Azospirillum–Methylobacterium Inoculation Enhances Olive Growth and Reshapes Root Exudate Chemistry: A Preliminary TD-GC-MS Characterization

Abstract Plant growth–promoting bacteria (PGPB) are increasingly applied to perennial crops, yet their chemical footprints at the root–soil interface remain poorly investigated in species such as olive. In the present study, greenhouse-grown plants of Olea europaea L. cv. Leccino were inoculated with Azospirillum spp. (single strain or consortium) and Methylobacterium symbioticum SB0023/3T. Soil inoculations included the single strain Sp245, a microbial consortium (MIX: Azospirillum spp– M. symbioticum ), and a water control. In addition, M. symbioticum was also applied as a foliar treatment. All treatments were performed monthly for a total of three applications over a 12-week greenhouse trial (early March–late May). Morpho-physiological traits of plants were recorded, and root exudates were chemically profiled via thermal desorption (TD)-GC-MS. In general, microbial inoculation markedly improved root morphology and biomass. The best performances were observed using the MIX which enhanced primary and secondary root growth and i the dry matter of hypogeal and epigeal organs (root, stem, and leaf dry weight increased by 70.2%, 98.6%, and 65.3%, respectively, relative to the control). Foliar M. symbioticum uniquely boosted leaf surface area and chlorophyll index (+ 83.8% and + 33.3% vs. control, respectively). TD-GC-MS of root exudates (in situ HMDS derivatization) revealed four consistent metabolite classes—organic acids (glycolic, lactic, hydroxybutyric acids), glycerol, carbohydrates (e.g., glucose, aldonic acid γ-lactones), and inositol (seven isomers). In each metabolite class, significant differences were found among treatments. In particular, the MIX induced the highest signals for acids, carbohydrates, and inositol (up to ~ 17-fold higher than the control for organic acids), with inositol undetectable in control samples. Collectively, these results demonstrate that (i) PGPB (especially the consortium) enhance roots olive growth and (ii) root exudate chemistry is remodelled toward acids, sugars, and inositol after inoculation. To our knowledge, this is the first chemically resolved TD-GC-MS characterization of olive root exudates under PGPB treatments.