Investigating the mechanisms underlying phytoprotection by plant growth-promoting rhizobacteria in Spartina densiflora under metal stress.

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.

Assessing the role of endophytic bacteria in the halophyte Arthrocnemum macrostachyum salt tolerance.

There is an increasing interest to use halophytes for revegetation of salt affected ecosystems, as well as in understanding their mechanisms of salt tolerance. We hypothesized that bacteria from the phyllosphere of these plants might play a key role in its high tolerance to excessive salinity. Eight endophytic bacteria belonging to Bacillus and closely related genera were isolated from phyllosphere of the halophyte Arthrocnemum macrostachyum growing in salty agricultural soils. The presence of plant-growth promoting (PGP) properties, enzymatic activities and tolerance towards NaCl was determined. Effects of inoculation on seeds germination and adult plant growth under experimental NaCl treatments (0, 510 and 1030 mM NaCl) were studied. Inoculation with a consortium including the best performing bacteria improved considerably the kinetics of germination and the final germination percentage of A. macrostachyum seeds. At high NaCl concentrations (1030 mM), inoculation of plants mitigated the effects of high salinity on plant growth and physiological performance and, in addition, this consortium appears to have increased the potential of A. macrostachyum to accumulate Na+ in its shoots, thus improving sodium phytoextraction capacity. Bacteria isolated from A. macrostachyum phyllosphere seem to play an important role in plant salt tolerance under stressing salt concentrations. The combined use of A. macrostachyum and its microbiome can be an adequate tool to enhance plant adaptation and sodium phytoextraction during restoration of salt degraded soils.

Isolation of plant-growth-promoting and metal-resistant cultivable bacteria from Arthrocnemum macrostachyum in the Odiel marshes with potential use in phytoremediation.

Arthrocnemum macrostachyum is a halophyte naturally growing in southwest coasts of Spain that can tolerate and accumulate heavy metals. A total of 48 bacteria (30 endophytes and 18 from the rhizosphere) were isolated from A. macrostachyum growing in the Odiel River marshes, an ecosystem with high levels of contamination. All the isolates exhibited plant-growth-promoting (PGP) properties and most of them were multiresistant to heavy metals. Although the presence of heavy metals reduced the capability of the isolates to exhibit PGP properties, several strains were able to maintain their properties or even enhance them in the presence of concrete metals. Two bacterial consortia with the best-performing endophytic or rhizospheric strains were selected for further experiments. Bacterial inoculation accelerated germination of A. macrostachyum seeds in both the absence and presence of heavy metals. These results suggest that inoculation of A. macrostachyum with the selected bacteria could ameliorate plant establishment and growth in contaminated marshes.

Scouting contaminated estuaries: heavy metal resistant and plant growth promoting rhizobacteria in the native metal rhizoaccumulator Spartina maritima.

Spartina maritima is a native endangered heavy metal rhizoaccumulator cordgrass naturally growing in southwest coasts of Spain, where is used as a biotool to rehabilitate degraded salt marshes. Fifteen bacterial strains were isolated from the rhizosphere of S. maritima growing in the estuary of the Tinto River, one of the most polluted areas in the world. A high proportion of bacteria were resistant towards several heavy metals. They also exhibited multiple plant growth promoting (PGP) properties, in the absence and the presence of Cu. Bacillus methylotrophicus SMT38, Bacillusaryabhattai SMT48, B. aryabhattai SMT50 and Bacilluslicheniformis SMT51 were selected as the best performing strains. In a gnobiotic assay, inoculation of Medicago sativa seeds with the selected isolates induced higher root elongation. The inoculation of S. maritima with these indigenous metal-resistant PGP rhizobacteria could be an efficient method to increase plant adaptation and growth in contaminated estuaries during restoration programs.

Prospecting metal-resistant plant-growth promoting rhizobacteria for rhizoremediation of metal contaminated estuaries using Spartina densiflora.

In the salt marshes of the joint estuary of Tinto and Odiel rivers (SW Spain), one of the most polluted areas by heavy metals in the world, Spartina densiflora grows on sediments with high concentrations of heavy metals. Furthermore, this species has shown to be useful for phytoremediation. The total bacterial population of the rhizosphere of S. densiflora grown in two estuaries with different levels of metal contamination was analyzed by PCR denaturing gradient gel electrophoresis. Results suggested that soil contamination influences bacterial population in a greater extent than the presence of the plant. Twenty-two different cultivable bacterial strains were isolated from the rhizosphere of S. densiflora grown in the Tinto river estuary. Seventy percent of the strains showed one or more plant growth-promoting (PGP) properties, including phosphate solubilization and siderophores or indolacetic acid production, besides a high resistance towards Cu. A bacterial consortium with PGP properties and very high multiresistance to heavy metals, composed by Aeromonas aquariorum SDT13, Pseudomonas composti SDT3, and Bacillus sp. SDT14, was selected for further experiments. This consortium was able to two-fold increase seed germination and to protect seeds against fungal contamination, suggesting that it could facilitate the establishment of the plant in polluted estuaries.

Taxonomic and symbiotic diversity of bacteria isolated from nodules of Acacia tortilis subsp. raddiana in arid soils of Tunisia.

A collection of rhizobia isolated from Acacia tortilis subsp. raddiana nodules from various arid soils in Tunisia was analyzed for their diversity at both taxonomic and symbiotic levels. The isolates were found to be phenotypically diverse. The majority of the isolates tolerated 3% NaCl and grew at 40 °C. Genetic characterization emphasized that most of the strains (42/50) belong to the genus Ensifer, particularly the species Ensifer meliloti, Ensifer garamanticus, and Ensifer numidicus. Symbiotic properties of isolates showed diversity in their capacity to nodulate their host plant and to fix atmospheric nitrogen. The most effective isolates were closely related to E. garamanticus. Nodulation tests showed that 3 strains belonging to Mesorhizobium genus failed to renodulate their host plant, which is surprising for symbiotic rhizobia. Furthermore, our results support the presence of non-nodulating endophytic bacteria belonging to the Acinetobacter genus in legume nodules.

Self-bioremediation of cork-processing wastewaters by (chloro)phenol-degrading bacteria immobilised onto residual cork particles.

Cork manufacturing is a traditional industry in Southern Europe, being the main application of this natural product in wine stoppers and insulation. Cork processing begins at boiling the raw material. As a consequence, great volumes of dark wastewaters, with elevated concentrations of chlorophenols, are generated, which must be depurated through costly physicochemical procedures before discarding them into public water courses. This work explores the potential of bacteria, isolated from cork-boiling waters storage ponds, in bioremediation of the same effluent. The bacterial population present in cork-processing wastewaters was analysed by DGGE; low bacterial biodiversity was found. Aerobic bacteria were isolated and investigated for their tolerance against phenol and two chlorophenols. The most tolerant strains were identified by sequencing 16S rDNA. The phenol-degrading capacity was investigated by determining enzyme activities of the phenol-degrading pathway. Moreover, the capacity to form biofilms was analysed in a microtitre plate assay. Finally, the capacity to form biofilms onto the surface of residual small cork particles was evaluated by acridine staining followed by epifluorescence microscopy and by SEM. A low-cost bioremediation system, using phenol-degrading bacteria immobilised onto residual cork particles (a by-product of the industry) is proposed for the remediation of this industrial effluent (self-bioremediation).

Characterization of root-nodulating bacteria associated to Prosopis farcta growing in the arid regions of Tunisia.

Diversity of 50 bacterial isolates recovered from root nodules of Prosopis farcta grown in different arid soils in Tunisia, was investigated. Characterization of isolates was assessed using a polyphasic approach including phenotypic characteristics, 16S rRNA gene PCR--RFLP and sequencing, nodA gene sequencing and MLSA. It was found that most of isolates are tolerant to high temperature (40°C) and salinity (3%). Genetic characterization emphasizes that isolates were assigned to the genus Ensifer (80%), Mesorhizobium (4%) and non-nodulating endophytic bacteria (16%). Forty isolates belonging to the genus Ensifer were affiliated to Ensifer meliloti, Ensifer xinjiangense/Ensifer fredii and Ensifer numidicus species. Two isolates belonged to the genus Mesorhizobium. Eight isolates failing to renodulate their host plant were endophytic bacteria and belonged to Bacillus, Paenibacillus and Acinetobacter genera. Symbiotic properties of nodulating isolates showed a diversity in their capacity to infect their host plant and fix atmospheric nitrogen. Isolate PG29 identified as Ensifer meliloti was the most effective one. Ability of Prosopis farcta to establish symbiosis with rhizobial species confers an important advantage for this species to be used in reforestation programs. This study offered the first systematic information about the diversity of microsymbionts nodulating Prosopis farcta in the arid regions of Tunisia.

Plasmid Transfer Detection in Soil using the Inducible lPR System Fused to Eukaryotic Luciferase Genes.

We report a model system for plasmid transfer analysis using the regulated lambda phage right promoter, lPR, fused to luc and lucOR as reporter genes. We have demonstrated that the systems cI857-lPR::luc and cI857-lPR::lucOR are temperature-inducible in Escherichia coli but not in other Gram-negative bacteria analyzed, enabling detection of luminescence when plasmids were mobilized from E. coli to those Gram-negative backgrounds. Using light for the detection, we have observed plasmid transfer from E. coli harboring RK2 and R388 derived plasmids to Pseudomonas putida KT2440 (co-introduced with donors) and to indigenous microorganisms, in vitro and in nonsterile soil microcosms. The importance of nutrients for an efficient plasmid transfer in nonsterile soil microcosms has been confirmed. When plasmid transfer experiments were carried out into nonsterile soil microcosms, significant populations of indigenous transconjugants arose. This system provides efficient marker genes and avoids the use of antibiotics for the selection of transconjugants.

Transformation of floral organs with GFP in Medicago truncatula.

A high frequency of embryogenesis and transformation from all parts of flowers of two lines of Medicago truncatula R-108-1 and Jemalong J5 were obtained. Using this flower system, we obtained transgenic plants expressing promoter-uidA gene fusions as well as the gfp living cell color reporter gene. Moreover, this method allows us to save time and to use a smaller greenhouse surface for the culture of donor plants. Southern hybridization showed that the internal gfp fragment had the expected size and the number of T-DNA copies integrated in the plant genome varied between one and three. These data suggest that the presence of the GFP protein has no toxic effects, since no rearrangement of the gfp reporter gene was detected in the regenerated plants.