Multiplex PCR for Detection of Deletions in and near the Aflatoxin Gene Cluster in Aspergillus flavus

K. A. Callicott, P. J. Cotty


Aflatoxins, potent naturally-occurring carcinogens, are a significant problem for agriculture and human health. These potent toxins contaminate crops during infection by Aspergillus flavus and closely related fungi. Currently, two atoxigenic vegetative compatibility groups (VCGs) of A. flavus are used commercially as biocontrols for the prevention of aflatoxin contamination in the United States. Additional atoxigenics for biocontrol may be needed for the most efficient and cost-effective management in diverse agro-ecosystems. Many atoxigenic strains are naturally distributed across agricultural production regions, with atoxigenicity often the result of deletions in the aflatoxin biosynthesis genes. Previous work has relied on amplification of key genes in the aflatoxin cluster either to monitor for very large deletions or to facilitate sequencing. This report presents a method that uses four panels of multiplexed PCR markers positioned regularly along one subtelomere of chromosome 3. The 32 markers cover a region that contains the sugar, aflatoxin, and cyclopiazonic acid (CPA) clusters. Diverse amplification patterns were observed in A. flavus VCGs isolated from Arizona and Texas. Non-amplification of markers in the aflatoxin cluster was associated with inability to produce aflatoxins. Cluster genes are sufficiently conserved to allow gene detection by the multiplex PCR even across A. flavus VCGs diverged at least 1 million years. This method allows for rapid characterization of potential biocontrol strains for deletions in the aflatoxin cluster and surrounding regions and thus provides insight on mechanisms of atoxigenicity.


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American Society for Microbiology General Meeting
    • ASM 112th (2012)