Genome sequence of the desert endophyte Pseudomonas granadensis R4-79 reveals potential for plant-growth promotion and disease suppression

Genome sequence of the desert endophyte Pseudomonas granadensis R4-79 reveals potential for plant-growth promotion and disease suppression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome sequence of the desert endophyte Pseudomonas granadensis R4-79 reveals potential for plant-growth promotion and disease suppression Linah Alghanmi, Ahmed Elhady, Sabiha Parween, Heribert Hirt This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6620576/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Oct, 2025 Read the published version in BMC Microbiology → Version 1 posted 10 You are reading this latest preprint version Abstract Desert ecosystems are limited by resources and optimal climatic conditions to support cropping in different farming models. Recently, microbial applications have emerged as promising strategies to enhance plant survival and adaptation in such extreme environments. However, identifying beneficial microbes and understanding their functional roles and adaptation mechanisms remains underexplored. This study reports, for the first time, the isolation and characterization of the Pseudomonas sp. R4-79 strain from the arid environment of Wadi Rum, Jordan, associated with Ifloga spicata . The genome of Pseudomonas sp. R4-79, sequenced at 275× PacBio coverage, consists of a 6.18 Mbp chromosome encoding 5,445 proteins, including gene clusters for siderophores, phenazines, hydrogen cyanide, and phytohormones, as well as advanced secretion systems (T2SS, T4SS, T6SS, and TAT). Genomic and phenotypic analyses revealed that Pseudomonas sp. R4-79 belongs to the genus P. granadensis and exhibits plant growth-promoting attributes and substantial biocontrol potential. Pseudomonas sp. R4-79 effectively suppresses key phytopathogens, including the necrotrophic fungus Botrytis cinerea in vitro , as well as Pseudomonas syringae pv. tomato DC3000 and root-knot nematodes ( Meloidogyne incognita ) in vivo in Arabidopsis and tomato. This work suggests that P. granadensis R4-79 might be a good biocontrol agent to improve crop yield and ecological restoration in arid systems. Genome Pseudomonas Desert agriculture Phytopathogens Biocontrol Plant Pathogen Suppression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Desert ecosystems are among the most fragile environments on Earth, where water is scarce and soils are not rich in nutrients (Coleine et al., 2024 ). These factors not only discourage ecological restoration but also create a limit to the scope of practicable models for sustainable farming (Rastgoo & Hasanfard, 2021 ). Conversely, while previously, water resources and minimum vegetation cover were sought to be the focus for the rejuvenation of the desert ecosystem, recent findings emphasize the significance of microbial communities in boosting the desert ecosystem (Alsharif et al., 2020 ; Islam et al., 2024 ). Microbes, particularly those adapted to arid conditions, hold substantial potential for enhancing soil fertility, promoting plant growth, and improving resilience to environmental stresses (Alsharif et al., 2020 ). However, identifying beneficial microbes, enhancing their growth, and expanding their community diversity continue to be major challenges. Moreover, investigating their functional traits and phylogenomic adaptations is necessary for advancement in ecological and agricultural applications. As abiotic and biotic stresses exist, they make ecological revegetation and farming in arid deserts highly challenging. Plants and crops in these environments are highly susceptible to infestations by parasitic nematodes and insects, as well as infections caused by bacterial and fungal phytopathogens (Elhady et al., 2024 ; Sulaiman & Bello, 2024 ). Meloidogyne incognita is among the most destructive plant-parasitic nematodes, with nearly 100% occurrence in the collected soil samples, often exceeding the economic threshold, particularly in the Sahara and Arabian deserts (Elhady et al., 2024 ). Additionally, pathogenic fungal species, including Fusarium spp., Alternaria spp., Pythium spp., Rhizoctonia solani , Phytophthora spp., Sclerotinia spp., and Verticillium spp., are widely distributed and cause significant damage, with losses in agricultural production reaching 50–75% in some cases (Panth et al., 2020 ). The harsh abiotic conditions, coupled with limited plant and microbial diversity, further exacerbate plant susceptibility to phytopathogens that hinder the establishment of resilient vegetation. Compared to other ecosystems, plant pathogens have a significantly greater impact on arid ecosystems, but efforts to control these pathogens have received minimal attention (Elhady et al., 2024 ; Jat et al., 2012 ). Desert plants depend on mutualistic interactions with specialized microbial species to persist in extreme environments. These interactions are vital for the fitness and survival of both partners. For example, the legume shrub Vachellia jacquemontii in the Thar Desert associates with the nitrogen-fixing bacterium Ensifer , which provides bioavailable nitrogen to support the plant's nutritional requirements (Ardley, 2017 ). Similarly, despite the extreme aridity of the Atacama Desert, arbuscular mycorrhizal (AM) fungi have been reported (Santander et al., 2021 ) to potentially play significant roles in phosphorus solubilization, plant stress tolerance, and growth promotion. The isolation and application of microbial strains from native desert plants has emerged as an effective strategy to enhance crop performance under harsh conditions. For instance, the halotolerant Bacillus cabrialesii from the Qatar desert improved seedling growth under salt stress and reduced tomato infections by gray mold disease ( Botrytis cinerea ) (Masmoudi et al., 2024 ). Similarly, two Klebsiella strains from Parastrephia quadrangularis in the Atacama Desert increased wheat seedling fresh weight by up to 60% under salinity stress (Acuña et al., 2019 ). Serratia marcescens from Capparis decidua in India’s Thar Desert enhanced wheat tolerance to salinity stress and reduced fungal disease severity caused by Fusarium graminearum (Singh & Jha, 2016 ). Additionally, Pseudomonas argentinensis SA190 from Indigofera argentea in Saudi Arabia's Jizan region improved drought tolerance in Arabidopsis thaliana seedlings (Alwutayd et al., 2023). In an attempt to identify beneficial bacterial strains from the Arabian desert to support arid farming and ecological restoration, Pseudomonas sp. R4-79 was tested for promoting plant growth and suppressing various phytopathogens. This strain was isolated from the desert plant Ifloga spicata in Wadi Rum, Jordan. In this work, we characterize the phenotypic properties of Pseudomonas sp. R4-79 which inhibited several phytopathogens such as the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, the root-knot nematode ( Meloidogyne incognita ) and the fungal pathogen Botrytis cinerea . Whole-genome sequencing was conducted to determine its taxonomic classification and explore its biochemical potential to promote plant growth and inhibit pathogens. Materials and Methods Root collection and bacterial strain isolation Root samples of the desert plant Ifloga spicata were collected from Wadi Rum, Jordan. To study the root endophytic bacteria, the root tissues were washed to remove soil debris and surface-sterilized by immersing them in 70% ethanol for 30 seconds, followed by treatment with 2% sodium hypochlorite for five minutes and thorough rinsing with sterile distilled water. The sterilized roots were macerated in a solution containing 0.8% saline and subjected to serial dilutions ranging from 10⁻² to 10⁻⁵. Each dilution was plated in duplicate on different culture media, including Tryptone-Yeast (TY), R2A, LB agar, and TSA. Bacterial colonies were selected based on distinctive morphological characteristics and pigmentation, followed by further purification to obtain individual strains. The purified bacterial strains were suspended in a 25% glycerol-LB solution and stored at − 80°C to serve as reference stocks. Genomic DNA extraction and strains identification To identify the bacterial strains, cells were streaked from 25% glycerol stock onto LB agar and incubated at 28°C overnight. A single bacterial colony was then inoculated into 10 mL of LB broth in a 50 mL Falcon tube and incubated at 28°C with shaking at 220 rpm. Genomic DNA was extracted using the Genelute Bacterial Genomic DNA Kit (Sigma-Aldrich). The 16S rRNA gene was amplified using Taq DNA Polymerase PCR Master Mix (Promega, Madison, WI, USA) and universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’). The amplified PCR products were purified using ExoSAP-IT (Affymetrix, Santa Clara, CA, USA) and sequenced via Sanger sequencing at the Core Labs, KAUST, Saudi Arabia. Strain identification was performed by comparing the sequences to NCBI’s GenBank database using BLAST. Strains were sorted and grouped into collections based on their genus-level taxonomy, including Pseudomonas spp. , Bacillus spp. , Enterobacter spp. , rhizobial strains, and others. Genome sequencing and annotation of Pseudomonas sp. R4-79 After screening 86 microbial strains for plant growth promotion, the Pseudomonas sp. R4-79 strain was selected for its high effectiveness in promoting plant growth. The strain was further analyzed to characterize its genetic features and beneficial potential using in silico , in vitro , and in vivo approaches. To analyze the genome of the Pseudomonas sp. R4-79 strain, the total genomic was shipped to Novogen Bioinformatics Technology Co., Ltd. (Singapore). Genomic sequencing and assembly were performed using Single-Molecule Real-Time (SMRT®) sequencing on the PacBio Sequel II/IIe platform. The whole genome assembly was performed using the Hierarchical Genome Assembly Process (HGAP). Genome polishing and circularization were carried out using Arrow (v2.3.3) and Circlator (v1.5.5), respectively. To assess the quality of the genome assembly, quantitative measurements were obtained using BUSCO (v4.0.2) (Benchmarking Universal Single-Copy Orthologs, https://busco.ezlab.org ). Genome annotation was performed for coding genes, repetitive sequences, non-coding RNAs, and pseudogenes using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). PGAP identifies structural annotations by comparing open reading frames (ORFs) against libraries containing protein hidden Markov models (HMMs), representative RefSeq proteins, and proteins from well-characterized reference genomes. For genomic regions lacking HMM or protein evidence, GeneMarkS-2 + was used to generate ab initio coding region predictions and to determine start sites for ORFs based on available HMM evidence. Phylogenetic analysis The genome sequence data were analyzed using the Type (Strain) Genome Server (TYGS), a free bioinformatics platform available at https://tygs.dsmz.de , for whole-genome-based taxonomic identification. Two methods were employed to identify the closest type strain genomes. First, the Pseudomonas sp. R4-79 strain genome was compared to all type strain genomes in the TYGS database using the MASH algorithm, which approximates intergenomic relatedness. The 10 closest type strains with the smallest MASH distances were selected. Second, 16S rDNA gene sequences were extracted from the Pseudomonas sp. R4-79 strain genome using RNAmmer, and each sequence was BLASTed against the 16S rDNA gene sequences of 18,357 type strains in the TYGS database. The 50 best-matching type strains were identified, and precise distances were calculated using the Genome BLAST Distance Phylogeny (GBDP) approach with the “coverage” algorithm and distance formula d5. These distances were used to determine the 10 closest type strain genomes to the Pseudomonas sp. R4-79 strain genome. For phylogenomic analysis, pairwise comparisons among the selected genomes were performed using GBDP, with intergenomic distances inferred using the ‘trimming’ algorithm and distance formula d5. One hundred distance replicates were calculated for each comparison. Digital DNA-DNA hybridization (dDDH) values and confidence intervals were computed using GGDC 3.0. The intergenomic distances were used to infer a balanced minimum evolution tree with branch support via FASTME 2.1.6.1, including SPR postprocessing and 100 pseudo-bootstrap replicates. The trees were rooted at the midpoint and visualized with PhyD3. Species clustering was based on a 70% digital DDH threshold, while subspecies clustering was done using a 79% dDDH threshold. Sequence accession number The WGS has been submitted to NCBI under Bioproject-PRJNA708169, Biosample-SAMN18220753, Genome accession-CP071650-CP071651. Phenotypic and biochemical properties of Pseudomonas sp. R4-79 Strain culturing . Cells of Pseudomonas sp. R4-79 strain were revived from a 25% glycerol stock stored at − 80°C by streaking onto King’s B agar (Sigma-Aldrich) and incubating at 28°C overnight. Then, a single pure colony was inoculated into 10 mL of sterilized LB broth (Invitrogen) in a 50 mL Falcon tube (Fisher Scientific) and incubated overnight on an orbital shaker (Innova 42, New Brunswick) at 28°C /220 rpm. A fresh culture was prepared by adding 5 ml of LB broth (Lennox L Broth Base, Invitrogen) to a 15 ml tube (Fisher Scientific), followed by the addition of 500 µl of bacterial culture. The mixture was incubated at 28°C with shaking at 220 rpm on an orbital shaker for 45 minutes to allow the culture to reach the exponential phase. The optical density at 600 nm (OD 600 ) was then measured and adjusted to 0.2 using a spectrophotometer (BioPhotometer, Eppendorf). Electron microscopy. The morphology of the bacterial strain Pseudomonas sp. R4-79 was visualized using the transmission electron microscopy (TEM). A 5-µl of the bacterial cells OD 600 = 0.2 was deposited onto carbon/Formvar-coated TEM grids (Electron Microscopy Sciences) and incubated for 2 minutes to allow adhesion. The grids were subsequently washed with distilled water and stained with 1% uranyl acetate for 1 minute. Imaging was performed using a Titan ST (Thermo Fisher Scientific) transmission electron microscope operated at an accelerating voltage of 300 kV. For scanning electron microscope (SEM), bacterial samples were fixed overnight at 4°C in 2.5% glutaraldehyde prepared in 0.1 M sodium cacodylate buffer (pH 7.4). After fixation, the samples were washed three times in 0.1 M sodium cacodylate buffer (pH 7.4) to remove residual fixative. Post-fixation was carried out using 1% osmium tetroxide in water for 1 hour at room temperature, followed by three washes with distilled water. The samples were dehydrated through a graded ethanol series (25%, 50%, 75%, 95%, and 100%) with a 10-minute incubation in each concentration. Critical point drying was performed using a Leica Microsystems critical point dryer to preserve structural integrity. The dried samples were sputter-coated with a 6 nm layer of platinum to enhance conductivity and imaged using a Zeiss Merlin Gemini II field emission scanning electron microscope (FE-SEM) operated at an accelerating voltage of 3 kV and a beam current of 30 pA. Motility assays. To assess the motility of the Pseudomonas sp. R4-79 strain, a motility test was conducted using Motility Test Media (SIGMA-ALDRICH) in solid agar form. A 10 µL aliquot of an overnight bacterial culture OD 600 = 0.2 was carefully inoculated onto the center of the motility agar plate. The plate was then incubated at 28°C for 48 hours to allow bacterial growth and potential motility. After the incubation period, the motility of the Pseudomonas sp. R4-79 strain was assessed by observing the spread of bacterial growth from the point of inoculation. A clear zone of growth extending beyond the initial inoculation point would indicate bacterial motility, whereas the absence of such spread would suggest the strain is non-motile. The results were recorded based on the extent of the growth diffusion from the inoculation site. Siderophore production . To assess the siderophore production potential of the Pseudomonas sp. R4-79 strain, the assay was performed using the method described by (Brian et al.,1990) with blue agar CAS media. A 10 µL aliquot of the overnight bacterial culture was inoculated onto the CAS agar plate, which contains a blue iron complex. The plate was incubated at 28°C for 48 hours. Siderophore production was indicated by a yellow-orange halo around the bacterial growth, caused by the chelation of iron from the CAS complex, resulting in a color change in the surrounding agar. Biofilm formation. 200 µl of diluted overnight bacterial culture was added to each well of a 96-well plate and incubated overnight at 28°C. The liquid culture was removed by inverting the plate, followed by two washes with water to eliminate excess liquid. Each well was then treated with 125 µl of 0.1% crystal violet solution and incubated for 15 minutes. After rinsing the plate 3–4 times with water, it was inverted and left to dry overnight on a paper towel. The following day, 125 µl of 30% acetic acid was added to each well and incubated for 15 minutes. Biofilm quantification was performed using a plate reader (Infinite M200 PRO, TECAN). Effect of Pseudomonas sp . R4-79 against Pseudomonas syringae ( P st) DC3000 infection of Arabidopsis thaliana Pst DC3000 seed germination assays. We used Arabidopsis thaliana Col-0 (wild type) to quantify the survival percentage affected by Pseudomonas sp. R4-79 and P st DC3000. Seeds of A. thaliana were surface sterilized with 70% ethanol containing 0.05% Triton X-100 and shaken for 10 minutes, followed by three washes with absolute ethanol. The sterilized seeds were placed in a 2 ml Eppendorf tube containing 1 ml sterilized water (H₂O) and kept at 4°C for two days to ensure uniform germination. In square Petri dishes (12 × 12 cm), 50 ml Murashige and Skoog (MS) Basal Salt Mixture containing 0.9% agar (½ MS agar) at pH 5.8 (Murashige and Skoog, 1962; M5524, Sigma Aldrich, Germany) were mixed with 100 µl of Pseudomonas sp. R4-79 cells (OD₆₀₀ = 0.2), 100 µl of DC3000 cells (OD₆₀₀ = 0.2), 100 µl of Pseudomonas sp. R4-79 and DC3000 cells together (OD₆₀₀ = 0.2), and ½ MS agar media alone as a mock. A. thaliana seeds were spotted individually onto the ½ MS agar plates, with approximately 36 seeds per plate. The plates were placed in growth chambers (Percival Scientific Inc., USA) vertically (~ 75° angle to the horizontal) and incubated for 16 days at 23°C with a 16/8 h (light/dark) photoperiod. After 16 days, the number of Arabidopsis seedlings was counted, and the survival percentage was measured using this formula: $$\:Survival\:percentage=number\:of\:Arabidopsis\:seedlings÷number\:of\:seeds\:\left(36\right)\times\:100$$ Pst DC3000 seedling growth assays. We further investigated the inhibitory effect of the Pseudomonas sp. R4-79 strain at different concentrations of the pathogenic P st DC3000 bacteria. Surface-sterilized seeds of A. thaliana were sown on ½ MS agar mixed with 100 µl of Pseudomonas sp. R4-79 strain (OD₆₀₀ = 0.2), sealed with micropore tape, covered with aluminum foil, and stratified for two days at 4°C. After stratification, the plates were placed vertically (~ 75° angle to the horizontal) in a growth chamber for five days at 23°C with a 16/8 h (light/dark) photoperiod to allow germination. After five days, ½ MS agar plates without or with P st DC3000 were prepared by mixing them with 100 µl of different CFU concentrations (2 × 10⁸ to 1 × 10⁹). Six seedlings colonized and non-colonized with root lengths of ~ 1–1.5 cm were transferred to the prepared plates. The plates were incubated under the same growth conditions for 16 days. At the end of the experiment, the fresh weight of the plants was measured. Three biological replicates were performed (Fig. 1 ). Suppressive effect of Pseudomonas sp. R4-79 against Meloidogyne incognita infection of tomato The inhibitory effect of the Pseudomonas sp. R4-79 strain was tested against various phytopathogens to assess its potential to control disease complexes. We evaluated its effects both in vitro and in vivo on the root-knot nematode ( Meloidogyne incognita , RKN), the fungal pathogen Botrytis cinerea , and the bacterial pathogen Pseudomonas syringae pv. Tomato ( Pst , DC3000 strain). In a greenhouse experiment, two-week-old tomato seedlings (Moneymaker cultivar) were grown in ½-liter pots filled with sand supplemented with 1 gram of Osmocote per liter. The plants were treated with Pseudomonas sp. R4-79 both via soil drenching or foliar spray at an OD 600 of 0.2. Control plants, which received no bacterial application, were also prepared. As positive controls, plants were treated with Pseudomonas fluorescens strain SBW25 or the nematicide Velum® Prime (Fluopyram 41.5% active ingredient; Bayer CropScience). Each pot was drenched once with 10 µL of Velum® Prime diluted in 50 mL of water. After 5 days of bacterial treatment, the pots were infected with 300 second-stage juvenile RKN (J2s) to assess the bacterial strain's potential to inhibit RKN. Three weeks post-infection, the plants were sampled, and the roots were washed to remove soil. Root galls were counted, and plant biomass was measured. The experiment was repeated twice with a total of n = 21 − 16 replicates. In an analogous experiment, tomato seedlings were grown in 2-liter pots filled with peat moss supplemented with 1 gram of Osmocote per liter. The bacterial-treated and untreated plants followed the same procedure but were not infected with any pathogens. This setup allowed for the quantification of the plant growth-promoting effects of the Pseudomonas sp. R4-79 strain on tomato plants. The plants were maintained under greenhouse conditions (25°C, 16-hour photoperiod) and watered every 5 days. The experiment was done once with 30 to 40 replicates. Growth inhibitory effect of Pseudomonas sp. R4-79 strain on the pathogenic fungus Botrytis cinerea To assess the antifungal activity of the Pseudomonas sp. R4-79 strain against Botrytis cinerea , a dual-culture assay was conducted. A 1 cm² plug of B. cinerea mycelium, grown on Potato Dextrose Agar (PDA), was placed at the center of a fresh PDA plate. Three individual spots of Pseudomonas sp. R4-79 culture were inoculated on the same plate; each positioned 1.5 cm away from the fungal plug. The plates were incubated at 25°C for 7 days, after which inhibition of fungal growth was assessed and visualized. The experiment was performed with three biological replicates. Results Genome assembly and functional annotation The genome analysis of the Pseudomonas sp. R4-79 strain using PacBio technology revealed a genome size with 6,175,612 base pairs chromosome and 5473 genes and gene clusters with no plasmids. The annotation pipeline generated 5445 protein-coding sequences (CDS), 96 exons, 73 tRNAs, 67 pseudo genes, 19 rRNAs, 9 riboswitches, and 1 ncRNA ( Table 1 ). Genome functional analysis of the Pseudomonas sp. R4-79 strain was performed using the Blast KOALA platform. Genome mining revealed the presence of several pathways related to metabolism, genetic and environmental information processing, cellular processes, and organismal systems. In addition, the Pseudomonas sp. R4-79 strain uses different systems, such as ATP-binding cassette (ABC) transporters, phosphotransferase system (PTS), and bacterial secretion systems for membrane transport. The ABC transporter can transport minerals and organic ions, oligosaccharides, polyols, lipids, monosaccharides, phosphate and amino acids, peptides and nickel, metallic cation, iron-siderophore, and vitamin B12. The PTS includes N-acetyl-D-glucosamine, trehalose, fructose, and nitrogen regulation. The Pseudomonas sp. R4-79 strain has three secretion systems (type 2, type 4, and type 6), twin arginine targeting (TAT), and Sec-SRP. The Pseudomonas sp. R4-79 strain produces secondary metabolites, such as hydrogen cyanide (HCN), which can confer several benefits in plant-microbe interactions (Sehrawat et al., 2022 ), and phenazine, which showed wide spectrum activity against numerous plant pathogenic bacteria and fungi (Karmegham et al., 2020). Phylogenetic analysis based on a partial 16S rRNA gene sequence or whole-genome sequence revealed that Pseudomonas sp. R4-79 forms a well-supported monophyletic group with Pseudomonas granadensis LMG 27940 (Fig. 2 A-B). P. granadensis LMG 27940 was isolated from soil samples collected in the Tejeda, Almijara, and Alhama Natural Park in Granada, Spain. Table 1 Summary of Pseudomonas sp. R4-79 genome features. Feature Chromosome Genome size (bp) 6175612 Genome coverage 275x Genes 5473 CDS 5445 Exon 96 tRNA 73 Pseudo genes 67 rRNA 19 Riboswitch 9 tmRNA 1 SRP_RNA 1 Rnase_P_RNA 1 ncRNA 1 Bacterial and biochemical features of Pseudomonas sp. R4-79 Pseudomonas sp. R4-79 is a gram-negative, rod-shaped bacterium equipped with lophotrichous flagella (Fig. 4 B-C). The colonies exhibit a mucoid and glossy appearance that suggests the production of extracellular polysaccharides (EPS) or biofilm formation (Fig. 4 A). Additionally, the colonies display spreading with irregular edges, which indicates notable motility. This observation was further supported by a motility assay, which demonstrated swarming behavior on agar plates (Fig. 4 D). A biofilm formation assay confirmed the capability of the Pseudomonas sp. R4-79 strain to produce biofilm (Fig. 4 E). Furthermore, the strain exhibited a fluorescent yellow-green color on King B agar, which suggests the potential production of the siderophore pyoverdine. To confirm this, a chrome azurol sulfonate (CAS) agar assay was performed. The Pseudomonas sp. R4-79 strain showed yellowish growth on CAS agar, further indicating siderophore production (Fig. 4 F). In the bacterial strain Pseudomonas sp. R4-79, antiSMASH analysis revealed the presence of siderophore-producing gene clusters, which are crucial for iron acquisition in iron-limited environments, as well as BGCs for potential antimicrobial compounds such as hydrogen cyanide (HCN), fragin, and lokisin (Supplementary Table.1). These metabolites not only enhance bacterial survival but also contribute to plant growth promotion and protection by inhibiting pathogenic microbes, highlighting their ecological and biotechnological significance. The Pseudomonas sp. R4-79 strain can produce quorum-sensing molecules such as toxoflavin and riboflavin. Additionally, genome functional analysis reveals that Pseudomonas sp. R4-79 possesses the same biofilm-associated genes found in Pseudomonas aeruginosa . Arabidopsis bacterial Pst DC3000 phytopathogen suppression by Pseudomonas sp. R4-79 Pst DC3000 seed germination assays. The Pseudomonas sp. R4-79 strain demonstrated a strong inhibitory effect against the pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 ( P st) in Arabidopsis thaliana . Plant survival percentage varied based on whether Arabidopsis was treated with the Pseudomonas sp. R4-79 strain, P st DC3000, or a combination of both. Whereas mock-inoculated plants showed a survival percentage of 83.34%, plants pre-colonized with the Pseudomonas sp. R4-79 strain showed full survival (100%). In contrast, infection with P st DC3000 completely inhibited the growth of A. thaliana immediately after germination, and no plants survived. Notably, A. thaliana plants infected with P st DC3000 but treated with the Pseudomonas sp. R4-79 strain afterward exhibited a high survival rate of 86.11% (Fig. 5 A). Pst DC3000 seedling growth assays. We further investigated whether the inhibitory effect of the Pseudomonas sp. R4-79 strain depended on the pathogen load of P st DC3000. The Pseudomonas sp. R4-79 strain maintained its ability to suppress P st DC3000 across bacterial concentrations ranging from 2×10 8 to 1×10 9 CFU, which confirmed its capacity to reduce disease severity even at higher pathogen loads (Fig. 5 B-C). Pseudomonas sp. R4-79 promotes tomato growth and suppresses the root-knot nematode M. incognita We investigated the efficacy of Pseudomonas sp. R4-79 in promoting plant growth and suppressing phytopathogens. In greenhouse experiments, tomato plants colonized by Pseudomonas sp. R4-79 exhibited a significant increase in fresh weight, with a 14% improvement compared to the control group (t-test: P < 0.0001) under normal conditions. Additionally, these plants demonstrated greater shoot length relative to the control (Fig. 6 ). Meanwhile, we evaluated the ability of the Pseudomonas sp. R4-79 strain to suppress the root-knot nematode M. incognita in tomato plants. Tomato plants colonized with Pseudomonas sp. R4-79 under greenhouse conditions exhibited a significant reduction up to 59.4% in gall numbers on the root system compared to control non-colonized tomato plants (P < 0.0001) (Fig. 7 A). Compared to Pseudomonas fluorescens SBW25, the Pseudomonas sp. R4-79 strain exhibited a comparable suppressive effect on M. incognita (Fig. 7 A). In contrast, the chemical commercial product Velum® Prime completely impeded gall formation on tomato roots (Fig. 7 A). The reduction in gall numbers by Pseudomonas sp. R4-79 was accompanied by a slight, non-significant increase (26%) in tomato fresh weight compared to control plants (Fig. 7 B). Pseudomonas granadensis , R4-79, inhibits the gray mold fungus , Botrytis cinerea To evaluate the suppressive and biocontrol potential of P. granadensis R4-79 against other phytopathogens, we examined its antagonistic activity toward the widespread necrotrophic fungus Botrytis cinerea. The dual culture assay revealed that the Pseudomonas sp. R4-79 strain effectively inhibits the growth of Botrytis cinerea . A clear zone of inhibition was observed around the site of bacterial inoculation, which demonstrates its ability to limit fungal proliferation. Within this inhibition zone, fungal growth exhibited a marked reduction or was entirely suppressed, whereas colonies situated beyond this zone displayed normal growth patterns (Fig. 8 ). These observations confirm that P. granadensis R4-79 effectively limits B. cinerea proliferation in vitro. Discussion Dual capability of Pseudomonas granadensis in growth promotion and disease suppression The application of beneficial microbes from arid environments, such as the Arabian desert, can significantly enhance sustainable farming practices and ecological restoration in water-limited regions. Pseudomonas sp. R4-79 demonstrated remarkable effectiveness in enhancing plant growth and pathogen suppression. Here, we provide a detailed analysis of the strain's phenotypic and genomic features along with its potential effect of suppressing plant diseases. The phylogenetic analysis showed that the genome of the Pseudomonas sp. R4-79 strain is very similar to that of Pseudomonas granadensis strain LMG 27940, which was previously isolated from soil (Cea-Torrescassana et al., 2024 ; Pascual et al., 2015). This study identifies Pseudomonas sp. R4-79 as the third strain reported under the P. granadensis species and the first to be isolated from an arid environment, specifically in Wadi Rum, Jordan. In addition to Pseudomonas granadensis strain LMG 27940, strain CT364—phylogenetically clustered in the same clade as LMG 27940 and R4-79 based on 16S rRNA analysis—exhibited notable plant growth-promoting traits (Supplementary Fig. 1). Specifically, it enhanced rooting in olive semi-hardwood cuttings more effectively than the synthetic auxin indole-3-butyric acid (IBA) (Cea-Torrescassana et al., 2024 ). CT364 is also a phosphorus-solubilizing and siderophore-producing. All these parameters led to a greater root length of canola or mung bean seeds and mung bean cuttings, respectively, in gnotobiotic conditions. Such observations delineate the plant growth-promoting activities of P. granadensis (Cea-Torrescassana et al., 2024 ). In this context, Pseudomonas sp. R4-79 exhibited comparable plant growth-promoting activity as demonstrated by the larger fresh weight and shoot length of tomato plants colonized by this strain as compared to non-colonized tomato plants. Furthermore, this study provides the first evidence of a P. granadensis strain with the ability to suppress phytopathogens. Targeting in particular, Pseudomonas sp. R4-79 inhibited root-knot nematode M. incognita in tomato and yielded the same inhibitory result as P. fluorescens SBW25. In addition to its nematode-suppressive properties, Pseudomonas sp. R4-79 demonstrated robust antagonistic activity against the bacterial pathogen P. syringae pv. tomato DC3000 ( P st) in Arabidopsis. The colonization of the Pseudomonas sp. R4-79 strain conferred plant survival under pathogen attack. The strain suppressed P st across varying concentrations, showing consistent efficacy in reducing disease severity. In addition, Pseudomonas sp. R4-79 also exhibited antifungal activity against B. cinerea , a fungal pathogen, in dual culture assays. These results suggest that Pseudomonas sp. R4-79 might represent a plant-growth-promoting and biocontrol option to control a broad range of phytopathogens attacking crops grown in arid environments and farm fields. Genetic features related to host-microbe interactions and pathogen suppression Exploring the genetic features of beneficial bacteria is essential for understanding their role in plant-microbe interactions. KEGG-based functional annotation of the Pseudomonas sp. R4-79 genome revealed key traits linked to plant growth promotion, stress tolerance, and pathogen suppression. For instance, Bacterial membrane transporters, such as ATP-binding cassette (ABC) transporters, phosphotransferase (PTS) systems, and bacterial secretion systems, mediate cellular homeostasis and host-microbe interactions. Pseudomonas sp. R4-79 strain uses these systems for the acquisition of nutrients, ion transport, and secretion of molecules relevant to its ecological interactions. For instance, in P. fluorescens Pf-5, ABC transporters are implicated in the secretion of antimicrobial metabolites, such as Pyoluteorin, which exhibit biocontrol activity against soilborne pathogens and improve rhizosphere colonization. In particular, pltI and pltJ genes that promote pyoluteorin export are both necessary for pyoluteorin production and efficient secretion (Brodhagen et al., 2005). Moreover, the Pseudomonas sp. R4-79 possesses a PTS, which serves as a sugar transport system that uptake and phosphorylation of sugars, such as glucose and fructose, maintains bacterial viability by tuning the fluxes of carbons. In P. putida , the system not only enables metabolic plasticity but also plays important roles in both bioremediation and metabolic engineering (Kremling et al., 2012 ). On the other hand, the KEGG analysis by BlastKOALA showed that Pseudomonas sp. R4-79 possess various secretion systems, including Type II (T2SS), Type IV (T4SS), and Type VI (T6SS), and arginine targeting (TAT). Such secretion systems allow the translocation of proteins, toxins, DNA, and small molecules. In Dyella japonica , T2SS was crucial in suppressing MAMP-triggered immunity (MTI) in plants, thus promoting root colonization. For example, mutations in key T2SS components, GspD and GspE, in Dyella japonica MF79 led to the loss of the ability to inhibit MTI (Teixeira et al., 2021 ). On the other hand, T4SS is involved in DNA conjugation systems and the secretion of protein complexes that help in bacterial competition and host manipulation. For example, T4SS in P. putida IsoF protects tomato plants against Ralstonia solanacearum by competitive exclusion and by disruption of biofilm (Purtschert-Montenegro et al., 2022 ). T6SS injects effector proteins into both prokaryotic and eukaryotic cells and is involved in competition and pathogen suppression processes. For instance, in P. putida KT2440, the isogenic triple mutant (ΔT6SS) inhibited its ability to suppress the pathogenic bacteria Xanthomonas campestris compared to the P. putida KT2440 wild-type (Bernal et al., 2017 ; Bernal et al., 2018). Along with that, the Pseudomonas sp. R4-79 strains are producing secondary metabolites, such as hydrogen cyanide (HCN), that can provide various advantages in plant-microbe interactions, especially in the suppression of a broad spectrum of phytopathogens (Sehrawat et al., 2022 ). For example, HCN produced by Pseudomonas chlororaphis O6 has been shown to have suppressive activity against a root-knot nematode, Meloidogyne hapla (Lee et al., 2011 ). Further, HCN has also demonstrated the ability to suppress mycelial growth of Phytophthora infestans fungal pathogen with inhibition rates of 57–80% (Anand et al., 2020 ). Another secondary metabolite that Pseudomonas sp. R4-79 strain showed capability to produce is phenazine. Pseudomonas sp. LBUM223 produces phenazine-1-carboxylic acid (PCA), which plays a significant role in controlling potato common scab caused by Streptomyces scabies (Arseneault et al., 2013 ). Conclusion Pseudomonas granadensis R4-79 has high activity not only as a plant growth-promoting bacterium (PGPB) but also as a biocontrol agent. Its varied genetic makeup, such as membrane transporters, secretion systems, and secondary metabolites production, is attributed to its ability to promote plant growth and inhibit phytopathogens. Isolated from an arid area, the strain's robustness in an extreme environment sheds light on its significance for use in farming in water-deficit areas. Genome functional analysis also shows advantageous properties, including biosynthetic gene clusters of siderophores, antimicrobial substances, and phytohormones, making P. granadensis R4-79 a valuable potential agent for increasing plant immunity and ecological restoration in dry ecosystems. Future studies should address its effect on abiotic stresses and optimize its application in field conditions to fully harness its capabilities. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Funding The work was supported by grant BAS/1/1062-01-01 from the University of Science and Technology to HH. Availability of data and materials The WGS has been submitted to NCBI under Bioproject-PRJNA708169, Biosample-SAMN18220753, Genome accession-CP071650-CP071651. Author contributions H.H. and A.E. conceptualized the project. L.A. extracted the R4-79 genomic DNA and carried out all plant- and pathogen-related work. S.P. annotated, did the taxonomy and functional analysis of the R4-79 genome. L.A., A.E. and H.H. wrote the paper and all authors reviewed and corrected the final version. Acknowledgements The work was supported by KAUST grant BAS/01/1062-01-01 to HH. We are also grateful to the Hirt lab members for technical assistance and fruitful discussions. References Acuña, J. J., Campos, M., Mora, M. d. l. L., Jaisi, D. P., & Jorquera, M. A. (2019, 2019/04/01/). ACCD-producing rhizobacteria from an Andean Altiplano native plant (Parastrephia quadrangularis) and their potential to alleviate salt stress in wheat seedlings. Applied Soil Ecology, 136 , 184-190. https://doi.org/https://doi.org/10.1016/j.apsoil.2019.01.005 Alsharif, W., Saad, M. M., & Hirt, H. (2020, 2020-July-22). Desert Microbes for Boosting Sustainable Agriculture in Extreme Environments [Review]. Frontiers in Microbiology, 11 . https://doi.org/10.3389/fmicb.2020.01666 Alwutayd, K. M., Rawat, A. A., Sheikh, A. 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Specific modulation of the root immune system by a community of commensal bacteria. Proceedings of the National Academy of Sciences, 118 (16), e2100678118. https://doi.org/doi:10.1073/pnas.2100678118 Additional Declarations No competing interests reported. Supplementary Files Supplementary.pptx Cite Share Download PDF Status: Published Journal Publication published 28 Oct, 2025 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 30 Jun, 2025 Reviews received at journal 24 Jun, 2025 Reviews received at journal 12 Jun, 2025 Reviewers agreed at journal 06 Jun, 2025 Reviewers agreed at journal 02 Jun, 2025 Reviewers invited by journal 21 May, 2025 Editor invited by journal 19 May, 2025 Editor assigned by journal 16 May, 2025 Submission checks completed at journal 16 May, 2025 First submitted to journal 08 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6620576","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":460331814,"identity":"1d4efb72-5652-4de4-b544-60a73a480c19","order_by":0,"name":"Linah Alghanmi","email":"","orcid":"","institution":"King Abdullah University of Sciences and Technology","correspondingAuthor":false,"prefix":"","firstName":"Linah","middleName":"","lastName":"Alghanmi","suffix":""},{"id":460331815,"identity":"d9ca1836-dc8b-4c9e-ac5f-93cb30d0e786","order_by":1,"name":"Ahmed Elhady","email":"","orcid":"","institution":"King Abdullah University of Sciences and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"","lastName":"Elhady","suffix":""},{"id":460331816,"identity":"a2ab0590-3669-4373-83e6-dc36e6007695","order_by":2,"name":"Sabiha Parween","email":"","orcid":"","institution":"King Abdullah University of Sciences and Technology","correspondingAuthor":false,"prefix":"","firstName":"Sabiha","middleName":"","lastName":"Parween","suffix":""},{"id":460331817,"identity":"c9a9db2c-63df-4f56-a707-40ff8ba5b1f0","order_by":3,"name":"Heribert Hirt","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIiWNgGAWjYBACAyA+DGZJMDYwMFQkQMXZCGuRAGs5cAakhZmwFmaIFiB5sI0ILebsvQ8PF1Tcq2OQbm77/HFemrzB+fMHGD6UHWaQbz+AVYtlz3GDwzPOFEswyBxsnnFwW47hhhvJDIwzzh1mMDiTgFWLwY00hsO8bQlAhyU2MxzcVsG44QYzAzNvG1ALAw4t958BtfyDaZlTYb/h/GEG5r9ALfL9D3DYwgbU0gDT0pCTuOFAMgMzI1ALww0ctpwBOmzGsQTJNpCWM8fSkmfeSDY42HMuncfgBg5bjh9j/lxQk8DPL5H+mKGiJtm27/zBhw9+lFnLyfdjtwUOUCLiABDz4Fc/CkbBKBgFowAfAADhbmMhosOwTAAAAABJRU5ErkJggg==","orcid":"","institution":"King Abdullah University of Sciences and Technology","correspondingAuthor":true,"prefix":"","firstName":"Heribert","middleName":"","lastName":"Hirt","suffix":""}],"badges":[],"createdAt":"2025-05-08 12:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6620576/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6620576/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-025-04431-4","type":"published","date":"2025-10-28T15:58:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83338810,"identity":"d8fc1897-55c5-4c0b-9ffa-eeb9cc9011b0","added_by":"auto","created_at":"2025-05-23 09:53:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of germination and plant growth assays using \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in response to bacterial treatments. \u003c/strong\u003eA) Germination Assay: seeds were sown on ½ MS agar with and without \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain under DC3000 stress. Survival rates were quantified. B) Plant Assay: 5-day-old pre-colonized seeds with \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain were transferred to ½ MS agar mixed with DC3000. After 16 days, the fresh weight was measured\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6620576/v1/241dee9ca5515290f4e1c8eb.png"},{"id":83338800,"identity":"d3de0c0c-9892-40d7-a7d7-f01f8f87457f","added_by":"auto","created_at":"2025-05-23 09:53:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":126206,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree shows that strain \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 forms a monophyletic group with \u003cem\u003ePseudomonas granadensis\u003c/em\u003e LMG 27940 based on A) partial 16S rRNA gene sequences and B) whole-genome sequence comparison. The scale bars represent the number of substitutions per site. Gradient blue and red squares denote GC% and Δ statistics and illustrate genomic and compositional variations between the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain and other \u003cem\u003ePseudomonas\u003c/em\u003especies within the phylogenetic tree.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6620576/v1/6a8fc9e958f7f7b510348938.png"},{"id":83338817,"identity":"d2b28e80-71a7-4535-9a9d-854a7abbb8d8","added_by":"auto","created_at":"2025-05-23 09:53:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":381326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCircular genome map of the Pseudomonas sp. R4-79 chromosome.\u003c/strong\u003e The tracks from inside to outside: GC skew [(G−C/ (G+C)] positive (red) and negative (purple), % GC content (black), Arrows on the GC skews indicate the origin of replication (oriC) and replication termination (terC) regions where the shifts occur.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6620576/v1/a251727c145e09f2460fee60.png"},{"id":83338927,"identity":"d9caf76d-acc0-4d80-9a37-5198ed759ada","added_by":"auto","created_at":"2025-05-23 10:01:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":562576,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological features of Pseudomonas granadensis sp. R4-79.\u003c/strong\u003e A) Colony formation of the Pseudomonas sp. R4-79 strain on a Petri dish, with slimy colonies on King’s B medium. B) Scanning Electron Microscope (SEM) and C) Transmission Electron Microscope (TEM) shows rod-shaped bacterium equipped with lophotrichous flagella. D) The Pseudomonas sp. R4-79 strain motility on motility test agar media. E) Biofilm formation of Pseudomonas sp. R4-79 strain with absorbance quantification biofilms using a plate reader at 630 nm. 1,2 and 3 are Pseudomonas sp. R4-79 strain replicates, and M is the mock. F) Siderophore production test of Pseudomonas sp. R4-79 strain on blue agar CAS assay.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6620576/v1/8f47b50cdb20c0a7c9c60909.png"},{"id":83338809,"identity":"f214aa7f-82ec-49d4-8de0-5ea56c861536","added_by":"auto","created_at":"2025-05-23 09:53:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":381795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePseudomonas\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esp. R4-79 on the pathogenicity of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePseudomonas syringae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e pv. tomato DC3000 (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003est) in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eA) Survival rates of \u003cem\u003eA. thaliana\u003c/em\u003e plants infected with Pst DC3000 after pre-treatment with \u003cem\u003ePseudomonas\u003c/em\u003esp. R4-79. Controls include mock plants, plants infected with \u003cem\u003eP\u003c/em\u003est DC3000 without \u003cem\u003ePseudomonas\u003c/em\u003e sp.\u003cem\u003e \u003c/em\u003eR4-79 pre-treatment, and plants pre-colonized with \u003cem\u003ePseudomonas\u003c/em\u003e sp.\u003cem\u003e \u003c/em\u003eR4-79 alone.\u003cstrong\u003e \u003c/strong\u003e(B-C) Effect of varying concentrations of Pst DC3000 (2 × 10⁸ to 1 × 10⁹ CFU) on the fresh weight of \u003cem\u003eA. thaliana\u003c/em\u003e plants pre-treated with \u003cem\u003ePseudomonas\u003c/em\u003esp.\u003cem\u003e \u003c/em\u003eR4-79 compared to untreated controls (n = 18). Error bars indicate standard deviation.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6620576/v1/e1e6809d1a68724bad33ec16.png"},{"id":83338801,"identity":"0c7c98ab-a6d0-4ff0-9779-a7f2c3236700","added_by":"auto","created_at":"2025-05-23 09:53:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":216497,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTomato growth promotion by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePseudomonas\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp. R4-79. \u003c/strong\u003eA) Boxplot illustrates the effect of the \u003cem\u003ePseudomonas\u003c/em\u003esp.\u003cem\u003e \u003c/em\u003eR4-79 strain on tomato fresh weight in comparison to untreated control plants. B) A picture of tomato plants treated with the \u003cem\u003ePseudomonas\u003c/em\u003e sp.\u003cem\u003e \u003c/em\u003eR4-79 strain versus untreated plants. The experiment was conducted in a greenhouse under a randomized complete block design (RCBD) with 40 replicates.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6620576/v1/309fffd1a205213ba4d015dd.png"},{"id":83338808,"identity":"641b8007-560c-472f-ae31-431df3e019df","added_by":"auto","created_at":"2025-05-23 09:53:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":30415,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePseudomonas\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp. R4-79 on tomato growth and infection by the root-knot nematode (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMeloidogyne incognita\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e).\u003c/strong\u003e (A) Plant growth effect of \u003cem\u003ePseudomonas\u003c/em\u003e sp.\u003cem\u003e \u003c/em\u003eR4-79 strain on infected plants. (B) Suppressive effect of \u003cem\u003ePseudomonas\u003c/em\u003e sp.\u003cem\u003e \u003c/em\u003eR4-79 strain against \u003cem\u003eM. incognita\u003c/em\u003e on tomato plants. Boxplots show root gall counts and plant biomass across treatments (n = 21–16). Tomato seedlings were grown in sand with Osmocote and treated with \u003cem\u003ePseudomonas\u003c/em\u003e sp.\u003cem\u003e \u003c/em\u003eR4-79 strain (OD\u003csub\u003e600\u003c/sub\u003e = 0.2), \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e SBW25, Velum® Prime, or left untreated. Plants were inoculated with 300 nematode juveniles after 5 days, and data were collected 3 weeks later. Each boxplot displays the median, interquartile range, and data points.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6620576/v1/35c3dc3c0145cf0cd46176a9.png"},{"id":83338812,"identity":"ffdde5cd-93e2-44e8-a59a-adb7902794e9","added_by":"auto","created_at":"2025-05-23 09:53:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":503090,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntifungal activity of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePseudomonas\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp. R4-79 against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBotrytis cinerea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e on Potato Dextrose Agar medium (PDA). \u003c/strong\u003eA dual-culture assay was carried out to observe the antifungal activity of the \u003cem\u003ePseudomonas\u003c/em\u003e sp.\u003cem\u003e \u003c/em\u003eR4-79 strain against \u003cem\u003eB. cinerea\u003c/em\u003e. The assay was replicated three times (n=3).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6620576/v1/930e880c7701d94a7faa6420.png"},{"id":95040414,"identity":"0ad706e6-8a42-4808-b4e2-f128d2612510","added_by":"auto","created_at":"2025-11-03 16:08:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3977057,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6620576/v1/a13f9293-826b-410b-a0d2-8028754d0590.pdf"},{"id":83338811,"identity":"113a8201-01d4-4b52-b1fd-f4c90c3f978b","added_by":"auto","created_at":"2025-05-23 09:53:41","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":69374,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6620576/v1/a828c6f2daf0578261d0fb86.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome sequence of the desert endophyte Pseudomonas granadensis R4-79 reveals potential for plant-growth promotion and disease suppression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDesert ecosystems are among the most fragile environments on Earth, where water is scarce and soils are not rich in nutrients (Coleine et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These factors not only discourage ecological restoration but also create a limit to the scope of practicable models for sustainable farming (Rastgoo \u0026amp; Hasanfard, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Conversely, while previously, water resources and minimum vegetation cover were sought to be the focus for the rejuvenation of the desert ecosystem, recent findings emphasize the significance of microbial communities in boosting the desert ecosystem (Alsharif et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Islam et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Microbes, particularly those adapted to arid conditions, hold substantial potential for enhancing soil fertility, promoting plant growth, and improving resilience to environmental stresses (Alsharif et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, identifying beneficial microbes, enhancing their growth, and expanding their community diversity continue to be major challenges. Moreover, investigating their functional traits and phylogenomic adaptations is necessary for advancement in ecological and agricultural applications.\u003c/p\u003e \u003cp\u003eAs abiotic and biotic stresses exist, they make ecological revegetation and farming in arid deserts highly challenging. Plants and crops in these environments are highly susceptible to infestations by parasitic nematodes and insects, as well as infections caused by bacterial and fungal phytopathogens (Elhady et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sulaiman \u0026amp; Bello, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eMeloidogyne incognita\u003c/em\u003e is among the most destructive plant-parasitic nematodes, with nearly 100% occurrence in the collected soil samples, often exceeding the economic threshold, particularly in the Sahara and Arabian deserts (Elhady et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, pathogenic fungal species, including \u003cem\u003eFusarium\u003c/em\u003e spp., \u003cem\u003eAlternaria\u003c/em\u003e spp., \u003cem\u003ePythium\u003c/em\u003e spp., \u003cem\u003eRhizoctonia solani\u003c/em\u003e, \u003cem\u003ePhytophthora\u003c/em\u003e spp., \u003cem\u003eSclerotinia\u003c/em\u003e spp., and \u003cem\u003eVerticillium\u003c/em\u003e spp., are widely distributed and cause significant damage, with losses in agricultural production reaching 50\u0026ndash;75% in some cases (Panth et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The harsh abiotic conditions, coupled with limited plant and microbial diversity, further exacerbate plant susceptibility to phytopathogens that hinder the establishment of resilient vegetation. Compared to other ecosystems, plant pathogens have a significantly greater impact on arid ecosystems, but efforts to control these pathogens have received minimal attention (Elhady et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Jat et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDesert plants depend on mutualistic interactions with specialized microbial species to persist in extreme environments. These interactions are vital for the fitness and survival of both partners. For example, the legume shrub \u003cem\u003eVachellia jacquemontii\u003c/em\u003e in the Thar Desert associates with the nitrogen-fixing bacterium \u003cem\u003eEnsifer\u003c/em\u003e, which provides bioavailable nitrogen to support the plant's nutritional requirements (Ardley, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Similarly, despite the extreme aridity of the Atacama Desert, arbuscular mycorrhizal (AM) fungi have been reported (Santander et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) to potentially play significant roles in phosphorus solubilization, plant stress tolerance, and growth promotion.\u003c/p\u003e \u003cp\u003eThe isolation and application of microbial strains from native desert plants has emerged as an effective strategy to enhance crop performance under harsh conditions. For instance, the halotolerant \u003cem\u003eBacillus cabrialesii\u003c/em\u003e from the Qatar desert improved seedling growth under salt stress and reduced tomato infections by gray mold disease (\u003cem\u003eBotrytis cinerea\u003c/em\u003e) (Masmoudi et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Similarly, two \u003cem\u003eKlebsiella\u003c/em\u003e strains from \u003cem\u003eParastrephia quadrangularis\u003c/em\u003e in the Atacama Desert increased wheat seedling fresh weight by up to 60% under salinity stress (Acu\u0026ntilde;a et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eSerratia marcescens\u003c/em\u003e from \u003cem\u003eCapparis decidua\u003c/em\u003e in India\u0026rsquo;s Thar Desert enhanced wheat tolerance to salinity stress and reduced fungal disease severity caused by \u003cem\u003eFusarium graminearum\u003c/em\u003e (Singh \u0026amp; Jha, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, \u003cem\u003ePseudomonas argentinensis\u003c/em\u003e SA190 from \u003cem\u003eIndigofera argentea\u003c/em\u003e in Saudi Arabia's Jizan region improved drought tolerance in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e seedlings (Alwutayd et al., 2023).\u003c/p\u003e \u003cp\u003eIn an attempt to identify beneficial bacterial strains from the Arabian desert to support arid farming and ecological restoration, \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 was tested for promoting plant growth and suppressing various phytopathogens. This strain was isolated from the desert plant \u003cem\u003eIfloga spicata\u003c/em\u003e in Wadi Rum, Jordan. In this work, we characterize the phenotypic properties of \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 which inhibited several phytopathogens such as the bacterial pathogen \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003etomato\u003c/em\u003e DC3000, the root-knot nematode (\u003cem\u003eMeloidogyne incognita\u003c/em\u003e) and the fungal pathogen \u003cem\u003eBotrytis cinerea\u003c/em\u003e. Whole-genome sequencing was conducted to determine its taxonomic classification and explore its biochemical potential to promote plant growth and inhibit pathogens.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRoot collection and bacterial strain isolation\u003c/h2\u003e \u003cp\u003eRoot samples of the desert plant \u003cem\u003eIfloga spicata\u003c/em\u003e were collected from Wadi Rum, Jordan. To study the root endophytic bacteria, the root tissues were washed to remove soil debris and surface-sterilized by immersing them in 70% ethanol for 30 seconds, followed by treatment with 2% sodium hypochlorite for five minutes and thorough rinsing with sterile distilled water. The sterilized roots were macerated in a solution containing 0.8% saline and subjected to serial dilutions ranging from 10⁻\u0026sup2; to 10⁻⁵. Each dilution was plated in duplicate on different culture media, including Tryptone-Yeast (TY), R2A, LB agar, and TSA. Bacterial colonies were selected based on distinctive morphological characteristics and pigmentation, followed by further purification to obtain individual strains. The purified bacterial strains were suspended in a 25% glycerol-LB solution and stored at \u0026minus;\u0026thinsp;80\u0026deg;C to serve as reference stocks.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenomic DNA extraction and strains identification\u003c/h3\u003e\n\u003cp\u003eTo identify the bacterial strains, cells were streaked from 25% glycerol stock onto LB agar and incubated at 28\u0026deg;C overnight. A single bacterial colony was then inoculated into 10 mL of LB broth in a 50 mL Falcon tube and incubated at 28\u0026deg;C with shaking at 220 rpm. Genomic DNA was extracted using the Genelute Bacterial Genomic DNA Kit (Sigma-Aldrich). The 16S rRNA gene was amplified using Taq DNA Polymerase PCR Master Mix (Promega, Madison, WI, USA) and universal primers 27F (5\u0026rsquo;-AGAGTTTGATCCTGGCTCAG-3\u0026rsquo;) and 1492R (5\u0026rsquo;-TACGGYTACCTTGTTACGACTT-3\u0026rsquo;). The amplified PCR products were purified using ExoSAP-IT (Affymetrix, Santa Clara, CA, USA) and sequenced via Sanger sequencing at the Core Labs, KAUST, Saudi Arabia. Strain identification was performed by comparing the sequences to NCBI\u0026rsquo;s GenBank database using BLAST. Strains were sorted and grouped into collections based on their genus-level taxonomy, including \u003cem\u003ePseudomonas spp.\u003c/em\u003e, \u003cem\u003eBacillus spp.\u003c/em\u003e, \u003cem\u003eEnterobacter spp.\u003c/em\u003e, rhizobial strains, and others.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenome sequencing and annotation of\u003c/b\u003e \u003cb\u003ePseudomonas sp.\u003c/b\u003e \u003cb\u003eR4-79\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter screening 86 microbial strains for plant growth promotion, the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain was selected for its high effectiveness in promoting plant growth. The strain was further analyzed to characterize its genetic features and beneficial potential using \u003cem\u003ein silico\u003c/em\u003e, \u003cem\u003ein vitro\u003c/em\u003e, and \u003cem\u003ein vivo\u003c/em\u003e approaches. To analyze the genome of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain, the total genomic was shipped to Novogen Bioinformatics Technology Co., Ltd. (Singapore). Genomic sequencing and assembly were performed using Single-Molecule Real-Time (SMRT\u0026reg;) sequencing on the PacBio Sequel II/IIe platform. The whole genome assembly was performed using the Hierarchical Genome Assembly Process (HGAP). Genome polishing and circularization were carried out using Arrow (v2.3.3) and Circlator (v1.5.5), respectively. To assess the quality of the genome assembly, quantitative measurements were obtained using BUSCO (v4.0.2) (Benchmarking Universal Single-Copy Orthologs, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://busco.ezlab.org\u003c/span\u003e\u003cspan address=\"https://busco.ezlab.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Genome annotation was performed for coding genes, repetitive sequences, non-coding RNAs, and pseudogenes using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). PGAP identifies structural annotations by comparing open reading frames (ORFs) against libraries containing protein hidden Markov models (HMMs), representative RefSeq proteins, and proteins from well-characterized reference genomes. For genomic regions lacking HMM or protein evidence, GeneMarkS-2\u0026thinsp;+\u0026thinsp;was used to generate ab initio coding region predictions and to determine start sites for ORFs based on available HMM evidence.\u003c/p\u003e\n\u003ch3\u003ePhylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eThe genome sequence data were analyzed using the Type (Strain) Genome Server (TYGS), a free bioinformatics platform available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tygs.dsmz.de\u003c/span\u003e\u003cspan address=\"https://tygs.dsmz.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, for whole-genome-based taxonomic identification. Two methods were employed to identify the closest type strain genomes. First, the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain genome was compared to all type strain genomes in the TYGS database using the MASH algorithm, which approximates intergenomic relatedness. The 10 closest type strains with the smallest MASH distances were selected. Second, 16S rDNA gene sequences were extracted from the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain genome using RNAmmer, and each sequence was BLASTed against the 16S rDNA gene sequences of 18,357 type strains in the TYGS database. The 50 best-matching type strains were identified, and precise distances were calculated using the Genome BLAST Distance Phylogeny (GBDP) approach with the \u0026ldquo;coverage\u0026rdquo; algorithm and distance formula d5. These distances were used to determine the 10 closest type strain genomes to the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain genome.\u003c/p\u003e \u003cp\u003eFor phylogenomic analysis, pairwise comparisons among the selected genomes were performed using GBDP, with intergenomic distances inferred using the \u0026lsquo;trimming\u0026rsquo; algorithm and distance formula d5. One hundred distance replicates were calculated for each comparison. Digital DNA-DNA hybridization (dDDH) values and confidence intervals were computed using GGDC 3.0. The intergenomic distances were used to infer a balanced minimum evolution tree with branch support via FASTME 2.1.6.1, including SPR postprocessing and 100 pseudo-bootstrap replicates. The trees were rooted at the midpoint and visualized with PhyD3. Species clustering was based on a 70% digital DDH threshold, while subspecies clustering was done using a 79% dDDH threshold.\u003c/p\u003e\n\u003ch3\u003eSequence accession number\u003c/h3\u003e\n\u003cp\u003eThe WGS has been submitted to NCBI under Bioproject-PRJNA708169, Biosample-SAMN18220753, Genome accession-CP071650-CP071651.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhenotypic and biochemical properties of\u003c/b\u003e \u003cb\u003ePseudomonas\u003c/b\u003e \u003cb\u003esp. R4-79\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eStrain culturing\u003c/b\u003e. Cells of \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain were revived from a 25% glycerol stock stored at \u0026minus;\u0026thinsp;80\u0026deg;C by streaking onto King\u0026rsquo;s B agar (Sigma-Aldrich) and incubating at 28\u0026deg;C overnight. Then, a single pure colony was inoculated into 10 mL of sterilized LB broth (Invitrogen) in a 50 mL Falcon tube (Fisher Scientific) and incubated overnight on an orbital shaker (Innova 42, New Brunswick) at 28\u0026deg;C /220 rpm. A fresh culture was prepared by adding 5 ml of LB broth (Lennox L Broth Base, Invitrogen) to a 15 ml tube (Fisher Scientific), followed by the addition of 500 \u0026micro;l of bacterial culture. The mixture was incubated at 28\u0026deg;C with shaking at 220 rpm on an orbital shaker for 45 minutes to allow the culture to reach the exponential phase. The optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) was then measured and adjusted to 0.2 using a spectrophotometer (BioPhotometer, Eppendorf).\u003c/p\u003e \u003cp\u003e \u003cb\u003eElectron microscopy.\u003c/b\u003e The morphology of the bacterial strain \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 was visualized using the transmission electron microscopy (TEM). A 5-\u0026micro;l of the bacterial cells OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.2 was deposited onto carbon/Formvar-coated TEM grids (Electron Microscopy Sciences) and incubated for 2 minutes to allow adhesion. The grids were subsequently washed with distilled water and stained with 1% uranyl acetate for 1 minute. Imaging was performed using a Titan ST (Thermo Fisher Scientific) transmission electron microscope operated at an accelerating voltage of 300 kV. For scanning electron microscope (SEM), bacterial samples were fixed overnight at 4\u0026deg;C in 2.5% glutaraldehyde prepared in 0.1 M sodium cacodylate buffer (pH 7.4). After fixation, the samples were washed three times in 0.1 M sodium cacodylate buffer (pH 7.4) to remove residual fixative. Post-fixation was carried out using 1% osmium tetroxide in water for 1 hour at room temperature, followed by three washes with distilled water. The samples were dehydrated through a graded ethanol series (25%, 50%, 75%, 95%, and 100%) with a 10-minute incubation in each concentration. Critical point drying was performed using a Leica Microsystems critical point dryer to preserve structural integrity. The dried samples were sputter-coated with a 6 nm layer of platinum to enhance conductivity and imaged using a Zeiss Merlin Gemini II field emission scanning electron microscope (FE-SEM) operated at an accelerating voltage of 3 kV and a beam current of 30 pA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMotility assays.\u003c/b\u003e To assess the motility of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain, a motility test was conducted using Motility Test Media (SIGMA-ALDRICH) in solid agar form. A 10 \u0026micro;L aliquot of an overnight bacterial culture OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.2 was carefully inoculated onto the center of the motility agar plate. The plate was then incubated at 28\u0026deg;C for 48 hours to allow bacterial growth and potential motility. After the incubation period, the motility of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain was assessed by observing the spread of bacterial growth from the point of inoculation. A clear zone of growth extending beyond the initial inoculation point would indicate bacterial motility, whereas the absence of such spread would suggest the strain is non-motile. The results were recorded based on the extent of the growth diffusion from the inoculation site.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSiderophore production\u003c/b\u003e. To assess the siderophore production potential of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain, the assay was performed using the method described by (Brian et al.,1990) with blue agar CAS media. A 10 \u0026micro;L aliquot of the overnight bacterial culture was inoculated onto the CAS agar plate, which contains a blue iron complex. The plate was incubated at 28\u0026deg;C for 48 hours. Siderophore production was indicated by a yellow-orange halo around the bacterial growth, caused by the chelation of iron from the CAS complex, resulting in a color change in the surrounding agar.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiofilm formation.\u003c/b\u003e 200 \u0026micro;l of diluted overnight bacterial culture was added to each well of a 96-well plate and incubated overnight at 28\u0026deg;C. The liquid culture was removed by inverting the plate, followed by two washes with water to eliminate excess liquid. Each well was then treated with 125 \u0026micro;l of 0.1% crystal violet solution and incubated for 15 minutes. After rinsing the plate 3\u0026ndash;4 times with water, it was inverted and left to dry overnight on a paper towel. The following day, 125 \u0026micro;l of 30% acetic acid was added to each well and incubated for 15 minutes. Biofilm quantification was performed using a plate reader (Infinite M200 PRO, TECAN).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003ePseudomonas\u003c/b\u003e \u003cb\u003esp\u003c/b\u003e. \u003cb\u003eR4-79 against\u003c/b\u003e \u003cb\u003ePseudomonas syringae\u003c/b\u003e \u003cb\u003e(\u003c/b\u003e\u003cb\u003eP\u003c/b\u003e\u003cb\u003est) DC3000 infection of\u003c/b\u003e \u003cb\u003eArabidopsis thaliana\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003ePst\u003c/b\u003e \u003cb\u003eDC3000 seed germination assays.\u003c/b\u003e We used \u003cem\u003eArabidopsis thaliana\u003c/em\u003e Col-0 (wild type) to quantify the survival percentage affected by \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 and \u003cem\u003eP\u003c/em\u003est DC3000. Seeds of \u003cem\u003eA. thaliana\u003c/em\u003e were surface sterilized with 70% ethanol containing 0.05% Triton X-100 and shaken for 10 minutes, followed by three washes with absolute ethanol. The sterilized seeds were placed in a 2 ml Eppendorf tube containing 1 ml sterilized water (H₂O) and kept at 4\u0026deg;C for two days to ensure uniform germination. In square Petri dishes (12 \u0026times; 12 cm), 50 ml Murashige and Skoog (MS) Basal Salt Mixture containing 0.9% agar (\u0026frac12; MS agar) at pH 5.8 (Murashige and Skoog, 1962; M5524, Sigma Aldrich, Germany) were mixed with 100 \u0026micro;l of \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 cells (OD₆₀₀ = 0.2), 100 \u0026micro;l of DC3000 cells (OD₆₀₀ = 0.2), 100 \u0026micro;l of \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 and DC3000 cells together (OD₆₀₀ = 0.2), and \u0026frac12; MS agar media alone as a mock. \u003cem\u003eA. thaliana\u003c/em\u003e seeds were spotted individually onto the \u0026frac12; MS agar plates, with approximately 36 seeds per plate. The plates were placed in growth chambers (Percival Scientific Inc., USA) vertically (~\u0026thinsp;75\u0026deg; angle to the horizontal) and incubated for 16 days at 23\u0026deg;C with a 16/8 h (light/dark) photoperiod. After 16 days, the number of \u003cem\u003eArabidopsis\u003c/em\u003e seedlings was counted, and the survival percentage was measured using this formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Survival\\:percentage=number\\:of\\:Arabidopsis\\:seedlings\u0026divide;number\\:of\\:seeds\\:\\left(36\\right)\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003ePst\u003c/b\u003e \u003cb\u003eDC3000 seedling growth assays.\u003c/b\u003e We further investigated the inhibitory effect of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain at different concentrations of the pathogenic \u003cem\u003eP\u003c/em\u003est DC3000 bacteria. Surface-sterilized seeds of \u003cem\u003eA. thaliana\u003c/em\u003e were sown on \u0026frac12; MS agar mixed with 100 \u0026micro;l of \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain (OD₆₀₀ = 0.2), sealed with micropore tape, covered with aluminum foil, and stratified for two days at 4\u0026deg;C. After stratification, the plates were placed vertically (~\u0026thinsp;75\u0026deg; angle to the horizontal) in a growth chamber for five days at 23\u0026deg;C with a 16/8 h (light/dark) photoperiod to allow germination. After five days, \u0026frac12; MS agar plates without or with \u003cem\u003eP\u003c/em\u003est DC3000 were prepared by mixing them with 100 \u0026micro;l of different CFU concentrations (2 \u0026times; 10⁸ to 1 \u0026times; 10⁹). Six seedlings colonized and non-colonized with root lengths of ~\u0026thinsp;1\u0026ndash;1.5 cm were transferred to the prepared plates. The plates were incubated under the same growth conditions for 16 days. At the end of the experiment, the fresh weight of the plants was measured. Three biological replicates were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSuppressive effect of\u003c/b\u003e \u003cb\u003ePseudomonas sp.\u003c/b\u003e \u003cb\u003eR4-79 against\u003c/b\u003e \u003cb\u003eMeloidogyne incognita\u003c/b\u003e \u003cb\u003einfection of tomato\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe inhibitory effect of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain was tested against various phytopathogens to assess its potential to control disease complexes. We evaluated its effects both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e on the root-knot nematode (\u003cem\u003eMeloidogyne incognita\u003c/em\u003e, RKN), the fungal pathogen \u003cem\u003eBotrytis cinerea\u003c/em\u003e, and the bacterial pathogen \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003eTomato\u003c/em\u003e (\u003cem\u003ePst\u003c/em\u003e, DC3000 strain). In a greenhouse experiment, two-week-old tomato seedlings (Moneymaker cultivar) were grown in \u0026frac12;-liter pots filled with sand supplemented with 1 gram of Osmocote per liter. The plants were treated with \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 both via soil drenching or foliar spray at an OD\u003csub\u003e600\u003c/sub\u003e of 0.2. Control plants, which received no bacterial application, were also prepared. As positive controls, plants were treated with \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e strain SBW25 or the nematicide Velum\u0026reg; Prime (Fluopyram 41.5% active ingredient; Bayer CropScience). Each pot was drenched once with 10 \u0026micro;L of Velum\u0026reg; Prime diluted in 50 mL of water. After 5 days of bacterial treatment, the pots were infected with 300 second-stage juvenile RKN (J2s) to assess the bacterial strain's potential to inhibit RKN. Three weeks post-infection, the plants were sampled, and the roots were washed to remove soil. Root galls were counted, and plant biomass was measured. The experiment was repeated twice with a total of n\u0026thinsp;=\u0026thinsp;21\u0026thinsp;\u0026minus;\u0026thinsp;16 replicates. In an analogous experiment, tomato seedlings were grown in 2-liter pots filled with peat moss supplemented with 1 gram of Osmocote per liter. The bacterial-treated and untreated plants followed the same procedure but were not infected with any pathogens. This setup allowed for the quantification of the plant growth-promoting effects of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain on tomato plants. The plants were maintained under greenhouse conditions (25\u0026deg;C, 16-hour photoperiod) and watered every 5 days. The experiment was done once with 30 to 40 replicates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth inhibitory effect of\u003c/b\u003e \u003cb\u003ePseudomonas\u003c/b\u003e \u003cb\u003esp. R4-79 strain on the pathogenic fungus\u003c/b\u003e \u003cb\u003eBotrytis cinerea\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess the antifungal activity of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain against \u003cem\u003eBotrytis cinerea\u003c/em\u003e, a dual-culture assay was conducted. A 1 cm\u0026sup2; plug of \u003cem\u003eB. cinerea\u003c/em\u003e mycelium, grown on Potato Dextrose Agar (PDA), was placed at the center of a fresh PDA plate. Three individual spots of \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 culture were inoculated on the same plate; each positioned 1.5 cm away from the fungal plug. The plates were incubated at 25\u0026deg;C for 7 days, after which inhibition of fungal growth was assessed and visualized. The experiment was performed with three biological replicates.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGenome assembly and functional annotation\u003c/h2\u003e \u003cp\u003eThe genome analysis of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain using PacBio technology revealed a genome size with 6,175,612 base pairs chromosome and 5473 genes and gene clusters with no plasmids. The annotation pipeline generated 5445 protein-coding sequences (CDS), 96 exons, 73 tRNAs, 67 pseudo genes, 19 rRNAs, 9 riboswitches, and 1 ncRNA \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenome functional analysis of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain was performed using the Blast KOALA platform. Genome mining revealed the presence of several pathways related to metabolism, genetic and environmental information processing, cellular processes, and organismal systems. In addition, the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain uses different systems, such as ATP-binding cassette (ABC) transporters, phosphotransferase system (PTS), and bacterial secretion systems for membrane transport. The ABC transporter can transport minerals and organic ions, oligosaccharides, polyols, lipids, monosaccharides, phosphate and amino acids, peptides and nickel, metallic cation, iron-siderophore, and vitamin B12. The PTS includes N-acetyl-D-glucosamine, trehalose, fructose, and nitrogen regulation. The \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain has three secretion systems (type 2, type 4, and type 6), twin arginine targeting (TAT), and Sec-SRP.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain produces secondary metabolites, such as hydrogen cyanide (HCN), which can confer several benefits in plant-microbe interactions (Sehrawat et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and phenazine, which showed wide spectrum activity against numerous plant pathogenic bacteria and fungi (Karmegham et al., 2020).\u003c/p\u003e \u003cp\u003ePhylogenetic analysis based on a partial 16S rRNA gene sequence or whole-genome sequence revealed that \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 forms a well-supported monophyletic group with \u003cem\u003ePseudomonas granadensis\u003c/em\u003e LMG 27940 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). \u003cem\u003eP. granadensis\u003c/em\u003e LMG 27940 was isolated from soil samples collected in the Tejeda, Almijara, and Alhama Natural Park in Granada, Spain.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of Pseudomonas sp. R4-79 genome features.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeature\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenome size (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6175612\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenome coverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e275x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5473\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5445\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePseudo genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003erRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRiboswitch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etmRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSRP_RNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRnase_P_RNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003encRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBacterial and biochemical features of\u003c/b\u003e \u003cb\u003ePseudomonas\u003c/b\u003e \u003cb\u003esp. R4-79\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 is a gram-negative, rod-shaped bacterium equipped with lophotrichous flagella (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). The colonies exhibit a mucoid and glossy appearance that suggests the production of extracellular polysaccharides (EPS) or biofilm formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Additionally, the colonies display spreading with irregular edges, which indicates notable motility. This observation was further supported by a motility assay, which demonstrated swarming behavior on agar plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). A biofilm formation assay confirmed the capability of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain to produce biofilm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Furthermore, the strain exhibited a fluorescent yellow-green color on King B agar, which suggests the potential production of the siderophore pyoverdine. To confirm this, a chrome azurol sulfonate (CAS) agar assay was performed. The \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain showed yellowish growth on CAS agar, further indicating siderophore production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the bacterial strain \u003cem\u003ePseudomonas sp.\u003c/em\u003e R4-79, antiSMASH analysis revealed the presence of siderophore-producing gene clusters, which are crucial for iron acquisition in iron-limited environments, as well as BGCs for potential antimicrobial compounds such as hydrogen cyanide (HCN), fragin, and lokisin (Supplementary Table.1). These metabolites not only enhance bacterial survival but also contribute to plant growth promotion and protection by inhibiting pathogenic microbes, highlighting their ecological and biotechnological significance. The \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain can produce quorum-sensing molecules such as toxoflavin and riboflavin. Additionally, genome functional analysis reveals that \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 possesses the same biofilm-associated genes found in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eArabidopsis\u003c/b\u003e \u003cb\u003ebacterial\u003c/b\u003e \u003cb\u003ePst\u003c/b\u003e \u003cb\u003eDC3000 phytopathogen suppression by\u003c/b\u003e \u003cb\u003ePseudomonas\u003c/b\u003e \u003cb\u003esp. R4-79\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003ePst\u003c/b\u003e \u003cb\u003eDC3000 seed germination assays.\u003c/b\u003e The \u003cem\u003ePseudomonas\u003c/em\u003e sp. \u003cem\u003eR4-79\u003c/em\u003e strain demonstrated a strong inhibitory effect against the pathogenic bacterium \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003etomato\u003c/em\u003e DC3000 (\u003cem\u003eP\u003c/em\u003est) in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Plant survival percentage varied based on whether \u003cem\u003eArabidopsis\u003c/em\u003e was treated with the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain, \u003cem\u003eP\u003c/em\u003est DC3000, or a combination of both. Whereas mock-inoculated plants showed a survival percentage of 83.34%, plants pre-colonized with the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain showed full survival (100%). In contrast, infection with \u003cem\u003eP\u003c/em\u003est DC3000 completely inhibited the growth of \u003cem\u003eA. thaliana\u003c/em\u003e immediately after germination, and no plants survived. Notably, \u003cem\u003eA. thaliana\u003c/em\u003e plants infected with \u003cem\u003eP\u003c/em\u003est DC3000 but treated with the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain afterward exhibited a high survival rate of 86.11% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePst\u003c/b\u003e \u003cb\u003eDC3000 seedling growth assays.\u003c/b\u003e We further investigated whether the inhibitory effect of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain depended on the pathogen load of \u003cem\u003eP\u003c/em\u003est DC3000. The \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain maintained its ability to suppress \u003cem\u003eP\u003c/em\u003est DC3000 across bacterial concentrations ranging from 2\u0026times;10\u003csup\u003e8\u003c/sup\u003e to 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU, which confirmed its capacity to reduce disease severity even at higher pathogen loads (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePseudomonas\u003c/b\u003e \u003cb\u003esp. R4-79 promotes tomato growth and suppresses the root-knot nematode\u003c/b\u003e \u003cb\u003eM. incognita\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe investigated the efficacy of \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 in promoting plant growth and suppressing phytopathogens. In greenhouse experiments, tomato plants colonized by \u003cem\u003ePseudomonas sp.\u003c/em\u003e R4-79 exhibited a significant increase in fresh weight, with a 14% improvement compared to the control group (t-test: P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) under normal conditions. Additionally, these plants demonstrated greater shoot length relative to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Meanwhile, we evaluated the ability of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain to suppress the root-knot nematode \u003cem\u003eM. incognita\u003c/em\u003e in tomato plants. Tomato plants colonized with \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 under greenhouse conditions exhibited a significant reduction up to 59.4% in gall numbers on the root system compared to control non-colonized tomato plants (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Compared to \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e SBW25, the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain exhibited a comparable suppressive effect on \u003cem\u003eM. incognita\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). In contrast, the chemical commercial product Velum\u0026reg; Prime completely impeded gall formation on tomato roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The reduction in gall numbers by \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 was accompanied by a slight, non-significant increase (26%) in tomato fresh weight compared to control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePseudomonas granadensis\u003c/b\u003e, \u003cb\u003eR4-79, inhibits the gray mold fungus\u003c/b\u003e, \u003cb\u003eBotrytis cinerea\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the suppressive and biocontrol potential of \u003cem\u003eP. granadensis\u003c/em\u003e R4-79 against other phytopathogens, we examined its antagonistic activity toward the widespread necrotrophic fungus \u003cem\u003eBotrytis cinerea.\u003c/em\u003e The dual culture assay revealed that the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain effectively inhibits the growth of \u003cem\u003eBotrytis cinerea\u003c/em\u003e. A clear zone of inhibition was observed around the site of bacterial inoculation, which demonstrates its ability to limit fungal proliferation. Within this inhibition zone, fungal growth exhibited a marked reduction or was entirely suppressed, whereas colonies situated beyond this zone displayed normal growth patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These observations confirm that \u003cem\u003eP. granadensis\u003c/em\u003e R4-79 effectively limits \u003cem\u003eB. cinerea\u003c/em\u003e proliferation in vitro.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eDual capability of\u003c/b\u003e \u003cb\u003ePseudomonas granadensis\u003c/b\u003e \u003cb\u003ein growth promotion and disease suppression\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe application of beneficial microbes from arid environments, such as the Arabian desert, can significantly enhance sustainable farming practices and ecological restoration in water-limited regions. \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 demonstrated remarkable effectiveness in enhancing plant growth and pathogen suppression. Here, we provide a detailed analysis of the strain's phenotypic and genomic features along with its potential effect of suppressing plant diseases. The phylogenetic analysis showed that the genome of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain is very similar to that of \u003cem\u003ePseudomonas granadensis\u003c/em\u003e strain LMG 27940, which was previously isolated from soil (Cea-Torrescassana et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Pascual et al., 2015). This study identifies \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 as the third strain reported under the \u003cem\u003eP. granadensis\u003c/em\u003e species and the first to be isolated from an arid environment, specifically in Wadi Rum, Jordan. In addition to \u003cem\u003ePseudomonas granadensis\u003c/em\u003e strain LMG 27940, strain CT364\u0026mdash;phylogenetically clustered in the same clade as LMG 27940 and R4-79 based on 16S rRNA analysis\u0026mdash;exhibited notable plant growth-promoting traits (Supplementary Fig.\u0026nbsp;1). Specifically, it enhanced rooting in olive semi-hardwood cuttings more effectively than the synthetic auxin indole-3-butyric acid (IBA) (Cea-Torrescassana et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). CT364 is also a phosphorus-solubilizing and siderophore-producing. All these parameters led to a greater root length of canola or mung bean seeds and mung bean cuttings, respectively, in gnotobiotic conditions. Such observations delineate the plant growth-promoting activities of \u003cem\u003eP. granadensis\u003c/em\u003e (Cea-Torrescassana et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this context, \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 exhibited comparable plant growth-promoting activity as demonstrated by the larger fresh weight and shoot length of tomato plants colonized by this strain as compared to non-colonized tomato plants. Furthermore, this study provides the first evidence of a \u003cem\u003eP. granadensis\u003c/em\u003e strain with the ability to suppress phytopathogens. Targeting in particular, \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 inhibited root-knot nematode \u003cem\u003eM. incognita\u003c/em\u003e in tomato and yielded the same inhibitory result as \u003cem\u003eP. fluorescens\u003c/em\u003e SBW25. In addition to its nematode-suppressive properties, \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 demonstrated robust antagonistic activity against the bacterial pathogen \u003cem\u003eP. syringae\u003c/em\u003e pv. tomato DC3000 (\u003cem\u003eP\u003c/em\u003est) in Arabidopsis. The colonization of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain conferred plant survival under pathogen attack. The strain suppressed \u003cem\u003eP\u003c/em\u003est across varying concentrations, showing consistent efficacy in reducing disease severity. In addition, \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 also exhibited antifungal activity against \u003cem\u003eB. cinerea\u003c/em\u003e, a fungal pathogen, in dual culture assays. These results suggest that \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 might represent a plant-growth-promoting and biocontrol option to control a broad range of phytopathogens attacking crops grown in arid environments and farm fields.\u003c/p\u003e\n\u003ch3\u003eGenetic features related to host-microbe interactions and pathogen suppression\u003c/h3\u003e\n\u003cp\u003eExploring the genetic features of beneficial bacteria is essential for understanding their role in plant-microbe interactions. KEGG-based functional annotation of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 genome revealed key traits linked to plant growth promotion, stress tolerance, and pathogen suppression. For instance, Bacterial membrane transporters, such as ATP-binding cassette (ABC) transporters, phosphotransferase (PTS) systems, and bacterial secretion systems, mediate cellular homeostasis and host-microbe interactions. \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain uses these systems for the acquisition of nutrients, ion transport, and secretion of molecules relevant to its ecological interactions. For instance, in \u003cem\u003eP. fluorescens\u003c/em\u003e Pf-5, ABC transporters are implicated in the secretion of antimicrobial metabolites, such as Pyoluteorin, which exhibit biocontrol activity against soilborne pathogens and improve rhizosphere colonization. In particular, pltI and pltJ genes that promote pyoluteorin export are both necessary for pyoluteorin production and efficient secretion (Brodhagen et al., 2005). Moreover, the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 possesses a PTS, which serves as a sugar transport system that uptake and phosphorylation of sugars, such as glucose and fructose, maintains bacterial viability by tuning the fluxes of carbons. In \u003cem\u003eP. putida\u003c/em\u003e, the system not only enables metabolic plasticity but also plays important roles in both bioremediation and metabolic engineering (Kremling et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). On the other hand, the KEGG analysis by BlastKOALA showed that \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 possess various secretion systems, including Type II (T2SS), Type IV (T4SS), and Type VI (T6SS), and arginine targeting (TAT). Such secretion systems allow the translocation of proteins, toxins, DNA, and small molecules. In \u003cem\u003eDyella japonica\u003c/em\u003e, T2SS was crucial in suppressing MAMP-triggered immunity (MTI) in plants, thus promoting root colonization. For example, mutations in key T2SS components, GspD and GspE, in \u003cem\u003eDyella japonica\u003c/em\u003e MF79 led to the loss of the ability to inhibit MTI (Teixeira et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). On the other hand, T4SS is involved in DNA conjugation systems and the secretion of protein complexes that help in bacterial competition and host manipulation. For example, T4SS in \u003cem\u003eP. putida\u003c/em\u003e IsoF protects tomato plants against \u003cem\u003eRalstonia solanacearum\u003c/em\u003e by competitive exclusion and by disruption of biofilm (Purtschert-Montenegro et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). T6SS injects effector proteins into both prokaryotic and eukaryotic cells and is involved in competition and pathogen suppression processes. For instance, in \u003cem\u003eP. putida\u003c/em\u003e KT2440, the isogenic triple mutant (ΔT6SS) inhibited its ability to suppress the pathogenic bacteria \u003cem\u003eXanthomonas campestris\u003c/em\u003e compared to the \u003cem\u003eP. putida\u003c/em\u003e KT2440 wild-type (Bernal et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bernal et al., 2018). Along with that, the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strains are producing secondary metabolites, such as hydrogen cyanide (HCN), that can provide various advantages in plant-microbe interactions, especially in the suppression of a broad spectrum of phytopathogens (Sehrawat et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For example, HCN produced by \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e O6 has been shown to have suppressive activity against a root-knot nematode, \u003cem\u003eMeloidogyne hapla\u003c/em\u003e (Lee et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Further, HCN has also demonstrated the ability to suppress mycelial growth of \u003cem\u003ePhytophthora infestans\u003c/em\u003e fungal pathogen with inhibition rates of 57\u0026ndash;80% (Anand et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Another secondary metabolite that \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain showed capability to produce is phenazine. \u003cem\u003ePseudomonas sp.\u003c/em\u003e LBUM223 produces phenazine-1-carboxylic acid (PCA), which plays a significant role in controlling potato common scab caused by \u003cem\u003eStreptomyces scabies\u003c/em\u003e (Arseneault et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cem\u003ePseudomonas granadensis\u003c/em\u003e R4-79 has high activity not only as a plant growth-promoting bacterium (PGPB) but also as a biocontrol agent. Its varied genetic makeup, such as membrane transporters, secretion systems, and secondary metabolites production, is attributed to its ability to promote plant growth and inhibit phytopathogens. Isolated from an arid area, the strain's robustness in an extreme environment sheds light on its significance for use in farming in water-deficit areas. Genome functional analysis also shows advantageous properties, including biosynthetic gene clusters of siderophores, antimicrobial substances, and phytohormones, making \u003cem\u003eP. granadensis\u003c/em\u003e R4-79 a valuable potential agent for increasing plant immunity and ecological restoration in dry ecosystems. Future studies should address its effect on abiotic stresses and optimize its application in field conditions to fully harness its capabilities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by grant BAS/1/1062-01-01 from the University of Science and Technology to HH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe WGS has been submitted to NCBI under Bioproject-PRJNA708169,\u0026nbsp;Biosample-SAMN18220753, Genome accession-CP071650-CP071651.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.H. and A.E. conceptualized the project. L.A. extracted the R4-79 genomic DNA and carried out all plant- and pathogen-related work. S.P. annotated, did the taxonomy and functional analysis of the R4-79 genome. L.A., A.E. and H.H. wrote the paper and all authors reviewed and corrected the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by KAUST grant BAS/01/1062-01-01 to HH. We are also grateful to the Hirt lab members for technical assistance and fruitful discussions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcu\u0026ntilde;a, J. J., Campos, M., Mora, M. d. l. L., Jaisi, D. P., \u0026amp; Jorquera, M. A. (2019, 2019/04/01/). 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Pseudomonas granadensis sp. nov., a new bacterial species isolated from the Tejeda, Almijara and Alhama Natural Park, Granada, Spain. \u003cem\u003eInt J Syst Evol Microbiol, 65\u003c/em\u003e(Pt 2), 625-632. https://doi.org/10.1099/ijs.0.069260-0 \u003c/li\u003e\n\u003cli\u003ePurtschert-Montenegro, G., C\u0026aacute;rcamo-Oyarce, G., Pinto-Carb\u0026oacute;, M., Agnoli, K., Bailly, A., \u0026amp; Eberl, L. (2022, 2022/10/01). Pseudomonas putida mediates bacterial killing, biofilm invasion and biocontrol with a type IVB secretion system. \u003cem\u003eNature Microbiology, 7\u003c/em\u003e(10), 1547-1557. https://doi.org/10.1038/s41564-022-01209-6 \u003c/li\u003e\n\u003cli\u003eRastgoo, M., \u0026amp; Hasanfard, A. (2021). Desertification in Agricultural Lands: Approaches to Mitigation. In. https://doi.org/10.5772/intechopen.98795 \u003c/li\u003e\n\u003cli\u003eSantander, C., Garc\u0026iacute;a, S., Moreira, J., Aponte, H., Araneda, P., Olave, J., Vidal, G., \u0026amp; Cornejo, P. (2021, 2021/06/01/). Arbuscular mycorrhizal fungal abundance in elevation belts of the hyperarid Atacama Desert. \u003cem\u003eFungal Ecology, 51\u003c/em\u003e, 101060. https://doi.org/https://doi.org/10.1016/j.funeco.2021.101060 \u003c/li\u003e\n\u003cli\u003eSehrawat, A., Sindhu, S. S., \u0026amp; Glick, B. R. (2022, 2022/02/01/). Hydrogen cyanide production by soil bacteria: Biological control of pests and promotion of plant growth in sustainable agriculture. \u003cem\u003ePedosphere, 32\u003c/em\u003e(1), 15-38. https://doi.org/https://doi.org/10.1016/S1002-0160(21)60058-9 \u003c/li\u003e\n\u003cli\u003eSingh, R. P., \u0026amp; Jha, P. N. (2016). The Multifarious PGPR Serratia marcescens CDP-13 Augments Induced Systemic Resistance and Enhanced Salinity Tolerance of Wheat (Triticum aestivum L.). \u003cem\u003ePLOS ONE, 11\u003c/em\u003e(6), e0155026. https://doi.org/10.1371/journal.pone.0155026 \u003c/li\u003e\n\u003cli\u003eSulaiman, M. A., \u0026amp; Bello, S. K. (2024, 2024/04/01). Biological control of soil-borne pathogens in arid lands: a review. \u003cem\u003eJournal of Plant Diseases and Protection, 131\u003c/em\u003e(2), 293-313. https://doi.org/10.1007/s41348-023-00824-7 \u003c/li\u003e\n\u003cli\u003eTeixeira, P. J. P. L., Colaianni, N. R., Law, T. F., Conway, J. M., Gilbert, S., Li, H., Salas-Gonz\u0026aacute;lez, I., Panda, D., Del Risco, N. M., Finkel, O. M., Castrillo, G., Mieczkowski, P., Jones, C. D., \u0026amp; Dangl, J. L. (2021). Specific modulation of the root immune system by a community of commensal bacteria. \u003cem\u003eProceedings of the National Academy of Sciences, 118\u003c/em\u003e(16), e2100678118. https://doi.org/doi:10.1073/pnas.2100678118 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Genome, Pseudomonas, Desert agriculture, Phytopathogens, Biocontrol, Plant Pathogen Suppression","lastPublishedDoi":"10.21203/rs.3.rs-6620576/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6620576/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDesert ecosystems are limited by resources and optimal climatic conditions to support cropping in different farming models. Recently, microbial applications have emerged as promising strategies to enhance plant survival and adaptation in such extreme environments. However, identifying beneficial microbes and understanding their functional roles and adaptation mechanisms remains underexplored. This study reports, for the first time, the isolation and characterization of the \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 strain from the arid environment of Wadi Rum, Jordan, associated with \u003cem\u003eIfloga spicata\u003c/em\u003e. The genome of \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79, sequenced at 275\u0026times; PacBio coverage, consists of a 6.18 Mbp chromosome encoding 5,445 proteins, including gene clusters for siderophores, phenazines, hydrogen cyanide, and phytohormones, as well as advanced secretion systems (T2SS, T4SS, T6SS, and TAT). Genomic and phenotypic analyses revealed that \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 belongs to the genus \u003cem\u003eP. granadensis\u003c/em\u003e and exhibits plant growth-promoting attributes and substantial biocontrol potential. \u003cem\u003ePseudomonas\u003c/em\u003e sp. R4-79 effectively suppresses key phytopathogens, including the necrotrophic fungus \u003cem\u003eBotrytis cinerea in vitro\u003c/em\u003e, as well as \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. tomato DC3000 and root-knot nematodes (\u003cem\u003eMeloidogyne incognita\u003c/em\u003e) \u003cem\u003ein vivo\u003c/em\u003e in Arabidopsis and tomato. This work suggests that \u003cem\u003eP. granadensis\u003c/em\u003e R4-79 might be a good biocontrol agent to improve crop yield and ecological restoration in arid systems.\u003c/p\u003e","manuscriptTitle":"Genome sequence of the desert endophyte Pseudomonas granadensis R4-79 reveals potential for plant-growth promotion and disease suppression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-23 09:53:22","doi":"10.21203/rs.3.rs-6620576/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-30T08:27:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-24T16:46:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-12T16:08:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305367763235791242497408505148097983147","date":"2025-06-06T08:04:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331795600467602605853389792861808651077","date":"2025-06-02T11:48:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-21T14:17:34+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-19T05:33:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-16T12:22:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-16T12:19:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-05-08T12:08:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c240f0c1-0d2a-45a4-b488-c2c3f36db0e0","owner":[],"postedDate":"May 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T16:03:45+00:00","versionOfRecord":{"articleIdentity":"rs-6620576","link":"https://doi.org/10.1186/s12866-025-04431-4","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2025-10-28 15:58:44","publishedOnDateReadable":"October 28th, 2025"},"versionCreatedAt":"2025-05-23 09:53:22","video":"","vorDoi":"10.1186/s12866-025-04431-4","vorDoiUrl":"https://doi.org/10.1186/s12866-025-04431-4","workflowStages":[]},"version":"v1","identity":"rs-6620576","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6620576","identity":"rs-6620576","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}