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Xylella fastidiosa is a xylem-limited plant pathogenic bacterium that is a menace to the agriculture worldwide threating economically relevant crops such as almond. The pathogen presents a dual lifestyle in the plant xylem, consisting of sessile microbial aggregates and mobile independent cells that move by twitching motility. The latter is essential for the systemic colonization of the host and is mediated through type IV pili. In previous reports, it has been demonstrated that peptides can affect different key processes of X. fastidiosa, but their effect on motility has never been assessed. In the present work, peptides previously identified and newly designed analogs were studied for its effect in vitro on the motility of X. fastidiosa and their protective effect against almond leaf scorch was determined. By assessing the twitching fringe width in colonies and using microfluidic chambers, the inhibitory effect of BP100 on twitching motility was demonstrated. Interestingly, type IV pili of BP100-treated cells were similar in frequency and length, and presented no morphological differences when compared to the non-treated control. The application of BP100 by endotherapy in almond plants inoculated with X. fastidiosa under greenhouse conditions significantly reduced population levels and showed less affected xylem vessels, which correlated with decreased disease symptoms. Therefore, BP100 is a promising candidate to manage almond leaf scorch caused by X. fastidiosa.

RESUMEN

KEY MESSAGE: New defense elicitor peptides have been identified which control Xylella fastidiosa infections in almond. Xylella fastidiosa is a plant pathogenic bacterium that has been introduced in the European Union (EU), threatening the agricultural economy of relevant Mediterranean crops such as almond (Prunus dulcis). Plant defense elicitor peptides would be promising to manage diseases such as almond leaf scorch, but their effect on the host has not been fully studied. In this work, the response of almond plants to the defense elicitor peptide flg22-NH2 was studied in depth using RNA-seq, confirming the activation of the salicylic acid and abscisic acid pathways. Marker genes related to the response triggered by flg22-NH2 were used to study the effect of the application strategy of the peptide on almond plants and to depict its time course. The application of flg22-NH2 by endotherapy triggered the highest number of upregulated genes, especially at 6 h after the treatment. A library of peptides that includes BP100-flg15, HpaG23, FV7, RIJK2, PIP-1, Pep13, BP16-Pep13, flg15-BP100 and BP16 triggered a stronger defense response in almond plants than flg22-NH2. The best candidate, FV7, when applied by endotherapy on almond plants inoculated with X. fastidiosa, significantly reduced levels of the pathogen and decreased disease symptoms. Therefore, these novel plant defense elicitors are suitable candidates to manage diseases caused by X. fastidiosa, in particular almond leaf scorch.

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Thirty peptide conjugates were designed by combining an antimicrobial peptide (BP16, BP100, BP143, KSL-W, BP387, or BP475) at the N- or C-terminus of a plant defense elicitor peptide (flg15, BP13, Pep13, or PIP1). These conjugates were highly active in vitro against six plant-pathogenic bacteria, especially against Xanthomonas arboricola pv. pruni, Xanthomonas fragariae and Xanthomonas axonopodis pv. vesicatoria. The most active peptides were those incorporating Pep13. The order of the conjugation influenced the antibacterial activity and the hemolysis. Regarding the former, peptide conjugates incorporating the elicitor peptide flg15 or Pep13 at the C-terminus were, in general, more active against Pseudomonas syringae pv. actinidiae and P. syringae pv. syringae, whereas those bearing these elicitor peptides at the N-terminus displayed higher activity against Erwinia. amylovora and the Xanthomonas species. The best peptide conjugates displayed MIC values between 0.8 and 12.5 µM against all the bacteria tested and also had low levels of hemolysis and low phytotoxicity. Analysis of the structural and physicochemical parameters revealed that a positive charge ranging from +5 to +7 and a moderate hydrophobic moment/amphipathic character is required for an optimal biological profile. Interestingly, flg15-BP475 exhibited a dual activity, causing the upregulation of the same genes as flg15 and reducing the severity of bacterial spot in tomato plants with a similar or even higher efficacy than copper oxychloride. Characterization by nuclear magnetic resonance (NMR) of the secondary structure of flg15-BP475 showed that residues 10 to 25 fold into an α-helix. This study establishes trends to design new bifunctional peptides useful against plant diseases caused by plant-pathogenic bacteria. IMPORTANCE The consequences of plant pathogens on crop production together with the lack of effective and environmentally friendly pesticides evidence the need of new agents to control plant diseases. Antimicrobial and plant defense elicitor peptides have emerged as good candidates to tackle this problem. This study focused on combining these two types of peptides into a single conjugate with the aim to potentiate the activity of the individual fragments. Differences in the biological activity of the resulting peptide conjugates were obtained depending on their charge, amphipathicity, and hydrophobicity, as well as on the order of the conjugation of the monomers. This work provided bifunctional peptide conjugates able to inhibit several plant-pathogenic bacteria, to stimulate plant defense responses, and to reduce the severity of bacterial spot in tomato plants. Thus, this study could serve as the basis for the development of new antibacterial/plant defense elicitor peptides to control bacterial plant pathogens.

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Xylella fastidiosa is a plant pathogenic bacterium that has been introduced in the European Union (EU), causing significant yield losses in economically important Mediterranean crops. Almond leaf scorch (ALS) is currently one of the most relevant diseases observed in Spain, and no cure has been found to be effective for this disease. In previous reports, the peptide BP178 has shown a strong bactericidal activity in vitro against X. fastidiosa and to other plant pathogens, and to trigger defense responses in tomato plants. In the present work, BP178 was applied by endotherapy to almond plants of cultivar Avijor using preventive and curative strategies. The capacity of BP178 to reduce the population levels of X. fastidiosa and to decrease disease symptoms and its persistence over time were demonstrated under greenhouse conditions. The most effective treatment consisted of a combination of preventive and curative applications, and the peptide was detected in the stem up to 60 days posttreatment. Priming plants with BP178 induced defense responses mainly through the salicylic acid pathway, but also overexpressed some genes of the jasmonic acid and ethylene pathways. It is concluded that the bifunctional peptide is a promising candidate to be further developed to manage ALS caused by X. fastidiosa.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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Xylella fastidiosa is a plant pathogen that was recently introduced in Europe and is causing havoc to its agriculture. This Gram-negative bacterium invades the host xylem, multiplies, and forms biofilm occluding the vessels and killing its host. In spite of the great research effort, there is no method that effectively prevents or cures hosts from infections. The main control strategies up to now are eradication, vector control, and pathogen-free plant material. Antimicrobial peptides have arisen as promising candidates to combat this bacterium due to their broad spectrum of activity and low environmental impact. In this work, peptides previously reported in the literature and newly designed analogs were studied for its bactericidal and antibiofilm activity against X. fastidiosa. Also, their hemolytic activity and effect on tobacco leaves when infiltrated were determined. To assess the activity of peptides, the strain IVIA 5387.2 with moderate growth, able to produce biofilm and susceptible to antimicrobial peptides, was selected among six representative strains found in the Mediterranean area (DD1, CFBP 8173, Temecula, IVIA 5387.2, IVIA 5770, and IVIA 5901.2). Two interesting groups of peptides were identified with bactericidal and/or antibiofilm activity and low-moderate toxicity. The peptides 1036 and RIJK2 with dual (bactericidal-antibiofilm) activity against the pathogen and moderate toxicity stand out as the best candidates to control X. fastidiosa diseases. Nevertheless, peptides with only antibiofilm activity and low toxicity are also promising agents as they could prevent the occlusion of xylem vessels caused by the pathogen. The present work contributes to provide novel compounds with antimicrobial and antibiofilm activity that could lead to the development of new treatments against diseases caused by X. fastidiosa.

RESUMEN

From a previous collection of lipopeptides derived from BP100, we selected 18 sequences in order to improve their biological profile. In particular, analogues containing a D-amino acid at position 4 were designed, prepared, and tested against plant pathogenic bacteria and fungi. The biological activity of these sequences was compared with that of the corresponding parent lipopeptides with all L-amino acids. In addition, the influence of the length of the hydrophobic chain on the biological activity was evaluated. Interestingly, the incorporation of a D-amino acid into lipopeptides bearing a butanoyl or a hexanoyl chain led to less hemolytic sequences and, in general, that were as active or more active than the corresponding all L-lipopeptides. The best lipopeptides were BP475 and BP485, both incorporating a D-Phe at position 4 and a butanoyl group, with MIC values between 0.8 and 6.2 µM, low hemolysis (0 and 24% at 250 µM, respectively), and low phytotoxicity. Characterization by NMR of the secondary structure of BP475 revealed that the D-Phe at position 4 disrupts the α-helix and that residues 6 to 10 are able to fold in an α-helix. This secondary structure would be responsible for the high antimicrobial activity and low hemolysis of this lipopeptide.

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