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

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 during early root colonization. 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, under in vitro conditions using polystyrene as a hydrophobic model surface. EPS fractions were quantified by Congo red and phenol–sulfuric acid assays, while hydrophobicity and adhesion were determined through xylene partitioning and crystal violet staining. The isolates exhibited wide phenotypic variability, with hydrophobicity index values ranging from 26.79% to 85.60% and relative adhesion capacity from 0.2 to 1.67. Bound EPS showed a strong negative correlation with both hydrophobicity (r = -0.730, p < 0.0001) and adhesion (r = -0.781, p < 0.0001), while hydrophobicity was positively associated with adhesion (r = 0.700, p < 0.0001). Regression analysis explained 66.4% of the variance in adhesion, identifying bound EPS (β = −0.6300, p < 0.0001) and hydrophobicity (β = 0.3193, p = 0.0196) as significant independent predictors, while released EPS was not significant. 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.