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

Elsevier Editorial System(tm) for Marine Pollution Bulletin Manuscript Draft Manuscript Number: MPB-D-16-00404R1 Title: Isolation of plant-growth promoting and metal resistant cultivable bacteria from Arthrocnemum macrostachyum in the Odiel marshes with potential use in phytoremediation Article Type: Research Paper Keywords: Arthrocnemum macrostachyum; contaminated marshes; PGPB; microbe-assisted phytoremediation; heavy metals Corresponding Author: Dr. IGNACIO DAVID RODRIGUEZ LLORENTE, Corresponding Author's Institution: UNIVERSIDAD DE SEVILLA First Author: Salvadora Navarro-Torre Order of Authors: Salvadora Navarro-Torre; Enrique Mateos-Naranjo, PhD; Miguel A Caviedes, PhD; Eloisa Pajuelo, PhD; IGNACIO DAVID RODRIGUEZ LLORENTE Abstract: Arthrocnemum macrostachyum is a halophyte naturally growing in southwest coasts of Spain able to tolerate and accumulate heavy metals. 48 bacteria (30 endophytes and18 from the rhizosphere) were isolated from A. macrostachyum growing in the Odiel River marshes, an ecosystem with high levels of contamination. All the isolates showed plant-growth promoting (PGP) properties and most of them were multi-resistant 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 to increase them in presence of concrete metals. Two bacterial consortia with the best performing endophytic or rizospheric strains were selected for further experiments. Bacterial inoculation accelerated A. macrostachyum seeds germination in 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. Arthrocnemum macrostachyum Endophytes Rhizospheric Cu2+ Zn2+ Pb2+ As(III) PGPB Pb2+ Cu2+ Zn2+ As(III) Accelerated germination Graphical Abstract Highlights - 48 bacteria isolated from A. macrostachyum with plant-growth promoting properties - Most of the isolates were multi-resistant to heavy metals - Several strains maintained their PGP properties in presence of concrete metals - Endophytic and rhizospheric bacterial consortia selected for further experiments - Inoculation accelerated seeds germination in absence and presence of heavy metals 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 Isolation of plant-growth promoting and metal resistant cultivable bacteria from Arthrocnemum macrostachyum in the Odiel marshes with potential use in phytoremediation S. Navarro-Torre1, E. Mateos-Naranjo2, M.A. Caviedes1, E. Pajuelo1, I.D. Rodríguez- Llorente1* 1Departamento de Microbiología, Facultad de Farmacia, Universidad de Sevilla, C/Profesor García González 2, 41012 Sevilla, Spain 2 Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, 41012 Sevilla, Spain *Correspondence: Dr. Ignacio David Rodríguez-Llorente, Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, C/Profesor García González 2, 41012 Sevilla, Spain. Phone +34 954556924. [email protected] *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 ABSTRACT Arthrocnemum macrostachyum is a halophyte naturally growing in southwest coasts of Spain able to tolerate and accumulate heavy metals. 48 bacteria (30 endophytes and18 from the rhizosphere) were isolated from A. macrostachyum growing in the Odiel River marshes, an ecosystem with high levels of contamination. All the isolates showed plantgrowth promoting (PGP) properties and most of them were multi-resistant 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 to increase them in presence of concrete metals. Two bacterial consortia with the best performing endophytic or rizospheric strains were selected for further experiments. Bacterial inoculation accelerated A. macrostachyum seeds germination in 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. Keywords: Arthrocnemum macrostachyum, contaminated marshes, PGPB, microbeassisted phytoremediation, heavy metals 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. INTRODUCTION Soil contamination with heavy metals is a global problem for environment and public health (Vangronsveld et al., 2009), mainly due to their low solubility and bioavailability and their capacity as carcinogenic and mutagenic agent (Davis et al., 2011). Industrial activities near to salt marshes have contributed to the introduction of a huge amount of contaminants, including heavy metals, in these environments. Metals discharged in the marshes are introduced into the food chain, affecting all the inhabitants of the ecosystem (Álvarez-Rogel et al., 2004). An example of this sort of contamination is the joint estuary of the Odiel and Tinto rivers in Huelva (SW, Spain), one of the most polluted areas in the world, due to the high concentration of metals (mainly As, Cu, Pb and Zn) found in their sediments (Nelson and Lamothe, 1993; Sáinz et al., 2004; Mesa et al., 2015a). For a hundred years, intense mining activity took place in this area, most recently, the beginning of the industrialization of the region in the late 60s has contributed to this contamination (Nelson and Lamothe, 1993; Davis et al., 2000). This estuary needs restorative intervention urgently. In order to regenerate this ecosystem, phytoremediation, the use of plants to remove or neutralize pollutants, could be an interesting biotechnological strategy, since it has several environmental advantages over traditional physicochemical approaches: it improves soil fertility, favors the ecological development of the ecosystem and maintains the esthetic of the landscapes (Vangronsveld et al., 2009). Phytostabilization, the phytoremediation technique aimed to immobilize metals into plant roots and rhizosphere, has been proposed as an appropriate tool to recover soils exposed to continuous mine drainage (Mendez and Maier, 2008; Yang et al., 2014). This technique involves the use of metal-resistant plants able to facilitate metal precipitation onto root surfaces and/or accumulation into them (Mendez and Maier, 2008). This is a long-term actuation that requires well-adapted and preferably autochthonous plants able to keep high growth ratios in presence of the contaminants (Mendez and Maier, 2008; Ali et al., 2013). Plant adaptation and growth in an ecosystem is intimately connected with complex soil ecological processes, highly influenced by microorganisms colonizing both the rizosphere and the phyllosphere. These microorganisms provide nutrients to the plants and can reduce harmful effects of contaminants in degraded soils (Newman and Reynolds, 2005; Yang et al., 2008). In this way, plant growth-promoting bacteria (PGPB) associated with plants used for 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 phytoremediation may contribute to metal plant tolerance, growth and uptake, thus accelerating the remediation process (Tak et al., 2013; Ullah et al., 2015). PGPB improve plant growth by different mechanisms, including among others atmospheric nitrogen fixation, production of auxins and other phytohormons, phosphorus solubilization, production of siderophores and ACC (1-aminocyclopropane- 1-carboxylic acid) deaminase synthesis (Glick, 2012). Through these mechanisms, PGPB increase plant health and robustness, thus facilitating plant adaptation in stressful conditions. Bacteria used to assist in the phytoremediation of heavy metals can contribute to this process direct or indirectly (Ullah et al., 2015). Direct mechanisms include enhanced metal solubilization and bioavailability, thus resulting in increased plant metal uptake (Glick, 2010). PGPB produce siderophores, chelating agents able to bind metals and enhance their bioavailability (Rajkumar et al., 2009; 2010). Low molecular weight organic acids and biosurfactants produced by PGPB have also the potential to improve metal mobilization (Mucha et al., 2005; Rajkumar et al., 2012). Other bacterial processes that modify plant metal availability are reduction/oxidization, methylation, precipitation and biosorption (Rajkumar et al., 2012). Concerning indirect processes, bacteria can enhance phytoremediation providing essential nutrients whose uptake is limited under metal stress (Ouzounidou et al., 2006). PGPB can fix atmospheric nitrogen, solubilize phosphorous and produce siderophores for iron chelation even under metal stress, thus facilitating the uptake of these nutrients (Ullah et al., 2015). Bacterial production of phytohormons under metal stress affects plant hormone levels and benefits plant response in such situations (Glick, 2012). Some PGPB are able to produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which hydrolizes ACC, the precursor of the plant hormone ethylene, whose overproduction under stressful conditions might inhibit plant growth (Glick, 2012). Arthrocnemum macrostachyum is a promising candidate to be used in phytoremediation, particularly phytostabilization, of metal contaminated estuaries. This halophytic shrub is distributed by the Mediterranean region and abundant in the Odiel marshes(Redondo-Gómez et al., 2010a) and has demonstrated ability for metal uptake and accumulation in mining wastes and polluted estuaries (Conesa and Schulin, 2010; Madejón et al., 2009). Nevertheless, nothing is known about the bacteria associated with this plant. Thus, the aims of this work were to: (1) isolate, identify and characterize bacteria associated with A. macrostachyum growing in a contaminated estuary, (2) characterize the resistance of these bacteria towards heavy metals, (3) study the 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 presence of plant growth-promoting properties in presence and absence of heavy metals and select the best performing isolates and (4) evaluate the effect of the selected bacteria on A. macrostachyum seeds germination under metal stress. 2. MATERIAL AND METHODS 2.1. Collection of samples and chemical analysis. Three plants of A. macrostachyum without a senescent appearance, and with similar size and age, were collected together with its rhizospheric soil (between 10-20 cm depth, which corresponds to the area of higher roots biomass) in February 2015 from a middlemarsh site in the Odiel marshes (Huelva, Spain) (37º 13’N - 6º 57’O). Each sample was placed in an individual plastic bag using a plastic shovel or gloves, for rhizosediments and plants samples respectively, according to Almeida et al. (2004), and immediately transported to the laboratory and stored at 4ºC until processing. Chemical analysis of soil samples and plant tissues was done exactly as described in Mesa et al., (2015b). Concentration of elements was measured by inductively coupled plasma (ICP-AES) spectroscopy (ARLFisons3410, USA). Conductivity was determined using a Crison-522 (Spain) conductivity meter and redox potential and pH with a Crison pH/mVp-506 (Spain) portable meter (Mesa et al., 2015b). Lastly, sand, silt and clay percentages were determined by means of the Bouyoucos hydrometer method (Bouyoucos, 1936). 2.2. Isolation of cultivable bacteria. Aerial parts and roots were separated and surface disinfected as follow: samples were treated with 70% (v/v) ethanol for 1 min by shaking, 15 min with 3% (v/v) sodium hypochlorite by slowly shaking, and then washed four times with sterile distilled water. Three samples of roots and three of aerial parts from each plant (each sample containing 3 fragments of plant material with 2-3 cm length) were crushed separately with a mortar in 1 ml 0.9% sterile saline solution and the generated mixture directly plated (100 µl per plate) into three Petri dishes with tryptic soy agar (TSA) medium supplemented with NaCl 0.3M and three plates supplemented with NaCl 0.6M. Plates were incubated at 28ºC for 72 h. To isolate rhizospheric bacteria, soil samples (near the root of the plant) were shaken with 0.9% sterile saline solution for 5 min and the suspension was plated into three TSA 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 NaCl 0.3M plates and three TSA NaCl 0.6M plates and incubated at 28ºC for 72 h. Bacteria were isolated according to the different colony morphology, Gram staining and motility. For motility, single colonies were transferred to 5 ml of liquid TSB (tryptic soybroth) and incubated at 28ºC for 30 minutes with gentle shaking. A drop of this culture was deposited over a slide, covered with a cover slip and observed using an Olympus CX41 microscope (100X objective). 2.3. DNA extraction, 16S rRNA amplification and Box-PCR fingerprinting analysis Genomic DNA was extracted using the i-genomic BYF DNA Extraction kit (Intron Biotechnology Ltd., Korea), according to the manufacturing’ instructions. 16S rRNA amplification was performed using 16F27 primer (5’- AGAGTTTGATCMTGGCTCAG-3’) and 16R1488 primer (5’- CGGTTACCTTGTTAGGACTTCACC-3’) and the following PCR conditions: an initial denaturation at 95ºC for 2 minutes, 30 cycles of denaturation at 95ºC for 45 seconds, an annealing at 58ºC for 45 seconds, an extension at 72ºC for 3 minutes and a final extension at 72ºC for 5 minutes. 16S rDNA sequences were compared with those located in the databases using the EzTaxon server (Chun et al., 2007). 16S rRNA sequences were deposited in the NCBI GenBank. Box-PCR fingerprinting analysis to assess genetic diversity of isolated bacteria was performed using BOX A1R primer following the conditions described in Mesa et al., 2015b. The gel was photographed and analysed with CLIQS 1D Pro Software (TotalLab, UK). Dendograms and similarities were determined by calculating the Pearson’s product moment correlation coefficient (Jobson, 1991). 2.4. Enzymatic activities. Pectinase and cellulase activities were examined according to the method described by Elbeltagy et al. (2000). Amylase activity was performed in starch agar plates incubated for 7 days at 28ºC and revealed with 10 ml of lugol. Lipase and protease activities were observed by the presence of halos around bacteria after incubation in Tween and casein agars, respectively, for 7 days at 28ºC (Prescott, 2002). Concerning DNAsa activity, bacteria were incubated 7 days at 28ºC in DNA agar plates. Plates were then revealed with HCl 1M. Halos in dark background were observed in bacteria with DNAsa activity. Chitinase activity was performed as described in Mesa et al. (2015a). Plates were supplemented with 0.3 M NaCl. 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 The PGP property most sensitive to the presence of metals was nitrogen fixation. In fact, only RA1 fixed nitrogen in presence of all metals and EAod10 in presence of As (Supplementary Table 5). Finally, the number of bacteria that solubilised phosphate increased in presence of Cd (Figure 6). Strains such as RA8, RA9, RA15 and endophytes EAR4 and EAR7, unable to solubilise phosphate in absence of Cd, showed this property in presence of this metal (Supplementary Table 4). 3.4. Effect of the selected consortia on seeds germination in presence and absence of heavy metals Among endophytes, Kushneria sp. EAod3, Micrococcus sp. EAod10, Bacillus sp. EAR8 and Halomonas sp. EAR18 were selected considering their characteristics. A second consortium composed by the rizospheric isolates Vibrio sp. RA1, Pseudoalteromonas sp. RA8 and RA15 and Staphylococcus sp. RA18 was also designed. A. macrostachyum seeds were inoculated separately with both selected consortia in the presence and absence of heavy metals. Although germination kinetics was monitored during 21 days, no changes in the number of germinated seeds were observed after one week (Figure 7). In absence of heavy metals, no significant differences in germination kinetics were observed between non-inoculated seeds and seeds inoculated with the consortium of endophytes (Figure 7A). Nevertheless, germination was accelerated in seeds inoculated with the rizospheric consortium, since it began between days 2 and 3, with more than 80% of germinated seeds in four days and close to 100% in five days, while non-inoculated seeds showed only 20% of germinated seeds in four days and needed one week to reach 100% of germination (Figure 7A). On the other hand, germination kinetics of seeds inoculated with either the rizospheric or the endophytic consortium was very similar in presence of small concentrations of heavy metals (Figure 7B). In that case, both groups of inoculants accelerated germination when compared with non-inoculated seeds, with 70% versus 20% of germinated seeds in four days, or 85% versus 55% in five days (Figure 7B). After one week, 97% of inoculated seeds germinated and close to 90% of non-inoculated seeds did. 4. DISCUSION 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 Arthrocnemum macrostachyum is a halophyte naturally growing in southwest coasts of Spain potentially useful for phytorremediation purposes, since it is able to tolerate and accumulate heavy metals (Conesa and Schulin, 2010; Madejón et al., 2009; Redondo- Gómez et al., 2010b). The ecological role of mycorrhizas in the rhizosphere of A. macrostachyum in polluted Mediterranean salt marshes has been described (Carrasco et al., 2006), but nothing is known about cultivable bacteria associated to this plant. In this work, 48 different bacterial strains were isolated from A. machrostachyum microbiome growing in a highly contaminated estuary. 40% of the rizospheric isolates were assigned to Bacillus genus. Identification of Bacillus strains in plant rizosphere is frequent in studies describing bacteria diversity of soils (Garbeva et al., 2003). Furthermore, the genus Bacillus is one of the most predominant in the rhizosphere of plants growing in metal contaminated soils (Abou-Shanab et al., 2007), including estuaries (Mesa et al., 2015b), due to its capacity to resist metals and NaCl (Nies and Silver, 2007). The colonization of endophytes into the plant takes place mainly through the secondary roots (Dong et al., 2003; Lamb et al., 1996), which explains why 39% of rhizospheric bacteria were also found as root endophytes. Concerning endophytes isolated from the phyllosphere, most of the strains belonged to halotolerant and halophilic genera, such as Halomonas and Kushneria. It is important to notice the low percentage of identity with previously described species of some isolates, suggesting a great unclassified diversity of cultivable bacteria associated to A. macrostachyum growing in this contaminated ecosystem. Although in some cases this low identity was probably due to partial sequencing with one single (forward) primer, the identity of some isolates was low despite the fact that the length of the sequence was almost complete (more than 1400 bp). Ongoing taxonomic studies of some of these strains will determine if they constitute new species. Most of the isolates were multi-resistant to heavy metals. A high percentage of strains were resistant towards elevated amounts of As, Cu, Pb and Zn, the most relevant contaminants in the Odiel marshes. The resistance towards Cu was lower among endophytes of phyllosphere than among the other two groups of isolates. Noteworthy, A. macrostachyum accumulates Cu in roots. On the contrary, although many strains grew in presence of high concentrations of Pb, the highest levels of resistance were observed in endophytes of phyllosphere. Previous reports indicated that A. macrostachyum accumulate high amounts of Pb (100-960 mg per kg) in the aerial part (Conesa and Schulin, 2010; Mendez and Maier, 2008; Alvarez-Rogel et al., 2004; 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 García et al., 2003) suggesting that endophytes could help the plant in the uptake of this metal. It has also been noted that this plant accumulates Cd in the roots (Redondo- Gómez et al., 2010b), so not surprisingly the strains with highest resistance towards this metal were found in the roots and the rizosphere of the plant. Concerning salt tolerance, most of strains were either halotolerant or halophilic, showing the highest levels of tolerance the endophytes isolated from the phyllosphere, where A. macrostachyum is able to accumulate high concentrations of NaCl (Redondo-Gómez et al., 2010a). The presence of enzymatic activities in the isolated bacteria is an important attribute for their use as inoculants. These enzymes are important, because they contribute to bacterial colonization (in the case of endophytes) and to degrade plant residues and acquire plant nutrients (Wang and Dai, 2010). Cellulase, pectinase and chitinase activities, the most relevant enzymatic activities, were present in both the rhizospheric and endophytic bacteria. Chitinase activity, found in root and rhizosphere isolates, could increment the resistance of the plant against fungus (Quecine et al., 2012; Trotel-Aziz et al., 2008). On the other hand, cellulase activity, mainly found in endophytes, could allow cell wall degradation and bacterial colonization. Motility, a characteristic present in most of endophytes, is responsible for the spreading of these bacteria through the plant (Compant et al., 2010). The roots of plants attract beneficial microorganisms, particularly under stress (Bais et al., 2006; Dzantor et al., 2007). At least one PGP property could be observed in each strain. Most of the isolates produced siderophores, which could influence heavy metals uptake by the plants (Gururani et al. 2012). In addition, it could also act as a biocontrol mechanism (Gaiero et al., 2013). Another property widely distributed among the isolated bacteria was IAA production, an essential hormone in root development (Lambrech et al., 2000). Phosphate is frequently found in the soil in the insoluble form, so bacteria able to solubilise phosphate make it available to the plant (Hameeda et al., 2008). Several strains showed this property. The capability to fix nitrogen, present in almost half of bacteria, may provide this element to the plant (Bhattacharyya and Jha, 2012). Finally, biofilm formation could help during bacterial attachment to the root and also to remove heavy metals, since extracellular polymeric substances involved in biofilm formation are able to immobilize and concentrate metals into their structure (Das et al., 2012). Few isolates were able to form biofilms. In general, the amount of bacteria with PGP properties decreased in presence of heavy metals. Interestingly, some strains were able to retain their PGP properties. Even though, in some cases, IAA 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 production increased in the presence of As and the number of isolates that solubilized phosphate was also higher in presence of Cd than in absence of the metal. The effect of Cu over the production of PGP properties was also studied by Mesa et al. (2015b) using strains isolated from the rizosphere of Spartina maritima in polluted marshes, reaching similar conclusions, that is, heavy metals clearly influence some PGP properties in an unpredictable way. These in vitro data could help to predict the behavior of the bacteria in polluted soils. With the prospect of future research, the best performing strains were selected among the isolated bacteria to form two bacterial consortia. The endophytes Kushneria sp. EAod3, Micrococcus sp. EAod10, Bacillus sp. EAR8 and Halomonas sp. EAR18 were chosen. EAod10 was the best strain, since it not only presented several PGP properties and maintained them in presence of heavy metals (nitrogen fixation, production of IAA and biofilm formation) but also was able to solubilize phosphate and produce siderophores with metals in the media. In addition, it showed the highest MTC values for most of metals. EAod3 produced siderophores and solubilized phosphate both in presence and in absence of heavy metals, complementing the PGP properties that were not present in EAod10 in absence of metals. This strain alsoproduced IAA in presence of some metals (including Cu) and had high MTC values for most of metals. Concerning root endophytes, EAR8 fixed nitrogen, solubilised phosphate in presence of Pb and produced the highest amount of siderophores, plus, it grew at high concentrations of heavy metals. Meanwhile, EAR18 was able to form biofilm and produce IAA in presence and absence of heavy metals. The second consortium contained the rhizospheric bacteria Vibrio sp. RA1, Pseudoalteromonas sp. RA8 and RA15 and Staphylococcus sp. RA18. These strains produced siderophores with and without heavy metals (except RA18, which only produced it in presence of heavy metals), RA1 being the best producer. They also produced IAA in presence of heavy metals, while RA15 and RA18 produced the hormone in absence of them. In addition, RA15 and RA18 solubilized phosphate with heavy metals in the culture media. RA1 provided the nitrogen fixation capacity to the consortium and RA8 the ability to form biofilms. Finally, these strains grew at high concentrations of heavy metals. Bacterial inoculation accelerated A. macrostachyum seed germination in absence and particularly in presence of heavy metals (As, Cu, Pb and Zn), reaching significantly higher germination rates in fewer days. Both consortia accelerated seeds germination in the presence of small concentrations of metals, while in the absence of metals, 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 acceleration was only relevant when seeds were inoculated with the rizospheric consortium. The beneficial effect of the rizospheric consortium on germination in absence of metals could be explained by the presence of enzymatic activities, such as pectinase and chitinase, which are important to break the cellular wall and could contribute to improve the hydration of the seeds. In presence of metals, IAA produced by both consortia may act as antagonist of ethylene produced by seeds in metal stressful conditions (Burdte al., 2000), thus contributing to improve germination. These results clearly suggest that selected bacteria could be useful as inoculants to ameliorate A. macrostachyum establishment in heavy metal contaminated marshes when using seeds to propagate the plant. Our global aim is to reduce and alleviate the effects of metal contamination in marsh soils using microbe-assisted phytoremediation techniques involving autochthonous plants. In a previous work, inoculation of the halophyte Spartina maritima in contaminated marsh soil with a rizospheric bacterial consortium increased plant growth and metal uptake in plant roots, thus improving the phytostabilization potential of the plant (Mesa et al., 2015c). On the other hand, endophytic cultivable bacteria improved S. maritima growth but not metal accumulation in plants that grew in the same soil (Mesa et al., 2015a). It looks clear that the behaviour of each consortium needs to be evaluated in planta, so the bacterial consortia described in this work will be used to inoculate A. macrostachyum plants in contaminated marsh soil in order to evaluate their ability to promote plant growth and/or metal uptake. The combined use of S. maritima, a fast growing grass that colonizes the lower marsh and A. macrostachyum, a shrub able to establish deep root networks into middle marsh soils, assisted by their respective selected PGPB, could be an appropriate strategy to implement in marsh restoration programmes aimed to recover contaminated coasts of southwest Spain. ACKNOWLEDGEMENT This work has been financed by Junta de Andalucía (P11-RNM-7274MO project) and INIA (RTA 2012-0006-C03-03 project). S. N-T also thanks Junta de Andalucía for personal financial support. English revision by Ms. Caroline Chiesa is acknowledged. 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 REFERENCES Abou-Shanab, R.A.I., van Berkum, P., Angle, J.S., 2007. 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A B Navarro-Torre et al., Figure 1 Figure 1 0 10 20 30 40 50 60 70 80 90 100 protease lipase amylase DNAse cellulase pectinase chitinase rhizosphere root aerial part Navarro-Torre et al., Figure 2 % bacteria Figure 2 0 10 20 30 40 50 60 70 80 90 100 As Cu Pb Zn Cd Co Hg Ni rhizosphere root aerial part % bacteria Navarro-Torre et al., Figure 3 0 20 40 60 80 100 120 0.6 1 1.5 2 2.5 3 % bacteria NaCl concentration (M) aerial part root rhizosphere A B Figure 3 As Zn Cu Pb 0 5 10 15 20 <2 mM 2-4 mM 4-6 mM 6-8 mM 8-10 mM >10 mM As Zn Cu Pb 0 5 10 15 <2 mM 2-4 mM 4-6 mM 6-8 mM 8-10 mM >10 mM As Zn Cu Pb 0 5 10 15 20 <2 mM 2-4 mM 4-6 mM 6-8 mM 8-10 mM >10 mM A B C Navarro-Torre et al., Figure 4 number of bacteria number of bacteria number of bacteria Figure 4 0 1 2 3 4 Number of PGP properites N fixation IAA biofilm phosphate siderophore 0 1 2 3 4 RA1 RA3 RA4 RA5 RA6 RA7 RA8 RA9 RA10 RA11 RA12 RA14 RA15 RA17 RA18 Number of PGP properties N fixation IAA biofilm phosphate siderophore B A Navarro-Torre et al., Figure 5 Figure 5 27% 73% 36% 64% 2% 98% 25% 75% 9% 91% 52% 48% 2% 98% 20% 80% 7% 93% 66% 34% 11% 89% 45% 55% 4% 96% 72% 28% 7% 93% 48% 52% 2% 98% 9% 91% 20% 80% 86% 14% 34% 66% 41% 59% +Cd +Zn +Pb +As +Cu Without metals Phosphate solubilization Siderophore IAA Nitrogen fixation 93% Navarro-Torre et al., Figure 6 Figure 6 Days 0 1 2 3 4 5 6 7 8 % germination 0 20 40 60 80 100 120 control +CR +CE A Days 0 1 2 3 4 5 6 7 8 % germination 0 20 40 60 80 100 120 B control +CR +CE Navarro-Torre et al., Figure 7 Figure 7 Supplementary Tables Click here to download Supplementary Data: Supplementary Tables.docx