Biocontrol of aflatoxins in Greece: Field efficacy and genetic diversity of endemic non-aflatoxigenic Aspergillus flavus strains in maize

Aflatoxins, produced mainly by Aspergillus flavus, pose a significant threat to maize safety and feed quality affecting both livestock health and the safety of animal-derived products. In this study, we evaluated the biocontrol potential of non-aflatoxigenic A. flavus isolates recovered from pistachios and maize kernels across Greece, in a three-year field trial (2022–2024), under both artificial inoculation and natural infection conditions. In 2022, the majority out of the 49 tested strains showed strong inhibitory activity against aflatoxin biosynthesis (>90 %), with several achieving complete suppression. The 14 most effective strains were further characterized using Cluster Amplification Pattern (CAP) analysis to assess structural variation in the aflatoxin and cyclopiazonic acid biosynthetic gene clusters, as well as by mating-type (MAT) profiling and vegetative compatibility group (VCG) analysis among corn and pistachio isolates to evaluate cross-host biocontrol potential. Three strains, selected on the basis of their outstanding field performance and extensive deletions in both the aflatoxin and cyclopiazonic acid gene clusters, were subsequently re-evaluated over the following two seasons. In 2023, these strains, applied individually and as a consortium, maintained 100 % inhibition of aflatoxin accumulation under artificial inoculation. In 2024, under natural infection conditions, their application via sorghum-coated seeds completely prevented detectable aflatoxin contamination. VCG analysis revealed overlaps between major maize- and pistachio-associated groups, suggesting cross-host biocontrol compatibility and broader ecological adaptation. MAT analysis revealed that the two most effective strains shared mating-type features with the highly aflatoxigenic strain, which may imply effects on biocontrol stability and persistence in the field. Overall, these findings highlight the strong potential of Greek endemic atoxigenic A. flavus strains as regionally adapted, sustainable biocontrol agents for aflatoxin mitigation in maize.