Identification and Characterization of Erwinia Phage IT22: A New Bacteriophage-Based Biocontrol against Erwinia amylovora.

Erwinia amylovora is a quarantine phytopathogenic bacterium that is the causal agent of fire blight, a destructive disease responsible for killing millions of fruit-bearing plants worldwide, including apple, pear, quince, and raspberry. Efficient and sustainable control strategies for this serious bacterial disease are still lacking, and traditional methods are limited to the use of antibiotics and some basic agricultural practices. This study aimed to contribute to the development of a sustainable control strategy through the identification, characterization, and application of bacteriophages (phages) able to control fire blight on pears. Phages isolated from wastewater collected in the Apulia region (southern Italy) were characterized and evaluated as antibacterial agents to treat experimental fire blight caused by E. amylovora. Transmission electron microscopy (TEM) conducted on purified phages (named EP-IT22 for Erwinia phage IT22) showed particles with icosahedral heads of ca. 90 ± 5 nm in length and long contractile tails of 100 ± 10 nm, typical of the Myoviridae family. Whole genome sequencing (WGS), assembly, and analysis of the phage DNA generated a single contig of 174.346 bp representing a complete circular genome composed of 310 open reading frames (ORFs). EP-IT22 was found to be 98.48% identical to the Straboviridae Erwinia phage Cronus (EPC) (GenBank Acc. n° NC_055743) at the nucleotide level. EP-IT22 was found to be resistant to high temperatures (up to 60 °C) and pH values between 4 and 11, and was able to accomplish a complete lytic cycle within one hour. Furthermore, the viability-qPCR and turbidity assays showed that EP-IT22 (MOI = 1) lysed 94% of E. amylovora cells in 20 h. The antibacterial activity of EP-IT22 in planta was evaluated in E. amylovora-inoculated pear plants that remained asymptomatic 40 days post inoculation, similarly to those treated with streptomycin sulphate. This is the first description of the morphological, biological, and molecular features of EP-IT22, highlighting its promising potential for biocontrol of E. amylovora against fire blight disease.

Exploring Active Peptides with Antimicrobial Activity In Planta against Xylella fastidiosa.

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.

Exploring Antimicrobial Peptides Efficacy against Fire Blight (Erwinia amylovora).

Antimicrobial peptides (AMPs) are a various group of molecules found in a wide range of organisms and act as a defense mechanism against different kinds of infectious pathogens (bacteria, viruses, and fungi, etc.). This study explored the antibacterial activity of nine candidates reported in the literature for their effect on human and animal bacteria, (i.e., Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa) against Erwinia amylovora (E. amylovora), the causal agent of fire blight disease on pome fruits. The antibacterial activity of these peptides against E. amylovora was evaluated in vitro using viable-quantitative PCR (v-qPCR), fluorescence microscopy (FM), optical density (OD), and transmission electron microscopy (TEM), while the in vivo control efficacy was evaluated in treating experimental fire blight on pear fruits. With a view to their safe and ecofriendly field use in the future, the study also used animal and plant eukaryotic cells to evaluate the possible toxicity of these AMPs. Results in vitro showed that KL29 was the most potent peptide in inhibiting E. amylovora cell proliferation. In addition, the results of v-qPCR, FM, and TEM showed that KL29 has a bifunctional mechanism of action (lytic and non-lytic) when used at different concentrations against E. amylovora. KL29 reduced fire blight symptoms by 85% when applied experimentally in vivo. Furthermore, it had no impact on animal or plant cells, thus demonstrating its potential for safe use as an antibacterial agent. This study sheds light on a new and potent antibacterial peptide for E. amylovora and its modes of action, which could be exploited to develop sustainable treatments for fire blight.

Phytochemical Analysis and Study of Antioxidant, Anticandidal, and Antibacterial Activities of Teucrium polium subsp. polium and Micromeria graeca (Lamiaceae) Essential Oils from Northern Morocco.

The protection of agricultural crops and the preservation of the organoleptic and health qualities of food products represent a major challenge for the agricultural and agro-food industries. Essential oils have received greater attention as alternatives to replace the control strategies based on pesticides against phytopathogenic bacteria and synthetic compounds in food preservation. The aims of this work were to study the chemical composition of Teucrium polium subsp. polium and Micromeria graeca essential oils and to examine their antioxidant and antimicrobial effects. To carry out this work, the chemical composition of the essential oil was determined using gas chromatography (GC) with the detection feature of mass spectrometry (MS). Subsequently, the antioxidant activity was investigated by DPPH and FRAPS assays. The antimicrobial effect was studied against phytopathogenic and foodborne pathogenic bacteria using the disc and the microdilution methods. Our results showed that GC-MS analysis of EOs allowed the identification of 30 compounds in T. polium EO (TPpEO), while 5 compounds were identified in M. graeca EO (MGEO). TPpEO had as major compounds ß-pinene (19.82%) and germacrene D (18.33%), while geranial (36.93%) and z-citral (18.25%) were the main components of MGEO. The most potent activity was obtained from MGEO (IC50 = 189.7 ± 2.62 µg/mL) compared to TPpEO (IC50 = 208.33 ± 3.51 µg/mL. For the FRAP test, the highest reducing power was obtained from 1.32 ± 0.1 mg AAE/g of TPpEO compared to MGEO 0.51 ± 0.13 mg AAE/g of EO. Both EOs exhibited varying degrees of antibacterial activities against all the tested strains with inhibition zones in the range of 9.33 ± 0.57 mm to >65 mm and MIC values from 0.19 to 12.5 mg/mL. However, MGEO exhibits an interesting anticandidal effect with inhibition zone 44.33 ± 0.57 mm. The findings of this research establish the riches of EOs on volatile compounds, their important antioxidant activity, and their antimicrobial effect against the bacteria tested.

GC-MS Analysis, Antioxidant and Antimicrobial Activities of Achillea Odorata Subsp. Pectinata and Ruta Montana Essential Oils and Their Potential Use as Food Preservatives.

In order to discover new natural resources with biological properties, the chemical composition, the antioxidant and antimicrobial activities, and the potential use as food preservative of essential oils of Moroccan Achillea odorata subsp. pectinata (AOpEO) and Ruta montana (RMEO) were studied. Gas chromatography-mass spectrometry (GC-MS) analysis revealed the presence of 21 and 25 compounds in AOpEO and RMEO, respectively. The results showed that the major compounds of AOpEO are camphor (45.01%), bornyl acetate (15.07%), borneol (11.33%), ß-eudesmol (4.74%), camphene (3.58%), and 1.8-cineole (eucalyptol) (2.96%), whereas 2-undecanone (63.97%), camphor (3.82%) and cyclopropanecarboxylic acid (3.66%) were the main components of RMEO. The antioxidant activities were evaluated by diphenylpicrylhydraziyl radical (DPPH) and reducing power assays. The antimicrobial activities of essential oils were tested against bacterial strains and food contaminant yeast using agar disc diffusion and microdilution methods. A significant antimicrobial activity of AOpEO was observed against Bacillus subtilis, Proteus mirabilis and Candida albicans, compared to RMEO. The efficacy of AOpEO was also evaluated in model food systems (cabbage and barley) artificially inoculated during storage. The results found that the adding of a minimal inhibitory concentration (MIC) and 4× MIC were potent in decreasing the Proteus mirabilis growth in food model systems. Our findings suggested that AOpEO may be potentially used as an alternative food preservative.

Molecular identification, in vitro copper resistance and antibiotics susceptibility of the causal agent of the olive knot disease in Morocco

Aims@#This study aimed to i) identify Pseudomonas savastanoi pv. savastanoi (Pss) as a causal agent of the olive knot on the basis of biochemical, pathogenicity and PCR technique ii) investigate in vitro bacterial resistance toward copper-based compounds and efficiency of some antibiotics on pathogen suppression. @*Methodology and results@#Biochemical, pathogenicity and molecular identification based on alkaline method for the DNA extraction were performed to identify possible causal agent of the olive knot. Copper resistance for Pss strains was evaluated by inoculation of bacterial suspensions into YPG medium, containing the cupric sulfate at 0, 100, 250 and 500 ppm. The efficiency of eight antibiotics on Pss strain was evaluated at different concentrations. Fifty-nine isolates caused typical knots at the site of inoculation with bacterial suspensions. All isolates have been identified as Pss using specific primers. No resistance to copper was detected with concentration of 500 ppm. In contrast, copper resistance was found during 48 h with lower concentration (100 or 250 ppm). The maximal inhibition of Pss 2102-4M was observed with the highest concentration (20 μg/mL) of the Aureomycin, Streptomycin and Novobiocin with inhibition diameters of 30, 24 and 10 mm, respectively. Whereas, Colchicine, Bacitracin, Cephalex, Ampicillin and Cycloserine have no inhibitory effect on the Pss 2102-4M strain. @*Conclusion, significance and impact of study@#The alkaline method for the DNA extraction from pure culture was reliable and rapid and can be recommended for molecular detection the causal agent of the olive knot. This is the first report determined copper resistance levels of Moroccan strains of Pss and in vitro evaluated for the susceptibility towards the antibiotics.

Essential oils of Origanum compactum and Thymus vulgaris exert a protective effect against the phytopathogen Allorhizobium vitis.

Allorhizobium (Agrobacterium) vitis is a host-specific pathogenic bacterium that causes grapevine crown gall disease, affecting vine growth and production worldwide. The antibacterial activities of different aromatic plant essential oils were tested in vitro and in planta against A. vitis. Among the essential oils tested, those of Origanum compactum and Thymus vulgaris showed the most significant in vitro antibacterial activities, with a MIC of 0.156 and 0.312 mg/mL, respectively. A synergistic effect of these two essential oils (1:1) was observed and confirmed by the checkerboard test. Carvacrol (61.8%) and thymol (47.8%) are, respectively, the major compounds in the essential oils of O. compactum and T. vulgaris and they have been shown to be largely responsible for the antibacterial activities of their corresponding essential oils. Results obtained in vitro were reinforced by an in planta pathogenicity test. A mixture of O. compactum and T. vulgaris essential oils (1:1), inoculated into the injured stem of a tomato plant and a grapevine at 0.312 mg/mL as a preventive treatment, reduced both the number of plants developing gall symptoms and the size of the tumors.

Adaptation of Phytophthora infestans to Partial Resistance in Potato: Evidence from French and Moroccan Populations.

ABSTRACT The use of partially resistant cultivars should become an essential component of a sustainable management strategy of potato late blight, caused by Phytophthora infestans. It is therefore important to determine to what extent P. infestans populations can be selected for increased aggressiveness by potato cultivars with different levels of partial resistance. To this end, we sampled P. infestans populations from France and Morocco, chosen as locations where late blight occurs regularly but which differ in the distribution of potato cultivars. Cross-inoculation experiments were used to determine the aggressiveness of all populations to potato cvs. Bintje (prevalent in France but not grown in Morocco) and Désirée (popular in Morocco but cultivated to a very small extent in France). French populations were more aggressive on cv. Bintje than on cv. Désirée, irrespective of the site they were sampled from. Their aggressiveness increased between early and late samplings, suggesting that both cultivars selected for increased aggressiveness during epidemics. By contrast, Moroccan populations were more aggressive on Désirée, regarded as partially resistant in Europe, than on Bintje, highly susceptible under European conditions. These data indicate that P. infestans populations adapt to locally dominant cultivars, irrespective of their resistance levels, and can therefore overcome polygenic, partial resistance. This adaptive pattern may render partial resistance nondurable if not properly managed.