Plant Pathogenic Bacteria Erwinia Amylovora Research Articles

First European Erwinia amylovora Lytic Bacteriophage Cocktails Effective in the Host: Characterization and Prospects for Fire Blight Biocontrol.

Fire blight, caused by the plant-pathogenic bacterium Erwinia amylovora, is a highly contagious and difficult-to-control disease due to its efficient dissemination and survival and the scarcity of effective control methods. Copper and antibiotics are the most used treatments but pose environmental and human health risks. Bacteriophages (phages) constitute an ecological, safe, and sustainable fire blight control alternative. The goal of this study was to search for specific E. amylovora phages from plant material, soil, and water samples in Mediterranean environments. A collection of phages able to specifically infect and lyse E. amylovora strains was generated from former fire blight-affected orchards in Eastern Spain. Following in vitro characterization, assays in immature fruit revealed that preventively applying some of the phages or their combinations delayed the onset of fire blight symptoms and reduced the disease's severity, suggesting their biocontrol potential in Spain and other countries. The morphological and molecular characterization of the selected E. amylovora phages classified them as members of the class Caudoviricetes (former Myoviridae family) and genus Kolesnikvirus. This study reveals Mediterranean settings as plausible sources of E. amylovora-specific bacteriophages and provides the first effective European phage cocktails in plant material for the development of sustainable fire blight management measures.

Biological Activity of Ag and Cu Monometallic Nanoparticles and Ag‐Cu Bimetallic Nanocomposites against Plant Pathogens and Seeds

The antibacterial activities of silver (Ag) and copper (Cu) monometallic and bimetallic nanoparticles (MNPs and BNPs) against the most economically important and common plant bacterial diseases in Kyrgyzstan were tested, as well as their phytotoxic effects on wheat and corn seeds. To achieve this, a range of Ag and Cu MNPs and BNPs were synthesized using different methods and were tested at different concentrations to compare their biological effects. The antibacterial activities of Ag and Cu MNPs and Ag‐Cu BNPs against the plant pathogenic bacteria Erwinia amylovora, Pseudomonas syringae, and Pectobacterium carotovorum were evaluated. Of the three pathogens, Ps. syringae was found to be the most sensitive to NPs. The antimicrobial activities of the tested NPs showed significant positive correlations with the concentration; hence, there was a dose‐dependent effect. Due to their antibacterial properties, these Ag‐Cu nanocomposites have high potential in plant protection applications.

Survey of apple collar and rootstock blight in Serbia

Apple production in Serbia is characterized by the introduction of new technologies and the establishment of intensive plantations based on modern systems. In intensive apple production systems, sudden and rapid decay of young apple trees exhibiting collar and rootstock blight symptoms in the first few years after planting is becoming a prevalent issue. However, knip or nursery trees with lateral branches (feathers) on rootstock M.9 i.e. oneto five-year old are most seriously affected. Based on a longitudinal survey of young apple plantations in Serbia and the results of pathogen detection and identification using conventional and molecular methods, the plant pathogenic bacterium Erwinia amylovora was found as the causative agent. By increasing the incidence of collar and rootstock blight in young apple trees, E. amylovora is gaining importance as the most destructive disease in apple production. In order to control E. amylovora effectively and apply appropriate control measures, it is necessary to survey the development of lesser-known symptoms caused by this pathogen, which are becoming prevalent in Serbian agroecological conditions.

Identification, genetic characterization and virulence of Serbian Erwinia amylovora isolates

Fire blight disease, caused by plant pathogenic bacteria Erwinia amylovora represents a global threat for pome fruit industry, affecting yield and causing serious economic damage. During six year period (2011–2016), a total of 45 isolates typical for E. amylovora were isolated from four different host plants (apple, pear, quince and medlar) in 21 localities within four Serbian regions (Southeast, Central, West, and North). Isolates were primarily identified using biochemical and serological tests, polymerase chain reaction (PCR) with species-specific primer pair (A/B) and their pathogenicity. Polyclonal antibodies used in DAS ELISA and IF were found to be specific and sensitive enough for rapid E. amylovora detection. In virulence test performed on immature pear fruitlets, higher variability (CV) was found between regions than among hosts from which isolates originated. Slighter virulence of isolates originated from North region of Serbia (Vojvodina) was found in comparison to the isolates from Central Serbia, which were the most variable. Principal components analysis (PCA) segregated quince as a host among the most prevalent isolates in group of highly virulent, but failed to identify difference related to geographic origin of Serbian isolates. The same results enabled multivariate statistical analysis. Different DNA fingerprinting techniques such as repetitive element sequence based PCR (rep-) with BOX, ERIC, REP, GTG5 and SERE primers, then Random Amplified Polymorphic DNA (RAPD) with M13-PCR primer revealed genetic homogeneity among all Serbian E. amylovora isolates. ERIC, SERE and M13 primers generated the most complex and visually perceptible patters. Restriction fragment length polymorphism analysis (RFLP) of gyrA and recA genes, digested with three restriction endonucleases BstUI, BgIII and BsmI and rpoS gene digested with BstUI gave the unuque, characteristic patterns for all Serbian isolates.

Isolation and characterization of Hena1 - a novel Erwinia amylovora bacteriophage.

Fire blight, caused by plant pathogenic bacterium Erwinia amylovora, is one of the most important diseases of Rosaceae plants. Due to the lack of effective control measures, fire blight infections pose a recurrent threat on agricultural production worldwide. Recently, bacterial viruses, or bacteriophages, have been proposed as environmentally friendly natural antimicrobial agents for fire blight control. Here, we isolated a novel bacteriophage Hena1 with activity against E. amylovora. Further analysis revealed that Hena1 is a narrow-host-range lytic phage belonging to Myoviridae family. Its genome consists of a linear 148,842bp dsDNA (48.42% GC content) encoding 240 ORFs and 23 tRNA genes. Based on virion structure and genomic composition, Hena1 was classified as a new species of bacteriophage subfamily Vequintavirinae. The comprehensive analysis of Hena1 genome may provide further insights into evolution of bacteriophages infecting plant pathogenic bacteria.

Setting-up a fast and reliable cytokinin biosensor based on a plant histidine kinase receptor expressed in Saccharomyces cerevisiae

Cytokinins (CK) have been extensively studied for their roles in plant development. Recently, they also appeared to ensure crucial functions in the pathogenicity of some bacterial and fungal plant pathogens. Thus, identifying cytokinin-producing pathogens is a prerequisite to gain a better understanding of their role in pathogenicity. Taking advantage of the cytokinin perception properties of Malus domestica CHASE Histidine Kinase receptor 2 (MdCHK2), we thereby developed a selective and highly sensitive yeast biosensor for the application of cytokinin detection in bacterial samples. The biosensor is based on the mutated sln1Δ Saccharomyces cerevisiae strain expressing MdCHK2. The biosensor does not require any extraction or purification steps of biological samples, enabling cytokinin analysis directly from crude bacterial supernatants. For the first time, the production of cytokinin was shown in the well-known plant pathogenic bacteria Erwinia amylovora and was also revealed in human pathogens Staphylococcus aureus and Streptococcus agalactiae. Importantly, this biosensor was shown to be an efficient tool for unraveling certain steps in cytokinin biosynthesis by micro-organisms since this it was successfully used to unveil the role of ygdH22, a LOG-like gene, that is probably involved in cytokinin biosynthesis pathway in Escherichia coli. Overall, we demonstrated that our biosensor displays several advantages including time- and cost-effectiveness by allowing a rapid and specific detection of cytokinins in bacterial supernatants These results also support its scalability to high-throughput formats.

Novel mechanism of metabolic co-regulation coordinates the biosynthesis of secondary metabolites in Pseudomonas protegens.

Metabolic co-regulation between biosynthetic pathways for secondary metabolites is common in microbes and can play an important role in microbial interactions. Here, we describe a novel mechanism of metabolic co-regulation in which an intermediate in one pathway is converted into signals that activate a second pathway. Our study focused on the co-regulation of 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin, two antimicrobial metabolites produced by the soil bacterium Pseudomonas protegens. We show that an intermediate in DAPG biosynthesis, phloroglucinol, is transformed by a halogenase encoded in the pyoluteorin gene cluster into mono- and di-chlorinated phloroglucinols. The chlorinated phloroglucinols function as intra- and inter-cellular signals that induce the expression of pyoluteorin biosynthetic genes, pyoluteorin production, and pyoluteorin-mediated inhibition of the plant-pathogenic bacterium Erwinia amylovora. This metabolic co-regulation provides a strategy for P. protegens to optimize the deployment of secondary metabolites with distinct roles in cooperative and competitive microbial interactions.

Development and evaluation of a real-time PCR assay targeting chromosomal DNA of Erwinia amylovora

To develop and evaluate a new and reliable real-time PCR detection protocol on chromosomal DNA of the contagious plant pathogenic bacterium Erwinia amylovora, the causal agent of fire blight. A Taqman minor-groove-binder real-time PCR assay targeting a hypothetical protein coding gene of Erw. amylovora has been developed. Colony PCR of 113 bacterial strains from different taxa was performed to prove specificity. Serial decimal dilutions of Erw. amylovora showed a consistent detection sensitivity of 2 bacterial units per microl. All strains of Erw. amylovora could be identified, and there were no cross-reactions with matrices or other bacteria also testing naturally contaminated samples. Rapid, reliable and sensitive detection of Erw. amylovora is important to avoid the spread of the disease within orchards, and the distribution by contaminated plant material or vectors carrying the pathogen. The selected conserved target gene allows relative quantitative detection of Erw. amylovora from different sources and host taxa. The newly developed protocol also enables the detection of recently found natural strains that lack the species-specific plasmid pEA29, which was so far widely used as target for detection and identification of this plant pathogen by PCR. This study demonstrates that the newly developed and evaluated real-time assay can specifically be used for identifying all known strains of the EU quarantine plant pathogen Erw. amylovora. Low concentrations of the bacteria can be detected and relatively quantified using a different target area than other real-time PCRs designed so far.

Improvement of cyclic decapeptides against plant pathogenic bacteria using a combinatorial chemistry approach

Cyclic decapeptides were developed based on the previously reported peptide c(LysLeuLysLeuLysPheLysLeuLysGln). These compounds were active against the economically important plant pathogenic bacteria Erwinia amylovora, Pseudomonas syringae and Xanthomonas vesicatoria. A library of 56 cyclic decapeptides was prepared and screened for antibacterial activity and eukaryotic cytotoxicity, and led to the identification of peptides with improved minimum inhibitory concentration (MIC) against P. syringae (3.1–6.2 μM) and X. vesicatoria (1.6–3.1 μM). Notably, peptides active against E. amylovora (MIC of 12.5–25 μM) were found, constituting the first report of cyclic peptides with activity towards this bacteria. A second library based on the structure c(X 1X 2X 3X 4LysPheLysLysLeuGln) with X being Lys or Leu yielded peptides with optimized activity profiles. The activity against E. amylovora was further improved (MIC of 6.2–12.5 μM) and the best peptides displayed a low eukaryotic cytotoxicity at concentrations 30–120 times higher than the MIC values. A design of experiments permitted to define rules for high antibacterial activity and low cytotoxicity, being the main rule X 2 ≠ X 3, and the secondary rule X 4 = Lys. The best analogs can be considered as good candidates for the development of effective antibacterial agents for use in plant protection.

Inhibition of plant-pathogenic bacteria by short synthetic cecropin A-melittin hybrid peptides.

Short peptides of 11 residues were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Pseudomonas syringae, and Xanthomonas vesicatoria and compared to the previously described peptide Pep3 (WKLFKKILKVL-NH(2)). The antimicrobial activity of Pep3 and 22 analogues was evaluated in terms of the MIC and the 50% effective dose (ED(50)) for growth. Peptide cytotoxicity against human red blood cells and peptide stability toward protease degradation were also determined. Pep3 and several analogues inhibited growth of the three pathogens and had a bactericidal effect at low micromolar concentrations (ED(50) of 1.3 to 7.3 microM). One of the analogues consisting of a replacement of both Trp and Val with Lys and Phe, respectively, resulted in a peptide with improved bactericidal activity and minimized cytotoxicity and susceptibility to protease degradation compared to Pep3. The best analogues can be considered as potential lead compounds for the development of new antimicrobial agents for use in plant protection either as components of pesticides or expressed in transgenic plants.

The ABI2-dependent abscisic acid signalling controls HrpN-induced drought tolerance in Arabidopsis

HrpN, a protein produced by the plant pathogenic bacterium Erwinia amylovora, has been shown to stimulate plant growth and resistance to pathogens and insects. Here we report that HrpN activates abscisic acid (ABA) signalling to induce drought tolerance (DT) in Arabidopsis thaliana L. plants grown with water stress. Spraying wild-type plants with HrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture and proline levels in leaves, and alleviated extents of damage to cell membranes and plant drought symptoms caused by water deficiency. In plants treated with HrpN, ABA levels increased; expression of several ABA-signalling regulatory genes and the important effector gene rd29B was induced or enhanced. Induced expression of rd29B, promotion of stomatal closure, and reduction in drought severity were observed in the abi1-1 mutant, which has a defect in the phosphatase ABI1, after HrpN was applied. In contrast, HrpN failed to induce these responses in the abi2-1 mutant, which is impaired in the phosphatase ABI2. Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN in promoting stomatal closure and reducing drought severity. Moreover, resistance to Pseudomonas syringae developed in abi2-1 as in wild-type plants following treatment with HrpN. Thus, an ABI2-dependent ABA signalling pathway is responsible for the induction of DT but does not affect pathogen defence under the circumstances of this study.

Identification of an RcsA/RcsB Recognition Motif in the Promoters of Exopolysaccharide Biosynthetic Operons from Erwinia amylovora and Pantoea stewartii Subspeciesstewartii

The regulation of capsule synthesis (Rcs) regulatory network is responsible for the induction of exopolysaccharide biosynthesis in many enterobacterial species. We have previously shown that two transcriptional regulators, RcsA and RcsB, do bind as a heterodimer to the promoter of amsG, the first reading frame in the operon for amylovoran biosynthesis in the plant pathogenic bacterium Erwinia amylovora. We now identified a 23-base pair fragment from position -555 to -533 upstream of the translational start site of amsG as sufficient for the specific binding of the Rcs proteins. In addition, we could detect an RcsA/RcsB-binding site in a corresponding region of the promoter of cpsA, the homologous counterpart to the E. amylovora amsG gene in the operon for stewartan biosynthesis of Pantoea stewartii. The specificity and characteristic parameters of the protein-DNA interaction were analyzed by DNA retardation, protein-DNA cross-linking, and directed mutagenesis. The central core motif TRVGAAWAWTSYG of the amsG promoter was found to be most important for the specific interaction with RcsA/RcsB, as evaluated by mutational analysis and an in vitro selection approach. The wild type P. stewartii Rcs binding motif is degenerated in two positions and an up-mutation according to our consensus motif resulted in about a 5-fold increased affinity of the RcsA/RcsB proteins.

Ultrastructural study ofErwinia amylovora strains: Effect of culture conditions and fixation procedures

Modifications of the ultrastructure of the plant pathogenic bacteriumErwinia amylovora were analyzed according to growth conditions and fixation procedures. Six bacterial strains with various virulence characteristics were examined. Cultures were grown either in Yeast Peptone Glucose medium (YPG) or in a medium containing asparagine (ASP) supplemented with sorbitol (1% or 5% sorbitol). When grown in ASP + 1% sorbitol or in YPG, the strains, structurally similar to each other in ASP + 5% sorbitol, presented different frequencies of small evaginations which were observed arising from the cell surface mainly after an OsO4 fixation step. There was no correlation between the frequency of evaginations and the virulence of the strain. An overnight storage at 4 °C considerably enhanced the frequency of the evaginations. It was suggested that the OsO4 fixation step visualized differences in the bacterial outer membrane structure.

Immuno gold staining (IGS) and immuno gold silver staining (IGSS) for the identification of the plant pathogenic bacterium Erwinia amylovora (Burrill) Winslow et al.

For the identification of the plant pathogenic bacterium Erwinia amylovora, the immuno gold staining (IGS) and immuno gold silver staining (IGSS) techniques are tested. The IGS and IGSS methods are at least as sensitive an indirect immunofluorescence and require less primary antiserum. Moreover they have the advantage that the preparations can be conserved permanently and unchanged. The preparation of the IGS can be observed with transmitted light or--with considerable better result--using epipolarization microscopy. The IGSS method deserves special attention because of its high contrast in normal brigth field microscopy with transmitted light.