Bactericidal and plant defense elicitation activities of Eucalyptus oil decrease the severity of infections by Xylella fastidiosa on almond plants.

The activity of Eucalyptus essential oil against eleven strains pertaining to six species of plant pathogenic bacteria was studied using growth inhibition and contact assays. All strains were susceptible to the formulation EGL2, and Xylella fastidiosa subspecies and Xanthomonas fragariae were the most sensitive. The bactericidal effect was strong causing 4.5 to 6.0 log reductions in survival in 30 min at concentrations in the range of 0.75 to 15.0 µl/ml depending on the bacteria tested. Transmission electron microscopy of the formulation EGL2 against the three X. fastidiosa subspecies studied allowed the observation of a strong lytic effect on bacterial cells. In addition, the preventive spray application of EGL2 to potted pear plants subsequently inoculated with Erwinia amylovora significantly decreased the severity of infections. Almond plants treated by endotherapy or soil drenching, and then inoculated with X. fastidiosa showed a significant decrease in disease severity as well as in the levels of the pathogen, depending on the strategy used (endotherapy/soil drenching, preventive/curative). The treatment by endotherapy in almond plants induced the expression of several genes involved in plant defense. It was concluded that the reduction of infections by the Eucalyptus oil treatments was due to the combination of its bactericidal and plant defense induction activities.

Nicotiana benthamiana as a model plant host for Xylella fastidiosa: Control of infections by transient expression and endotherapy with a bifunctional peptide.

Transient expression of genes encoding peptides BP134 and BP178 by means of a Potato virus X (PVX) based-vector system, and treatment with synthetic peptides by endotherapy, were evaluated in the control of Xylella fastidiosa infections, in the model plant Nicotiana benthamiana. Transient production of BP178 significantly decreased disease severity compared to PVX and non-treated control (NTC) plants, without adverse effects. Plants treated with synthetic BP134 and BP178 showed consistently lower levels of disease than NTC plants. However, the coinfection with PVX-BP134 and X. fastidiosa caused detrimental effects resulting in plant death. The levels of X. fastidiosa in three zones sampled, upwards and downwards of the inoculation/treatment point, significantly decreased compared to the NTC plants, after the treatment with BP178, but not when BP178 was produced transiently. The effect of treatment and transient production of BP178 in the induction of defense-related genes was also studied. Synthetic BP178 applied by endotherapy induced the expression of ERF1, PR1a, PAL, PALII and WRKY25, while the transient expression of BP178 overexpressed the Cath, Cyc, PR4a, 9-LOX and Endochitinase B genes. Both treatments upregulated the expression of PR1, PR3, PR4 and CycT9299 genes compared to the NTC or PVX plants. It was concluded that the effect of BP178, either by endotherapy or by transient expression, on the control of the X. fastidiosa infections in N. benthamiana, was due in part to the induction of the plant defense system in addition to its bactericidal activity reported in previous studies. However, the protection observed when BP178 was transiently produced seems mainly mediated by the induction of plant defense, because the levels of X. fastidiosa were not significantly affected.

Induction of Defense Responses and Protection of Almond Plants Against Xylella fastidiosa by Endotherapy with a Bifunctional Peptide.

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.

A Bifunctional Peptide Conjugate That Controls Infections of Erwinia amylovora in Pear Plants.

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.

Aggressiveness of Spanish Isolates of Xylella fastidiosa to Almond Plants of Different Cultivars Under Greenhouse Conditions.

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.

Screening and identification of BP100 peptide conjugates active against Xylella fastidiosa using a viability-qPCR method.

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.

Differential Susceptibility of Xylella fastidiosa Strains to Synthetic Bactericidal Peptides.

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 (<10 µM), resulting in pore formation in cell membranes, abundant production of outer membrane vesicles, and lysis. A threshold ratio of 109 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.

Antimicrobial peptide KSL-W and analogues: Promising agents to control plant diseases.

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.

Antimicrobial activity of linear lipopeptides derived from BP100 towards plant pathogens.

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.