used three in vitro models to characterize how different Trichoderma species interact with two aflatoxin producers, Aspergillus flavus and Aspergillus parasiticus , to help develop a climate-resilient biological control strategy against aflatoxigenic Aspergillus species. The growth rate of Trichoderma species is a critical factor in suppressing aflatoxigenic strains via physical interactions. The dual plate assay suggests that Trichoderma mainly suppresses A. flavus via antibiosis, whereas the suppression of A. parasiticus occurs through mycoparasitism. Volatile organic compounds (VOCs) produced by Trichoderma inhibited the growth of A. parasiticus (34.6 ± 3.3%) and A. flavus (20.9 ± 1.6%). The VOCs released by T. asperellum BTU and T. harzianum OSK-34 were most effective in suppressing A. flavus growth. Metabolites secreted by T. asperellum OSK-38, T. asperellum BTU, T. virens OSK-13, and T. virens OSK-36 reduced the growth of both aflatoxigenic species. Overall, T. asperellum BTU was the most effective at suppressing the growth and aflatoxin B1 production of both species across all models. This work will guide efforts to screen for effective biological control agents to mitigate aflatoxin accumulation.

The target of rapamycin (TOR) signaling pathway is highly conserved and plays a crucial role in diverse biological processes in eukaryotes. Despite its significance, the underlying mechanism of the TOR pathway in Aspergillus flavus remains elusive. In this study, we comprehensively analyzed the TOR signaling pathway in A. flavus by identifying and characterizing nine genes that encode distinct components of this pathway. The FK506-binding protein Fkbp3 and its lysine succinylation are important for aflatoxin production and rapamycin resistance. The TorA kinase plays a pivotal role in the regulation of growth, spore production, aflatoxin biosynthesis, and responses to rapamycin and cell membrane stress. As a significant downstream effector molecule of the TorA kinase, the Sch9 kinase regulates aflatoxin B 1 (AFB 1 ) synthesis, osmotic and calcium stress response in A. flavus, and this regulation is mediated through its S&#95;TKc, S&#95;TK&#95;X domains, and the ATP-binding site at K340. We also showed that the Sch9 kinase may have a regulatory impact on the high osmolarity glycerol (HOG) signaling pathway. TapA and TipA, the other downstream components of the TorA kinase, play a significant role in regulating cell wall stress response in A. flavus . Moreover, the members of the TapA-phosphatase complexes, SitA and Ppg1, are important for various biological processes in A. flavus , including vegetative growth, sclerotia formation, AFB 1 biosynthesis, and pathogenicity. We also demonstrated that SitA and Ppg1 are involved in regulating lipid droplets (LDs) biogenesis and cell wall integrity (CWI) signaling pathways. In addition, another phosphatase complex, Nem1/Spo7, plays critical roles in hyphal development, conidiation, aflatoxin production, and LDs biogenesis. Collectively, our study has provided important insight into the regulatory network of the TOR signaling pathway and has elucidated the underlying molecular mechanisms of aflatoxin biosynthesis in A. flavus ., Competing Interests: GL, XC, ET, QZ, SW No competing interests declared, (© 2023, Li et al.)

Root-knot nematodes (RKNs) are a vital pest that causes significant yield losses and economic damage to potato plants. The use of chemical pesticides to control these nematodes has led to environmental concerns and the development of resistance in the nematode populations. Endophytic fungi offer an eco-friendly alternative to control these pests and produce secondary metabolites that have nematicidal activity against RKNs. The objective of this study is to assess the efficacy of Aspergillus flavus (ON146363), an entophyte fungus isolated from Trigonella foenum-graecum seeds, against Meloidogyne incognita in filtered culture broth using GC-MS analysis. Among them, various nematicidal secondary metabolites were produced: Gadoleic acid, Oleic acid di-ethanolamide, Oleic acid, and Palmitic acid. In addition, biochemical compounds such as Gallic acid, Catechin, Protocatechuic acid, Esculatin, Vanillic acid, Pyrocatechol, Coumarine, Cinnamic acid, 4, 3-indol butyl acetic acid and Naphthyl acetic acid by HPLC. The fungus was identified through morphological and molecular analysis, including ITS 1-4 regions of ribosomal DNA. In vitro experiments showed that culture filtrate of A. flavus had a variable effect on reducing the number of egg hatchings and larval mortality, with higher concentrations showing greater efficacy than Abamectin. The fungus inhibited the development and multiplication of M. incognita in potato plants, reducing the number of galls and eggs by 90% and 89%, respectively. A. flavus increased the activity of defense-related enzymes Chitinas, Catalyse, and Peroxidase after 15, 45, and 60 days. Leaching of the concentrated culture significantly reduced the second juveniles' stage to 97% /250 g soil and decreased the penetration of nematodes into the roots. A. flavus cultural filtrates via soil spraying improved seedling growth and reduced nematode propagation, resulting in systemic resistance to nematode infection. Therefore, A. flavus can be an effective biological control agent for root-knot nematodes in potato plants. This approach provides a sustainable solution for farmers and minimizes the environmental impact., (© 2024. The Author(s).)

Aspergillus flavus and its carcinogenic secondary metabolites, aflatoxins, not only cause serious losses in the agricultural economy, but also endanger human health. Rhein, a compound extracted from the Chinese herbal medicine Rheum palmatum L. (Dahuang), exhibits good anti-inflammatory, anti-tumor, and anti-oxidative effects. However, its effect and underlying mechanisms against Aspergillus flavus have not yet been fully illustrated. In this study, we characterized the inhibition effect of rhein on A. flavus mycelial growth, sporulation, and aflatoxin B 1 (AFB 1 ) biosynthesis and the potential mechanism using RNA-seq analysis. The results indicate that A. flavus mycelial growth and AFB 1 biosynthesis were significantly inhibited by 50 μM rhein, with a 43.83% reduction in colony diameter and 87.2% reduction in AFB 1 production. The RNA-seq findings demonstrated that the differentially expressed genes primarily participated in processes such as spore formation and development, the maintenance of cell wall and membrane integrity, management of oxidative stress, the regulation of the citric acid cycle, and the biosynthesis of aflatoxin. Biochemical verification experiments further confirmed that 50 μM rhein effectively disrupted cell wall and membrane integrity and caused mitochondrial dysfunction through disrupting energy metabolism pathways, leading to decreased ATP synthesis and ROS accumulation, resulting in impaired aflatoxin biosynthesis. In addition, a pathogenicity test showed that 50 μM rhein inhibited A. flavus spore growth in peanut and maize seeds by 34.1% and 90.4%, while AFB 1 biosynthesis was inhibited by 60.52% and 99.43%, respectively. In conclusion, this research expands the knowledge regarding the antifungal activity of rhein and provides a new strategy to mitigate A. 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 AFB 1 biosynthesis, exhibiting a promising potential for food industry applications as a biopreservative and biocontrol agent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s).)

Aspergillosis is one of the most common fungal infections that can threaten individuals with immune compromised condition. Due to the increasing resistance of pathogens to the existing antifungal drugs, it is difficult to tackle such disease conditions. Whereas, nikkomycin is an emerging safe and effective antifungal drug which causes fungal cell wall disruption by inhibiting chitin synthase. Hence, the study aims at the development of nikkomycin loaded PEG coated PLGA nanoparticles for its increased antifungal efficiency and inhibiting Aspergillus infections. The P-PLGA-Nik NPs were synthesized by w/o/w double emulsification method which resulted in a particle size of 208.3 ± 15 nm with a drug loading of 52.97 %. The NPs showed first order diffusion-controlled drug release which was sustained for 24 h. These nanoparticle's antifungal efficacy was tested using the CLSI - M61 guidelines and the MIC 50 defined against Aspergillus flavus and Aspergillus fumigatus was found to be >32 μg/ml which was similar to the nikkomycin MIC. The hyphal tip bursting showed the fungal cell wall disruption. The non-cytotoxic and non-haemolytic nature highlights the drug safety profile., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

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

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 B 1 and B 2 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 B 1 and B 2 , 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., (© 2024. The Author(s).)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aflatoxins are carcinogens produced by the fungi Aspergillus flavus and A. parasiticus that contaminate pistachio crops. International markets reject pistachio when aflatoxins exceed permitted maximum levels. Releasing the atoxigenic strain AF36 of A. flavus is the leading aflatoxin pre-harvest control method. The product AF36 Prevail, sorghum grains coated with AF36 propagules, has been used in California since 2017. However, a high percentage of grains of the Prevail fail to sporulate in orchards. Here, the effect of soil moisture on the percentage of AF36 product grains sporulating (SG) and the quantity of spores per grain using a sporulation index (SI) was determined. Under controlled conditions, SG was higher than 85% when soil moisture was 13% or more, and SI increased with increasing soil moisture from 8.4 to 21%. The highest AF36 sporulation occurred near the micro-sprinklers when the grains were not impacted by the irrigation water drops. Arthropod predation was responsible for lost product grains, which was more pronounced in non-tilled soil than in tilled soil. Dispersal of the AF36 spores decreased markedly with the height and distance from the inoculum source, following a pattern of diffusion equations. However, AF36 spores easily reached canopies of pistachios located 10 m from the inoculum source. Our results indicate that AF36 Prevail should be applied close to the irrigation line in the moist soil area but avoiding the areas where excess irrigation causes water accumulation. The biocontrol of aflatoxins in California's pistachio production areas was optimized by improving the field realization of the biological control agent., Competing Interests: The author(s) declare no conflict of interest.

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&#95;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 Δ vepN 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, Δ vepN 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 .

Aspergillus flavus is a common saprophytic aerobic fungus in oil crops that poses a serious threat worldwide with the carcinogenic aflatoxin. Prevention of aflatoxin B1 contamination has great significance to ensure food safety and reduce the economic loss. The present work focuses on the antagonistic activity against A. flavus growth in peanuts by fumigation with dimethyl trisulfide. The results indicated that dimethyl trisulfide exhibits great antifungal activity against A. flavus. The conidial germination and mycelial growth of A. flavus were completely suppressed after exposure to 15 and 20 µl/l of dimethyl trisulfide, respectively. Numerous deformed conidia were found after exposure to dimethyl trisulfide at high concentration (≥20 µl/l). Scanning electron microscope observation demonstrated that dimethyl trisulfide induced severely shrinking mycelia of A. flavus. The results of OD-260 nm absorption and rhodamine-123 fluorescent staining indicated that cell membrane and mitochondria may be legitimate antifungal targets of dimethyl trisulfide. Dimethyl triethyl has a significant inhibitory effect on A. flavus infection in peanuts. In addition, dimethyl trisulfide could reduce the production of aflatoxin B1 via down-regulation of toxin synthesis and regulatory gene expression. Dimethyl trisulfide can be a tremendous potential agent for the biological control of A. flavus and deepened our understanding of the anti-fungal mechanisms of volatile organic compounds., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Aspergillus flavus aflR , a gene encoding a Zn(II) 2 Cys 6 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 Δ aflR 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 B 1 (AFB 1 ) of the Δ aflR strain was 180 ng/mL and aflatoxin B 2 (AFB 2 ) 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 Δ aflR 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 Δ aflR 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 Δ aflR 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 Δ aflR 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 Δ aflR and OE strains to demonstrate the role of aflR in growth and development of A. flavus. For the first time, we confirmed that the Δ aflR 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 Δ aflR 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 .

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 B 1 (AfB 1 ) content of 15-day-old Af-9 cultures; 4 were ineffective, and 2 increased AfB 1 . Reduction of AfB 1 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 AfB 1 production and vice versa, we investigated the mechanism of reduction of AfB 1 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 AfB 1 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.

Mimosa tenuiflora aqueous extract (MAE) is rich in phenolic compounds. Among them, condensed tannins have been demonstrated to exhibit a strong antioxidant and antiaflatoxin B1 activities in Aspergillus flavus . Since antioxidant capacity can change with time due to environmental interactions, this study aimed to evaluate the ability of encapsulation by spray-drying of Mimosa tenuiflora aqueous extract to preserve their biological activities through storage. A dry formulation may also facilitate transportation and uses. For that, three different wall materials were used and compared for their efficiency. Total phenolic content, antioxidant activity, antifungal and antiaflatoxin activities were measured after the production of the microparticles and after one year of storage at room temperature. These results confirmed that encapsulation by spray-drying using polysaccharide wall materials is able to preserve antiaflatoxin activity of Mimosa tenuiflora extract better than freezing.

Background: The majority of studies with essential oils in foods focus mainly on improving the shelf life of products; however, the present study goes further and demonstrates not only the effect of essential oil on conservation properties, but also the effect of free and encapsulated orange essential oil (OEO) on the technological, sensorial and digestibility properties of bakery products., Results: OEO was encapsulated into β-cyclodextrin (β-CD) by inclusion complex formation (β-CD/OEO 97.4% of encapsulation efficiency). OEO demonstrated in vitro antifungal activity against Aspergillus flavus (inhibition zone of 11.33 mm on mycelial growth). In situ antifungal activity against A. flavus confirmed that free OEO can effectively delay the fungal growth, unlike encapsulated OEO. Regarding texture profile and starch digestibility: cake with β-CD/OEO showed lower hardness (31.64 N) and lower starch digestibility (69.10%) than cake with free OEO (44.30 N; 82.10%, respectively) and the addition of OEO (both free and encapsulated) decreased the adhesiveness of the cakes. Cake with free OEO showed a higher intensity of orange aroma, being preferred by 60% of panelists, whereas cake with β-CD/OEO presented a very slight orange taste and aroma., Conclusion: The encapsulation of OEO into β-CD improved the crumb texture of cakes and promoted a lower starch digestibility in the cakes. On the other hand, the encapsulation process was not effective under the conditions tested (OEO concentration and baking temperatures), compromising the action of the OEO as a natural flavoring and preservative agent. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Climate change will increase the co-occurrence of Fusarium verticillioides and Aspergillus flavus , along with their mycotoxins, in European maize. In this study, the expression profiles of two pathogenesis-related ( PR ) genes and four mycotoxin biosynthetic genes, FUM1 and FUM13 , fumonisin pathway, and aflR and aflD , aflatoxin pathway, as well as mycotoxin production, were examined in kernels and in artificial medium after a single inoculation with F. verticillioides or A. flavus or with the two fungi in combination. Different temperature regimes (20, 25 and 30 °C) over a time-course of 21 days were also considered. In maize kernels, PR genes showed the strongest induction at 25 °C in the earlier days post inoculation (dpi)with both fungi inoculated singularly. A similar behaviour was maintained with fungi co-occurrence, but with enhanced defence response at 9 dpi under 20 °C. Regarding FUM genes, in the kernels inoculated with F. verticillioides the maximal transcript levels occurred at 6 dpi at 25 °C. At this temperature regime, expression values decreased with the co-occurrence of A. flavus , where the highest gene induction was detected at 20 °C. Similar results were observed in fungi grown in vitro, whilst A. flavus presence determined lower levels of expression along the entire time-course. As concerns afl genes, considering both A. flavus alone and in combination, the most elevated transcript accumulation occurred at 30 °C during all time-course both in infected kernels and in fungi grown in vitro. Regarding mycotoxin production, no significant differences were found among temperatures for kernel contamination, whereas in vitro the highest production was registered at 25 °C for aflatoxin B1 and at 20 °C for fumonisins in the case of single inoculation. In fungal co-occurrence, both mycotoxins resulted reduced at all the temperatures considered compared to the amount produced with single inoculation.