Exploring Active Peptides with Antimicrobial Activity In Planta against Xylella fastidiosa

Simple Summary Xylella fastidiosa is one of the most harmful plant bacteria in the world, which could lead to significant economic losses. The lack of direct and efficient therapies to treat plants infected with this bacterium has involved researchers in the search for new approaches and compounds capable of countering this alarming pathogen. Antimicrobial peptides (AMPs) are one of the promising candidates for sustainable and eco-friendly treatment for agricultural applications. Accordingly, nine AMPs against Xf were evaluated, three of which were shown to be promising in limiting Xf growth and its biofilm formation in in vivo and in vitro experiments, and thus, are considered as emerging biocontrol agents against Xf infection. Abstract Xylella fastidiosa (Xf) is a xylem-limited quarantine plant bacterium and one of the most harmful agricultural pathogens across the world. Despite significant research efforts, neither a direct treatment nor an efficient strategy has yet been developed for combatting Xylella-associated diseases. Antimicrobial peptides (AMPs) have been gaining interest as a promising sustainable tool to control pathogens due to their unique mechanism of action, broad spectrum of activity, and low environmental impact. In this study, we disclose the bioactivity of nine AMPs reported in the literature to be efficient against human and plant pathogen bacteria, i.e., Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, against Xf, through in vitro and in vivo experiments. Based on viable-quantitative PCR (v-qPCR), fluorescence microscopy (FM), optical density (OD), and transmission electron microscopy (TEM) assays, peptides Ascaphin-8 (GF19), DASamP1 (FF13), and DASamP2 (IL14) demonstrated the highest bactericidal and antibiofilm activities and were more efficient than the peptide PB178 (KL29), reported as one of the most potent AMPs against Xf at present. Furthermore, these AMPs showed low to no toxicity when tested on eukaryotic cells. In in planta tests, no Xf disease symptoms were noticed in Nicotiana tabacum plants treated with the AMPs 40 days post inoculation. This study highlighted the high antagonistic activity of newly tested AMP candidates against Xf, which could lead to the development of promising eco-friendly management of Xf-related diseases.


Antibiogram Assays
Due to the particular nature of Xf, and to establish an approximate starting concentration of peptides with which Xf cells could be afterward challenged, prior screening was performed using the surrogate Xa, a phylogenetically close and relatively swift-growing organism [26].
Six different peptide concentrations (150, 100, 50, 25, 12, and 6 µM) were tested and 5 µL from the 6 concentrations of each AMP were applied directly on Xa cells cultured on LPGA medium. Effects of AMPs on Xa, i.e., inhibition halos, were checked after 48 h of incubation at 25 • C, using the Gel Doc XR+ (Bio-Rad Laboratories, Hercules, CA, USA). Results were considered positive (+) when halo formation occurred, and negative (−) when there was no observable halo.
For Xf, bacteria and AMPs were co-plated in the following order: Three drops of Xf suspension (10 8 CFU/mL), each containing 30 µL, were placed at the top of the petri dish, 1 cm apart, and allowed to slowly flow down to the opposite side of the plate, resulting in three parallel rows of Xf. After drying under the laminar flow hood, 5 µL of 50, 25, and 12 µM of AMPs were placed on top of each row, and PBS was used as a negative control. The growth inhibition zones were checked after 10-15 days post incubation at 28 • C and evaluated as the distance between the top of the plate and the edges of Xf growth. Three independent replicates were performed for both bacteria.

Effect of Selected AMPs on Xylella fastidiosa Growth
Effects of AMPs on Xf growth were assessed by contact test assays, coupled with OD600 measurements over 24 h. Xf suspension (180 µL, 10 8 CFU/mL) in PD2 (Pierce disease 2 media) was mixed with 20 µL of the corresponding peptide dilutions (50, 25, and 12 µM). Bacterial growth was measured by monitoring the OD600 using the NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA), after 0 min, 0.5 h, 1.5 h, 3 h, 18 h, and 24 h of incubation at 28 • C. Experiments were performed two times, with three replicates for each treatment.

Viable-Quantitative PCR (v-qPCR) and Fluorescence Microscopy (FM)
The bactericidal activity of the AMPs was assessed by contact test, coupled with viable-quantitative PCR (v-qPCR) [30]. AMP-treated Xf cells, together with heated Xf cells (95 • C for 10 min) used as positive controls, were subject to v-qPCR assay using PMAxx™ (Biotium, Rome, Italy), which is a photo-reactive dye that binds with high affinity to DNA templates. Upon peptide lysis activity, the PMAxx™ dye becomes covalently attached to disrupted DNA that cannot be amplified by PCR. PMAxx™ dye is designed to interact with cell membrane permeability; in a population of live and dead cells, only dead cells are susceptible to DNA modification due to compromised cell membranes. This unique feature makes PMAxx™ highly useful in selective detection of live bacteria by qPCR. Therefore, dilutions of a homogeneous cell suspension (from 10 8 to 10 2 CFU/mL) of viable or dead cells to a total volume of 200 µL in DNA low-binding tubes were prepared. Samples were kept in the dark for 8 min at room temperature (25 • C), following a 15-min photoactivation with PMA-LiteTM LED Photolysis Device, at a final concentration of 7.5 µM. The DNA of Xf was extracted using the CTAB protocol [30] and was analyzed in duplicate by a TaqMan-based qPCR assay using the target-specific primers Xf -F: 5 -CACGGCTGGTAACGGAAGA-3 and Xf -R: 5 -GGGTTGCGTGGTGAAATCAAG-3 and the probe Xf -Prb: 5 6FAM-TCGCATCCCGTGGCTCAGTCC-BHQ-1-3 [30]. Three replicates for each dilution and AMP experiment were performed.
The reduction in viability, expressed as log10 CFU/mL, was obtained by interpolating the CT value from each sample against the respective standard curve for each strain and subtracting it from the non-treated control (Log10 (N0/N). Results were analyzed with a one-way analysis of variance (ANOVA) followed by the post-hoc Student-Newman-Keuls test. SPSS statistical package software (SPSS for Windows, Version 20, SPSS Inc., Chicago, IL, USA) was used for the statistical analysis of data.
For the fluorescence microscopy (FM), aliquots of Xf suspension were incubated with AMPs at 50 µM for 1 h at room temperature. The untreated control reaction consisted of a bacterial suspension with only sterile distilled water. The LIVE/DEAD ® BacLight™ (Molecular Probes) viability kit was used to assess the viability of bacteria cells treated with peptides. The kit contains two nucleic acid dyes SYTO 9 and propidium iodide (PI) that allow distinguishing live cells with intact plasma membranes (green channel) from dead bacteria with compromised membranes by the peptide's activity (red channel). Photomicrographs were taken with a Nikon E800 microscope using fluorescein isothiocyanate (480/30 excitation filter, DM505 dichroic mirror, 535/40 emission filter) and tetramethyl rhodamine isocyanate (546/10 excitation filter, DM575 dichroic mirror, 590 emission filter) fluorescence filter sets.

Transmission Electron Microscopy (TEM) of AMPs against Xylella fastidiosa
Xylella fastidiosa cells in suspension (10 8 CFU/mL) were incubated with peptides at 50 µM for 1 h at room temperature. NAC-treated Xf suspensions were used as control reactions for the lytic activity. Preparations were observed under TEM (FEI MORGAGNI 282D, Hillsboro, OR, USA) by the dip method; i.e., the carbon-coated copper/rhodium grids were incubated with treated and untreated bacterial suspension for 5 min and afterward were rinsed with 200 µL of distilled water. Negative staining was obtained by floating

Antibiofilm and Planktonic Cell Activity
The effects of AMPs on Xf biofilm formation and planktonic cells were further investigated. NAC, which has been reported to reduce biofilm formation of Xf, was used at 25 and 50 µM as a positive control reaction. Xf suspension (180 µL) in PD2 was mixed with 20 µL of the corresponding peptide dilution in 96-well plates. Microplates were incubated for 20 days at 28 • C under constant shaking (140 rpm). Afterward, planktonic cells were recovered from the media and transferred into a new microplate, and their OD600 was measured.
To assess the biofilm formation, the original 96-well plate was rinsed 3 times carefully with sterile distilled water, stained with 250 µL of crystal violet (0.1%) for 20 min, and rinsed with sterile distilled water 3 times to discard excess dye. Eventually, with 250 µL of a mixture of ethanol/acetone (4:6) for 10 min, crystal violet adhered to the biofilm was solubilized and the OD595 was measured. DNA extraction was carried out for each sample and analyzed in duplicate by a TaqMan-based qPCR. For dose-response modelling of biofilm formation, the percentage of biofilm (B) was calculated according to the following formula: B = (Oi/Oc) × 100, where Oi is the OD595 of the treatment and Oc is the OD595 of the untreated control [21].

Toxicity Evaluation of AMPs
The eventual toxicity of the AMPs was evaluated on plant (Nicotiana tabacum cv. Xanthi) and animal (horse) eukaryotic cells. Using a syringe, 100 µL of peptide solutions (50 and 25 µM) were injected into fully expanded tobacco leaves. Three infiltrations per plant were performed, for a total of six replicates. The negative control reaction consisted of using sterile distilled water instead of AMPs. Plants were maintained in a glasshouse at 25 • C for 48 h. The possible toxicity on leaves was determined by measuring the diameter of lesions caused by the AMPs.
Erythrocytes from a horse blood sample were provided by the University of Bari, Department of Veterinary Medicine (Valenzano, Italy). The erythrocytes were challenged with AMPs at a final concentration of 50 µM. The hemolytic activity was observed under a light microscope.

In Planta Antagonistic Activity of AMPs against Xylella fastidiosa
To evaluate the antagonistic activity of AMPs that could interfere with the Xf virulence inside the plant, Xf infection and AMP application in the stem were carried out in Nicotiana tabacum (var. Xanthi) plants, maintained in a quarantine laboratory. Ten groups of six plants each were mechanically syringe-injected with 100 µL (10 7 CFU/mL) of Xf subsp. pauca isolate De Donno [21]. Immediately after the drops dried, 100 µL of AMPs (at concentrations of 50 and 25 µM) was administered at the same points using sterile distilled water as mock. Two groups of six plants were only injected with peptides at the two different concentrations. Leaf lesions, a characteristic symptom of tobacco plants infected with Xf, were inspected for 60 days post inoculation (dpi). Two independent experiments were conducted. TaqMan-based qPCR assays were carried out to detect the presence of AMP-treated Xf infection in the leaves above the inoculation point. Briefly, 1 g of leaf tissue was ground with 2 mL of CTAB buffer [30] and heated at 65 • C for 30 min. The plant extract was centrifuged at 13,000× g for 15 min, and the supernatant was twice washed with chloroform and precipitated in cold isopropyl alcohol. The pellet (total DNA) was eluted in 100 µL of sterile water, whilst 50 ng of DNA was used in qPCR assays.

Screening of Potent AMP Activities against Xanthomonas albilineans and Xylella fastidiosa
In this study, nine selected peptides were evaluated for their antimicrobial activities against the surrogate Xa as part of an initial in vitro screening. Of note, Xa and Xf show two different growths (in time and shape) on petri dishes and their antibiograms resulting from the AMP challenge are somehow incomparable. However, the results showed that all peptides were active against Xa, with MICs ranging from 6 µM to 50 µM (Table 2). This result provides an information about which concentrations and AMPs can be utilized against Xf. In addition, all AMPs were able to reduce the growth of Xf at varying degrees at 50, 25, and 12 µM. Three AMPs (GF19, FF13, and IL14) were particularly highly active and were able to strongly curtail Xf growth. FF13 and IL14 showed high antimicrobial activity, causing very large inhibition zones ( Figure 1), with mean growth zones of 15.6 and 13.7 mm, respectively. GF19 exhibited distinct inhibitory activity, whereas KL29, which is currently considered to be the most potent peptide against Xf, showed a clearing zone of 11.8 mm, representing lower activity than the three AMPs newly tested here. leaf tissue was ground with 2 mL of CTAB buffer [30] and heated at 65 °C for 30 min. The plant extract was centrifuged at 13.000 × g for 15 min, and the supernatant was twice washed with chloroform and precipitated in cold isopropyl alcohol. The pellet (total DNA) was eluted in 100 μL of sterile water, whilst 50 ng of DNA was used in qPCR assays.

Screening of Potent AMP Activities against Xanthomonas albilineans and Xylella fastidiosa
In this study, nine selected peptides were evaluated for their antimicrobial activities against the surrogate Xa as part of an initial in vitro screening. Of note, Xa and Xf show two different growths (in time and shape) on petri dishes and their antibiograms resulting from the AMP challenge are somehow incomparable. However, the results showed that all peptides were active against Xa, with MICs ranging from 6 μM to 50 μM ( Table 2). This result provides an information about which concentrations and AMPs can be utilized against Xf. In addition, all AMPs were able to reduce the growth of Xf at varying degrees at 50, 25, and 12 μM. Three AMPs (GF19, FF13, and IL14) were particularly highly active and were able to strongly curtail Xf growth. FF13 and IL14 showed high antimicrobial activity, causing very large inhibition zones (Figure 1), with mean growth zones of 15.6 and 13.7 mm, respectively. GF19 exhibited distinct inhibitory activity, whereas KL29, which is currently considered to be the most potent peptide against Xf, showed a clearing zone of 11.8 mm, representing lower activity than the three AMPs newly tested here.

Optical Density of AMP-Treated Xylella fastidiosa Cells
The dose-effect reactions of Xf to the various AMPs were evaluated over 24 h of incubation. In general, all AMPs at different concentrations (12,25, and 50 µM) showed reductive activity against Xf cells within the first 30 min of contact, and were still particularly highly functional up to 3 h post contact ( Figure 2). Furthermore, this reductive activity persisted for 24 h post contact, until the total exhaustion of Xf cells, where GF19, FF13, and IL14 each showed the greatest activity at 50 µM. At 24 h of incubation and at 12 µM or higher concentration, all peptides showed significant cell reduction activity (up to 99% of reduction) (Figure 2). ductive activity against Xf cells within the first 30 min of contact, and were still particularly highly functional up to 3 h post contact ( Figure 2). Furthermore, this reductive activity persisted for 24 h post contact, until the total exhaustion of Xf cells, where GF19, FF13, and IL14 each showed the greatest activity at 50 μM. At 24 h of incubation and at 12 µ M or higher concentration, all peptides showed significant cell reduction activity (up to 99% of reduction) (Figure 2).

Effect of AMPs on Xylella fastidiosa: V-qPCR, Fluorescence, and Transmission Electron Microscopy
The reduction in viability of AMP-treated Xf was evaluated at 12, 25, and 50 µ M. The PMAxx™ assay, used for testing the viability of bacteria and consequently only intercepts dead bacteria with compromised cell membranes, showed that GF19 was highly active at 25 and 50 µ M concentrations, leading to a more-than-fourfold reduction in viability compared to untreated Xf cells (Figure 3).

Effect of AMPs on Xylella fastidiosa: V-qPCR, Fluorescence, and Transmission Electron Microscopy
The reduction in viability of AMP-treated Xf was evaluated at 12, 25, and 50 µM. The PMAxx™ assay, used for testing the viability of bacteria and consequently only intercepts dead bacteria with compromised cell membranes, showed that GF19 was highly active at 25 and 50 µM concentrations, leading to a more-than-fourfold reduction in viability compared to untreated Xf cells (Figure 3).  In addition, FF13, IL14, and KL29 exhibited high activity, with a twofold reduction in cell viability. The AMPs' activity against Xf cells was also examined under FM using two distinct dyes able to distinguish between live cells with intact plasma membranes (green channel) and dead bacteria with membranes compromised by the peptide activity (red channel). The examined micrographs of all tested peptides showed that GF19 and KL29 had the highest lytic activities, inducing intense red channels indicating the huge number of dead cells. However, the IL14 and FF13 micrographs showed a discreet green fluorescence, highlighting a moderate lytic activity of these peptides against Xf (Figure 4).
Under EM, micrographs of all AMP-treated Xf cells showed different morphological  In addition, FF13, IL14, and KL29 exhibited high activity, with a twofold reduction in cell viability. The AMPs' activity against Xf cells was also examined under FM using two distinct dyes able to distinguish between live cells with intact plasma membranes (green channel) and dead bacteria with membranes compromised by the peptide activity (red channel). The examined micrographs of all tested peptides showed that GF19 and KL29 had the highest lytic activities, inducing intense red channels indicating the huge number of dead cells. However, the IL14 and FF13 micrographs showed a discreet green fluorescence, highlighting a moderate lytic activity of these peptides against Xf (Figure 4).  Under EM, micrographs of all AMP-treated Xf cells showed different morphological changes at the structural and cell wall membrane levels. GF19-treated Xf cells had damaged and fragmented cell walls, with complete destruction of the outer and inner bacterial membranes ( Figure 5). IL14-and FF13-treated Xf cells showed cytoplasmic condensation, loss of interior appearance, and alteration of the outer membrane. KL29-and NAC (a bactericidal compound used as a control)-treated cells showed pore and protrusion formation and condensed cytoplasmic outflow from the outer membrane ( Figure 5).

Effects of AMPs against Xylella fastidiosa Biofilm Formation
Xf biofilm formation was evaluated upon exposure to 50 μM of GF19, FF13, IL14, reference peptide KL29, and NAC. The results show that all tested peptides were able to reduce biofilm formation by 40% to 50% compared to the NTC ( Figure 6). GF19 interfered with biofilm formation in the same manner as NAC. GF19, FF13, and IL14 each showed greater performance than KL29, which is notoriously known to have high antagonistic activity against Xf biofilm formation. Furthermore, these peptides conditioned the ratios of planktonic cells within a range of 13.8% ( Figure 6).

Evaluation of AMPs' Toxicity on Eukaryotic Cells
The toxicity of AMPs was explored on model cells of animal (horse erythrocytes) and plant (tobacco leaves), for possible future application of these AMPs as eco-friendly candidates in the control of Xf. The reason for using such a heterologous system (horse erythrocytes) was to investigate whether these AMPs can differentiate between animal and plant cells regarding their possible toxicities, and to ensure that they do not pose any danger to users. At 25 and 50 μM, all AMPs induced neither a hypersensitivity reaction in the tobacco leaves nor morphological abnormalities in the cellular structures of the erythro-

Effects of AMPs against Xylella fastidiosa Biofilm Formation
Xf biofilm formation was evaluated upon exposure to 50 µM of GF19, FF13, IL14, reference peptide KL29, and NAC. The results show that all tested peptides were able to reduce biofilm formation by 40% to 50% compared to the NTC ( Figure 6). GF19 interfered with biofilm formation in the same manner as NAC. GF19, FF13, and IL14 each showed greater performance than KL29, which is notoriously known to have high antagonistic activity against Xf biofilm formation. Furthermore, these peptides conditioned the ratios of planktonic cells within a range of 13.8% ( Figure 6).

Effects of AMPs against Xylella fastidiosa Biofilm Formation
Xf biofilm formation was evaluated upon exposure to 50 μM of GF19, FF13, IL14 reference peptide KL29, and NAC. The results show that all tested peptides were able t reduce biofilm formation by 40% to 50% compared to the NTC ( Figure 6). GF19 interfere with biofilm formation in the same manner as NAC. GF19, FF13, and IL14 each showe greater performance than KL29, which is notoriously known to have high antagonisti activity against Xf biofilm formation. Furthermore, these peptides conditioned the ratio of planktonic cells within a range of 13.8% ( Figure 6).

Evaluation of AMPs' Toxicity on Eukaryotic Cells
The toxicity of AMPs was explored on model cells of animal (horse erythrocytes) an plant (tobacco leaves), for possible future application of these AMPs as eco-friendly can didates in the control of Xf. The reason for using such a heterologous system (horse eryth

Evaluation of AMPs' Toxicity on Eukaryotic Cells
The toxicity of AMPs was explored on model cells of animal (horse erythrocytes) and plant (tobacco leaves), for possible future application of these AMPs as eco-friendly candidates in the control of Xf. The reason for using such a heterologous system (horse erythrocytes) was to investigate whether these AMPs can differentiate between animal and plant cells regarding their possible toxicities, and to ensure that they do not pose any danger to users. At 25 and 50 µM, all AMPs induced neither a hypersensitivity reaction in the tobacco leaves nor morphological abnormalities in the cellular structures of the erythrocytes (Figure 7). However, at 50 µM, GF19 generated a light hypersensitive reaction in tobacco leaves, i.e., halo-shaped tissue dissolving at 7 mm in diameter (data not shown). These findings were taken as prospective proof for their possible safe use; however, further assessments are needed to scrutinize their possible wide application in different hosts and environments. These findings were taken as prospective proof for their possible safe use; however, further assessments are needed to scrutinize their possible wide application in different hosts and environments.

AMPs' Efficiency in the Control of Xylella fastidiosa In Planta
To contrast the Xf disease infection in planta, the efficiency of the AMPs was evaluated in Xf-infected tobacco plants. At 30 dpi, the untreated Xf-infected tobacco plants showed marginal and apical scorched areas on leaves, whereas AMP-treated Xf-infected tobacco plants did not develop any symptoms, similarly to those treated with N-acetyl-Lcysteine, known for its bactericidal activity against Xf (Figure 8). AMP-treated Xf-infected tobacco plants maintained a healthy appearance until 50 dpi. However, at 25 µ M and 45 dpi, KL29-treated Xf-infected tobacco plants developed light leaf scorch symptoms that were less intense than those in the untreated Xf-infected tobacco plants (Figure 8). These in planta results, i.e., presence and absence of Xf infection and symptoms, were confirmed by qPCR assays conducted on different leaves situated above the inoculation point ( Figure  9).

AMPs' Efficiency in the Control of Xylella fastidiosa In Planta
To contrast the Xf disease infection in planta, the efficiency of the AMPs was evaluated in Xf -infected tobacco plants. At 30 dpi, the untreated Xf -infected tobacco plants showed marginal and apical scorched areas on leaves, whereas AMP-treated Xf -infected tobacco plants did not develop any symptoms, similarly to those treated with N-acetyl-L-cysteine, known for its bactericidal activity against Xf (Figure 8). AMP-treated Xf -infected tobacco plants maintained a healthy appearance until 50 dpi. However, at 25 µM and 45 dpi, KL29-treated Xf -infected tobacco plants developed light leaf scorch symptoms that were less intense than those in the untreated Xf -infected tobacco plants (Figure 8). These in planta results, i.e., presence and absence of Xf infection and symptoms, were confirmed by qPCR assays conducted on different leaves situated above the inoculation point ( Figure 9).

Discussion and Conclusions
The extreme difficulties of managing Xf infections have led to the search for new bactericides able to treat the disease with an eco-friendly approach without compromising the contrasting efficiencies. Many active compounds including toxins, phenolic acids, antibiotics, and AMPs have been reported to be effective against numerous Xf subspecies with minimal bacteriostatic/bactericide concentrations [31][32][33]. Moreover, some of these AMPs have been successfully expressed in grapevines to control Xf in greenhouses [34][35][36]. Thus, AMPs are considered to be promising candidates for controlling this plant pathogen because of their antibacterial activity, low toxicity to the host plants, low propensity to incite bacteria-acquired resistance, and ability to be expressed in plants [37,38]. To search and explore whether there are more effective AMPs against Xf infection, this study

Discussion and Conclusions
The extreme difficulties of managing Xf infections have led to the search for new bactericides able to treat the disease with an eco-friendly approach without compromising the contrasting efficiencies. Many active compounds including toxins, phenolic acids, antibiotics, and AMPs have been reported to be effective against numerous Xf subspecies with minimal bacteriostatic/bactericide concentrations [31][32][33]. Moreover, some of these AMPs have been successfully expressed in grapevines to control Xf in greenhouses [34][35][36]. Thus, AMPs are considered to be promising candidates for controlling this plant pathogen because of their antibacterial activity, low toxicity to the host plants, low propensity to incite bacteria-acquired resistance, and ability to be expressed in plants [37,38]. To search and explore whether there are more effective AMPs against Xf infection, this study

Discussion and Conclusions
The extreme difficulties of managing Xf infections have led to the search for new bactericides able to treat the disease with an eco-friendly approach without compromising the contrasting efficiencies. Many active compounds including toxins, phenolic acids, antibiotics, and AMPs have been reported to be effective against numerous Xf subspecies with minimal bacteriostatic/bactericide concentrations [31][32][33]. Moreover, some of these AMPs have been successfully expressed in grapevines to control Xf in greenhouses [34][35][36]. Thus, AMPs are considered to be promising candidates for controlling this plant pathogen because of their antibacterial activity, low toxicity to the host plants, low propensity to incite bacteria-acquired resistance, and ability to be expressed in plants [37,38]. To search and explore whether there are more effective AMPs against Xf infection, this study has examined for the first time the efficiency of a set of nine potential peptides reported in the literature for their antimicrobial activity against Gram-negative and Gram-positive bacteria with biofilm activity [24,39], contrasting Xf in planta. All in vitro assays (antibiogram, OD, v-qPCR, biofilm inhibition, FM, and TEM) showed the high antimicrobial activity of the selected AMPs, among which GF19, FF13, and IL14 were found to be the most active at different concentrations. Most importantly, the selected AMPs showed supremacy in contrasting Xf infection in planta, demonstrating their prospects of being future antagonistic compounds for Xf. In addition, they had an innocuous impact on the eukaryotic plant and animal cells tested in this study, a finding that could trigger concrete AMP field experimental trials on Xf -infected trees.
Digging deeper into the efficiency of these AMPs, this study was able to distinguish between the biostatic and biocidal activities of the selected AMPs and their perfect ability to contrast biofilm formation. Furthermore, the efficiency of the selected AMPs against Xf at low concentrations was promising, representing an advantageous trait for their commercial production and application. N-acetylcysteine is a thiol compound that has been used as an antibacterial agent and inhibitor of biofilm formation which affects Xf strain 9a5c adhesion to glass surfaces, consequently reducing the biofilm biomass at 0, 2.0, and 6.0 mg/mL [18]; at 1 mg/mL, it was biocidal against Xf [18].
In general, several drawbacks limit the development of naturally occurring AMPs into useful phytopathogenic control agents [40], including stability issues, where the proteolytic degradation and the potential interaction of AMPs with enzymes might result in decreased antimicrobial activity [40]. Nanoparticle encapsulation systems have been extensively developed to enhance the bioavailability AMPs (such as natural polymerics [41,42], solid lipids [43], synthetic polymerics [44], liposomes [45], and inorganic nanoparticles [46]) in order to solve the limit of application of AMPs in plants.
High manufacturing costs are also another problem to be overcome. Researchers have been developing mimics or peptidomimetics with improved properties while maintaining the basic properties of membrane-active natural AMPs, for example, with amphipathic design and cationic charge. Multimeric (dendrimeric) peptides, protein epitope mimetics, lipidated peptides (synthetic), peptoids, oligoacyllysines, ceragenins, and other foldamers are some of the other approaches developed so far [47].
In this study, new bactericidal peptides, along with KL29 as previously described, have been identified, and their potential regarding new treatments for plant diseases caused by Xylella fastidiosa is realistic. Furthermore, these AMPs could be suitable candidates for environmentally and biologically friendly control measures in tandem with the preventive measures currently applied, contributing to greener agriculture, in view of European Union rules for quarantine organisms, especially for Xf. Furthermore, their ability to be expressed in plants makes them potentially useful in technologies through which transgenic plants can produce peptides to kill pathogens [48,49]. These AMPs need to be studied further in the future, covering a variety of combinations (e.g., mixtures of AMPs and combinations of AMPs and other antimicrobial agents). The effects of peptides on hormonal response, in gene expression in the preferred hosts of Xf, and in the field (whether alone or combined) should also be studied.