Molecular characterization of aflatoxigenic Aspergillus section Flavi isolates from wheat grains

Wheat, the most extensively produced cereal in Turkey, is subject to contamination by members of Aspergillus section Flavi (Aspergillus flavus, A. parasiticus, A. nominus, A. niger) during harvesting, transportation, storage, and processing. This study aimed to determine the presence and toxin-producing potential of A. section Flavi members in wheat. For this purpose, wheat grains were first placed in PDA medium and incubated at 25℃ for 5-7 days. After incubation, suspected Aspergillus isolates were purified in PDA medium. Morphological identification determined that all 25 isolates were presumptive as A. flavus. Molecular identification of these isolates was conducted by screening ITS, β-tubulin, and IGS gene regions. Additionally, aflatoxin biosynthesis-related genes (norA, norB, pksA, ver-1, and afIR) were evaluated. PCR analyses yielded bands of 600 bp, 340 bp, and 674 bp for the ITS, β-tubulin, and IGS gene regions, respectively, while norA, norB, pksA, ver-1, and afIR genes produced bands of 759 bp, 452 bp, 416 bp, 785 bp, and 798 bp, respectively. The study was continued with RFLP analysis and for this purpose, 25 PCR products amplified with ITS1/ITS4 primers were cut with HhaI restriction enzyme. As a result, four fragments of different sizes were obtained from the ITS gene region. These PCR products were evaluated as A. flavus. Subsequently, β-tubulin and ITS gene regions of five randomly selected A. flavus isolates were amplified and subjected to BLAST (Basic Local Alignment Search Tool) analysis. When compared with the species in the GenBank database, these isolates exhibited 99-100% nucleotide similarity. DNA sequences of the β-tubulin and ITS gene regions were used to construct a phylogenetic tree employing the Neighbor-Joining method in MEGA X software to determine phylogenetic relationships. Based on the analyses, Aspergillus isolates obtained from wheat grains were identified as A. flavus. Furthermore, the presence of aflatoxin biosynthesis-related gene regions suggested the potential aflatoxigenicity of these isolates. Keywords: Aspergillus section Flavi, wheat, isolation, aflatoxin biosynthesis genes