In Vitro Evaluation of Five Antimicrobial Peptides against the Plant Pathogen Erwinia amylovora

Abstract

Fire blight is a major pome fruit trees disease that is caused by the quarantine phytopathogenic Erwinia amylovora, leading to major losses, namely, in pear and apple productions. Nevertheless, no effective sustainable control treatments and measures have yet been disclosed. In that regard, antimicrobial peptides (AMPs) have been proposed as an alternative biomolecule against pathogens but some of those AMPs have yet to be tested against E. amylovora. In this study, the potential of five AMPs (RW-BP100, CA-M, 3.1, D4E1, and Dhvar-5) together with BP100, were assessed to control E. amylovora. Antibiograms, minimal inhibitory, and bactericidal concentrations (minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), growth and IC50 were determined and membrane permeabilization capacity was evaluated by flow cytometry analysis and colony-forming units (CFUs) plate counting. For the tested AMPs, the higher inhibitory and bactericidal capacity was observed for RW-BP100 and CA-M (5 and 5–8 µM, respectively for both MIC and MBC), whilst for IC50 RW-BP100 presented higher efficiency (2.8 to 3.5 µM). Growth curves for the first concentrations bellow MIC showed that these AMPs delayed E. amylovora growth. Flow cytometry disclosed faster membrane permeabilization for CA-M. These results highlight the potential of RW-BP100 and CA-M AMPs as sustainable control measures against E. amylovora.

Evaluation of Three Antimicrobial Peptides Mixtures to Control the Phytopathogen Responsible for Fire Blight Disease

“…To mitigate this problem, and also to respond to the call for a more sustainable agriculture, AMPs have taken the spotlight as good candidates to substitute conventional antibiotics and other chemical compounds with associated health and environmental risks. In a previous study, the bioactivity of five AMPs against E. amylovora was determined, alongside the previously studied AMP BP100 [17]. However, only one study has been performed with the equimolar mixture of AMPs [15].…”

“…Current control measures are mainly based on preventive action against fire blight (cultural control, such as, pruning of early-stage infected trees for example), and/or copperbased phytosanitary compounds and antibiotic applications (chemical control), which is prohibited in the European Union with a few exceptions, due to the dramatic rise of antibiotic resistance [7][8][9][10][11]. Alternative sustainable control methods of E. amylovora have been explored extensively in the last few years, such as, the use of antagonistic bacteria [12], bacteriophages [13], essential oils [14], and antimicrobial peptides (AMPs) [15][16][17].…”

“…pruni, Xanthomonas fragariae, Xanthomonas axonopodis pv. vesicatoria [25], and E. amylovora [17,25,27].…”

“…The effects of AMPs on E. amylovora and their use as a control measure for this bacterium have been reported in several studies over the last 15 years [16,[27][28][29][30][31]. Recently, we have demonstrated the bioactivity of two AMPs against E. amylovora [17], namely, RW-BP100 and CA-M, which had already been reported to display activity against pathogenic bacteria but never tested against the causative agent of the fire blight disease [32][33][34]. RW-BP100 is an analogue of BP100, a well-known AMP presenting high antimicrobial activity against E. amylovora, and low phytotoxicity and haemolysis [27,28,35], and CA-M is a cecropin A-melittin (CA-M) hybrid peptide, also known as CA(1-7)M (2)(3)(4)(5)(6)(7)(8)(9).…”

“…In connection with the above, the aim of this work was to study the putative synergic effect of three AMPs mixtures (BP100, RW-BP100, and CA-M) which were previously shown to be particularly promising against E. amylovora when tested individually [17]. To this end, three different AMPs mixtures at different equimolar concentrations were used against three different strains of the pathogen representing distinct genotypes and virulence (LMG 2024 as the type strain, Ea 630 a highly virulent strain, and Ea 680 a mild virulent strain [6].…”