Dual culture of atoxigenic and toxigenic strains of Aspergillus flavus to gain insight into repression of aflatoxin biosynthesis and fungal interaction
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Application of atoxigenic strains to compete against toxigenic strains of Aspergillus flavus strains has emerged as one of the practical strategies for reducing aflatoxin contamination in corn, peanut, and tree nuts. The actual mechanism that results in aflatoxin reduction is not fully understood. Real-time RT-PCR and relative quantification of gene expression protocol were applied to elucidate the molecular mechanism. Transcriptional analyses of aflatoxin biosynthetic gene cluster in dual culture of toxigenic and atoxigenic A. flavus strains were carried out. Six targeted genes, aflR, aflJ, omtA, ordA, pksA, and vbs, were downregulated to variable levels depending on paired strains of toxigenic and atoxigenic A. flavus. Consistent with the decreased gene expression levels, the aflatoxin concentrations in dual cultures were reduced significantly in comparison with toxigenic cultures. Fluorescent images showed fungal hyphae in dual culture displayed green fluorescent, and contacts of live hyphae were seen. A coconut agar plate assay was used to show that toxigenic A. flavus colony produced blue fluorescence under long UV exposure, suggesting that aflatoxin is exported outside fungal hyphae. Furthermore, the assay was applied to demonstrate the potential role of thigmo-regulation in fungal interaction.
KeywordsBiocontrol Aflatoxin Gene regulation Aspergillus flavus Food safety
This work is supported by in-house research program funds from U.S. Department of Agriculture, Agricultural Research Services (Project Numbers: 5325-42000-039-00D).
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Conflict of interest
The authors declare that they have no conflicts of interest.
- Almeida MC, Brand AC (2017) Thigmo responses: the fungal sense of touch. Microbiol Spectrum 5(2):FUNK-0040-2016. https://doi.org/10.1128/microbiolspec.FUNK-0040-2016 Google Scholar
- Cary JW, Ehrlich KC, Bland JM, Montalbano BG (2006) The aflatoxin biosynthesis cluster gene, aflX, encodes an oxidoreductase involved in conversion of versicolorin a to demethylsterigmatocystin. Appl Environ Microbiol 72:1096–1101. https://doi.org/10.1128/AEM.72.2.1096-1101.2006 CrossRefGoogle Scholar
- FAO (2009) Declaration of the World Summit on Food Security. WFSFS 2009/2, Rome, 16–18 November 2009Google Scholar
- Flaherty JE, Weaver ME, Payne GA, Woloshuk CP (1995) A beta-glucuronidase reporter gene construct for monitoring aflatoxin biosynthesis in A. flavus. Appl Environ Microbiol 61:2482–2486Google Scholar
- Hruska Z, Rajasekaran K, Yao H, Kincaid R, Darlington D, Brown RL, Bhatnagar D, Cleveland TE (2014) Co-inoculation of aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus to study fungal invasion, colonization, and competition in maize kernels. Front Microbiol 5(122). https://doi.org/10.3389/fmicb.2014.00122
- Hua SST, Chang PK, Palumbo JD (2017) Mycotoxins. In: Witczak ASZ (ed) Toxins and other harmful compounds in foods. CRC Press, Taylor & Francis Group, Boca Raton, pp 153–168Google Scholar
- Meyers DM, O’Brian G, Du WL, Bhatnagar D, Payne GA (1998) Characterization of aflJ, a gene required for conversion of pathway intermediates to aflatoxin. Appl Environ Microbiol 64:3713–3717Google Scholar
- Pfaffl MW (2004) Quantification strategies in real-time PCR, in: A-Z of quantitative PCR. 87–112. https://doi.org/10.1007/s10551-011-0963-1
- Pfaffl MW (2012) Quantification strategies in real-time polymerase chain reaction. https://www.gene.quantification.de/pfaffl-chapter-3-quan-strategies-appl-microbiol-2012.pdf
- Woloshuk CP, Foutz KR, Brewer JF, Bhatnagar D, Cleveland TE, Payne GA (1994) Molecular characterization of aflR, a regulatory locus for aflatoxin biosynthesis. Appl Environ Microbiol 60:2408–2414Google Scholar