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Beyond Xylella, Integrated Management Strategies for Mitigating Xylella fastidiosa Impact in Europe (BeXyl) (Grant Agreement 101060593). Partner/Coordinador principal: Blanca B. Landa del Castillo, Investigadora Científica del Instituto de Agricultura Sostenible (IAS-CSIC)., 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., This work was supported by grants from Spain Ministerio de Ciencia e Innovación (MCIU)/AEI and EU FEDER (RTI2018-099410-B-C21, and FPU19/01434 to L. Moll) and from the European Commission XF-ACTORS (grant 727987) and BeXyL (grant 101060593). A. Bar o was recipient of a research grantfrom the Secretaria d’Universitats i Recerca, Departament d'Innovaci o, Universitatsi Empresa, Generalitat de Catalunya (grant 2018 FI B00334).

The aggressiveness of Spanish isolates of Xylella fastidiosa, representing different sequence types, were studied in almond plants of several cultivars by means of the dynamics of the population levels and symptoms, colonization and spread, and dose–effect relationships. Pathogen dynamics in almond plants under greenhouse conditions showed doubling times of 2.1 to 2.5 days during the exponential growth phase, with a maximum population size of about 35 days postinoculation (dpi). Differences in patterns in population dynamics were observed between sap and xylem tissue after the exponential growth, as population levels in the xylem tissue remained stable while viable cells in sap decreased. Population levels were higher in two upward zones than in the downward zone with respect to the inoculation area. The first symptoms were observed between 20 and 60 dpi, and disease severity increased over time at doubling times of 30 days, with a maximum observed at 120 dpi. Strains tested showed differences in population levels in the cultivars studied and were able to spread with different intensity from contaminated plant parts to new growing shoots after pruning. Two almond isolates showed different performance in dose–effect relationships when inoculated in cultivar Avijor. Whereas IVIA 5387.2 reached high population levels but showed high median effective dose (ED50) and minimal infective dose (MID) values, IVIA 5901.2 showed low population levels and low ED50 and MID values. This study has implications for the epidemiology of X. fastidiosa in almond crops, estimating doubling times of the pathogen in planta and of symptom development and showing differences in aggressiveness between strains.

In this paper, peptide conjugates were designed and synthesized by incorporating the antimicrobial undecapeptide BP16 at the C- or N-terminus of the plant defense elicitor peptide flg15, leading to BP358 and BP359, respectively. The evaluation of their in vitro activity against six plant pathogenic bacteria revealed that BP358 displayed MIC values between 1.6 and 12.5 μM, being more active than flg15, BP16, BP359, and an equimolar mixture of BP16 and flg15. Moreover, BP358 was neither hemolytic nor toxic to tobacco leaves. BP358 triggered the overexpression of 6 out of the 11 plant defense-related genes tested. Interestingly, BP358 inhibited Erwinia amylovora infections in pear plants, showing slightly higher efficacy than the mixture of BP16 and flg15, and both treatments were as effective as the antibiotic kasugamycin. Thus, the bifunctional peptide conjugate BP358 is a promising agent to control fire blight and possibly other plant bacterial diseases This research was funded by MINECO/FEDER, UE, grant number AGL2015-69876-C2-2-R, by the Universitat de Girona, grant number MPCUdG2016/038, and by MCIU/AEI/FEDER, UE, grant number RTI2018-099410-B-C22

The aggressiveness of Spanish isolates of

The kinetics of cell inactivation and the susceptibility of

The kinetics of cell inactivation and the susceptibility of Xylella fastidiosa subspecies fastidiosa, multiplex, and pauca to synthetic antimicrobial peptides from two libraries (CECMEL11 and CYCLO10) were studied. The bactericidal effect was dependent on the relative concentrations of peptide and bacterial cells, and was influenced by the diluent, either buffer or sap. The most bactericidal and lytic peptide was BP178, an enlarged derivative of the amphipathic cationic linear undecapeptide BP100. The maximum reduction in survivors after BP178 treatment occurred within the first 10 to 20 min of contact and at micromolar concentrations (9 molecules of peptide per bacterial cell was estimated to be necessary to initiate cell inactivation. There was a differential susceptibility to BP178 among strains, with DD1 being the most resistant and CFBP 8173 the most susceptible. Moreover, strains showed a proportion of cells under the viable but nonculturable state, which was highly variable among strains. These findings may have implications for managing the diseases caused by X. fastidiosa.

Recent strong restrictions on the use of pesticides has prompted the search for safer alternatives, being antimicrobial peptides promising candidates. Herein, with the aim of identifying new agents, 15 peptides reported as plant defense elicitors, promiscuous, multifunctional or antimicrobial were selected and tested against six plant pathogenic bacteria of economic importance. Within this set, KSL-W (KKVVFWVKFK-NH2) displayed high antibacterial activity against all the tested pathogens, low hemolysis and low phytotoxicity in tobacco leaves. This peptide was taken as a lead and 49 analogues were designed and synthesized, including N-terminal deletion sequences, peptides incorporating a d-amino acid and lipopeptides. The screening of these sequences revealed that a nine amino acid length was the minimum for activity. The presence of a d-amino acid significantly decreased the hemolysis and endowed KSL-W with the capacity to induce the expression of defense-related genes in tomato plants. The incorporation of an acyl chain led to sequences with high activity against Xanthomonas strains, low hemolysis and phytotoxicity. Therefore, this study demonstrates that KSL-W constitutes an excellent candidate as new agent to control plant diseases and can be considered as a lead to develop derivatives with multifunctional properties, including antimicrobial and plant defense elicitation C.C. was the recipient of a predoctoral fellowship from the MINECO of Spain. This work was financed by grants AGL2012-39880-C02-02, AGL2015-69876-C2-2-R (MINECO/FEDER, EU) and MPCUdG2016/038. The authors acknowledge the Serveis Tècnics de Recerca of the University of Girona for the mass spectrometry analysis

A collection of 36 lipopeptides were designed from the cecropin A-melittin hybrid peptide BP100 (H-Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-NH2) previously described with activity against phytopathogenic bacteria. These lipopeptides were synthesized on solid-phase and screened for their antimicrobial activity, toxicity and proteolytic stability. They incorporated a butanoyl, a hexanoyl or a lauroyl group at the N-terminus or at the side chain of a lysine residue placed at each position of the sequence. Their antimicrobial activity and hemolysis depended on the fatty acid length and its position. In particular, lipopeptides containing a butanoyl or a hexanoyl chain exhibited the best biological activity profile. In addition, we observed that the incorporation of the acyl group did not induce the overexpression of defense-related genes in tomato. Best lipopeptides were BP370, BP378, BP381, BP387 and BP389, which were highly active against all the pathogens tested (minimum inhibitory concentration of 0.8 to 12.5 μM), low hemolytic, low phytotoxic and significantly stable to protease degradation. This family of lipopeptides might be promising functional peptides useful for plant protection The work was supported by the following: Ministerio de Economía y Competitividad (ES); AGL2012-39880-C02-02; LM, EB, EM, LF, MP; Ministerio de Economía y Competitividad (ES); AGL2015-69876-C2-2-R; AO, LM, EB, EM, LF, MP; University of Girona (ES); MPCUDG2016/038; AO, LF, MP

Background Xylella fastidiosa is one of the most harmful bacterial plant pathogens worldwide, causing a variety of diseases, with huge economic impact to agriculture and environment. Although it has been extensively studied, there are no therapeutic solutions to suppress disease development in infected plants. In this context, antimicrobial peptides represent promising alternatives to traditional compounds due to their activity against a wide range of plant pathogens, their low cytotoxicity, their mode of action that make resistance more difficult and their availability for being expressed in plants. Results Peptide conjugates derived from the lead peptide BP100 and fragments of cecropin, magainin or melittin were selected and tested against the plant pathogenic bacteria X. fastidiosa. In order to screen the activity of these antimicrobials, and due to the fastidious nature of the pathogen, a methodology consisting of a contact test coupled with the viability-quantitative PCR (v-qPCR) method was developed. The nucleic acid-binding dye PEMAX was used to selectively quantify viable cells by v-qPCR. In addition, the primer set XF16S-3 amplifying a 279 bp fragment was selected as the most suitable for v-qPCR. The performance of the method was assessed by comparing v-qPCR viable cells estimation with conventional qPCR and plate counting. When cells were treated with peptide conjugates derived from BP100, the observed differences between methods suggested that, in addition to cell death due to the lytic effect of the peptides, there was an induction of the viable but non-culturable state in cells. Notably, a contact test coupled to v-qPCR allowed fast and accurate screening of antimicrobial peptides, and led to the identification of new peptide conjugates active against X. fastidiosa. Conclusions Antimicrobial peptides active against X. fastidiosa have been identified using an optimized methodology that quantifies viable cells without a cultivation stage, avoiding underestimation or false negative detection of the pathogen due to the viable but non-culturable state, and overestimation of the viable population observed using qPCR. These findings provide new alternative compounds for being tested in planta for the control of X. fastidiosa, and a methodology that enables the fast screening of a large amount of antimicrobials against this plant pathogenic bacterium.

Xylella fastidiosa (Xf) diseases have emerged in recent years as several outbreaks of considerable intensity and extension in Europe. Their management is currently based on the use of pathogen-free propagation plant material, quarantine and eradication measures, and vector control. However, no cure of the disease exists up to now. Antimicrobial peptides have been identified as potential candidates for plant disease control, and have demonstrated a strong potential for the control of Xf. Specifically, it has been reported that the BP100 derivative peptide, BP178, synthetized for being expressed in plants, show high in vitro antibacterial activity against a collection of Xf strains. Although the application of bactericidal compounds to control xylem and phloem limited bacterial diseases has been tested by spray or trunk injection, the production of transgenic plant lines expressing antimicrobial peptides has been reported as promising alternative in the control of Xf. We have developed a transient-expression system based on Potato virus X (PVX) in Nicotiana benthamiana plants, for the production of the BP178 peptide in plants and evaluation of their potential in controlling Xf. The BP178 sequence was engineered in a PVX viral vector to be expressed in fusion with the NH2 Coat protein terminus. The release of the peptide occurs by the proteolytic activity of a Foot and Mouth Disease Virus 2A sequence. Western blot analysis showed that the BP178-CP fused proteins were expressed in N. benthamiana plants infected with the recombinant PVX-BP178-CP. Co-inoculations of N. benthamiana plants with PVX-BP178-CP and Xff strain IVIA 5387.2 are ongoing with initial results showing efficacy in controlling the pathogen over time., ES; PPT; laura.montesinos@udg.edu