Complementary functionalities of extracellular polymeric substances, adhesion ability and hydrophobicity in Pseudomonas isolates may help the selection of strategically advantageous microbial inoculants

Complementary functionalities of extracellular polymeric substances, adhesion ability and hydrophobicity in Pseudomonas isolates may help the selection of strategically advantageous microbial inoculants | 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 Complementary functionalities of extracellular polymeric substances, adhesion ability and hydrophobicity in Pseudomonas isolates may help the selection of strategically advantageous microbial inoculants Federico Rossi, Arianna Grassi, Caterina Cristani, Irene Pagliarani, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8583662/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Mar, 2026 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted 9 You are reading this latest preprint version Abstract Microbial persistence and colonization in the rhizosphere rely on traits that control how cells attach, interact, and organize into biofilms. Among these traits, extracellular polymeric substances (EPS), cell surface hydrophobicity, and adhesion play central roles in the early stages of root surface colonization. Here, we examined 48 isolates of the Pseudomonas fluorescens group associated with Tuber borchii fruiting bodies to explore the relationships among bound and released EPS fractions, hydrophobicity and adhesion ability. All experiments were carried out under controlled in vitro conditions using polystyrene as an abiotic model that mimics the hydrophobic interfaces occurring in soil–root environments. EPS fractions were quantified by Congo red and phenol–sulfuric acid assays, while surface hydrophobicity and adhesion were determined through xylene partitioning and crystal violet staining. The isolates exhibited wide phenotypic variability. Bound EPS showed a strong negative relationship with both hydrophobicity and adhesion, while hydrophobicity was positively associated with adhesion strength. Regression models confirmed that bound EPS and hydrophobicity independently modulate the adhesion response. These findings suggest that thick, hydrated EPS layers can hinder early attachment, while thinner EPS coatings enhance cell–surface interactions. Understanding this functional trade-off provides a basis for the informed selection of microbial inoculants, combining stable biofilm formers with highly adhesive strains to improve persistence and colonization efficiency in rhizosphere environments. Biofilm formation Cell surface hydrophobicity Extracellular polymeric substances (EPS) Microbial adhesion Pseudomonas fluorescens group Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION In soil ecosystems, bacteria can exist as free-living cells, microcolonies, or as biofilms adhering to a wide range of mineral and biological substrates, including plant tissues (Mina et al. 2019 ), fungal mycelia (Kjeldgaard et al. 2019 ), and decomposing organic matter (Cai et al. 2019 ). Biofilms are structured microbial communities that colonize surfaces in patchy, thin, or multilayered arrangements, embedded within a self-produced matrix of extracellular polymeric substances (EPS) (Li et al. 2024 ). The shift from a free-living to a biofilm lifestyle represents a key microbial strategy for persistence and adaptation in the complex and dynamic soil environment. The EPS matrix provides a stable microhabitat that protects microbial cells from a wide range of physical, chemical, and biological stressors, including pH fluctuations, salinity, antibiotics, and predation (Flemming 2016 ). Compared to planktonic cells—typically characterized by slower growth, reduced motility, and increased sensitivity to stress—biofilm-associated bacteria exhibit enhanced resilience and ecological fitness (Olson et al. 2002 ). Biofilm development generally proceeds through four stages: reversible adhesion, irreversible adhesion, microcolony formation, and maturation (Rumbaugh and Sauer 2020 ). Throughout these stages—particularly during maturation—bacteria secrete EPS, composed of polysaccharides, proteins, lipids, and extracellular DNA (Costa et al. 2018 ). EPS represent the dominant component of the biofilm structure, accounting for 75–90% of microbial aggregates (Garrett et al. 2008 ) and up to 80% of the biofilm dry mass (Ganesan et al. 2013 ). Their biochemical properties—such as water retention capacity, sorptive behavior, charge, and hydrophobicity—are critical in shaping biofilm architecture and function. The EPS matrix forms a hydrated, three-dimensional network surrounding the cells (Costerton 1999 ), stabilizing the biofilm through dispersion forces, electrostatic interactions, and hydrogen bonding (Flemming et al. 2016 ), while also serving as a protective barrier against environmental stressors, including UV radiation, pH shifts, and desiccation (Mari and Vrane 2007 ). EPS around microbial cells are usually categorized into two main fractions: bound EPS (b-EPS), tightly attached, sometimes organized as capsules or sheaths (Kachlany et al. 2001 ; Whitfield and Paiment 2003 ; Harimawan and Ting 2016 ), and released EPS (r-EPS), loosely associated with the cell surface, more water-soluble than b-EPS, and easily released into the extracellular environment (Wang et al. 2014 ). The reversible adhesion is crucial as it represents the transition from the planktonic to the sessile (attached) state. It marks the initiation of bacterial-host interactions and lays the foundation for subsequent biofilm development. However, not all bacteria that initially adhere proceed to form fully mature biofilms. In some cases, only microcolonies are formed in plant rhizosphere (Pearce et al. 1995 ) and bacterial adhesion does not always result in fully sessile growth (Chagnot et al. 2013 ). Therefore, adhesion and biofilm formation should be considered distinct processes, each requiring separate investigation. The reversible attachment is governed by van der Waals forces, steric and electrostatic interactions, collectively known as DLVO (Derjaguin, Verwey, Landau, and Overbeek) forces (Hermansson 1999 ). According to DLVO theory, bacterial attachment is a balance between attractive van der Waals forces and repulsive forces arising from the ionic charges on the bacterial cell surface. If the repulsive forces exceed the attractive forces, detachment is likely. At this stage, the hydrophobic properties of both the bacterial cell surface and the substrate play a significant role (Wheatley and Poole 2018 ), as reflected in the extended DLVO theory (Chang and Chang 2002 ). EPS production is recognized as a key factor in microbial adhesion and biofilm formation (Tsuneda et al. 2003 ; Hwang et al. 2012 ; Harimawan and Ting 2016 ). However, the diverse and heterogeneous physicochemical properties of EPS influence microbial interactions with different surfaces. Microbial adhesion represents the initial step of biofilm development, acting as the gateway for colonization and the establishment of complex ecological interactions, which may be either beneficial or detrimental to the host. Beneficial biofilms, such as those formed by plant-growth-promoting bacteria (PGPB), function as specialized microbial consortia capable of synthesizing key secondary metabolites, including ACC deaminase, siderophores, antibiotics, plant hormones, acetoin, 2,3-butanediol, and proline. These compounds directly contribute to plant growth promotion, improved disease resistance (Bhattacharyya et al. 2023 ), and enhanced tolerance to abiotic stresses, such as drought (Karimi et al. 2022 ). As a result, PGPB are increasingly considered eco-friendly biological tools able to reduce the use of chemical inputs in sustainable agriculture (Lopes et al. 2021 ). However, the practical use of these bacteria is often limited by their low survival and colonization efficiency in heterogeneous soil environments (Basu et al. 2021 ). Although biofilm formation enhances microbial persistence in soil, this trait is frequently overlooked during strain selection, despite its central role in ensuring stable establishment within key rhizosphere niches (Li et al. 2024 ). Despite the central role attributed to EPS in biofilm development, their specific contribution to early adhesion and surface hydrophobicity remains debated, particularly with respect to b- versus r-EPS. In this work, we investigated bound and released EPS, adhesion ability and hydrophobicity in 48 selected bacterial strains belonging to the Pseudomonas fluorescens group, isolated from Tuber borchii fruiting bodies and characterized for their PGP traits for their possible use as novel rhizosphere inoculants with beneficial effects on plant health (Cristani et al., under submission ). MATERIAL AND METHODS Bacterial isolates and culture media In this study we tested 48 selected bacterial isolates belonging to the Pseudomonas fluorescens group, maintained in our collection of the Department of Agriculture, Food and Environment of the University of Pisa (IMA, International Microbial Archives), and isolated from the fruiting bodies of Tuber borchii collected in three Tuscan geographic areas with high truffle vocation (Cristani et al., under submission). The isolates were initially maintained on Nutrient Agar (NA; Thermo Fisher Scientific, USA). For experimental assays, isolates were cultured in ATCC No. 14 mineral medium (Mu’minah et al. 2015 ; composition detailed in Online Resource Table S1 a). Overnight cultures were grown at 25°C in liquid ATCC No. 14, then adjusted to an optical density of OD₆₀₀ = 0.3. Aliquots (20 µL) of each culture were spotted in triplicate onto ATCC No. 14 agar plates and incubated at 25°C for 48 hours. Colony morphology and EPS production were evaluated at the end of the incubation. Congo Red agar assay and Congo Red-binding assay The CR-binding assay was performed following the method of (An et al. 2010 ) with minor modifications. Isolates were grown overnight in ATCC No. 14 broth at 25°C and adjusted to OD₆₀₀ = 0.3 with sterile culture medium. Then, 20 µL of each culture was spotted in triplicate onto ATCC No. 14 agar supplemented with 0.08% (w/v) Congo Red (CR; Sigma-Aldrich). The CR stock solution was autoclaved separately from the medium and added after cooling to 55°C. On CRA plates, EPS-overproducing isolates formed black colonies, while non-producers exhibited red to pink coloration. For the CR-binding assay, isolates were grown in ATCC No. 14 broth at 25°C for 48 hours. Cultures were centrifuged at 5,000 × g, and pellets were resuspended in 3 mL of a 40 µg mL⁻¹ CR solution. The suspensions were incubated at room temperature for 90 minutes under stirring to allow dye binding. After incubation, samples were centrifuged at 4,900 × g , and the absorbance of the supernatant was measured at 490 nm (Shimadzu UV-1800 spectrophotometer). CR binding was quantified using a standard curve prepared with CR concentrations ranging from 0 to 40 µg mL⁻¹. Quantification of released EPS (r-EPS) Released EPS were quantified via ethanol precipitation (Sirajunnisa et al. 2016 ) followed by phenol-sulfuric acid assay (DuBois et al. 1956). Isolates were cultured in ATCC No. 14 broth at 25°C for 48 hours. Supernatants were obtained by centrifugation at 6,000 × g and mixed with two volumes of 95% ethanol. Samples were incubated at 4°C overnight to allow polymer precipitation. The precipitate was resuspended in 10 mL of distilled water and re-precipitated with ethanol under the same conditions. After drying at 45°C, the precipitate was dissolved again in 10 mL of distilled water. For quantification, 1 mL of sample was mixed with 1 mL of 5% (w/v) phenol and 1 mL of 96% sulfuric acid. After 10 minutes at room temperature and further cooling, absorbance was measured at 488 nm. EPS content was calculated using a D-glucose calibration curve (0-200 µg mL⁻¹) and expressed as mg glucose equivalents per liter of culture. All measurements were performed in triplicate. Cell surface hydrophobicity (MATH assay) Cell surface hydrophobicity was assessed using the microbial adhesion to hydrocarbons (MATH) assay, with modifications from Zabielska et al. ( 2017 ) and Krishnamoorthy et al. ( 2018 ). Isolates were cultured in ATCC No. 14 broth at 25°C for 48 hours. Cells were washed and resuspended in phosphate-buffered urea magnesium (PUM) solution (Table S1 b), which reduces electrostatic effects and enhances hydrophobic interaction measurements (Rosenberg 2006 ). The initial absorbance (A₁) at 520 nm was recorded. Then, 3 mL of bacterial suspension was mixed with 0.5 mL of xylene and incubated at 37°C for 10 minutes. After vortexing for 60 seconds and incubating for 45 minutes to allow phase separation, the absorbance of the aqueous phase (A₂) was measured. The hydrophobicity index (HI%) was calculated as: HI (%) = [(A₁ - A₂)/A₁] × 100. Strains were classified as highly hydrophobic (HI > 60%), hydrophobic (HI = 50–60%), moderately hydrophobic (HI = 20–50%), or hydrophilic (HI < 20%) according to (Krepsky et al. 2003 ). All tests were performed in triplicate. Adhesion potential Adhesion to abiotic surfaces was evaluated using the crystal violet (CV) microplate assay. Overnight cultures were diluted to OD₆₀₀ = 0.3 in ATCC No. 14 medium, and 200 µL aliquots were inoculated into 96-well microtiter plates. After 48 hours at 25°C, non-adherent cells were discarded, and wells were gently rinsed with distilled water. Each well received 200 µL of 0.01% CV solution and was incubated at room temperature for 30 minutes. Excess dye was removed, and wells were rinsed twice with water. Bound dye was solubilized in 200 µL of 33% acetic acid, and absorbance was measured at 595 nm (Bio-Rad Model 680 microplate reader). The Relative Adhesion Capacity (RAC) was calculated for each isolate using the formula: RAC = (Aₓ – A c )/[Σ(Aₙ – A c )/48]. Where Aₓ is the OD₅₉₅ of the isolate, A c is the control absorbance, and Aₙ is the OD₅₉₅ of each isolate (Basson et al. 2008 ). Statistical analysis All experiments were performed in biological triplicate with at least three technical replicates per sample. One-way ANOVA was used to identify significant differences among variables, followed by Tukey’s HSD post-hoc test (P ≤ 0.05). Pearson’s correlation coefficients (r) were calculated to assess relationships among adhesion (Adh), hydrophobicity (HI%), bound EPS (CR-binding), and released EPS (r-EPS). Correlations were classified as strong (|r| ≥ 0.7), moderate (0.4 ≤ |r| < 0.7), or weak (|r| < 0.4), with statistical significance set at p < 0.05. A generalized linear regression model was used to evaluate the independent effects of CR-binding, r-EPS, and HI% on adhesion. The model assumed a normal distribution for the response variable and was fitted using the least squares method. Model fit was assessed via R² and adjusted R². A hierarchical cluster analysis was performed to explore global patterns and groupings among isolates based on their phenotypic profiles. The variables included in the analysis were hydrophobicity (HI), adhesion (Adh), bound CR (bound-CR), and released EPS (r-EPS). All data were standardized (z-scores) prior to clustering to ensure equal weighting of variables. Clustering was based on Ward’s minimum variance method using Euclidean distances as the dissimilarity metric. Results were visualized as a heatmap combined with a dendrogram to facilitate the identification of phenotypic clusters. Colour gradients represented standardized values for each variable, and both isolates, and variables were ordered according to the hierarchical tree structure. All analyses were conducted using JMP Pro v.17.0.0 (SAS Institute Inc., Cary, NC, USA). RESULTS EPS-associated morphology of isolates The 48 isolates grown on ATCC No. 14 agar exhibited diverse colony morphologies, which were grouped into five distinct types based on colony shape, surface texture, margin structure, and the presence or absence of extracellular material (Fig. 1 ; Online Resource Fig. S1 ). Type I colonies (Fig. 1 a), represented by eight isolates (C10, C21, C22, C32, S11, S18, S20, S31), were circular, raised and mucoid, with a diffuse layer of loosely associated exocellular material surrounding the colony. Type II colonies (Fig. 1 b), the most frequent phenotype (39.5% of isolates: C27, C28, C33, C37, S8, S12, S22, S23, S25, S34, S35, S38, S42, S44, S54, S56, S58 and S60), were crateriform, raised and round with well-defined margins. These colonies exhibited abundant and more condensed extracellular material than Type I. Type III colonies (Fig. 1 c), observed in five isolates (C14, C42, S3, S40 and S50), were translucent, flat and dry with a mat-like appearance, and showed no visible extracellular material. Type IV colonies (Fig. 1 d), observed in isolates C4, C9, C44, C47, C59, S10, and S48, were flat, round and displayed curled margins. These colonies lacked any evident exocellular matrix. Type V colonies (Fig. 1 e), identified in isolates C1, C17, C25, C41, C48, C60, S2, S21, S28 and S29, appeared round with minimal extracellular material localized primarily at the colony edges. [Near Fig. 1 ] Congo red agar (CRA) and Congo red-binding assay (CR-BA) Inoculation on Congo Red Agar (CRA) revealed that 20 out of the 48 bacterial strains were Congo Red (CR)-positive, producing dark colonies after 48 hours of incubation (Online Resource Fig. S1 ). All CR-positive isolates produced type II colonies on ATCC No. 14 agar (Fig. 2 ), with two exceptions: isolate C42, which displayed type III morphology, and isolate S29, which exhibited type V morphology. [Near Fig. 2 ] Congo Red binding assays (CR-BA) showed that all isolates were capable of accumulating CR, although in varying amounts (Fig. 3 ). Following supernatant separation during the assay, CR was found to bind preferentially to bound EPS (b-EPS) rather than to bacterial cells (Online Resource Fig. S2). Most isolates (62.5%) accumulated relatively low levels of CR (< 10 ppm), whereas 33% exhibited significantly higher CR removal capabilities (P < 0.05), with bound CR levels exceeding 20 ppm (Fig. 3 ). Notably, this group consisted entirely of CRA-positive isolates forming type II colonies on ATCC No. 14 agar, characterized by mucoid morphology and condensed EPS. ANOVA confirmed that the isolate factor explained most of the variation in CR binding (F = 82.323, p < 0.0001), indicating highly significant differences in b-EPS production among isolates (Online Resource Table S2). [Near Fig. 3 ] Production of released EPS (r-EPS) The production of released EPS (r-EPS) by the bacterial isolates in ATCC No. 14 liquid medium is shown in Fig. 4 . Based on average yields, the isolates were grouped into three categories: low, medium and high EPS producers. Most isolates were classified as low producers, with glucose-equivalent concentrations below 0.5 mg mL⁻¹, while 27% were medium producers, yielding between 0.5 and 1.0 mg mL⁻¹. Sixteen percent of the isolates were identified as high producers, with yields exceeding 1.0 mg mL⁻¹. Notably, eight isolates - C27, C33, C37, S22, S44, S54, S58, and S60 - showed the highest r-EPS production levels. Among the isolates, C27 stood out for its significantly higher yield, 2.57 ± 0.03 mg mL⁻¹. One-way ANOVA revealed statistically significant differences in r-EPS production among the isolates (Online Resource Table S3), with the model explaining 97.55% of the variance (adjusted R² = 96.35%). [Near Fig. 4 ] Cell Surface hydrophobicity The hydrophobicity index (HI) values of the bacterial isolates are presented in Fig. 5 . The isolates exhibited a wide range of HI values, from a minimum of 26.79% (S60) to a maximum of 85.60% (C22). Most isolates showed HI values between 37% and 84%, indicating a general trend toward hydrophobic behaviour. None of the isolates displayed low hydrophobicity (HI < 20%). One-way ANOVA followed by post-hoc analysis revealed significant variation in HI values among the 48 isolates (Online Resource Table S4), with a highly significant p-value (< 0.0001). The overall mean HI was 62.86%, and the model’s F-ratio of 62.8393 indicated strong statistical support for the observed differences. Based on HI values, the isolates were classified into different hydrophobicity categories: moderately hydrophobic isolates (e.g., S60, 26.79%; S8, 28.01%), hydrophobic isolates (e.g., C33, 54.75%), and strongly hydrophobic isolates (HI > 60%) such as C22 (85.60%) and C47 (81.78%). [Near Fig. 5 ] Adhesion potential of the isolates All bacterial isolates were evaluated for their ability to adhere to polystyrene microtiter plates (Fig. 6 ). Each isolate exhibited at least a minimal level of adhesion, with relative adhesion capacity (RAC) values ranging from 0.2 (C28) to 1.67 (C59). A total of 22 isolates displayed RAC values greater than 1, indicating relatively strong adhesive capabilities. Among them, isolates C10, C47, C48, C59, and S31 showed particularly high RAC values, suggesting high adhesion potential. One-way ANOVA revealed significant variations in adhesion potential among the isolates (Online Resource Table S5), with an F-ratio of 45.6109 and a highly significant p-value (< 0.0001). The R-squared value of 0.9571 indicates that the model explains a substantial proportion of the total variance, while the adjusted R-squared value of 0.9362 further supports the robustness of the analysis. [Near Fig. 6 ] Correlations among b-CR, r-EPS, HI and Adh values A preliminary exploratory analysis was carried out to examine the relationships among b-CR, r-EPS, HI and Adh values. The correlation matrix and scatterplot matrix are reported in Table 1 and Fig. 7 , respectively. The results revealed a strong and significant positive correlation between adhesion and HI (r = 0.700, p < 0.0001), as well as a strong negative correlation between adhesion and b-CR (r = -0.781, p < 0.0001). Moreover, a moderate negative correlation was observed between adhesion and r-EPS production (r = -0.417, p = 0.0032). Table 1 Pearson’s correlation coefficients (r) among bound EPS (b-CR), released EPS (r-EPS), hydrophobicity index (HI), and adhesion capacity (Adh) of Pseudomonas fluorescens group isolates. Values in bold indicate significant relationships (|r| ≥ 0.7). Variable 1 Variable 2 Correlation coefficient (r) p -value Adh HI 0.700 < 0.0001 Adh b-CR -0.781 < 0.0001 Adh r-EPS -0.417 0.00320 HI b-CR -0.730 < 0.0001 HI r-EPS -0.537 < 0.0001 b-CR r-EPS -0.432 < 0.0027 [Near Table 1 ] [Near Fig. 7 ] In order to provide a more precise assessment of the independent contribution of each predictor (b-EPS, r-EPS. HI) to the dependent variable (Adh), we conducted a regression analysis (Online Resource Table S6). The model explained 66.42% of the variance in adhesion (R-squared = 0.6642, adjusted R-squared = 0.6408). While b-CR (β = -0.6300, p < 0.0001) and HI% (β = 0.3193, p = 0.0196) were both significant predictors of adhesion, r-EPS (β = 0.1438, p = 0.2044) did not emerge as a significant predictor in the regression model. This suggests that, although a moderate correlation between r-EPS and adhesion was observed initially, the relationship is no longer significant when the effects of other predictors are considered in the regression model. A generalized linear regression model was also performed to investigate the effects of EPS production on hydrophobicity index (HI). The model was fitted using the least squares method, with HI as the dependent variable. The model explained 56.09% of the variance in HI (R-squared = 0.5609, adjusted R-squared = 0.5409) (Online Resource Table S7). In the regression model, b-CR was a significant negative predictor of HI (β = -0.6546, p < 0.0001). On the other hand, r-EPS was not a significant predictor of HI (β = -0.1513, p = 0.2350), indicating that the production of r-EPS did not have a significant impact on the hydrophobicity of the bacterial surface when b-CR was considered. To further explore the diversity of EPS-associated traits among isolates, a hierarchical cluster analysis was performed based on standardized values of b-CR, r-EPS, HI, and Adh. The clustered heatmap (Fig. 8 ) revealed distinct groupings among isolates. Notably, strains with high b-EPS production (as indicated by high and positive b-CR values) clustered separately from those exhibiting high Adh and HI values, further supporting the distinction between EPS-producing and adhesion capable isolates. [Near Fig. 8 ] DISCUSSION This study, for the first time, revealed a large variability among bound and released EPS, adhesion ability and hydrophobicity in 48 fluorescent Pseudomonas isolated from the same ecological niche - Tuber borchii fruiting bodies. Correlation analyses among EPS-associated traits revealed complex interrelationships, as bacterial EPS production and surface adhesion capability are distinct microbial traits requiring independent evaluation. The bacterial isolates exhibited a marked variability in EPS production, both in terms of yields and aspect of the extracellular material. Some isolates formed mucous colonies rich in exocellular material (type II morphology), while others produced slimy, less structured colonies (type I morphology), suggesting distinct strategies in EPS secretion and surface organization. All isolates with type II colony morphology tested positive on Congo Red Agar (CRA) and accumulated high amounts of CR (> 20 µg mL⁻¹), as quantified by the CR-binding assay. CRA positivity is typically associated with enhanced EPS production and biofilm-forming ability (Jones and Wozniak 2017 ), while the CR-binding assay provides a quantitative measure of CR retention by the EPS (Ghitti et al. 2024 ). The combination of CRA and CR-binding assays provides complementary insights: while the former reflects overall biofilm-associated phenotypes, the latter allows for the estimation of the b-EPS fraction. Moreover, the binding affinity of CR to specific extracellular components, such as amyloid fibers (e.g., curli; Reichhardt et al. 2015 ), fimbriae (Spiers and Rainey 2005 ), and β-linked glucans including cellulose (Weiner et al. 1999 ; Semedo et al. 2015 ; Heredia-Ponce et al. 2020 ), suggests that CRA and CR-based assays may indirectly inform on the biochemical composition of b-EPS, encompassing both polysaccharidic and proteinaceous elements. In line with this, nearly half of the isolates in this study can be regarded as strong biofilm formers, exhibiting both CRA positivity and high b-CR accumulation in amounts comparable with those observed by for other biofilm-forming Pseudomonas strains (Quintieri et al. 2020 ). While b-EPS are compositionally heterogeneous, r-EPS are generally composed almost exclusively of polysaccharides, with minor contributions (2–3%) from other biomolecules (Mahto et al. 2022 ). This supports the use of the phenol-sulfuric acid method for their quantification. Interestingly, a subset of isolates (C27, C33, C37, S22, S44, S54, S58, and S60) exhibited high levels of both b-EPS and r-EPS, indicating a robust capacity for producing both fractions. This dual production of tightly cell-associated and freely soluble EPS is particularly noteworthy. While b-EPS primarily contributes to cell cohesion, microcolony stability, and protection against abiotic stress (Wang et al. 2020 ), r-EPS exerts broader effects in the extracellular environment. Due to its solubility and diffusibility, r-EPS can serve as a carbon source for surrounding soil microbes (Chen et al. 2014 ), facilitate soil particle aggregation, and influence water retention and nutrient dynamics. The eight isolates showing the highest r-EPS production yielded quantities equally or exceeding those of Pseudomonas fluorescens AF814, a strain recognized for EPS production ability and quantity under drought stress (Ajijah et al. 2024 ). Isolates capable of producing both b-EPS and r-EPS in substantial amounts may fulfil a dual ecological function, i.e., stabilizing local microbial niches through matrix formation while enhancing ecosystem-level resilience and resource availability within the rhizosphere or soil microhabitats. The adhesion assay revealed a heterogeneous adhesive capacity among the isolates. Notably, 48% of them exhibited a Relative Adhesion Capacity (RAC) greater than 1, indicating enhanced adherence to the polystyrene surface under the experimental conditions. However, this adhesive potential showed a significant inverse relationship with EPS production. Specifically, a strong negative correlation was observed between adhesion and b-EPS (r = -0.781, p < 0.001), while r-EPS showed a moderate but still significant negative correlation with adhesion (r = -0.417, p = 0.003). These results suggest that higher EPS production, particularly of the cell-associated b-EPS fraction, tends to hinder initial adhesion to abiotic surfaces such as polystyrene. This inverse relationship may be particularly relevant during the early, reversible stage of bacterial adhesion, where physical-chemical interactions dominate. At this stage, electrostatic and hydrophobic forces govern the approach and initial contact of bacterial cells with surfaces. Hydrophobic interactions are often longer-ranged and more stable than electrostatic forces, exerting a stronger and more persistent influence, particularly in microbe–plant root interactions (Janczarek et al. 2015 ). Cell surface hydrophobicity itself is a complex trait influenced by multiple structural and compositional factors, including the presence of microbial fimbriae or hydrophobic adhesins (Otto et al. 1999 ), as well as the amount and nature of EPS (Vidhyalakshmi et al. 2018 ). An overall hydrophilic EPS matrix, by modifying surface charge and masking hydrophobic domains, may act as a barrier that reduces the effective contact of cells with the substrate. Microbial EPS can range from strongly hydrophilic to amphiphilic or even neutral, depending on their sugar composition, molecular weight, and the presence of charged side groups (Marvasi et al. 2010 ). Therefore, the overall effect of EPS on adhesion is not only dependent on quantity but also on their physicochemical quality. In our dataset, isolates with abundant b-EPS, which likely formed a more cohesive and hydrated cell envelope, were generally less adherent, supporting the hypothesis that thick EPS layers reduce effective cell-surface interactions during the early phases of attachment. However, EPS production could also be associated with alterations in the hydrophobicity of the bacterial cell surface. To test this, we used polystyrene microtiter plates, a widely accepted model substrate for studying biological interactions and microbial adhesion (Lyklema et al. 1989 ; Nowak et al. 2014 ). Although polystyrene surfaces carry a slight negative charge in aqueous environments, their overall physicochemical profile is predominantly hydrophobic, with a contact angle of approximately 85° (Thormann et al. 2008 ). Therefore, we expected isolates with more hydrophobic surfaces to exhibit stronger adhesion. To evaluate cell surface hydrophobicity, we used the xylene partition assay to calculate a hydrophobicity index (HI). As expected, bacterial adhesion was significantly and positively correlated with HI (r = 0.700, p < 0.0001), supporting the idea that hydrophobic interactions facilitate bacterial attachment to the polystyrene surface. Interestingly, HI showed a strong inverse correlation with b-EPS production (r = -0.730, p < 0.001) and a moderate but significant negative correlation with r-EPS production (r = -0.538, p < 0.001). These results reinforce the view that EPS production plays a major role in reducing cell surface hydrophobicity. Two non-mutually exclusive mechanisms may explain this effect: (1) EPS may act as a physical barrier, effectively masking hydrophobic structures on the cell surface; (2) EPS may possess a predominantly hydrophilic or ionic nature, which can interfere with hydrophobic interactions at the cell-substrate interface. These findings are consistent with previous studies showing that high EPS production can impair surface adhesion. Dertli et al. ( 2015 ), for example, reported that overproduction of EPS in Lactobacillus johnsonii resulted in decreased adhesion to polystyrene under comparable experimental conditions. More recently, Mougin et al. ( 2024 ) demonstrated that hydrophilic EPS derived from microalgae inhibited the adhesion of various foodborne bacteria to polystyrene surfaces, further supporting the inhibitory role of hydrophilic extracellular polymers in early-stage adhesion. Overall, our results highlight an antagonistic relationship between EPS synthesis and cell surface hydrophobicity, a feature that may play a central role in regulating microbial adhesion dynamics in both natural and artificial environments. When the relationships between adhesion and the investigated predictors were analyzed using a generalized linear regression model, both b-CR and HI emerged as significant independent predictors of bacterial adhesion. In contrast, r-EPS no longer exerted a significant effect. This suggests that, while a moderate bivariate correlation between r-EPS and adhesion was observed, its contribution is likely indirect and mediated by stronger, more proximal factors such as b-EPS (as inferred from b-CR) and surface hydrophobicity. These results underscore the pivotal roles of cell-associated EPS and hydrophobic interactions in mediating initial adhesion to hydrophobic substrates like polystyrene. Further, when HI was modelled as the dependent variable, b-CR again emerged as a significant negative predictor, reinforcing its central role in modulating surface hydrophobicity. The explanatory power of the regression models—66.42% of the variance in adhesion and 56.09% in HI—highlights the robustness of these relationships, while also pointing to the existence of additional factors, potentially including environmental cues, surface proteins, lipopolysaccharide composition, or regulatory pathways, that may modulate surface traits. Importantly, our findings support the view that EPS production and surface adhesion are distinct microbial traits that require independent evaluation. In our dataset, isolates tended to cluster into two functional groups: low-EPS producers with strong adhesion capacity, and high-EPS producers with poor adhesion. In conclusion, in our study case b-EPS appear to play a dominant role in modulating cell-surface hydrophobicity and adhesion, contrary to the commonly assumed view that EPS universally promote attachment. Our results indicate that b-EPS, rather than r-EPS, represent the dominant structural determinant modulating cell–surface interactions during the early stages of adhesion. Moreover, by linking our in vitro results to ecologically relevant processes, such as rhizosphere colonization, we provide a valuable framework for selecting microbial strains with tailored properties for use in sustainable agriculture and biofertilizer formulations. From an applied perspective, the separate consideration of the processes of b-EPS production (positively related to biofilm-forming ability) and adhesion capability can be strategically advantageous. In designing microbial consortia for soil inoculants or plant growth-promoting formulations, a combinatory approach may prove beneficial—pairing biofilm-forming strains with robust EPS production (to ensure structural stability of the microbial aggregate, protection, and moisture retention) alongside strains with superior adhesive capabilities (to ensure rapid and efficient surface colonization). Such complementary functionalities could enhance the persistence and efficacy of microbial inoculants in the rhizosphere or on soil particles and may be particularly relevant for the design of next-generation microbial inoculants, where persistence, surface colonization and matrix formation need to be balanced rather than maximized individually. Declarations Competing interests The authors have no relevant financial or non-financial interests to disclose. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and formal analysis were performed by F.R., A.G., I.P. and C.C. Data curation was performed by F.R. and A.G. The first draft of the manuscript was written by F.R. and all authors commented on previous versions of the manuscript. M.A. and M.G. supervised the work, revised the manuscript and contributed to the final editing. All authors read and approved the final version of the manuscript. Data Availability Data are available on reasonable request from the corresponding authors. References Ajijah N, Fiodor A, Dziewit L, Pranaw K (2024) Biological amelioration of water stress in rapeseed (Brassica napus L.) by exopolysaccharides-producing Pseudomonas protegens ML15. Physiol Plant 176:e70012. https://doi.org/10.1111/ppl.70012 An S, Wu J, Zhang L-H (2010) Modulation of Pseudomonas aeruginosa Biofilm Dispersal by a Cyclic-Di-GMP Phosphodiesterase with a Putative Hypoxia-Sensing Domain. Appl Environ Microbiol 76:8160–8173. https://doi.org/10.1128/AEM.01233-10 Basson A, Flemming LA, Chenia HY (2008) Evaluation of Adherence, Hydrophobicity, Aggregation, and Biofilm Development of Flavobacterium johnsoniae -Like Isolates. Microb Ecol 55:1–14 Basu A, Prasad P, Das SN et al (2021) Plant Growth Promoting Rhizobacteria (PGPR) as Green Bioinoculants: Recent Developments, Constraints, and Prospects. Sustainability 13:1140. https://doi.org/10.3390/su13031140 Bhattacharyya A, Mavrodi O, Bhowmik N et al (2023) Chap. 1 - Bacterial biofilms as an essential component of rhizosphere plant-microbe interactions. In: Gurtler V, Patrauchan M (eds) Methods in Microbiology. Academic Press, pp 3–48 Cai P, Sun X, Wu Y et al (2019) Soil biofilms: microbial interactions, challenges, and advanced techniques for ex-situ characterization. Soil Ecol Lett 1:85–93. https://doi.org/10.1007/s42832-019-0017-7 Chagnot C, Zorgani MA, Astruc T, Desvaux M (2013) Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective. Front Microbiol 4:303. https://doi.org/10.3389/fmicb.2013.00303 Chang Y-I, Chang P-K (2002) The role of hydration force on the stability of the suspension of Saccharomyces cerevisiae –application of the extended DLVO theory. Colloids Surf Physicochem Eng Asp 211:67–77. https://doi.org/10.1016/S0927-7757(02)00238-8 Chen L, Rossi F, Deng S et al (2014) Macromolecular and chemical features of the excreted extracellular polysaccharides in induced biological soil crusts of different ages. Soil Biol Biochem 78:1–9. https://doi.org/10.1016/j.soilbio.2014.07.004 Costa OYA, Raaijmakers JM, Kuramae EE (2018) Microbial Extracellular Polymeric Substances: Ecological Function and Impact on Soil Aggregation. Front Microbiol 9 Costerton JW (1999) Introduction to biofilm. Int J Antimicrob Agents 11:217–221 discussion 237–239. https://doi.org/10.1016/s0924-8579(99)00018-7 Dertli E, Mayer MJ, Narbad A (2015) Impact of the exopolysaccharide layer on biofilms, adhesion and resistance to stress in Lactobacillus johnsonii FI9785. BMC Microbiol 15:8. https://doi.org/10.1186/s12866-015-0347-2 DuBois Michel, Gilles KA, Hamilton JK et al (1956) Colorimetric method for the determination of sugars and related substances. Anal Chem 28:350–356. https://doi.org/10.1021/ac60111a017 Flemming H-C (2016) EPS—Then and Now. Microorganisms 4:41. https://doi.org/10.3390/microorganisms4040041 Flemming H-C, Wingender J, Szewzyk U et al (2016) Biofilms: an emergent form of bacterial life. Nat Rev Microbiol 14:563–575. https://doi.org/10.1038/nrmicro.2016.94 Ganesan M, Stewart EJ, Szafranski J et al (2013) Molar mass, entanglement and associations of the biofilm polysaccharide of Staphylococcus epidermidis . Biomacromolecules 14:1474–1481. https://doi.org/10.1021/bm400149a Garrett TR, Bhakoo M, Zhang Z (2008) Bacterial adhesion and biofilms on surfaces. Prog Nat Sci 18:1049–1056. https://doi.org/10.1016/j.pnsc.2008.04.001 Ghitti E, Rolli E, Vergani L, Borin S (2024) Flavonoids influence key rhizocompetence traits for early root colonization and PCB degradation potential of Paraburkholderia xenovorans LB400. Front Plant Sci 15. https://doi.org/10.3389/fpls.2024.1325048 Harimawan A, Ting Y-P (2016) Investigation of extracellular polymeric substances (EPS) properties of P. aeruginosa and B. subtilis and their role in bacterial adhesion. Colloids Surf B Biointerfaces 146:459–467. https://doi.org/10.1016/j.colsurfb.2016.06.039 Heredia-Ponce Z, Gutiérrez-Barranquero JA, Purtschert-Montenegro G et al (2020) Biological role of EPS from Pseudomonas syringae pv. syringae UMAF0158 extracellular matrix, focusing on a Psl-like polysaccharide. Npj Biofilms Microbiomes 6:1–13. https://doi.org/10.1038/s41522-020-00148-6 Hermansson M (1999) The DLVO theory in microbial adhesion. Colloids Surf B Biointerfaces 14:105–119. https://doi.org/10.1016/S0927-7765(99)00029-6 Hwang G, Kang S, El-Din MG, Liu Y (2012) Impact of an extracellular polymeric substance (EPS) precoating on the initial adhesion of Burkholderia cepacia and Pseudomonas aeruginosa . Biofouling 28:525–538. https://doi.org/10.1080/08927014.2012.694138 Janczarek M, Rachwał K, Cieśla J et al (2015) Production of exopolysaccharide by Rhizobium leguminosarum bv. trifolii and its role in bacterial attachment and surface properties. Plant Soil 388:211–227. https://doi.org/10.1007/s11104-014-2320-5 Jones CJ, Wozniak DJ (2017) Congo Red Stain Identifies Matrix Overproduction and Is an Indirect Measurement for c-di-GMP in Many Species of Bacteria. Methods Mol Biol Clifton NJ 1657:147–156. https://doi.org/10.1007/978-1-4939-7240-1_12 Kachlany SC, Levery SB, Kim JS et al (2001) Structure and carbohydrate analysis of the exopolysaccharide capsule of Pseudomonas putida G7. Environ Microbiol 3:774–784. https://doi.org/10.1046/j.1462-2920.2001.00248.x Karimi E, Aliasgharzad N, Esfandiari E et al (2022) Biofilm forming rhizobacteria affect the physiological and biochemical responses of wheat to drought. AMB Express 12:93. https://doi.org/10.1186/s13568-022-01432-8 Kjeldgaard B, Listian SA, Ramaswamhi V et al (2019) Fungal hyphae colonization by Bacillus subtilis relies on biofilm matrix components. Biofilm 1:100007. https://doi.org/10.1016/j.bioflm.2019.100007 Krepsky N, Ferreira RBR, Nunes APF et al (2003) Cell Surface Hydrophobicity and Slime Production of Staphylococcus epidermidis Brazilian Isolates. Curr Microbiol 46:0280–0286. https://doi.org/10.1007/s00284-002-3868-5 Krishnamoorthy M, Nayak BK, Nanda A (2018) In vivo and in vitro characterization of probiotic organisms for their microbial adhesion property isolated from Coconut toddy. Karbala Int J Mod Sci 4:341–346. https://doi.org/10.1016/j.kijoms.2018.08.001 Li Y, Narayanan M, Shi X et al (2024) Biofilms formation in plant growth-promoting bacteria for alleviating agro-environmental stress. Sci Total Environ 907:167774. https://doi.org/10.1016/j.scitotenv.2023.167774 Lopes MJdosS, Dias-Filho MB, Gurgel ESC (2021) Successful Plant Growth-Promoting Microbes: Inoculation Methods and Abiotic Factors. Front Sustain Food Syst 5. https://doi.org/10.3389/fsufs.2021.606454 Lyklema J, Norde W, van Loosdrecht MCM, Zehnder AJB (1989) Adhesion of bacteria to polystyrene surfaces. Colloids Surf 39:175–187. https://doi.org/10.1016/0166-6622(89)80186-6 Mahto KU, Vandana, Priyadarshanee M et al (2022) Bacterial biofilm and extracellular polymeric substances in the treatment of environmental pollutants: Beyond the protective role in survivability. J Clean Prod 379:134759. https://doi.org/10.1016/j.jclepro.2022.134759 Mari S, Vrane J (2007) Characteristics and significance of microbial biofilm formation. Period Biol 109:115–121 Marvasi M, Visscher PT, Casillas Martinez L (2010) Exopolymeric substances (EPS) from Bacillus subtilis : polymers and genes encoding their synthesis. FEMS Microbiol Lett 313:1–9. https://doi.org/10.1111/j.1574-6968.2010.02085.x Mina IR, Jara NP, Criollo JE, Castillo JA (2019) The critical role of biofilms in bacterial vascular plant pathogenesis. Plant Pathol 68:1439–1447. https://doi.org/10.1111/ppa.13073 Mougin J, Pavaux A-S, Fanesi A et al (2024) Bacterial adhesion inhibition by microalgal EPSs from Cylindrotheca closterium and Tetraselmis suecica biofilms. Appl Microbiol Biotechnol 108:168. https://doi.org/10.1007/s00253-023-12960-z Mu’minah B, Subair H, Fahruddin (2015) Isolation and Screening Bacterial Exopolysaccharide (EPS) from Potato Rhizosphere in Highland and the Potential as a Producer Indole Acetic Acid (IAA). Procedia Food Sci 3:74–81. https://doi.org/10.1016/j.profoo.2015.01.007 Nowak J, Watala C, Boncler M (2014) Antibody binding, platelet adhesion, and protein adsorption on various polymer surfaces. Blood Coagul Fibrinolysis 25:52. https://doi.org/10.1097/MBC.0b013e328364a802 Olson ME, Ceri H, Morck DW et al (2002) Biofilm bacteria: formation and comparative susceptibility to antibiotics. Can J Vet Res 66:86–92 Otto K, Elwing H, Hermansson M (1999) The role of type 1 fimbriae in adhesion of Escherichia coli to hydrophilic and hydrophobic surfaces. Colloids Surf B Biointerfaces 15:99–111. https://doi.org/10.1016/S0927-7765(99)00050-8 Pearce D, Bazin MJ, Lynch JM (1995) The Rhizosphere as a Biofilm. In: Lappin-Scott HM, Costerton JW (eds) Microbial Biofilms. Cambridge University Press, Cambridge, pp 207–220 Quintieri L, Fanelli F, Zühlke D et al (2020) Biofilm and Pathogenesis-Related Proteins in the Foodborne P. fluorescens ITEM 17298 With Distinctive Phenotypes During Cold Storage. Front Microbiol 11 Reichhardt C, Jacobson AN, Maher MC et al (2015) Congo Red Interactions with Curli-Producing E. coli and Native Curli Amyloid Fibers. PLoS ONE 10:e0140388. https://doi.org/10.1371/journal.pone.0140388 Rosenberg M (2006) Microbial adhesion to hydrocarbons: twenty-five years of doing MATH. FEMS Microbiol Lett 262:129–134. https://doi.org/10.1111/j.1574-6968.2006.00291.x Rumbaugh KP, Sauer K (2020) Biofilm dispersion. Nat Rev Microbiol 18:571–586. https://doi.org/10.1038/s41579-020-0385-0 Semedo MC, Karmali A, Fonseca L (2015) A high throughput colorimetric assay of β-1,3-d-glucans by Congo red dye. J Microbiol Methods 109:140–148. https://doi.org/10.1016/j.mimet.2014.12.020 Sirajunnisa AR, Vijayagopal V, Sivaprakash B et al (2016) Optimization, kinetics and antioxidant activity of exopolysaccharide produced from rhizosphere isolate. Pseudomonas fluorescens CrN6 Carbohydr Polym 135:35–43. https://doi.org/10.1016/j.carbpol.2015.08.080 Spiers AJ, Rainey PB (2005) The Pseudomonas fluorescens SBW25 wrinkly spreader biofilm requires attachment factor, cellulose fibre and LPS interactions to maintain strength and integrity. Microbiol Read Engl 151:2829–2839. https://doi.org/10.1099/mic.0.27984-0 Thormann E, Simonsen AC, Hansen PL, Mouritsen OG (2008) Interactions between a Polystyrene Particle and Hydrophilic and Hydrophobic Surfaces in Aqueous Solutions. Langmuir 24:7278–7284. https://doi.org/10.1021/la8005162 Tsuneda S, Aikawa H, Hayashi H et al (2003) Extracellular polymeric substances responsible for bacterial adhesion onto solid surface. FEMS Microbiol Lett 223:287–292. https://doi.org/10.1016/S0378-1097(03)00399-9 Vidhyalakshmi R, Valli Nachiyar C, Narendra Kumar G et al (2018) Production, characterization and emulsifying property of exopolysaccharide produced by marine isolate of Pseudomonas fluorescens . Biocatal Agric Biotechnol 16:320–325. https://doi.org/10.1016/j.bcab.2018.08.023 Wang K, Li W, Rui X et al (2014) Structural characterization and bioactivity of released exopolysaccharides from Lactobacillus plantarum 70810. Int J Biol Macromol 67:71–78. https://doi.org/10.1016/j.ijbiomac.2014.02.056 Wang S, Zhi L, Shan W et al (2020) Correlation of extracellular polymeric substances and microbial community structure in denitrification biofilm exposed to adverse conditions. Microb Biotechnol 13:1889–1903. https://doi.org/10.1111/1751-7915.13633 Weiner R, Seagren E, Arnosti C, Quintero E (1999) Bacterial survival in biofilms: probes for exopolysaccharide and its hydrolysis, and measurements of intra- and interphase mass fluxes. Methods Enzymol 310:403–426. https://doi.org/10.1016/s0076-6879(99)10032-6 Wheatley RM, Poole PS (2018) Mechanisms of bacterial attachment to roots. FEMS Microbiol Rev. https://doi.org/10.1093/femsre/fuy014 Whitfield C, Paiment A (2003) Biosynthesis and assembly of Group 1 capsular polysaccharides in Escherichia coli and related extracellular polysaccharides in other bacteria. Carbohydr Res 338:2491–2502. https://doi.org/10.1016/j.carres.2003.08.010 Zabielska J, Kunicka-Styczyńska A, Otlewska A (2017) Adhesive and hydrophobic properties of Pseudomonas aeruginosa and Pseudomonas cedrina associated with cosmetics. Ecol Quest 28:41–46. https://doi.org/10.12775/EQ.2017.037 Additional Declarations No competing interests reported. Supplementary Files Rossietal.ESMWJMB.pdf Cite Share Download PDF Status: Published Journal Publication published 23 Mar, 2026 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted Editorial decision: Revision requested 22 Feb, 2026 Reviews received at journal 21 Feb, 2026 Reviews received at journal 11 Feb, 2026 Reviewers agreed at journal 07 Feb, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers invited by journal 14 Jan, 2026 Editor assigned by journal 14 Jan, 2026 Submission checks completed at journal 14 Jan, 2026 First submitted to journal 12 Jan, 2026 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8583662","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":576146589,"identity":"a4136460-fc11-4f90-8b69-03872935fa02","order_by":0,"name":"Federico Rossi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIie3QuwrCMBSA4SOFdjnqWhHrKxwJ1EXqq7QUOjkUXBwLvlCdxK0ScPLyAI6uDu2m0MGIdyVVN5H8dAr5kpwCqFQ/mBEBwfE7RR0ADSC5rbyGySMJLkRu8OlAfsVyYgxHm0HoWFDnPNuFK29slKcJhLmc4KzfmpPPoBIEdaS1NxlW3MKHdc2eXYso8SJEWwNBYo5UPEtz296fCct2tPyAmGiXzoRMsfkDgkFfPMxnOurHWXwWczGLS0xODD7KotyxqqiJP5Y7jXi14GmaW1JyTb87BsB9Dx5v/nK/SqVS/XsH2pdL+mV4tKYAAAAASUVORK5CYII=","orcid":"","institution":"University of Pisa","correspondingAuthor":true,"prefix":"","firstName":"Federico","middleName":"","lastName":"Rossi","suffix":""},{"id":576146592,"identity":"b9756bb8-c1c4-4655-9266-8b1c519edcca","order_by":1,"name":"Arianna Grassi","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Arianna","middleName":"","lastName":"Grassi","suffix":""},{"id":576146594,"identity":"337a5219-261e-4f0c-8aa4-6229a7e0400e","order_by":2,"name":"Caterina Cristani","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Caterina","middleName":"","lastName":"Cristani","suffix":""},{"id":576146596,"identity":"8ea88660-2896-4705-9dd1-24bc829b49ff","order_by":3,"name":"Irene Pagliarani","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Irene","middleName":"","lastName":"Pagliarani","suffix":""},{"id":576146598,"identity":"4ce4d0a1-7f88-4e62-8c8b-130385012a89","order_by":4,"name":"Manuela Giovannetti","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Manuela","middleName":"","lastName":"Giovannetti","suffix":""},{"id":576146600,"identity":"c1032ef4-8e49-429d-8398-f8e4ab8b05f7","order_by":5,"name":"Monica Agnolucci","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Monica","middleName":"","lastName":"Agnolucci","suffix":""}],"badges":[],"createdAt":"2026-01-12 16:11:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8583662/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8583662/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11274-026-04899-w","type":"published","date":"2026-03-23T16:10:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":100595414,"identity":"d0e92571-7472-42f4-a6da-485b52bf26a6","added_by":"auto","created_at":"2026-01-19 13:48:25","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9829734,"visible":true,"origin":"","legend":"","description":"","filename":"Rossietal.completemanuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/abc7ce2cce61384f02dd8e5f.docx"},{"id":100595946,"identity":"d3b9b2ef-b9dc-4376-a3f5-684212fc539a","added_by":"auto","created_at":"2026-01-19 13:49:48","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7744,"visible":true,"origin":"","legend":"","description":"","filename":"8a39d2c601ec463fad6b8f0c167345e0.json","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/b6ee672194f30d31a847ef87.json"},{"id":100595068,"identity":"6b69edb1-fb3c-41ed-8933-2537a548d8c1","added_by":"auto","created_at":"2026-01-19 13:47:17","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":412635,"visible":true,"origin":"","legend":"","description":"","filename":"Rossietal.ESMWJMB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/d8ff85b45711adf978df34f5.pdf"},{"id":100567633,"identity":"7460ebad-cd39-4ddc-a43c-4ebb3a4238c7","added_by":"auto","created_at":"2026-01-19 09:12:28","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138186,"visible":true,"origin":"","legend":"","description":"","filename":"8a39d2c601ec463fad6b8f0c167345e01enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/ea4d9cf54a8e2afe15140e9f.xml"},{"id":100595228,"identity":"8705efe3-d939-492b-8f02-7abb9baa9e51","added_by":"auto","created_at":"2026-01-19 13:47:59","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":223317,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/27d0e22c4c40a84f85f5ce7e.jpeg"},{"id":100595940,"identity":"800a250d-b5de-47a7-bd99-c7354f23d0aa","added_by":"auto","created_at":"2026-01-19 13:49:47","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":280230,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/e4bd32e6f63f00e70b971dda.jpeg"},{"id":100567647,"identity":"961bfe1d-fdb0-4b05-a403-6d2b71181cb0","added_by":"auto","created_at":"2026-01-19 09:12:28","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":412333,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/dcc118b8258fcddbe0403ad7.jpeg"},{"id":100595490,"identity":"b9b25451-51e0-4ea3-bc6f-9776d25e9b06","added_by":"auto","created_at":"2026-01-19 13:48:35","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":347439,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/62491c746bba98765b06fec4.jpeg"},{"id":100594841,"identity":"7c102808-0b8c-433e-983a-38c8b0af9423","added_by":"auto","created_at":"2026-01-19 13:45:33","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":299913,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/b55e47d4f54b291b9ad29797.jpeg"},{"id":100567645,"identity":"26b35556-0a9a-4552-a335-7c1219479ee1","added_by":"auto","created_at":"2026-01-19 09:12:28","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":262818,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/9d20257b2de316e21cba749a.jpeg"},{"id":100595183,"identity":"24f419bc-1f5d-4eba-b2bd-d5f0cb546f80","added_by":"auto","created_at":"2026-01-19 13:47:47","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":413772,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/9df482c2a9c9d4458cd39e1e.jpeg"},{"id":100595404,"identity":"6794a767-9949-4380-a270-1677d4429a64","added_by":"auto","created_at":"2026-01-19 13:48:24","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4071498,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/ce9ed100e61a930bd584af0a.jpeg"},{"id":100567655,"identity":"959f2a5b-705c-4e87-b1f9-2eac14a11613","added_by":"auto","created_at":"2026-01-19 09:12:28","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":88427,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/ac888689b58bea0e9df79329.png"},{"id":100594777,"identity":"d661ac53-a154-472c-8817-d25d11c01069","added_by":"auto","created_at":"2026-01-19 13:44:54","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":58981,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/337c0ea05f24c5b657855667.png"},{"id":100595715,"identity":"c81c603a-84b2-4a70-bc77-214507482c9e","added_by":"auto","created_at":"2026-01-19 13:49:14","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114457,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/bad119df082aca9670aab75f.png"},{"id":100594888,"identity":"eeda9288-3021-4f18-85b6-0b6692b565a4","added_by":"auto","created_at":"2026-01-19 13:46:12","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":205741,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/83a6179517bfa020d4ad6100.png"},{"id":100595719,"identity":"5d587014-faf1-4eed-ae54-0b927a9861d6","added_by":"auto","created_at":"2026-01-19 13:49:15","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115236,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/9d29cff909f0f1409c9b2746.png"},{"id":100567650,"identity":"be5433d6-cd07-4b4a-b768-0200c6ea0f99","added_by":"auto","created_at":"2026-01-19 09:12:28","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":99624,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/2a224c1282c2b3e5b313961f.png"},{"id":100595263,"identity":"ef8117de-6e82-4f94-ae58-0c0905681c04","added_by":"auto","created_at":"2026-01-19 13:48:05","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106553,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/a73ec7bf388e092fe98fed45.png"},{"id":100594813,"identity":"d8088d81-b079-4f6b-b781-41675c8c54d7","added_by":"auto","created_at":"2026-01-19 13:45:19","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":378647,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/6bb08cf77763a23ce2017c15.png"},{"id":100567657,"identity":"fc7ab01f-68ad-4df9-9bab-f7c37bcd9ba3","added_by":"auto","created_at":"2026-01-19 09:12:28","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139054,"visible":true,"origin":"","legend":"","description":"","filename":"8a39d2c601ec463fad6b8f0c167345e01structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/5f8a179edaf59220954b97ab.xml"},{"id":100567661,"identity":"9af9b784-b7ad-4095-ad04-27d68ee90528","added_by":"auto","created_at":"2026-01-19 09:12:28","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":149436,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/9d7313e086ba6cac69fda6cf.html"},{"id":100595445,"identity":"bb4ffedd-bf97-494d-8d7a-21643522e406","added_by":"auto","created_at":"2026-01-19 13:48:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3878919,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative colony morphotypes of \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e group isolates grown on ATCC No. 14 agar. (a) Type I: mucoid, raised colonies with diffuse EPS layers; (b) Type II: crateriform colonies with abundant condensed EPS; (c) Type III: flat, dry colonies without evident EPS; (d) Type IV: curled-margin colonies with no evident EPS accumulation; (e) Type V: colonies with minimal peripheral EPS accumulation\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/e902d0e2c54ad20d0a16261d.png"},{"id":100567634,"identity":"199e9dfb-fbcb-440d-af4a-7d90fcdd45b7","added_by":"auto","created_at":"2026-01-19 09:12:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1898025,"visible":true,"origin":"","legend":"\u003cp\u003eCR-negative and CR-positive reaction after spot inoculation on CRA. Colony appearance was evaluated after 48 h of incubation at 25°C. CR-positive strains formed solid black colonies, whereas CR-negative strains appeared translucent red to pink\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/1647aaafdb9cfab5a8f2f14f.png"},{"id":100595918,"identity":"8a528c1e-758a-4fbc-9228-ee5508d990bc","added_by":"auto","created_at":"2026-01-19 13:49:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9203346,"visible":true,"origin":"","legend":"\u003cp\u003eAmount of Congo Red (CR) bound to the biomass of bacterial isolates (b-CR). Values were expressed as µg mL⁻¹ CR removed from an initial concentration of 40 µg mL⁻¹. Average values are based on at least three independent experimental replicates. Error bars indicate standard deviation. Dotted lines mark the thresholds of 10 and 20 µg mL⁻¹. Values marked with an asterisk (*) are significantly different from unmarked values (P \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/80f58a5028c9b88f22ce369d.png"},{"id":100567643,"identity":"df879728-3a4b-4750-a0ea-54d4ab5cbbbb","added_by":"auto","created_at":"2026-01-19 09:12:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1485269,"visible":true,"origin":"","legend":"\u003cp\u003eReleased EPS (r-EPS) by the bacterial isolates. Values were expressed as mg mL\u003csup\u003e-1\u003c/sup\u003e glucose equivalents and are means of at least three independent experimental replicates. Error bars represent the standard deviation. Dotted lines indicate the 0.5 and 1.0 mg mL⁻¹ thresholds for r-EPS production. Values marked with different letters are significantly different (P \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/ff093684f469e2fad798a5df.png"},{"id":100567638,"identity":"061081ee-ebf4-4b20-a41a-b7f61e9803cf","added_by":"auto","created_at":"2026-01-19 09:12:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":641716,"visible":true,"origin":"","legend":"\u003cp\u003eHydrophobicity index (HI) of bacterial isolates biomass. Values are expressed as % and are determined through partitioning in xylene. Each value represents the mean of at least three independent replicates, with error bars indicating the standard deviation. HI values between 50% and 60% indicate hydrophobicity, while values between 50% and 20% represent moderate hydrophobicity, according to Krepsky et al. (2003). Values of HI of 60% or higher indicate strong hydrophobicity. Dotted lines evidence the HI thresholds of 20%, 50%, and 80%\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/87cb4f3c33ba60c64a1a7edc.png"},{"id":100595240,"identity":"bb92758f-9e61-4622-9b08-0c0e3c1ee101","added_by":"auto","created_at":"2026-01-19 13:48:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":669315,"visible":true,"origin":"","legend":"\u003cp\u003eRelative adhesion capability (RAC) of the 48 bacterial isolates. A value of 1 (highlighted with the dotted line in the graph) corresponds to the average adhesion capability across all isolates. Values are means of at least three independent replicates and error bars indicate standard deviation\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/3b63a40b3b493c278258f7cd.png"},{"id":100567662,"identity":"c55dff54-a7f6-4894-a04e-eada2c044a1d","added_by":"auto","created_at":"2026-01-19 09:12:29","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":39841660,"visible":true,"origin":"","legend":"\u003cp\u003eScatterplot matrix of EPS production, hydrophobicity, and adhesion. Scatterplot matrix showing the pairwise linear relationships among adhesion capacity (Adh), released EPS (r-EPS), bound EPS measured as bound Congo Red (b-CR), and hydrophobicity index (HI). The plots illustrate the strength and direction of the correlations among the four traits\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/3a1e3e36da66c3f8e2d325ea.png"},{"id":100595121,"identity":"ec6a5bcd-8339-42e0-bbd7-dbb393fcebee","added_by":"auto","created_at":"2026-01-19 13:47:30","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":20720675,"visible":true,"origin":"","legend":"\u003cp\u003eCluster heatmap showing the distribution of released EPS (r-EPS), bound EPS (b-EPS), hydrophobicity index (HI), and adhesion capacity (Adh) across the bacterial isolates. Values were standardized as z-scores prior to clustering to enable direct comparison among traits\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/9fac3b551df1eb102d3feeb6.png"},{"id":100595919,"identity":"cbb86ceb-a0e2-4640-b522-7989a7ee6547","added_by":"auto","created_at":"2026-01-19 13:49:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":412635,"visible":true,"origin":"","legend":"","description":"","filename":"Rossietal.ESMWJMB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8583662/v1/4acdbac2df7cc1668a8243d4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Complementary functionalities of extracellular polymeric substances, adhesion ability and hydrophobicity in Pseudomonas isolates may help the selection of strategically advantageous microbial inoculants","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIn soil ecosystems, bacteria can exist as free-living cells, microcolonies, or as biofilms adhering to a wide range of mineral and biological substrates, including plant tissues (Mina et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), fungal mycelia (Kjeldgaard et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and decomposing organic matter (Cai et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Biofilms are structured microbial communities that colonize surfaces in patchy, thin, or multilayered arrangements, embedded within a self-produced matrix of extracellular polymeric substances (EPS) (Li et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The shift from a free-living to a biofilm lifestyle represents a key microbial strategy for persistence and adaptation in the complex and dynamic soil environment. The EPS matrix provides a stable microhabitat that protects microbial cells from a wide range of physical, chemical, and biological stressors, including pH fluctuations, salinity, antibiotics, and predation (Flemming \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Compared to planktonic cells\u0026mdash;typically characterized by slower growth, reduced motility, and increased sensitivity to stress\u0026mdash;biofilm-associated bacteria exhibit enhanced resilience and ecological fitness (Olson et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiofilm development generally proceeds through four stages: reversible adhesion, irreversible adhesion, microcolony formation, and maturation (Rumbaugh and Sauer \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Throughout these stages\u0026mdash;particularly during maturation\u0026mdash;bacteria secrete EPS, composed of polysaccharides, proteins, lipids, and extracellular DNA (Costa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). EPS represent the dominant component of the biofilm structure, accounting for 75\u0026ndash;90% of microbial aggregates (Garrett et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and up to 80% of the biofilm dry mass (Ganesan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Their biochemical properties\u0026mdash;such as water retention capacity, sorptive behavior, charge, and hydrophobicity\u0026mdash;are critical in shaping biofilm architecture and function. The EPS matrix forms a hydrated, three-dimensional network surrounding the cells (Costerton \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), stabilizing the biofilm through dispersion forces, electrostatic interactions, and hydrogen bonding (Flemming et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), while also serving as a protective barrier against environmental stressors, including UV radiation, pH shifts, and desiccation (Mari and Vrane \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). EPS around microbial cells are usually categorized into two main fractions: bound EPS (b-EPS), tightly attached, sometimes organized as capsules or sheaths (Kachlany et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Whitfield and Paiment \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Harimawan and Ting \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and released EPS (r-EPS), loosely associated with the cell surface, more water-soluble than b-EPS, and easily released into the extracellular environment (Wang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe reversible adhesion is crucial as it represents the transition from the planktonic to the sessile (attached) state. It marks the initiation of bacterial-host interactions and lays the foundation for subsequent biofilm development. However, not all bacteria that initially adhere proceed to form fully mature biofilms. In some cases, only microcolonies are formed in plant rhizosphere (Pearce et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) and bacterial adhesion does not always result in fully sessile growth (Chagnot et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, adhesion and biofilm formation should be considered distinct processes, each requiring separate investigation. The reversible attachment is governed by van der Waals forces, steric and electrostatic interactions, collectively known as DLVO (Derjaguin, Verwey, Landau, and Overbeek) forces (Hermansson \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). According to DLVO theory, bacterial attachment is a balance between attractive van der Waals forces and repulsive forces arising from the ionic charges on the bacterial cell surface. If the repulsive forces exceed the attractive forces, detachment is likely. At this stage, the hydrophobic properties of both the bacterial cell surface and the substrate play a significant role (Wheatley and Poole \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), as reflected in the extended DLVO theory (Chang and Chang \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). EPS production is recognized as a key factor in microbial adhesion and biofilm formation (Tsuneda et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Hwang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Harimawan and Ting \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, the diverse and heterogeneous physicochemical properties of EPS influence microbial interactions with different surfaces.\u003c/p\u003e \u003cp\u003eMicrobial adhesion represents the initial step of biofilm development, acting as the gateway for colonization and the establishment of complex ecological interactions, which may be either beneficial or detrimental to the host. Beneficial biofilms, such as those formed by plant-growth-promoting bacteria (PGPB), function as specialized microbial consortia capable of synthesizing key secondary metabolites, including ACC deaminase, siderophores, antibiotics, plant hormones, acetoin, 2,3-butanediol, and proline. These compounds directly contribute to plant growth promotion, improved disease resistance (Bhattacharyya et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and enhanced tolerance to abiotic stresses, such as drought (Karimi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As a result, PGPB are increasingly considered eco-friendly biological tools able to reduce the use of chemical inputs in sustainable agriculture (Lopes et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the practical use of these bacteria is often limited by their low survival and colonization efficiency in heterogeneous soil environments (Basu et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although biofilm formation enhances microbial persistence in soil, this trait is frequently overlooked during strain selection, despite its central role in ensuring stable establishment within key rhizosphere niches (Li et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Despite the central role attributed to EPS in biofilm development, their specific contribution to early adhesion and surface hydrophobicity remains debated, particularly with respect to b- versus r-EPS.\u003c/p\u003e \u003cp\u003eIn this work, we investigated bound and released EPS, adhesion ability and hydrophobicity in 48 selected bacterial strains belonging to the \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e group, isolated from \u003cem\u003eTuber borchii\u003c/em\u003e fruiting bodies and characterized for their PGP traits for their possible use as novel rhizosphere inoculants with beneficial effects on plant health (Cristani et al., \u003cem\u003eunder submission\u003c/em\u003e).\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial isolates and culture media\u003c/h2\u003e \u003cp\u003eIn this study we tested 48 selected bacterial isolates belonging to the \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e group, maintained in our collection of the Department of Agriculture, Food and Environment of the University of Pisa (IMA, International Microbial Archives), and isolated from the fruiting bodies of \u003cem\u003eTuber borchii\u003c/em\u003e collected in three Tuscan geographic areas with high truffle vocation (Cristani et al., under submission). The isolates were initially maintained on Nutrient Agar (NA; Thermo Fisher Scientific, USA). For experimental assays, isolates were cultured in ATCC No. 14 mineral medium (Mu\u0026rsquo;minah et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; composition detailed in Online Resource Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). Overnight cultures were grown at 25\u0026deg;C in liquid ATCC No. 14, then adjusted to an optical density of OD₆₀₀ = 0.3. Aliquots (20 \u0026micro;L) of each culture were spotted in triplicate onto ATCC No. 14 agar plates and incubated at 25\u0026deg;C for 48 hours. Colony morphology and EPS production were evaluated at the end of the incubation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCongo Red agar assay and Congo Red-binding assay\u003c/h3\u003e\n\u003cp\u003eThe CR-binding assay was performed following the method of (An et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) with minor modifications. Isolates were grown overnight in ATCC No. 14 broth at 25\u0026deg;C and adjusted to OD₆₀₀ = 0.3 with sterile culture medium. Then, 20 \u0026micro;L of each culture was spotted in triplicate onto ATCC No. 14 agar supplemented with 0.08% (w/v) Congo Red (CR; Sigma-Aldrich). The CR stock solution was autoclaved separately from the medium and added after cooling to 55\u0026deg;C. On CRA plates, EPS-overproducing isolates formed black colonies, while non-producers exhibited red to pink coloration.\u003c/p\u003e \u003cp\u003eFor the CR-binding assay, isolates were grown in ATCC No. 14 broth at 25\u0026deg;C for 48 hours. Cultures were centrifuged at 5,000 \u0026times; g, and pellets were resuspended in 3 mL of a 40 \u0026micro;g mL⁻\u0026sup1; CR solution. The suspensions were incubated at room temperature for 90 minutes under stirring to allow dye binding. After incubation, samples were centrifuged at 4,900 \u0026times; \u003cem\u003eg\u003c/em\u003e, and the absorbance of the supernatant was measured at 490 nm (Shimadzu UV-1800 spectrophotometer). CR binding was quantified using a standard curve prepared with CR concentrations ranging from 0 to 40 \u0026micro;g mL⁻\u0026sup1;.\u003c/p\u003e\n\u003ch3\u003eQuantification of released EPS (r-EPS)\u003c/h3\u003e\n\u003cp\u003eReleased EPS were quantified via ethanol precipitation (Sirajunnisa et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) followed by phenol-sulfuric acid assay (DuBois et al. 1956). Isolates were cultured in ATCC No. 14 broth at 25\u0026deg;C for 48 hours. Supernatants were obtained by centrifugation at 6,000 \u0026times; \u003cem\u003eg\u003c/em\u003e and mixed with two volumes of 95% ethanol. Samples were incubated at 4\u0026deg;C overnight to allow polymer precipitation. The precipitate was resuspended in 10 mL of distilled water and re-precipitated with ethanol under the same conditions. After drying at 45\u0026deg;C, the precipitate was dissolved again in 10 mL of distilled water. For quantification, 1 mL of sample was mixed with 1 mL of 5% (w/v) phenol and 1 mL of 96% sulfuric acid. After 10 minutes at room temperature and further cooling, absorbance was measured at 488 nm. EPS content was calculated using a D-glucose calibration curve (0-200 \u0026micro;g mL⁻\u0026sup1;) and expressed as mg glucose equivalents per liter of culture. All measurements were performed in triplicate.\u003c/p\u003e\n\u003ch3\u003eCell surface hydrophobicity (MATH assay)\u003c/h3\u003e\n\u003cp\u003eCell surface hydrophobicity was assessed using the microbial adhesion to hydrocarbons (MATH) assay, with modifications from Zabielska et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and Krishnamoorthy et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Isolates were cultured in ATCC No. 14 broth at 25\u0026deg;C for 48 hours. Cells were washed and resuspended in phosphate-buffered urea magnesium (PUM) solution (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb), which reduces electrostatic effects and enhances hydrophobic interaction measurements (Rosenberg \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The initial absorbance (A₁) at 520 nm was recorded. Then, 3 mL of bacterial suspension was mixed with 0.5 mL of xylene and incubated at 37\u0026deg;C for 10 minutes. After vortexing for 60 seconds and incubating for 45 minutes to allow phase separation, the absorbance of the aqueous phase (A₂) was measured. The hydrophobicity index (HI%) was calculated as: HI (%) = [(A₁ - A₂)/A₁] \u0026times; 100. Strains were classified as highly hydrophobic (HI\u0026thinsp;\u0026gt;\u0026thinsp;60%), hydrophobic (HI\u0026thinsp;=\u0026thinsp;50\u0026ndash;60%), moderately hydrophobic (HI\u0026thinsp;=\u0026thinsp;20\u0026ndash;50%), or hydrophilic (HI\u0026thinsp;\u0026lt;\u0026thinsp;20%) according to (Krepsky et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). All tests were performed in triplicate.\u003c/p\u003e\n\u003ch3\u003eAdhesion potential\u003c/h3\u003e\n\u003cp\u003eAdhesion to abiotic surfaces was evaluated using the crystal violet (CV) microplate assay. Overnight cultures were diluted to OD₆₀₀ = 0.3 in ATCC No. 14 medium, and 200 \u0026micro;L aliquots were inoculated into 96-well microtiter plates. After 48 hours at 25\u0026deg;C, non-adherent cells were discarded, and wells were gently rinsed with distilled water. Each well received 200 \u0026micro;L of 0.01% CV solution and was incubated at room temperature for 30 minutes. Excess dye was removed, and wells were rinsed twice with water. Bound dye was solubilized in 200 \u0026micro;L of 33% acetic acid, and absorbance was measured at 595 nm (Bio-Rad Model 680 microplate reader). The Relative Adhesion Capacity (RAC) was calculated for each isolate using the formula: RAC = (Aₓ \u0026ndash; A\u003csub\u003ec\u003c/sub\u003e)/[Σ(Aₙ \u0026ndash; A\u003csub\u003ec\u003c/sub\u003e)/48]. Where Aₓ is the OD₅₉₅ of the isolate, A\u003csub\u003ec\u003c/sub\u003e is the control absorbance, and Aₙ is the OD₅₉₅ of each isolate (Basson et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed in biological triplicate with at least three technical replicates per sample. One-way ANOVA was used to identify significant differences among variables, followed by Tukey\u0026rsquo;s HSD post-hoc test (P\u0026thinsp;\u0026le;\u0026thinsp;0.05). Pearson\u0026rsquo;s correlation coefficients (r) were calculated to assess relationships among adhesion (Adh), hydrophobicity (HI%), bound EPS (CR-binding), and released EPS (r-EPS). Correlations were classified as strong (|r| \u0026ge; 0.7), moderate (0.4 \u0026le; |r| \u0026lt; 0.7), or weak (|r| \u0026lt; 0.4), with statistical significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eA generalized linear regression model was used to evaluate the independent effects of CR-binding, r-EPS, and HI% on adhesion. The model assumed a normal distribution for the response variable and was fitted using the least squares method. Model fit was assessed via R\u0026sup2; and adjusted R\u0026sup2;. A hierarchical cluster analysis was performed to explore global patterns and groupings among isolates based on their phenotypic profiles. The variables included in the analysis were hydrophobicity (HI), adhesion (Adh), bound CR (bound-CR), and released EPS (r-EPS). All data were standardized (z-scores) prior to clustering to ensure equal weighting of variables. Clustering was based on Ward\u0026rsquo;s minimum variance method using Euclidean distances as the dissimilarity metric. Results were visualized as a heatmap combined with a dendrogram to facilitate the identification of phenotypic clusters. Colour gradients represented standardized values for each variable, and both isolates, and variables were ordered according to the hierarchical tree structure. All analyses were conducted using JMP Pro v.17.0.0 (SAS Institute Inc., Cary, NC, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEPS-associated morphology of isolates\u003c/h2\u003e \u003cp\u003eThe 48 isolates grown on ATCC No. 14 agar exhibited diverse colony morphologies, which were grouped into five distinct types based on colony shape, surface texture, margin structure, and the presence or absence of extracellular material (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Online Resource Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Type I colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), represented by eight isolates (C10, C21, C22, C32, S11, S18, S20, S31), were circular, raised and mucoid, with a diffuse layer of loosely associated exocellular material surrounding the colony. Type II colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), the most frequent phenotype (39.5% of isolates: C27, C28, C33, C37, S8, S12, S22, S23, S25, S34, S35, S38, S42, S44, S54, S56, S58 and S60), were crateriform, raised and round with well-defined margins. These colonies exhibited abundant and more condensed extracellular material than Type I. Type III colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), observed in five isolates (C14, C42, S3, S40 and S50), were translucent, flat and dry with a mat-like appearance, and showed no visible extracellular material. Type IV colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), observed in isolates C4, C9, C44, C47, C59, S10, and S48, were flat, round and displayed curled margins. These colonies lacked any evident exocellular matrix. Type V colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), identified in isolates C1, C17, C25, C41, C48, C60, S2, S21, S28 and S29, appeared round with minimal extracellular material localized primarily at the colony edges.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e[Near Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCongo red agar (CRA) and Congo red-binding assay (CR-BA)\u003c/h2\u003e \u003cp\u003eInoculation on Congo Red Agar (CRA) revealed that 20 out of the 48 bacterial strains were Congo Red (CR)-positive, producing dark colonies after 48 hours of incubation (Online Resource Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All CR-positive isolates produced type II colonies on ATCC No. 14 agar (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), with two exceptions: isolate C42, which displayed type III morphology, and isolate S29, which exhibited type V morphology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e[Near Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eCongo Red binding assays (CR-BA) showed that all isolates were capable of accumulating CR, although in varying amounts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Following supernatant separation during the assay, CR was found to bind preferentially to bound EPS (b-EPS) rather than to bacterial cells (Online Resource Fig. S2). Most isolates (62.5%) accumulated relatively low levels of CR (\u0026lt;\u0026thinsp;10 ppm), whereas 33% exhibited significantly higher CR removal capabilities (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with bound CR levels exceeding 20 ppm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, this group consisted entirely of CRA-positive isolates forming type II colonies on ATCC No. 14 agar, characterized by mucoid morphology and condensed EPS. ANOVA confirmed that the isolate factor explained most of the variation in CR binding (F\u0026thinsp;=\u0026thinsp;82.323, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating highly significant differences in b-EPS production among isolates (Online Resource Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e[Near Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProduction of released EPS (r-EPS)\u003c/h2\u003e \u003cp\u003eThe production of released EPS (r-EPS) by the bacterial isolates in ATCC No. 14 liquid medium is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Based on average yields, the isolates were grouped into three categories: low, medium and high EPS producers. Most isolates were classified as low producers, with glucose-equivalent concentrations below 0.5 mg mL⁻\u0026sup1;, while 27% were medium producers, yielding between 0.5 and 1.0 mg mL⁻\u0026sup1;. Sixteen percent of the isolates were identified as high producers, with yields exceeding 1.0 mg mL⁻\u0026sup1;. Notably, eight isolates - C27, C33, C37, S22, S44, S54, S58, and S60 - showed the highest r-EPS production levels. Among the isolates, C27 stood out for its significantly higher yield, 2.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mg mL⁻\u0026sup1;. One-way ANOVA revealed statistically significant differences in r-EPS production among the isolates (Online Resource Table S3), with the model explaining 97.55% of the variance (adjusted R\u0026sup2; = 96.35%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e[Near Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell Surface hydrophobicity\u003c/h2\u003e \u003cp\u003eThe hydrophobicity index (HI) values of the bacterial isolates are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The isolates exhibited a wide range of HI values, from a minimum of 26.79% (S60) to a maximum of 85.60% (C22). Most isolates showed HI values between 37% and 84%, indicating a general trend toward hydrophobic behaviour. None of the isolates displayed low hydrophobicity (HI\u0026thinsp;\u0026lt;\u0026thinsp;20%). One-way ANOVA followed by post-hoc analysis revealed significant variation in HI values among the 48 isolates (Online Resource Table S4), with a highly significant p-value (\u0026lt;\u0026thinsp;0.0001). The overall mean HI was 62.86%, and the model\u0026rsquo;s F-ratio of 62.8393 indicated strong statistical support for the observed differences. Based on HI values, the isolates were classified into different hydrophobicity categories: moderately hydrophobic isolates (e.g., S60, 26.79%; S8, 28.01%), hydrophobic isolates (e.g., C33, 54.75%), and strongly hydrophobic isolates (HI\u0026thinsp;\u0026gt;\u0026thinsp;60%) such as C22 (85.60%) and C47 (81.78%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e[Near Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAdhesion potential of the isolates\u003c/h2\u003e \u003cp\u003eAll bacterial isolates were evaluated for their ability to adhere to polystyrene microtiter plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Each isolate exhibited at least a minimal level of adhesion, with relative adhesion capacity (RAC) values ranging from 0.2 (C28) to 1.67 (C59). A total of 22 isolates displayed RAC values greater than 1, indicating relatively strong adhesive capabilities. Among them, isolates C10, C47, C48, C59, and S31 showed particularly high RAC values, suggesting high adhesion potential. One-way ANOVA revealed significant variations in adhesion potential among the isolates (Online Resource Table S5), with an F-ratio of 45.6109 and a highly significant p-value (\u0026lt;\u0026thinsp;0.0001). The R-squared value of 0.9571 indicates that the model explains a substantial proportion of the total variance, while the adjusted R-squared value of 0.9362 further supports the robustness of the analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e[Near Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCorrelations among b-CR, r-EPS, HI and Adh values\u003c/h2\u003e \u003cp\u003eA preliminary exploratory analysis was carried out to examine the relationships among b-CR, r-EPS, HI and Adh values. The correlation matrix and scatterplot matrix are reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, respectively. The results revealed a strong and significant positive correlation between adhesion and HI (r\u0026thinsp;=\u0026thinsp;0.700, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), as well as a strong negative correlation between adhesion and b-CR (r = -0.781, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Moreover, a moderate negative correlation was observed between adhesion and r-EPS production (r = -0.417, p\u0026thinsp;=\u0026thinsp;0.0032).\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\u003ePearson\u0026rsquo;s correlation coefficients (r) among bound EPS (b-CR), released EPS (r-EPS), hydrophobicity index (HI), and adhesion capacity (Adh) of \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e group isolates. Values in bold indicate significant relationships (|r| \u0026ge; 0.7).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVariable 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCorrelation coefficient (r)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.700\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eb-CR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-0.781\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003er-EPS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.417\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00320\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eb-CR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-0.730\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003er-EPS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eb-CR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003er-EPS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.432\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0027\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[Near Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e[Near Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn order to provide a more precise assessment of the independent contribution of each predictor (b-EPS, r-EPS. HI) to the dependent variable (Adh), we conducted a regression analysis (Online Resource Table S6). The model explained 66.42% of the variance in adhesion (R-squared\u0026thinsp;=\u0026thinsp;0.6642, adjusted R-squared\u0026thinsp;=\u0026thinsp;0.6408). While b-CR (β = -0.6300, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and HI% (β\u0026thinsp;=\u0026thinsp;0.3193, p\u0026thinsp;=\u0026thinsp;0.0196) were both significant predictors of adhesion, r-EPS (β\u0026thinsp;=\u0026thinsp;0.1438, p\u0026thinsp;=\u0026thinsp;0.2044) did not emerge as a significant predictor in the regression model. This suggests that, although a moderate correlation between r-EPS and adhesion was observed initially, the relationship is no longer significant when the effects of other predictors are considered in the regression model.\u003c/p\u003e \u003cp\u003eA generalized linear regression model was also performed to investigate the effects of EPS production on hydrophobicity index (HI). The model was fitted using the least squares method, with HI as the dependent variable. The model explained 56.09% of the variance in HI (R-squared\u0026thinsp;=\u0026thinsp;0.5609, adjusted R-squared\u0026thinsp;=\u0026thinsp;0.5409) (Online Resource Table S7).\u003c/p\u003e \u003cp\u003eIn the regression model, b-CR was a significant negative predictor of HI (β = -0.6546, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). On the other hand, r-EPS was not a significant predictor of HI (β = -0.1513, p\u0026thinsp;=\u0026thinsp;0.2350), indicating that the production of r-EPS did not have a significant impact on the hydrophobicity of the bacterial surface when b-CR was considered.\u003c/p\u003e \u003cp\u003eTo further explore the diversity of EPS-associated traits among isolates, a hierarchical cluster analysis was performed based on standardized values of b-CR, r-EPS, HI, and Adh. The clustered heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) revealed distinct groupings among isolates. Notably, strains with high b-EPS production (as indicated by high and positive b-CR values) clustered separately from those exhibiting high Adh and HI values, further supporting the distinction between EPS-producing and adhesion capable isolates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e[Near Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study, for the first time, revealed a large variability among bound and released EPS, adhesion ability and hydrophobicity in 48 fluorescent \u003cem\u003ePseudomonas\u003c/em\u003e isolated from the same ecological niche - \u003cem\u003eTuber borchii\u003c/em\u003e fruiting bodies. Correlation analyses among EPS-associated traits revealed complex interrelationships, as bacterial EPS production and surface adhesion capability are distinct microbial traits requiring independent evaluation.\u003c/p\u003e \u003cp\u003eThe bacterial isolates exhibited a marked variability in EPS production, both in terms of yields and aspect of the extracellular material. Some isolates formed mucous colonies rich in exocellular material (type II morphology), while others produced slimy, less structured colonies (type I morphology), suggesting distinct strategies in EPS secretion and surface organization. All isolates with type II colony morphology tested positive on Congo Red Agar (CRA) and accumulated high amounts of CR (\u0026gt;\u0026thinsp;20 \u0026micro;g mL⁻\u0026sup1;), as quantified by the CR-binding assay. CRA positivity is typically associated with enhanced EPS production and biofilm-forming ability (Jones and Wozniak \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), while the CR-binding assay provides a quantitative measure of CR retention by the EPS (Ghitti et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The combination of CRA and CR-binding assays provides complementary insights: while the former reflects overall biofilm-associated phenotypes, the latter allows for the estimation of the b-EPS fraction. Moreover, the binding affinity of CR to specific extracellular components, such as amyloid fibers (e.g., curli; Reichhardt et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), fimbriae (Spiers and Rainey \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and β-linked glucans including cellulose (Weiner et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Semedo et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Heredia-Ponce et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), suggests that CRA and CR-based assays may indirectly inform on the biochemical composition of b-EPS, encompassing both polysaccharidic and proteinaceous elements. In line with this, nearly half of the isolates in this study can be regarded as strong biofilm formers, exhibiting both CRA positivity and high b-CR accumulation in amounts comparable with those observed by for other biofilm-forming \u003cem\u003ePseudomonas\u003c/em\u003e strains (Quintieri et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile b-EPS are compositionally heterogeneous, r-EPS are generally composed almost exclusively of polysaccharides, with minor contributions (2\u0026ndash;3%) from other biomolecules (Mahto et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This supports the use of the phenol-sulfuric acid method for their quantification. Interestingly, a subset of isolates (C27, C33, C37, S22, S44, S54, S58, and S60) exhibited high levels of both b-EPS and r-EPS, indicating a robust capacity for producing both fractions. This dual production of tightly cell-associated and freely soluble EPS is particularly noteworthy. While b-EPS primarily contributes to cell cohesion, microcolony stability, and protection against abiotic stress (Wang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), r-EPS exerts broader effects in the extracellular environment. Due to its solubility and diffusibility, r-EPS can serve as a carbon source for surrounding soil microbes (Chen et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), facilitate soil particle aggregation, and influence water retention and nutrient dynamics. The eight isolates showing the highest r-EPS production yielded quantities equally or exceeding those of \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e AF814, a strain recognized for EPS production ability and quantity under drought stress (Ajijah et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Isolates capable of producing both b-EPS and r-EPS in substantial amounts may fulfil a dual ecological function, i.e., stabilizing local microbial niches through matrix formation while enhancing ecosystem-level resilience and resource availability within the rhizosphere or soil microhabitats.\u003c/p\u003e \u003cp\u003eThe adhesion assay revealed a heterogeneous adhesive capacity among the isolates. Notably, 48% of them exhibited a Relative Adhesion Capacity (RAC) greater than 1, indicating enhanced adherence to the polystyrene surface under the experimental conditions. However, this adhesive potential showed a significant inverse relationship with EPS production. Specifically, a strong negative correlation was observed between adhesion and b-EPS (r = -0.781, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while r-EPS showed a moderate but still significant negative correlation with adhesion (r = -0.417, p\u0026thinsp;=\u0026thinsp;0.003). These results suggest that higher EPS production, particularly of the cell-associated b-EPS fraction, tends to hinder initial adhesion to abiotic surfaces such as polystyrene. This inverse relationship may be particularly relevant during the early, reversible stage of bacterial adhesion, where physical-chemical interactions dominate. At this stage, electrostatic and hydrophobic forces govern the approach and initial contact of bacterial cells with surfaces. Hydrophobic interactions are often longer-ranged and more stable than electrostatic forces, exerting a stronger and more persistent influence, particularly in microbe\u0026ndash;plant root interactions (Janczarek et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Cell surface hydrophobicity itself is a complex trait influenced by multiple structural and compositional factors, including the presence of microbial fimbriae or hydrophobic adhesins (Otto et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), as well as the amount and nature of EPS (Vidhyalakshmi et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). An overall hydrophilic EPS matrix, by modifying surface charge and masking hydrophobic domains, may act as a barrier that reduces the effective contact of cells with the substrate. Microbial EPS can range from strongly hydrophilic to amphiphilic or even neutral, depending on their sugar composition, molecular weight, and the presence of charged side groups (Marvasi et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Therefore, the overall effect of EPS on adhesion is not only dependent on quantity but also on their physicochemical quality. In our dataset, isolates with abundant b-EPS, which likely formed a more cohesive and hydrated cell envelope, were generally less adherent, supporting the hypothesis that thick EPS layers reduce effective cell-surface interactions during the early phases of attachment.\u003c/p\u003e \u003cp\u003eHowever, EPS production could also be associated with alterations in the hydrophobicity of the bacterial cell surface. To test this, we used polystyrene microtiter plates, a widely accepted model substrate for studying biological interactions and microbial adhesion (Lyklema et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Nowak et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although polystyrene surfaces carry a slight negative charge in aqueous environments, their overall physicochemical profile is predominantly hydrophobic, with a contact angle of approximately 85\u0026deg; (Thormann et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Therefore, we expected isolates with more hydrophobic surfaces to exhibit stronger adhesion. To evaluate cell surface hydrophobicity, we used the xylene partition assay to calculate a hydrophobicity index (HI). As expected, bacterial adhesion was significantly and positively correlated with HI (r\u0026thinsp;=\u0026thinsp;0.700, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), supporting the idea that hydrophobic interactions facilitate bacterial attachment to the polystyrene surface.\u003c/p\u003e \u003cp\u003eInterestingly, HI showed a strong inverse correlation with b-EPS production (r = -0.730, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and a moderate but significant negative correlation with r-EPS production (r = -0.538, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These results reinforce the view that EPS production plays a major role in reducing cell surface hydrophobicity. Two non-mutually exclusive mechanisms may explain this effect: (1) EPS may act as a physical barrier, effectively masking hydrophobic structures on the cell surface; (2) EPS may possess a predominantly hydrophilic or ionic nature, which can interfere with hydrophobic interactions at the cell-substrate interface.\u003c/p\u003e \u003cp\u003eThese findings are consistent with previous studies showing that high EPS production can impair surface adhesion. Dertli et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), for example, reported that overproduction of EPS in \u003cem\u003eLactobacillus johnsonii\u003c/em\u003e resulted in decreased adhesion to polystyrene under comparable experimental conditions. More recently, Mougin et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) demonstrated that hydrophilic EPS derived from microalgae inhibited the adhesion of various foodborne bacteria to polystyrene surfaces, further supporting the inhibitory role of hydrophilic extracellular polymers in early-stage adhesion.\u003c/p\u003e \u003cp\u003eOverall, our results highlight an antagonistic relationship between EPS synthesis and cell surface hydrophobicity, a feature that may play a central role in regulating microbial adhesion dynamics in both natural and artificial environments.\u003c/p\u003e \u003cp\u003eWhen the relationships between adhesion and the investigated predictors were analyzed using a generalized linear regression model, both b-CR and HI emerged as significant independent predictors of bacterial adhesion. In contrast, r-EPS no longer exerted a significant effect. This suggests that, while a moderate bivariate correlation between r-EPS and adhesion was observed, its contribution is likely indirect and mediated by stronger, more proximal factors such as b-EPS (as inferred from b-CR) and surface hydrophobicity. These results underscore the pivotal roles of cell-associated EPS and hydrophobic interactions in mediating initial adhesion to hydrophobic substrates like polystyrene.\u003c/p\u003e \u003cp\u003eFurther, when HI was modelled as the dependent variable, b-CR again emerged as a significant negative predictor, reinforcing its central role in modulating surface hydrophobicity. The explanatory power of the regression models\u0026mdash;66.42% of the variance in adhesion and 56.09% in HI\u0026mdash;highlights the robustness of these relationships, while also pointing to the existence of additional factors, potentially including environmental cues, surface proteins, lipopolysaccharide composition, or regulatory pathways, that may modulate surface traits.\u003c/p\u003e \u003cp\u003eImportantly, our findings support the view that EPS production and surface adhesion are distinct microbial traits that require independent evaluation. In our dataset, isolates tended to cluster into two functional groups: low-EPS producers with strong adhesion capacity, and high-EPS producers with poor adhesion.\u003c/p\u003e \u003cp\u003eIn conclusion, in our study case b-EPS appear to play a dominant role in modulating cell-surface hydrophobicity and adhesion, contrary to the commonly assumed view that EPS universally promote attachment. Our results indicate that b-EPS, rather than r-EPS, represent the dominant structural determinant modulating cell\u0026ndash;surface interactions during the early stages of adhesion. Moreover, by linking our \u003cem\u003ein vitro\u003c/em\u003e results to ecologically relevant processes, such as rhizosphere colonization, we provide a valuable framework for selecting microbial strains with tailored properties for use in sustainable agriculture and biofertilizer formulations. From an applied perspective, the separate consideration of the processes of b-EPS production (positively related to biofilm-forming ability) and adhesion capability can be strategically advantageous. In designing microbial consortia for soil inoculants or plant growth-promoting formulations, a combinatory approach may prove beneficial\u0026mdash;pairing biofilm-forming strains with robust EPS production (to ensure structural stability of the microbial aggregate, protection, and moisture retention) alongside strains with superior adhesive capabilities (to ensure rapid and efficient surface colonization). Such complementary functionalities could enhance the persistence and efficacy of microbial inoculants in the rhizosphere or on soil particles and may be particularly relevant for the design of next-generation microbial inoculants, where persistence, surface colonization and matrix formation need to be balanced rather than maximized individually.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and formal analysis were performed by F.R., A.G., I.P. and C.C. Data curation was performed by F.R. and A.G. The first draft of the manuscript was written by F.R. and all authors commented on previous versions of the manuscript. M.A. and M.G. supervised the work, revised the manuscript and contributed to the final editing. All authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData are available on reasonable request from the corresponding authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAjijah N, Fiodor A, Dziewit L, Pranaw K (2024) Biological amelioration of water stress in rapeseed (Brassica napus L.) by exopolysaccharides-producing \u003cem\u003ePseudomonas protegens\u003c/em\u003e ML15. Physiol Plant 176:e70012. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/ppl.70012\u003c/span\u003e\u003cspan address=\"10.1111/ppl.70012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAn S, Wu J, Zhang L-H (2010) Modulation of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e Biofilm Dispersal by a Cyclic-Di-GMP Phosphodiesterase with a Putative Hypoxia-Sensing Domain. Appl Environ Microbiol 76:8160\u0026ndash;8173. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.01233-10\u003c/span\u003e\u003cspan address=\"10.1128/AEM.01233-10\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBasson A, Flemming LA, Chenia HY (2008) Evaluation of Adherence, Hydrophobicity, Aggregation, and Biofilm Development of \u003cem\u003eFlavobacterium johnsoniae\u003c/em\u003e-Like Isolates. Microb Ecol 55:1\u0026ndash;14\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBasu A, Prasad P, Das SN et al (2021) Plant Growth Promoting Rhizobacteria (PGPR) as Green Bioinoculants: Recent Developments, Constraints, and Prospects. Sustainability 13:1140. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/su13031140\u003c/span\u003e\u003cspan address=\"10.3390/su13031140\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhattacharyya A, Mavrodi O, Bhowmik N et al (2023) Chap. 1 - Bacterial biofilms as an essential component of rhizosphere plant-microbe interactions. In: Gurtler V, Patrauchan M (eds) Methods in Microbiology. Academic Press, pp 3\u0026ndash;48\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai P, Sun X, Wu Y et al (2019) Soil biofilms: microbial interactions, challenges, and advanced techniques for ex-situ characterization. Soil Ecol Lett 1:85\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s42832-019-0017-7\u003c/span\u003e\u003cspan address=\"10.1007/s42832-019-0017-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChagnot C, Zorgani MA, Astruc T, Desvaux M (2013) Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective. Front Microbiol 4:303. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2013.00303\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2013.00303\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang Y-I, Chang P-K (2002) The role of hydration force on the stability of the suspension of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e\u0026ndash;application of the extended DLVO theory. Colloids Surf Physicochem Eng Asp 211:67\u0026ndash;77. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0927-7757(02)00238-8\u003c/span\u003e\u003cspan address=\"10.1016/S0927-7757(02)00238-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Rossi F, Deng S et al (2014) Macromolecular and chemical features of the excreted extracellular polysaccharides in induced biological soil crusts of different ages. Soil Biol Biochem 78:1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.soilbio.2014.07.004\u003c/span\u003e\u003cspan address=\"10.1016/j.soilbio.2014.07.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosta OYA, Raaijmakers JM, Kuramae EE (2018) Microbial Extracellular Polymeric Substances: Ecological Function and Impact on Soil Aggregation. Front Microbiol 9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosterton JW (1999) Introduction to biofilm. Int J Antimicrob Agents 11:217\u0026ndash;221 discussion 237\u0026ndash;239. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0924-8579(99)00018-7\u003c/span\u003e\u003cspan address=\"10.1016/s0924-8579(99)00018-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDertli E, Mayer MJ, Narbad A (2015) Impact of the exopolysaccharide layer on biofilms, adhesion and resistance to stress in \u003cem\u003eLactobacillus johnsonii\u003c/em\u003e FI9785. BMC Microbiol 15:8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12866-015-0347-2\u003c/span\u003e\u003cspan address=\"10.1186/s12866-015-0347-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuBois Michel, Gilles KA, Hamilton JK et al (1956) Colorimetric method for the determination of sugars and related substances. Anal Chem 28:350\u0026ndash;356. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ac60111a017\u003c/span\u003e\u003cspan address=\"10.1021/ac60111a017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlemming H-C (2016) EPS\u0026mdash;Then and Now. Microorganisms 4:41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms4040041\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms4040041\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlemming H-C, Wingender J, Szewzyk U et al (2016) Biofilms: an emergent form of bacterial life. Nat Rev Microbiol 14:563\u0026ndash;575. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrmicro.2016.94\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro.2016.94\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGanesan M, Stewart EJ, Szafranski J et al (2013) Molar mass, entanglement and associations of the biofilm polysaccharide of \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e. Biomacromolecules 14:1474\u0026ndash;1481. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/bm400149a\u003c/span\u003e\u003cspan address=\"10.1021/bm400149a\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarrett TR, Bhakoo M, Zhang Z (2008) Bacterial adhesion and biofilms on surfaces. Prog Nat Sci 18:1049\u0026ndash;1056. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pnsc.2008.04.001\u003c/span\u003e\u003cspan address=\"10.1016/j.pnsc.2008.04.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhitti E, Rolli E, Vergani L, Borin S (2024) Flavonoids influence key rhizocompetence traits for early root colonization and PCB degradation potential of \u003cem\u003eParaburkholderia xenovorans\u003c/em\u003e LB400. Front Plant Sci 15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2024.1325048\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2024.1325048\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarimawan A, Ting Y-P (2016) Investigation of extracellular polymeric substances (EPS) properties of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eB. subtilis\u003c/em\u003e and their role in bacterial adhesion. Colloids Surf B Biointerfaces 146:459\u0026ndash;467. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.colsurfb.2016.06.039\u003c/span\u003e\u003cspan address=\"10.1016/j.colsurfb.2016.06.039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeredia-Ponce Z, Guti\u0026eacute;rrez-Barranquero JA, Purtschert-Montenegro G et al (2020) Biological role of EPS from \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003esyringae\u003c/em\u003e UMAF0158 extracellular matrix, focusing on a Psl-like polysaccharide. Npj Biofilms Microbiomes 6:1\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41522-020-00148-6\u003c/span\u003e\u003cspan address=\"10.1038/s41522-020-00148-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHermansson M (1999) The DLVO theory in microbial adhesion. Colloids Surf B Biointerfaces 14:105\u0026ndash;119. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0927-7765(99)00029-6\u003c/span\u003e\u003cspan address=\"10.1016/S0927-7765(99)00029-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHwang G, Kang S, El-Din MG, Liu Y (2012) Impact of an extracellular polymeric substance (EPS) precoating on the initial adhesion of \u003cem\u003eBurkholderia cepacia\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. Biofouling 28:525\u0026ndash;538. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/08927014.2012.694138\u003c/span\u003e\u003cspan address=\"10.1080/08927014.2012.694138\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanczarek M, Rachwał K, Cieśla J et al (2015) Production of exopolysaccharide by \u003cem\u003eRhizobium leguminosarum\u003c/em\u003e bv. trifolii and its role in bacterial attachment and surface properties. Plant Soil 388:211\u0026ndash;227. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11104-014-2320-5\u003c/span\u003e\u003cspan address=\"10.1007/s11104-014-2320-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones CJ, Wozniak DJ (2017) Congo Red Stain Identifies Matrix Overproduction and Is an Indirect Measurement for c-di-GMP in Many Species of Bacteria. Methods Mol Biol Clifton NJ 1657:147\u0026ndash;156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-4939-7240-1_12\u003c/span\u003e\u003cspan address=\"10.1007/978-1-4939-7240-1_12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKachlany SC, Levery SB, Kim JS et al (2001) Structure and carbohydrate analysis of the exopolysaccharide capsule of \u003cem\u003ePseudomonas putida\u003c/em\u003e G7. Environ Microbiol 3:774\u0026ndash;784. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1462-2920.2001.00248.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1462-2920.2001.00248.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarimi E, Aliasgharzad N, Esfandiari E et al (2022) Biofilm forming rhizobacteria affect the physiological and biochemical responses of wheat to drought. AMB Express 12:93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13568-022-01432-8\u003c/span\u003e\u003cspan address=\"10.1186/s13568-022-01432-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKjeldgaard B, Listian SA, Ramaswamhi V et al (2019) Fungal hyphae colonization by \u003cem\u003eBacillus subtilis\u003c/em\u003e relies on biofilm matrix components. Biofilm 1:100007. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bioflm.2019.100007\u003c/span\u003e\u003cspan address=\"10.1016/j.bioflm.2019.100007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrepsky N, Ferreira RBR, Nunes APF et al (2003) Cell Surface Hydrophobicity and Slime Production of \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e Brazilian Isolates. Curr Microbiol 46:0280\u0026ndash;0286. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00284-002-3868-5\u003c/span\u003e\u003cspan address=\"10.1007/s00284-002-3868-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrishnamoorthy M, Nayak BK, Nanda A (2018) \u003cem\u003eIn vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e characterization of probiotic organisms for their microbial adhesion property isolated from Coconut toddy. Karbala Int J Mod Sci 4:341\u0026ndash;346. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.kijoms.2018.08.001\u003c/span\u003e\u003cspan address=\"10.1016/j.kijoms.2018.08.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Narayanan M, Shi X et al (2024) Biofilms formation in plant growth-promoting bacteria for alleviating agro-environmental stress. Sci Total Environ 907:167774. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2023.167774\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2023.167774\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLopes MJdosS, Dias-Filho MB, Gurgel ESC (2021) Successful Plant Growth-Promoting Microbes: Inoculation Methods and Abiotic Factors. Front Sustain Food Syst 5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fsufs.2021.606454\u003c/span\u003e\u003cspan address=\"10.3389/fsufs.2021.606454\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLyklema J, Norde W, van Loosdrecht MCM, Zehnder AJB (1989) Adhesion of bacteria to polystyrene surfaces. Colloids Surf 39:175\u0026ndash;187. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0166-6622(89)80186-6\u003c/span\u003e\u003cspan address=\"10.1016/0166-6622(89)80186-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahto KU, Vandana, Priyadarshanee M et al (2022) Bacterial biofilm and extracellular polymeric substances in the treatment of environmental pollutants: Beyond the protective role in survivability. J Clean Prod 379:134759. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jclepro.2022.134759\u003c/span\u003e\u003cspan address=\"10.1016/j.jclepro.2022.134759\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMari S, Vrane J (2007) Characteristics and significance of microbial biofilm formation. Period Biol 109:115\u0026ndash;121\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarvasi M, Visscher PT, Casillas Martinez L (2010) Exopolymeric substances (EPS) from \u003cem\u003eBacillus subtilis\u003c/em\u003e : polymers and genes encoding their synthesis. FEMS Microbiol Lett 313:1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1574-6968.2010.02085.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1574-6968.2010.02085.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMina IR, Jara NP, Criollo JE, Castillo JA (2019) The critical role of biofilms in bacterial vascular plant pathogenesis. Plant Pathol 68:1439\u0026ndash;1447. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/ppa.13073\u003c/span\u003e\u003cspan address=\"10.1111/ppa.13073\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMougin J, Pavaux A-S, Fanesi A et al (2024) Bacterial adhesion inhibition by microalgal EPSs from \u003cem\u003eCylindrotheca closterium\u003c/em\u003e and \u003cem\u003eTetraselmis suecica\u003c/em\u003e biofilms. Appl Microbiol Biotechnol 108:168. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-023-12960-z\u003c/span\u003e\u003cspan address=\"10.1007/s00253-023-12960-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMu\u0026rsquo;minah B, Subair H, Fahruddin (2015) Isolation and Screening Bacterial Exopolysaccharide (EPS) from Potato Rhizosphere in Highland and the Potential as a Producer Indole Acetic Acid (IAA). Procedia Food Sci 3:74\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.profoo.2015.01.007\u003c/span\u003e\u003cspan address=\"10.1016/j.profoo.2015.01.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNowak J, Watala C, Boncler M (2014) Antibody binding, platelet adhesion, and protein adsorption on various polymer surfaces. Blood Coagul Fibrinolysis 25:52. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/MBC.0b013e328364a802\u003c/span\u003e\u003cspan address=\"10.1097/MBC.0b013e328364a802\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlson ME, Ceri H, Morck DW et al (2002) Biofilm bacteria: formation and comparative susceptibility to antibiotics. Can J Vet Res 66:86\u0026ndash;92\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOtto K, Elwing H, Hermansson M (1999) The role of type 1 fimbriae in adhesion of \u003cem\u003eEscherichia coli\u003c/em\u003e to hydrophilic and hydrophobic surfaces. Colloids Surf B Biointerfaces 15:99\u0026ndash;111. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0927-7765(99)00050-8\u003c/span\u003e\u003cspan address=\"10.1016/S0927-7765(99)00050-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePearce D, Bazin MJ, Lynch JM (1995) The Rhizosphere as a Biofilm. In: Lappin-Scott HM, Costerton JW (eds) Microbial Biofilms. Cambridge University Press, Cambridge, pp 207\u0026ndash;220\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuintieri L, Fanelli F, Z\u0026uuml;hlke D et al (2020) Biofilm and Pathogenesis-Related Proteins in the Foodborne \u003cem\u003eP. fluorescens\u003c/em\u003e ITEM 17298 With Distinctive Phenotypes During Cold Storage. Front Microbiol 11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReichhardt C, Jacobson AN, Maher MC et al (2015) Congo Red Interactions with Curli-Producing \u003cem\u003eE. coli\u003c/em\u003e and Native Curli Amyloid Fibers. PLoS ONE 10:e0140388. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0140388\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0140388\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenberg M (2006) Microbial adhesion to hydrocarbons: twenty-five years of doing MATH. FEMS Microbiol Lett 262:129\u0026ndash;134. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1574-6968.2006.00291.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1574-6968.2006.00291.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRumbaugh KP, Sauer K (2020) Biofilm dispersion. Nat Rev Microbiol 18:571\u0026ndash;586. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41579-020-0385-0\u003c/span\u003e\u003cspan address=\"10.1038/s41579-020-0385-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSemedo MC, Karmali A, Fonseca L (2015) A high throughput colorimetric assay of β-1,3-d-glucans by Congo red dye. J Microbiol Methods 109:140\u0026ndash;148. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.mimet.2014.12.020\u003c/span\u003e\u003cspan address=\"10.1016/j.mimet.2014.12.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSirajunnisa AR, Vijayagopal V, Sivaprakash B et al (2016) Optimization, kinetics and antioxidant activity of exopolysaccharide produced from rhizosphere isolate. Pseudomonas fluorescens CrN6 Carbohydr Polym 135:35\u0026ndash;43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.carbpol.2015.08.080\u003c/span\u003e\u003cspan address=\"10.1016/j.carbpol.2015.08.080\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpiers AJ, Rainey PB (2005) The \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e SBW25 wrinkly spreader biofilm requires attachment factor, cellulose fibre and LPS interactions to maintain strength and integrity. Microbiol Read Engl 151:2829\u0026ndash;2839. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/mic.0.27984-0\u003c/span\u003e\u003cspan address=\"10.1099/mic.0.27984-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThormann E, Simonsen AC, Hansen PL, Mouritsen OG (2008) Interactions between a Polystyrene Particle and Hydrophilic and Hydrophobic Surfaces in Aqueous Solutions. Langmuir 24:7278\u0026ndash;7284. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/la8005162\u003c/span\u003e\u003cspan address=\"10.1021/la8005162\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsuneda S, Aikawa H, Hayashi H et al (2003) Extracellular polymeric substances responsible for bacterial adhesion onto solid surface. FEMS Microbiol Lett 223:287\u0026ndash;292. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0378-1097(03)00399-9\u003c/span\u003e\u003cspan address=\"10.1016/S0378-1097(03)00399-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVidhyalakshmi R, Valli Nachiyar C, Narendra Kumar G et al (2018) Production, characterization and emulsifying property of exopolysaccharide produced by marine isolate of \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e. Biocatal Agric Biotechnol 16:320\u0026ndash;325. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bcab.2018.08.023\u003c/span\u003e\u003cspan address=\"10.1016/j.bcab.2018.08.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang K, Li W, Rui X et al (2014) Structural characterization and bioactivity of released exopolysaccharides from \u003cem\u003eLactobacillus plantarum\u003c/em\u003e 70810. Int J Biol Macromol 67:71\u0026ndash;78. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2014.02.056\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2014.02.056\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Zhi L, Shan W et al (2020) Correlation of extracellular polymeric substances and microbial community structure in denitrification biofilm exposed to adverse conditions. Microb Biotechnol 13:1889\u0026ndash;1903. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1751-7915.13633\u003c/span\u003e\u003cspan address=\"10.1111/1751-7915.13633\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeiner R, Seagren E, Arnosti C, Quintero E (1999) Bacterial survival in biofilms: probes for exopolysaccharide and its hydrolysis, and measurements of intra- and interphase mass fluxes. Methods Enzymol 310:403\u0026ndash;426. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0076-6879(99)10032-6\u003c/span\u003e\u003cspan address=\"10.1016/s0076-6879(99)10032-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWheatley RM, Poole PS (2018) Mechanisms of bacterial attachment to roots. FEMS Microbiol Rev. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/femsre/fuy014\u003c/span\u003e\u003cspan address=\"10.1093/femsre/fuy014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhitfield C, Paiment A (2003) Biosynthesis and assembly of Group 1 capsular polysaccharides in \u003cem\u003eEscherichia coli\u003c/em\u003e and related extracellular polysaccharides in other bacteria. Carbohydr Res 338:2491\u0026ndash;2502. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.carres.2003.08.010\u003c/span\u003e\u003cspan address=\"10.1016/j.carres.2003.08.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZabielska J, Kunicka-Styczyńska A, Otlewska A (2017) Adhesive and hydrophobic properties of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003ePseudomonas cedrina\u003c/em\u003e associated with cosmetics. Ecol Quest 28:41\u0026ndash;46. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.12775/EQ.2017.037\u003c/span\u003e\u003cspan address=\"10.12775/EQ.2017.037\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Biofilm formation, Cell surface hydrophobicity, Extracellular polymeric substances (EPS), Microbial adhesion, Pseudomonas fluorescens group","lastPublishedDoi":"10.21203/rs.3.rs-8583662/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8583662/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrobial persistence and colonization in the rhizosphere rely on traits that control how cells attach, interact, and organize into biofilms. Among these traits, extracellular polymeric substances (EPS), cell surface hydrophobicity, and adhesion play central roles in the early stages of root surface colonization. Here, we examined 48 isolates of the \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e group associated with \u003cem\u003eTuber borchii\u003c/em\u003e fruiting bodies to explore the relationships among bound and released EPS fractions, hydrophobicity and adhesion ability. All experiments were carried out under controlled in vitro conditions using polystyrene as an abiotic model that mimics the hydrophobic interfaces occurring in soil\u0026ndash;root environments. EPS fractions were quantified by Congo red and phenol\u0026ndash;sulfuric acid assays, while surface hydrophobicity and adhesion were determined through xylene partitioning and crystal violet staining. The isolates exhibited wide phenotypic variability. Bound EPS showed a strong negative relationship with both hydrophobicity and adhesion, while hydrophobicity was positively associated with adhesion strength. Regression models confirmed that bound EPS and hydrophobicity independently modulate the adhesion response. These findings suggest that thick, hydrated EPS layers can hinder early attachment, while thinner EPS coatings enhance cell\u0026ndash;surface interactions. Understanding this functional trade-off provides a basis for the informed selection of microbial inoculants, combining stable biofilm formers with highly adhesive strains to improve persistence and colonization efficiency in rhizosphere environments.\u003c/p\u003e","manuscriptTitle":"Complementary functionalities of extracellular polymeric substances, adhesion ability and hydrophobicity in Pseudomonas isolates may help the selection of strategically advantageous microbial inoculants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 09:12:23","doi":"10.21203/rs.3.rs-8583662/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-22T08:34:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-21T21:41:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-11T13:51:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235297808447819787760602989616614962941","date":"2026-02-07T19:55:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194940922776958175439479967940597039600","date":"2026-01-26T16:37:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-14T17:07:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-14T12:27:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-14T09:30:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2026-01-12T15:46:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1b2905ad-67a3-4ef2-a15a-8a9806864ced","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:18:24+00:00","versionOfRecord":{"articleIdentity":"rs-8583662","link":"https://doi.org/10.1007/s11274-026-04899-w","journal":{"identity":"world-journal-of-microbiology-and-biotechnology","isVorOnly":false,"title":"World Journal of Microbiology and Biotechnology"},"publishedOn":"2026-03-23 16:10:36","publishedOnDateReadable":"March 23rd, 2026"},"versionCreatedAt":"2026-01-19 09:12:23","video":"","vorDoi":"10.1007/s11274-026-04899-w","vorDoiUrl":"https://doi.org/10.1007/s11274-026-04899-w","workflowStages":[]},"version":"v1","identity":"rs-8583662","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8583662","identity":"rs-8583662","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}