Lamia Tafifet
email: lamia.tafifet@hotmail.fr

Aida Raio

Maria C Holeva

Redhouane Dikhai

Ould Chawki

Sophie Kouskoussa

Zoulikha Cesbron

Krimi

C O Kouskoussa

: Z Krimi

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

Keywords: Biocontrol, Erwinia amylovora, Fire blight, MLVA-VNTR

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Erwinia amylovora, the pathogen responsible for fire blight disease of many Rosaceae plants, is ranked among the most destructive bacterial plant pathogens [START_REF] Mansfield | Top 10 plant pathogenic bacteria in molecular plant pathology: top 10 plant pathogenic bacteria[END_REF]. This quarantine pest, assigned to the A2 list of the European and Mediterranean Plant Protection Organisation (EPPO), belongs to enterobacteria which includes Gram-negative, mobile, and facultative aero-anaerobic bacteria (EPPO 2013).

E. amylovora was the first bacterium described as the causative agent of a plant disease [START_REF] Burrill | New species of Micrococcus (bacteria)[END_REF]).

E. amylovora enters hosts mainly through the nectarthodes of flowers and establishes systemic infections [START_REF] Norelli | Fire blight management in the twenty-first century: Using new technologies that enhance host resistance in apple[END_REF]. The pathogen moves through the intercellular spaces of parenchyma, then along the xylem vessels, and incites necrosis [START_REF] Dellagi | Dual role of desferrioxamine in Erwinia amylovora pathogenicity[END_REF][START_REF] Piqué | Virulence factors of Erwinia amylovora: a review[END_REF]. The symptoms include dieback and blight of inflorescences, shoots, leaves, fruits, trunks as well as production of bacterial exudate [START_REF] Van Der Zwet | Fire blight: Its nature, prevention and control. A practical guide to integrated disease management[END_REF]. Symptoms resembling those of fire blight were observed in several pear orchards in different provinces in north Algeria and the presence of E. amylovora in these orchards was confirmed in 2011 by the national official control services (The Agriculture Ministry and National Institute of Plant Protection INPV). Phytosanitary measures have been introduced to control the disease in these areas [START_REF] Laala | Fire blight survey and first characterization of Erwinia amylovora isolates from Algeria[END_REF].

Treatment of plants with antibiotics could be employed for the control of fire blight, but their use in plant protection within the European Union has only been permitted on an emergency basis, under tightly restricted conditions [START_REF] Fried | Control of fire blight in Baden-Württenberg at the end of the streptomycin era[END_REF]. In Algeria, antibiotics are also prohibited. Their use is linked to an increased risk for selection of resistant strains of E. amylovora and other bacteria inhabiting orchards and the possible negative impact on the environment. Because of these limitations, the research interest in finding new solutions for fire blight control has increased, including the use of bacterial biocontrol agents [START_REF] Mikiciński | Antagonistic potential of Pseudomonas graminis 49M against Erwinia amylovora, the causal agent of fire blight[END_REF].

Several biopesticides using bacteria are available, such as Bacillus subtilis QST 713 [START_REF] Aldwinckle | Evaluation of control of fire blight infection of apple blossoms and shoots with SAR inducers, biological agents, a growth regulator, copper compounds, and other materials[END_REF]), B. subtilis BD170 (Broggini-Schärer et al. 2005), Pseudomonas fluorescens A506 [START_REF] Wilson | Interactions between the biological control agent Pseudomonas fluorescens strain A506 and Erwinia amylovora in pear flowers[END_REF] and Lactobacillus plantarum, however; they have a low efficacy against E. amylovora [START_REF] Roselló | Biological control of fire blight of apple and pear with antagonistic Lactobacillus plantarum[END_REF]. The main mechanism of action of these strains to control fire blight is based on competition through nutrient depletion and site colonization [START_REF] Cabrefiga | Mechanism of antagonism of Pseudomonas fluorescens EPS62e against Erwinia amylovora, the causal agent of fire blight[END_REF]. Research is also being carried out with the aim of improving the efficacy of these biopesticides as well as the selection of new effective strains with different mechanisms of action [START_REF] Pusey | Potential of osmoadaptation for improving Pantoea agglomerans E325 as biocontrol agent for fire blight of apple and pear[END_REF].

In this context, the aim of this work was to study the molecular diversity of Algerian E. amylovora population in pear orchards and identify possible biocontrol agents against them. To this end, we isolated and identified E. amylovora strains from different pear orchards of Northern Algeria. Analysis of genomic sequences of E. amylovora isolates was performed using VNTR markers, which have high discriminatory power that allows distinguishing different strains within a population [START_REF] Bühlmann | Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites[END_REF]. Multiple-Locus Variable number tandem repeat Analysis (MLVA) using VNTRs have been already used successfully to perform molecular typing of E. amylovora [START_REF] Bühlmann | Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites[END_REF]) and several other bacterial species such as Staphylococcus aureus [START_REF] Schouls | Multiple-locus variable number tandem repeat analysis of Staphylococcus Aureus: Comparison with pulsed-field gel electrophoresis and spatyping[END_REF]), Acinetobacter baumannii [START_REF] Pourcel | Identification of variablenumber tandem-repeat (VNTR) sequences in acinetobacter baumannii and interlaboratory validation of an optimized multiple-locus VNTR analysis typing scheme[END_REF], Mycobacterium tuberculosis [START_REF] Aandahl | A model-based Bayesian estimation of the rate of evolution of VNTR loci in Mycobacterium tuberculosis[END_REF]) and Pseudomonas aeruginosa [START_REF] Lashgarian | Multiple locus variable number tandem repeat analysis (MLVA) for typing Pseudomonas Aeruginosa isolated from urine samples of different patients[END_REF]. For these species, the VNTR technique has proved to be highly valuable in ecological, epidemiological and evolutionary studies. Knowledge of the E. amylovora population structure contributes to our understanding on how fire blight spreads over a geographical region.

Fire blight is difficult to control, as E. amylovora is able to rapidly spread in the plant and effective control methods are still lacking. Various mechanisms were proposed to explain the inhibitory effect of different bacterial antagonists against E. amylovora, including the production of toxic secondary metabolites and competition for nutrients and space [START_REF] Cabrefiga | Mechanism of antagonism of Pseudomonas fluorescens EPS62e against Erwinia amylovora, the causal agent of fire blight[END_REF]). Moreover, Pseudomonas fluorescens and Pantoea agglomerans strains showing high potential as biological control agents against fire blight, were shown to inhibit growth of E. amylovora through a mechanism related to the production of antibiotics [START_REF] Anderson | Emerging infectious diseases of plants: Pathogen pollution, climate change and agrotechnology drivers[END_REF][START_REF] Temple | Bioavailability of iron to Pseudomonas fluorescens A506 on flowers of pear and apple[END_REF]. With the aim to select effective biocontrol agents of fire blight, native Algerian strains that were isolated as endophytes of different spontaneous plants [START_REF] Krimi | Bacterial endophytes of weeds are effective biocontrol agents of Agrobacterium spp., Pectobacterium spp., and promote growth of tomato plants[END_REF] were tested in vitro as well as in vivo on pear fruits in this study to evaluate their efficacy against E. amylovora.

Material and methods

Field surveys and collection of samples from diseased plants

Surveys were carried out during spring and summer from 2014 to 2017 in orchards of Pyrus communis cv. Santa Maria located in an area of pear fruit production in the Mitidja zone (Northern Algeria) which was officially declared affected by fire blight in 2011 by the Ministry of Agriculture and National Institute of Plant Protection.

In these orchards, about 70% of trees, 15 to 20 years old, showed various typical fire blight symptoms, such as necrosis of shoots, flowers, leaf petioles and fruits, oozed shoots, and mummified fruits.

Oozing shoots, blossoms and leaves were collected from 14 orchards (farms) located in seven different provinces: Sidi Yahia, Oued El Alleug, Bensalah, L'Arbaa, Birtouta, Tessala El Merdja and Attatba (nine farms at Blida, three at Algiers and two at Tipaza) (Fig. 1). Plant material was placed in sterile plastic bags, labeled, transported to the laboratory and stored at 4 °C until isolation of bacteria that was performed during the same week of sampling.

Isolation and purification of E. amylovora strains

The collected samples of infected shoots and twigs were examined for the presence of exudates that developed during storage on the plant surface allowing easy isolation. Part of these exudates were suspended in 2 ml of sterile distilled water (SDW) and streaked on Levan and King B media [START_REF] Schaad | In plant pathologic bacteria, laboratory guide for identification[END_REF]. Plates were kept for 1 week at 27 °C and observed daily for bacterial growth. Single colonies of putative E. amylovora isolates showing mucoid, circular, white and domed morphology [START_REF] Yaich | Fire blight (Erwinia amylovora [Burrill] Winslow) in Morocco: importance, geographical distribution and characterization[END_REF] were selected and transferred to Levan medium [START_REF] Schaad | In plant pathologic bacteria, laboratory guide for identification[END_REF]).The purification step was repeated three to four times in order to obtain pure cultures [START_REF] Jones | Erwinia amylovora group[END_REF]. Pure isolates were maintained at 4 °C on Levan agar medium slants for further testing and in 30% glycerol solution at -80 °C for long term storage.

Biochemical characterization of bacterial isolates

Gram staining [START_REF] Suslow | Application of a rapid method for gram differenciation of plant pathogenic and saprophytic bacteria without staining[END_REF]) and a series of assays to determine biochemical features of the bacterial isolates, i.e. gelatin liquefaction, fluorescent pigment production on King's B medium, reducing substances from sucrose, utilization of citrate, oxidation/fermentation of glucose (O/F), catalase production, oxidase production, growth at 39 °C and indole prodution were carried out using the EPPO identification protocol (EPPO 2013). The phenotypic properties were determined according to the methods of [START_REF] Jones | Erwinia amylovora group[END_REF].

Serological test

The isolates were tested by the commercially available rapid lateral flow immunosorbent assay named 'AgriStrip test' (BIOREBA). For this assay, strips were inserted into tubes containing bacterial suspensions at the recommended concentration (10 6 cfu ml -1 ). Strains producing a red band on the strip were considered positive and identified as E. amylovora species.

Tobacco hypersensitivity test

The tobacco hypersensitivity (HR) test was performed on young leaves of Nicotiana tabacum cv. xanthi. The test was carried out using 24 h old bacterial cultures grown on Levan medium. For each isolate, a bacterial suspension (10 8 cfu ml -1 ) was infiltrated using a needle into parenchymatic tissues of tobacco leaves [START_REF] Vanneste | Bacteriophage mu as a genetic tool to study Erwinia amylovora pathogenicity and hypersensitive reaction on tobacco[END_REF]). Tobacco plants were kept at room temperature. A complete collapse of the infiltrated tissue within 24-48 h was considered a positive response.

Pathogenicity test on immature pear fruits Immature fruits collected from symptomless pear trees were inoculated following the protocol described by [START_REF] Zhao | Identification of Erwinia amylovora genes induced during infection of immature pear tissue[END_REF] with some modifications. Pear fruits were surface sterilized using 75% ethanol, wounded with a sterile scalpel (5 wounds/fruit) and inoculated with 5 μl of suspension (10 6 cfu ml -1 ) of the putative E. amylovora 37 pathogenic strains. As negative control, pear fruits were inoculated with 5 μl of SDW. Fruits were placed in plastic boxes containing sterile Whatman paper on the bottom, daily sprayed with SDW and incubated at 25-26 °C. The experiment was repeated four times. Symptoms were recorded over a period of 5 to 7 days. Production of bacterial exudates and/or development of necrotic areas around or underneath the inoculation point was recorded as a positive response [START_REF] Billing | Laboratory diagnosis of fire blight and bacterial blossom blight of pears[END_REF].

Duplex PCR

Thirty-seven strains with Erwinia amylovora biochemical features, identified as HR positive, and pathogenic on pear fruits, were tested by duplex PCR [START_REF] Hannou | Characterization of Erwinia amylovora strains from middle Atlas Mountains in Morocco by PCR based on tandem repeat sequences[END_REF]. Forty-eight hour-old bacterial colonies were picked-up from Levan agar medium, transferred into Levan broth and incubated at 28 °C overnight on a rotary shaker. The bacterial cultures were centrifuged for 10 min at 14000 rpm and the pelleted bacterial cells were resuspended in SDW. Cells suspensions in SDW were lysed by heating for 15 min at 95 °C, quickly cooled on ice and centrifuged for 5 min at 7000×g. The obtained supernatants were used to perform amplification by duplex PCR. The duplex PCR protocol was carried out using two pairs of primers: a) AJ75 (5-CGTATTCACGGCTTCGCAGAT) and AJ76 (5-ACCCGCCAGGATAGTCGCATA), a pair that amplifies an 844-bp fragment of the 29-kb plasmid pEA29 of E. amylovora [START_REF] Mcmanus | Detection of Erwinia amylovora by nested PCR and PCR-dot-blot and reverse-blot hybridizations[END_REF], and b) AMSbL (5-GCTACCAGCAGGGTGAG-3) and AMSbR (5-TCATCACGATGGTGTAG -3), a pair that amplifies a 1.6-Kb fragment of the chromosomic amsregion of E. amylovora [START_REF] Bereswill | Identification of the fire blight pathogen, Erwinia amylovora, by PCR assays with chromosomal DNA[END_REF]. Sterile water and genomic DNA extracted from the French E. amylovora strain CFBP 1430 were used as negative and positive amplification controls, respectively. PCR amplification was performed as described in [START_REF] Hannou | Characterization of Erwinia amylovora strains from middle Atlas Mountains in Morocco by PCR based on tandem repeat sequences[END_REF].

The PCR program included an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 45 s at 94 °C for denaturation, 45 s at 52 °C for annealing, and 1 min and 45 s at 72 °C for extension; with a final extension at 72 °C for 10 min [START_REF] Hannou | Characterization of Erwinia amylovora strains from middle Atlas Mountains in Morocco by PCR based on tandem repeat sequences[END_REF]. Amplified fragments were loaded on 1,5% agarose gel to be separated by electrophoresis. VNTR analyses.

Twenty-five selected E. amylovora strains isolated in this study were analyzed using six VNTRs primers described in [START_REF] Bühlmann | Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites[END_REF] applying a Multiple-Locus VNTR Analysis (MLVA). Bacterial DNA templates were obtained using a direct lysis method, as previously described in [START_REF] Bühlmann | Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites[END_REF], of overnight cultures grown on King's B medium at 28 °C. DNA-containing supernatants (1 μl) were used as the template in the PCR reaction mixtures containing 12.8 μl ultrapure water, 4 μl GoTaq Flexi 5X buffer (Promega, Fitchburg, WI, USA), 1.2 μl MgCl 2 25 mM, 0.5 μl dNTP 10 mM, 0.25 μl each primer 10 μM, 0.05 μl (0.25 u) GoTaq Flexi DNA polymerase (Promega) in a final volume of 20.05 μl. The amplification was performed in a GeneAmp PCR system model 9700 (Applied Biosystems, Courtaboeuf, France) following the steps: 95 °C for 5 min, 32 cycles denaturation at 95 °C for 30 s, annealing for 30 s at a temperature depending on the VNTR primer, elongation at 72 °C for 30 s, and a final elongation step at 72 °C for 10 min.

The VNTR primers described by [START_REF] Bühlmann | Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites[END_REF] were used at the following annealing temperatures: 60 °C for VNTR A, B and H, 50 °C for VNTR C, 52 °C for VNTR D and 56 °C for VNTR F (Online Resource 1).

The VNTR primers were labelled with one of the following fluorescent dyes: Atto550 for VNTR A, HEX for VNTR B and D, FAM for VNTR C and H and Atto565 for VNTR F (Eurofins Genomics, France) at the 5′ end. Up to four differently labelled PCR products were diluted 1/30 and pooled to prepare the DNA samples. Then, 2.4 μL aliquots of the DNA samples were mixed with 9.35 μL HiDi formamide (SigmaAldrich, Saint Quentin, France) and 0.15 μL GeneScan 500 Liz internal lane size standard (Applied Biosystems) and denatured at 95 °C for 5 min prior to capillary electrophoresis analysis with an ABI PRISM 3130 analyzer (Applied Biosystems).

Peak data were analyzed using Peakscanner™ Software V1.0 (Life Technologies) to calculate the repeat number for each VNTR locus on the basis of fragment length and fluorescent label [START_REF] Bühlmann | Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites[END_REF]). Simpson's index of diversity [START_REF] Hunter | Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity[END_REF] was calculated using Bionumerics Software V7.6.3 (Applied Maths). Minimum-spanning-trees (MST) were generated using Bionumerics Software V7.6.3 using the standard categorical coefficient and the maximum number of single-locus variants as a priority rule.

The categorical coefficient discriminates between different repeat numbers, however it does not quantify the difference. Equal weight was assigned to each of the retained VNTR loci.

Selection of effective antagonistic strains against E. amylovora and their characterization

Potential antagonistic Pseudomonas and Bacillus species evaluated in this study belong to the collection of the phytobacteriology laboratory of Blida University, Algeria. All strains were endophytes isolated from various spontaneous weeds (Calendula arvensis, Euphorbia helioscopia, Euphorbia peplus, Plantago lanceolata and Urtica dioica) showing antagonistic activity against different phytopathogenic bacteria [START_REF] Krimi | Bacterial endophytes of weeds are effective biocontrol agents of Agrobacterium spp., Pectobacterium spp., and promote growth of tomato plants[END_REF].

Twenty Pseudomonas and Bacillus strains from the previously mentioned bacterial collection (Blida University, Algeria) were screened for in vitro antagonistic activity against pathogenic E. amylovora strains by the dual culture assay, i.e. a plate-spraying technique [START_REF] Stonier | Agrobacterium tumefaciens Conn. II. Production of an antibiotic substance[END_REF] as modified by [START_REF] Cooksey | Biological control of crown gall with fungal and bacterial antagonists[END_REF]. A volume of 100 μl of suspension (10 7 cfu ml -1 ) of each potential antagonistic strain was spotted at the center of YPGA medium [START_REF] Hildebrand | Pseudomonas[END_REF]). The plates were incubated for 48 h at room temperature (RT). After incubation, the plates were sprayed with cell suspensions of two E. amylovora strains E1 and E5, isolated and characterized in this study. Inoculum of pathogens was prepared from late log-phase cultures grown in YPG broth on a rotary shaker at 25 ± 2 °C and diluted to about 1 × 10 8 cells ml -1 in SDW. The plates were let again at RT for 24-48 h. Generally in this kind of assay, the antibacterial agent from the antagonistic strain diffuses into the agar and inhibits growth of the test microorganism. Antagonism is detected as the presence of a zone of growth inhibition of the test microorganism around the growth of the spotted antagonistic strain [START_REF] Cabrefiga | Mechanism of antagonism of Pseudomonas fluorescens EPS62e against Erwinia amylovora, the causal agent of fire blight[END_REF], and then the diameter of growth inhibition zones are measured using a graduated precision rule (mm) [START_REF] Lindow | Lack of correlation of in vitro antibiosis with antagonism of ice nucleation active bacteria on leaf surfaces by non-ice nucleation active bacteria[END_REF].

The test was repeated three times for each of the twenty antagonistic strain tested. Antagonistic strains able to inhibit both pathogenic strains E1 and E5 were tested for biocontrol of fire blight on immature pear fruits. For the negative control, the plates were sprayed with cell suspensions of E. amylovora strains E1 and E5 without any antagonistic strain.

Bacteria motility, siderophore, biofilm and biosurfactant production Motility helps bacteria to reach the most favorable environment and to successfully compete with other microorganisms [START_REF] Harshey | Bacterial motility on a surface: many ways to a common goal[END_REF] and then may represent a remarkable competitive advantage for putative biocontrol agents. Tests to evaluate swimming and swarming motility of the four selected antagonistic strains (CR2, EHF5, PF3, and PS1) were performed according to the protocol of [START_REF] Déziel | Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with the emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming and twitching motilities[END_REF]. A volume of 2 μl of each bacterial suspension (10 8 cfu ml -1 ) was spotted in the center of plates containing tryptone swim and swarm agar media, respectively [START_REF] Déziel | Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with the emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming and twitching motilities[END_REF]. For each strain, three plates per medium were seeded. After 24 and 48 h of incubation at 25 ± 2 °C, two radius measurements per colony were made. Pseudomonas chlororaphis M71 and E. coli DH5α were used as positive and negative controls, respectively. The experiment was carried out twice.

To evaluate the ability to produce siderophores, a volume of 200 μl of overnight cultures of the four selected antagonistic bacterial strains (10 8 cfu ml -1 ) was added to 50 ml of iron-free succinate and KB broth media respectively [START_REF] Sasirekha | Siderophore production by Pseudomonas aeruginosa FP6, a biocontrol strain for Rhizoctonia solani and Colletotrichum gloeosporioides causing diseases in chilli[END_REF]. The flasks were incubated at room temperature for 72 h on a horizontal shaker. After centrifugation for 10 min at 10,000 rpm, the absorbance of supernatants were measured at 400 nm using spectrophotometer [START_REF] Meyer | The fluorescent pigment of Pseudomonas fluorescens: biosynthesis, purification, and physicochemical properties[END_REF]. The experiment was performed twice. The medium without bacteria was used as negative control. Bacterial growth and siderophore production in KB medium were compared to those in succinate medium containing ammonium sulphate [START_REF] Tailor | Characterization and optimization of siderophore production from Pseudomonas fluorescens strain isolated from sugarcane rhizosphere[END_REF]. The negative control was used as reference to quantify the difference between absorbance in KB medium and succinate medium. To characterize the nature of siderophores, 5 ml of 2% aqueous solution of FeCl3 were added to each supernatant and OD was determined at 490-515 nm for catecholate [START_REF] Jalal | Isolation and spectroscopic identification of fungal siderophores[END_REF] and at 420 to 450 nm for hydroxamate [START_REF] Neilands | Microbial iron compounds[END_REF]. Non-inoculated media were used as reference.

The ability of the antagonistic strains to produce biofilm was tested by adding 15 μL of an overnight culture of strains EHF5, PF3, CR2 and PS1 (10 8 cfu ml -1 ) to 0.5 ml of Luria-Bertani (LB) broth. Suspensions were incubated without agitation at 28 °C and biofilm was quantified after 3, 5 and 20 h. LB medium without bacteria was used as negative control. The experiment was repeated 3 times. Cultures were stained with 1 ml of 0.4% (w/v) crystal violet (CV) for 20 min at RT and then rinsed thoroughly and vigorously with distilled water to remove unattached cells and residual dye. One volume of ethanol (70%) was used to solubilize the dye. The absorbance of the solubilized dye was determined by a spectrophotometer at 570 nm wavelength (O'Toole and Kolter 1998; [START_REF] Stepanovic | A modified microtiter-plate test for quantification of staphylococcal biofilm formation[END_REF].

The ability to produce biosurfactant compounds was determined by the drop collapsing test described by [START_REF] Bunster | Effect of surface-active Pseudomonas spp. on leaf wettability[END_REF] with some modifications. Strains were grown in KB broth amended with 1% of yeast extract for 48 h at 28 °C on a rotary shaker. Bacterial cultures were then centrifuged and the supernatant was filter-sterilized. An amount of 10 μl of each supernatant was then placed on a parafilm strip and mixed with 1 μl of 1% bromophenol blue solution. P. chlororaphis M71 strain and sterile KB broth were used as positive and negative controls respectively. The strip was covered with a Petri dish and kept in humid and sterile conditions. The diameter, size, and shape of drops were observed after 24 and 36 h. Flat drops with a diameter size wider than negative control indicates biosurfactant production by the tested strains.

Effect of environmental factors on bacterial growth

The effect of temperature, pH and NaCl concentration was determined by growing the antagonists on LB agar plates. Bacteria were streaked on agar surface and incubated at 4 °C, 30 °C, 35 °C, 42 °C and 45 °C for 24 to 48 h and then checked for growth [START_REF] Rhodes | The cytology of Pseudomonas spp. as revealed by a silver-plating staining method[END_REF]. Bacterial growth at 27 °C was used as reference temperature. To determine the effect of pH, bacteria were streaked on LB agar plates with pH 4, 6, 8 and 10. PH 7 was considered as the reference value. To test salt tolerance, the following amounts of NaCl were added to LB agar: 0.3 M (17.58 g l -1 ), 0.5 M (29.3 g l -1 ), 0.8 M (46.88 g l -1 ) and 1 M (58.6 g l -1 ) NaCl. Three replicates were used per sample. LB medium supplemented with 0.17 M (10 g l -1 ) of NaCl [START_REF] Rabhi | Isolement de Pseudomonas spp. fluorescents d'un sol salé. Effet d'osmoprotecteurs naturels[END_REF] was used as reference.

To study the effect of osmotic stress on antagonist growth, nutrient broth (NB) supplemented with different concentrations of PEG 8000 (20%, 40% and 60%) was used. Tubes containing 5 ml of supplemented NB were inoculated with 100 μl of each bacterial suspension and incubated at RT on a horizontal shaker for 72 h. Three replications per sample were maintained. Non-supplemented NB broth was used as negative control. Strain growth was determined by measuring the O.D. at 600 nm with a spectrophotometer [START_REF] Manjunatha | Evaluation of endophytic bacteria for their influence on plant growth and seed germination under water stress conditions[END_REF].

Fire blight control by Bacillus and Pseudomonas strains Immature pear fruits cv. Santa Maria were used to determine the effectiveness of the bacterial antagonists in controlling locally pathogenic E. amylovora strains. Fruits were washed under running tap water, superficially disinfected with ethanol and then rinsed three times in SDW. Five wounds of 10 mm long and 3 mm deep were made on each fruit by using a sterile syringe needle. The fruits for each antagonist/pathogen combination were inoculated with 50 μL of 10 8 cfu ml -1 suspension of each antagonistic strain and 50 μl of 10 7 cfu ml -1 of the suspension of the pathogenic strains. Pear fruits wounded and treated with SDW were used as negative control. Treatments were arranged in a completely randomized design with two replications per treatment, five pear fruits per replicate and five wounds per fruit. Disease incidence was assessed for each repetition.

The biocontrol activity was tested by treating the wounds with the antagonists 24 h before or 24 h after inoculation with two strains of E. amylovora (E1, E5), to evaluate their preventive and curative activity, respectively. After inoculation, pear fruits were placed in sterile containers at RT. One week after inoculation the presence or absence of the necrotic lesions and exudate production were recorded. Disease severity was evaluated considering the size of fruit necrosis and production of bacterial exudates according to the following disease index: 0: no necrosis, 1: necrosis formation around the wound without ooze production, 2: necrosis of less than 50% of fruit surface with ooze production, 3: necrosis of 50%-80% of fruit surface with ooze production, 4: 100% fruit necrosis and ooze production [START_REF] Ohike | In vitro and in vivo assay for assessment of the biological control potential of Streptomyces sp[END_REF]).

Results

Isolation of E. amylovora strains and their characterization by biochemical, serological and pathogenicity assays Thirty-seven bacterial strains isolated from the samples of pear shoots and twigs collected during the field survey, which formed E. amylovora-like colonies, were selected for further study. The colonies were mucoid, white, shiny, domed, with radial stripes and dense flocculent centers on Levan medium [START_REF] Billing | Characteristics of English isolates of Erwinia amylovora (Burrill) Winslow et al[END_REF][START_REF] Ashmawy | Pathological and molecular studies on Erwinia amylovora the causal agent of fire blight disease[END_REF],. The isolates were Gram-negative, oxidase-negative, catalase-positive, citrate utilizers, gelatin degraders, non-fluorescent on King's B medium and sucrose reducers. Moreover, they did not grow at 39 °C (Online Resource 2). Serological test of the 37 isolates were positive for E. amylovora, and all isolates produced a positive HR reaction on tobacco leaves. Pathogenicity test on all isolates induced necrosis and brownish coloration of tissues surrounding the artificially inoculated wounds. Within two to three days after inoculation, symptoms spread to the whole fruit and ooze production was observed. SDW negative control did not cause any symptom on the pear fruits.

Molecular identification of E. amylovora isolates

There was amplification of the 844-bp fragment of pEA29 plasmid with AJ75/AJ76 primers (McManus and Jones 1995) in all 37 isolates, suggesting the ubiquitous presence of this plasmid in the tested strains. In addition, all strains (Online Resource 3) produced a clearly visible 1.6-kb band corresponding to the ams chromosomal region with the AMSbL/AMSbR primers [START_REF] Bereswill | Identification of the fire blight pathogen, Erwinia amylovora, by PCR assays with chromosomal DNA[END_REF].

A variable number of tandem repeat sequences (VNTR) assay was used successfully to genotype 25 selected E. amylovora strains. Only two loci (A and F) were polymorphic with repeats per locus ranging from five to seven for locus A and from six to seven for locus F (Table 1).

Minimal spanning tree analysis (MST) showed that four haplotypes were present within the 25 tested isolates that differed for one VNTR repeat number. The analysis did not originate distinct groups on the basis of year of isolation. (Fig. 2). One distinct haplotype was instead found only from one farm of Attatba province when the analysis was performed considering the geographical origin of strains (Fig. 3).

Compared to data published in [START_REF] Bühlmann | Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites[END_REF], strains 40b, 44, 45, 46 shared the same haplotype with strain NIBZ-1058 isolated from Pyrus in 2007 in Slovenia. Other Algerian strains harboured new haplotypes when compared to Bühlmann's data. In vitro selection of bacteria antagonistic to E. amylovora strains All twenty potential antagonistic strains screened in this study by the dual culture assay inhibited the growth of both E. amylovora strains (E1 and E5) in vitro, with inhibition zones ranging from 18 mm for Bacillus amyloliquefaciens CR1 to 53 mm for B. methylotrophicus PF3 (Online Resource 4. Among this group of antagonistic strains, four were selected for further study, including a) two Bacillus s t r a i n s ( B . a m y l o l i q u e f a c i e n s E H F 5 a n d B. methylotrophicus PF3) that produced wide inhibition zones as well as presenting traits such as rapid growth and colonization, aggressiveness towards other microorganisms and adaptation to environmental stress, making them good potential biocontrol a g e n t s , a n d b ) t w o P s e u d o m o n a s s t r a n s (P. brassicacearum CR2 and PS1) that had been shown also in previous work [START_REF] Krimi | Bacterial endophytes of weeds are effective biocontrol agents of Agrobacterium spp., Pectobacterium spp., and promote growth of tomato plants[END_REF]) to be interesting beneficial strains with potential biocontrol and growth promotion activities.

Characterization of antagonists

The swimming ability of EHF5, PF3 and CR2 strains at 24 h was comparable to that of the negative control (E. coli), while PS1 showed a swimming activity significantly higher than the negative control but lower than the positive control (P. chlororaphis) (Fig. 4a). After 48 h of incubation all four strains were able to swim, even though Pseudomonas strains were slower than Bacillus and P. chlororaphis. There were no significant differences in swarming ability among the four antagonistic strains at 24 h of incubation, however; after 48 h of incubation, Bacillus colonies covered the whole agar surface while Pseudomonas colonies did not show any swarming ability (Fig. 4b).

The four strains showed a different ability to produce siderophores on KB and succinate media. Strain EHF5 was the most active siderophore producer on KB medium without ferric chloride while strains CR2 and PS1 produced catecholate and hydroxamate siderophore type on KB medium amended with FeCl 3 respectively (Fig. 5). On succinate medium amended with FeCl 3, all strains produced fewer siderophores than on succinate without ferric chloride (Online Resource 5). The four antagonistic strains produced biofilm in vitro. No significant difference was observed among the strains after 3 and 5 h of incubation. EHF5 was the most active after 5 h of incubation and did not differ from PS1 strain at 20 h of incubation. After 20 h of incubation, strain CR2 was the most active biofilm producer. Conversely, PF3 showed a low ability to produce biofilm (Fig. 6).

The drop collapsing test showed that only the two Bacillus strains were able to produce biosurfactant compounds determining the spread of bacterial suspension over the solid surface (results not shown). The drops for these two strains appeared larger and flatter than the negative control (up to 7-8 mm diameter vs 4-5 mm of the negative control). On the contrary, both Pseudomonas strains did not produce biosurfactant compounds in this test since the diameter and shape of their drops were similar to those of the negative control.

Effect of environmental factors on the growth of antagonists

The antagonistic strains grew at pH values 4-8 (except for CR2 strain that did not grow at pH 4.0) and did not grow at pH 10. All strains grew at temperature ranging between 30 and 42 °C, while at the temperatures of 4 °C and 45 °C, no growth was recorded. The two Pseudomonas strains where not inhibited by concentrations of NaCl ranging between 0,3 M and 1,0 M, while both Bacillus strains did not grow at 1.0 M NaCl (Table 2).

Strain tolerance to osmotic stress decreased with increasing concentrations of PEG. In the presence of 20% PEG, growth of the two pseudomonads was reduced more than 80% while growth of Bacillus spp. was reduced by approximately 70%, indicating that all strains were very sensitive to osmotic stress. At a concentration of 40% PEG, bacterial growth was further reduced, and at a concentration of 60% PEG, absorbance values of broth cultures were close to zero. No significant differences were found among the different strains (Fig. 7).

Fire blight control by Bacillus and Pseudomonas strains

The strains Bacillus methylotrophicus PF3, B. amyloliquefaciens EHF5, P. brassicacearum CR2 and PS1 were selected to test their ability to control fire blight. These strains were selected based on results for the in vitro evaluation of their antagonistic activity, their bio-control linked performances, and their resistance to abiotic factors.

A significant reduction of disease symptoms (P ≤ 0.05) was recorded when E. amylovora was inoculated on pear fruits after wound protection with the Bacillus and Pseudomonas strains under study (Fig. 8). Pseudomonas strains CR2 and PS1 were the most effective against both E. amylovora strains used, with symptoms on pear fruits reduced to a necrotic area of few millimeters surrounding the wounds and without bacterial exudate (disease index 1) (Fig. 9). Bacillus strains also caused a significant reduction of disease symptoms, but the efficacy was lower than Pseudomonads (disease index 2). The effect of antagonists on the necrosis spread and ooze production was not significant when the E. amylovora strains (E1 and E5) were inoculated 24 h before the wound protection (data not shown).

Discussion

Fire blight has been reported since 2010 in Tipaza and Oued El Alleug provinces of Algeria on pear, apple and [START_REF] Laala | Fire blight survey and first characterization of Erwinia amylovora isolates from Algeria[END_REF].

Several plasmids (pEA29, pEI70, pEA34, pEA8.7, pCPP60, pEA72, pEL60, pEA2.8, and pEU30) have been identified in E. amylovora strains (McGhee and Jones 2000; [START_REF] Foster | Nucleotide sequences, genetic organization, and distribution of pEU30 and pEL60 from Erwinia amylovora[END_REF]). However, pEA29 is the plasmid playing a significant quantitative role in pathogenicity [START_REF] Laurent | Characterization of an ubiquitous plasmid in Erwinia amylovora[END_REF]. Indeed, sequences of this plasmid are used for genetic differentiation of E. amylovora strains [START_REF] Sebaihia | Complete genome sequence of the plant pathogen Erwinia amylovora strain ATCC 49946[END_REF].

In this study, 37 strains isolated from diseased pear trees in orchards located in different areas of North Algeria were identified as E. amylovora species by biochemical tests, PCR based methods, a serological identification test (Agristrip) and pathogenicity assay. AJ75 /AJ76 primers annealing to plasmid pEA29 and AMSbL /AMSbR primers annealing to ams chromosomal region were used for molecular identification of these strains. At the same time, the ubiquitous presence of the plasmid pEA29 in the Algerian strains tested was confirmed.

Genetic diversity of E. amylovora is low given the relatively recent global spread of this pathogen. Indeed, at genome level, 99.4% identity was found within the Rubus-infecting strains and 99.98% within the Spiraeoideae-infecting strains [START_REF] Mann | Comparative genomics of 12 strains of Erwinia amylovora identifies a pan-genome with a large conserved core[END_REF]. The genetic diversity of E. amylovora has been studied in Morocco, Tunisia and Iran by VNTR analysis [START_REF] Hannou | Characterization of Erwinia amylovora strains from middle Atlas Mountains in Morocco by PCR based on tandem repeat sequences[END_REF][START_REF] Dardouri | Molecular characterization of Tunisian strains of Erwinia amylovora[END_REF][START_REF] Refahi | Exploring possible variation among Iranian Erwinia amylovora strains using multilocus typing and tandem repeat analysis[END_REF]) using the same VNTRs as described in [START_REF] Hannou | Characterization of Erwinia amylovora strains from middle Atlas Mountains in Morocco by PCR based on tandem repeat sequences[END_REF]. Additional MLVA analysis [START_REF] Alnaasan | Modification of a multiple-locus variable number tandem repeat analysis (MLVA) for typing isolates of Erwinia amylovora[END_REF]) was performed with the primers described by [START_REF] Bühlmann | Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites[END_REF] to study strains from the Mediterranean area. No database is publicly available for MLVA on Erwinia amylovora. In this study, VNTR analysis differentiated four haplotypes within the Algerian E. amylovora strains identified. Previous analysis performed on strains isolated in North Africa showed that five different genotypes were present within the Tunisian collection of E. amylovora [START_REF] Dardouri | Molecular characterization of Tunisian strains of Erwinia amylovora[END_REF] while two distinct patterns were found among the Moroccan E. amylovora strains [START_REF] Hannou | Characterization of Erwinia amylovora strains from middle Atlas Mountains in Morocco by PCR based on tandem repeat sequences[END_REF]. [START_REF] Bühlmann | Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites[END_REF] showed the presence of 227 haplotypes by studying a total of 833 E. amylovora strains of worldwide origin in a work based on MLVA analysis. One of these strains originated from Slovenia shared the same haplotype as four Algerian isolates of the present study. According to [START_REF] Alnaasan | Modification of a multiple-locus variable number tandem repeat analysis (MLVA) for typing isolates of Erwinia amylovora[END_REF], Italian isolates of E. amylovora showed a high diversity which let hypothesize multiple events of introduction of the bacterium from several countries. Two of these isolates were closely related to strains from Algeria and Egypt [START_REF] Alnaasan | Modification of a multiple-locus variable number tandem repeat analysis (MLVA) for typing isolates of Erwinia amylovora[END_REF]). This suggested that E. amylovora could have been introduced in Italy and Algeria during the same period even though no official report from Algeria is available. It is possible that the high volume of traded plants and the inherent difficulty to intercept latently infected plants, have contributed to the introduction and spread of E. amylovora in these Mediterranean countries. Further comparisons with isolates from other countries in North Africa are needed to conclude on the routes and the number of introductions in Algeria.

Regardless of the E. amylovora diversity present in each country and the pathways of pathogen's introduction in new areas, sustainable management measures to address plant infections by this disastrous pathogen are urgently needed. To this respect, research on biological control agents has been initiated in the past, including various Pseudomonas and Bacillus strains.Pseudomonas spp. are very ubiquitous, colonize a wide variety of ecological niches, and can be isolated from water, soil and plants. They grow in temperatures ranging from 4 °C to 42 °C [START_REF] Chakravarty | The genus Pseudomonas[END_REF]. In this study, the Pseudomonas strains did not grow at 4 °C and 45 °C, which are extreme temperatures, apart from CR2 (P. brassicacearum) which grew at 4 °C. Species of the genus Bacillus possess significant physiological and metabolic diversity that allow them to survive in extreme habitats, they can be thermophilic and psychrophilic and are capable at growing at pH, temperatures, and salt concentrations [START_REF] Awais | Isolation, identification and optimization of Bacillus sp[END_REF].

Pseudomonas accumulate from the external environment or by synthesis of several varieties of compatible solutes. Glutamate and trehalose are used by Pseudomonas species during growth in a medium of high osmolarity [START_REF] Souza-Ault | Roles of N-Acetylglutaminylglutamine amide and glycine betaine in adaptation of Pseudomonas aeruginosa to osmotic stress[END_REF][START_REF] Pocard | A prominent role for glucosylglycerol in the adaptation of Pseudomonas mendocina SKB70 to osmotic stress[END_REF]. Pseudomonads are able to survive and multiply in pH between 6 and 8. However, their survival is low in acidic medium such as pH 4 [START_REF] Garrity | The Proteobacteria -part B: The Gammaproteobacteria[END_REF]. The Bacillus have physiological properties and varied habitats [START_REF] Awais | Isolation, identification and optimization of Bacillus sp[END_REF] shown with PF3 and EHF5 (acidophilic) but alkaline-sensitive at basic pH 10 for all strains.

Strains of Bacillus were moderately halotolerant, tolerating up to 0.8 M NaCl, However, Psedomonas strains were more halotolerant they grow at 1 M NaCl. Psedomonas are particularly resistant to hyper-osmotic shock in LB medium. There is a synergistic effect between the different osmoprotective agents of the LB medium which explains the high yield of growth at high osmolarity [START_REF] Ghoul | Halotolerance de Escherichia coli: effet des osmoprotecteurs naturels[END_REF]). For Bacillus, the sudden decrease in the activity of cytoplasmic water induces severe plasmolysis. It inhibits cellular functions such as nutrient absorbtion, DNA replication and biosynthesis of macromolecules [START_REF] Csonka | Physiological and genetic responses of bacteria to osmotic stress[END_REF].

In this study, four strains were identified that strongly inhibited E. amylovora in vitro (dual culture) and in vivo (on pear fruits), and their biocontrol performances characterized. Even though several studies have shown that the best performing strains in the in vitro assay to evaluate antagonistic effect, are not always the most effective biocontrol agents in vivo [START_REF] Ülke | Biological control studies on fire blight caused by Erwinia amylovora (Burr.) Winslow et al[END_REF][START_REF] Mikiciński | Selection of bacteria from epiphytic populations on apple trees and soil environment for ability to control fire blight (Erwinia amylovora)[END_REF], this in vitro assay still represents the best technique to screen among large number of putative antagonistic bacterial strains. The two P. brassicacearum strains as well as the B. amyloliquefaciens and B. methyloptrophicus strains, selected based on their in vitro antagonistic activity and other known characteristics, showed a variable biocontrol efficacy against E. amylovora on artificially inoculated immature pear fruits. This pathogenicity test is a very reliable tool both for biocontrol and virulence studies [START_REF] Cabrefiga | Analysis of aggressiveness of Erwinia amylovora using disease-dose and time relationships[END_REF]. Both P. brassicacearum strains were highly effective (more than the Bacillus strains) in reducing symptom development (small necrosis size and suppression of bacterial exudates production) on pear fruits, even though in dual culture test the Bacillus strains. Had shown the best inhibiting activity. [START_REF] Gerami | Evaluation of some bacterial antagonists for biological control of fire blight disease[END_REF] also showed that the bacterial antagonists Pseudomonas putida and Serratia marcescens were effective in planta but not in vitro. The discrepancy may be related to biocontrol mechanisms other than antibiosis such as competition for space and nutrients, siderophore and nonvolatile inhibitory metabolites production [START_REF] Stockwell | Enhancement of biocontrol of fire blight with an iron chelate[END_REF][START_REF] Temple | Bioavailability of iron to Pseudomonas fluorescens A506 on flowers of pear and apple[END_REF]. Competition is a mechanism effective against E. amylovora reported both for P. fluorescens A506 [START_REF] Wilson | Interactions between the biological control agent Pseudomonas fluorescens strain A506 and Erwinia amylovora in pear flowers[END_REF] as well as P. fluorescens EPS62e [START_REF] Cabrefiga | Mechanism of antagonism of Pseudomonas fluorescens EPS62e against Erwinia amylovora, the causal agent of fire blight[END_REF]. Siderophores produced by strain Pseudomonas graminis 49 M strongly inhibited E. amylovora growth in vitro, but clear evidence of their role in the biocontrol of the pathogen in vivo have not yet been found [START_REF] Mikiciński | Antagonistic potential of Pseudomonas graminis 49M against Erwinia amylovora, the causal agent of fire blight[END_REF]. Similarly to our results, there are several reports on the ability of antagonistic bacteria of Pseudomonas spp. in suppressing fire blight disease [START_REF] Mercier | Field performance of antagonistic bacteria identified in a novel laboratory assay for biological control of fire blight of pear[END_REF][START_REF] Bahadou | New potential bacterial antagonists for the biocontrol of fire blight disease (Erwinia amylovora) in Morocco[END_REF].

In this research line, further work is under way to determine the mechanisms of action underlying the in vivo biocontrol of E. amylovora. Pseudomonas CR2 and PS1 strains, that were more effective than Bacillus in controlling the pathogen on immature pear fruits, produced hydroxamate and catecholate siderophores on KB medium, were able to swim but not swarm on appropriate agar media and were more active producers of biofilm than Bacillus strains. The high siderophore production in succinate medium may be related to the production of pyoverdine in which the three amino moieties of the chromophore are replaced by various acyl groups derived from succinate, malate, α-keto-glutarate [START_REF] Linget | Bacterial siderophores: the structure of a desferriferribactin produced by P. fluorescens ATCC 13525[END_REF][START_REF] Sasirekha | Siderophore production by Pseudomonas aeruginosa FP6, a biocontrol strain for Rhizoctonia solani and Colletotrichum gloeosporioides causing diseases in chilli[END_REF]. This kind of siderophore may act also as an antibiotic [START_REF] Kraus | Characterization of a genomic region required for production of the antibiotic pyoluteorin by the biological control agent Pseudomonas fluorescens Pf-5[END_REF]. Biofilm formation is another important feature for bacteria to succeed during colonization of plant surfaces and intercellular spaces since it increases their survival and activity and allows water retention [START_REF] Zhang | Measurement of polysaccharides and proteins in biofilm extracellular polymers[END_REF]. Biofilms are made up mostly of exopolysaccharide (EPS), proteins, lipids and nucleic acids [START_REF] Davey | Microbial biofilms: from ecology to molecular genetics[END_REF] and protects bacteria against unfavorable environmental conditions (Flemming 1993). The significantly higher ability of the two Pseudomonas strains to form biofilm in comparison with the Bacillus spp. strains may be one important trait linked to its biocontrol activity. Biofilm formation, production of siderophores and antibiotic synthesis are regulated by the quorum sensing (QS) system. This is a mechanism of intercellular signaling among bacteria that operates through small molecular weight QS signals to regulate the transcription of target genes [START_REF] Maddula | Quorum sensing and phenazines are involved in biofilm formation by Pseudomonas chlororaphis (aureofaciens) strain 30-84[END_REF].

QS also affects motility and biosurfactants production of bacteria that are considered two important activities for a bacterial biocontrol agents. In particular, swarming motility is involved in colonization process of leaf and root surface and is described as a movement of bacteria in groups of tightly bound cells [START_REF] Harshey | Bacterial motility on a surface: many ways to a common goal[END_REF]. Both Bacillus strains showed higher swimming and swarming activity than the two Pseudomonas strains and were able to produce biosurfactants; nevertheless their in vivo biocontrol activity was less effective than that of Pseudomonas strains. The in vivo biocontrol efficacy of microbial antagonists is the result of the activation of several mechanisms which interact with the target pathogen, the autochthonous microflora, the host genotype and the environmental conditions. Given the complexity of these interactions, the result is not always predictable on the basis of the information provided by the experimental in vitro trials. On the other hand, it is also possible that further powerful mechanisms as 2,4-diacetylphloroglucinol (DAPG) and hydrogen cyanide (HCN), not investigated in this work, are involved in biocontrol of E. amylovora by P. brassicacearum strains (Paulin et al. 2017).

Extreme conditions of pH, temperature, salt concentration and osmolarity were equally tolerated by the Bacillus spp. and P. brassicacearum strains showing their ability to face stressing environmental conditions.

In the light of the results obtained during this study, it is reported here that E. amylovora is widespread in Northern Algeria orchards and that P. brassicacearum could constitute a potential biocontrol agent to be exploited against this pathogen. P. brassicacearum has been already reported as an effective biological control agent against Sclerotinia sclerotiorum, Phytophthora cactorum, Verticillium dahliae and also Clavibacter michiganensis subsp. michiganensis. [START_REF] Loewen | Genome sequence of Pseudomonas brassicacearum DF41[END_REF][START_REF] Novinscak | C o m p l e t e g e n o m e s e q u e n c e o f P s e u d o m o n a s brassicacearum LBUM300, a disease-suppressive bacterium with antagonistic activity toward fungal, oomycete, and bacterial plant pathogens[END_REF]. Further studies are needed t o i d e n t i f y t h e m e c h a n i s m s i n v o l v e d i n P. brassicacearum interaction with E. amylovora and to assess its practical use in field. In addition, there is a need to optimize the formulation for maximum activity and survival of the bacterial cells, and to assess the vegetative stage and rate of application. To this end, monitoring viable cells in planta could aid the design of a suitable application scheme [START_REF] Daranas | Monitoring viable cells of the biological control agent Lactobacillus plantarum PM411 in aerial plant surfaces by means of a strain-specific viability quantitative PCR method[END_REF]. Likewise, the improvement of ecological fitness of P. brassicacearum PS1 and CR2 could be investigated and further studies under different agricultural and climatic conditions are needed to confirm their performance in the field.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interests.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Fig. 1

 1 Fig. 1 Geographical location of pear orchards in North Algeria

Fig. 2

 2 Fig. 2 Minimal spanning tree of Algerian E. amylovora isolates colored according to year of isolation. Each circle represents an MLVA type with a size corresponding to the number of strains that share an identical MLVA type. MLVA types connected by a thick solid line differ by at most one VNTR locus

Fig. 4 Fig. 5

 45 Fig. 4 Swimming (a) and swarming (b) activity of antagonists at 24 and 48 h of incubation. Data represent the average measure of the half diameter of the bacterial colonies. Two half diameters for each colony were measured on three repetitions. The experiment was carried out twice. The bars represent the standard error. Significant differences (p ≤ 0,05) at 24 and 48 h were determined by Tukey test

Fig. 6

 6 Fig.6Biofilm production by the four antagonistic strains at 3, 5 and 20 h post incubation. Bacterial suspension had a concentration of 10 8 cfu ml -1 . The columns represent the average of 3 repeats

Fig. 7

 7 Fig.7Effect of osmotic stress on the growth of the antagonistic bacteria. Bacterial suspension had a concentration of 10 8 cfu ml -1 . The columns represent the average of 3 repeats and the bars represent standard error. No significant differences were obtained by Tukey test

Fig. 9

 9 Fig.9Disease symptoms on pear fruits developed around wounds protected with the antagonistic strains (CR2, PS1, EHF5, PF3) and inoculated with E. amylovora (E1 or E5 strain).

Table 1

 1 Results of MLVA analysis performed on twenty-five isolates of E. amylovora

	loci	Number of	Number of	Number of	Diversity index
		different alleles	repeats min	repeats max	(Simpson)
	A	3	5	7	0 . 4 4 3 2
	B	1	3	3	0
	C	1	7	7	0
	D	1	9	9	0
	F	2	6	7	0 . 8 5 2 8
	H	1	8	8	0

Table 2

 2 Effect of different pH, temperature and salt concentrations on the growth of antagonistic strains The disease spread quickly to other fruit growing regions in the north of Algeria such as Blida and Algiers. Infected areas in Blida province reached 400 Ha 1 year after the first E. amylovora record in this area

	Antagonistic Strain	pH					Temperature (°C)			NaCl concentration (M)
		4	6	7	8	10	4	30	35	42	45	0,3	0,5	0,8	1,0
	CR2 (P. brassicacearum)	-	+	+	+	-	-	+	+	+	-	+	+	+	+
	PS1 (P. brassicacearum)	+	+	+	+	-	-	+	+	+	-	+	+	+	+
	EHF5 (B. amyloliquefaciens)	+	+	+	+	-	-	+	+	+	-	+	+	+	-
	PF3 (B. methylotrophicus)	+	+	+	+	-	-	+	+	+	-	+	+	+	-
	+: bacterial growth occurred

-: no bacterial growth occurred quince trees.