X-ray Irradiation Reduces Live Aspergillus flavus Viability but Not Aflatoxin B1 in Naturally Contaminated Maize.

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.

Unraveling the antifungal and anti-aflatoxin B1 mechanisms of piperitone on Aspergillus flavus.

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.

Peanut Rhizosphere Achromobacter xylosoxidans Inhibits Aspergillus flavus Development and Aflatoxin Synthesis by Inducing Apoptosis through Targeting the Cell Membrane.

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.

Rhein Inhibits Cell Development and Aflatoxin Biosynthesis via Energy Supply Disruption and ROS Accumulation in Aspergillus flavus.

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 B1 (AFB1) biosynthesis and the potential mechanism using RNA-seq analysis. The results indicate that A. flavus mycelial growth and AFB1 biosynthesis were significantly inhibited by 50 µM rhein, with a 43.83% reduction in colony diameter and 87.2% reduction in AFB1 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 AFB1 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.

Mitigating fungal contamination of cereals: The efficacy of microplasma-based far-UVC lamps against Aspergillus flavus and Fusarium graminearum.

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/cm2, and a 5.11-log reduction of Fusarium graminearum conidia in suspension with a dose of 619.2 mJ/cm2. 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/cm2 and 460.8 mJ/cm2, 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/cm2 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.

Self-assembled thymol-betaine co-crystals with controlled release and hygroscopic properties as green preservatives for aflatoxin prevention.

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 H2O 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 B1. In practical terms, the THY-TMG co-crystal was successful in preventing AFB1 contamination and nutrients loss in peanuts, thereby prolonging their shelf-life under conditions of 28 °C and 70% RH.

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

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

The potential of antifungal peptides derived from Lactiplantibacillus plantarum WYH for biocontrol of Aspergillus flavus contamination.

Aspergillus flavus is a notorious fungus that contaminates food crops with toxic aflatoxins, posing a serious threat to human health and the agricultural economy. To overcome the inadequacy of traditional control methods and meet consumer preferences for natural-sources additives, there is an urgent demand for novel biocontrol agents that are safe and efficient. This study aims to investigate the antifungal properties of a novel antifungal agent derived from the biologically safe Lactiplantibacillus plantarum WYH. Firstly, antifungal peptides (AFPs) with a molecular weight of less than 3kD, exhibiting remarkable temperature stability and effectively retarding fungal growth in a dose-dependent manner specifically against A. flavus, were concentrated from the fermentation supernatant of L. plantarum WYH and were named as AFPs-WYH. Further analysis demonstrated that AFPs-WYH might exert antifungal effects through the induction of oxidative stress, disruption of mitochondrial function, alteration of membrane permeability, and cell apoptosis in A. flavus. To further validate our findings, a transcriptomics analysis was conducted on A. flavus treated with 2 and 5 mg/mL of AFPs-WYH, which elucidated the potential effect of AFPs-WYH administration on the regulation of genes involved in impairing fungal development and preventing aflatoxin biosynthesis pathways. Overall, AFPs-WYH reduced the A. flavus proliferation and affected the AFB1 biosynthesis, exhibiting a promising potential for food industry applications as a biopreservative and biocontrol agent.

Antifungal mechanisms investigation of lactic acid bacteria against Aspergillus flavus: through combining microbial metabolomics and co-culture system.

AIMS: To develop antifungal lactic acid bacteria (LAB) and investigate their antifungal mechanisms against Aspergillus flavus in aflatoxin (AF) production. METHODS AND RESULTS: We isolated 179 LABs from cereal-based fermentation starters and investigated their antifungal mechanism against A. flavus through liquid chromatography-mass spectrometry and co-culture analysis techniques. Of the 179 isolates, antifungal activity was identified in Pediococcus pentosaceus, Lactobacillus crustorum, and Weissella paramesenteroides. These LABs reduced AF concentration by (i) inhibiting mycelial growth, (ii) binding AF to the cell wall, and (iii) producing antifungal compounds. Species-specific activities were also observed, with P. pentosaceus inhibiting AF production and W. paramesenteroides showing AF B1 binding activity. In addition, crucial extracellular metabolites for selecting antifungal LAB were involved in the 2',3'-cAMP-adenosine and nucleoside pathways. CONCLUSIONS: This study demonstrates that P. pentosaceus, L. crustorum, and W. paramesenteroides are key LAB strains with distinct antifungal mechanisms against A. flavus, suggesting their potential as biological agents to reduce AF in food materials.

Application of Cystoseira myrica phycosynthesized selenium nanoparticles incorporated with nano-chitosan to control aflatoxigenic fungi in fish feed.

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.

Mimosa tenuiflora Aqueous Extract: Role of Condensed Tannins in Anti-Aflatoxin B1 Activity in Aspergillus flavus.

Aflatoxin B1 (AFB1) is a potent carcinogenic mycotoxin that contaminates numerous crops pre- and post-harvest. To protect foods and feeds from such toxins without resorting to pesticides, the use of plant extracts has been increasingly studied. The most interesting candidate plants are those with strong antioxidative activity because oxidation reactions may interfere with AFB1 production. The present study investigates how an aqueous extract of Mimosa tenuiflora bark affects both the growth of Aspergillus flavus and AFB1 production. The results reveal a dose-dependent inhibition of toxin synthesis with no impact on fungal growth. AFB1 inhibition is related to a down-modulation of the cluster genes of the biosynthetic pathway and especially to the two internal regulators aflR and aflS. Its strong anti-oxidative activity also allows the aqueous extract to modulate the expression of genes involved in fungal oxidative-stress response, such as msnA, mtfA, atfA, or sod1. Finally, a bio-guided fractionation of the aqueous extract demonstrates that condensed tannins play a major role in the anti-aflatoxin activity of Mimosa tenuiflora bark.

Inhibition of Aspergillus flavus growth and aflatoxins production on peanuts over α-Fe2O3 nanorods under sunlight irradiation.

Peanut is an important resource of edible oil and digestible protein in daily life, which is rich in the nutriments and antioxidants such as vitamins, minerals and polyphenols. However, peanut is susceptible to the contamination of Aspergillus flavus (A. flavus), which can produce highly carcinogenic toxins that brings serious threats to human health and food safety. Exploring green and effective methods to control A. flavus is meaningful. Herein, a green and economical way to control A. flavus on peanuts was demonstrated. It was found that the growth of A. flavus hyphae and germination of its spores could be inhibited in the presence of α-Fe2O3 nanorods under sunlight irradiation according to the agar diffusion method, flat colony counting method and fluorescence-based live/dead test. The diameter of inhibition zone was 22.3 ± 0.2 mm and the inhibition rate of spores germination was about 60 ± 5%, when the concentration of α-Fe2O3 was 10 mg/mL for 7 h sunlight irradiation. Most important, α-Fe2O3 showed the photocatalytic inhibition of A. flavus on peanuts under sunlight irradiation with the inhibition rate of about 90 ± 5%, and the production of aflatoxin B1 and aflatoxin B2 were reduced by 90 ± 2% and 70 ± 3%, respectively. By comparing the fat contents, protein contents, acid value, peroxide value and antioxidative compositions of peanuts, it was found that there was no obvious effect on the quality of peanuts after inhibition treatment. The findings provide a green, safe and economical strategy to control A. flavus on peanuts, which may be as a promising way to be used in food and agro-food preservation.

Aspergillus flavus Growth Inhibition and Aflatoxin B1 Decontamination by Streptomyces Isolates and Their Metabolites.

Aflatoxin B1 is a potent carcinogen produced by Aspergillus flavus, mainly during grain storage. As pre-harvest methods are insufficient to avoid mycotoxin presence during storage, diverse curative techniques are being investigated for the inhibition of fungal growth and aflatoxin detoxification. Streptomyces spp. represent an alternative as they are a promising source of detoxifying enzymes. Fifty-nine Streptomyces isolates and a Streptomyces griseoviridis strain from the commercial product Mycostop®, evaluated against Penicillium verrucosum and ochratoxin A during previous work, were screened for their ability to inhibit Aspergillus flavus growth and decrease the aflatoxin amount. The activities of bacterial cells and cell-free extracts (CFEs) from liquid cultures were also evaluated. Fifty-eight isolates were able to inhibit fungal growth during dual culture assays, with a maximal reduction going down to 13% of the control. Aflatoxin-specific production was decreased by all isolates to at least 54% of the control. CFEs were less effective in decreasing fungal growth (down to 40% and 55% for unheated and heated CFEs, respectively) and aflatoxin-specific production, with a few CFEs causing an overproduction of mycotoxins. Nearly all Streptomyces isolates were able to degrade AFB1 when growing in solid and liquid media. A total degradation of AFB1 was achieved by Mycostop® on solid medium, as well as an almost complete degradation by IX20 in liquid medium (6% of the control). CFE maximal degradation went down to 37% of the control for isolate IX09. The search for degradation by-products indicated the presence of a few unknown molecules. The evaluation of residual toxicity of the tested isolates by the SOS chromotest indicated a detoxification of at least 68% of AFB1's genotoxicity.

Effect of Temperature during Drying and Storage of Dried Figs on Growth, Gene Expression and Aflatoxin Production.

Dried fig is susceptible to infection by Aspergillus flavus, the major producer of the carcinogenic mycotoxins. This fruit may be contaminated by the fungus throughout the entire chain production, especially during natural sun-drying, post-harvest, industrial processing, storage, and fruit retailing. Correct management of such critical stages is necessary to prevent mould growth and mycotoxin accumulation, with temperature being one of the main factors associated with these problems. The effect of different temperatures (5, 16, 25, 30, and 37 °C) related to dried-fig processing on growth, one of the regulatory genes of aflatoxin pathway (aflR) and mycotoxin production by A. flavus, was assessed. Firstly, growth and aflatoxin production of 11 A. flavus strains were checked before selecting two strains (M30 and M144) for in-depth studies. Findings showed that there were enormous differences in aflatoxin amounts and related-gene expression between the two selected strains. Based on the results, mild temperatures, and changes in temperature during drying and storage of dried figs should be avoided. Drying should be conducted at temperatures >30 °C and close to 37 °C, while industry processing, storage, and retailing of dried figs are advisable to perform at refrigeration temperatures (<10 °C) to avoid mycotoxin production.

Impact of encapsulated orange essential oil with ß-cyclodextrin on technological, digestibility, sensory properties of wheat cakes as well as Aspergillus flavus spoilage.

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 ß-cyclodextrin (ß-CD) by inclusion complex formation (ß-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 ß-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 ß-CD/OEO presented a very slight orange taste and aroma. CONCLUSION: The encapsulation of OEO into ß-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.

Synthetic antimicrobial agents inhibit aflatoxin production.

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

Computational modelling of survival of Aspergillus flavus in peanut kernels during hot air-assisted radio frequency pasteurization.

In recent years, radio frequency (RF) heating is getting popular as an alternative pasteurization method for agricultural commodities and low moisture foods. Computer simulation is an effective way to help understand RF interactions with food components and predict temperature distributions among food samples after RF treatments. In this study, a computer model based on Joule heating and thermal inactivation kinetic of A. flavus was established to predict both temperature distribution and microbial reduction among peanut kernels after RF processing. For the process validation, three 2-g peanut samples inoculated with 40 µL A. flavus were placed at three representative locations among 2.17 kg peanut kernels and subjected to various processing conditions in a 27.12 MHz, 6 kW RF heating unit together with hot air system. Results showed that the average difference of the sample temperature and microbial reduction between simulation and experiment was small with RMSE values of 0.009 °C and 0.012 °C, and 0.31 log CFU/g and 0.42 log CFU/g for peanut moisture contents of 7.56% and 12.02% w. b., respectively. Nonuniform RF heating resulted in the least lethality of A. flavus at the cold spot. The validated computer model was further used to estimate microbial reduction distributions at other target temperatures based on predicted temperature profiles. This computer model may help design the RF pasteurization protocols for peanut kernels without extensive experiments in food industry.

Dimethylformamide Inhibits Fungal Growth and Aflatoxin B1 Biosynthesis in Aspergillus flavus by Down-Regulating Glucose Metabolism and Amino Acid Biosynthesis.

Aflatoxins (AFs) are secondary metabolites produced by plant fungal pathogens infecting crops with strong carcinogenic and mutagenic properties. Dimethylformamide (DMF) is an excellent solvent widely used in biology, medicine and other fields. However, the effect and mechanism of DMF as a common organic solvent against fungal growth and AFs production are not clear. Here, we discovered that DMF had obvious inhibitory effect against A. flavus, as well as displayed complete strong capacity to combat AFs production. Hereafter, the inhibition mechanism of DMF act on AFs production was revealed by the transcriptional expression analysis of genes referred to AFs biosynthesis. With 1% DMF treatment, two positive regulatory genes of AFs biosynthetic pathway aflS and aflR were down-regulated, leading to the suppression of the structural genes in AFs cluster like aflW, aflP. These changes may be due to the suppression of VeA and the subsequent up-regulation of FluG. Exposure to DMF caused the damage of cell wall and the dysfunction of mitochondria. In particular, it is worth noting that most amino acid biosynthesis and glucose metabolism pathway were down-regulated by 1% DMF using Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Taken together, these RNA-Seq data strongly suggest that DMF inhibits fungal growth and aflatoxin B1 (AFB1) production by A. flavus via the synergistic interference of glucose metabolism, amino acid biosynthesis and oxidative phosphorylation.

Genetic map and heritability of Aspergillus flavus.

The carcinogenic aflatoxins are a human health concern as well as an economic burden to corn, peanut and other crops grown within the United States and globally. Aflatoxins are produced by fungi species in Aspergillus section Flavi, primarily Aspergillus flavus. Though previously thought of as only asexual, A. flavus has recently been found to undergo sexual reproduction both in laboratory crosses and in the field. To elucidate the consequences of genetic exchange through a single generation of the sexual cycle within A. flavus, we constructed genetic maps based on three mapping populations, each composed of the parental strains and approximately 70 F1 progeny. Genome-wide data using double digest Restriction Associated DNA sequencing identified 496, 811, and 576 significant polymorphisms differentiating parents across eight linkage groups; these polymorphisms served as markers. Average spacing between marker loci was 3.1, 2.1, and 3.5 map units and overall map length was 1504.4, 1669.2, and 2001.3 cM. Recombination was non-randomly distributed across chromosomes with an average rate of recombination of about 46.81 cM per Mbp. We showed inheritance of mitochondrial loci from the sclerotial (female) parent in crosses, whereas nuclear loci showed a 1:1 segregation ratio from both parents. The linkage map will be useful in QTL analyses to identify traits that increase sexual fertility in A. flavus and modulate aflatoxin production, both of which have significant implications for sustainable reduction of aflatoxin contamination using biological control agents.

MAPK pathway-related tyrosine phosphatases regulate development, secondary metabolism and pathogenicity in fungus Aspergillus flavus.

Mitogen-activated protein kinase (MAPK) cascades are highly conserved in eukaryotic cells and are known to play crucial roles in the regulation of various cellular processes. However, compared with kinase-mediated phosphorylation, dephosphorylation catalysed by phosphatases has not been well characterized in filamentous fungi. In this study, we identified five MAPK pathway-related phosphatases (Msg5, Yvh1, Ptp1, Ptp2 and Oca2) and characterized their functions in Aspergillus flavus, which produces aflatoxin B1 (AFB1 ), one of the most toxic and carcinogenic secondary metabolites. These five phosphatases were identified as negative regulators of MAPK (Slt2, Fus3 and Hog1) pathways. Deletion of Msg5 and Yvh1 resulted in significant defects in conidiation, sclerotia formation, aflatoxin production and crop infection. Additionally, double knockout mutants (ΔMsg5/ΔPtp1, ΔMsg5/ΔPtp2 and ΔMsg5/ΔOca2) displayed similar defects to those observed in the ΔMsg5 single mutant, indicating that Msg5 plays a major role in the regulation of development and pathogenicity in A. flavus. Importantly, we found that the active site at C439 is essential for the function of the Msg5 phosphatase. Furthermore, the MAP kinase Fus3 was found to be involved in the regulation of development, aflatoxin biosynthesis and pathogenicity, and its conserved phosphorylation residues (Thr and Tyr) were critical for the full range of its functions in A. flavus. Overall, our results reveal that MAPK related tyrosine phosphatases play important roles in the regulation of development, secondary metabolism and pathogenicity in A. flavus, and could be developed as potential targets for preventing damage caused by this fungal pathogen.