Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/15/2004
Publication Date: 7/2/2004
Citation: Ehrlich, K., Cotty, P.J. 2004. An isolate of Aspergillus flavus used to reduce aflatoxin contamination in cottonseed has a defective polyketide synthase gene. Applied Microbiology and Biotechnology. 65:473-478. Interpretive Summary: Aspergillus flavus AF36 is an EPA registered biocompetitive pesticide now being used to treat cotton fields in Arizona to prevent contamination of cottonseed with the highly carcinogenic and toxic chemical aflatoxin. This fungal strain does not produce aflatoxin. Previous work showed that there is a defect in the ability to produce aflatoxin that occurs very early in the biosynthesis process. Aflatoxin biosynthesis requires two proteins for the initial steps. The first is a fatty acid synthase and the second is a polyketide synthase. We have now found where the defect is in the gene. Both AF36 aflatoxin genes and genes from an aflatoxin-producing Aspergillus flavus were compared, by aligning their sequences. Based on the alignment, we found a defect in the polyketide synthase sequence that prevents the gene from being used to make the protein necessary for formation of the aflatoxin precursor. All fungal isolates of the same type (vegetative compatibility group) have the same defect. This work now shows that this strain of Aspergillus flavus is truly atoxigenic by virtue of the defect and not just inhibited from producing aflatoxin. Such a finding validates the use of this type of fungus for further field treatments.
Technical Abstract: Aflatoxins are toxic and highly carcinogenic metabolites produced by several species of Aspergillus. A. flavus is the most common causal agent of preharvest aflatoxin contamination in cottonseed. In order to minimize the potential hazard to humans, in the United States, the maximum aflatoxin levels in cottonseed fed to dairy cows and non-lactating cattle is required to be less than 20 ppb and 300 ppb, respectively. In certain cotton-growing areas where aflatoxin contamination is an endemic problem, levels in the seed often exceed the permitted amounts. A. flavus is an asexual species divided into numerous clonal lineages by a vegetative incompatibility system that serves as a barrier to genetic exchange between all but like individuals. The vegetative compatibility groups (VCGs) delimited by this system vary widely in aflatoxin-producing ability. VCGs which produce no aflatoxins are called "atoxigenic strains." Atoxigenic strains can potentially reduce aflatoxin contamination through competitive exclusion of aflatoxin producers. Atoxigenic strains vary in their utility in aflatoxin management. One atoxigenic strain, AF36, is an EPA registered pesticide selected for the management, by competitive exclusion, of aflatoxin-producing fungi during cotton production. AF36 has been used to decrease aflatoxin contamination of cottonseed on over 80,000 acres in Arizona and Texas since 1999. None of the tested isolates of the AF36 vegetative compatibility group made aflatoxin. Nonetheless, most of the aflatoxin-biosynthetic pathway must be intact in AF36 because aflatoxin is produced when AF36 is fed the aflatoxin precursor, norsolorinic acid. Elucidating the molecular mechanism of atoxigenicity in AF36 may lead to improved understanding of the stability and distribution of the AF36 atoxigenic phenotype and may be useful for the continued EPA registration of this novel biopesticide. We now report that a nucleotide polymorphism in the coding region of the polyketide synthase gene in the AF36 aflatoxin pathway gene cluster introduces a stop codon that causes the truncation of the polyketide synthase necessary for conversion of a precursor hexanoylCoA to norsolorinic acid.