Comparative analysis of the sensitivity to distinct antimicrobials among Penicillium spp. causing fruit postharvest decay

Abstract

The postharvest fungal pathogens Penicillium digitatum, P. italicum and P. expansum are an increasing problem for the Mediterranean orchards and fruit industry. This study was designed to gain knowledge on factors affecting susceptibility of Penicillium spp. to antimicrobial peptides (AMPs) as new antifungal compounds for plant protection. The previously characterized PAF26 is a novel penetratin-type AMP with activity against phytopathogenic fungi. Comparative analyses were conducted on the sensitivity of Penicillium spp. to PAF26, to the cytolytic peptide melittin and to other antimicrobials. The research included microscopic observations, chitin quantification, virulence assays on citrus and apple fruits, and molecular phylogenetic relationships within Penicillium isolates from citrus fruit. Virulence analysis and phylogenetic reconstruction confirmed the host specificity and monophyletic origin for P. digitatum, contrary to the closely-related species P. expansum and P. italicum. A parallelism was found between sensitivity to PAF26 of Penicillium isolates and to the chitin dye calcofluor white (CFW). No such correlation was found between sensitivity to PAF26 and to the membrane perturbing compound SDS or the oxidizing agent H₂O₂. Microscopy studies showed that mycelium and conidia from the PAF26-sensitive fungi were also prone to CFW staining, but no direct correlation with the mycelial chitin content was found. The data are consistent with the fact that fungal cell walls influence the outcome of the interaction of AMPs with fungi, and that PAF26 is more active towards Penicillium citrus fruit pathogens. In this context, CFW could help both to elucidate AMPs mode of action and in studies of the mechanisms of virulence and host specificity within Penicillium spp.