Synergistic antibacterial activity of Lactococcus lactis and Xylella phage MATE 2 for an effective biocontrol strategy against black rot disease in broccoli

Black rot, caused by <i>Xanthomonas campestris</i> pv. <i>campestris</i> (<i>Xcc</i>), is considered the most destructive disease affecting cruciferous vegetables, resulting in significant losses worldwide. The need for biocontrol agents against <i>Xcc</i> that can reduce reliance on chemical pesticides, enhance sustainability, and ensure crops and environmental health is crucial. Combining phages with other antibacterial agents (i.e., antibiotics and bacteriocins) to treat bacterial infections is gaining increased attention due to the frequently observed synergistic effects. This study introduces for the first time the combination of a lytic phage, i.e., <i>Xylella</i> phage MATE 2 (MATE 2) with nisin-producing <i>Lactococcus lactis</i> subsp. <i>lactis</i> (<i>L. lactis</i>) bacterium as an eco-friendly, cost-effective, and practical strategy for controlling <i>Xcc</i> in cruciferous vegetables. The antibacterial efficacy of MATE 2 and <i>L. lactis</i>, individually and in combination, against <i>Xcc</i> was investigated through a series of <i>in vitro</i> assays and <i>in planta</i> experiments conducted on broccoli plants. The time-killing curves results showed that under conditions of reduced <i>Xcc</i> population concentration (10³ CFU/mL), MATE 2 at 10⁸ PFU/mL exerted a persistent inhibitory effect on <i>Xcc</i> growth for 7 days. The Spot assays and v-qPCR analysis showed that both <i>L. lactis</i> and its bacteriocin nisin have significant antibacterial potential to contrast <i>Xcc</i>. Furthermore, combined application of MATE 2 and <i>L. lactis</i> in broccoli plants by foliar spraying generated significant synergistic efficacy in preventing <i>Xcc</i> infections, achieving a 71% reduction in symptoms, compared with 64 and 38% for single applications, respectively. In this study, the positive synergistic effect of the combined application of phage and beneficial bacteria in preventing black rot disease underscores this eco-friendly and cost-effective approach as a promising control measure against plant bacterial diseases.