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.

Prospecting metal-tolerant rhizobia for phytoremediation of mining soils from Morocco using Anthyllis vulneraria L.

The aim of this work was using the legume plant Anthyllis vulneraria L. (ecotype metallicolous) as a trap plant, in order to isolate metal-tolerant rhizobial strains from metal-contaminated soils from Morocco, with pollution indexes spanning three orders of magnitude. As bioindicator, soil bacterial density was inversely correlated to the pollution index. Forty-three bulk soil bacteria and sixty two bacteria from nodules were isolated. The resistance of bacteria from nodules to heavy metals was four to ten times higher than that of bulk soil bacteria, reaching high maximum tolerable concentrations for Cd (2 mM), Cu (2 mM), Pb (7 mM), and Zn (3 mM). Besides, some strains show multiple metal-tolerant abilities and great metal biosorption onto the bacterial surface. Amplification and restriction analysis of ribosomal 16S rDNA (ARDRA) and 16S ribosomal DNA (rDNA) sequencing were used to assess biodiversity and phylogenetic position among bacteria present in nodules. Our results suggest that a great diversity of non-rhizobial bacteria (alpha- and gamma-proteobacteria) colonize nodules of Anthyllis plants in contaminated soils. Taking together, our results evidence that, in polluted soils, rhizobia can be displaced by non-rhizobial (and hence, non-fixing) strains from nodules. Thus, the selection of metal-resistant rhizobia is a key step for using A. vulneraria symbioses for in situ phytoremediation.

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.

Rhizostabilization of metals in soils using Lupinus luteus inoculated with the metal resistant rhizobacterium Serratia sp. MSMC541.

The aim of this work was to test Lupinus luteus plants, inoculated with metal resistant rhizobacteria, in order to phytostabilise metals in contaminated soils. The resistance to heavy metals of strains isolated from nodules of Lupinus plants was evaluated. The strain MSMC541 showed multi-resistance to several metals (up to 13.3 mM As, 2.2 mM Cd, 2.3 mM Cu, 9 mM Pb and 30 mM Zn), and it was selected for further characterization. Furthermore, this strain was able to biosorb great amounts of metals in cell biomass. 16S rDNA sequencing positioned this strain within the genus Serratia. The presence of arsenic resistance genes was confirmed by southern blot and PCR amplification. A rhizoremediation pot experiment was conducted using Lupinus luteus grown on sand supplemented with heavy metals and inoculated with MSMC541. Plant growth parameters and metal accumulation were determined in inoculated vs. non-inoculated Lupinus luteus plants. The results showed that inoculation with MSMC541 improved the plant tolerance to metals. At the same time, metal translocation to the shoot was significantly reduced upon inoculation. These results suggest that Lupinus luteus plants, inoculated with the metal resistant strain Serratia sp. MSMC541, have a great potential for phytostabilization of metal contaminated soils.

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.

"In situ" phytostabilisation of heavy metal polluted soils using Lupinus luteus inoculated with metal resistant plant-growth promoting rhizobacteria.

The aim of this work is the evaluation of metal phytostabilisation potential of Lupinus luteus inoculated with Bradyrhizobium sp. 750 and heavy metal resistant PGPRs (plant-growth promoting rhizobacteria), for in situ reclamation of multi-metal contaminated soil after a mine spill. Yellow lupines accumulated heavy metals mainly in roots (Cu, Cd and especially Pb were poorly translocated to shoots). This indicates a potential use of this plant in metal phytostabilisation. Furthermore, As accumulation was undetectable. On the other hand, zinc accumulation was 10-100 times higher than all other metals, both in roots and in shoots. Inoculation with Bradyrhizobium sp. 750 increased both biomass and nitrogen content, indicating that nitrogen fixation was effective in soils with moderate levels of contamination. Co-inoculation of lupines with a consortium of metal resistant PGPR (including Bradyrhizobium sp., Pseudomonas sp. and Ochrobactrum cytisi) produced an additional improvement of plant biomass. At the same time, a decrease in metal accumulation was observed, both in shoots and roots, which could be due to a protective effect exerted on plant rhizosphere. Our results indicate the usefulness of L. luteus inoculated with a bacterial consortium of metal resistant PGPRs as a method for in situ reclamation of metal polluted soils.

Evaluation of the metal phytoextraction potential of crop legumes. Regulation of the expression of O-acetylserine (thiol)lyase under metal stress.

The metal phytoextraction potential of three legumes belonging to different genera has been studied under greenhouse conditions. Legumes accumulate As and metals mainly in roots, although translocation to shoot is observed. Alfalfa did accumulate the highest concentrations of As and metals in shoots and aerial biomass was less affected by the toxic elements, indicating its good behaviour in phytoextraction. Clover accumulated less metal, but showed larger biomass. EDTA addition enhanced Pb phytoextraction up to levels similar to those described for plants proposed in phytoremediation. The regulation of O-acetylserine (thiol)lyase from legumes under metal stress has been analysed to test the possibility of establishing a possible correlation between the expression of OASTL in the presence of the metals and the metal accumulation in legume plant tissues. Cd and Pb(EDTA) produce the strongest increases of OASTL activity, with the higher enhancement seen in roots, in parallel with the higher metal accumulation. Arsenic produced an increase of root enzyme activity, whereas Cu produced a decrease, mainly in shoots. Western blots using antibodies against an A. THALIANA cytosolic OAS-TL recognised up to five protein bands in crude extracts from LOTUS and clover. A low molecular weight isoform of 32 kDa was induced in the presence of Cd and Pb. A partial RT-PCR sequence from clover has been obtained, showing 86 - 97 % identity with other described OASTLs. The PCR fragment has been used to analyse OASTL mRNA levels of legumes under metal stress. OASTL transcripts were increased by As, Cd, and Pb, especially in roots, where metal accumulation was maximal, while Cu produced a decrease in the transcript levels.

Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype.

Legume plants carefully control the extent of nodulation in response to rhizobial infection. To examine the mechanism underlying this process we conducted a detailed analysis of the Lotus japonicus hypernodulating mutants, har1-1, 2 and 3 that define a new locus, HYPERNODULATION ABERRANT ROOT FORMATION (Har1), involved in root and symbiotic development. Mutations in the Har1 locus alter root architecture by inhibiting root elongation, diminishing root diameter and stimulating lateral root initiation. At the cellular level these developmental alterations are associated with changes in the position and duration of root cell growth and result in a premature differentiation of har1-1 mutant root. No significant differences between har1-1 mutant and wild-type plants were detected with respect to root growth responses to 1-aminocyclopropane1-carboxylic acid, the immediate precursor of ethylene, and auxin; however, cytokinin in the presence of AVG (aminoetoxyvinylglycine) was found to stimulate root elongation of the har1-1 mutant but not the wild-type. After inoculation with Mesorhizobium loti, the har1 mutant lines display an unusual hypernodulation (HNR) response, characterized by unrestricted nodulation (hypernodulation), and a concomitant drastic inhibition of root and shoot growth. These observations implicate a role for the Har1 locus in both symbiotic and non-symbiotic development of L. japonicus, and suggest that regulatory processes controlling nodule organogenesis and nodule number are integrated in an overall mechanism governing root growth and development.

Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus.

Nitrogen-fixing root nodules develop on legumes as a result of an interaction between host plants and soil bacteria collectively referred to as rhizobia. The organogenic process resulting in nodule development is triggered by the bacterial microsymbiont, but genetically controlled by the host plant genome. Using T-DNA insertion as a tool to identify novel plant genes that regulate nodule ontogeny, we have identified two putatively tagged symbiotic loci, Ljsym8 and Ljsym13, in the diploid legume Lotus japonicus. The sym8 mutants are arrested during infection by the bacteria early in the developmental process. The sym13 mutants are arrested in the final stages of infection, and ineffective nodules are formed. These two plant mutant lines were identified in progeny from 1112 primary transformants obtained after Agrobacterium tumefaciens T-DNA-mediated transformation of L. japonicus and subsequent screening for defects in the symbiosis with Mesorhizobium loti. Additional nontagged mutants arrested at different developmental stages were also identified and genetic complementation tests assigned all the mutations to 16 monogenic symbiotic loci segregating recessive mutant alleles. In the screen reported here independent symbiotic loci thus appeared with a frequency of approximately 1.5%, suggesting that a relatively large set of genes is required for the symbiotic interaction.

Regulation of the expression of ferredoxin-glutamate synthase in barley.

We have investigated the regulation of ferredoxin-glutamate synthase (Fd-GOGAT) in leaves of barley (Hordeum vulgare L. cv. Maris Mink) at the mRNA, protein and enzyme activity levels. Studies of the changes in Fd-GOGAT during plant development showed that the activity in shoots increases rapidly after germination to reach a maximum (on a fresh-weight basis) at day 10 and then declines markedly to less than 50% of the maximal activity by day 30, this decline being correlated with an equivalent loss of Fd-GOGAT protein. Growing the plants in darkness reduced the maximum activity attained in the shoots, but did not affect the overall pattern of the changes or their timing. The activity of Fd-GOGAT increased two- to three-fold within 48 h when etiolated leaves were exposed to light, and Northern blots indicated that the induction occurred at the mRNA level. However, whilst a carbon source could at least partially substitute for light in the induction of nitrate reductase activity, no induction of Fd-GOGAT activity was seen when etiolated leaves were treated with either sucrose or glucose. Interestingly, the levels of Fd-GOGAT mRNA and activity remained high up to a period of 16 h or 72 h darkness, respectively. Compared with plants grown in N-free medium, light-grown plants supplied with nitrate had almost two-fold higher Fd-GOGAT activities and increased Fd-GOGAT mRNA levels, but nitrate had no effect on the abundance of the enzyme or its mRNA in etiolated plants, indicating that light is required for nitrate induction of barley Fd-GOGAT.

Regulation of the expression of ferredoxin-nitrite reductase in synchronous cultures of Chlamydomonas reinhardtii.

The regulation of ferredoxin-nitrite reductase--the second enzyme involved in the nitrate assimilatory pathway--in synchronous cultures of C. reinhardtii has been studied both at the activity and protein levels using specific antibodies. During a cycle of 12 h light/12 h dark (12L:12D), ferredoxin-nitrite reductase activity shows a 24-h fluctuation with a maximum in the middle of the light period. The increase of activity during the first few hours of the light phase is due to de novo synthesis of the enzyme. This synthesis occurs in the absence of NH4+ and it is highly induced by either nitrate or nitrite, but it does not require light so long as carbon skeletons are available. The decrease of ferredoxin-nitrite reductase activity during the last hours of the light period and during the dark phase is suggested to be due to protein degradation, although this process is slow because of the high stability of the enzyme. The changes in the level of ferredoxin-nitrite reductase seem to be related to events in the cell cycle under the illumination conditions used. Thus, synthesis of the enzyme correlates to growth periods within the cell cycle, and it does not seem to be under the control of a circadian rhythm.