Strain B22^T was isolated from the rhizospheric soil of Salicornia hipanica in El Saladar del Margen, Cúllar, Granada, Spain (37° 38’ 50.6’’N, 2° 37’ 22.2’’ W). Pseudomonas tehranensis SWRI 196^T (LMG 32044), P. alvandae SWRI 17^T (LMG 32056) and P. canavaninivorans HB 002^T (LMG 32336) were used for taxonomic identification. The AHL-producing plant pathogenic strains used in this work were Dickeya solani LMG 25993^T, Pectobacterium atrosepticum CECT 314^T, P. carotovorum subsp. carotovorum CECT 225^T, Pseudomonas syringae pv. tomato DC3000 and Pantoea agglomerans CFBP 11141. All of them were routinely grown in Luria Bertani (LB) media at 28°C.

The AHL biosensor strains Chromobacterium subtsugae CV026 (Harrison and Soby, 2020) and C. violaceum VIR07 (Morohoshi et al., 2008) were grown in LB medium while Agrobacterium tumefaciens NTL4 (pZLR4) (Shaw et al., 1997) was cultured in LB medium supplemented with 2.5 mmol L^-1 CaCl₂ x 2H₂0 and 2.5 mmol L^-1 MgSO₄ x 7H₂0 (LB/MC) or Agrobacterium broth (AB) medium (Chilton et al., 1974). The antibiotics kanamycin (Km) and gentamicin (Gm) were used in final concentrations of 50 μg mL⁻¹ for the growth of the biosensor strains. All strains were grown at 28°C and at 120 rpm in an orbital shaker.

Genomic DNA of strain B22^T was extracted using the X-DNA Purification Kit (Xtrem Biotech S.L., Granada, Spain). The 16S rRNA gene was then amplified using universal bacterial primers 16F27 and 16R1488. The resulting PCR product was purified and cloned into the pGEM^®-T vector (Promega).

Direct sequencing of the PCR-amplified DNA was performed, and the DNA sequence was compared with reference 16S rRNA gene sequences from GenBank and EMBL databases via the NCBI Genome Database using EzBioCloud server (Yoon et al., 2017).

Phylogenetic analysis was conducted using the Molecular Evolutionary Genetics Analysis (MEGA) software version X (Kumar et al., 2018), incorporating multiple sequence alignments with CLUSTAL OMEGA (Sievers et al., 2011). Evolutionary distances and clustering were determined using the neighbor-joining and maximum-likelihood methods. Cluster stability was assessed through bootstrap analysis with 1,000 replications.

To construct a more robust phylogenetic tree, a multigene approach was used. MLSA was conducted by concatenating the sequences of four housekeeping genes (16S rRNA, gyrB, rpoD, and rpoB). Housekeeping gene sequences from other Pseudomonas species were retrieved from the genomes deposited in NCBI GenBank database. Multiple sequence alignment of the concatenated gene sequences was performed using the MUSCLE algorithm embedded in MEGA X, followed by manual verification to identify and correct alignment errors.

A phylogenetic tree was generated using the neighbor-joining (NJ) algorithm, with 1000 bootstrap replicates to assess the robustness of the branching.

Genomic DNA of strain B22^T, which was extracted as described above, was sequenced by the Illumina MiSeq methodology at the STAB VIDA facility (Caparica, Portugal) with 2 x 150-bp paired-end reads. The reads were trimmed using software tools implemented in the BBMap project (https://sourceforge.net/project/bbmap/) (Bushnell, 2016) to remove the adapters and low-quality bases and de novo assembled using SPADES v3.11.1 (Bankevich et al., 2012). CheckM v1.0.18 (Parks et al., 2015) and Quast v5.0.2 (Gurevich et al., 2013) were used for assembly quality checked. The genome of strain B22^T was annotated using RASTtk v1.073 (Aziz et al., 2008; Overbeek et al., 2014; Brettin et al., 2015) and deposited in GenBank/EMBL/DDBJ under the accession number JBCNTJ010000000.

Average nucleotide identity (ANI) was determined by using OrthoANI software (Lee et al., 2016). For digital DNA–DNA hybridization (dDDH), values were calculated using the BLAST+ algorithm via the DSMZ Genome-to-Genome Distance Calculator (GGDC 3.0) web service at http://ggdc.dsmz.de/ (Meier-Kolthoff et al., 2013, 2022). The results presented in this study are based on the recommended Formula 2 (identities/HSP length), which is independent of genome length, ensuring robustness even when using incomplete draft genomes. The results of the strain described in this study were additionally validated using the Type (Strain) Genome Server [TYGS (Meier-Kolthoff et al., 2022)] in January 2025.

A core genome analysis of strain B22^T and 14 closest related bacteria based on their 16S rRNA percentage of similarity for which their genome was available, downloaded from the NCBI RefSeq database, was also performed using Bacterial Pan Genome Analysis (BPGA) software (Chaudhari et al., 2016) with the default parameters. After obtaining the core of these 15 bacterial genomes, all protein orthologs belonging to the core genome were concatenated and aligned by MAFFT (Katoh and Standley, 2013). A phylogenomic tree of the core genes of the species was then constructed using MEGA X software according to the maximum-likelihood method, incorporating 1,000 bootstrap replicates to evaluate the robustness of the branching.

The colonies were observed on LB agar after 48 h of incubation at 28°C. The oxidase and catalase were determined. Salinity tolerance and optimal growth conditions of strain B22^T were determined at 28°C on LB agar plates supplemented with 0; 0.5; 2.5; 5; 7.5; 12 and 25% (w/v) of NaCl adjusting the pH 7. The pH growth range and optimum pH were analyzed on LB agar plates, testing from 4 to 10 pH unit intervals. The temperature range for growth and the optimum temperature were determined on LB plates at 4; 10; 15; 20; 25; 30; 37 and 40°C. In both cases 1% NaCl w/v was selected.

Phenotypic characteristics of strain B22^T related with the in vitro plant-growth-promoting (PGP) activities and rhizosphere competence were evaluated: amylase (Barrow and Feltham, 1993), caseinase (Barrow and Feltham, 1993), cellulase (Tasse et al., 2010), acid phosphatase (Baird-Parker, 1963), alkaline phosphatase (Pikovskaya, 1948), phytase (Hosseinkhani and Hosseinkhani, 2009), gelatinase (Tindall et al., 2007), lecithinase (Larpent and Larpent-Gourgand, 1957), quitinase, hydrolysis of Tween 20 and Tween 80 (Sierra, 1957), production of indole-3-acetic (IAA) (Naik et al., 2008) and siderophores (Alexander and Zuberer, 1991).

Other biochemical tests were performed using inoculum of B22^T grown in optimal conditions in API 20NE, API 50CH, API ZYM and BIOLOG GEN III according to the manufacturer’s instructions. In addition, DNAase (Jeffris et al., 1957) and hemolysin (Columbia blood agar plates, Difco) were detected.

The cellular fatty acids were analyzed at the Spanish Type Culture Collection (CECT) in Valencia, Spain, following the instruction of the Microbial Identification System Operating Manual (MIDI, 2008). For this, the cell mass of the B22^T strain was obtained after growing for 24 h in LB at 28°C and 1% NaCl w/v.

The analysis of polar lipids and respiratory quinones in strain B22^T was performed by the Identification Service at DSMZ in Braunschweig, Germany.

To test the PGP activity of B22^T, an in vivo assay was performed by inoculating tomato plants (Solanum lycopersicum L. var. Roma) with this bacterium. After the sterilization of the seeds, they were planted in seedbeds (20 x 7 cm) using sterile vermiculite as a substrate. Sterile water was added until the field capacity was reached, and it was covered with plastic film to maintain humidity. The seedlings were maintained in a greenhouse throughout the duration of the experiment, with a relative humidity (RH) of 60%, temperatures of 25°C during the day and 20°C at night, and a long-day photoperiod (light:dark, 16:8 hours). During a month, the plant seedlings were irrigated weekly with 250 μL of a bacterial suspension (10⁸ CFU mL^-1), while the control group was irrigated with sterile distilled water. The experiment was carried out with 12 replicates. Once the treatment had concluded, the plants were harvested and the length, dry weight and thickness of the stem were determined (Sánchez et al., 2023).

Another assay to investigate the colonization properties of B22^T was also performed using the model plant Arabidopsis thaliana (Lee et al., 2020; Furci et al., 2021). Five sterile seeds were placed 2 cm apart in a square Petri dish (10 x 10 cm) containing Murashige & Skoog medium at 0.8% (w/v) agar and a final sucrose concentration of 2.5 g L^-1. In vitro cultures were maintained under controlled conditions in a phytotron (Hederahelix Ing.) with a long day photoperiod (light:dark, 16:8 h), at 25°C day and 20°C night, relative humidity 60-80% and a luminosity of 250 microsiemens/cm²/sg. After one week of growth, the seedlings were inoculated with 1 μL of the bacterial suspension (10⁸ CFU mL⁻¹) at 1 cm from the base of the plant stem. The same volume of sterile distilled water was inoculated in the control and three replicates were made per treatment. The plates were maintained under controlled conditions as mentioned above for another 7 days. After this period, root colonization was observed by differential interference contrast microscopy between slide and coverslip, using 70% (v/v) glycerol as mounting medium in a LEICA DM5500B microscope.

The QQ activity of the B22^T strain against synthetic AHLs was assessed through a well-diffusion agar-plate assay (Torres et al., 2013). In brief, each AHL was added to an overnight culture in LB medium of the B22^T strain, at a final concentration of 10μM, followed by a 24-hour incubation at 28°C. A sterile LB medium supplemented with AHLs served as negative control. The remaining AHLs were identified in the supernatant of each sample, which was then placed in wells on LB agar plates covered with C. subtsugae CV026 or C. violaceum VIR07, or on AB agar plates added with 80 μg mL⁻¹ of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (Xgal) and overlaid with A. tumefaciens NTL4 (pZLR4). The plates were then incubated at 28°C for 24 hours to observe the appearance of purple or blue coloration around each well. This assay was conducted three times for validation.

The synthetic AHLs (Sigma-Aldrich, Saint Louis, USA) used were C6-HSL (N-hexanoyl-DL-homoserine lactone), C8-HSL (N-octanoyl-DL-homoserine lactone), 3-O-C8-HSL (N-3-oxo-octanoyl-DL-homoserine lactone), C10-HSL (N-decanoyl-DL-homoserine lactone), 3-OH-C10-HSL (N-3-hydroxydecanoyl-DL-homoserine lactone), C12-HSL (N-dodecanoyl-DL-homoserine lactone) and 3-O-C12-HSL (N-3-oxo-dodecanoyl-DL-homoserine lactone).

To determine whether the QQ enzyme of B22^T was a lactonase type, an acidification assay was carried out (Yates et al., 2002). Briefly, C12-HSL was added to a 24-hour incubation at 28°C. A sterile LB medium supplemented with C12-HSL served as the negative control. Then, the culture and the control were centrifuged, and the pH of the supernatants was adjusted to 2.0 with HCl 1N. Acidified supernatants were then incubated for 24 h at 28°C. The remaining amount of C12-HSL was extracted twice with 1 volume of dichloromethane. Dried extracts were resuspended in acetonitrile and analyzed by HPLC/MS (Torres et al., 2013) as well as by well-diffusion agar-plate assay using A. tumefaciens NTL4 (pZLR4) as biosensor strain.

The cellular localization of QQ activity was identified in the supernatant and crude cellular extract (CCE) fractions from a 24 h culture of strain B22^T following the methodology previously described. Supernatant and CCE were obtained as described previously and filtered through a 0.22 μm-pore membrane filter (Romero et al., 2014).

To identify genes encoding for QQ activity in strain B22^T, the whole genome annotated using RAST was used, and potential genes were tested. A protein encoding a predicted acylhomoserine lactone acylase was selected WP_343468083.1 (782 aa) and used to design the specific primers B22-acilgen-Fw: 5’atgtccgggcagttatcgagg-3’ and B22-acilgen-Rev: 5’-ttagtgcgcggccaccctgc-3’. A PCR fragment was amplified and cloned into pGEM-T cloning vector (Promega) following the manufacturer’s instructions. Then, the plasmid construction was transferred into Escherichia coli DH5α and the AHL-degradation activity was tested in LB media supplemented with IPTG and AHLs (C10-HSL and C12-HSL) as described above.

Previous to determine the QQ activity, we determine if the strain B22^T interferes the growth of the five plant pathogenic bacteria in an antagonist assay based in a well diffusion method (Frikha-Gargouri et al., 2017). Briefly, 24 h culture of each pathogen was used to prepare an overlay on LB agar plates and 100 μL of aliquots of the filtered supernatant from a 5-day culture of strain B22^T were placed on previously prepared wells. After 48 h incubation at 28°C, plates were examined for the appearance of growth inhibition halos surrounding the wells.

QQ activity of strain B22^T upon phytopathogenic bacteria was determined by co-culture assays (Torres et al., 2016; Rodríguez et al., 2020). Briefly, 24 h cultures of each pathogen (10⁹ CFU mL^-1) were co-cultured with 24 h culture of strain B22^T (10⁹ CFU mL^-1) at a ratio of 1:100 in LB medium and incubated for 24 h. As controls, each bacterium was cultured in similar conditions. Then, AHLs from each co-cultures y monocultures were detected using the well-diffusion agar-plate assay as described above, and C. subtsugae CV026 and A. tumefaciens NTL4 (pZLR4) as bioindicator strains. The abundance of strain B22^T and each pathogen in the co-cultures were quantified by serial dilutions and plate counts, using LB medium and LB medium supplemented with 5% (w/v) NaCl and King B agar.

The interference of strain B22 with virulence-associated cellular functions controlled by QS in the pathogens was evaluated by spotting 10 μL of co-cultures and monocultures on different media. Particularly, the following phenotypic characteristics were evaluated: production of acid and alkaline phosphatase (Pikovskaya, 1948; Baird-Parker, 1963); siderophore production (Alexander and Zuberer, 1991); production of hydrolytic enzymes such as amylase and caseinase (Barrow and Feltham, 1993), DNAase (Jeffris et al., 1957), lipases (hydrolysis of Tween 20 and Tween 80) and gelatinase (Tindall et al., 2007).

The interference of strain B22^T with the virulence of D. solani LMG 25993^T and P. atrosepticum CECT 314^T was evaluated on potato slices (Solanum tuberosum). Infection of P. carotovorum subsp. carotovorum CECT 225^T was tested on potato and carrot (Daucus carota) slices while P. agglomerans CFBP 11141 was evaluated in cherry tomatoes (S. lycopersicum L. var. cerasiforme) (Garge and Nerurkar, 2016; Rodríguez et al., 2020). Briefly, vegetables were surface sterilized. Then, potatoes and carrots were cut into slices and placed in Petri dishes while cherry tomatoes were placed entirely with wet filter paper to maintain moisture. The following treatments were conducted for each pathogen: sterilized distillated water, monoculture of pathogen, monoculture of strain B22^T, pathogen: B22^T co-culture (1:100). Each bacterial culture (10⁹ CFU mL^-1) was centrifuged, resuspended in sterilized distillated water and 15 μL of each condition was inoculated into the tomatoes, carrot and potato slices. Nine replicates of each treatment were conducted, and the assay was repeated three times. After 2–5 days of incubation (depending on each pathogen) at 28°C, maceration areas were calculated by image analysis using ImageJ software (Schneider et al., 2012).

The impact of AHL degradation of P. syringae pv. tomato DC3000 by strain B22^T was evaluated in tomato plants (S. lycopersicum L. var. Roma) following the technique described elsewhere (Yan et al., 2008). Briefly, tomato seeds were surface-sterilized and sown in pots as described above (section 2.7.). Three pots containing 50 seeds per pot were used for each treatment: sterile distilled water (negative control), strain B22^T, the pathogen (positive control) and the pathogen: B22^T co-culture. The pots were kept in an indoor greenhouse during a long-day photoperiod (16:8h, light:dark) at 25°C and watered with 50 mL sterile distilled water twice a week. After 3 weeks, the pots were exposed to 100% humidity for 16 h to induces stomatal opening and then sprayed with a 5 mL washed cell suspension of P. syringae pv. tomato DC3000 (10⁹ CFU mL^-1), co-culture of pathogen with strain B22^T (ratio 1:100), strain B22^T (10⁹ CFU mL^-1) and sterile distilled water. Relative humidity was maintained at 100% for a further 24h to facilitate pathogen infection. Finally, after 4 weeks post-inoculation, affected (necrotic and chlorotic leaves, dead leaves) and unaffected shoot (healthy leaves) were counted and followed by plant harvesting (López-Escudero et al., 2004). The unaffected and affected shoot and roots length total dry weight of 20 plants per treatment were determined.

The chlorophyll fluorescence (Fv/Fm ratio) in tomato leaves was determined using a fluorometer (Handy PEA Hansatech Instruments Ltd.) by measuring the fluorescence on the adaxial side of the leaf for a time of 30 minutes. The parameters of basal fluorescence (F0), maximum fluorescence (Fm), variable fluorescence (Fv), (Fv= Fm-F0), and the ratio (Fv/Fm), an indicator of the functionality of the photochemical conversion of light energy in photosystem II, were obtained (Passari et al., 2019).

The data analysis for this study was conducted using GraphPad Prism 9, Statistical Package for the Social Sciences (SPSS) software. The normality of the data was evaluated through either the Shapiro-Wilk test or the D’Agostino and Pearson test. A one-way ANOVA followed by Tukey’s post hoc test was employed for comparing group means. A 95% confidence interval was applied in all analyses.

Strain B22^T was isolated from the rhizospheric soil of the halophyte Salicornia hispanica. The almost complete 16 rRNA gene sequence ( ̴1,500 bp) of strain B22^T was obtained by PCR, which was identical to that extracted from its genomic sequence. Strain B22^T showed the highest sequence identity to Pseudomonas tehranensis SWRI 196^T (99.86%), P. alvandae SWRI 17^T (99,66%) and P. canavaninivorans HB 002^T (99,59%), while identities below 97% were obtained with other species from Pseudomonas genus. Phylogenetic analysis of its 16S rRNA gene sequences and other related strains through a phylogenetic tree reconstruction using the maximum-likelihood algorithm showed that this strain is a member of the genus Pseudomonas and forms a cluster with the P. tehranensis species, which showed the highest sequence similarity ( Figure 1 ). A similar phylogenetic distribution was obtained when the neighbor-joining and maximum parsimony algorithms were used ( Supplementary Figure S1 ).

The MLSA resulting Neighbor-joining tree, obtained by concatenating the sequences of four housekeeping genes (16S rRNA, gyrB, rpoD, and rpoB) also indicated that B22^T and Pseudomonas tehranensis SWRI196^T forms a cluster with a bootstrap value of 100% ( Supplementary Figure S2 ).

The draft genome of strain B22^T was manually curated, resulting in a genome size of over 5.7 Mbp across 32 contigs. The assembly quality was evaluated using the Quality Assessment Tool for Genome Assemblies (QUAST), yielding an N50 value of 237,4 kb, an L50 of 9, and approximately 300X coverage, indicating high-quality sequencing. Annotation of the draft genome using PGAP (Tatusova et al., 2016) identified 5014 protein-coding genes (PCGs). This genome sequence was deposited in the GenBank/EMBL/DDBJ database under accession number JBCNTJ010000000 and was used for further analysis.

The in silico analysis of the G+C content in the draft genome of strain B22^T revealed a value of 60.5 mol%. Average nucleotide identity (ANI) values, calculated using OrthoANI, yielded a similarity between strain B22^T and P. tehranensis SWRI 196^T of 95.61%, within the limit of the species delimitation threshold of 95–96% (Chun et al., 2018). The values for strain B22^T and other related species were below this threshold ( Supplementary Table S1 ).

Digital DNA-DNA hybridization (dDDH) analysis of the whole-genome sequences of strain B22^T and closely related species also produced values below the species delineation threshold (70%) ( Supplementary Table S1 ).

The concatenated alignment of 3093 core orthologous proteins from strain B22^T and related species of the genus Pseudomonas, was used to construct a maximum-likelihood phylogenetic tree. The resulting tree is shown in Figure 2 .

The predominant cellular fatty acids of the strain B22^T were summed feature 3 (contains C_16:1 ω7c and/or C_16:1 ω6c) (41.07%), C_16:0 (28.87%), and summed feature 8 (contains C_18:1 ω7c and C_18:1 ω6c) (12.04%). The major polar lipids of strain B22^T were diphosphatidylglycerol and phosphatidylethanolamine. Other polar lipids present were phosphatidylglycerol, an unidentified glycolipid, an unidentified phospholipid and an unidentified lipid ( Supplementary Figure S3 ). The predominant respiratory quinone of strain B22^T was ubiquinone-9 (Q-9).

Cells of strain B22^T were Gram-negative, rod-shape and motile ( Supplementary Figure S4 ). It forms creamy small colonies after 48 h of incubation in LB agar plates. Growth was positive on Simmons citrate agar and fluorescent colonies were detected on King B agar. Strain B22^T did not produce endospores. It was catalase and oxidase positive. It was aerobe, with growth temperatures ranging from 4 to 40°C and with an optimum temperature at 28°C. It grew in pH range from 5 to 10 with pH 7 as optimum. Strain B22^T resulted to be halotolerant as it grew from 0 to 5% (w/v) of NaCl, with 0.5-2.5% (w/v) being the optimum concentration. The differential characteristics of this strain with respect to the most closely related species, P. tehranensis SWRI 196^T, P. alvandae SWRI 17^T and P. canavaninivorans HB 002^T were listed in Table 1 . For example, strain B22^T doesn’t assimilate D-trehalose, D-serine, D-sorbitol, ß-hydroxy-phenylacetic acid while P. tehranensis SWRI 196^T does. B22^T is sensitive to fusidic acid while P. tehranensis SWRI 196^T is resistant. More phenotypical characteristics of strain B22^T were detailed in the species description and in Supplementary Tables S2 , S3 .

Concerning the PGP activity, strain B22^T resulted positive for the hydrolysis of Tween 20 and Tween 80 as well as for phytase and acid phosphatase. It also produced IAA and siderophores. Nevertheless, it did not produce amylase, cellulase, caseinase, lecithinase, alkaline phosphatase and chitinase ( Supplementary Table S3 ).

To assess the PGP activity of the halotolerant bacteria in tomato plants 28 days post-inoculation, the plants’ morphological characteristics were evaluated. Plant dry weight and length were significantly higher compared to non-inoculated plants 114.73 ± 62.07 mg vs 19.34 ± 2.83 cm and 27.6 ± 3.51 cm vs 19.34 ± 2.83 cm, respectively ( Figures 3A, B ). A similar pattern was observed for stem diameter, with plants inoculated with the B22^T strain showing the largest stem diameters compared to the non-inoculated plants 0.9 ± 0.35 mm vs 0.31 ± 0.1 mm, respectively.

The colonization of plant roots was studied in Arabidopsis thaliana through microscopy. The micrographs obtained by differential interference contrast showed small bacterial masses at the base of the root hairs of plants treated with the strain B22^T strain, and the absence of bacteria in the control roots ( Figure 3C ).

The ability of AHL degradation of strain B22^T was tested against a wide range of synthetic AHLs through a well-diffusion agar-plate assay. Strain B22^T was able to completely degrade the following AHLs: C6-HSL, C10-HSL, 3-OH-C10-HSL, C12-HSL and 3-oxo-C12-HSL as the corresponding biosensor strains (C. subtsugae CV026, C. violaceum VIR07 and A. tumefaciens NTL4 pZLR4) were not activated. Nevertheless, C8-HSL and 3-oxo-C8-HSL were not degraded ( Supplementary Figure S5 ).

To determine whether the enzymatic activity of strain B22^T was due to a AHL lactonase type, an acidification assay to test the lactone ring closure was conducted using C12-HSL, an AHL totally degraded by this strain. The remaining AHLs in the acidified cell-free supernatant (pH 2) as well as in the control (cell-free supernatant at pH 7) were detected by a well-diffusion agar-plate assay using A. tumefaciens NTL4 (pLR4) as biosensor strain. The AHL concentration was not restored in the acid conditions. This result was confirmed by HPLC-MRM, suggesting that QQ activity in this strain was not caused by an AHL lactonase ( Supplementary Figure S6A ).

The cellular localization of the enzyme was assessed by testing C10-HSL and C12-HSL degrading activity of strain B22^T in cell-free supernatant (SN) and crude cellular extract (CCE). Well-diffusion agar-plate assay with C. violaceum VIR07 and A. tumefaciens NTL4 (pZLR4) showed that QQ activity was detected in SN, whilst no AHL degradation was seen in CCE, suggesting that the enzyme was secreted ( Supplementary Figure S6B ).

To identify the gene responsible for AHL-degradation activity in strain B22^T, the automatic annotation of the whole genome was used to select a gene that encodes potential acylase protein WP_343468083.1 (782 aa). A DNA fragment of ~2349 bp was amplified by PCR, using designed specific primers, purified and cloned in the pGEM-T vector and then, expressed in E. coli DH5α. The QQ activity of the plasmid construction pGEMT-B22 against C10-HSL and C12-HSL was confirmed by the well-diffusion agar-plate assay ( Supplementary Figure S6C ). In addition, BLASTp analysis revealed that the predicted protein encoded by the identified gene WP_343468083.1 (782 aa) shared 95.27% and 93.48% identity with the acylase sequences from P. fluorescens and P. tehranensis, respectively, both of which are annotated as members of the penicillin acylase family.

The QQ activity of strain B22^T was tested against the AHLs produced by D. solani LMG 25993^T, P. atrosepticum CECT 314^T, P. carotovorum subsp. carotovorum CECT 225^T, P. agglomerans CFBP 11141 and P. syringae pv. tomato DC3000 to evaluate its potential use for biocontrol. In these pathogens, AHLs have been characterized and correlated to the expression of virulence factors. Firstly, an antagonism assay was conducted to evaluate whether strain B22^T interferes with the growth of each pathogen and no inhibitory effect was detected (data not shown). The five plant pathogens were co-cultured with strain B22^T in LB medium at a ratio of 1:100 for 24 h. Monocultures of each bacterium were used in each case as controls. The concentration of each bacterium was determined by counting plate using selective media and they remained stable through the experiment. Then, AHLs were detected by well-diffusion agar-plate assay with A. tumefaciens NTL4 (pLRZ4) and C. subtsugae CV026 as bioindicator strains. The results indicated that under our assay conditions, strain B22^T degraded almost completely the AHLs produced by D. solani LMG 25993^T and P. agglomerans CFBP 11141 as no activation of the biosensors were visualized in their respectively co-cultures. In respect of co-cultures of strain B22^T and the pathogens P. atrosepticum CECT 314^T, P. carotovorum subsp. carotovorum CECT 225^T and P. syringae pv. tomato DC3000, a partial AHL degradation was observed ( Supplementary Figure S7A ).

Based on the capacity of strain B22^T to degrade AHLs produced by the tested phytopathogens, similar co-cultures were conducted and used to evaluate the effect on the production of cellular functions regulated by QS in each pathogen. Since strain B22^T produced numerous PGP traits, the possible reduction of some phenotypic characteristics in the pathogens could not detected ( Supplementary Table S3 ). As results, some phenotypes of pathogens were inhibited when co-cultured with strain B22^T. The production of alkaline phosphatase and lecithinase in D. solani; the production of Tween 20 in P. atrosepticum and the production of amylase in P. agglomerans was inhibited in the presence of strain B22^T while a reduction in DNAase production was observed in P. carotovorum. Regarding P. syringae pv. tomato, no traits were found to be affected in our phenotypic assays ( Supplementary Figure 7B ).

To evaluate whether the AHL degradation produced by strain B22^T in co-culture with the plant pathogens has an impact on their in vivo virulence, different assays on fruits and plants were conducted. Co-cultures of strain B22^T with the pathogens were prepared as described above. Additionally, sterile water and monoculture of each strain were used as controls. Mono and co-cultures of D. solani, P. atrosepticum and P. carotovorum were inoculated on the surface of potato slices. In the case of P. carotovorum, cultures were also inoculated on the surface of carrot slices. Mono and co-cultures of B22^T with P. agglomerans were inoculated in cherry tomatoes. As it is shown in Figure 4 and Supplementary Figure S8 , under our assay conditions, strain B22^T drastically reduced the virulence capacity of D. solani and P. atrosepticum to produce soft rot due to maceration area of 5.46 ± 1.79% and 5.22 ± 0.52% respectively, while a maceration area of 65.38 ± 3.21% and 14.96 ± 1.25% was originated by each pathogen in monoculture respectively. Regarding P. carotovorum, strain B22^T significantly diminished the capacity of the pathogen to induce soft rot in slice carrots (7.06 ± 0.58%) and potatoes (18.19 ± 0.06%) as compared to the 46.17 ± 3.12% and 34.44 ± 8.40% observed by the pathogen in monoculture respectively. In the case of P. agglomerans, cherry tomatoes inoculated with the pathogen exhibited tissue damage (25 ± 0.01%) while a significant reduction of the infection symptoms were observed in the presence of strain B22^T (2.5 ± 0.01%). In all of assays conducted with the different types of vegetables, the inoculation of strain B22^T did not originate any infection symptoms.

The use of strain B22^T as biocontrol agent was also evaluate by conducting in vivo assays of tomato plants infected with P. syringae pv. tomato DC3000. As it is shown in Figure 5 , tomato plants treated with the pathogen:strain B22^T co-culture showed less infection symptoms than those infected with the pathogen alone. Indeed, tomato plants treated with the co-culture showed a significative increase of 31.49% in healthy leaves as compared to plants inoculated with the pathogen. Moreover, the number of necrotic/chlorotic leaves in plants treated with the co-culture was 0 while the value was 37.5% in those plants treated with the pathogen alone. Negative control and strain B22^T-treated plants showed some damaged leaves associated with natural senescence.

Regarding fluorescence parameters, plants exposed to the monoculture of B22^T and the pathogen:strain B22^T co-culture showed an increase of the optimum quantum yield of PSII, represented by the Fv/Fm ratio, compared with plants inoculated with the pathogen (7 and 15% respectively) (data not shown). These results indicated the benefits of the strain B22^T that clearly attenuated the virulence of the pathogen.

Pseudomonas halotolerans (ha.lo.to′le.rans. Gr. n. hals salt; L. pres. part. tolerans tolerating; N.L. part. adj. halotolerans referring to the ability of the organism to tolerate high salt concentrations).

Cells of this species are aerobic, Gram-negative, non-sporeforming, motile and rod-shaped. In LB agar plates, cells can grow forming creamy and small colonies. Growth occurs in 0-5% (w/v) NaCl (optimum 0.5-2.5%), at pH varying from pH 5 to 10 (optimum pH 7), and within the temperature range of 4-40° C (optimum 28° C). Catalase and oxidase tests are both positive. Results obtained with Biolog GEN III microplates indicate that cells can use the following substrates as carbon and energy sources: D-trehalose, sucrose, α-D-glucose, D-mannose, D-fructose, D-galactose, D-fucose, inosine, D-mannitol, glycerol, D-fructose 6-PO₄, L-alanine, L-aspartic acid, L-histidine, L-pyroglutamic acid, L-serine, pectin, D-galacturonic acid, L-galacturonic acid lactone, D-gluconic acid, D-glucuronic acid, glucuronamide, mucic acid, quinic acid, D-saccharic acid, methyl pyruvate, L-lactic acid, citric acid, L-malic acid, bromo-succinic acid, gamma-amino-butyric acid β-hydroxyl-D L-butyric acid, propionic acid and acetic acid. All other substrates in the Biolog GEN III panel did not or only weakly support cell growth. Based on Biolog Gen III sensitivity assays, cells of this species can grow at 1% w/v sodium lactate, and are resistant to troleandomycin, rifamycin SV, lincomycin, guanidine HCl, niaproof 4, vancomycin, tetrazolium violet, tetrazolium blue, lithium chloride, potassium tellurite, nalidixic acid, aztreonam and sodium butyrate. Cell growth was inhibited by all other compounds tested within the Biolog GEN III sensitivity assays.

Results obtained with API 50 CH and API 20NE strips indicate that cells of this species oxidize or assimilate of the following substrates: L-arabinose, D-glucose, aesculin, sucrose, D-mannose, D-mannitol, potassium gluconate, trisodium citrate, capric acid and malic acid. Cells of this species exhibited the following enzymatic activities detected by API 20NE and API ZYM: reduction of nitrates to nitrites, gelatine hydrolysis, leucine arylamidase, esterase (C8) and weakly positive for esterase (C4), lipase (C14), valine arylamide, trypsin and naphtol-AS-BI-phosphohydrolase, acid phosphatase and α-fucosidase. Acids from glucose were negative. Strain B22^T resulted positive for the hydrolysis of Tween 20 and Tween 80 as well as for phytase and acid phosphatase. It also produced IAA and siderophores. It did not produce amylase, cellulase, caseinase, lecithinase, alkaline phosphatase and chitinase.

The main cellular fatty acids of strain B22^T were C_16:0, summed feature 3 (C_16:1 ω7c/C_16:1 ω6c) and summed feature 8 (C_18:1 ω7c/C_18:1 ω6c). The major polar lipids identified were diphosphatidylglycerol and phosphatidylethanolamine, while the predominant respiratory quinone was ubiquinone (Q-9).

The genome of P. halotolerans B22^T consists of a circular chromosome of 5.7 Mbp, G+C value of 60.5 mol% and 5014 protein-coding genes. The whole genome sequence of P. halotolerans B22^T has been deposited al NCBI and is publicly available at NCBI GeneBank accs. no. JBCNTJ010000000.

The type strain is B22^T (= CECT31209 = LMG33902), isolated from the rhizospheric soil of the halophyte plant Salicornia hispanica.