Azole resistance in Aspergillus flavus and associated fitness cost.

BACKGROUND: The resistance of Aspergillus flavus to the azole antifungal drugs is an emerging problem. Mutations in the molecular targets of the azole antifungals - CYP 51 A, B and C - are possible mechanisms of resistance, but data to confirm this hypothesis are scarce. In addition, the behaviour of resistant strains in vitro and in vivo is not yet understood. OBJECTIVES: This study had 3 objectives. The first was to compare the sequences of CYP51 A, B and C in resistant and susceptible strains of A. flavus. The second was to look for the existence of a fitness cost associated with resistance. The third was to evaluate the activity of voriconazole and posaconazole on resistant strains in the Galleria mellonella model. METHODS: The CYP51 A, B and C sequences of seven resistant strains with those of four susceptible strains are compared. Fitness costs were assessed by growing the strains in RPMI medium and testing their virulence in G. mellonella larvae. In addition, G. mellonella larvae infected with strains of A. flavus were treated with voriconazole and posaconazole. RESULTS: In the CYP51A sequences, we found the A91T, C708T and A1296T nucleotide substitutions only in the resistant strains. The resistant strains showed a fitness cost with reduced in vitro growth and reduced virulence in G. mellonella. In vivo resistance to posaconazole is confirmed in a strain with the highest MIC for this antifungal agent. CONCLUSIONS: These results allow to conclude that some substitutions in CYP51 genes, in particular CYP51A, contribute to resistance to azole drugs in A. flavus. The study of the relationship between drug dosage and treatment duration with resistance and the reduction of fitness costs in resistant strains is a major perspective of this study. This work could help to establish recommendations for the treatment of infections with resistant strains of A. flavus.

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.

Cold atmospheric plasma inactivates Aspergillus flavus and Fusarium keratoplasticum biofilms and conidia in vitro.

Introduction. Aspergillus flavus and Fusarium keratoplasticum are common causative pathogens of fungal keratitis (FK), a severe corneal disease associated with significant morbidity and vision loss. Escalating incidence of antifungal resistance to available antifungal drugs poses a major challenge to FK treatment. Cold atmospheric plasma (CAP) is a pioneering nonpharmacologic antimicrobial intervention that has demonstrated potential as a broad-spectrum antifungal treatment.Gap statement. Previous research highlights biofilm-associated resistance as a critical barrier to effective FK treatment. Although CAP has shown promise against various fungal infections, its efficacy against biofilm and conidial forms of FK pathogens remains inadequately explored.Aim. This study aims to investigate the antifungal efficacy of CAP against clinical fungal keratitis isolates of A. flavus and F. keratoplasticum in vitro.Methodology. Power parameters (22-27 kVpp, 300-400 Hz and 20-80 mA) of a dielectric barrier discharge CAP device were optimized for inactivation of A. flavus biofilms. Optimal applied voltage and total current were applied to F. keratoplasticum biofilms and conidial suspensions of A. flavus and F. keratoplasticum. The antifungal effect of CAP treatment was investigated by evaluating fungal viability through means of metabolic activity, c.f.u. enumeration (c.f.u. ml-1) and biofilm formation.Results. For both fungal species, CAP exhibited strong time-dependent inactivation, achieving greater than 80 % reduction in metabolic activity and c.f.u. ml-1 within 300 s or less, and complete inhibition after 600 s of treatment.Conclusion. Our findings indicate that CAP is a promising broad-spectrum antifungal intervention. CAP treatment effectively reduces fungal viability in both biofilm and conidial suspension cultures of A. flavus and F. keratoplasticum, suggesting its potential as an alternative treatment strategy for fungal keratitis.

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.

Three Ecological Models to Evaluate the Effectiveness of Trichoderma spp. for Suppressing Aflatoxigenic Aspergillus flavus and Aspergillus parasiticus.

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.

Biocontrol potential of endophytic fungi against phytopathogenic nematodes on potato (Solanum tuberosum L.).

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.

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

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

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.

Biogenic silver nanoparticles from fungal sources: Synthesis, characterization, and antifungal potential.

Nano-biotechnology is quickly developing as an important field of modern research, generating the most promising applications in medicine and agriculture. Biosynthesis of silver nanoparticles using biogenic or green approach provide ecofriendly, clean and effective way out for the synthesis of nanoparticles. The main aim of the study was to synthesize silver nanoparticles (AgNPs) from Aspergillus niger, Aspergillus flavus and Pencillium chrysogenum using a green approach and to test the antifungal activity of these synthesized AgNPs against a variety of pathogenic fungi. The characterization of samples was done by using UV-visible spectroscopy, SEM (scanning electron microscopy), FTIR (Fourier transmission infrared spectroscopy), and XRD (X-ray diffractometry). The investigation confirmed the creation of AgNPs by the fungi Aspergillus niger, Aspergillus flavus and Pencillium chrysogenum, as evidenced by prominent plasmon absorbance bands at 420 and 450 nm.The biosynthesized AgNPs were 80-100 nm in size, asymmetrical in shape and became spherical to sub-spherical when aggregated. Agar well diffusion method was performed to evaluate the antifungal activity of AgNPs against various plant pathogenic fungi. An efficient and strong antifungal activity was shown by these biosynthesized nanoparticles against serious plant pathogenic fungi, viz. Aspergillus terreus, Fusarium oxysporum, Penicillium citrinum, Rhizopus stolonifer and Mucor mucedo. The biosynthesized AgNPs at various concentrations caused significant zone of inhibition in the test fungal pathogens. Silver nanoparticles (AgNPs) biosynthesized from Aspergillus niger at highest concentrations showed maximum zone of inhibition against Penicillium citrinum (19.33 ± 0.57 mm) followed by Rhizopus stolonifer (17.66 ± 0.57), Aspergillus terreus (16.33 ± 1.54 mm), Fusarium oxysporum (14.00 ± 1.00 mm) and Mucor mucedo (13.33 ± 1.15 mm) respectively. Therefore, the findings clearly indicate that silver nanoparticles could play a significant role in managing diverse plant diseases caused by fungi.

Controlled delivery of nikkomycin by PEG coated PLGA nanoparticles inhibits chitin synthase to prevent growth of Aspergillus flavus and Aspergillus fumigatus.

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 MIC50 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.

HPLC, NMR Based Characterization, Antioxidant and Anticancer Activities of Chemical Constituents from Therapeutically Active Fungal Endophytes.

Fungi generate different metabolites some of which are intrinsically bioactive and could therefore serve as templates for drug development. In the current study, six endophytic fungi namely Aspergillus flavus, Aspergillus tubigenesis, Aspergillus oryzae, Penicillium oxalicum, Aspergillus niger, and Aspergillus brasiliensis were isolated and identified from the medicinal plant, Silybum marianum. These endophytic fungi were identified through intra transcribed sequence (ITS) gene sequencing. The bioactive potentials of fungal extracts were investigated using several bioassays such as antibacterial activity by well-diffusion, MIC, MBC, anti-biofilm, antioxidant, and haemolysis. The Pseudomonas aeruginosa PAO1 was used to determine the antibiofilm activity. The ethyl acetate extract of Aspergillus flavus showed strong to moderate efficacy against Staphylococcus aureus, Escherichia coli, P. aeruginosa, and Bacillus spizizenii. Aspergillus flavus and Aspergillus brasiliensis exhibited significant antibiofilm activity with IC50 at 4.02 and 3.63 mg/ml, while A. flavus exhibited maximum antioxidant activity of 50.8%. Based on HPLC, LC-MS, and NMR experiments kojic acid (1) and carbamic acid (methylene-4, 1-phenylene) bis-dimethyl ester (2) were identified from A. flavus. Kojic acid exhibited DPPH free radical scavenging activity with an IC50 value of 99.3 µg/ml and moderate activity against ovarian teratocarcinoma (CH1), colon carcinoma (SW480), and non-small cell lung cancer (A549) cell lines. These findings suggest that endophytic fungi are able to produce promising bioactive compounds which deserve further investigation.

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.

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.

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.

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

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

Investigation of Azole Resistance Involving cyp51A and cyp51B Genes in Clinical Aspergillus flavus Isolates.

This study aimed to investigate azole resistance mechanisms in Aspergillus flavus, which involve cyp51A and cyp51B genes. Real-time Reverse Transcriptase qPCR method was applied to determine the overexpression of cyp51A and cyp51B genes for 34 A. flavus isolates. PCR sequencing of these two genes was used to detect the presence of gene mutations. Susceptibility test found sensitivity to voriconazole (VOR) in all strains. 14.7% and 8.8% of isolates were resistant to itraconazole (IT) and posaconazole (POS), respectively, with a cross-resistance in 5.8%. For the double resistant isolates (IT/POS), the expression of cyp51A was up to 17-fold higher. PCR sequencing showed the presence of 2 mutations in cyp51A: a synonymous point mutation (P61P) in eight isolates, which did not affect the structure of CYP51A protein, and another non synonymous mutation (G206L) for only the TN-33 strain (cross IT/POS resistance) causing an amino acid change in the protein sequence. However, we noted in cyp51B the presence of the only non-synonymous mutation (L177G) causing a change in amino acids in the protein sequence for the TN-31 strain, which exhibits IT/POS cross-resistance. A short single intron of 67 bp was identified in the cyp51A gene, whereas three short introns of 54, 53, and 160 bp were identified in the cyp51B gene. According to the models provided by PatchDock software, the presence of non-synonymous mutations did not affect the interaction of CYP51A and CYP51B proteins with antifungals. In our study, the overexpression of the cyp51A and cyp51B genes is the primary mechanism responsible for resistance in A. flavus collection. Nevertheless, other resistance mechanisms can be involved.

Potential fungicidal and antiaflatoxigenic effects of cinnamon essential oils on Aspergillus flavus inhabiting the stored wheat grains.

Wheat is one of the essential crops for the human and animal nutrition, however, contamination with aflatoxigenic fungi, due to the improper storage conditions and high humidity, was the main global threats. So, preventing the growth of aflatoxigenic fungi in stored wheat grains, by using different essential oils was the main objective of this work. Aspergillus flavus EFBL-MU12 PP087400, EFBL-MU23 PP087401 and EFBL-MU36 PP087403 isolates were the most potent aflatoxins producers inhabiting wheat grains. The effect of storage conditions of wheat grains "humidity, temperature, incubation period, and pH" on growth of A. flavus, was assessed by the response surface methodology using Plackett-Burman design and FCCD. The highest yield of aflatoxins EFBL-MU12 B1 and B2 by A. flavus grown on wheat grains were 145.3 and 7.6 µg/kg, respectively, at incubation temperature 35°C, 16% moisture contents, initial pH 5.0, and incubated for 14 days. The tested oils had a powerful antifungal activity for the growth and aflatoxins production by A. flavus in a concentration-dependent manner. Among these oils, cinnamon oil had the highest fungicidal activity for A. flavus at 0.125%, with about 85-90 % reduction to the aflatoxins B1 and B2, conidial pigmentation and chitin contents on wheat grains. From the SEM analysis, cinnamon oils had the most deleterious effect on A. flavus with morphological aberrations to the conidial heads, vegetative mycelia, alteration in conidiophores identity, hyphae shrank, and winding. To emphasize the effect of the essential oils on the aflatoxins producing potency of A. flavus, the molecular expression of the aflatoxins biosynthetic genes was estimated by RT-qPCR. The molecular expression of nor-1, afLR, pKsA and afLJ genes was suppressed by 94-96%, due to cinnamon oil at 0.062% compared to the control. Conclusively, from the results, cinnamon oils followed by the peppermint oils displayed the most fungicidal activity for the growth and aflatoxins production by A. flavus grown on wheat grains.

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

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

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.