Assessment of Aflatoxin Gene Cluster and Interaction of Toxigenic and Atoxigenic Aspergillus Flavus Strains of Pistachio in Vitro

Aflatoxin is the most significant mycotoxin hazard to human health, a secondary metabolite characterized by mutagenicity, teratogenicity, and carcinogenicity. It is produced by some species of Aspergillus in susceptible agricultural crops under certain environmental conditions. Different strategies, such as physical, cultural, biological, and chemical, have been suggested to manage the contamination of various crops with Aspergillus fungus. Using atoxigenic isolates of Aspergillus flavus is considered an effective and successful method in reducing aflatoxin contamination.

In the present study, 58 atoxigenic isolates of Aspergillus flavus obtained from pistachio orchards in different agro-ecological zones of Iran were used. Coconut agar medium (CAM) Petri plates were co-inoculated by an equal number of spores (4×103 ) of 58 atoxigenic and one toxigenic strain. The interactions between toxigenic and atoxigenic strains to produce aflatoxin were evaluated by exposing them to ammonia vapor. Potato dextrose broth (PDB) medium was used to produce mycelium, and the CTAB modified method was used to extract DNA. Species-specific primer was used for molecular identification. Four genes, including nor1, avnA, estA, aflR, and C3 region of the aflatoxin gene cluster, were detected in 24 atoxigenic isolates using specific primers and PCR reaction.

Overall, the isolates were divided into six groups based on the color intensity of the back of the colony. Atoxigenic isolates were placed into 12 groups on the basis of the gene deletion pattern.

In vitro competition test showed a potential for limiting toxigenic A. flavus strains growth and aflatoxin control among atoxigenic strains