The aim of our work was the isolation and characterization of bacteria from the rhizosphere of Spartina maritima in the metal contaminated Odiel estuary (Huelva, SW Spain). From 25 strains, 84 % were identified as gram-positive, particularly Staphylococcus and Bacillus. Gram-negative bacteria were represented by Pantoea and Salmonella. Salt and heavy metal tolerance, metal bioabsorption, plant growth promoting (PGP) properties, and biofilm formation were investigated in the bacterial collection. Despite the higher abundance of gram-positive bacteria, gram-negative isolates displayed higher tolerance toward metal(loid)s (As, Cu, Zn, and Pb) and greater metal biosorption, as deduced from ICP-OES and SEM-EDX analyses. Besides, they exhibited better PGP properties, which were retained in the presence of metals and the ability to form biofilms. Gram-negative strains Pantoea agglomerans RSO6 and RSO7, together with gram-positive Bacillus aryabhattai RSO25, were selected for a bacterial consortium aimed to inoculate S. maritima plants in metal polluted estuaries for phytoremediation purposes.

Pollution of coasts by toxic metals and metalloids is a worldwide problem for which phytoremediation using halophytes and associated microbiomes is becoming relevant. Metal(loid) excess is a constraint for plant establishment and development, and plant growth promoting rhizobacteria (PGPR) mitigate plant stress under these conditions. However, mechanisms underlying this effect remain elusive. The effect of toxic metal(loid)s on activity and gene expression of ROS-scavenging enzymes in roots of the halophyte Spartina densiflora grown on real polluted sediments in a greenhouse experiment was investigated. Sediments of the metal-polluted joint estuary of Tinto and Odiel rivers and control, unpollutred samples from the Piedras estuary were collected and submitted to ICP-OES. Seeds of S. densiflora were collected from the polluted Odiel marshes and grown in polluted and unpolluted sediments. Rhizophere biofilm-forming bacteria were selected based on metal tolerance and inoculated to S. densiflora and grown for 4 months. Fresh or frozen harvested plants were used for enzyme assays and gene expression studies, respectively. Metal excess induced SOD (five-fold increase), whereas CAT and ascorbate peroxidase displayed minor induction (twofold). A twofold increase of TBARs indicated membrane damage. Our results showed that metal-resistant PGPR (P. agglomerans RSO6 and RSO7 and B. aryabhattai RSO25) contributed to alleviate metal stress, as deduced from lower levels of all antioxidant enzymes to levels below those of non-exposed plants. The oxidative stress index (OSI) decreased between 50 and 75% upon inoculation. The results also evidenced the important role of PAL, involved in secondary metabolism and/or lignin synthesis, as a pathway for metal stress management in this halophyte upon inoculation with appropriate PGPR, since the different inoculation treatments enhanced PAL expression between 3.75- and five-fold. Our data confirm, at the molecular level, the role of PGPR in alleviating metal stress in S. densiflora and evidence the difficulty of working with halophytes for which little genetic information is available.

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