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Background Fire blight, caused by Erwinia amylovora, is the most destructive bacterial disease affecting plants in the Rosaceae family, leading to significant economic losses. In Algeria, this disease has been reported since 2010. This study aimed to investigate the origin of fire blight in Algeria, in order to increase knowledge of the epidemiology of this serious disease and contribute to its management. A comprehensive characterization of 18 E. amylovora isolates recovered from northern Algeria between 2016 and 2021 to evaluate their phenotypical and genotypical diversity was conducted. Results Phenotypic differences, particularly in growth kinetics, virulence, and fatty acid profiles, allowed differentiation of strains into five groups, possibly indicating distinct introduction events. Genetic characterization revealed that only one strain lacked the ubiquitous plasmid pEA29, which is correlated with reduced virulence, while none harbored the pEI70 plasmid. Phylogenetic analysis using concatenated sequences of the recA, groEL, rpoS, ams, and hrpN genes grouped Algerian strains with those from a broadly prevalent clade. CRISPR genotyping identified a novel CR1 pattern and three genotypes, two of them previously unreported. Conclusions This study represents the first phenotypic, genetic, and phylogenetic investigation of E. amylovora strains in the region, and provides valuable information on the possible pathways of the introduction of this fire blight pathogen in northern Africa. The findings suggest one or more introduction events from a common ancestor, likely originating in northern Italy, followed by dispersal in various regions of Algeria.

In May 2023, characteristic symptoms of fire blight, suspected to be caused by Erwinia amylovora , were observed infected wild Cotoneaster plants in the secondary forest along the lower reaches of the Jilgilang River, Xinjiang, China. Symptomatic tissues were collected, and bacterial isolation and purification were performed. The bacterium was identified through morphological characterization of the colonies, pathogenicity assays, tobacco hypersensitivity tests, and specific polymerase chain reaction (PCR) amplification, followed by sequence analysis. The isolated bacterium exhibited morphological features consistent with E. amylovora , and pathogenicity tests conducted under greenhouse conditions confirmed its pathogenicity, as evidenced by bacterial dissemination from the main leaf veins to surrounding tissues and the presence of bacterial exudates at the petioles. Furthermore, a pronounced hypersensitive response was observed in tobacco. PCR followed by sequencing revealed over 99.6% similarity with E. amylovora plasmid pEa29 repeat region. These findings confirm the E. amylovora is the causal agent of the fire blight disease in wild Cotoneaster plants. This is the first documented case of the fire blight affecting Cotoneaster plants in China. The detection of this pathogen has significant implications for the conservation of wild Rosaceae germplasm in the Tianshan Mountains wild fruit forests in Xinjiang.

Phage therapy has garnered significant attention due to the rise of life-threatening multidrug-resistant pathogenic bacteria and the growing awareness of the transfer of resistance genes between pathogens. Considering this, phage therapy applications are being extended to target plant pathogenic bacteria, such as Erwinia amylovora, which causes fire blight in apple and pear orchards. Understanding the mechanisms involved in phage resistance is crucial for enhancing the effectiveness of phage therapy. Despite the challenges of naturally developing a bacteriophage-insensitive mutant (BIM) of E. amylovora (without traditional mutagenesis methods), this study successfully created a BIM against the podovirus ϕEa46-1-A1. The parent strain, E. amylovora D7, and the BIM B6-2 were extensively compared at genomic, transcriptomic, and phenotypic levels. The phenotypic comparison included the metabolic behavior, biofilm formation, and in planta evaluations of pathogenicity. The results revealed a mutation in strain B6-2 in the rcsB gene, which encodes a second regulator in the Rcs two-component phosphorelay system (TCS). This mutation resulted in significant changes in the B6-2 BIM, including downregulation of amylovoran gene expression (e.g., an average log2 fold change of −4.35 across amsA-L), visible alterations in biofilm formation, increased sensitivity to antibiotics (22.4% more sensitive to streptomycin), and a loss of pathogenicity as assessed in an apple seedling virulence model in comparison to the wildtype strain. The findings presented in this study highlight the critical role of the Rcs phosphorelay system in phage resistance in E. amylovora. Based on these findings, we have proposed a model that explains the effect of the B6-2 rcsB mutation on the Rcs phosphorelay system and its contribution to the development of phage resistance in E. amylovora.

Erwinia amylovora is the causative agent of fire blight. Resistance to streptomycin, the main antibiotic in fire blight management, has led to an urgent requirement to develop alternative biological control agents, such as the phage-carrier system (PCS). Previous studies have focused on the dynamic interactions between the carrier ( Pantoea agglomerans ), lytic phages, and the pathogen. However, crucial information about phage receptors on these hosts is still lacking. Here, a biochemical approach was used and the phage receptors of two  E. amylovora  phages (ϕEa21-4 and ϕEa46-1-A1) on both hosts, have been identified as LPS and OmpA on E. amylovora  and OmpA only on P. agglomerans.  Interestingly, this work uncovered for the first time that amylovoran is tightly attached to the LPS of E. amylovora . Confirmation of this interaction and an infection model are presented that have far reaching implications for additional PCS improvement and pathogen-host interaction details.

Erwinia amylovora , a Gram-negative phytopathogenic bacterium, is the etiological agent of fire blight in apples and pears. Key virulence factors include the exopolysaccharide amylovoran, the type III secretion system, and siderophore-mediated iron uptake. Within the iron uptake pathway, the periplasmic siderophore binding protein FhuD, unique to Erwinia species infecting Rosaceae, plays a vital role in transporting iron-loaded siderophores to the inner periplasmic membrane, making it a crucial target for structural and functional characterization. This article presents the predicted 3D model of FhuD from E. amylovora (FhuD_Ea), along with the sequence analyses and structural comparison of its homologs from eight organisms whose structures are available in the PDB. We also performed bioinformatics analysis on protein sequences of 145 orthologs. Despite the low sequence identity, the homologs exhibited similar structures, with consistent ligand binding clefts. Nine conserved residues, primarily located in the N-terminal domain, were identified, with the exception of GLY 202 (in the C-terminal domain of FhuD_Ea). Among orthologs, ILE 88 emerged as a notably conserved residue in the N-terminal region, while TRP 64, though often positioned in the binding cleft, was not universally conserved. A phylogenetic tree based on 145 orthologs revealed no distinct grouping between Gram-positive and Gram-negative bacteria, suggesting that the periplasmic binding protein retains similar structural and functional characteristics across diverse bacterial lineages. The apparent lack of universally conserved residues in the ligand-binding pocket suggests functional flexibility, allowing FhuD to recognize siderophores with similar chemical features rather than identical structures. Molecular docking analyses further supported this hypothesis, showing that FhuD_Ea preferentially binds hydroxamate-type siderophores like ferrioxamine, but also accommodates structurally related ligands such as coprogen, with even greater binding affinity. These findings point to an adaptable binding mechanism that may enhance iron acquisition under varying environmental conditions.

Erwinia amylovora is a highly destructive phytopathogenic bacterium that causes fire blight in Rosaceae plants, particularly apple and pear trees. Due to its status as a quarantine organism in many countries, extensive epidemiological and genotypic studies have been conducted to better understand and manage this pathogen. This study presents a novel workflow for identifying strain-specific simple sequence repeats (SSRs) in bacterial genomes, focusing on E. amylovora . Leveraging the abundance of available genome data, the workflow involves identifying single-copy genes via BLASTX searches, followed by SSR identification using the MISA tool and validation through sequence alignment. Utilizing the genome of E. amylovora strain CFBP 1430 as a reference, 27 SSRs within 26 single-copy genes were identified, displaying diverse repeat patterns and lengths. Five genes containing SSRs were selected, offering valuable markers for strain differentiation. Phylogenetic analysis of 114 E. amylovora strains revealed three distinct groups: widely prevalent, Western NA, and Eastern NA clades, in line with previous findings. A comparative analysis with clustered regularly interspaced short palindromic repeat (CRISPR) analysis and multiple-locus variable-number tandem repeat analysis (MLVA) highlighted differences in marker selection strategies and underscored the need to validate findings using multiple genotyping approaches. While limitations remain due to incomplete nucleotide sequences in public databases, this study advances our understanding of the genetic diversity, population structure, and evolutionary dynamics of E. amylovora strains, and positions SSR-based methods as a complementary tool alongside other established techniques.

Plant microbiomes have important roles in plant health and productivity. However, despite flowers being directly linked to reproductive outcomes, little is known about the microbiomes of flowers and their potential interaction with pathogen infection. Here, we investigated the temporal spatial dynamics of the apple stigma microbiome when challenged with a phytopathogen Erwinia amylovora , the causal agent of fire blight disease. We profiled the microbiome from the stigmas of individual flowers, greatly increasing the resolution at which we can characterize shifts in the composition of the microbiome. Individual flowers harbored unique microbiomes at the operational taxonomic unit level. However, taxonomic analysis of community succession showed a population gradually dominated by bacteria within the families Enterobacteriaceae and Pseudomonadaceae . Flowers inoculated with E. amylovora established large populations of the phytopathogen, with pathogen-specific gene counts of >3.0 × 10 7 in 90% of the flowers. Yet, only 42% of inoculated flowers later developed fire blight symptoms. This reveals that pathogen abundance on the stigma is not sufficient to predict disease outcome. Our data demonstrate that apple flowers represent an excellent model in which to characterize how plant microbiomes establish, develop, and correlate with biological processes such as disease progression in an experimentally tractable plant organ.

Main conclusion Certain apple cultivars accumulate to high levels in their nectar and stigma exudate an acidic chitinase III protein that can protect against pathogens including fire blight disease causing Erwinia amylovora. To prevent microbial infections, flower nectars and stigma exudates contain various antimicrobial compounds. Erwinia amylovora , the causing bacterium of the devastating fire blight apple disease, is the model pathogen that multiplies in flower secretions and infects through the nectaries. Although Erwinia -resistant apples are not available, certain cultivars are tolerant. It was reported that in flower infection assay, the ‘Freedom’ cultivar was Erwinia tolerant, while the ‘Jonagold’ cultivar was susceptible. We hypothesized that differences in the nectar protein compositions lead to different susceptibility. Indeed, we found that an acidic chitinase III protein (Machi3-1) selectively accumulates to very high levels in the nectar and the stigma exudate of the ‘Freedom’ cultivar. We show that three different Machi3 - 1 alleles exist in apple cultivars and that only the 5B - Machi3 - 1 allele expresses the Machi3-1 protein in the nectar and the stigma exudate. We demonstrate that the 5B - Machi3 - 1 allele was introgressed from the Malus floribunda 821 clone into different apple cultivars including the ‘Freedom’. Our data suggest that MYB-binding site containing repeats of the 5B- Machi3 - 1 promoter is responsible for the strong nectar- and stigma exudate-specific expression. As we found that in vitro, the Machi3-1 protein impairs growth and biofilm formation of Erwinia at physiological concentration, we propose that the Machi3-1 protein could partially protect 5B - Machi3 - 1 allele containing cultivars against Erwinia by inhibiting the multiplication and biofilm formation of the pathogen in the stigma exudate and in the nectar.

Fire blight disease continues to plague the commercial production of apples and pears despite more than a century of research into disease epidemiology and disease control. The causative agent of fire blight, Erwinia amylovora coordinates turning on or off specific virulence-associated traits at the appropriate time during disease development. The development of novel control strategies requires an in-depth understanding of E. amylovora regulatory mechanisms, including regulatory control of virulence-associated traits. This study investigates how the small RNA ArcZ regulates motility at the transcriptional level and identifies the transcription factor Lrp as a novel participant in the regulation of several virulence-associated traits. We report that ArcZ and Lrp together affect key virulence-associated traits through integration of transcriptional and posttranscriptional mechanisms. Further understanding of the topology of virulence regulatory networks can uncover weak points that can subsequently be exploited to control E. amylovora . Erwinia amylovora causes the devastating fire blight disease of apple and pear trees. During systemic infection of host trees, pathogen cells must rapidly respond to changes in their environment as they move through different host tissues that present distinct challenges and sources of nutrition. Growing evidence indicates that small RNAs (sRNAs) play an important role in disease progression as posttranscriptional regulators. The sRNA ArcZ positively regulates the motility phenotype and transcription of flagellar genes in E. amylovora Ea1189 yet is a direct repressor of translation of the flagellar master regulator, FlhD. We utilized transposon mutagenesis to conduct a forward genetic screen and identified suppressor mutations that increase motility in the Ea1189 ΔarcZ mutant background. This enabled us to determine that the mechanism of transcriptional activation of the flhDC mRNA by ArcZ is mediated by the leucine-responsive regulatory protein, Lrp. We show that Lrp contributes to expression of virulence and several virulence-associated traits, including production of the exopolysaccharide amylovoran, levansucrase activity, and biofilm formation. We further show that Lrp is regulated posttranscriptionally by ArcZ through destabilization of lrp mRNA. Thus, ArcZ regulation of FlhDC directly and indirectly through Lrp forms an incoherent feed-forward loop that regulates levansucrase activity and motility as outputs. This work identifies Lrp as a novel participant in virulence regulation in E. amylovora and places it in the context of a virulence-associated regulatory network. IMPORTANCE Fire blight disease continues to plague the commercial production of apples and pears despite more than a century of research into disease epidemiology and disease control. The causative agent of fire blight, Erwinia amylovora coordinates turning on or off specific virulence-associated traits at the appropriate time during disease development. The development of novel control strategies requires an in-depth understanding of E. amylovora regulatory mechanisms, including regulatory control of virulence-associated traits. This study investigates how the small RNA ArcZ regulates motility at the transcriptional level and identifies the transcription factor Lrp as a novel participant in the regulation of several virulence-associated traits. We report that ArcZ and Lrp together affect key virulence-associated traits through integration of transcriptional and posttranscriptional mechanisms. Further understanding of the topology of virulence regulatory networks can uncover weak points that can subsequently be exploited to control E. amylovora .

Background Type VI secretion systems (T6SS) are widespread among Gram-negative bacteria and have a potential role as essential virulence factors or to maintain symbiotic interactions. Three T6SS gene clusters were identified in the genome of E . amylovora CFBP 1430, of which T6SS-1 and T6SS-3 represent complete T6SS machineries, while T6SS-2 is reduced in its gene content. Results To assess the contribution of T6SSs to virulence and potential transcriptomic changes of E. amylovora CFBP 1430, single and double mutants in two structural genes were generated for T6SS-1 and T6SS-3. Plant assays showed that mutants in T6SS-3 were slightly more virulent in apple shoots while inducing less disease symptoms on apple flowers, indicating that T6SSs have only a minor effect on virulence of E . amylovora CFBP 1430. The mutations led under in vitro conditions to the differential expression of type III secretion systems, iron acquisition, chemotaxis, flagellar, and fimbrial genes. Comparison of the in planta and in vitro transcriptome data sets revealed a common differential expression of three processes and a set of chemotaxis and motility genes. Additional experiments proved that T6SS mutants are impaired in their motility. Conclusion These results suggest that the deletion of T6SSs alters metabolic and motility processes. Nevertheless, the difference in lesion development in apple shoots and flower necrosis of T6SS mutants was indicative that T6SSs influences the disease progression and the establishment of the pathogen on host plants.