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 also is a health hazard to humans and animals. Previously, we observed inhibition of A. flavus aflatoxin biosynthesis upon 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 to control samples at 40 h. But no statistical difference in fungal biomass was observed upon these treatments. The Vg treatments were most effective on aflatoxin biosynthesis as was reflected in significant downregulation of majority of the 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 may inhibit other secondary metabolites as well. Finally, a weighted gene correlation network analysis identified an upregulation of ten genes that were most strongly associated with Vg-dependent aflatoxin inhibition and provide a novel start-point in understanding the mechanisms that result in this phenomenon.

Screening of Bacillus velezensis E2 and the Inhibitory Effect of Its Antifungal Substances on Aspergillus flavus

Aspergilus flavus is the main pathogenic fungus that causes food mold. Effective control of A. flavus contamination is essential to ensure food safety. The lipopeptides (LPs) produced by Bacillus strains have been shown to have an obvious antifungal effect on molds. In this study, an antagonist strain of Bacillus velezensis with obvious antifungal activity against A. flavus was isolated from the surface of healthy rice. Using HPLC-MS analysis, the main components of LPs produced by strain E2 were identified as fengycin and iturins. Further investigations showed that LPs could inhibit the spore germination, and even cause abnormal expansion of hyphae and cell rupture. Transcriptomic analyses showed that some genes, involved in ribosome biogenesis in eukaryotes (NOG1, KRE33) and aflatoxin biosynthesis (aflK, aflR, veA, omtA) pathways in A. flavus were significantly down-regulated by LPs. In conclusion, this study provides novel insights into the cellular and molecular antifungal mechanisms of LPs against grain A. flavus contamination.

Analysis of the competitiveness between a non-aflatoxigenic and an aflatoxigenic Aspergillus flavus strain on maize kernels by droplet digital PCR

Non-aflatoxigenic Aspergillus flavus strains are used as a biocontrol system on maize fields to decrease the aflatoxin biosynthesis of aflatoxigenic A. flavus strains. A. flavus strain AF36 was the first commercially available biocontrol strain and is authorized for use on maize fields by the US Environmental Protection Agency, e.g., in Texas and Arizona. A droplet digital PCR (ddPCR) assay was developed to analyze the mechanisms of competition and interaction of aflatoxigenic and non-aflatoxigenic A. flavus strains. This assay enables the parallel identification and quantification of the biocontrol strain A. flavus AF36 and the aflatoxigenic A. flavus strain MRI19. To test the assay, spores of both strains were mixed in varying ratios and were incubated on maize-based agar or maize kernels for up to 20 days. Genomic equivalent ratios (genome copy numbers) of both strains were determined by ddPCR at certain times after incubation and were compared to the spore ratios used for inoculation. The aflatoxin biosynthesis was also measured. In general, A. flavus MRI19 had higher competitiveness in the tested habitats compared to the non-aflatoxigenic strain, as indicated by higher final genomic equivalent ratios of this strain compared to the spore ratios used for inoculation. Nevertheless, A. flavus AF36 effectively controlled aflatoxin biosynthesis of A. flavus MRI19, as a clear aflatoxin inhibition was already seen by the inoculation of 10% spores of the biocontrol strain mixed with 90% spores of the aflatoxigenic strain compared to samples inoculated with only spores of the aflatoxigenic A. flavus MRI19.

aflN Is Involved in the Biosynthesis of Aflatoxin and Conidiation in Aspergillus flavus

Aspergillus flavus poses a threat to society economy and public health due to aflatoxin production. aflN is a gene located in the aflatoxin gene cluster, but the function of AflN is undefined in Aspergillus flavus. In this study, aflN is knocked out and overexpressed to study the function of AflN. The results indicated that the loss of AflN leads to the defect of aflatoxin biosynthesis. AflN is also found to play a role in conidiation but not hyphal growth and sclerotia development. Moreover, AlfN is related to the response to environmental oxidative stress and intracellular levels of reactive oxygen species. At last, AflN is involved in the pathogenicity of Aspergillus flavus to host. These results suggested that AflN played important roles in aflatoxin biosynthesis, conidiation and reactive oxygen species generation in Aspergillus flavus, which will be helpful for the understanding of aflN function, and will be beneficial to the prevention and control of Aspergillus flavus and aflatoxins contamination.

In silico analysis to contemplate the chemistry behind gallic acid-mediated inhibition of Polyketide Synthase A from aflatoxin biosynthesis pathway of Aspergillus flavus

Aspergillus flavus is known for producing the potent carcinogenic agent aflatoxin. Food contamination with aflatoxins is an important safety concern for agricultural yields. To identify and develop anti-aflatoxigenic agents, studies on phytochemicals as anti-aflatoxigenic agents have been documented including gallic acid. Thus, interaction studies using in-silico tools have been explored to understand the molecular mechanism behind inhibition of aflatoxin biosynthesis by studying the chemical interactions of gallic acid with polyketide synthase A (PksA) of A. flavus. The 3D structure of PksA consisting of seven domains was modeled using a Swiss-Model server followed by docking using Autodock tools-1.5.6 with substrate hexanoic acid and with that to gallic acid. The binding energy (electrostatic, inter-molecular or total internal energy) for gallic acid was lower (-6.09 to -4.79 kcal/mol) in comparison to hexanoic acid (-5.05 to -3.36 kcal/mol). During an interaction with the acyl transferase domain of PksA, both ligands showed H-bond formation at Glu36, Arg8, Thr11 positions. Ligplot analysis showed the formation of 7-H bonds in gallic acid and 3-H bonds in hexanoic acid. In addition, gallic acid showed stable binding with the active site of PksA indicated by steady root mean square deviation through molecular dynamic simulations. The chemistry between gallic acid and polyketide synthase A(PksA) exhibited that Gallic Acid possesses the highest level of binding potential (more number of hydrogen bonds) with PksA domain in comparison to hexanoic acid, a precursor for aflatoxin biosynthesis. Thus, we suggest enzymes from the aflatoxin biosynthetic pathway in aflatoxin-producing Aspergilli could be an important target for potential inhibitors.

Aspergillus Goes Viral: Ecological Insights from the Geographical Distribution of the Mycovirome within an Aspergillus flavus Population and Its Possible Correlation with Aflatoxin Biosynthesis

Microbial multi-level interactions are essential to control the success of spreading and survival of most microbes in natural environments. Phytopathogenic mycotoxigenic fungal species, such as Aspergillus flavus, represent an important issue in food safety. Usually, non-toxigenic strains are exploited for biocontrol strategies to mitigate infections by toxigenic strains. To comprehend all the biological variables involved in the aflatoxin biosynthesis, and to possibly evaluate the interplay between A. flavus toxigenic and non-toxigenic strains during intraspecific biocompetition, the “virological” perspective should be considered. For these reasons, investigations on mycoviruses associated to A. flavus populations inhabiting specific agroecosystems are highly desirable. Here, we provide the first accurate characterization of the novel mycovirome identified within an A. flavus wild population colonizing the maize fields of northern Italy: a selection of A. flavus strains was biologically characterized and subjected to RNAseq analysis, revealing new mycoviruses and a peculiar geographic pattern distribution in addition to a 20% rate of infection. More interestingly, a negative correlation between viral infection and aflatoxin production was found. Results significantly expanded the limited existent data about mycoviruses in wild A. flavus, opening new and intriguing hypotheses about the ecological significance of mycoviruses.

Molecular analysis of aflR gene in Aspergillus flavus isolated from Iran

Aspergillus flavus produces the most potent carcinogens, aflatoxins, when it contaminates agricultural crops. aflR gene regulates aflatoxin-related genes and it has been identified in four species of A. flavus, A. parasiticus, A. sojae and A. oryzae. Contamination of agricultural commodities with aflatoxin is a grave risk to humans and animals’ health. Aflatoxin related genes are clustered in a 75 kb region of genome in A. flavus. Investigations obviously demonstrated that aflatoxin biosynthesis needs the aflR gene product and an entirely functional aflatoxin biosynthetic cluster. The purpose of the current study was to investigate the presence of the aflR gene in A. flavus. Material and methods. Forty-two A. flavus isolates including 10 references, 25 clinical and 7 environmental isolates were analyzed in this study. The isolates were identified by morphology. To characterize morphologically, the conidial arrangement, philiades, vesicles and conidiophores were observed microscopically. Using PCR, the aflR gene was amplified with primers aflR1 and aflR2. PCR were carried out to amplify an 800 bp DNA fragment of aflR gene. Some amplicons were sequenced. The sequences were searched in NCBI database and analyzed with MEGA5 software. Results and discussion. Out of 42 A. flavus isolates, an 800 bp band was amplified for 35 isolates. No band was observed for seven isolates including 4 clinical and 3 environmental isolates. Data analysis demonstrated that 100% of reference strains and 84% of clinical strains produced the expected fragment while it was only 57.14% for environmental isolates. The sequences had 100% identity with A. flavus aflR gene which was deposited in the NCBI database. Conclusion. In conclusion, molecular analysis of the aflR gene showed that this gene was not amplified from some strains of A.flavus; therefore, perhaps it lacks the gene or it is greatly abnormal. Additional researches are needed to verify whether the strains with lack of aflR gene have a loss of function in production of aflatoxin or other mechanisms of regulation exist