AflaILVB/G/I and AflaILVD are involved in mycelial production, aflatoxin biosynthesis, and fungal virulence in Aspergillus flavus.

Aflatoxins (AFs) are produced by fungi such as Aspergillus flavus and A. parasiticus and are one of the most toxic mycotoxins found in agricultural products and food. Aflatoxin contamination, which requires the control of A. flavus, remains problematic because of the lack of effective strategies and the exploration of new compounds that can inhibit A. flavus growth and mycotoxin production is urgently required to alleviate potential deleterious effects. Acetohydroxy acid synthase (AHAS) and dihydroxy acid dehydratase are important enzymes in the biosynthetic pathways of branched-chain amino acids (BCAAs), including isoleucine, leucine, and valine. Enzymes involved in BCAA biosynthesis are present in bacteria, plants, and fungi, but not in mammals, and are therefore, attractive targets for antimicrobial and herbicide development. In this study, we characterized AflaILVB/G/I and AflaILVD, which encode the catalytic and regulatory subunits of AHAS and dihydroxy acid dehydratase, from the pathogenic fungus Aspergillus flavus. The AflaILVB/G/I and AflaILVD deletion mutant grew slower and produced smaller colonies than the wild-type strain when grown on glucose minimal medium, potato dextrose agar, and yeast extract medium for three days at 28°C, and disruption of AflaILVB/G/I caused a significant reduction in conidia production when grown on all kinds of media. Cellular stress assays determined that all strains were sensitive to H2O2. Importantly, the pathogenicity and aflatoxin production were affected when AflaILVB/G/I and AflaILVD were knocked out, particularly AflaILVB/G/I. A series of genes that encoded enzymes involved in aflatoxin synthesis were downregulated, meaning that the knockout of AflaILVB/G/I influenced aflatoxin synthesis in A. flavus strain WT. Collectively, our results demonstrate the potential value of antifungals targeting AflaILVB/G/I in A. flavus.

m6A methyltransferase AflIme4 orchestrates mycelial growth, development and aflatoxin B1 biosynthesis in Aspergillus flavus.

Aflatoxin B1 (AFB1), a highly toxic secondary metabolite produced by Aspergillus flavus, poses a severe threat to agricultural production, food safety and human health. The methylation of mRNA m6A has been identified as a regulator of both the growth and AFB1 production of A. flavus. However, its intracellular occurrence and function needs to be elucidated. Here, we identified and characterized a m6A methyltransferase, AflIme4, in A. flavus. The enzyme was localized in the cytoplasm, and knockout of AflIme4 significantly reduced the methylation modification level of mRNA. Compared with the control strains, ΔAflIme4 exhibited diminished growth, conidial formation, mycelial hydrophobicity, sclerotium yield, pathogenicity and increased sensitivity to CR, SDS, NaCl and H2O2. Notably, AFB1 production was markedly inhibited in the A. flavus ΔAflIme4 strain. RNA-Seq coupled with RT-qPCR validation showed that the transcriptional levels of genes involved in the AFB1 biosynthesis pathway including aflA, aflG, aflH, aflK, aflL, aflO, aflS, aflV and aflY were significantly upregulated. Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) analysis demonstrated a significant increase in m6A methylation modification levels of these pathway-specific genes, concomitant with a decrease in mRNA stability. These results suggest that AflIme4 attenuates the mRNA stability of genes in AFB1 biosynthesis by enhancing their mRNA m6A methylation modification, leading to impaired AFB1 biosynthesis. Our study identifies a novel m6A methyltransferase AflIme4 and highlights it as a potential target to control A. flavus growth, development and aflatoxin pollution.

Inclusion Complexes of Litsea cubeba (Lour.) Pers Essential Oil into ß-Cyclodextrin: Preparation, Physicochemical Characterization, Cytotoxicity and Antifungal Activity.

The uses of natural compounds, such as essential oils (EOs), are limited due to their instability to light, oxygen and temperature, factors that affect their application. Therefore, improving stability becomes necessary. The objective of this study was to prepare inclusion complexes of Litsea cubeba essential oil (LCEO) with ß-cyclodextrin (ß-CD) using physical mixing (PM), kneading (KN) and co-precipitation (CP) methods and to evaluate the efficiency of the complexes and their physicochemical properties using ATR-FTIR, FT-Raman, DSC and TG. The study also assessed cytotoxicity against human colorectal and cervical cancer cells and antifungal activity against Aspergillus flavus and Fusarium verticillioides. The complexation efficiency results presented significant evidence of LCEO:ß-CD inclusion complex formation, with KN (83%) and CP (73%) being the best methods used in this study. All tested LCEO:ß-CD inclusion complexes exhibited toxicity to HT-29 cells. Although the cytotoxic effect was less pronounced in HeLa tumor cells, LCEO-KN was more active against Hela than non-tumor cells. LCEO-KN and LCEO-CP inclusion complexes were efficient against both toxigenic fungi, A. flavus and F. verticillioides. Therefore, the molecular inclusion of LCEO into ß-CD was successful, as well as the preliminary biological results, evidencing that the ß-CD inclusion process may be a viable alternative to facilitate and increase future applications of this EO as therapeutic medication, food additive and natural antifungal agent.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Toxigenic Aspergillus flavus and A. carbonarius in Nuts.

Aspergillus species create major postharvest problems due to the food losses caused by their mere presence and the hazardous mycotoxins they produce, such as aflatoxin B1 (AFB1) and ochratoxin A (OTA). These mycotoxins are mainly produced by A. flavus and A. carbonarius, respectively. In this study, we developed a rapid detection method for the two aforementioned species based on loop-mediated isothermal amplification (LAMP). The primers were designed to target genes belonging to the mycotoxin clusters pks and aflT for A. carbonarius and A. flavus, respectively. Result visualization was carried out in real time via the detection of fluorescent signals. The method developed showed high sensitivity and specificity, with detection limits of 0.3 and 0.03 pg/reaction of purified DNA of A. carbonarius and A. flavus, respectively. The assays were further implemented on inoculated nuts, including pistachios and almonds, after one-step crude DNA extraction. These tests revealed a detection level of 0.5 spore/g that shows the effectiveness of LAMP as a rapid method for detecting potentially toxigenic Aspergillus spp. directly in food. The validation of the assays included tests on a larger scale that further confirmed their sensitivity and specificity, as well as enabling the production of ready-to-use LAMP prototype kits. These kits are easy to use and aim to simplify the screening of food samples in order to monitor the presence of specific Aspergillus contaminations.

Aflatoxin profiles of Aspergillus flavus isolates in Sudanese fungal rhinosinusitis.

Aspergillus flavus is a commonly encountered pathogen responsible for fungal rhinosinusitis (FRS) in arid regions. The species is known to produce aflatoxins, posing a significant risk to human health. This study aimed to investigate the aflatoxin profiles of A. flavus isolates causing FRS in Sudan. A total of 93 clinical and 34 environmental A. flavus isolates were studied. Aflatoxin profiles were evaluated by phenotypic (thin-layer and high-performance chromatography) and genotypic methods at various temperatures and substrates. Gene expression of aflD and aflR was also analyzed. A total of 42/93 (45%) isolates were positive for aflatoxin B1 and AFB2 by HPLC. When the incubation temperature changed from 28°C to 36°C, the number of positive isolates decreased to 41% (38/93). Genetic analysis revealed that 85% (79/93) of clinical isolates possessed all seven aflatoxin biosynthesis-associated genes, while 27% (14/51) of non-producing isolates lacked specific genes (aflD/aflR/aflS). Mutations were observed in aflS and aflR genes across both aflatoxin-producers and non-producers. Gene expression of aflD and aflR showed the highest expression between the 4th and 6th days of incubation on the Sabouraud medium and on the 9th day of incubation on the RPMI (Roswell Park Memorial Institute) medium. Aspergillus flavus clinical isolates demonstrated aflatoxigenic capabilities, influenced by incubation temperature and substrate. Dynamic aflD and aflR gene expression patterns over time enriched our understanding of aflatoxin production regulation. The overall findings underscored the health risks of Sudanese patients infected by this species, emphasizing the importance of monitoring aflatoxin exposure.

Aspergillus flavus, mainly causing fungal rhinosinusitis in Sudan, poses health risks due to aflatoxin production. This study revealed diverse levels of aflatoxin and gene expression of clinical isolates by pheno- and genotypic methods, emphasizing the need for vigilant monitoring in the region.

Melanin depletion affects Aspergillus flavus conidial surface proteins, architecture, and virulence.

Melanin is an Aspergillus flavus cell wall component that provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile, and virulence of melanized conidia (MC) and non-melanized conidia (NMC) of A. flavus. Kojic acid treatment inhibited melanin synthesis in A. flavus, and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathways were found only in the formic acid extracts of NMC. Scanning electron microscopy (SEM) analysis showed the conidial surface morphology difference between the NMC and MC, indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor white staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining between MC and NMC. Evaluation of the virulence of MC and NMC in the Galleria mellonella model showed NMC was less virulent compared to MC. Our findings showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer, and hence, melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. The G. mellonella virulence assay also confirmed that the NMC were susceptible to host defense as in other Aspergillus pathogens. KEY POINTS: • l-DOPA melanin production was inhibited in A. flavus isolates by kojic acid, and for the first time, scanning electron microscopy (SEM) analysis revealed morphological differences between MC and NMC of A. flavus strains • Proteome profile of non-melanized conidia showed more conidial surface proteins and these proteins were mainly involved in the virulence, oxidative stress, and metabolism pathways • Non-melanized conidia of A. flavus strains were shown to be less virulent than melanised conidia in an in vivo virulence experiment with the G. melonella model.

Structure of Aspergillus flavus populations associated with maize in Greece, Spain, and Serbia: Implications for aflatoxin biocontrol on a regional scale.

Aspergillus flavus is the most frequently identified producer of aflatoxins. Non-aflatoxigenic members of the A. flavus L strains are used in various continents as active ingredients of bioprotectants directed at preventing aflatoxin contamination by competitive displacement of aflatoxin producers. The current research examined the genetic diversity of A. flavus L strain across southern Europe to gain insights into the population structure and evolution of this species and to evaluate the prevalence of genotypes closely related to MUCL54911, the active ingredient of AF-X1. A total of 2173L strain isolates recovered from maize collected across Greece, Spain, and Serbia in 2020 and 2021 were subjected to simple sequence repeat (SSR) genotyping. The analysis revealed high diversity within and among countries and dozens of haplotypes shared. Linkage disequilibrium analysis indicated asexual reproduction and clonal evolution of A. flavus L strain resident in Europe. Moreover, haplotypes closely related to MUCL54911 were found to belong to the same vegetative compatibility group (VCG) IT006 and were relatively common in all three countries. The results indicate that IT006 is endemic to southern Europe and may be utilized as an aflatoxin mitigation tool for maize across the region without concern for potential adverse impacts associated with the introduction of an exotic microorganism.

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.

Synergistic antifungal mechanism of cinnamaldehyde and nonanal against Aspergillus flavus and its application in food preservation.

Aspergillus flavus colonization on agricultural products during preharvest and postharvest results in tremendous economic losses. Inspired by the synergistic antifungal effects of essential oils, the aims of this study were to explore the mechanism of combined cinnamaldehyde and nonanal (SCAN) against A. flavus and to evaluate the antifungal activity of SCAN loading into diatomite (DM). Shriveled mycelia were observed by scanning electron microscopy, especially in the SCAN treatment group. Calcofluor white staining, transmission electron microscopy, dichloro-dihydro-fluorescein diacetate staining and the inhibition of key enzymes in tricarboxylic acid cycle indicated that the antifungal mechanism of SCAN against A. flavus was related to the cell wall damage, reactive oxygen species accumulation and energy metabolism interruption. RNA sequencing revealed that some genes involved in antioxidation were upregulated, whereas genes responsible for cell wall biosynthesis, oxidative stress, cell cycle and spore development were significantly downregulated, supporting the occurrence of cellular apoptosis. In addition, compared with the control group, conidia production in 1.5 mg/mL DM/cinnamaldehyde, DM/nonanal and DM/SCAN groups were decreased by 27.16%, 48.22% and 76.66%, respectively, and the aflatoxin B1 (AFB1) contents decreased by 2.00%, 73.02% and 84.15%, respectively. These finding suggest that DM/SCAN complex has potential uses in food preservation.

Inhibition of Aflatoxin Production in Aspergillus flavus by a Klebsiella sp. and Its Metabolite Cyclo(l-Ala-Gly).

During an experiment where we were cultivating aflatoxigenic Aspergillus flavus on peanuts, we accidentally discovered that a bacterium adhering to the peanut strongly inhibited aflatoxin (AF) production by A. flavus. The bacterium, isolated and identified as Klebsiella aerogenes, was found to produce an AF production inhibitor. Cyclo(l-Ala-Gly), isolated from the bacterial culture supernatant, was the main active component. The aflatoxin production-inhibitory activity of cyclo(l-Ala-Gly) has not been reported. Cyclo(l-Ala-Gly) inhibited AF production in A. flavus without affecting its fungal growth in a liquid medium with stronger potency than cyclo(l-Ala-l-Pro). Cyclo(l-Ala-Gly) has the strongest AF production-inhibitory activity among known AF production-inhibitory diketopiperazines. Related compounds in which the methyl moiety in cyclo(l-Ala-Gly) is replaced by ethyl, propyl, or isopropyl have shown much stronger activity than cyclo(l-Ala-Gly). Cyclo(l-Ala-Gly) did not inhibit recombinant glutathione-S-transferase (GST) in A. flavus, unlike (l-Ala-l-Pro), which showed that the inhibition of GST was not responsible for the AF production-inhibition of cyclo(l-Ala-Gly). When A. flavus was cultured on peanuts dipped for a short period of time in a dilution series bacterial culture broth, AF production in the peanuts was strongly inhibited, even at a 1 × 104-fold dilution. This strong inhibitory activity suggests that the bacterium is a candidate for an effective biocontrol agent for AF control.

Genome-Wide Characterization of the Phenylalanine Ammonia-Lyase Gene Family and Their Potential Roles in Response to Aspergillus flavus L. Infection in Cultivated Peanut (Arachis hypogaea L.).

Phenylalanine ammonia-lyase (PAL) is an essential enzyme in the phenylpropanoid pathway, in which numerous aromatic intermediate metabolites play significant roles in plant growth, adaptation, and disease resistance. Cultivated peanuts are highly susceptible to Aspergillus flavus L. infection. Although PAL genes have been characterized in various major crops, no systematic studies have been conducted in cultivated peanuts, especially in response to A. flavus infection. In the present study, a systematic genome-wide analysis was conducted to identify PAL genes in the Arachis hypogaea L. genome. Ten AhPAL genes were distributed unevenly on nine A. hypogaea chromosomes. Based on phylogenetic analysis, the AhPAL proteins were classified into three groups. Structural and conserved motif analysis of PAL genes in A. hypogaea revealed that all peanut PAL genes contained one intron and ten motifs in the conserved domains. Furthermore, synteny analysis indicated that the ten AhPAL genes could be categorized into five pairs and that each AhPAL gene had a homologous gene in the wild-type peanut. Cis-element analysis revealed that the promoter region of the AhPAL gene family was rich in stress- and hormone-related elements. Expression analysis indicated that genes from Group I (AhPAL1 and AhPAL2), which had large number of ABRE, WUN, and ARE elements in the promoter, played a strong role in response to A. flavus stress.

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.

The potential for reducing aflatoxin B1 contamination of stored peanuts by soil disinfection.

Aflatoxins from the fungus Aspergillus flavus (A. flavus) that contaminate stored peanuts is a major hazard to human health worldwide. Reducing A. flavus in soil can decrease the risk of aflatoxins in stored peanuts. In this experiment, we determined whether peanuts grown on soil fumigated with dazomet (DZ), metham sodium (MS), allyl isothiocyanate (AITC), chloropicrin (PIC) or dimethyl disulfide (DMDS) would reduce of the quantity of A. flavus and its toxin's presence. The results of bioassays and field tests showed that PIC was the most effective fumigant for preventing and controlling A. flavus, followed by MS. PIC and MS applied to the soil for 14 d resulted in LD50 values against A. flavus of 3.558 and 4.893 mg kg-1, respectively, leading to almost 100% and 98.82% effectiveness of A. flavus, respectively. Peanuts harvested from fumigated soil and then stored for 60 d resulted in undetectable levels of aflatoxin B1 (AFB1) compared to unfumigated soil that contained 0.64 ug kg-1 of AFB1, which suggested that soil fumigation can reduce the probability of aflatoxin contamination during peanut storage and showed the potential to increase the safety of peanuts consumed by humans. Further research is planned to determine the practical value of our research in commercial practice.

Bacteria-mediated green synthesis of silver nanoparticles and their antifungal potentials against Aspergillus flavus.

The best biocontroller Bacillus subtilis produced silver nanoparticles (AgNPs) with a spherical form and a 62 nm size through green synthesis. Using UV-vis spectroscopy, PSA, and zeta potential analysis, scanning electron microscopy, and Fourier transform infrared spectroscopy, the properties of synthesized silver nanoparticles were determined. Silver nanoparticles were tested for their antifungicidal efficacy against the most virulent isolate of the Aspergillus flavus fungus, JAM-JKB-BHA-GG20, and among the 10 different treatments, the treatment T6 [PDA + 1 ml of NP (19: 1)] + Pathogen was shown to be extremely significant (82.53%). TG-51 and GG-22 were found to be the most sensitive groundnut varieties after 5 and 10 days of LC-MS QTOF infection when 25 different groundnut varieties were screened using the most toxic Aspergillus flavus isolate JAM- JKB-BHA-GG20, respectively. In this research, the most susceptible groundnut cultivar, designated GG-22, was tested. Because less aflatoxin (1651.15 g.kg-1) was observed, treatment T8 (Seed + Pathogen + 2 ml silver nanoparticles) was determined to be much more effective. The treated samples were examined by Inductively Coupled Plasma Mass Spectrometry for the detection of metal ions and the fungicide carbendazim. Ag particles (0.8 g/g-1) and the fungicide carbendazim (0.025 g/g-1) were found during Inductively Coupled Plasma Mass Spectrometry analysis below detectable levels. To protect plants against the invasion of fungal pathogens, environmentally friendly green silver nanoparticle antagonists with antifungal properties were able to prevent the synthesis of mycotoxin by up to 82.53%.

Synthesis and toxicological study of chitosan-pirul (Schinus molle L.) essential oil nanoparticles on Aspergillus flavus.

In recent years, the study of essential oils as antifungal alternatives and their encapsulation to increase their properties for greater effects has been tested. In this work, nanoparticles of chitosan-Schinus molle L. essential oil (CS-PEO-Np) with a size of 260 ± 31.1 nm were obtained by ionic gelation and evaluated in some growth phases of Aspergillus flavus, a toxigenic fungus. At a concentration of 250 µg/mL of CS-PEO-Np, the A. flavus mycelial growth was inhibited at 97.1% with respect to control, at 96 h of incubation; the germination and viability of spores were inhibited at 74.8 and 40%, respectively, after exposure to 500 µg/mL of these nanomaterials, at 12 h of incubation. The fluorescence images of stained spores with DAPI showed the affectations caused by nanoparticles in the cell membrane, vacuoles and vacuolar content, cell wall, and nucleic acids. For both nanoparticles, CS-Np and CS-PEO-Np, no mutagenic effect was observed in Salmonella Typhimurium; also, the phytotoxic assay showed low-to-moderate toxicity toward seeds, which was dependent on the nanoparticle's concentration. The acute toxicity of CS-PEO-Np to A. salina nauplii was considered low in comparison to CS-Np (control), which indicates that the incorporation of Schinus molle essential oil into nanoparticles of chitosan is a strategy to reduce the toxicity commonly associated with nanostructured materials. The nanoparticulated systems of CS-PEO-Np represent an effective and non-toxic alternative for the control of toxigenic fungi such as A. flavus by delaying the initial growth stage.

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.

Assessment of Trachyspermum ammi essential oil against Aspergillus flavus, aflatoxin B1 contamination, and post-harvest quality of Sorghum bicolor.

The present investigation explored the antifungal effectiveness of Trachyspermum ammi essential oil (TAEO) against Aspergillus flavus, aflatoxin B1 (AFB1) contamination, and its mechanism of action using biochemical and computational approaches. The GC-MS result revealed the chemical diversity of TAEO with the highest percentage of γ-terpinene (39 %). The TAEO exhibited minimum inhibitory concentration against A. flavus growth (0.5 µL/mL) and AFB1 (0.4 µL/mL) with radical scavenging activity (IC50 = 2.13 µL/mL). The mechanism of action of TAEO was associated with the alteration in plasma membrane functioning, antioxidative defense, and carbon source catabolism. The molecular dynamic result shows the multi-regime binding of γ-terpinene with the target proteins (Nor1, Omt1, and Vbs) of AFB1 biosynthesis. Furthermore, TAEO exhibited remarkable in-situ protection of Sorghum bicolor seed samples against A. flavus and AFB1 contamination and protected the nutritional deterioration. Hence, the study recommends TAEO as a natural antifungal agent for food protection against A. flavus mediated biodeterioration.

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.

Evaluation of the efficacy of cinnamon oil on Aspergillus flavus and Fusarium proliferatum growth and mycotoxin production on paddy and polished rice: Towards a mitigation strategy.

In the present investigation, the effect of cinnamon oil (CO) (10, 30, 50 and 70 %) on the growth rate (mm/day) and aflatoxin B1 (AFB1) and fumonisin B1 (FB1) production of Aspergillus flavus (AF01) and Fusarium proliferatum (FP01) isolates, respectively was determined at optimum water activities (0.95 and 0.99 aw) and temperatures (25, 30 and 35 °C) on paddy and polished rice grains. The results showed that the growth rate, AFB1 and FB1 production of all the fungal isolates decreased with an increase in CO concentrations on both matrices. AF01 and FP01 failed to grow under all conditions on paddy at 50 % of CO concentration whereas both fungi were completely inhibited (No Growth-NG) at 70 % of CO on polished rice. Regarding mycotoxin production, 30 % of CO concentrations could inhibit AFB1 and FB1 production in both matrices (No Detection-ND). In this study, the production of mycotoxins was significantly influenced by cinnamon oil compared to the growth of both fungi. These results indicated the promising potential of CO in improving the quality of rice preservation in post-harvest; however, further investigations should be evaluated on the effects on the qualitative characteristics of grains. Especially, the prospective application of CO in rice storage in industry scales to mitigate mycotoxin contamination need also to be further researched. Moreover, collaboration between researchers, agricultural experts, and food industry should be set up to achieve effective and sustainable strategies for preserving rice.

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.