Anti-fungal properties of Puroindoline A against Aspergillus glaucus

Abstract

Aspergillus glaucus can grow in low moisture grain, and is one of the main fungi responsible for agricultural product losses. Puroindoline A (PINA) is a tryptophan-rich alkaline adiponectin that can effectively inhibit numerous plant bacteria and fungi. However, the mechanism of PINA against A. glaucus remains unclear. Herein, we found that recombinant PINA (rPINA) could inhibit A. glaucus mycelia growth on salt Czapek dox agar (SCDA) medium and spore germination on Czapek dox (CD) medium. Scanning electron microscopy revealed that incomplete morphological characteristics of both A. glaucus spores and mycelia occurred following rPINA treatment. Laser scanning confocal microscopy (LSCM) showed that rPINA could enter the interior of spores. Flow cytometry and propidium iodide (PI) staining illustrated membranes of spores were severely damaged, especially after treatment with 0.9 mg/mL rPINA for 12 h, and spores with intact membranes were reduced by 30.7%. Additionally, rPINA reduced the mitochondrial membrane potential (MMP) by 5,5′,6,6′-tetrachlorr-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) staining, and caused DNA damage by 4′,6-diamidino-2-phenylindole (DAPI) staining. These results indicated that rPINA may damage cell membranes and DNA structure and reduce MMP, thereby inhibiting the growth of A. glaucus. The antifungal mechanism has been demonstrated in this study, and results show that rPINA has application potential in preventing postharvest loss in the agricultural industry. Keywords: Puroindoline A, Aspergillus glaucus, Membrane integrity, Mitochondrial membrane potential, DNA damage