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.

Volatiles of antagonistic soil yeasts inhibit growth and aflatoxin production of Aspergillus flavus.

Minimizing Aspergillus flavus growth is an effective strategy to mitigate aflatoxin contamination in food and agricultural products. In the present investigation, we attempted to utilize soil-associated yeasts from the Western and Eastern Ghats of India against A. flavus to reduce aflatoxin contamination. Forty-five yeast isolates were screened against A. flavus using overlay and dual plate assays. Among them, 12 isolates effectively inhibited the growth of A. flavus. The 18S rDNA gene sequence analysis identified the twelve antagonistic isolates as belonging to Saccharomyces cerevisiae, Suhomyces xylopsoci, Pichia kudriavzevii, and Candida tropicalis. From the isolated yeasts, S. cerevisiae strains were selected for further evaluation based on the potential antagonistic activity. Volatiles of S. cerevisiae effectively suppressed the mycelial growth of A. flavus (P < 0.05) up to 92.1 % at 7 DAI. Scanning electron microscopic images of the fungus exposed to volatiles showed hyphal deformity and mycelial damage. Aflatoxin B1 (AFB1) production was drastically reduced up to 99.0 % in the volatile-exposed fungus compared to the control. The yeast strain YKK1 showed consistent Aspergillus flavus growth inhibition (80.7 %) and AFB1 production (98.1 %) for 14 days. Gas chromatography-mass spectrophotometry analysis of the yeast volatiles revealed the presence of antimicrobial compounds, including 1-pentanol, 1-propanol, ethyl hexanol, ethanol, 2-methyl-1-butanol, ethyl acetate, dimethyl trisulfide, p-xylene, styrene, and 1,4-pentadiene. The evaluated compounds of yeast volatiles, including ethyl acetate, hexanal, 1-propanol, 1-heptanol, 1-butanol, and benzothiazole, inhibited the fungal growth and AFB1 production of Aspergillus flavus when applied as pure chemicals. Benzothiazole at 5 mM was responsible for a high level of growth inhibition (23.6 %) and reduction of AFB1 synthesis (93.5 %). Hence, volatile compounds produced by soil yeast strains could be a potential biocontrol mechanism against aflatoxin contamination.

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

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

Unveiling the effect of interacting forecasted abiotic factors on growth and aflatoxin B1 production kinetics by Aspergillus flavus.

The aim was to decipher the temporal impact of key interacting climate change (CC) abiotic factors of temperature (30 vs 37 °C), water activity (aw; 0.985 vs 0.930) and CO2 exposure (400 vs 1000 ppm) on (a) growth of Aspergillus flavus and effects on (b) gene expression of a structural (aflD) and key regulatory gene (aflR) involved in aflatoxin B1 (AFB1) biosynthesis and (c) AFB1 production on a yeast extract sucrose medium over a period of 10 days. A. flavus grew and produced AFB1 very early with toxin detected after only 48 h. Both growth and toxin production were significantly impacted by the interacting abiotic factors. The relative expression of the aflD gene was significantly influenced by temperature; aflR gene expression was mainly modulated by time. However, no clear relationship was observed for both genes with AFB1 production over the experimental time frame. The optimum temperature for AFB1 production was 30 °C. Maximum AFB1 production occurred between days 4-8. Exposure to higher CO2 conditions simulating forecasted CC conditions resulted in the amount of AFB1 produced in elevated temperature (37 °C) being higher than with the optimum temperature (30 °C) showing a potential for increased risk for human/animal health due to higher accumulation of this toxin.

Unveiling the spatial distribution of aflatoxin B1 and plant defense metabolites in maize using AP-SMALDI mass spectrometry imaging.

In order to cope with the presence of unfavorable compounds, plants can biotransform xenobiotics, translocate both parent compounds and metabolites, and perform compartmentation and segregation at the cellular or tissue level. Such a scenario also applies to mycotoxins, fungal secondary metabolites with a pre-eminent role in plant infection. In this work, we aimed to describe the effect of the interplay between Zea mays (maize) and aflatoxin B1 (AFB1) at the tissue and organ level. To address this challenge, we used atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) to investigate the biotransformation, localization and subsequent effects of AFB1 on primary and secondary metabolism of healthy maize plants, both in situ and from a metabolomics standpoint. High spatial resolution (5 µm) provided fine localization of AFB1, which was located within the root intercellular spaces, and co-localized with its phase-I metabolite aflatoxin M2. We provided a parallel visualization of maize metabolic changes, induced in different organs and tissues by an accumulation of AFB1. According to our untargeted metabolomics investigation, anthocyanin biosynthesis and chlorophyll metabolism in roots are most affected. The biosynthesis of these metabolites appears to be inhibited by AFB1 accumulation. On the other hand, metabolites found in above-ground organs suggest that the presence of AFB1 may also activate the biochemical response in the absence of an actual fungal infection; indeed, several plant secondary metabolites known for their antimicrobial or antioxidant activities were localized in the outer tissues, such as phenylpropanoids, benzoxazinoids, phytohormones and lipids.

Impact of Sequencing Depth and Library Preparation on Toxicological Interpretation of RNA-Seq Data in a "Three-Sample" Scenario.

While RNA-sequencing (RNA-seq) has emerged as a standard approach in toxicogenomics, its full potential in gaining underlying toxicological mechanisms is still not clear when only three biological replicates are used. This "three-sample" study design is common in toxicological research, particularly in animal studies during preclinical drug development. Sequencing depth (the total number of reads in an experiment) and library preparation are critical to the resolution and integrity of RNA-seq data and biological interpretation. We used aflatoxin b1 (AFB1), a model toxicant, to investigate the effect of sequencing depth and library preparation in RNA-seq on toxicological interpretation in the "three-sample" scenario. We also compared different gene profiling platforms (RNA-seq, TempO-seq, microarray, and qPCR) using identical liver samples. Well-established mechanisms of AFB1 toxicity served as ground truth for our comparative analyses. We found that a minimum of 20 million reads was sufficient to elicit key toxicity functions and pathways underlying AFB1-induced liver toxicity using three replicates and that identification of differentially expressed genes was positively associated with sequencing depth to a certain extent. Further, our results showed that RNA-seq revealed toxicological insights from pathway enrichment with overall higher statistical power and overlap ratio, compared with TempO-seq and microarray. Moreover, library preparation using the same methods was important to reproducing the toxicological interpretation.

Degradation and Detoxification of Aflatoxin B1 by Tea-Derived Aspergillus niger RAF106.

Microbial degradation is an effective and attractive method for eliminating aflatoxin B1 (AFB1), which is severely toxic to humans and animals. In this study, Aspergillus niger RAF106 could effectively degrade AFB1 when cultivated in Sabouraud dextrose broth (SDB) with contents of AFB1 ranging from 0.1 to 4 µg/mL. Treatment with yeast extract as a nitrogen source stimulated the degradation, but treatment with NaNO3 and NaNO2 as nitrogen sources and lactose and sucrose as carbon sources suppressed the degradation. Moreover, A. niger RAF106 still degraded AFB1 at initial pH values that ranged from 4 to 10 and at cultivation temperatures that ranged from 25 to 45 °C. In addition, intracellular enzymes or proteins with excellent thermotolerance were verified as being able to degrade AFB1 into metabolites with low or no mutagenicity. Furthermore, genomic sequence analysis indicated that the fungus was considered to be safe owing to the absence of virulence genes and the gene clusters for the synthesis of mycotoxins. These results indicate that A. niger RAF106 and its intracellular enzymes or proteins have a promising potential to be applied commercially in the processing and industry of food and feed to detoxify AFB1.

Aflatoxin B1 and Sterigmatocystin Binding Potential of Non-Lactobacillus LAB Strains.

Research on the ability of lactic acid bacteria (LAB) to bind aflatoxin B1 (AFB1) has mostly been focusing on lactobacilli and bifidobacteria. In this study, the AFB1 binding capacities of 20 Enterococcus strains belonging to E. casseliflavus, E. faecalis, E. faecium, E. hirae, E. lactis, and E. mundtii, 24 Pediococcus strains belonging to species P. acidilactici, P. lolii, P. pentosaceus, and P. stilesii, one strain of Lactococcus formosensis and L.garviae, and 3 strains of Weissella soli were investigated in MRS broth at 37 °C at 0.2 µg/mL mycotoxin concentration. According to our results, among non-lactobacilli LAB, the genera with the best AFB1 binding abilities were genus Pediococcus, with a maximum binding percentage of 7.6% by P. acidilactici OR83, followed by genus Lactococcus. For AFB1 bio-detoxification purposes, beside lactobacilli, pediococci can also be chosen, but it is important to select a strain with better binding properties than the average value of its genus. Five Pediococcus strains have been selected to compare their sterigmatocystin (ST) binding abilities to AFB1 binding, and a 2-3-fold difference was obtained similar to previous findings for lactobacilli. The best strain was P. acidilactici OR83 with 18% ST binding capacity. This is the first report on ST binding capabilities of non-Lactobacillus LAB strains.

Cellular, physiological and molecular approaches to investigate the antifungal and anti-aflatoxigenic effects of thyme essential oil on Aspergillus flavus.

Several approaches, including the detection of apoptotic-like cell death, aflatoxin B1 (AFB1) production and gene expression analysis, were carried out to provide insights into the antifungal and anti-aflatoxigenic effects of thyme essential oil (EO) on Aspergillus flavus. At 0.5 µL mL-1, thyme EO completely inhibited A. flavus growth. Furthermore, this antifungal activity triggered significant apoptosis, via nuclear condensation (87.5% of nuclei analyzed) and plasma membrane damage (in 100% of treated hyphae). Further analysis of AFB1 production and gene expression related to secondary metabolism (laeA) and the mechanism of virulence (lipA and meT) of A. flavus in the presence of thyme EO indicated important physiological changes related to its anti-aflatoxigenic property. These results highlight the potent antifungal abilities of thyme EO in controlling A. flavus and AFB1 production, especially the abilities that operate by exerting changes at the molecular level and inducing significant apoptotic-like cell death.

Inhibition of aflatoxin B1 biosynthesis and down regulation of aflR and aflB genes in presence of benzimidazole derivatives without impairing the growth of Aspergillus flavus.

Aflatoxins are mutagenic secondary metabolites produced by certain ubiquitous saprophytic fungi. These contaminate agricultural crops and pose a serious health threat to humans and livestock all over the world. Benzimidazole and its derivatives are biologically active heterocyclic compounds known for their fungicidal activity. In the present study, second and sixth position substituted benzimidazole derivatives are tested for their antifungal and anti-aflatoxigenic activity. Aflatoxigenic strain of Aspergillus flavus cultured in Yeast extract sucrose (YES) medium as well as in rice in the presence and absence of test compounds. 2-(2-Furyl) benzimidazole (FBD) showed complete inhibition of fungal growth at 50 µg/mL. However, the polar derivatives of FBD viz. 6-NFBD, 6-AFBD, 6-CAFBD, and 6-CFBD did not impair the fungal growth but effectively inhibited aflatoxin B1 biosynthesis. Significant down-regulation of aflR gene involved in regulation and aflB structural gene for aflatoxin B1 biosynthesis was observed in presence of 6-NFBD. These benzimidazole derivatives also showed good anti-aflatoxigenic activity in rice, though the IC50 concentrations in rice were comparatively higher than those in YES medium. This study summarizes the most notable structure-activity relationship (SAR) of 2-(2-Furyl) benzimidazoles for anti-aflatoxigenic and anti-fungal activities. These molecules can be further studied for their applications in industrial fermentation processes vulnerable to mold growth and subsequent aflatoxin B1 synthesis like koji fermentation, cheese production, etc.

Could aflatoxin B1 production by Aspergillus flavus affect the severity of keratitis: an experience in two tertiary health care centers, Egypt.

To investigate the expression of AFB1 gene in isolates obtained from corneal scrapping samples from keratitis patients and to correlate the quantity of AFB1 to the severity of keratitis. An observational study was undertaken in Medical Microbiology and Immunology department, Mansoura University, Egypt, over corneal scrapping samples that were cultured aiming to isolate fungal causes of infective keratitis followed by AFB1 gene detection in Aspergillus flavus isolates by nested PCR then quantitation of the toxin by TLC. Out of 843 corneal scrapping samples collected from patients with infective keratitis, positive fungal growth was identified in 277 cases (32.9%). A. flavus was the commonest fungal agent isolated in 93 cases (33.6%). The AFB1 toxin-encoding gene was detected in 63.4% of A. flavus isolates. There was a positive correlation between the quantity of produced AFB1 toxin and the degree of severity of keratitis (P value < 0.0001*). Aspergillus flavus was the most common cause of fungal keratitis, with the AFB1 toxin-encoding gene detected in more than half of the isolates. A significant correlation between the degree of severity of keratitis and the quantity of produced AFB1 toxin was detected. Therefore, exploring presence or absence of AFB1 toxin is important for the clinicians in their diagnostic assessment and selection of proper treatment choices.

Biocontrol activity of volatile organic compounds from Streptomyces alboflavus TD-1 against Aspergillus flavus growth and aflatoxin production.

Aspergillus flavus is a saprophytic fungus that contaminates crops with carcinogenic aflatoxin. In the present work, the antifungal effects of volatile organic compounds (VOCs) from Streptomyces alboflavus TD-1 against A. flavus were investigated. VOCs from 8-day-old wheat bran culture of S. alboflavus TD-1 displayed strong inhibitory effects against mycelial growth, sporulation, and conidial germination of A. flavus. Severely misshapen conidia and hyphae of A. flavus were observed by scanning electron microscopy after exposure to VOCs for 6 and 12 h, respectively. Rhodamine 123 staining of mitochondria indicated that mitochondria may be a legitimate antifungal target of the VOCs from S. alboflavus TD-1. Furthermore, the VOCs effectively inhibited aflatoxin B1 production by downregulating genes involved in aflatoxin biosynthesis. Dimethyl trisulfide and benzenamine may play important roles in the suppression of A. flavus growth and production of aflatoxin. The results indicate that VOCs from S. alboflavus TD-1 have tremendous potential to be developed as a useful bio-pesticide for controlling A. flavus.

Multiple CH/π Interactions Maintain the Binding of Aflatoxin B1 in the Active Cavity of Human Cytochrome P450 1A2.

Human cytochrome P450 1A2 (CYP1A2) is one of the key CYPs that activate aflatoxin B1 (AFB1), a notorious mycotoxin, into carcinogenic exo-8,9-epoxides (AFBO) in the liver. Although the structure of CYP1A2 is available, the mechanism of CYP1A2-specific binding to AFB1 has not been fully clarified. In this study, we used calculation biology to predict a model of CYP1A2 with AFB1, where Thr-124, Phe-125, Phe-226, and Phe-260 possibly participate in the specific binding. Site-directed mutagenesis was performed to construct mutants T124A, F125A, F226A, and F260A. Escherichia coli-expressed recombinant proteins T124A, F226A, and F260A had active structures, while F125A did not. This was evidenced by Fe2+∙Carbon monoxide (CO)-reduced difference spectra and circular dichroism spectroscopy. Mutant F125A was expressed in HEK293T cells. Steady kinetic assays showed that T124A had enhanced activity towards AFB1, while F125A, F226A, and F260A were significantly reduced in their ability to activate AFB1, implying that hydrogen bonds between Thr-124 and AFB1 were not important for substrate-specific binding, whereas Phe-125, Phe-226, and Phe-260 were essential for the process. The computation simulation and experimental results showed that the three key CH/π interactions between Phe-125, Phe-226, or Phe-260 and AFB1 collectively maintained the stable binding of AFB1 in the active cavity of CYP1A2.

Aflatoxin B1 metabolism: Regulation by phase I and II metabolizing enzymes and chemoprotective agents.

Aflatoxin B1 (AFB1) widely contaminates staple food and feed crops and is well-known as the most potent natural hepatocarcinogen in humans and domesticated animals. This review highlights significant advances in our understanding of the pivotal role of phase I and II metabolizing enzymes in the bioactivation and detoxification of AFB1 and its metabolites across species. In humans, cytochrome P450 (CYP) 1A2, CYP3A4, CYP3A5, and CYP3A7 in liver and CYP2A13 in lung are essential for the bioactivation of AFB1 to the extremely toxic exo-AFB1-8,9-epoxide (AFBO), whereas CYP1A1, CYP1A2, CYP2A6, and CYP3A4 are important in the turkey and duck, CYP1A1 and CYP2A6 are important in the chicken and quail, CYP3A11 and CYP3A13 are important in mice, and CYP2A5 are important in the hamster. In contrast, glutathione-S-transferase (GST) M1 and GSTT1 are primary responsible for detoxification of the AFB1 by catalyzing the conjugation of GSH to AFBO in humans, whereas GSTM2 in a nonhuman primate, GSTA3 in mice, GSTA5 in rats, and GSTA1, GSTA2, GSTA3 and GSTA4 in the turkey are important. Additionally, microsomal epoxide hydrolase (mEH) and aflatoxin-aldehyde reductase (AFAR) have also been shown to play key roles in AFB1 detoxification in the human, rat, and pig. Moreover, an overview of the chemoprotective agents, including synthetic compounds and naturally occurring plant compounds, which can be used to reduce aflatoxicosis is provided based on their ability to regulate these key enzymes. Collectively, this review summarizes the pivotal enzymes in the metabolism of AFB1 among humans, experimental and farm animals, as well as the chemoprotective agents that can be used to minimize risk of aflatoxicosis.

PbsB Regulates Morphogenesis, Aflatoxin B1 Biosynthesis, and Pathogenicity of Aspergillus flavus.

As an opportunistic pathogen, Aspergillus flavus is one of the major causes of food contamination around the world. In this study, pbsB gene knockout mutant (ΔpbsB) and pbsB overexpression strain (OE) of A. flavus were constructed by homologous recombination. The results showed that the mycelia growth, conidiation, and the formation of sclerotia in ΔpbsB mutant were significantly suppressed, and up-regulated in OE strian compared to wild-type strain (WT). Q-PCR analysis showed that PbsB regulated the sclerotia formation through sclerotia related gene nsdC. With TLC and qRT-PCR analysis, it was found that PbsB up-regulated the bio-synthesis of aflatoxin B1 (AFB1) through regulatory gene aflR and structural gene aflC, aflD, aflK, and aflQ in the aflatoxin gene cluster. In osmotic stress response analysis, ΔpbsB mutant was significantly more sensitive to osmotic pressure with 1.2 mol/L sorbitol, compared to WT and OE strains. In virulence analysis, the infection capacity of ΔpbsB strain to peanut and maize kernels decreased dramatically, and significantly fewer spores and lesser mycelia were produced in ΔpbsB strain on the surface of peanut and maize kernels, and the infection capacity of OE strain to kernels increased significantly compared with WT strain. The AFB1 bio-synthesis ability of A. flavus in crop invasion models was also found to be coincide with the expression level of pbsB. All the results of the study shows that, as a MAPKK, PbsB is critical for growth and virulence in A. flavus, and lay a theoretical foundation for the prevention and control of A. flavus contamination.

An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus.

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ∼ 60 positive transformants per 106 conidia using our protocol. A small-scale insertional mutant library (∼ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

Nanoparticle based bio-bar code technology for trace analysis of aflatoxin B1 in Chinese herbs.

A novel and sensitive assay for aflatoxin B1 (AFB1) detection has been developed by using bio-bar code assay (BCA). The method that relies on polyclonal antibodies encoded with DNA modified gold nanoparticle (NP) and monoclonal antibodies modified magnetic microparticle (MMP), and subsequent detection of amplified target in the form of bio-bar code using a fluorescent quantitative polymerase chain reaction (FQ-PCR) detection method. First, NP probes encoded with DNA that was unique to AFB1, MMP probes with monoclonal antibodies that bind AFB1 specifically were prepared. Then, the MMP-AFB1-NP sandwich compounds were acquired, dehybridization of the oligonucleotides on the nanoparticle surface allows the determination of the presence of AFB1 by identifying the oligonucleotide sequence released from the NP through FQ-PCR detection. The bio-bar code techniques system for detecting AFB1 was established, and the sensitivity limit was about 10-8 ng/mL, comparable ELISA assays for detecting the same target, it showed that we can detect AFB1 at low attomolar levels with the bio-bar-code amplification approach. This is also the first demonstration of a bio-bar code type assay for the detection of AFB1 in Chinese herbs.

Isolation and characterization of Aspergillus flavus strains in China.

Important staple foods (peanuts, maize and rice) are susceptible to contamination by aflatoxin (AF)-producing fungi such as Aspergillus flavus. The objective of this study was to explore non-aflatoxin-producing (atoxigenic) A. flavus strains as biocontrol agents for the control of AFs. In the current study, a total of 724 A. flavus strains were isolated from different regions of China. Polyphasic approaches were utilized for species identification. Non-aflatoxin and non-cyclopiazonic acid (CPA)-producing strains were further screened for aflatoxin B1 (AFB1) biosynthesis pathway gene clusters using a PCR assay. Strains lacking an amplicon for the regulatory gene aflR were then analyzed for the presence of the other 28 biosynthetic genes. Only 229 (32%) of the A. flavus strains were found to be atoxigenic. Smaller (S) sclerotial phenotypes were dominant (51%) compared to large (L, 34%) and non-sclerotial (NS, 15%) phenotypes. Among the atoxigenic strains, 24 strains were PCR-negative for the fas-1 and aflJ genes. Sixteen (67%) atoxigenic A. flavus strains were PCRnegative for 10 or more of the biosynthetic genes. Altogether, 18 new PCR product patterns were observed, indicating great diversity in the AFB1 biosynthesis pathway. The current study demonstrates that many atoxigenic A. flavus strains can be isolated from different regions of China. In the future laboratory as well as field based studies are recommended to test these atoxigenic strains as biocontrol agents for aflatoxin contamination.

The 14-3-3 Protein Homolog ArtA Regulates Development and Secondary Metabolism in the Opportunistic Plant Pathogen Aspergillus flavus.

The opportunistic plant-pathogenic fungus Aspergillus flavus produces carcinogenic mycotoxins termed aflatoxins (AF). Aflatoxin contamination of agriculturally important crops, such as maize, peanut, sorghum, and tree nuts, is responsible for serious adverse health and economic impacts worldwide. In order to identify possible genetic targets to reduce AF contamination, we have characterized the artA gene, encoding a putative 14-3-3 homolog in A. flavus The artA deletion mutant presents a slight decrease in vegetative growth and alterations in morphological development and secondary metabolism. Specifically, artA affects conidiation, and this effect is influenced by the type of substrate and culture condition. In addition, normal levels of artA are required for sclerotial development. Importantly, artA negatively regulates AF production as well as the concomitant expression of genes in the AF gene cluster. An increase in AF is also observed in seeds infected with the A. flavus strain lacking artA Furthermore, the expression of other secondary metabolite genes is also artA dependent, including genes in the cyclopiazonic acid (CPA) and ustiloxin gene clusters, in this agriculturally important fungus.IMPORTANCE In the current study, artA, which encodes a 14-3-3 homolog, was characterized in the agriculturally and medically important fungus Aspergillus flavus, specifically, its possible role governing sporulation, formation of resistant structures, and secondary metabolism. The highly conserved artA is necessary for normal fungal morphogenesis in an environment-dependent manner, affecting the balance between production of conidiophores and the formation of resistant structures that are necessary for the dissemination and survival of this opportunistic pathogen. This study reports a 14-3-3 protein affecting secondary metabolism in filamentous fungi. Importantly, artA regulates the biosynthesis of the potent carcinogenic compound aflatoxin B1 (AFB1) as well as the production of other secondary metabolites.

Aflatoxin B1 inhibition in Aspergillus flavus by Aspergillus niger through down-regulating expression of major biosynthetic genes and AFB1 degradation by atoxigenic A. flavus.

Twenty Aspergillus niger strains were isolated from peanuts and 14 strains were able to completely inhibit AFB1 production with co-cultivation. By using a Spin-X centrifuge system, it was confirmed that there are some soluble signal molecules or antibiotics involved in the inhibition by A. niger, although they are absent during the initial 24h of A. flavus growth when it is sensitive to inhibition. In A. flavus, 19 of 20 aflatoxin biosynthetic genes were down-regulated by A. niger. Importantly, the expression of aflS was significantly down-regulated, resulting in a reduction of AflS/AflR ratio. The results suggest that A. niger could directly inhibit AFB1 biosynthesis through reducing the abundance of aflS to aflR mRNAs. Interestingly, atoxigenic A. flavus JZ2 and GZ15 effectively degrade AFB1. Two new metabolites were identified and the key toxic lactone and furofuran rings both were destroyed and hydrogenated, meaning that lactonase and reductase might be involved in the degradation process.