Leuconostoc mesenteroides strain MS4-derived bacteriocins: A potent antimicrobial arsenal for controlling Xylella fastidiosa infection.

Abstract

Xylella fastidiosa subsp. pauca (Xfp) currently presents a serious threat to agriculture in Europe and in the Mediterranean, following its discovery in several countries. Addressing this bacterial plant disease with traditional agricultural practices and management strategies has proven inadequate, highlighting the urgent need for effective and environmentally safe antibacterial solutions. In this study, we explored the antibacterial activity of the lactic acid bacterium Leuconostoc mesenteroides strain MS4-derived bacteriocins against Xfp, utilizing a combination of in vitro and in planta experiments. In particular, the cell-free precipitate (CFP) derived from strain MS4 culture in MRS broth, suppressed Xfp growth on BCYE agar plate, whereas protease K-treated CFP was inactive, highlighting the presence of antimicrobial compounds of proteinaceous nature. Additionally, fluorescence and transmission electron microscopy analyses showed that the CFP exhibits a bactericidal effect on Xfp cells, characterized by membrane disruption and subsequent cellular damage. The whole-genome sequencing and bioinformatic analysis revealed that MS4 genome consists of a circular chromosome of 1860,891 bp and a circular plasmid of 37,317 bp and most importantly to encompass six bacteriocin-encoding genes, with a peptide size ranging from 45 to 59 amino acids. MALDI-TOF/TOF MS and RPLC-ESI-MS assays performed on cell-free supernatant (CFS) confirmed the secretion of four (out of 6) bacteriocins (denoted MK-45, MR-53, MW-56, and MG-58) by MS4 in MRS broth. In spot assays, these bacteriocins displayed significant lethality against Xfp, with a minimum lethal concentration between 0.2 and 0.4 mg/mL. The application of CFP on Xfp-infected Nicotiana benthamiana plants, implemented both as preventive and curative approach, successfully controlled the infection, resulting in no visible symptoms 40 days post-inoculation. The finding of MS4 as a natural source of various potent bacteriocins against Xfp, coupled with a significant production under low-cost and uncomplicated laboratory conditions, make of MS4 a cost-effective and realistic option for sustainable management of Xf-related diseases.