title: Interaction of antifungal peptide BP15 with Stemphylium vesicarium, the causal agent of brown spot of pear.
abstract: Peptide BP15 has shown antifungal activity against several plant pathogenic fungi, including Stemphylium vesicarium, the causal agent of brown spot of pear. BP15 inhibits the germination, growth and sporulation of S. vesicarium and displays post-infection activity by stopping fungal infection in pear leaves. In this work, live-cell imaging was undertaken to understand the antifungal mechanism of BP15. A double-staining method based on the combination of calcofluor white and SYTOX green coupled with epifluorescence microscopy was used to investigate fungal cell permeabilization and alterations in fungal growth induced by BP15. GFP-transformants of S. vesicarium were obtained and exposed to rhodamine-labelled BP15. Confocal laser microscopy provided evidence of peptide internalization by hyphae, resulting in fungal cell disorganization and death. S. vesicarium membrane permeabilization by BP15 was found to be peptide-concentration dependent. BP15 at MIC and sub-MIC concentrations (10 and 5 μM, respectively) inhibited S. vesicarium growth and produced morphological alterations to germ tubes, with slow and discontinuous compromise of fungal cell membranes. Fungal cell membrane disruption was immediately induced by BP15 at 100 μM, and this was accompanied by rapid peptide internalization by S. vesicarium hyphae. Peptide BP15 interacted with germ tubes and hyphae of S. vesicarium but not with conidial cells.
authors.affiliation: Institute of Food and Agricultural Technology- XaRTA-CIDSAV, University of Girona, C/ Maria Aurèlia Capmany, 61. 17071 Girona, Spain.
authors.forename: Mireia
authors.initials: M
authors.lastname: Puig
authors.affiliation: Institute of Food and Agricultural Technology- XaRTA-CIDSAV, University of Girona, C/ Maria Aurèlia Capmany, 61. 17071 Girona, Spain. Electronic address: concepcio.moragrega@udg.edu.
authors.forename: Concepció
authors.initials: C
authors.lastname: Moragrega
authors.affiliation: Institute of Food and Agricultural Technology- XaRTA-CIDSAV, University of Girona, C/ Maria Aurèlia Capmany, 61. 17071 Girona, Spain.
authors.forename: Lídia
authors.initials: L
authors.lastname: Ruz
authors.affiliation: Departamento de Microbiología, Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", IHSM-UMA-CSIC, Universidad de Málaga, Facultad de Ciencias, Campus de Teatinos s/n. 29071 Málaga, Spain.
authors.forename: Claudia E
authors.initials: CE
authors.lastname: Calderón
authors.affiliation: Departamento de Microbiología, Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", IHSM-UMA-CSIC, Universidad de Málaga, Facultad de Ciencias, Campus de Teatinos s/n. 29071 Málaga, Spain.
authors.forename: Francisco M
authors.initials: FM
authors.lastname: Cazorla
authors.affiliation: Institute of Food and Agricultural Technology- XaRTA-CIDSAV, University of Girona, C/ Maria Aurèlia Capmany, 61. 17071 Girona, Spain.
authors.forename: Emilio
authors.initials: E
authors.lastname: Montesinos
authors.affiliation: Institute of Food and Agricultural Technology- XaRTA-CIDSAV, University of Girona, C/ Maria Aurèlia Capmany, 61. 17071 Girona, Spain.
authors.forename: Isidre
authors.initials: I
authors.lastname: Llorente
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D001203:Ascomycota / Q000187:drug effects* / Q000254:growth & development; D025301:Hyphae / Q000187:drug effects / Q000254:growth & development; D008826:Microbial Sensitivity Tests; D010455:Peptides / Q000494:pharmacology*; D010935:Plant Diseases / Q000382:microbiology*; D031989:Pyrus / Q000382:microbiology*
chemical_list: D000935:Antifungal Agents; D010455:Peptides
keywords: Agrobacterium tumefaciens-Mediated transformation; Green fluorescent protein; Growth inhibition; Membrane permeabilization; Mode of action