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

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

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

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

Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Chitin Deacetylase Homologous Gene cda Contributes to Development and Aflatoxin Synthesis in Aspergillus flavus.

The fungal cell wall serves as the primary interface between fungi and their external environment, providing protection and facilitating interactions with the surroundings. Chitin is a vital structural element in fungal cell wall. Chitin deacetylase (CDA) can transform chitin into chitosan through deacetylation, providing various biological functions across fungal species. Although this modification is widespread in fungi, the biological functions of CDA enzymes in Aspergillus flavus remain largely unexplored. In this study, we aimed to investigate the biofunctions of the CDA family in A. flavus. The A. flavus genome contains six annotated putative chitin deacetylases. We constructed knockout strains targeting each member of the CDA family, including Δcda1, Δcda2, Δcda3, Δcda4, Δcda5, and Δcda6. Functional analyses revealed that the deletion of CDA family members neither significantly affects the chitin content nor exhibits the expected chitin deacetylation function in A. flavus. However, the Δcda6 strain displayed distinct phenotypic characteristics compared to the wild-type (WT), including an increased conidia count, decreased mycelium production, heightened aflatoxin production, and impaired seed colonization. Subcellular localization experiments indicated the cellular localization of CDA6 protein within the cell wall of A. flavus filaments. Moreover, our findings highlight the significance of the CBD1 and CBD2 structural domains in mediating the functional role of the CDA6 protein. Overall, we analyzed the gene functions of CDA family in A. flavus, which contribute to a deeper understanding of the mechanisms underlying aflatoxin contamination and lay the groundwork for potential biocontrol strategies targeting A. flavus.

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

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

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

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

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

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

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

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

Efficacy of atoxigenic Aspergillus flavus from southern China as biocontrol agents against aflatoxin contamination in corn and peanuts.

Aspergillus flavus is a ubiquitous facultative pathogen that routinely infects important crops leading to formation of aflatoxins during crop development and after harvest. Corn and peanuts in warm and/or drought-prone regions are highly susceptible to aflatoxin contamination. Controlling aflatoxin using atoxigenic A. flavus is a widely adopted strategy. However, no A. flavus genotypes are currently approved for use in China. The current study aimed to select atoxigenic A. flavus endemic to Guangxi Zhuang Autonomous Region with potential as active ingredients of aflatoxin biocontrol products. A total of 204 A. flavus isolates from corn, peanuts, and field soil were evaluated for ability to produce the targeted mycotoxins. Overall, 57.3% could not produce aflatoxins while 17.15% were incapable of producing both aflatoxins and CPA. Atoxigenic germplasm endemic to Guangxi was highly diverse, yielding 8 different gene deletion patterns in the aflatoxin and CPA biosynthesis gene clusters ranging from no deletion to deletion of both clusters. Inoculation of corn and peanuts with both an aflatoxin producer and selected atoxigenic genotypes showed significant reduction (74 to 99%) in aflatoxin B1 (AFB1) formation compared with inoculation with the aflatoxin producer alone. Atoxigenic genotypes also efficiently degraded AFB1 (61%). Furthermore, atoxigenic isolates were also highly efficient at reducing aflatoxin concentrations even when present at lower concentrations than aflatoxin producers. The use of multiple atoxigenics was not always as effective as the use of a single atoxigenic. Effective atoxigenic genotypes of A. flavus with known mechanisms of atoxigenicity are demonstrated to be endemic to Southern China. These A. flavus may be utilized as active ingredients of biocontrol products without concern for detrimental impacts that may result from introduction of exotic fungi. Field efficacy trials in the agroecosystems of Southern China are needed to determine the extent to which such products may allow the production of safer food and feed.

Fus3 Interacts with Gal83, Revealing the MAPK Crosstalk to Snf1/AMPK to Regulate Secondary Metabolic Substrates in Aspergillus flavus.

Aflatoxins (AFs), highly carcinogenic natural products, are produced by the secondary metabolism of fungi such as Aspergillus flavus. Essential for the fungi to respond to environmental changes and aflatoxin synthesis, the pheromone mitogen-activated protein kinase (MAPK) is a potential regulator of aflatoxin biosynthesis. However, the mechanism by which pheromone MAPK regulates aflatoxin biosynthesis is not clear. Here, we showed Gal83, a new target of Fus3, and identified the pheromone Fus3-MAPK signaling pathway as a regulator of the Snf1/AMPK energy-sensing pathway modulating aflatoxins synthesis substrates. The screening for Fus3 target proteins identified the ß subunit of Snf1/AMPK complexes using tandem affinity purification and multiomics. This subunit physically interacted with Fus3 both in vivo and in vitro and received phosphorylation from Fus3. Although the transcript levels of aflatoxin synthesis genes were not noticeably downregulated in both gal83 and fus3 deletion mutant strains, the levels of aflatoxin B1 and its synthesis substrates and gene expression levels of primary metabolizing enzymes were significantly reduced. This suggests that both the Fus3-MAPK and Snf1/AMPK pathways respond to energy signals. In conclusion, all the evidence unlocks a novel pathway of Fus3-MAPK to regulate AFs synthesis substrates by cross-talking with the Snf1/AMPK complexes.

The Gene vepN Regulated by Global Regulatory Factor veA That Affects Aflatoxin Production, Morphological Development and Pathogenicity in Aspergillus flavus.

Velvet (VeA), a light-regulated protein that shuttles between the cytoplasm and the nucleus, serves as a key global regulator of secondary metabolism in various Aspergillus species and plays a pivotal role in controlling multiple developmental processes. The gene vepN was chosen for further investigation through CHIP-seq analysis due to significant alterations in its interaction with VeA under varying conditions. This gene (AFLA_006970) contains a Septin-type guanine nucleotide-binding (G) domain, which has not been previously reported in Aspergillus flavus (A. flavus). The functional role of vepN in A. flavus was elucidated through the creation of a gene knockout mutant and a gene overexpression strain using a well-established dual-crossover recombinational technique. A comparison between the wild type (WT) and the ΔvepN mutant revealed distinct differences in morphology, reproductive capacity, colonization efficiency, and aflatoxin production. The mutant displayed reduced growth rate; dispersion of conidial heads; impaired cell wall integrity; and decreased sclerotia formation, colonization capacity, and aflatoxin levels. Notably, ΔvepN exhibited complete growth inhibition under specific stress conditions, highlighting the essential role of vepN in A. flavus. This study provides evidence that vepN positively influences aflatoxin production, morphological development, and pathogenicity in A. flavus.

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

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

HacA, a key transcription factor for the unfolded protein response, is required for fungal development, aflatoxin biosynthesis and pathogenicity of Aspergillus flavus.

Aspergillus flavus is a fungus notorious for contaminating food and feed with aflatoxins. As a saprophytic fungus, it secretes large amounts of enzymes to access nutrients, making endoplasmic reticulum (ER) homeostasis important for protein folding and secretion. The role of HacA, a key transcription factor in the unfolded protein response pathway, remains poorly understood in A. flavus. In this study, the hacA gene in A. flavus was knockout. Results showed that the absence of hacA led to a decreased pathogenicity of the strain, as it failed to colonize intact maize kernels. This may be due to retarded vegetable growth, especially the abnormal development of swollen tips and shorter hyphal septa. Deletion of hacA also hindered conidiogenesis and sclerotial development. Notably, the mutant strain failed to produce aflatoxin B1. Moreover, compared to the wild type, the mutant strain showed increased sensitivity to ER stress inducer such as Dithiothreitol (DTT), and heat stress. It also displayed heightened sensitivity to other environmental stresses, including cell wall, osmotic, and pH stresses. Further transcriptomic analysis revealed the involvement of the hacA in numerous biological processes, including filamentous growth, asexual reproduction, mycotoxin biosynthetic process, signal transduction, budding cell apical bud growth, invasive filamentous growth, response to stimulus, and so on. Taken together, HacA plays a vital role in fungal development, pathogenicity and aflatoxins biosynthesis. This highlights the potential of targeting hacA as a novel approach for early prevention of A. flavus contamination.

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

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

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

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

Macro-micro exploration on dynamic interaction between aflatoxigenic Aspergillus flavus and maize kernels using Vis/NIR hyperspectral imaging and SEM technology.

Aspergillus flavus and its toxic metabolites-aflatoxins infect and contaminate maize kernels, posing a threat to grain safety and human health. Due to the complexity of microbial growth and metabolic processes, dynamic mechanisms among fungal growth, nutrient depletion of maize kernels and aflatoxin production is still unclear. In this study, visible/near infrared (Vis/NIR) hyperspectral imaging (HSI) combined with the scanning electron microscope (SEM) was used to elucidate the critical organismal interaction at kernel (macro-) and microscopic levels. As kernel damage is the main entrance for fungal invasion, maize kernels with gradually aggravated damages from intact to pierced to halved kernels with A. flavus were cultured for 0-120 h. The spectral fingerprints of the A. flavus-maize kernel complex over time were analyzed with principal components analysis (PCA) of hyperspectral images, where the pseudo-color score maps and the loading plots of the first three PCs were used to investigate the dynamic process of fungal infection and to capture the subtle changes in the complex with different hardness of the maize matrix. The dynamic growth process of A. flavus and the interactions of fungus-maize complexes were explained on a microscopic level using SEM. Specifically, fungus morphology, e.g., hyphae, conidia, and conidiophore (stipe) was accurately captured on the microscopic level, and the interaction process between A. flavus and nutrient loss from the maize kernel tissues (i.e., embryo, and endosperm) was described. Furthermore, the growth stage discrimination models based on PLSDA with the results of CCRC = 100 %, CCRV = 97 %, CCRIV = 93 %, and the prediction models of AFB1 based on PLSR with satisfactory performance (R2C = 0.96, R2V = 0.95, R2IV = 0.93 and RPD = 3.58) were both achieved. In conclusion, the results from both macro-level (Vis/NIR-HSI) and micro-level (SEM) assessments revealed the dynamic organismal interactions in A. flavus-maize kernel complex, and the detailed data could be used for modeling, and quantitative prediction of aflatoxin, which would establish a theoretical foundation for the early detection of fungal or toxin contaminated grains to ensure food security.

Liamocins overproduction via the two-pH stage fermentation and anti-Aspergillus flavus activity of Massoia lactone.

Aureobasidium melanogenum was found to be grown the best at the constant pH 7.0 and to produce the highest amount of liamocins at the constant pH 3.0. Therefore, the wild type strain A. melanogenum 9-1 and the engineered strain V33 constructed in the laboratory were grown at the constant pH 7.0 for 48 h, then, they were continued to be cultivated at the constant pH 3.0. Under such conditions, A. melanogenum 9-1 produced 36.51 ± 0.55 g L-1 of liamocin and its cell mass was 27.43 ± 0.63 and 6.00 ± 0.11 g L-1 of glucose was left in the finished medium within 168 h while the engineered strain V33 secreted 70.86 ± 2.04 g L-1 of liamocin, its cell mass was 31.63 ± 0.74 g L-1 , 0.16 ± 0.01 g L-1 of glucose was maintained in the finished medium. Then, Massoia lactone was released from the produced liamocins. The released Massoia lactone loaded in the nanoemulsions could be used to actively damage cell wall and cell membrane of both spores and mycelia of Aspergillus flavus, leading to its cell necrosis. Massoia lactone loaded in the nanoemulsions also actively inhibited cell growth of A. flavus, its conidia production and aflatoxin biosynthesis on peanuts, indicating that Massoia lactone loaded in the nanoemulsions had highly potential application in controlling cell growth of A. flavus and aflatoxin biosynthesis in foods and feedstuffs.

Biotransformation of the azo dye reactive orange 16 by Aspergillus flavus A5P1: Performance, genetic background, pathway, and mechanism.

This study reports the strain Aspergillus flavus A5P1 (A5P1), which is with the capable of degrading the azo dye reactive orange 16 (RO16). The mechanism of RO16 degradation by A5P1 was elucidated through genomic analysis, enzymatic analysis, degradation pathway analysis and oxidative stress analysis. Strain A5P1 exhibited aerobic degradation of RO16, with optimal degradation at an initial pH of 3.0. Genomic analysis indicates that strain A5P1 possesses the potential for acid tolerance and degradation of azo dye. Enzymatic analysis, combined with degradation product analysis, demonstrated that extracellular laccase, intracellular lignin peroxidase, and intracellular quinone reductase were likely key enzymes in the RO16 degradation process. Oxidative stress analysis revealed that cell stress responses may participate in the RO16 biotransformation process. The results indicated that the biotransformation of RO16 may involves biological processes such as transmembrane transport of RO16, cometabolism of the strain with RO16, and cell stress responses. These findings shed light on the biodegradation of RO16 by A5P1, indicating A5P1's potential for environmental remediation.

Effects of Tripleurospermum caucasicum, Salvia rosmarinus and Tanacetum fruticulosum essential oils on aflatoxin B1 production and aflR gene expression in Aspergillus flavus.

Aflatoxin B1 (AFB1) is one of the most hazardous mycotoxins for humans and livestock that mainly produced by members of the genus Aspergillus in a variety of food commodities. In this study, the effect of S. rosmarinus, T. fruticulosum, and T. caucasicum essential oils (EOs) was studied on fungal growth, AFB1 production and aflR gene expression in toxigenic A. flavus IPI 247. The AFB1 producer A. flavus strain was cultured in YES medium in presence of various two-fold concentrations of the plant EOs (62.5-500 µg/mL) for 4 days at 28 °C. EO composition of plants was analyzed by Gas Chromatography/Mass Spectrometry (GC/MS). The amount of fungal growth, ergosterol content of fungal mycelia and AFB1 content of EO-treated and non-treated controls were measured. The expression of aflR gene was evaluated using Real-time PCR in the fungus exposed to minimum inhibitory concentration (MIC50) of EOs. The main constituents of the oils analyzed by GC/MS analysis were elemicin (33.80 %) and 2,3-dihydro farnesol (33.19 %) in T. caucasicum, 1,8-cineole (17.87 %), trans-caryophyllene (11.14 %), α and ẞ-pinene (10.92 and 8.83 %) in S. rosmarinus, and camphor (17.65 %), bornyl acetate (15.08 %), borneol (12.48 %) and camphene (11.72 %) in T. fruticulosum. The results showed that plant EOs at the concentration of 500 µg/mL suppressed significantly the fungal growth by 35.24-71.70 %, while mycelial ergosterol content and AFB1 production were inhibited meaningfully by 36.20-65.51 % and 20.61-89.16 %. T. caucasicum was the most effective plant, while T. fruticulosum showed the lowest effectiveness on fungal growth and AFB1 production. The expression of aflR in T. caucasicum and S. rosmarinus -treated fungus was significantly down-regulated by 2.85 and 2.12 folds, respectively, while it did not change in T. fruticulosum-treated A. flavus compared to non-treated controls. Our findings on the inhibitory activity of T. caucasicum and S. rosmarinus EOs toward A. flavus growth and AFB1 production could promise these plants as good candidates to control fungal contamination of agricultural crops and food commodities and subsequent contamination by AFB1. Down-regulation of aflR as the key regulatory gene in AF biosynthesis pathway warrants the use of these plants in AF control programs. Further studies to evaluate the inhibitory activity of studied plants EOs in food model systems are recommended.

Response of broilers to dietary inclusion of atoxigenic Aspergillus flavus strain as a biocontrol strategy of aflatoxin.

The objective of this trial was to evaluate how broilers responded to Aspergillus flavus strains that are toxigenic and atoxigenic. The study included four treatments in a 2 × 2 factorial design, with six replicates of 10 birds each. As a result of this study measuring feed intake (FI), weight gain (WG), feed conversion ratio (FCR), crude protein, ether extract, and crude fibre, the interaction was insignificant between the toxigenic and atoxigenic diets (P > 0.05). Consumption of toxigenic aflatoxin B1-500 ppb diet decreased FI and WG but increased FCR, and cost to produce live broiler weight (P < 0.05) compared to the control diets. The addition of atoxigenic strains to contaminated diets significantly offset (P < 0.05) the effects. Diets with or without 500 ppb toxigenic and atoxigenic A. flavus did not affect the relative weight g/100gBW of pancreas, gizzard and bursa of Fabricius. Dietary inclusion of 500 ppb toxigenic Aspergillus spp. increased the relative weight (P < 0.05) of the kidney, liver, spleen and thymus while atoxigenic dietary addition reduced the relative weight of the same organs (P < 0.05). Dietary inclusion of toxigenic and atoxigenic Aspergillus spp. did not significantly affect the haematological parameters measured (P < 0.05). Dietary inclusion of 500 ppb toxigenic Aspergillus elevated the urea, creatine, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in the serum of the broilers (P < 0.05). A decrease was observed when atox igenic A. flavus was used in the intervention for urea, creatinine and AST (P < 0.05), whereas an insignificant reduction was observed for ALT and ALP (P ≤ 0.05). This study concluded that dietary atoxigenic strain improved broiler performance, digestibility, and blood parameters.