A critical evaluation of cultural methods for the identification of atoxigenic Aspergillus flavus isolates for aflatoxin mitigation in pistachio orchards of Iran

Aflatoxin contamination of tree nuts is a growing concern for pistachio producing countries. Development of competitive exclusion strategies through application of atoxigenic Aspergillus flavus isolates is a highly effective route of natural aflatoxin mitigation. Aflatoxin assays conducted on a high number of native A. flavus isolates are a first step to identify potential biological control isolates. Many cultural methods for the rapid and visual identification of atoxigenic A. flavus isolates have been described. The current study identified atoxigenic A. flavus isolates from Iranian pistachio orchards using and contrasting cultural, analytical and molecular methods. Ammonium vapour (AV) and fluorescence detection (FD), two rapid aflatoxin assays, were directly compared using various media preparations to screen 524 A. flavus isolates obtained from Iranian pistachio orchards. Percentages of false negatives were high using FD assays for all media preparations ranging from 13 to 15 %. This in contrast to AV assays. Here incidences of false negatives ranged from 0 % (using coconut agar medium) to 7.2 % (using potato dextrose agar). Aflatoxin-producing ability of all isolates was further confirmed using thin layer- and high-performance liquid chromatography. Sixty three atoxigenic A. flavus isolates were identified as atoxigenic in all assays. For these isolates, five loci across the aflatoxin biosynthesis cluster pathway were compared to identify genetic defects explaining atoxigenicity. Genetic deletions in at least one of five loci in the aflatoxin biosynthesis pathway were found for 97 % of isolates. Frequencies of atoxigenic strains ranged from 7.1 to 37.5 % with the lowest incidence detected in the Kerman province. Proper identification of atoxigenic isolates is considered a first step in the development of biological control strategies. Ability of identified isolates to competitively exclude aflatoxin-producing fungi has to be further investigated.