Aflatoxin Pathway Research Articles

Aspergillus population diversity and its role in aflatoxin contamination of cashew nuts from coastal Kenya.

Cashew nuts are among the main cash crops in coastal Kenya, due in large part to their high nutritional value. Unfortunately, they also make them highly susceptible to mold contamination, resulting in biodeterioration of the nutritional value and potential contamination with toxic secondary metabolites, such as aflatoxins, that cause them to be rejected for sale at the market. We determined the population diversity of the Aspergillus species and their role in aflatoxin contamination in cashew nuts in selected coastal regions of Kenya. Fifty raw cashew nut samples were collected from post-harvest storage facilities across three counties in Kenya's coastal region and examined for moisture content and the presence of Aspergillus fungi. About 63 presumptive isolates were recovered from the cashew nuts. ITS and 28S rDNA regions were sequenced. The aflD, aflM and aflR genes were amplified to identify the potentially aflatoxigenic from the Aspergillus isolates. The Aflatoxins' presence on the isolates was screened using UV and the ammonia vapour test on coconut milk agar and validated using ELISA assay. A comparison of cashew moisture content between the three counties sampled revealed a significant difference. Sixty-three isolates were recovered and identified to section based on morphological characters and their respective ITS regions were used to obtain species identifications. Three sections from the genus were represented, Flavi and Nigri, and Terrei with isolates from the section Nigri having slightly greater abundance (n = 35). The aflD, aflM and aflR genes were amplified for all isolates to assess the presence of the aflatoxin biosynthesis pathway, indicating the potential for aflatoxin production. Less than half of the Aspergillus isolates (39.68%) contained the aflatoxin pathway genes, while 22.22% isolates were aflatoxigenic, which included only the section Flavi isolates. Section Flavi isolates identification was confirmed by calmodulin gene. The presence of species from Aspergillus section Flavi and section Nigri indicate the potential for aflatoxin or ochratoxin in the cashew nuts. The study established a foundation for future investigations of the fungi and mycotoxins contaminating cashew nuts in Kenya, which necessitates developing strategies to prevent infection by mycotoxigenic fungi, especially during the storage and processing phases.

Control of aflatoxin biosynthesis by sulfur containing benzimidazole derivatives: In-silico interaction, biological activity, and gene regulation of Aspergillus flavus.

Aspergillus flavus producing aflatoxins is one of the potent contaminants of raw food commodities during pre-and post-harvest crops. Aflatoxins are the group of secondary metabolites a subset of natural polyketides. Our major focus is on the inhibition of the biosynthesis pathway of aflatoxin by targeting the enzymes involved. Benzimidazoles are known antimicrobial compounds. In this study the sulfur containing benzimidazole derivatives were tested for their antifungal and antiaflatoxigenic activity. The fungal growth and aflatoxin production was analysed in culture medium as well as in the rice. Inhibition of specific genes was studied in terms of mRNA expression and the interaction of test compound with polyketide synthases by in-silico molecular docking. Substitution at the 6th position of 2-(2-thienyl) benzimidazole (2-TBD) reduced the antifungal property of benzimidazole but effectively inhibited the aflatoxin synthesis in the culture medium as well as in the rice from the toxigenic strain of A. flavus. Among the derivatives tested, the methyl group containing 2-(2-thienyl)-6-methylbenzimidazole (6-MTBD) inhibited aflatoxin B1 most effectively followed by carboxylic group containing 2-(2-thienyl) benzimidazole-6-carboxylic acid (6-TBCA) with IC50 value of 12.36 and 18.25µg/mL respectively. Molecular docking study shows that 2-(2-thienyl) benzimidazole-6-carbonitrile (6-CTBD) and 6-MTBD occupy same pocket on TE domain of PksA with similar range of binding energy, however the experimental data show a different effect on the biosynthesis of AFB1. 6-MTBD effectively inhibited the AFB1 synthesis (97%) while 6-CTBD could not (39.5%). Data obtained from the expression study also supports the experimental observations. These compounds are non-toxic to mammalian cells. These benzimidazole derivatives inhibit toxic secondary metabolites without affecting the growth of the fungi hence can be used during fermentation to avoid mycotoxin contamination.

Multiplexed Host-Induced Gene Silencing of Aspergillus flavus Genes Confers Aflatoxin Resistance in Groundnut.

Aflatoxins are immunosuppressive and carcinogenic secondary metabolites, produced by the filamentous ascomycete Aspergillus flavus, that are hazardous to animal and human health. In this study, we show that multiplexed host-induced gene silencing (HIGS) of Aspergillus flavus genes essential for fungal sporulation and aflatoxin production (nsdC, veA, aflR, and aflM) confers enhanced resistance to Aspergillus infection and aflatoxin contamination in groundnut (<20 ppb). Comparative proteomic analysis of contrasting groundnut genotypes (WT and near-isogenic HIGS lines) supported a better understanding of the molecular processes underlying the induced resistance and identified several groundnut metabolites that might play a significant role in resistance to Aspergillus infection and aflatoxin contamination. Fungal differentiation and pathogenicity proteins, including calmodulin, transcriptional activator-HacA, kynurenine 3-monooxygenase 2, VeA, VelC, and several aflatoxin pathway biosynthetic enzymes, were downregulated in Aspergillus infecting the HIGS lines. Additionally, in the resistant HIGS lines, a number of host resistance proteins associated with fatty acid metabolism were strongly induced, including phosphatidylinositol phosphate kinase, lysophosphatidic acyltransferase-5, palmitoyl-monogalactosyldiacylglycerol Δ-7 desaturase, ceramide kinase-related protein, sphingolipid Δ-8 desaturase, and phospholipase-D. Combined, this knowledge can be used for groundnut pre-breeding and breeding programs to provide a safe and secure food supply.

Vibrio gazogenes Inhibits Aflatoxin Production Through Downregulation of Aflatoxin Biosynthetic Genes in Aspergillus flavus

Aspergillus flavus is an opportunistic pathogen of oilseed crops such as maize, peanut, cottonseed, and tree nuts and produces carcinogenic secondary metabolites known as aflatoxins during seed colonization. Aflatoxin contamination not only reduces the value of the produce but is also a health hazard to humans and animals. Previously, we observed inhibition of A. flavus aflatoxin biosynthesis on exposure to the marine bacterium Vibrio gazogenes ( Vg). In this study, we used RNA sequencing to examine the transcriptional profiles of A. flavus treated with both live and heat-inactivated dead Vg and control samples. Fungal biomass, total accumulated aflatoxins, and expression profiles of genes constituting secondary metabolite biosynthetic gene clusters were determined at 24, 30, and 40 h after treatment. Statistically significant reductions in total aflatoxins were detected in Vg-treated samples as compared with control samples at 40 h. However, no statistical difference in fungal biomass was observed on these treatments. The Vg treatments were most effective on aflatoxin biosynthesis, as was reflected in significant downregulation of most genes in the aflatoxin gene cluster, including the aflatoxin pathway regulator gene, aflR. Along with aflatoxin genes, we also observed significant downregulation in some other secondary metabolite gene clusters, including cyclopiazonic acid and aflavarin, suggesting that the treatment could inhibit other secondary metabolites as well. Finally, a weighted gene correlation network analysis identified an upregulation of 10 genes that were most strongly associated with Vg-dependent aflatoxin inhibition and provide a novel starting point in understanding the mechanisms that result in this phenomenon. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022.

Detoxification of Aflatoxin B1 by a Potential Probiotic Bacillus amyloliquefaciens WF2020

Microbial degradation is considered as an attractive method to eliminate exposure to aflatoxin B1 (AFB1), the most toxic mycotoxin that causes great economic losses and brings a serious threat to human and animal health, in food and feed. In this study, Bacillus amyloliquefaciens WF2020, isolated from naturally fermented pickles, could effectively degrade AFB1 ranging from 1 to 8 μg/ml, and the optimum temperature and pH value were 37–45°C and 8.0, respectively. Moreover, B. amyloliquefaciens WF2020 was considered to be a potential probiotic due to the synthesis of active compounds, absence of virulence genes, susceptibility to various antibiotics, and enhanced lifespan of Caenorhabditis elegans. Extracellular enzymes or proteins played a major role in AFB1 degradation mediated by B. amyloliquefaciens WF2020 into metabolites with low or no mutagenicity and toxicity to C. elegans. AFB1 degradation by the cell-free supernatant was stable up to 70°C, with an optimal pH of 8.0, and the cell-free supernatant could still degrade AFB1 by 37.16% after boiling for 20 min. Furthermore, B. amyloliquefaciens WF2020 caused a slight defect in fungal growth and completely inhibited AFB1 production when co-incubated with Aspergillus flavus. Additionally, B. amyloliquefaciens WF2020 suppressed the expression of 10 aflatoxin pathway genes and 2 transcription factors (alfR and alfS), suggesting that B. amyloliquefaciens WF2020 might inhibit AFB1 synthesis in A. flavus. These results indicate that B. amyloliquefaciens WF2020 and/or its extracellular enzymes or proteins have a promising potential to be applied in protecting food and feed from AFB1 contamination.

Fusarium verticillioides and Aspergillus flavus Co-Occurrence Influences Plant and Fungal Transcriptional Profiles in Maize Kernels and In Vitro.

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

In Vitro Biological Control of Aspergillus flavus by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793, Producers of Antifungal Volatile Organic Compounds.

Aspergillus flavus is a toxigenic fungal colonizer of fruits and cereals and may produce one of the most important mycotoxins from a food safety perspective, aflatoxins. Therefore, its growth and mycotoxin production should be effectively avoided to protect consumers’ health. Among the safe and green antifungal strategies that can be applied in the field, biocontrol is a recent and emerging strategy that needs to be explored. Yeasts are normally good biocontrol candidates to minimize mold-related hazards and their modes of action are numerous, one of them being the production of volatile organic compounds (VOCs). To this end, the influence of VOCs produced by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793 on growth, expression of the regulatory gene of the aflatoxin pathway (aflR) and mycotoxin production by A. flavus for 21 days was assessed. The results showed that both yeasts, despite producing different kinds of VOCs, had a similar effect on inhibiting growth, mycotoxin biosynthetic gene expression and phenotypic toxin production overall at the mid-incubation period when their synthesis was the greatest. Based on the results, both yeast strains, H. opuntiae L479 and H. uvarum L793, are potentially suitable as a biopreservative agents for inhibiting the growth of A. flavus and reducing aflatoxin accumulation.

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

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

In silico Targeting, inhibition and analysis of polyketide synthase enzyme in Aspergillus ssp.

Aflatoxins are toxic and carcinogenic components produced by some Aspergillus species such as Aspergillus flavus. Polyketide synthases enzyme (PKS) plays a central role in aflatoxin s biosynthesis of in Aspergillus flavus, especially the product template (PT) domain, which controls the aldol cyclization of the polyketide forerunner during the biosynthesis of the aflatoxin pathway process. Here, we apply the in silico approaches to validate 623 natural components obtained from the South African Natural Compound Database (SANCDB), to distinguish the PT domain s prospected inhibitors. From the 623 compounds, docking results showed that there are 330 different compounds with energy binding lower than the natural substrate (palmitic acid or PLM) of the Product Templet domain (PT). Three factors were selected to determine the best 10 inhibiting components; 1) energy binding, 2) the strengthen chemical interactions, 3) the drug-likeness. The top ten inhibiting components are kraussianone 6, kraussianone 1, neodiospyrin, clionamine D, bromotopsentin, isodiospyrin, spongotine A, kraussianone 3, 14β-Hydroxybufa-3,5,20,22-tetraenolide and kraussianone 7. The chemical interactions between 3HRQ domain and the natural substrate in the active site amino acids are highly similar to the 3HRQ with the top ten components, but the main differences are in the binding energy which is the best in the top ten ligands. Those ten components give successful inhibition with PT domain which will lead to the formula to be used for inhibition and control aflatoxin contamination of agriculture crop yields and lessen the degree of harming and sicknesses that are coming about because of acquiring measures of aflatoxin.

Fungal contamination spices from Indonesia with emphasis on Aspergillus flavus

Filamentous fungi were isolated from ten spices collected from markets in Indonesia. The aim was to enumerate fungal contamination and to determine the toxigenicity of Aspergillus flavus strains on each of the spices. Viable fungal populations were determined using a dilution method. Toxigenicity of Aspergillus flavus was determined by culture on a quick screening coconut agar and by PCR using four sets of primers specific for aflatoxin pathway genes. All the tested spices were contaminated by storage fungi, Species of Aspergillus was the most commonly isolated moulds followed by species of Fusarium, Mucor, Penicillium and Rhizopus. The greatest number of Aspergillus flavus isolates were found on white pepper, followed by nutmeg, cardamom, and black pepper. The greatest number of Aspergillus chevalieri isolates were found on coriander, followed by nutmeg. Fifty strains of A. flavus were isolated, all of the strains produced large sclerotia and biseriate conidiophores. Their toxigenicity was assayed by the presence of yellow pigment on a quick screening coconut agar medium and PCR amplification of regulatory and structural genes in the aflatoxin pathway.

Computer-aid molecular docking technology in cereal mycotoxin analysis.

Computer-aid molecular docking is a simulative process that receptors and ligands recognize each other through energy matching and geometric matching. It is widely used in bioactive compounds simulative screening and preliminary exploring the bioactivity and toxicity of molecular, which plays important guiding role in toxicity and bioactivity study of molecular entities. In our study, we used the computer-aid molecular docking software-discovery studio 3.1 client to test the mechanism of aflatoxins such as aflatoxin B1, B2, M1, M2, G1, G2 and the results of our experiment help to illustrate the pathway of aflatoxin’s toxication. We also used this technology to test the preliminary toxicity of zearalenone (ZEN) and its two degradation products: a- zearalenol (a-ZOL) and s-zearalenol (s-ZOL), which indicates that these three products possessed significant estrogenic activity. The order of the estrogenic activity is: a-zearalenol > zearalenone >s-zearalenol.

Fusion expression and anti-Aspergillus flavus activity of a novel inhibitory protein DN-AflR

The regulatory gene (aflR) encodes AflR, a positive regulator of transcriptional pathway that activates aflatoxin biosynthesis. It has been demonstrated in our laboratory that L-Asp-L-Asn (DN) extracted from Bacillus megaterium inhibited the growth of Aspergillus flavus. We fused gene encoding DN with the gene encoding specific dinuclear zinc finger cluster protein of AflR, then fusion protein competed with the AflS-AflR complex for the AflR binding site and significantly improved anti-A. flavus activity (growth of A. flavus and biosynthesis of aflatoxin B1) of DN. The fusion gene dn-aflR was cloned into pET32a and recombinant plasmid was introduced into Escherichia coli BL21. The highest expression was observed after 10 h induction and fusion protein was purified by affinity chromatography column. Compared with DN, the novel fusion protein DN-AflR significantly inhibited the growth of A. flavus and biosynthesis of aflatoxin B1 (P < 0.05). This study promoted the use of competitive inhibition of fusion proteins to reduce the expression of regulatory genes in the biosynthetic pathway of aflatoxin. Moreover, it provided more supports for deep research and industrialization of such novel anti-A. flavus bio-inhibitors and biological control of microbial contamination.

Characterization of Aspergilli from dried red chilies (Capsicum spp.): Insights into the etiology of aflatoxin contamination

Aflatoxins are toxic carcinogens produced by several species of Aspergillus section Flavi, with some aflatoxin producers associated with specific crops. Red chilies (Capsicum spp.) are grown in warm regions that also favor aflatoxin-producers. Aflatoxins in red chilies may result in serious health concerns and severe economic losses. The current study sought to gain insight on causal agents of aflatoxin contamination in red chilies. Naturally contaminated chilies from markets in Nigeria (n = 55) and the United States (US) (n = 169) were examined. The A. flavus L strain was the predominant member of Aspergillus section Flavi (84%) in chilies. Highly toxigenic fungi with S strain morphology were also detected in chilies from both countries (11%), followed by A. tamarii (4.6%) and A. parasiticus (0.4%). Fungi with L morphology produced significantly lower quantities of aflatoxins (mean = 43 μg g−1) compared to S morphology fungi (mean = 667 μg g−1; p < 0.01) in liquid fermentation. Eighty-one percent of S morphology fungi from chilies in US markets produced only B aflatoxins, whereas 20%, all imported from Nigeria, produced both B and G aflatoxins; all S morphology fungi from Nigerian chilies produced both B and G aflatoxins. Multi-gene phylogenetic analyses of partial gene sequences for nitrate reductase (niaD, 2.1 kb) and the aflatoxin pathway transcription factor (aflR, 1.9 kb) resolved Aspergilli recovered from chilies into five highly supported distinct clades: 1) A. parasiticus; 2) A. flavus with either L or S morphology; 3) A. minisclerotigenes; 4) A. aflatoxiformans, and 5) a new lineage. Aspergillus aflatoxiformans and the new lineage produced the highest concentrations of total aflatoxins in chilies, whereas A. flavus L strains produced the least. The results suggest etiology of aflatoxin contamination of chili is complex and may vary with region. Knowledge of causal agents of aflatoxin contamination of chilies will be helpful in developing mitigation strategies to prevent human exposure.

Large-Scale Comparative Analysis of Eugenol-Induced/Repressed Genes Expression in Aspergillus flavus Using RNA-seq.

Aflatoxin B1 (AFB1), which is mainly produced by Aspergillus flavus and Aspergillus parasiticus, is the most toxic and hepatocarcinogenic polyketide known. Chemical fungicides are currently utilized to reduce this fungal contaminant, but they are potentially harmful to human health and the environment. Therefore, natural anti-aflatoxigenic products are used as sustainable alternatives to control food and feed contamination. For example, eugenol, presents in many essential oils, has been identified as an aflatoxin inhibitor. However, its exact mechanism of inhibition is yet to be clarified. In this study, the anti-aflatoxigenic mechanism of eugenol in A. flavus was determined using a comparative transcriptomic approach. Twenty of twenty-nine genes in the aflatoxin biosynthetic pathway were down-regulated by eugenol. The most strongly down-regulated gene was aflMa, followed by aflI, aflJ, aflCa, aflH, aflNa, aflE, aflG, aflM, aflD, and aflP. However, the expression of the regulator gene aflR did not change significantly and the expression of aflS was slightly up-regulated. The down-regulation of the global regulator gene veA resulted in the up-regulation of srrA, and the down-regulation of ap-1 and mtfA. The early developmental regulator brlA was profoundly up-regulated in A. flavus after eugenol treatment. These results suggested a model in which eugenol improves fungal development by up-regulating the expression of brlA by the suppression of veA expression and inhibits aflatoxin production through the suppression of veA expression. Exposure to eugenol also caused dysregulated transcript levels of the G protein-coupled receptors (GPCRs) and oxylipins genes. A Gene Ontology analysis indicated that the genes that were highly responsive to eugenol were mainly enriched in RNA-binding functions, suggesting that post-transcriptional modification plays a pivotal role in aflatoxin biosynthesis. KEGG analysis showed that ribosome biogenesis was the most dysregulated pathway, suggesting that eugenol dysregulates ribosome biogenesis, which then interrupts the biosynthesis of Nor-1, Ver-1, and OmtA, and prevents aflatoxisomes performing their normal function in aflatoxin production. In conclusion, our results indicated that eugenol inhibited AFB1 production by modulating the expression of structural genes in aflatoxin pathway, fungal antioxidant status, post-transcriptional modifications and biosynthesis of backbone enzymes in A. flavus.

The Aspergillus flavus Homeobox Gene, hbx1, is Required for Development and Aflatoxin Production.

Homeobox proteins, a class of well conserved transcription factors, regulate the expression of targeted genes, especially those involved in development. In filamentous fungi, homeobox genes are required for normal conidiogenesis and fruiting body formation. In the present study, we identified eight homeobox (hbx) genes in the aflatoxin-producing ascomycete, Aspergillus flavus, and determined their respective role in growth, conidiation and sclerotial production. Disruption of seven of the eight genes had little to no effect on fungal growth and development. However, disruption of the homeobox gene AFLA_069100, designated as hbx1, in two morphologically different A. flavus strains, CA14 and AF70, resulted in complete loss of production of conidia and sclerotia as well as aflatoxins B1 and B2, cyclopiazonic acid and aflatrem. Microscopic examination showed that the Δhbx1 mutants did not produce conidiophores. The inability of Δhbx1 mutants to produce conidia was related to downregulation of brlA (bristle) and abaA (abacus), regulatory genes for conidiophore development. These mutants also had significant downregulation of the aflatoxin pathway biosynthetic genes aflC, aflD, aflM and the cluster-specific regulatory gene, aflR. Our results demonstrate that hbx1 not only plays a significant role in controlling A. flavus development but is also critical for the production of secondary metabolites, such as aflatoxins.

A Network Approach of Gene Co-expression in the Zea mays/Aspergillus flavus Pathosystem to Map Host/Pathogen Interaction Pathways.

A gene co-expression network (GEN) was generated using a dual RNA-seq study with the fungal pathogen Aspergillus flavus and its plant host Zea mays during the initial 3 days of infection. The analysis deciphered novel pathways and mapped genes of interest in both organisms during the infection. This network revealed a high degree of connectivity in many of the previously recognized pathways in Z. mays such as jasmonic acid, ethylene, and reactive oxygen species (ROS). For the pathogen A. flavus, a link between aflatoxin production and vesicular transport was identified within the network. There was significant interspecies correlation of expression between Z. mays and A. flavus for a subset of 104 Z. mays, and 1942 A. flavus genes. This resulted in an interspecies subnetwork enriched in multiple Z. mays genes involved in the production of ROS. In addition to the ROS from Z. mays, there was enrichment in the vesicular transport pathways and the aflatoxin pathway for A. flavus. Included in these genes, a key aflatoxin cluster regulator, AflS, was found to be co-regulated with multiple Z. mays ROS producing genes within the network, suggesting AflS may be monitoring host ROS levels. The entire GEN for both host and pathogen, and the subset of interspecies correlations, is presented as a tool for hypothesis generation and discovery for events in the early stages of fungal infection of Z. mays by A. flavus.

Molecular and mycotoxigenic biodiversity of Aspergillus flavus isolated from Brazil nuts

The objective of this study was to carry out a transcription analysis of eight genes belonging to the aflatoxin (AF) and cyclopiazonic acid (CPA) biosynthesis pathway, and to detect aflatoxin B1 (AFB1) and CPA production in Aspergillus flavus strains isolated from Brazil nuts. Additionally, these genes were correlated with the different mycotoxigenic profiles of the same strains. Four previously identified A. flavus strains (ICB-01, ICB-151, ICB-161, and ICB-165) were grown on Brazil nut agar at 25°C for 10days. Mycotoxins were separated by high-performance liquid chromatography. Transcriptional analysis was performed by real-time RT-PCR using specific primers designed based on the conserved regions of two regulatory genes (aflR and aflS), three structural genes of the AFB1 biosynthesis pathway (aflH, aflJ and aflP), and three structural genes of the CPA biosynthesis pathway (maoA, dmaT and pks-nrps). The expression of most genes in the A. flavus isolates varied according to the mycotoxin profile of each strain. The most expressed genes in the aflatoxigenic strain ICB-151 were aflJ (77.11%) and aflH (32.75%), while the CPA-producing strain ICB-161 mainly expressed dmaT (100%), maoA (63.72%), aflS (43.52%), and aflR (42.63%). The ICB-01 isolate was a producer of AFB1 and CPA and the most expressed genes were aflS (47.79%), dmaT (42.77%), aflP (39.5%), and aflR (38.02%). ICB-198 did not produce any mycotoxin and exhibited lower expression of almost all genes analyzed. Furthermore, the ratio of aflS/aflR expression was correlated with the biosynthesis of AF and CPA in A. flavus strains producing exclusively AF or CPA or producing both AF and CPA. The ratio of aflS/aflR expression therefore seems to be related to the production of mycotoxins in Brazil nuts. Our results provide important data for the development of innovative and more cost-effective strategies to reduce and prevent AFB and CPA contamination in Brazil nuts.

Aflatoxin-Exposure of Vibrio gazogenes as a Novel System for the Generation of Aflatoxin Synthesis Inhibitors.

Aflatoxin is a mycotoxin and a secondary metabolite, and the most potent known liver carcinogen that contaminates several important crops, and represents a significant threat to public health and the economy. Available approaches reported thus far have been insufficient to eliminate this threat, and therefore provide the rational to explore novel methods for preventing aflatoxin accumulation in the environment. Many terrestrial plants and microbes that share ecological niches and encounter the aflatoxin producers have the ability to synthesize compounds that inhibit aflatoxin synthesis. However, reports of natural aflatoxin inhibitors from marine ecosystem components that do not share ecological niches with the aflatoxin producers are rare. Here, we show that a non-pathogenic marine bacterium, Vibrio gazogenes, when exposed to low non-toxic doses of aflatoxin B1, demonstrates a shift in its metabolic output and synthesizes a metabolite fraction that inhibits aflatoxin synthesis without affecting hyphal growth in the model aflatoxin producer, Aspergillus parasiticus. The molecular mass of the predominant metabolite in this fraction was also different from the known prodigiosins, which are the known antifungal secondary metabolites synthesized by this Vibrio. Gene expression analyses using RT-PCR demonstrate that this metabolite fraction inhibits aflatoxin synthesis by down-regulating the expression of early-, middle-, and late- growth stage aflatoxin genes, the aflatoxin pathway regulator, aflR and one global regulator of secondary metabolism, laeA. Our study establishes a novel system for generation of aflatoxin synthesis inhibitors, and emphasizes the potential of the under-explored Vibrio’s silent genome for generating new modulators of fungal secondary metabolism.

Effects of Zinc Chelators on Aflatoxin Production in Aspergillus parasiticus.

Zinc concentrations strongly influence aflatoxin accumulation in laboratory media and in food and feed crops. The presence of zinc stimulates aflatoxin production, and the absence of zinc impedes toxin production. Initial studies that suggested a link between zinc and aflatoxin biosynthesis were presented in the 1970s. In the present study, we utilized two zinc chelators, N,N,N′,N′-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and 2,3-dimercapto-1-propanesulfonic acid (DMPS) to explore the effect of zinc limitation on aflatoxin synthesis in Aspergillus parasiticus. TPEN but not DMPS decreased aflatoxin biosynthesis up to six-fold depending on whether A. parasiticus was grown on rich or minimal medium. Although we observed significant inhibition of aflatoxin production by TPEN, no detectable changes were observed in expression levels of the aflatoxin pathway gene ver-1 and the zinc binuclear cluster transcription factor, AflR. Treatment of growing A. parasiticus solid culture with a fluorescent zinc probe demonstrated an increase in intracellular zinc levels assessed by increases in fluorescent intensity of cultures treated with TPEN compared to controls. These data suggest that TPEN binds to cytoplasmic zinc therefore limiting fungal access to zinc. To investigate the efficacy of TPEN on food and feed crops, we found that TPEN effectively decreases aflatoxin accumulation on peanut medium but not in a sunflower seeds-derived medium. From an application perspective, these data provide the basis for biological differences that exist in the efficacy of different zinc chelators in various food and feed crops frequently contaminated by aflatoxin.

Suppression of Aflatoxin Biosynthesis in Aspergillus flavus by 2-Phenylethanol Is Associated with Stimulated Growth and Decreased Degradation of Branched-Chain Amino Acids.

The saprophytic soil fungus Aspergillus flavus infects crops and produces aflatoxin. Pichia anomala, which is a biocontrol yeast and produces the major volatile 2-phenylethanol (2-PE), is able to reduce growth of A. flavus and aflatoxin production when applied onto pistachio trees. High levels of 2-PE are lethal to A. flavus and other fungi. However, at low levels, the underlying mechanism of 2-PE to inhibit aflatoxin production remains unclear. In this study, we characterized the temporal transcriptome response of A. flavus to 2-PE at a subinhibitory level (1 µL/mL) using RNA-Seq technology and bioinformatics tools. The treatment during the entire 72 h experimental period resulted in 131 of the total A. flavus 13,485 genes to be significantly impacted, of which 82 genes exhibited decreased expression. They included those encoding conidiation proteins and involved in cyclopiazonic acid biosynthesis. All genes in the aflatoxin gene cluster were also significantly decreased during the first 48 h treatment. Gene Ontology (GO) analyses showed that biological processes with GO terms related to catabolism of propionate and branched-chain amino acids (valine, leucine and isoleucine) were significantly enriched in the down-regulated gene group, while those associated with ribosome biogenesis, translation, and biosynthesis of α-amino acids were over-represented among the up-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that metabolic pathways negatively impacted among the down-regulated genes parallel to those active at 30 °C, a condition conducive to aflatoxin biosynthesis. In contrast, metabolic pathways positively related to the up-regulated gene group resembled those at 37 °C, which favors rapid fungal growth and is inhibitory to aflatoxin biosynthesis. The results showed that 2-PE at a low level stimulated active growth of A. flavus but concomitantly rendered decreased activities in branched-chain amino acid degradation. Since secondary metabolism occurs after active growth has ceased, this growth stimulation resulted in suppression of expression of aflatoxin biosynthesis genes. On the other hand, increased activities in degradation pathways for branched-chain amino acids probably are required for the activation of the aflatoxin pathway by providing building blocks and energy regeneration. Metabolic flux in primary metabolism apparently has an important role in the expression of genes of secondary metabolism.