Bioaugmentation with bacteria selected from the microbiome enhances Arthrocnemum macrostachyum metal accumulation and tolerance

Depósito de Investigación de la Universidad de Sevilla https://idus.us.es/ This is an Accepted Manuscript of an article published by ELSEVIER in MARINE POLLUTION BULLETIN, Vol. 117 , on 2017, available at: https://doi.org/10.1016/j.marpolbul.2017.02.008 Copyright 2017 Elsevier. En idUS Licencia Creative Commons CC BY-NC-ND Elsevier Editorial System(tm) for Marine Pollution Bulletin Manuscript Draft Manuscript Number: MPB-D-16-01358R1 Title: Bioaugmentation with bacteria selected from the microbiome enhances Arthrocnemum macrostachyum metal accumulation and tolerance Article Type: Research Paper Keywords: Arthrocnemum macrostachyum; endophytic bacteria; metal pollution; PGPB; phytoremediation; rizospheric bacteria Corresponding Author: Dr. Enrique Mateos-Naranjo, Corresponding Author's Institution: First Author: Salvadora Navarro-Torre Order of Authors: Salvadora Navarro-Torre; José M Barcia-Piedras; Miguel A Caviedes; Eloisa Pajuelo; Susana Redondo-Gómez; Ignacio D Rodríguez-Llorente; Enrique Mateos-Naranjo Abstract: A glasshouse experiment was designed to investigate the role of bacterial consortia isolated from the endosphere (CE) and rizosphere (CR) of Arthrocnemum macrostachyum on its metal uptake capacity and tolerance in plants grown in metal polluted sediments. A. macrostachyum plants were randomly assigned to three bioaugmentation treatments (CE, CR and without inoculation) during 120 days. Bioaugmentation with both bacterial consortia enhanced A. macrostachyum capacity to accumulate ions in its roots, while shoot ions concentration only increased with CE treatment. Furthermore bioaugmentation ameliorated the phytotoxicity levels, which was reflected in an increment of plant growth of 59 and 113% for shoots and 52 and 98% for roots with CE and CR treatments, respectively. This effect was supported by bacteria beneficial effect on photochemical apparatus and the modulation of its oxidative stress machinery. These findings indicated that bacteria selected from the microbiome can be claimed to improve A. macrostachyum metal remediation efficiency. Highlights  Endoand rizosphere bacterial role on A. macrostachyum metal response was analysed  Bacterial bioaugmentation enhanced the efficiency of metal phytoextraction  Bacterial inoculation mitigated the impact of pollution on A. macrostachyum grown  They positively affect its photochemical apparatus  They favoured the modulation of its oxidative stress machinery *Highlights 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 Bioaugmentation with bacteria selected from the microbiome enhances Arthrocnemum macrostachyum metal accumulation and tolerance Salvadora Navarro-Torre1, José M. Barcia-Piedras2,3, Miguel A. Caviedes1, Eloísa Pajuelo1, Susana Redondo-Gómez2, Ignacio D. Rodríguez-Llorente1, Enrique MateosNaranjo2* 1Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla. c/ Profesor García González, 2, 41012-Sevilla, Spain. 2Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla. 1095, 41012-Sevilla, Spain. 3IFAPA, Centro Las Torres –Tomejil. Ctra Sevilla-Cazalla, km 12.200. 41200 Alcalá del Río, Sevilla, Spain. *Corresponding autor: Dr. Enrique Mateos Naranjo e-mail: [email protected] Tel.: + 34 – 954557064 Fax: + 34 – 954-4615780 *Manuscript (clean - no tracked changes) Click here to view linked References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 Abstract A glasshouse experiment was designed to investigate the role of bacterial consortia isolated from the endosphere (CE) and rizosphere (CR) of Arthrocnemum macrostachyum on its metal uptake capacity and tolerance in plants grown in metal polluted sediments. A. macrostachyum plants were randomly assigned to three bioaugmentation treatments (CE, CR and without inoculation) during 120 days. Bioaugmentation with both bacterial consortia enhanced A. macrostachyum capacity to accumulate ions in its roots, while shoot ions concentration only increased with CE treatment. Furthermore bioaugmentation ameliorated the phytotoxicity levels, which was reflected in an increment of plant growth of 59 and 113% for shoots and 52 and 98% for roots with CE and CR treatments, respectively. This effect was supported by bacteria beneficial effect on photochemical apparatus and the modulation of its oxidative stress machinery. These findings indicated that bacteria selected from the microbiome can be claimed to improve A. macrostachyum metal remediation efficiency. Key words: Arthrocnemum macrostachyum, endophytic bacteria, metal pollution, PGPB, phytoremediation, rizospheric bacteria. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 1. Introduction Phytoremediation is a promising clean-up solution to act upon of the organic and inorganic pollutants in a wide variety of contaminated sites, by extracting, degrading or immobilizing them through the use of plants (Marques et al., 2011; Weis and Weis, 2004). Plants used for metal remediation should be chosen on the basis of their potential to uptake, accumulate and transport large amount of metals, as well as taking into account its ability to maintain high growth rates under metal excess (Marques et al., 2011). Sometimes phytoremediation efficiency is compromised by growth inhibition as a consequence of the increment of metal phytotoxicity levels in plant tissues (Khan et al., 2015). Recently a number of scientists have begun to explore the possibility of using the microbial populations which colonizing the rhizosphere (Mesa et al., 2015b; Pajuelo et al. 2014) or the endosphere (Doty, 2008; Mesa et al., 2015a; Newman and Reynolds, 2005; Rajkumar et al., 2009) in order to make this technology more efficacious. Among these bacterial populations the interest has been particularly focused plant growth promoting bacteria (PGPB), which have demonstrated the ability to promote plant growth under stress conditions (Ahemad and Kibret, 2014; de Bashan et al., 2012; Glick, 2010; Mesa et al., 2015a,b; Mateos-Naranjo et al., 2015; Navarro-Torre et al., 2016a; Weyens et al., 2009). However the effect of microbial inoculation on plant metal uptake does not follow a clear pattern, being highly dependent of sediment and contaminant characteristics, as well as of plant-microbe partnerships (Phieler et al., 2014; Sessitsch et al., 2013). Therefore, this study was designed to contribute to fill these gaps of knowledge. Arthrocnemum macrostachyum (Moric) C. Koch is a common halophytic shrub in SW Iberian Peninsula, which is distributed in coastal areas through Mediterranean 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 basin to the Middle East and Asia (Redondo-Gómez et al., 2010b). In the marshes of the south-west of Spain develops populations distributed from the middle to high elevations in the tidal range, which are occasionally subject to tidal inundations and seasonal hypersalinity (Redondo-Gómez et al., 2010b). This species represent suitable model plant to study plant-bacterial interactions and specifically in terms of metal phytoremediation efficiency, since this species has demonstrated a great capacity for accumulating metals in its tissues (Redondo-Gómez et al., 2010a), being considered a suitable candidate for remediation of metal polluted coastal sediments (Conesa and Schulin, 2010; Redondo-Gómez et al., 2010a). In addition, Navarro-Torre et al. (2016b) recently isolated different bacterial strains from the rhizosphere and endosphere of A. macrostachyum plants from the Odiel estuary, which exhibited several plant growth promoting (PGP) properties, even in the presence of heavy metals. Among all the isolates, the strains Vibrio kanaloae RA1, Pseudoalteromonas distinct RA8, Pseudoalteromonas prydzensis RA15 and Staphylococcus warneri RA18 isolated from the rhizosphere of A. macrostachyum and the strains Kushneria marisflavi EAod3, Micrococcus aloeverae EAod10, Bacillus vietnamensis EAR8 and Halomonas zincidurans EAR18 isolated from the endosphere, were proposed as the best candidates to design two bacterial consortia aimed to inoculate A. macrostachyum plants to improve its metal remediation potential (Navarro-Torre et al., 2016b). Thus, in the present study, a continuation of this previous one, we hypothesized that these selected bacterial consortia could play an important role on metal uptake, accumulation and transport, as well as on the mitigation of phytotoxicity effects arising from metal accumulation in plant tissues. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 The aim of this study was to investigate the role of bacteria isolated from the microbiome of A. macrostachyum in its metal uptake capacity and tolerance to metal excess. 2. Material and methods 2.1. Plant and sediment source Seeds of A. macrostachyum and samples of the first 15 cm of sediment around the plants were collected in March 2015 from the Odiel marshes (37º15´N, 6º58´W; SW Spain) and subsequently transported to the laboratory. Then, seeds were disinfected with 10% sodium hypochlorite for 10 min and washed six times with sterile distillated water. These seeds were plated in 9% agar plates and placed in a growth chamber (AGP-700-HR ESP; Radiber, Barcelona, Spain) with a regime of 10 h of light (20 ºC, 50% HR and 35 μmol m-2 s-1, 400-700 nm) and 14 h dark (5 ºC and 50% HR) for 20 days. After, germinated seedlings were transferred to individual pots (9 cm high x 11 cm diameter, n = 30) filled with perlite. Seedlings were maintained in a greenhouse with the following conditions: temperature between 21-25 ºC, 40-60% relative humidity, natural daylight of 250 as minimum and 1000 μmol m-2 s-1 as maximum light and supplemented with 20% Hoagland solution (Hoagland and Arnon, 1938). On the other hand, sediment texture, conductivity, pH, redox potential and total sediment metal concentrations (n = 5) were measured using the method described by Mateos-Naranjo et al. (2011) to determine the physicochemical characteristic of the collected sediment. The physicochemical properties of the sediments are given in Table 1. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 6 2.2. Inoculation and plant assays in polluted soils Two bacterial consortia were selected for this work on basis on their PGP properties according with the recent work by Navarro-Torre et al. (2016b). They represented the best-performing strains among all the isolates characterized in that study. Thus we employed a consortium with bacteria isolated from the rhizosphere (CR) of A. macrostachyum (integrated by the strains Vibrio kanaloae RA1, Pseudoalteromonas distinct RA8, Pseudoalteromonas prydzensis RA15 and Staphylococcus warneri RA18) and a consortium with four endophytic strains (CE), two isolated from plant phyllosphere (Kushneria marisflavi EAod3 and Micrococcus aloeverae EAod10) and two from roots of A. macrostachyum (Bacillus vietnamensis EAR8 and Halomonas zincidurans EAR18). Each strain was cultivated separately in 50 mL of TSB (Tryptic Soy Broth) medium supplemented with 0.3 M NaCl at 28 ºC during 24 h in continuous shaking to reach 108 cells/mL. Then, cultures were centrifuged for 10 min at 8000 rpm and pellets were washed with 50 mL of 0.9% saline solution, centrifuging at the same conditions. Pellets were re-suspended in 5 mL of 0.9% saline solution and mixed in 50-mL Falcon tubes, forming the CR and CE consortia respectively. Finally, both consortia were mixed separately with 250 mL of tap water for plant inoculation. In October 2015, after four months of A. macrostachyum plant growth under glasshouse conditions described above, the perlite of A. macrostachyum plants was washed off and plants were transferred to individual 250 mL plastic pots (one plant per pot and each pot was placed in an individual shallow tray) filled with a homogenate of Odiel sediment. Pots were randomly assigned to three inoculation treatments (non- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 13 form roots to shoots, which could contribute to improve its accumulator potential. The endophytic consortium used in this experiment (EAod3, EAod10, EAR8 and EAR18) showed the presence of a variety of enzymatic activities, such as proteases, pectinases and amylases (Navarro-Torre et al., 2016b). Hence, these endophytic enzymatic activities may aid in penetration and colonization of the host plant, and also it is possible that endophytic bacteria could have certain metal uptake capacity through nonspecific binding of the cation to cell surface or by metabolic-dependent intracellular uptake mechanism (Gadd and Griffiths, 1978), which could contribute to improve the transport of metals inside the plants, since some of them presented also mobility (Navarro-Torre et al., 2016b). 3.2. Impact of sediment bioaugmentation on plant tolerance to metal stress Sometimes the potential of plants used as biological tools for metal remediation is often compromised through growth inhibition by increased toxicity of metals in its tissues, and especially when this accumulation occurs in its photosynthetic parts which are more sensitive to metal excess (Mateos-Naranjo et al., 2008; Perez-Romero et al., 2016). In this concern, Redondo-Gómez et al. (2010a) found that the increment of Cd concentration in the tissues of A. macrostachyum plants was followed by 25% of biomass reduction after a month in plants grown at 1.35 mM Cd under glasshouse conditions. By contrast, the increase of metal accumulation in inoculated plants observed in our work did not correlate with plant growth inhibition, despite that these concentrations were greater than toxicity threshold for plants in its tissues (KabataPendias and Pendias, 2000). In fact, the relative growth rate (RGR) of A. macrostachyum plants grown in polluted sediment from Odiel marshes tended to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 14 increase in similar degree with both bioaugmentation treatments (ANOVA, P < 0.05; Fig. 2A). Furthermore this positive trend was reflected in both shoot and root dry mass, which showed a mean increase of 59% and 113% for shoots and of 52% and 98% for roots in CE and CR inoculation treatments, respectively (Fig. 2B). Several factors could contribute to explain this response, such as the presence of PGP properties in the bacterial consortiums used in this study (Navarro-Torre et al., 2016b) and/or by bacterial reduction of metal phytotoxicity in the host plant (Haferburg and Kothe 2007). Our rhizosphere bacterial consortium was integrated by the strains RA1, RA8, RA15 and RA18, while the endophytic consortium was formed by the strains EAod3, EAod10, EAR8 and EAR18. Both bacterial consortia have a mix of bacteria isolated from the rizosphere and from the endosphere of A. macrostachym with the best PGP properties, such as indolacetic acid (IAA) and siderophores production, phosphate solubilisation, biofilm formation and nitrogen fixation in presence of heavy metals (Navarro-Torre et al., 2016b). In particular IAA is a key compound in cell division, elongation and plant tissues differentiation (Patten and Glick 2002) and auxins have a major role in root growth and lateral root formation (Ljung, 2013; Remans et al., 2008; Zhao, 2010), which could contribute to explain the better growth of A. macrostachyum plants with the bioaugmentation treatments. Our results are consistent with others where an enhanced in plant growth was recorded after inoculation with rizospheric and endophytic bacterial strains (Abou-Shanab et al. 2003; Mesa et al., 2015a; NavarroTorre et al. 2016a). Moving to bacterial reduction of metal phytotoxicity in plants, there are increasing reports indicating that metal resistant bacteria can diminish the metal stress of plants (Ahemad and Kibret, 2014; Mesa et al., 2015a,b; Rakjumar et al., 2012; Ullah et al., 2015; Yang et al., 2009). In this particular, the Micrococcus aloeverae EAod10, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 15 Halomonas zincidurans EAR18 and Pseudoalteromonas distinct RA8 strains used in this work have shown the ability to form biofilms (Navarro-Torre et al., 2016b), which could contribute to decrease phytotoxicity through to sequester of metals and to minimize the translocation of excessive metals into shoots. Furthermore, it has been described that toxic pollutants taken up by the plant may be degraded into plant by endophytes (Khan and Doty, 2011), although the mechanism are not well understood. On the top of that, other bacterial beneficial effects on plant metal tolerance have been related to osmotic adjustment, antioxidant system modulation and enhanced uptake of minerals (Compant et al., 2005; Khan et al., 2015; Rajkumar et al., 2009), as well as with the protection of the photosynthetic functions (Mesa et al., 2015a; Rincon et al., 2008). Regarding to bioaugmentation impact on photosynthetic apparatus of A. macrostachyum, our results revealed that net photosynthetic rate (AN) values were greater in plants grown in sediment bioaugmented with the bacterial consortiums, showing in both cases (CE and CR treatments) a increment c. 65% respect to noninoculated treatment after 120 days of treatment (one-way ANOVA, P < 0.01; Fig. 3A). Stomatal conductante (gs) and intercellular CO2 concentration (Ci) showed trends that were highly similar to that of AN, with increments around 125% and 170% for gs and 33% and 42% for Ci, respectively (one-way ANOVA, P < 0.01; Fig. 3B and C). In addition, maximum quantum efficiency of PSII photochemistry (Fv/Fm) and quantum efficiency of PSII (  PSII) values at midday did not vary with the bioaugmentation treatments respect to the control after 120 days of experiment, with values around 0.82 and 0.57 for Fv/Fm and  PSII, respectively (Table 1. Supplementary material). Obtained results suggested that the increase in growth can also be attributed to the improvement in the photosynthetic carbon assimilation of A.macrostachyum plants grown in bioaugmented sediments. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 16 Furthermore other physiological effect recorded in this study was the differential modulation of the antioxidant responses of A. macrotachyum plants grown in bioaugmentated sediments. Metal toxicity in plants is known to induce ROS generation and as consequence oxidative damage, which injury plant grown and development (Redondo-Gómez et al., 2011). However plants have the ability to module antioxidative enzymes activities, such as ascorbate peroxidase (APx), catalase (CAT), guaiacol peroxidise (GPx) and superoxide dismutase (SOD), which contribute to maintain the homeostasis again the high levels of ROS (Dazy et al., 2009; Qiu et al., 2008; RedondoGómez et al., 2011; Mesnoua et al., 2016). In our experiment the antioxidative enzymes activities in shoots and roots were significantly affected by sediment bioaugmentation treatments after 120 days of treatment (one-way ANOVA, P < 0.01; Fig. 4). Thus in shoots, APx activity was significantly enhanced in both inoculation treatments, and GPx and SOD activities increased only in plants grown in sediment inoculated with CE treatment, while CAT activity did not significantly vary with the bioaugmentation treatments (Fig. 4A-D). The increment of SOD and GPx activities in CE treatment, suggested that the inoculation with endophytic bacteria could contribute to reduce the oxidative stress derived of the greater amount of metal recorded in this plants. Thus, SOD can convert superoxide radicals to H2O2, while GPx can catalyze decomposition of H2O2 into H2O (Dazy et al., 2009; Qiu et al., 2008), which could contribute to maintain the photosynthetic function (Redondo-Gómez et al., 2011), despite the high level of metals recorded in A. macrostachyum plants. On the other hand, our results showed that in roots APx, CAT, GPx and SOD activities were significantly lower in plants grown in CE treatment, compared with the control and CR treatments (one-way ANOVA, P < 0.01; Fig. 4). This response suggests that endophytic could contributed in the modulation of metal stress at roots levels, as was previously indicated in Solanum 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 17 nigrum plants inoculated with the endophytic strain Serratia sp. RSC-14 (Khan et al., 2015). So it is possible, as we previously indicated, that in certain degree the metals inside of roots could be sequestered by endophytic bacteria avoiding its interaction with essential metabolic processes of A. macrostachyum plants; however this area deserves further research. Conclusion We can conclude that sediment bioaugmentation with appropriate PGPB, isolated from the microbiome of A. macrostachyum plants, contribute to improve its phytoremediation efficiency. Inoculation with both bacterial consortia improved metal uptake and accumulation in roots. This enhancement could be ascribed to bacterial effects on metal mobility and bioavailability. Also our results showed that CE consortium increased the capacity of A. macrostachyum to accumulate metals in its shoots, which could be linked with the penetration and colonization capacities, as well as with metal uptake of endophytic bacteria. Concerning bioaugmentation effects on plant tolerance, both bacterial consortia contributed to mitigate phytotoxicity level related with metal accumulation in plants tissues. This effect was evident in terms of growth and CO2 assimilation capacity, as indicated the greater RGR and photosynthetic assimilation values compared with its non-inoculated counterparts. Likewise, unaffected efficiency of PSII photochemistry apparatus might indicate that bioaugmentation treatments mitigated metal impact on the functionality and integrity of the photosynthetic apparatus of A. macrostachyum. Finally our results indicated that bioaugmentation effect on A. macrostachyum metal tolerance was also ascribed to the modulation of antioxidative enzyme machinery in its roots and shoots. Consequently, the inoculation with selected 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 18 rhizospheric and endospheric bacteria is a suitable method which would allow to enhance the efficiency of A. macrostachyum during restoration programs of polluted coastal sediments. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 19 Acknowledgements This work was funded by Junta de Andalucía (P11-RNM-7274MO project), INIA (RTA 2012-0006-C03-03 project) and Ministerio de Economía y Competitividad (CGL201675550-R project). S. Navarro-Torre thanks Junta de Andalucía and J.M. Barcia-Piedras thanks INIA for their personal financial supports. Authors are grateful to University of Seville Greenhouse General Services (CITIUS) for its collaboration. We also thank the useful comments of reviewers who helped to improve the final version of the manuscript. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 20 REFERENCES Abou-Shanab, R.A.I., Angle, J., Delorme, T., Chaney, R., Van Berkum, P., Moawad, H., Ghanem, K., Ghozlan, H., 2003. Rhizobacterial effects on nickel extraction from soil and uptake by Alyssum murale. New Phytol. 158, 219-224. doi:10.1046/j.1469-8137.2003.00721.x. Ahemad, M., Kibret, M., 2014. Mechanisms and applications of plant growth promoting rhizobacteria: Current perspective. J. King Saud Univ – Sci. 26, 1-20. doi:10.1016/j.jksus.2013.05.001. 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Safe 74, 2040-2049. doi: 10.1016/j.ecoenv.2011.06.019. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 29 Legends for figures Fig. 1. Effect of bioaugmentation treatments (control, without inoculation; CE, inoculation with a bacterial consortium from the endosphere and CR, inoculation with a bacterial consortium from the rhizosphere; details in material and methods) on total arsenic, As (A), chromium, Cr (B), copper, Cu (C), lead, Pb (D), nickel, Ni (E) and zinc (F) for shoots and roots of Arthrocnemum macrostachyum plants grown in natural sediments from Odiel marsh for 120 days. Values are means ± S.E. (n = 7). Different letters indicate means that are significantly different from each other (P < 0.05). Fig. 2. Effect of bioaugmentation treatments (control, without inoculation; CE, inoculation with a bacterial consortium from the endosphere and CR, inoculation with a bacterial consortium from the rhizosphere; details in material and methods) on relative growth rate, RGR (A) and total dry mass (B) in Arthrocnemum macrostachyum plants grown in natural sediments from Odiel marsh for 120 days. Values are means ± S.E. (n = 7). Different letters indicate means that are significantly different from each other (P < 0.05). Fig. 3. Effect of bioaugmentation treatments (control, without inoculation; CE, inoculation with a bacterial consortium from the endosphere and CR, inoculation with a bacterial consortium from the rhizosphere; details in material and methods) on net photosynthetic rate, AN (A), stomatal conductance, gs (B) and intercellular CO2 concentration, Ci (C) in random primary branches of Arthrocnemum macrostachyum plants grown in natural sediments from Odiel marsh for 120 days. Values are means ± S.E. (n = 7). Different letters indicate means that are significantly different from each other (P < 0.05). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 30 Fig. 4. Effect of bioaugmentation treatments (control, without inoculation; CE, inoculation with a bacterial consortium from the endosphere and CR, inoculation with a bacterial consortium from the rhizosphere; details in material and methods) on Ascorbato peroxidase, APx (A), catalase, CAT (B), guaiacol peroxidase, GPx (C) and superoxide dismutase, SOD (D) in shoots and roots of Arthrocnemum macrostachyum plants grown in natural sediments from Odiel marsh for 120 days. Values are means ± S.E. (n = 3). Different letters indicate means that are significantly different from each other (P < 0.05). 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