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.

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 nonculturable 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.

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 20 selected and tested against six plant pathogenic bacteria of economic importance. Within this set, KSL-W (KKVVFWVKFK-NH 2 ) 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 Damino acid and lipopeptides. The screening of these sequences revealed that a nine amino acid length 25 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 30 derivatives with multifunctional properties, including antimicrobial and plant defense elicitation. Keywords35 D-Amino acids; Lipopeptides; Plant pathogens; Plant defense elicitors have identified linear undecapeptides, cyclic decapeptides and triazolyl or acylated derivatives with 55high antibacterial activity, low hemolysis and phytotoxicity, and reasonable susceptibility to protease degradation [19][20][21][22]. Moreover, we have recently investigated a new strategy for plant disease management based on the use of peptides as plant defense elicitors. Interestingly, we have found sequences from the aforementioned families that are able to induce defense responses on tobacco cells and tomato plants, and that efficiently control fire blight infections on pear [23]. 60 4 The finding of new antimicrobial peptides active in vivo against plant pathogens demands the accessibility to a wide range of sequences with high activity in vitro. Realizing this need, we selected sequences described in the literature as plant defense elicitors, promiscuous, multifunctional or antimicrobial to be further tested against our target plant pathogens.Among peptide elicitors, we chose PIP-1 (YGIHTH-NH 2 ), identified through combinatorial 65 chemistry, and Pep-13 (VWNQPVRGFKVYE-OH), a pathogen-associated molecular pattern (PAMP) from Phytophthora sojae, which trigger multiple defense responses in plant cells [24][25][26][27][28]. The promiscuous peptides studied were the AMPs Cn-AMP1 (SVAGRAQGM-NH 2 ), Cn-AMP2 (TESYFVFSVGM-NH 2 ) and Cn-AMP3 (YCSYTMEA-NH 2 ) identified from green coconut (Cocos nucifera) water [29,30]. 70Regarding multifunctional peptides, we considered QKALNEINQF-NH 2 (p10) and TKKTKLTEEEKNRL-NH 2 (p14) which were isolated from bovine...

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.

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 cv. 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 post-treatment. 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.