The antifungal activity of puroindoline A protein and its biocontrol potential for inhibiting Aspergillus flavus infection in peanut and corn

The Puroindoline A (PINA) from soft wheat endosperm is a broad-spectrum antimicrobial protein. Herein, we investigated the action of the PINA protein on Aspergillus flavus mycelia growth, conidia and sclerotia development, and its ability to control pathogenicity in infected crop seeds. The PINA protein strongly inhibited spore germination, mycelia growth, and conidia and sclerotia formation at 128 μg/mL. Furthermore, scanning electron microscopy revealed altered mycelia morphology, mycelia were dented, shrivelled and holes were observed, suggesting that the cell wall and membrane were damaged, and this was confirmed by calcofluor white fluorescent probe assays and propidium iodide staining. Additionally, there were significant changes in mycelia intramembranous structure based on 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide and 4′,6-diamidino-2-phenylindole staining, which indicates decreased mitochondrial membrane potential, excessive nuclear condensation, and DNA fragmentation. These results demonstrated that the defects of A. flavus pathogenicity in peanut and corn appeared to be related to cell wall and membrane destruction, which may trigger mitochondrial and DNA damage, leading to cell death. These findings provide visual evidence that the PINA protein inhibits A. flavus pathogenicity, and may assist food preservation applications in future.