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 Xanthomonas campestris pv. campestris (Xcc), is considered the most destructive disease affecting cruciferous vegetables, resulting in significant losses worldwide. The need for biocontrol agents against Xcc 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., Xylella phage MATE 2 (MATE 2) with nisin-producing Lactococcus lactis subsp. lactis (L. lactis) bacterium as an eco-friendly, cost-effective, and practical strategy for controlling Xcc in cruciferous vegetables. The antibacterial efficacy of MATE 2 and L. lactis, individually and in combination, against Xcc was investigated through a series of in vitro assays and in planta experiments conducted on broccoli plants. The time-killing curves results showed that under conditions of reduced Xcc population concentration (103 CFU/mL), MATE 2 at 108 PFU/mL exerted a persistent inhibitory effect on Xcc growth for 7 days. The Spot assays and v-qPCR analysis showed that both L. lactis and its bacteriocin nisin have significant antibacterial potential to contrast Xcc. Furthermore, combined application of MATE 2 and L. lactis in broccoli plants by foliar spraying generated significant synergistic efficacy in preventing Xcc 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.


Introduction
Xanthomonas campestris pv.campestris (Xcc) is a Gram-negative bacterium belonging to the family Xanthomonadaceae, which includes the most devastating plant pathogens that continually threaten food security (Dimkić et al., 2023).Xcc is an economically important phytopathogen that causes black rot disease in many Brassicaceae species (Alvarez, 2000;Papaianni et al., 2020).Black rot is considered the main yield-limiting and destructive disease of brassica vegetable crops worldwide, including cabbage, radish, cauliflower, broccoli, kale, and Brussels sprouts (Vicente and Holub, 2013;Hakalová et al., 2022).Characteristic symptoms of black rot are V-shaped yellow lesions starting from the leaf margins and blackening of the veins that results from Xcc movement in the vascular system (Vega-Álvarez et al., 2021).The affected leaves can fall prematurely, and the disease can cause stunted growth and the death of young plants, significantly affecting crop biomass and quality (Vicente and Holub, 2013).The pathogen can live epiphytically on leaf surfaces or as a saprophyte in necrotic tissues and generally enters the plant through hydathodes on leaf margins or through wounds and colonizes the plant's vascular system (Danhorn and Fuqua, 2007;Boulanger et al., 2014).
Historically, the control of Xcc has been challenging and primarily relies on the use of Xcc-free planting material (seeds or transplants), crop rotation, and the elimination of other potential inoculum sources such as residues of infected crops and cruciferous weeds (Iglesias-Bernabé et al., 2019).The development and use of resistant cultivars has had limited success due to the scarcity of resistance sources and the existence of nine races of the pathogen (Vicente and Holub, 2013;Iglesias-Bernabé et al., 2019).Additionally, chemical treatments and the use of antibiotics in crop production are being hampered by the emergence of resistant bacterial strains, soil accumulation, as well as growing concerns about their environmental and health implications (Behlau et al., 2020;Holtappels et al., 2022).Nowadays, bacteriophages (viruses that specifically infect and lyse bacteria) are being evaluated as alternative biocontrol agents to be integrated into the management of plant diseases (Anyaegbunam et al., 2022).Lytic bacteriophages are responsible for the destruction of the host bacterial population by infecting and replicating within the bacteria, causing lysis and release of new phage particles, which can then infect other bacterial cells (Greer et al., 2024).In the case of black rot disease, numerous studies have highlighted the potential usefulness of implementing phage therapy to combat Xcc infections (Papaianni et al., 2020;Holtappels et al., 2022).
Phage therapy is currently one of the recommended therapeutic options, preferably in combination with another antibacterial agent, a practice thought to improve efficacy (Bhargava et al., 2021).Several studies have reported the potential of bacteriophages to act synergistically with other antimicrobial agents such as antibiotics and bacteriocins, reinforcing the concept that when combined, they are more efficient (Gu Liu et al., 2020;Bhargava et al., 2021;Rendueles et al., 2022).Among antibacterial agents, Lactococcus lactis subsp.lactis (L.lactis) represents one of the most extensively studied lactic acid bacteria (LAB) due to its aptitude to produce nisin, a bacteriocin that exhibits potent antibacterial activity against both Gram-positive and Gram-negative disease-associated pathogens (Shin et al., 2016;Bukvicki et al., 2020).This LAB and its cell-free supernatant have been shown to exhibit antibacterial activity against Xanthomonas spp.strains (Mačionienė et al., 2022).
Additionally, Serna-Cock et al. (2013) had also showed that L. lactis exerts an in vitro antimicrobial activity against Xanthomonas albilineans.Nevertheless, research on the efficacy of L. lactis against Xcc infections is still lacking.
Nisin, recognized as an FDA-approved bacteriocin, is widely used in food preservation and veterinary medicine for the treatment of dermatological conditions and intramammary infections in animals (Greer et al., 2024).It exerts an antibacterial activity by generating pores in the cell membrane and interrupting cell wall biosynthesis through specific lipid II interaction (Prince et al., 2016).In the field of plant protection, nisin has been demonstrated to be effective in vitro and in planta against plant pathogenic bacteria belonging to the Xanthomonadaceae family, namely Xylella fastidiosa (Xf) (Sabri et al., 2024a).Additionally, the combination of nisin with lytic phages has demonstrated high potential to simultaneously control Salmonella typhimurium, Salmonella enteritidis, and Escherichia coli (Duc et al., 2023).This encourages the exploration of the potential use of L. lactis in combination with Xcc lytic phages as a cost-efficient biocontrol strategy for combating Xcc infections in planta.Accordingly, this study offers a viable alternative to conventional pesticides by integrating the Xylella phage MATE 2 (Sabri et al., 2024b) with L. lactis and explores for the first time their antibacterial activities against Xcc in vitro and in planta.

Bacteriophage, bacterial strains, and culture conditions
Xcc strain CFBP 1710 and L. lactis strain ATCC 11454 were grown either in liquid yeast extract peptone glucose broth (YPG) (5.0 g/L yeast extract, 5.0 g/L peptone and 10.0 g/L glucose) or on yeast extract peptone glucose agar (YPGA, i.e., YPG supplemented with 1.5% agar).The strains were stored at −80°C in 25% glycerol in YPG broth.Before use, the strains were plated from glycerol stocks onto YPGA agar plates and incubated at 28°C for 24 h.The bacteriophage named Xylella phage MATE 2 (MATE 2) from our collection, which possessed a broad spectrum of antibacterial activity against various phytobacteria of the Xanthomonadaceae family (Sabri et al., 2024b), including Xcc, was used in this study.
Nisin (ITSISLCTPGCKTGALMGCNMKTATCHCSIHVSK), extracted and purified from L. lactis, was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).According to the manufacturer, the formulation contains 2.5% (w/w) pure nisin with potency ≥900 IU/mg.Lyophilized nisin was solubilized in sterile Milli-Q water to a stock concentration of 12.5 mg/mL and filter sterilized through a 0.22 μm nylon Acrodisc ® syringe filter (Merck, Rome, Italy).

MATE 2 inhibitory effect on Xcc growth
To evaluate the lysis potential of MATE 2 in suppressing the growth of Xcc in liquid culture over a 7-day growth period, a bacterial growth reduction assay was performed.Briefly, 100 μL of MATE 2 suspensions (10 8 and 10 4 PFU/mL) were separately mixed with 100 μL of each concentration of Xcc (ranging from 10 8 to 10 2 CFU/mL) in triplicates.The mixture was then inoculated into 2 mL of YPG broth in a 10 mL centrifuge tube and incubated at 28°C for 7 days.During incubation, 11 optical density measures (0, 4, 8, 18, 24, 48, 72, 96, 120, 144, and 168 h) at OD 600 were obtained using the UV spectrophotometer (UV -1800, Shimadzu Corporation, Kyoto, Japan).All the data are presented as mean ± standard deviation (SD) of three independent experiments.

In-vitro investigation for antagonism between Lactococcus lactis and Xcc
The antagonistic potential of L. lactis ATCC 11454 against Xcc was assessed through a spot assay as follows: 300 μL of Xcc suspension (OD 600 = 0.1) were equally spread on YPGA plates and allowed to dry under the laminar flow hood.Subsequently, drops of 10 μL of L. lactis suspension (OD 600 = 0.2) were spotted onto the surface of the plates.Spots were dried at room temperature and the plates were incubated for up to 2 days at 28°C.Sterile distilled water served as the negative control.The antagonistic activity of L. lactis was perceived as an inhibition zone of Xcc growth, which was measured using a digital caliper.This experiment was conducted in triplicates.

Fluorescence microscopy
To further evaluate the bactericidal activity of nisin against Xcc cells, 100 μL of Xcc suspension (OD600 = 0.2) was mixed with 100 μL of nisin at 2 mg/mL and incubated at room temperature for 3 h.The LIVE/DEAD ® BacLight™ viability kit (ThermoFisher Scientific, Milan, Italy) was used according to the manufacturer's instructions to assess the viability of Xcc cells treated with nisin at 15 min, 30 min, 1 h, 2 h and 3 h post-incubation.Photomicrographs were taken using a Nikon E800 microscope equipped with fluorescein isothiocyanate (480/30 excitation filter, DM505 dichroic mirror, 535/40 emission filter) and tetramethyl rhodamine isothiocyanate (546/10 excitation filter, DM575 dichroic mirror, 590 emission filter) fluorescence filter sets.

Transmission electron microscopy
The antibacterial effect of nisin against Xcc cells at the ultrastructural level was assessed using a TEM.Briefly, 100 μL of Xcc suspension (OD600 = 0.2) was treated with 100 μL of nisin at 2 mg/mL for 3 h at room temperature.Micrographs were taken after 3 h via TEM (FEI MORGAGNI 282D, United States) using the dip method.Briefly, carbon-coated copper/rhodium grids were immersed in both the nisin-treated and untreated bacterial suspensions for 5 min, followed by a rinse with 200 μL of distilled water.To achieve negative staining, the grids were floated on 200 μL of a 0.5% w/v UA-zero EM stain solution (Agar-Scientific Ltd., Stansted, United Kingdom), and observed under an accelerating voltage of 80 kV.

Nisin-MATE 2 combination against Xcc
To study the synergistic effects, 50 μL of phage suspension (10 8 PFU/mL) was added to 50 μL of nisin at the sublethal concentration (1 mg/mL) and mixed with 100 μL of Xcc suspension (OD600 = 0.2).Additionally, 100 μL of MATE 2 (10 8 PFU/mL) and 100 μL of nisin (1 mg/mL) were individually mixed with 100 μL of Xcc suspension (OD600 = 0.2) and served as controls.The mixtures were then inoculated into 2 mL of YPG broth in a 10 mL centrifuge tube and incubated at 28°C for 3 days.Optical density measures were determined using the UV spectrophotometer and compared to the controls.

In planta antagonistic effects of Lactococcus lactis and MATE 2 against Xcc
To evaluate the efficacy of L. lactis and MATE 2, individually and in combination, in contrasting Xcc infections within plant tissues, an in-planta assay was carried out on one-month-old broccoli plants (Brassica oleracea var.italica) in a greenhouse under controlled conditions (25 ± 2°C and ~ 70% humidity).Three leaves per plant were wounded by multi-needle prickers and then sprayed with the Xcc suspension (0.66 mL/plant; OD600 = 0.2) and the biocontrol agents.L. lactis (2.5 mL/plant; OD600 = 0.7) and MATE 2 (2.5 mL/plant; 10 8 PFU/mL), individually and in combination, were applied preventively, i.e., 24 h prior to Xcc inoculation, and curatively (24 h after Xcc inoculation).Ten broccoli plants were used for each treatment.Untreated plants inoculated with sterile water or with Xcc only were included as negative and positive controls, respectively.Fourteen days post-infection (dpi), disease symptoms were quantified by measuring the area of necrosis on the treated leaf according to an arbitrary six-level disease scale whereby: 0 = no symptoms, 1 = symptoms begin to appear, 2 = ¼ of leaf area showing symptoms, 3 = ⅓ of leaf area showing symptoms, 4 = ½ or more of leaf area showing symptoms, 5 = the entire leaf showing symptoms.The efficacy of each treatment in reducing Xcc infections was calculated as follows: Where P = severity score, T = number of treated leaves having the same score, and C = number of infected leaves (positive control) having the same score.

Statistical data analysis
To test the antagonistic effect of L. lactis and MATE 2 on Xcc infections in broccoli plants, a one-way analysis of variance (ANOVA) was performed using the IBM SPSS Statistics 26 software.The significance of differences was calculated by Duncan's post-hoc test and a p-value less than 0.05 was considered statistically significant.All data are presented as mean ± standard deviation (SD) of two independent experiments.

Persistence of Lactococcus lactis and MATE 2 in broccoli plants
To investigate the persistence of L. lactis and MATE 2 within broccoli plants, the treated leaves were analyzed at the end of the in-planta experiment (14 dpi).Briefly, two leaves from each treatment were ground in 4 mL of sterile water and centrifuged at 1,000 g for 5 min to remove plant debris.In the case of L. lactis samples, the resultant supernatant was diluted six times using a 10-fold serial dilution and 50 μL were plated on YPGA plates.The plates were incubated at 28°C for 24 h and L. lactis-like colonies were purified on YPGA plates and subjected to PCR testing using the L. lactis subsp.lactis-specific primers Nis-F: 5′-CGGCTCTGATTAAATTCTGAAG-3′ and Nis-R: 5′-GGATTAGCTAGTAGTAACTGTTC-3′ reported in Alegría et al. (2010).For MATE 2 samples, the macerate was filtered through 0.22-μm filter and 500 μL of the filtrate was added to 200 μL of Xcc suspension (OD600 = 0.2).The mixture was then inoculated into 2 mL of YPG broth in a 10 mL centrifuge tube and incubated for 24 h at 28°C for phage enrichment.The phage was then tested against Xcc using a spot assay and subjected to PCR using MATE 2-specific primers MATE 2-F: 5′-CCCGCCCAGGATCGCAAGCCGT-3′ and MATE 2-R: 5′-TCGTGCTCTCAGATTGTTGACGC-3′ designed in this study.The PCR cycles consisted of an initial denaturation step at 94°C for 5 min; 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and a final elongation step at 72°C for 5 min.

Spot assay
The lytic activity of MATE 2 against Xcc was initially examined through a spot assay.The results showed that MATE 2 produced clear lysis zones on the Xcc lawn at 10 8 , 10 7 , 10 6 , and 10 5 PFU/mL (Figure 1), indicating complete lysis of Xcc bacteria.At 10 4 PFU/mL, the lysis zone turned semi-translucent, demonstrating reduced antibacterial activity.Beyond this titer, no distinct lysis zones were observed, indicating that the phage titer was insufficient to induce distinct effect on Xcc lawn.These results highlight the lytic effectiveness of MATE 2 against Xcc and shed light on phage concentration requirements for combating Xcc infections.

Bacteriolytic effect of MATE 2 on Xcc growth
To examine the dynamics of MATE 2 virulence, killing curves were generated by infecting Xcc at concentrations of 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , and 10 2 CFU/mL with MATE 2 at two titers of 10 8 and 10 4 PFU/mL.These latter were selected based on the results of the spot assay, wherein a titer of 10 4 PFU/mL was identified as the minimum titer of MATE 2 capable of inducing a lytic effect on Xcc lawn.The results showed that both phage titers effectively inhibited the growth of all Xcc cultures for 24 h (Figure 2).However, after 24 h, MATE 2-infected bacteria at high concentrations (10 8 , 10 7 , and 10 6 CFU/mL) started to display an increase in OD reading, indicating the emergence of phage resistance (Figure 2).Despite the resistance development, MATE 2 showed significant antibacterial activity against Xcc growth, with an inhibition rate of 60%, 7 dpi.Xcc at 10 5 and 10 4 CFU/mL concentrations developed resistance to both phage titers after 48 h, similar to Xcc concentrations (10 3 and 10 2 CFU/mL) treated with MATE 2 at 10 4 PFU/mL (Figure 2).Most importantly, the infection of Xcc at 10 3 and 10 2 CFU/mL with MATE 2 at 10 8 PFU/mL resulted in a persisting inhibitory effect for 7 days, leading to the complete inhibition of Xcc growth (OD600 = 0) (Figure 2).At the end of the experiments, MATE 2-resistant mutants were isolated on YPGA agar plates and resistance was confirmed by spot assay.These results provide insights into the Xcc-MATE 2 interaction, revealing that a 10 8 PFU/mL titer appears to be suitable for phage preparation to reduce the development of bacterial resistance.The results also indicated that at controversial concentrations, i.e., low Xcc population and high MATE 2 titer, Xcc is unable to develop resistance to MATE 2 during 7 days of co-incubation.This suggests that application of MATE 2 at high concentrations could significantly contrast Xcc infections and delay the emergence of phage resistance.

Antibacterial activities of Lactococcus lactis and nisin against Xcc
The antagonistic activity of L. lactis on Xcc cells was examined through in-vitro culture assay, resulting in an inhibition zone diameter of 22.5 mm for Xcc growth (Figure 3).This inhibitory effect highlights the efficacy of L. lactis in contrasting Xcc, suggesting its potential use as a biocontrol agent or a source of antimicrobial compounds targeting Xcc in agricultural applications.Time-killing curves showing the bacteriolytic effect of MATE 2 at 10 8 and 10 4 PFU/mL on Xcc growth at various concentrations (10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , and 10 2 CFU/mL).The optical density of treated and untreated-bacterial culture with MATE 2 at 24-h interval for 7 days is compared.The bars indicate the error means for three replicates.Spot assay showing the antibacterial activity of nisin at various concentrations against Xcc.SDW: sterile distilled water used as a negative control.
The bactericidal activity of nisin against Xcc was also determined using v-qPCR and spot assays.Results of the v-qPCR showed that nisin exerted antibacterial activity against Xcc in a dose-dependent manner (Figure 4).Xcc bacteria treated with nisin at 6, 4, 3, and 2 mg/mL exhibited no amplification, similarly to dead Xcc cells used as control (Figure 4), indicating complete bacterial lysis.Below these concentrations, nisin showed reduced activity against Xcc, with 1 mg/mL as the sublethal concentration.These results were consistent with the spot assay results, which showed clear lysis zones on the Xcc lawn induced by nisin at 6, 4, 3, and 2 mg/mL, while 1 mg/mL produced a clouded lysis zone (Figure 5).These findings provide valuable insight into the bactericidal activity of nisin against Xcc and suggest that nisin is involved in the antagonistic activity exhibited by L. lactis against Xcc.

Nisin activity against Xcc cells under electron and fluorescence microscopy
FM and TEM analyses were performed to visually evaluate the bactericidal activity of nisin against Xcc.FM micrographs showed that the staining of nisin-treated Xcc cells at 2 mg/mL shifted progressively from green (live bacterial cells) to red (dead bacterial cells), indicating damages in the cell membrane (Figure 6).Three hours after contact, intense red channels were observed (Figure 6), indicating robust lysis of Xcc and confirming the spot assay and v-qPCR results.At the ultrastructural level, TEM analyses revealed that nisin treatment causes significant structural changes in Xcc cells compared to untreated cells (Figure 7A).Electron microscope analysis highlighted the disrupting action of nisin on the bacterial double membrane (Figure 7C, arrows) and the cytoplasm alterations through the production of vesicles (Figure 7B, asterisks), which impair bacterial viability.

Combined effect of nisin and MATE 2 against Xcc
The combined effect of nisin and MATE 2 on Xcc growth was determined by measuring variations in optical density at OD 600 over a 3-day incubation period.The results showed that the nisin alone at its sublethal concentration (1 mg/mL) was less effective against Xcc growth than MATE 2, resulting in a 45% reduction in Xcc growth after 72 h of treatment (Figure 8).Additionally, as previously demonstrated, MATE 2 alone maintained high inhibitory efficacy against Xcc for 24 h, after which bacterial growth slightly resumed, resulting in an inhibition rate of 67% after 72 h of co-incubation (Figure 8).Remarkably, the combination of MATE 2 with nisin was more effective against Xcc than single treatments, achieving a 76% reduction in bacterial growth (Figure 8).This suggested a synergistic inhibitory  V-qPCR assay showing DNA amplification curves obtained from: (A) untreated Xcc, (B) nisin-treated Xcc at 0.1 mg/mL, (C) at 0.2 mg/mL, (D) at 0.4 mg/mL, (E) at 0.6 mg/mL, (F) at 0.8 mg/mL, (G) at 1 mg/ mL, (H) DNA amplification curves obtained from nisin-treated Xcc at 2, 3, 4, 6 mg/mL, dead Xcc cells, and sterile distilled water used as negative control.
10. 3389/fmicb.2024.1468792Frontiers in Microbiology frontiersin.org effect against Xcc and the potential use of MATE 2 and nisinproducing L. lactis bacteria to cost-efficiently tackle Xcc infections in cruciferous vegetables.

Efficiency of Lactococcus lactis and MATE 2 as individual and combined applications in controlling Xcc infections in broccoli
The in-planta antibacterial potentials of MATE 2 and L. lactis, individually and in combination, against Xcc were evaluated in broccoli plants.The untreated Xcc-infected plants (positive control) displayed severe infection symptoms 14 dpi, namely V-shaped yellow lesions on leaf margins, chlorosis, blackening of veins, and loss of leaves, while uninfected plants (negative control) showed no symptoms of Xcc (Figure 9).Interestingly, plants treated with MATE 2 and L. lactis, individually and in combination, showed reduced Xcc symptoms compared to the positive control (Figure 9).In terms of the efficacy of each treatment in reducing Xcc symptoms, which was calculated based on the symptom severity scores as described in section 2.9, analysis of our data revealed a very highly significant difference between the treatments (p = 0.000) (Figure 10).Once applied preventively, the combination of MATE 2 and L. lactis significantly decreased disease symptoms on broccoli leaves by 71% 14 dpi, while their individual applications reduced disease by 64 and 38%, respectively.Curatively, MATE 2 and L. lactis exhibited modest efficacy in mitigating Xcc symptoms in broccoli plants, achieving respective efficacy rates of 30 and 15%, while their combination resulted in a 31% disease reduction (Figure 10).At the end of the experiment, MATE 2 and L. lactis were successfully reisolated from treated broccoli plants and their identity was confirmed by PCR analyses (Figure 11).Furthermore, their persistent antibacterial activity against Xcc was validated afterward by spot assays (data not shown).These results clearly indicated the suitability of MATE 2 and L. lactis for controlling Xcc in cruciferous vegetables and demonstrated their effective combined application in preventing black rot disease.In agriculture, the use of phages and LAB offers significant advantages, characterized by cost-effectiveness, sustainability, and ease of application (Costa et al., 2023;Ojo and de Smidt, 2023).Phages provide targeted antimicrobial activity against specific bacterial pathogens, thereby lowering the need for broad-spectrum antimicrobials and limiting environmental impacts (Strathdee et al., 2023).Their capacity to multiply inside bacterial hosts enables selfrenewal and scalability, adding to cost efficiency in production and application (Ling et al., 2022).Furthermore, phages can be applied in diverse forms, including sprays, drenching, seed treatment and irrigation, facilitating their adoption in agricultural settings (Holtappels et al., 2021).LAB, known for their safety use and production of bacteriocins, offer natural antibacterial properties that improve food preservation and agricultural safety (Ayivi et al., 2020).Their ease of application through fermentation processes and incorporation into probiotic formulations supports practical integration into food production (Timothy et al., 2021).Together, phages and LAB provide cost-effective solutions that support sustainable practices by decreasing the use of chemicals, improving food quality, and managing risks related to pathogens; thus, promoting and building resilience in agricultural and food sectors.To this end, this study reports for the first time the successful use of phage MATE 2 and L. lactis in combination to prevent black rot in broccoli plants.
The dynamic of MATE 2 virulence indicated that a high titer of this phage is more efficacious against Xcc, maintaining a persistent inhibitory effect on low bacterial populations for 7 days.Similarly, in our previous study we found that MATE 2 was able to significantly inhibit the growth of Xf over 7 days with a titer of 10 8 PFU/mL (Sabri et al., 2024b).However, the emergence bacterial resistance is a common challenge in phage therapy that is conditioned by many aspects, i.e., phage and bacterial concentrations, lysis potential, etc.Therefore, employing MATE 2 in conjunction with other antimicrobial compounds (bacteriocins) would help target bacteria more effectively, preserving the therapeutic potential of MATE 2, and reducing the risk of resistance development.These findings emphasize the importance of conducting a preliminary evaluation of bacterial concentration to intervene with accurate phage concentration, thereby ensuring an effective biocontrol.
On the other hand, L. lactis showed for the first time an antagonistic activity against Xcc, effectively inhibiting its growth on YPGA plates.This aligns with previous reports demonstrating the antibacterial effect of L. lactis on both Gram-negative and Gram-positive bacteria, strengthening its role as a possible biocontrol agent in agricultural practices (Settier-Ramírez et al., 2021;Sabri et al., 2024a).Subsequent in vitro tests revealed that nisin exerts bactericidal activity against Xcc cells, suggesting its involvement in the antagonistic effect of L. lactis.The TEM analyses showed that nisin induces severe structural changes in Xcc cells, including alterations in the outer membrane structure and cell cytoplasm, compromising the viability.This activity against Xcc can be added to the long list of nisin's antimicrobial effects against pathogenic bacteria (Villarreal et al., 2024).Notably, the MATE 2-nisin combination resulted in over 76% reduction in Xcc growth after 72 h of treatment, whereas phage and nisin alone showed a 67 and 45% reduction, respectively, underscoring the enhanced effectiveness of the antimicrobial combination.Indeed, the synergistic effect of phage-nisin combination has been previously reported, demonstrating that it is based on the complementary action of phages, which specifically lyse bacterial cells, and of nisin, which disrupts resistance mechanisms and bacterial physiology, resulting in a significant increase in their individual actions (Rendueles et al., 2022).This is in addition to a possible role of phages in constraining bacterial defense mechanisms against  For biocontrol purposes, the stability and the safety of biocontrol agents are mandatory.In this context, MATE 2 has already demonstrated stability over a wide range of pH and temperatures, and its genome is devoid of genes encoding virulence factors, antibiotic resistance, toxins, or genes related to lysogenic action (Sabri et al., 2024b).Additionally, L. lactis has "generally recognized as safe" (GRAS) status and is found in a wide range of environments and a large variety of food products (de Lacerda et al., 2016).L. lactis strains have already been isolated from stems of Eucalyptus, from the interior of aquatic plants, and from the inner tissues and leaves of sugarcane (de Lacerda et al., 2016).Furthermore, Golomb and Marco (2015) showed that certain strains of L. lactis are well adapted for growth on plants.Likewise, in our in-planta experiments, MATE 2 and L. lactis successfully remained in broccoli leaves for 14 dpi and their persistent antibacterial activity against Xcc was confirmed, indicating their suitability in controlling black rot disease.
In the management of black rot disease in broccoli plants, preventive applications of MATE 2 and L. lactis were more effective in reducing disease symptoms than their curative applications.These findings align with those of a prior study, which demonstrated that applying phages as a preventative agent could reduce bacterial infections more effectively than post-challenge application (Ahmadi et al., 2016).The results also showed that MATE 2 was more effective than L. lactis in controlling black rot.This differential efficacy could be attributed to their behaviors and interactions within the host plant.Phages, unlike other types of antimicrobial compounds, their post-application levels will increase at the expense of bacterial host survival.Additionally, their replication within bacterial cells enables them to maintain proximity to Xcc throughout the plant, which may allow them to effectively track and lyse Xcc cells within the plant.Consistent with the in-vitro results, the combined application of MATE 2 and L. lactis preventively against Xcc resulted in increased effectiveness, successfully preventing black rot symptoms by 71% in broccoli plants, while their individual applications reduced disease by 64 and 38%, respectively.These findings validate the potential of MATE 2 and L. lactis to act synergistically and boost significantly their individual actions for enhanced black rot control.In addition to nisin production, L. lactis can produce various antimicrobial compounds such as organic acids, hydrogen peroxide, diacetyl, reuterin, acetaldehyde, acetoin, carbon dioxide, and other bacteriocins (Siroli et al., 2020), which may in our case be involved in the antibacterial activity against Xcc and may positively interact with MATE 2 against Xcc infections in plant tissues.Whether this is the case, is yet to be determined.
Beyond their antimicrobial activities, LAB and phages have also demonstrated synergistic benefits in enhancing plant production.Papaianni et al. (2020) highlighted the diverse potential advantages of phages in influencing plant metabolism and enhancing defense mechanisms.Their research underscores the role of phages in agricultural systems by potentially boosting plant resilience and response to stress factors.Furthermore, Jaffar et al. (2023) demonstrated the broadspectrum activities of L. lactis and its bacteriocin nisin, which contribute positively to plant growth, soil health, and overall crop productivity.Additionally, Raman et al. (2022) reported that L. lactis significantly promoted the root and shoot length of rice, as well as cabbage growth and production.Integrating these properties, the combination of MATE 2 and L. lactis emerges as a pivotal strategy in integrated pest management of black rot disease.However, further research is needed to explore their potential applications in field conditions against Xcc and their effects on cruciferous vegetable production.
This study extends previous research on Xf, reaffirming the broadspectrum of antibacterial efficacy of MATE 2 and nisin-producing L. lactis (Sabri et al., 2024a,b) and their suitability for being used in plant systems; thus, supporting their application in Xf-infected plants.These attributes, together with ease of production, make them a practical and cost-effective strategy for dealing with Xf and Xcc infections in plants.Such investigations will also be crucial to translating these promising results into viable solutions for farmers, thus contributing to more resilient and productive agricultural systems.

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FIGURE 1
FIGURE 1YPGA agar plate showing the lytic activity of MATE 2 at different titers (10 8 -10 1 PFU/mL) against Xcc.SDW: sterile distilled water used as a negative control.

FIGURE 3
FIGURE 3YPGA plate showing the antagonistic effect of L. lactis on Xcc.

FIGURE 6
FIGURE 6Fluorescent micrographs showing nisin-treated Xcc cells at 2 mg/mL.Green and red fluorescence represent live and dead cells, respectively.Magnification 20X, bar 20 μm.

FIGURE 7
FIGURE 7 Transmission electron micrographs showing the bactericidal effect of nisin at 2 mg/mL on Xcc cells.(A) untreated Xcc cells, used as control.(B,C) Nisin-treated Xcc cells showing structural, cell wall, and cytoplasm alterations.Bar (A,B) 100 nm; (C) 50 nm.

FIGURE 8
FIGURE 8In-vitro killing curves displaying the antibacterial activity of MATE 2 and nisin, individually and in combination, against Xcc.The bars show the average for three replicates.

FIGURE 10
FIGURE 10Histograms showing the severity of Xcc symptoms on broccoli plants treated preventively and curatively with MATE 2 and L. lactis, individually and in combination.