Immature Pear Fruits Research Articles

Novel Single Nucleotide Variations Alter Pathogenicity in Korean Isolates of Erwinia amylovora.

Erwinia amylovora, the causal agent of fire blight disease, has become a serious threat to the pome fruit industry in Korea since 2015. In this study, we showed that two new isolates of E. amylovora, Ea17-2187 and Ea19-7, obtained from pear orchards in Anseong, Korea, exhibited unique pathogenicity compared with other isolates thus far. Both were nonpathogenic to immature apple fruits but occasionally caused disease on immature pear fruits at varying reduced rates. Bioinformatic analyses revealed that their genomes are highly similar to those of the type strains TS3128 and ATCC49946 but have different mutations in essential virulence regulatory genes. Ea17-2187 has a single nucleotide substitution in rcsC, which encodes the core components of the Rcs system that activates the exopolysaccharide amylovoran production. In contrast, Ea19-7 contains a single nucleotide insertion in hrpL, which encodes a master regulator of the type III secretion system. In both cases, the mutation can cause premature termination and production of truncated gene products, disrupting virulence regulation. Introduction of the nonmutated rcsC and hrpL genes into Ea17-2187 and Ea19-7, respectively, fully recovered pathogenicity, comparable with that of TS3128; hence, these mutations were responsible for the altered pathogenicity observed. Interestingly, virulence assays on immature pear fruits showed that the hrpL mutant of Ea19-7 was still pathogenic, although its virulence level was markedly reduced. Taken together, these results suggest that the two new isolates might act as opportunistic pathogens or cheaters and that some Korean isolates might have evolved to acquire alternative pathways for activating pathogenicity factors.

Genomic characterization of Pseudomonas syringae pv. syringae from Callery pear and the efficiency of associated phages in disease protection.

The Pseudomonas syringae species complex is a heterogeneous group of plant pathogenic bacteria associated with a wide distribution of plant species. Advances in genomics are revealing the complex evolutionary history of this species complex and the wide array of genetic adaptations underpinning their diverse lifestyles. Here, we genomically characterize two P. syringae isolates collected from diseased Callery pears (Pyrus calleryana) in Berkeley, California in 2019 and 2022. We also isolated a lytic bacteriophage, which we characterized and evaluated for biocontrol efficiency. Using a multilocus sequence analysis and core genome alignment, we classified the P. syringae isolates as members of phylogroup 2, related to other strains previously isolated from Pyrus and Prunus. An analysis of effector proteins demonstrated an evolutionary conservation of effectoromes across isolates classified in PG2 and yet uncovered unique effector profiles for each, including the two newly identified isolates. Whole-genome sequencing of the associated phage uncovered a novel phage genus related to Pseudomonas syringae pv. actinidiae phage PHB09 and the Flaumdravirus genus. Finally, using in planta infection assays, we demonstrate that the phage was equally useful in symptom mitigation of immature pear fruit regardless of the Pss strain tested. Overall, this study demonstrates the diversity of P. syringae and their viruses associated with ornamental pear trees, posing spill-over risks to commercial pear trees and the possibility of using phages as biocontrol agents to reduce the impact of disease.IMPORTANCEGlobal change exacerbates the spread and impact of pathogens, especially in agricultural settings. There is a clear need to better monitor the spread and diversity of plant pathogens, including in potential spillover hosts, and for the development of novel and sustainable control strategies. In this study, we characterize the first described strains of Pseudomonas syringae pv. syringae isolated from Callery pear in Berkeley, California from diseased tissues in an urban environment. We show that these strains have divergent virulence profiles from previously described strains and that they can cause disease in commercial pears. Additionally, we describe a novel bacteriophage that is associated with these strains and explore its potential to act as a biocontrol agent. Together, the data presented here demonstrate that ornamental pear trees harbor novel P. syringae pv. syringae isolates that potentially pose a risk to local fruit production, or vice versa-but also provide us with novel associated phages, effective in disease mitigation.

Screening for Novel Beneficial Environmental Bacteria for an Antagonism-Based Erwinia amylovora Biological Control.

Erwinia amylovora, the bacterial species responsible for fire blight, causes major economic losses in pome fruit crops worldwide. Chemical control is not always effective and poses a serious threat to the environment and human health. Social demands for eco-sustainable and safe control methods make it necessary to search for new biocontrol strategies such as those based on antagonists. A bacterial collection from different fire blight-free Mediterranean environments was tested for antagonistic activity against Spanish strains of E. amylovora. Antagonistic assays were carried out in vitro in culture medium and ex vivo in immature loquat and pear fruits. Results revealed that 12% of the 82 bacterial isolates tested were able to inhibit the growth of several strains of the pathogen. Some of the isolates also maintained their antagonistic activity even after chloroform inactivation. Selected isolates were further tested ex vivo, with several of them being able to delay and/or reduce fire blight symptom severity in both loquats and pears and having activity against some E. amylovora strains. The isolates showing the best antagonism also produced different hydrolases linked to biocontrol (protease, lipase, amylase, and/or DNAse) and were able to fix molecular nitrogen. Based on this additional characterization, four biocontrol strain candidates were further selected and identified using MALDI-TOF MS. Three of them were Gram-positive bacteria belonging to Bacillus and Paenarthrobacter genera, and the fourth was a Pseudomonas strain. Results provide promising prospects for an improvement in the biological control strategies against fire blight disease.

Isolation and characterization of three bacteriophages infecting Erwinia amylovora and their potential as biological control agent

BackgroundFire Blight, incited by Erwinia amylovora, is one of the most damaging pear and apple diseases in the world. Fire blight was introduced to Egypt in the 1960 and threatens the Egypt’s costs for pear industry. Currently, Phage therapy is considered to be secured biological method for controlling plant bacterial diseases. This investigation aimed to isolate and identify molecularly for bacteria causing fire bright disease. As well as isolation and identification bacteriophages via spot and plaque assay techniques from pear fire blight lesions and soil. On the other hand, bacteriophages were identified based on plaque morphology, virion morphology, physical characters, profile of DNA restriction and protein.ResultsPathogenicity test revealed that healthy seedlings and pear fruits were responsive to fire blight E. amylovora. Considering the relatively wide host range and greatest protein and genetic variability, using restriction enzyme pattern, the three diversity phage isolates named, EAP1, EAP2 and EAP3 showed a lack of diversity out of five were fatherly characterized. The phages confirmed the close relation of EAP1, EAP2 to Siphoviridae (hexagonal head and long flexible non-contractile tail) and EAP3 to Myoviridae (icosahedral head and contractile tail). The phages retained higher lytic competence of 90.4; 92.68 and 95.25% for EAP1, EAP2 and EAP3, respectively. The phages were stable at strong alkaline (pH 10) 2% salt solution conditions and UV spectra. While EAP3 phage revealed the hexagonal head and very short tail that belongs to Myoviridae family. Bacteriophages were characterized by digestion of the phage DNA with three restriction endonucleases and were placed into three groups based on the patterns. Bacteriophages were 9 used for reducing bacterial infection populations and severity on pear. In a bioassay, the biocontrol of E. amylovora was evaluated using disks of immature pear fruit. On the pear disk surface, bacterial exudate was considerably suppressed by all phage isolates. According to measurements of the bacterial population still present on the disk surface, phage therapy could reduce it by up to 97%. Bacteriophages reduced pear fire blight disease severity on pear fruit trails.ConclusionThe results indicated that bacteriophage isolates may demonstrate variable reactivity against E. amylovora. Bacteriophages reduced pear fire blight disease severity on pear fruit trials. The results indicated that bacteriophage isolates may demonstrate variable reactivity against E. amylovora.

First Report of Fire Blight Caused by Erwinia amylovora on Korean Mountain Ash (Sorbus alnifolia) in Korea

During the nationwide survey of fire blight, the typical shoot blight symptoms were found on Korean mountain ash (Sorbus alnifolia) which was located near an orchard that produced fire blight on pear trees in Eumseong, Korea, May 2021. To identify the causal agent, we progressed isolation from the symptomatic leaves and shoots. Two white and mucoid colonies were isolated into the pure culture. Two isolates were identified as Erwinia amylovora according to the colony–polymerase chain reaction (PCR) with amsB primers and the phylogenetic tree using 16S rRNA sequences. To test of pathogenicity of two isolates, we inoculated immature pear fruits and understock of apple. We observed necrosis and oozes on immature pear fruits and shoot blight resulting in necrosis on apple shoots six days after inoculation. Colonies were recovered from the inoculated pears and apples, and identity was confirmed through colony PCR for amsB genes. To our knowledge, E. amylovora was first reported on Korean mountain ash native to South Korea.

First Report of Fire Blight Caused by Erwinia amylovora on Korean Mountain Ash (Sorbus alnifolia) in Korea

During the nationwide survey of fire blight, the typical shoot blight symptoms were found on Korean mountain ash (Sorbus alnifolia) which was located near an orchard that produced fire blight on pear trees in Eumseong, Korea, May 2021. To identify the causal agent, we progressed isolation from the symptomatic leaves and shoots. Two white and mucoid colonies were isolated into the pure culture. Two isolates were identified as Erwinia amylovora according to the colony–polymerase chain reaction (PCR) with amsB primers and the phylogenetic tree using 16S rRNA sequences. To test of pathogenicity of two isolates, we inoculated immature pear fruits and understock of apple. We observed necrosis and oozes on immature pear fruits and shoot blight resulting in necrosis on apple shoots six days after inoculation. Colonies were recovered from the inoculated pears and apples, and identity was confirmed through colony PCR for amsB genes. To our knowledge, E. amylovora was first reported on Korean mountain ash native to South Korea.

Development of the Droplet Digital PCR Method for the Detection and Quantification of Erwinia pyrifoliae.

Black shoot blight disease caused by Erwinia pyrifoliae has serious impacts on quality and yield in pear production in Korea; therefore, rapid and accurate methods for its detection are needed. However, traditional detection methods require a great deal of time and fail to achieve absolute quantification. In the present study, we developed a droplet digital polymerase chain reaction (ddPCR) method for the detection and absolute quantification of E. pyrifoliae using a pair of species-specific primers. The detection range was 103 - 107 copies/ml (DNA templates) and cfu/ml (cell culture templates). This new method exhibited good linearity and repeatability and was validated by absolute quantification of E. pyrifoliae DNA copies from samples of artificially inoculated immature pear fruits. Here, we present the first study of ddPCR assay for the detection and quantification of E. pyrifoliae. This method has potential applications in epidemiology and for the early prediction of black shoot blight outbreaks.

Antagonistic effect of Leuconostoc mesenteroides on grapevine crown gall and fire blight

ABSTRACT Grapevine crown gall, caused by Allorhizobium vitis (A. vitis) and fire blight, caused by Erwinia amylovora (E. amylovora) of rosaceous plants, have been recognized as serious bacterial diseases and responsible for major crop losses in grapevine (Vitis vinifera L.), pear (Pyrus communis L.) and apple (Malus domestica Borkh.) worldwide. To develop an effective biocontrol method against the two bacterial pathogens, lactic acid bacteria (LAB) isolated from agricultural irrigation canals were tested as biocontrol agents. Thirty-two bacterial isolates were screened for their in vitro antagonistic activity. Four isolates (MS-1, MS-2, MS-3, and MS-4), identified by partial sequencing of 16S rRNA as Leuconostoc mesenteroides subsp. Jonggajibkimchii, were found to significantly inhibit the growth of A. vitis and E. amylovora in vitro. The biocontrol efficiency of these strains was tested against E. amylovora on immature pear fruits under laboratory conditions, and against A. vitis in tomato (Solanum lycopersicum L.) and kalanchoe (Kalanchoe daigremontiana) plants, under greenhouse conditions. Results showed that all selected antagonists were able to suppress fire blight disease on immature pear fruits. On tomato plants, the MS-1 and MS-2 strains were able to reduce the A. vitis infection by 82% and 99%, respectively. Similarly, the MS-2 strain reduced gall formation on kalanchoe plants by 99%. We concluded that Leuconostoc mesenteroides subsp. Jonggajibkimchii strains could serve as a potential biocontrol agent against fire blight and grapevine crown gall diseases.

Chemical composition and antibacterial activity of Moroccan Artemisia mesatlantica and A. absinthium essential oils against fire blight caused by Erwinia amylovora

The aim of this study was to find an alternative to chemical products used in the control of fire blight disease of rosaceous plants. In this work, essential oils of two Moroccan Artemisia species: Artemisia mesatlantica and Artemisia absinthium were screened for their antibacterial activity against Erwinia amylovora the causal agent of this disease. The EOs extraction was carried out by water distillation method in Clevenger- type apparatus and analyzed by gas Chromatography-Flame Ionization Detector (GC/FID) and by Gas Chromatography-Mass Spectrometry (GC/MS). The antibacterial activity of extracted EOs was tested in vitro based on the agar diffusion method and broth microdilution method and in vivo on detached immature pear fruits. The results revealed that the yields of extracted EOs were 1.25 and 0.58% respectively for A. mesatlantica and A. absinthium. The chemical composition analysis showed that investigated EOs were characterized with a high amount of β-Thujone (40.42% for A. mesatlantica and 36.25% for A. absinthium). Both EOs have a similar MIC value (2.08%). A. absinthium EOs were found to be more effective than A. mesatlantica EOs since they show the lowest MBC. These results indicate that EOs of A. absinthium and A. mesatlantica contained compounds with antibacterial potential against Erwinia amylovora. In the bioassay test, the application of A. absinthium EOs on detached immature pear fruits infected by E. amylovora resulted in total suppression of disease severity. Such natural products may represent a sustainable alternative to the use of chemical pesticides.

The Arac-like transcriptional regulator YqhC is involved in pathogenicity of Erwinia amylovora.

This study aimed to identify virulence-associated genes and functions that affect disease development on pear caused by Erwinia amylovora EaUMG3 isolated from Iran. A mini-Tn5 transposon library was generated in EaUMG3. An E. amylovora mutant that had lost its ability to cause lesions on immature pear fruits, was selected for further analysis. This mutant was shown to have a transposon insertion in yqhC, a gene belongs to the AraC family of transcriptional regulators. A mutant of the wild-type EaUMG3 carrying an unmarked deletion of the yqhC gene was created using pDMS197. The Ea∆yqhC mutant showed reduced disease progression on immature pear fruits and pear plants, reduced motility and significantly lower levels of the virulence factors siderophore and amylovoran. Complementation with yqhC cloned in pBBR1MCS restored disease progression and the level of virulence factors to near wild type. YqhC transcriptional regulator is necessary for full virulence of E. amylovora. In addition, this regulator affects virulence factors such as siderophore production, amylovoran production, and motility. The identification of a novel transcriptional regulator with strong impact in the pathogenesis of E. amylovora, an organism causing significant economic losses in fruit production.

Isolation and efficacy of two bacterial strains antagonists of Erwinia amylovora and Pectobacterium carotovorum

BackgroundThe development of ecofriendly tools against plant diseases is an important issue in crop protection. Screening and selection process of bacterial strains antagonists of 2 pathogenic bacterial species that limit very important crops, Erwinia amylovora, the causal agent of the fire blight disease, and Pectobacterium carotovorum, the causal agent of bacterial potato soft rot, were reported. Bacterial colonies were isolated from different ecological niches, where both pathogens were found: rhizosphere of potato tubers and fruits and leaves of pear trees from the northwest region of Algeria. Direct and indirect confrontation tests against strains of E. amylovora and P. carotovorum were performed.ResultsResults showed a significant antagonistic activity against both phytopathogenic species, using direct confrontation method and supernatants of cultures (p<0.005). In vitro assays showed growth inhibitions of both phytopathogenic species. Furthermore, results revealed that the strains of S. plymuthica had a better inhibitory effect than the strains of P. fluorescens against both pathogens. In vivo results on immature pear fruits showed a significant decrease in the progression of the fire blight symptoms, with a variation in the infection index from one antagonistic strain to another between 31.3 and 50%, and slice of potato showed total inhibition of the pathogen (P. carotovorum) by the antagonistic strains of Serratia plymuthica (p<0.005).ConclusionThis study highlighted that the effective bacteria did not show any infection signs towards plant tissue, and considered as a potential strategy to limit the fire blight and soft rot diseases.

Anti-Aging Effects of Hydrophobic and Hydrophilic Components From Immature Pear Fruits Extract

Cellular senescence, the decline of cellular function due to aging, causes gradual loss of physiological functions and induces some chronic diseases, which negatively affect the quality of human life. Intervention in the cellular senescence process may reduce these incidences and delay the progression of age-related diseases, thereby contributing to the longevity of human lifespan. The budding yeast, Saccharomyces cerevisiae, is a model system that can provide significant insights into the genetics and molecular biology of senescence and is a suitable cellular model for research on mammalian cells. In the 2019 GSA meeting, we had revealed that the prolongation of yeast cell lifespan was induced by the addition of immature pear fruits extracts (iPE). In this study, we have focused on investigating the anti-senescence effects of hydrophilic (WiPE) and hydrophobic (OiPE) components of iPE on yeast cells and their genes and their possible application in extending human lifespan. The anti-aging effects of iPE were investigated using a chronological lifespan assay on S. cerevisiae cells. The chronological lifespan of the yeast was significantly extended in those treated with both WiPE and OiPE at 1% (v/v). The expression of sirtuin-related genes, which regulate cellular senescence, was examined by RT-PCR. Interestingly, gene expression was found to be significantly increased only in WiPE treated cells. The results suggested that the different polarity components from iPE exhibited anti-aging effects on the cells via different mechanisms. Research on the identification of useful components in iPE and the possibility of application to mammalian cells is ongoing.

Orchestration of virulence factor expression and modulation of biofilm dispersal in Erwinia amylovora through activation of the Hfq-dependent small RNA RprA.

Erwinia amylovora is the causative agent of the devastating disease fire blight of pome fruit trees. After infection of host plant leaves at apple shoot tips, E. amylovora cells form biofilms in xylem vessels, restrict water flow, and cause wilting symptoms. Although E. amylovora is well known to be able to cause systemic infection, how biofilm cells of E. amylovora transit from the sessile mode of growth in xylem to the planktonic mode of growth in cortical parenchyma remains unknown. Increasing evidence has suggested the important modulatory roles of Hfq‐dependent small RNAs (sRNAs) in the pathogenesis of E. amylovora. Here, we demonstrate that the sRNA RprA acts as a positive regulator of amylovoran exopolysaccharide production, the type III secretion system (T3SS), and flagellar‐dependent motility, and as a negative regulator of levansucrase activity and cellulose production. We also show that RprA affects the promoter activity of multiple virulence factor genes and regulates hrpS, a critical T3SS regulator, at the posttranscriptional level. We determined that rprA expression can be activated by the Rcs phosphorelay, and that expression is active during T3SS‐mediated host infection in an immature pear fruit infection model. We further showed that overexpression of rprA activated the in vitro dispersal of E. amylovora cells from biofilms. Thus, our investigation of the varied role of RprA in affecting E. amylovora virulence provides important insights into the functions of this sRNA in biofilm control and systemic infection.

Characterization of Paenibacillus polymixa N179 as a robust and multifunctional biocontrol agent

The strain N179 was isolated from agricultural fields and selected through screening, based on its antagonistic potential, PGPR activity and robust biofilm formation. This strain was identified as Paenibacillus polymyxa based on morphological, biochemical and molecular characteristics. It was found that this strain has strong antagonistic activity against a wide variety of plant pathogens including fungi, bacteria, Phytophthora drechsleri, and root-knot nematode. The strain N179 was able to produce PGPR-related metabolites and it was able to solubilize mineral phosphate in vitro condition. Results showed that strain N179 is able to promote growth of bean, cucumber, wheat and corn seedlings in growth chamber condition. We also checked the effect of this strain on wheat yield in field condition. Highest yield was achieved when the bacterium was applied by seed coating method with 4 × 106 cells/ml concentration. Detached pear blossom and immature pear fruit assays were used to check the ability of N179 to suppress a phytopathogen in vivo condition. In both bioassays strain N179 was able to drastically suppress Erwinia amylovora. Inoculation of turnip cabbage roots with strain N179 resulted in reduction of growth rate of Xanthomonas campestris pv. campestris strain 8004 in leaves and developed symptoms were lower in these plants in comparison to infected control plants.

Functional characterization of the adenine transporter EaAdeP from the fire blight pathogen Erwinia amylovora and its effect on disease establishment in apples and pears.

Erwinia amylovora is the causal agent of fire blight, an economically important disease of apples and pears. As part of the infection process, Er. amylovora propagates on different plant tissues each with distinct nutrient environments. Here, the biochemical properties of the Er. amylovora adenine permease (EaAdeP) are investigated. Heterologous expression of EaAdeP in nucleobase transporter-deficient Escherichia coli strains, coupled with radiolabel uptake studies, revealed that EaAdeP is a high affinity adenine transporter with a Km of 0.43± 0.09 μM. Both Es. coli and Er. amylovora carrying extra copies of EaAdeP are sensitive to growth on the toxic analog 8-azaadenine. EaAdeP is expressed during immature pear fruit infection. Immature pear and apple fruit virulence assays reveal that an E. amylovora ΔadeP::Camr mutant is still able to cause disease symptoms, however, with growth at a lower level, indicating that external adenine is utilized in disease establishment.

Genome-wide identification of the Sec-dependent secretory protease genes in Erwinia amylovora and analysis of their expression during infection of immature pear fruit.

The general secretory (Sec) pathway represents a common mechanism by which bacteria secrete proteins, including virulence factors, into the extracytoplasmic milieu. However, there is little information about this system, as well as its associated secretory proteins, in relation to the fire blight pathogen Erwinia amylovora. In this study, data mining revealed that E. amylovora harbors all of the essential components of the Sec system. Based on this information, we identified putative Sec-dependent secretory proteases in E. amylovora on a genome-wide scale. Using the programs SignalP, LipoP, and Phobius, a total of 15 putative proteases were predicted to contain the N-terminal signal peptides (SPs) that might link them to the Sec-dependent pathway. The activities of the predicted SPs were further validated using an Escherichia coli-based alkaline phosphatase (PhoA) gene fusion system that confirmed their extracytoplasmic property. Transcriptional analyses showed that the expression of 11 of the 15 extracytoplasmic protease genes increased significantly when E. amylovora was used to inoculate immature pears, suggesting their potential roles in plant infection. The results of this study support the suggestion that E. amylovora might employ the Sec system to secrete a suite of proteases to enable successful infection of plants, and shed new light on the interaction of E. amylovora with host plants.

Characterization of genes involved in (p)ppGpp precursor biosynthesis in Erwinia amylovora

Previous studies have shown that the hypersensitive response and pathogenicity (hrp)-type III secretion system (T3SS) is activated by the bacterial alarmone (p)ppGpp, which mediates the stringent response in nutrient starvation conditions in Erwinia amylovora. ATP and GTP, the precursors of (p)ppGpp biosynthesis, are synthesized from AMP/adenine and GMP/guanine, respectively, by the de novo pathway of purine biosynthesis. In this study, we examined the role of the purA/purB and guaA/guaB genes, which are involved in the AMP/adenine and GMP/guanine biosynthesis, respectively. Our results showed that growth of the purA, purB, and guaAB mutants was much slower in minimal media, but could be restored by addition of exogenous AMP/adenine and GMP/guanine, respectively. However, mixed results were obtained for their contribution to virulence on immature pear fruits. These results suggested that the de novo purine biosynthesis pathway might be required for proliferation, colonization and infection of E. amylovora.

Antagonistic activity and mechanism of a novel Bacillus amyloliquefaciens MB40 strain against fire blight

Fire blight caused by Erwinia amylovora is an important disease affecting most types of rosaceous trees and represents a significant threat to fruit cultivation in many parts of the world. The effectiveness of antagonistic bacteria Bacillus amyloliquefaciens strain MB40 to inhibit the growth of Erwinia amylovora strain 1E IMIV was evaluated under in vitro conditions. B. amyloliquefaciens MB40 was isolated from leaves of fire blight host mature apple trees (cultivar ‘Zarya Alatau’) sampled in the Almaty region of Kazakhstan. Local isolate of the fire blight causative agent E. amylovora was used as pathogen inoculum in laboratory trials. Erwinia amylovora and Bacillus amyloliquefaciens were identified using morphological and biochemical tests, and the pathogenicity of E. amylovora 1E IMIV was demonstrated in a bioassay on immature pear fruits. B. amyloliquefaciens MB40 showed high antagonistic activity against the pathogen. Biological efficiency of the applied culture broth of B. amyloliquefaciens was 90.6% (95% confidence interval [CI] = 74.1 to 107.2). Analysis of active substances produced by the antagonistic bacterium disclosed high amounts of acetoin (3-hydroxybutanone) - 53.7 ± 1.8% and and 2,3-butanedione - 34.4 ± 3.6%. The inhibitory activity of the latter was demonstrated. The possible mechanism of its action is discussed, suggesting that B. amyloliquefaciens MB40 is a novel antibacterial strain with potential application in controlling E. amylovora in fruit growing.

Molecular characterization of Algerian Erwinia amylovora strains by VNTR analysis and biocontrol efficacy of Bacillus spp. and Pseudomonas brassicacearum antagonists

Samples of pear shoots, blossoms and leaves showing typical fire blight symptoms were collected from diseased pear trees during spring and summer over a period of 3 years from orchards situated in the Mitidja region, a main pear producing area in Northern Algeria. From all collected plant samples, thirty-seven putative Erwinia amylovora strains were isolated and identified by biochemical, serological and molecular tests. Molecular typing of these strains was performed using six variable number of tandem repeats sequences (VNTRs). Minimal spanning trees showed that four different haplotypes were present within the bacterial population analyzed. The efficacy of twenty potential bacterial antagonists was evaluated in vitro against two E. amylovora pathogenic strains. Four antagonists (Bacillus amyloliquefaciens, Bacillus methylotrophicus and two Pseudomonas brassicaceaurum) were selected to perform a biocontrol assay on immature pear fruits. Results showed that the two Pseudomonas strains were the most effective for preventive treatment on pear fruits, leading to a necrosis reduction of up to 90%. No curative effect was observed. The four selected antagonists were characterized for features linked to biocontrol activity, such as biofilm, siderophores and biosurfactant-production, motility, and effect of environmental conditions on bacterial growth.

Effects of plant essential oils on growth and virulence factors of Erwinia amylovora

Erwinia amylovora causes fire blight in plants of economic importance in Rosaceae family. In this study, 11 essential oils were screened for their antibacterial activities and capabilities to influence the growth and virulence factors by the E. amylovora, such as amylovoran production, biofilm formation, and motility at non-lethal concentrations. Essential oils from Foeniculum vulgare and Pimpinella anisum showed strong antibacterial activity, those of Artemisia aucheri and Heracleum persicum had a moderate antibacterial activity. Oils of seven other plant species did not show substantial growth inhibition but could reduce the production of virulence factors in E. amylovora at non-lethal concentrations. Determining bacteriostatic and bactericidal concentrations enabled us to gather precise data about the antibacterial characteristics. The results suggested that at non-lethal concentrations, some essential oils reduced the effect of virulence factors of E. amylovora. An inverse correlation was found between amylovoran and biofilm production when the bacterium was treated with oils of H. persicum, P. anisum, C. arabica, J. horizentalis, C. aurantifolia and V. agnus-castus. The oils with low antibacterial activity had an insignificant effect on motility at non-lethal concentrations. Generally, Apium graveolens (celery seed) and Curcuma longa (turmeric) essential oils showed the optimum reduction in the effects of virulence factors of E. amylovora. These oils led to 41.71% and 30.17% reduction in the disease progression of E. amylovora on immature pear fruit, respectively and reduced disease progression in the shoot of pear seedlings with 26.9% and 16.7%. This study indicated that essential oils individually may be applied as environmentally safe compounds for controlling the fire blight disease.