Plant Biology ISSN 1435-8603

RESEARCH PAPER

Bacteria associated with yellow lupine grown on a metal-contaminated soil: in vitro screening and in vivo evaluation for their potential to enhance Cd phytoextraction N. Weyens1, M. Gielen1, B. Beckers1, J. Boulet1, D. van der Lelie2, S. Taghavi2, R. Carleer1 & J. Vangronsveld1 1 Centre for Environmental Sciences, Hasselt University, Diepenbeek, Belgium 2 Research Triangle Institute, Research Triangle Park, NC, USA

Keywords Cadmium; natural selection; phytoremediation; plant-associated bacteria; plant–bacteria interactions. Correspondence N. Weyens, Centre for Environmental Sciences, Hasselt University, Agoralaan Building D, B-3590 Diepenbeek, Belgium. E-mail: [email protected] Editor H. Papen Received: 22 April 2013; Accepted: 7 November 2013 doi:10.1111/plb.12141

ABSTRACT In order to stimulate selection for plant-associated bacteria with the potential to improve Cd phytoextraction, yellow lupine plants were grown on a metal-contaminated field soil. It was hypothesised that growing these plants on this contaminated soil, which is a source of bacteria possessing different traits to cope with Cd, could enhance colonisation of lupine with potential plant-associated bacteria that could then be inoculated in Cd-exposed plants to reduce Cd phytotoxicity and enhance Cd uptake. All cultivable bacteria from rhizosphere, root and stem were isolated and genotypically and phenotypically characterised. Many of the rhizobacteria and root endophytes produce siderophores, organic acids, indole-3-acetic acid (IAA) and aminocyclopropane-1-carboxylate (ACC) deaminase, as well as being resistant to Cd and Zn. Most of the stem endophytes could produce organic acids (73.8%) and IAA (74.3%), however, only a minor fraction (up to 0.7%) were Cd or Zn resistant or could produce siderophores or ACC deaminase. A siderophore- and ACC deaminase-producing, highly Cd-resistant Rhizobium sp. from the rhizosphere, a siderophore-, organic acid-, IAA- and ACC deaminase-producing highly Cd-resistant Pseudomonas sp. colonising the roots, a highly Cd- and Zn-resistant organic acid and IAA-producing Clavibacter sp. present in the stem, and a consortium composed of these three strains were inoculated into non-exposed and Cd-exposed yellow lupine plants. Although all selected strains possessed promising in vitro characteristics to improve Cd phytoextraction, inoculation of none of the strains (i) reduced Cd phytotoxicity nor (ii) strongly affected plant Cd uptake. This work highlights that in vitro characterisation of bacteria is not sufficient to predict the in vivo behaviour of bacteria in interaction with their host plants.

INTRODUCTION Soil and water contamination with metals and other harmful substances has become a growing concern in our industrialised world. This concern primarily relies on the potential adverse effects of contaminants on human health. An example of largescale metal contamination can be found in the NE of Belgium and the SE of the Netherlands (Campine region). In this region, an area of about 700 km² is contaminated with metals, mainly cadmium (Cd; 0.8–17.0 mg kg 1), zinc (Zn) and lead (Pb; both several hundreds of mg kg 1), due to the presence of several zinc smelters since about 1885 (Meers et al. 2007; Ruttens et al. 2011). This historical metal contamination is responsible for continued metal exposure of people and ecosystems in the area (Nawrot et al. 2006, 2008; Hogervorst et al. 2007). Furthermore, a considerable part of the contaminated Campine region is currently used for agriculture, although the Belgian Federal Agency for Food Safety (FAVV) has already confiscated several local vegetable harvests cultivated for the food industry due to Cd concentrations in crops exceeding

legal threshold values for human consumption (Ruttens et al. 2011). Consequently, Cd contamination and its remediation can be considered as a major issue for that region. Since it is virtually impossible to adopt conventional remediation methods, such as excavation and land filling, soil washing, solidification, vitrification or soil capping (Vangronsveld & Cunningham 1998; Vangronsveld et al. 2009), throughout the area, the Flemish government has expressed a major interest in potential applications of alternative remediation technologies, including phytoextraction. In these plant-based technologies, plant–bacteria interactions can play a key role (Weyens et al. 2009a; Mei & Flinn 2010). Plant-associated bacteria can promote plant growth and development: (a) directly by (a1) fixing nitrogen; (a2) supplying unavailable nutrients such as phosphorus and other mineral nutrients; (a3) producing plant growth regulators such as auxins, cytokinins and gibberellins; and (a4) suppressing stress ethylene production through 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity (Glick et al. 2007; Weyens et al. 2011). And (b) indirectly by preventing the growth or activity of plant

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Lupine bacteria and Cd phytoextraction

Weyens, Gielen, Beckers, Boulet, van der Lelie, Taghavi, Carleer & Vangronsveld

pathogens through (b1) competition for space and nutrients; (b2) antibiosis; (b3) production of hydrolytic enzymes; (b4) inhibition of pathogen-produced enzymes or toxins; and (b5) induction of plant defence mechanisms (Zhuang et al. 2007; van der Lelie et al. 2009; Taghavi et al. 2009; Weyens et al. 2009a,b; Yang et al. 2009; Francis et al. 2010). During phytoextraction of metal-contaminated soils, plants and their associated microorganisms have a role in mobilising, extracting and translocating metals to the aerial plant parts (Weyens et al. 2009a,b). The plant biomass containing the toxic metals should then be harvested, reduced in volume, e.g. by composting, low temperature ashing or other methods, stored, land-filled or ideally used for retrieval of the metals. Metal phytoextraction is a promising remediation technology, but for most elements (except for Ni; Chaney et al. 2007) the market value of the metals at this moment is too low to make phytoextraction economically profitable (Vangronsveld et al. 2009). The efficiency of phytoextraction is mainly determined by the amount of biomass produced, together with the metal uptake and translocation and the metal phytotoxicity (McGrath & Zhao 2003; Vassilev et al. 2004; Vangronsveld et al. 2009). Beside their direct and indirect positive effect on biomass production, plant-associated bacteria can also contribute to an increase in metal availability and uptake and a decrease in metal phytotoxicity (Valls & de Lorenzo 2002; Lebeau et al. 2008; Kidd et al. 2009; Mastretta et al. 2009; Rajkumar et al. 2009; Weyens et al. 2009a,b). Rhizosphere bacteria and root endophytes that produce natural metal chelators (such as siderophores) and/or organic acids can enhance metal availability and uptake, while endophytes that are equipped with a metal sequestration system can reduce metal phytotoxicity and increase metal translocation to the aerial parts. Bacteria possessing the above characteristics are frequently naturally abundant on metal-contaminated sites (Sessitsch & Puschenreiter 2008). Furthermore, a possible strategy to enhance phytoextraction efficiency could be enrichment through inoculation of these naturally abundant bacteria with the appropriate characteristics. Where these bacteria are not naturally colonising the plant, after isolation, the bacteria can also be equipped with metabolic pathways for the synthesis of natural chelators and metal sequestration systems (Top et al. 1992). Proof of this concept was provided in Lodewyckx et al. (2001), who inoculated Ni-exposed yellow lupine plants with a constructed Ni-resistant endophyte. After inoculation a 30% increase in Ni concentration in the roots was achieved. In this work, all cultivable bacteria were isolated from the rhizosphere, roots and stems of yellow lupine plants grown on a metal-contaminated soil originating from a former maize field in the Campine region. Bacteria were identified and tested for their production of siderophores, organic acids, indole-3acetic acid (IAA) and aminocyclopropane-1-carboxylate (ACC) deaminase, and for their Cd and Zn resistance. From all bacteria, one rhizosphere strain, one root endophyte and one stem endophyte presumed to possess potential to improve Cd phytoextraction were selected. To test if enrichment of these naturally abundant bacteria can effectively enhance phytoextraction, the selected strains were inoculated separately, as well as together as a consortium, into non-exposed and Cd-exposed yellow lupine plants. Phytoextraction efficiency was evaluated by investigating plant biomass, metal phytotoxicity and metal uptake. 2

MATERIAL AND METHODS Plant cultivation on metal-contaminated soil Since yellow lupine was the model plant chosen to prove the concepts of using endophytes to improve phytoremediation of toxic metals (Lodewyckx et al. 2001), organic contaminants (Barac et al. 2004) and mixed contaminations (Weyens et al. 2010a), it was also chosen as the host plant for this study. To obtain lupine-associated bacteria with the potential to improve Cd phytoextraction, yellow lupine (Lupinus luteus L.) plants were grown on metal-contaminated soil. The soil was collected at a former maize field in the Campine region and thoroughly homogenised. The total metal concentrations were: 6.5  0.8 mg Cd kg 1 dry weight (DW), 377  70 mg Zn kg 1 DW and 198  17 mg Pb kg 1 DW. Yellow lupine seeds were planted in 500-ml plastic pots filled with the contaminated soil and saturated with tapwater. Plants were grown for 4 weeks under greenhouse conditions before harvest. Isolation of cultivable lupine-associated rhizosphere, root and stem bacteria After 4 weeks of growth on metal-contaminated soil, all cultivable bacteria were isolated from the rhizosphere, roots and stems. Since the objective of this work was to obtain lupineassociated bacteria that are metal resistant, produce siderophores and/or organic acids that can be enriched through inoculation, only cultivable bacteria were characterised. The rhizosphere as well as roots and stems were sampled from three different lupine plants and were pooled for further analysis. To isolate rhizosphere bacteria, roots with their surrounding soil were sampled. Samples were stored in sterile Falcon tubes (50 ml) filled with 20 ml sterile 10 mM MgSO4. After vortexing the tubes, roots were removed, serial dilutions up to 10 5 were prepared in 10 mM MgSO4 solution, this was plated on 1/10 diluted 869 solid medium (Mergeay et al. 1985), and the plates were incubated for 7 days at 30 °C. Root and stem bacteria were isolated as described earlier (Barac et al. 2004), and serial dilutions up to 10 5 were plated on the same 1/10 diluted 869 solid medium and incubated for 7 days at 30 °C. After 7 days of incubation, colony-forming units (CFU) were counted and for each morphotype calculated per gram rhizosphere soil, fresh root sample or fresh stem sample. From all morphologically different bacteria, (if possible) five replicates were purified three times on 1/10 diluted 869 solid medium. Characterisation of lupine-associated rhizosphere, root and stem bacteria Genotypic characterisation The DNeasy Blood and Tissue kit (Qiagen, Venlo, the Netherlands) was used to extract total genomic DNA from five replicates of all morphologically different purified bacteria. The quantity and the quality of the extracted DNA were analysed using a Nanodrop ND-1000 Spectrophotometer (Isogen Life Science, De Meern, the Netherlands). To amplify 16SrDNA, aliquots (1 ll) of the extracted DNA were used directly. The universal 1392R (5′-ACGGGCGGTGTGTGTRC-3′) and the bacteria-specific 26F (5′-AGAGTTTGATCCTGGCTCAG-3′)

Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

Weyens, Gielen, Beckers, Boulet, van der Lelie, Taghavi, Carleer & Vangronsveld

primers were used for prokaryotic 16SrDNA amplification as described previously in Weyens et al. (2009c). The PCR products of the 16SrDNA amplification were directly used for Amplified rDNA Restriction Analysis (ARDRA), performed as described in Weyens et al. (2009c). The PCR purification kit (Qiagen) was used to purify the amplified 16SrDNA of strains with distinct ARDRA patterns. Sequencing was performed at the VIB Genetic Service Facility (University of Antwerp), and consensus sequences were obtained with the Staden Package. Sequence Match at the Ribosome Database Project II was used for species identification. Phenotypic characterisation The five replicates of all purified, morphologically different bacteria (below) were screened for their Cd and Zn resistance and capacity to produce siderophores (Sid), organic acids (OA), IAA and ACC-deaminase (ACC-D). Bacteria were plated on selective 284 medium (Weyens et al. 2009c) with addition of a carbon mix (CMIX; per liter medium: 0.52 g glucose, 0.35 g lactate, 0.66 g gluconate, 0.54 g fructose, 0.81 g succinate) and 0, 0.4 and 0.8 mM CdSO4 or 0, 0.6 and 1.0 mM ZnSO4 in order to test, respectively, Cd resistance and Zn resistance. Growth was evaluated after an incubation period of 7 days. For the detection of siderophore production, bacteria were first grown in liquid 869 medium. In a next step, 20 ll of this bacterial solution were transferred to microplate wells each containing 800 ll of culture medium. To detect siderophore production, the bacterial suspension was transferred to two microplate wells, one containing 800 ll of 284 medium (Weyens et al. 2009c) supplemented with CMIX, but without Fe(III) NH4 citrate, and one containing 800 ll of 284 medium (Weyens et al. 2009c) supplemented with CMIX and 4.8 ml l 1 Fe(III)NH4 citrate. After 5 days of incubation at 25 °C, siderophores were detected according to the method of Schwyn & Neilands (1987) by adding 100 ll of the blue chromium-azurol S (CAS) reagent to all microplate wells. After 4 h, orange wells were considered as positive. For detection of organic acids, the culture medium was a sucrose tryptone (ST) medium containing 20 g l 1 sucrose, 5 g l 1 tryptone and 10 ml l 1 trace elements (per liter: 20 mg NaMoO4, 200 mg H3BO4, 20 mg CuSO45H2O, 100 mg FeCl3, 20 mg MnCl24H2O, 280 mg ZnCl2). Organic acids were detected according to the method of Cunningham & Kuiack (1992) by adding 100 ll of the pH indicator alizarine red S. After 15 min, yellow wells were considered as positive. Bacteria were tested for their IAA production capacity using the Salkowski assay (adapted from Patten & Glick 2002). Bacteria were grown in darkness in 5 ml 869 medium (Mergeay et al. 1985) with 0.5 g l 1 tryptophan (precursor of IAA) for 4 days at 28 °C at 110 rpm. Bacterial suspensions were centrifuged (30 min at 3220 g) and 0.5 ml of the supernatant was mixed with 1 ml Salkowski’s reagent (50 ml 35% HClO4, 1 ml 0.5 M FeCl3). After 20 min a pink colour was considered positive for IAA production. The ACC deaminase activity was determined by monitoring the amount of a-ketobutyrate generated in the enzymatic hydrolysis of ACC (Saleh & Glick 2001). Washed bacterial pellets were resuspended in 1 ml SMN medium (Belimov et al. 2005) with 10 mM ACC as sole nitrogen source. Subsequently, bacterial cells were incubated for 3 days at 30 °C, centrifuged

Lupine bacteria and Cd phytoextraction

(30 min at 3220 g), resuspended in 0.5 ml Tris–HCl buffer (pH 8.5), disrupted by addition of 20 ll toluene and vigorously vortexing. An amount of 100 ll broken cell suspension was added to 10 ll 0.5 M ACC and 100 ll 0.1 M Tris–HCl buffer (pH 8.5), and incubated for 45 min at 30 °C. Possible ACC deaminase activity was stopped by adding 0.5 ml 0.56 N HCl. Then, a mixture of 400 ll 0.56 N HCl, 150 ll 0.2% 2.4-dinitrophenylhydrazine in 2 N HCl and 500 ll supernatant, obtained by centrifugation of the previous mixture, were incubated for 45 min at 30 °C. Bacterial strains that induced a colour change from yellow to brown after addition of 1 ml 2 N NaOH were considered as capable of producing ACC deaminase (Belimov et al. 2005). Inoculation, growth, Cd exposure and harvest of plants From all characterised lupine-associated bacteria, one rhizosphere strain, one root and one stem endophyte with potential to improve Cd phytoextraction were selected for inoculation. The bacteria were inoculated separately as well as together in a consortium. Fresh cultures of all strains were grown in 869 medium (Mergeay et al. 1985) at 30 °C until an approximate absorbance (A660) value of 1 was reached. The bacterial cells were collected by centrifugation (30 min at 3220 g), washed in 10 mM MgSO4 and resuspended in the original volume of 10 mM MgSO4 (final CFU ml 1: 109). Seeds were surface sterilised as described earlier in Weyens et al. (2010b) and planted in 400-ml plastic jars filled with sterilised perlite and saturated with half strength Hoagland’s nutrient solution with addition of CdSO4 (final concentration: 25 lM). Perlite was chosen as substrate during inoculation because preliminary experiments (N. Weyens, M. Gielen, J. Vangronsveld, unpublished data) revealed that the inoculation of yellow lupine was most successful using this substrate. The half strength Hoagland’s solution contains per litre distilled water, 50 ml macroelements, 500 ll microelements and 300 ll Fe-EDTA (macroelements (g l 1): 10.2 HNO3, 7.08 Ca(NO3)24H2O, 2.30 NH4H2PO4, 4.9 MgSO47H2O; microelements (g l 1): 2.86 H3BO3, 1.81 MnCl24H2O, 0.08 CuSO45H2O, 0.09 H2MoO4H2O, 0.22 ZnSO47H2O; Fe-EDTA (g l 1): 5.00 EDTA-Na, 7.60 FeSO47H2O). The bacterial inocula (in 10 mM MgSO4) were added to each jar at a final concentration of 108 CFU ml 1. Non-inoculated plants were used as controls and were supplied with the same volume of 10 mM MgSO4 as the inoculated plants, but without bacteria. Only after 2 weeks were plants transferred to 400-ml pots filled with sterile sand and saturated with half strength Hoagland’s nutrient solution containing CdSO4 (final concentration: 25 lM). Plants were watered with half strength Hoagland nutrient solution (without Cd) every other day until saturation level was reached again. In this experiment, sterilised Cd-enriched sand was chosen as a substrate, instead of the contaminated soil, because it was best to work under conditions that were as sterile as possible to be able to exclusively evaluate the effect of the inoculated strain. For each condition, at least 15 biologically independent replicates were tested. After 4 weeks of growth and exposure, plants were harvested. At harvest, root and shoot were separated and their fresh biomass determined to evaluate the effect of Cd on the growth of yellow lupine. Subsequently, from at least six plants from each condition, roots were washed twice for 10 min at 4 °C with 10 mM Pb(NO3)2 and deionised water to exchange surface-bound elements (Cuypers et al. 2000). The shoots were

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Lupine bacteria and Cd phytoextraction

Weyens, Gielen, Beckers, Boulet, van der Lelie, Taghavi, Carleer & Vangronsveld

washed twice with deionised water. All samples were oven-dried (48 h at 65 °C) and used for the determination of Cd uptake in the root and shoot. Re-isolation of inoculated bacteria from lupine plants To ensure the inoculation was successful, after 4 weeks of growth and exposure, the inoculated strains were re-isolated from three pooled rhizosphere–root–shoot samples for each condition. For a pooled rhizosphere–root–shoot sample, a rhizosphere sample was prepared (above), the root and shoot samples were surface sterilised and mixed with the rhizosphere sample, as described in Barac et al. (2004). Samples were plated on 284 medium (Weyens et al. 2009c) with the addition of CMIX and 0.4 mM CdSO4, which is selective for the inoculated strains. After 7 days of incubation at 30 °C, CFU were counted and calculated per gram fresh plant sample. To confirm that the isolated strains are identical to the inoculated strains, the BOX1 primer was used for BOX-PCR DNA fingerprinting, which was carried out as described earlier (Barac et al. 2004). Cadmium uptake in roots and shoots Oven-dried root and shoot samples were used to determine Cd in the roots and the shoots. Cd was extracted from the samples and measured with flame atomic absorption spectrometry as described previously (Weyens et al. 2010a). Statistical analysis All data sets were statistically analysed using one-way or twoway ANOVA and post-hoc multiple comparison testing (Tukey– Kramer). When necessary, log transformations were applied to approximate normality and/or homoscedasticity. The statistical analyses were performed in SAS version 9.1.3 (SAS Institute, Cary, NC, USA). Further, all results shown in this work were confirmed in an additional independent experiment. RESULTS Characterisation of lupine-associated rhizosphere, root and stem bacteria From all cultivable bacteria colonising the rhizosphere, the roots or the stem of yellow lupine growing on the metal-contaminated soil that were isolated, five replicates of all morphologically different strains (in total 141 strains) were purified and characterised genotypically and phenotypically. The non-cultivable strains were not investigated since the ultimate aim of this study was to obtain lupine-associated bacteria that can be enriched through inoculation to improve Cd phytoextraction. The total numbers of cultivable bacteria recovered from the rhizosphere (98.01 9 105 CFU g 1), the roots (82.65 9 105 CFU g 1) and the stems (79.69 9 105 CFU g 1) were very similar (Table 1). Only a slightly decreasing trend could be observed from the rhizosphere to the roots to the stems. Genotypic characterisation In the rhizosphere of the yellow lupine plants, high diversity was observed, without any strongly dominant taxon. The culti4

vable rhizosphere bacterial community was represented by Pseudomonas spp. (15.9%), Bacillus spp. (13.3%), Clavibacter spp. (12.3%), Rhizobium spp. (10.5%), Dyella spp. (9.5%), Burkholderia spp. (9.4%), Arthrobacter spp. (8.0%), Variovorax sp. (5.9%), Luteibacter spp. (4.5%), Enterobacter spp. (4.5%), Caulobacter sp. (4.1%), Agromyces sp. (1.1%) and Mycobacterium sp. (0.6%; Fig. 1c). The diversity of the cultivable endophytic bacteria was strikingly lower than that found in the rhizosphere. In roots, Pseudomonas spp. accounted for the majority (78.7%) of the cultivable strains; the remaining part consisted of Bacillus spp. (14.6%), Rhizobium spp. (4.8%) and Serratia spp. (1.9%; Fig. 1b). The stem endophytes were dominated by Clavibacter spp. (73.8%). Rhodococcus spp. (25.7%) and Arthrobacter spp. (0.5%), accounted for the remaining 26.2% of cultivable endophytic bacteria (Fig. 1a). Phenotypic characterisation All isolated, purified cultivable strains that were morphologically different were tested for their Cd and Zn resistance and their capacity to produce siderophores, organic acids, IAA and ACC deaminase. From the total number of isolated rhizosphere strains (98.0 9 105 CFU g 1 rhizosphere), 34.6% were able to produce siderophores, 18.0% organic acids, 49.2% IAA and 26.8% ACC deaminase. A considerable portion (12.1% and 22.0%, respectively) of the isolated rhizosphere community was resistant to the lowest Cd (0.4 mM) and Zn (0.6 mM) concentrations, while only 1.7% of the strains resisted the highest Cd concentration (0.8 mM) while no strains survived on the highest Zn concentration (1.0 mM). The majority (92.0%) of all isolated root endophytes (82.65 9 105 CFU g 1 root) were able to produce siderophores. Moreover, a significant part (49.3%) of the root-associated population was able to produce IAA. However, only 17.8% and 18.2%, respectively, tested positive for organic acid and ACC deaminase production. In contrast to the rhizosphere, a high percentage of the root endophytes were resistant to 0.4 mM Cd (43.1%) and 0.6 mM Zn (73.3%), and 13.7% resisted 0.8 mM Cd. Although, for the rhizosphere and root bacteria, a significant amount of siderophore- and ACC deaminase-producing strains was isolated, the opposite was found for the stem endophytes (only 0.5%). In the stem, the majority of strains (73.8% and 74.3%, respectively) produced organic acids and IAA. Furthermore, only 0.2% resisted 0.4 and 0.8 mM Cd and only 0.7% could grow on 0.6 mM Zn. From each compartment (rhizosphere, root, stem), the strain with the most promising characteristics for improving Cd phytoextraction was selected for inoculation of lupine plants (Table 1: *?). From the rhizosphere strains, a siderophore- and ACC deaminase-producing, highly Cd-resistant Rhizobium sp. was selected. A siderophore-, organic acid-, IAA- and ACC deaminase-producing highly Cd-resistant Pseudomonas sp. was chosen as a root endophyte. Furthermore, a highly Cd- and Znresistant organic acid- and IAA-producing Clavibacter sp. was selected as a stem endophyte. The selected strains were inoculated separately as well as combined as a consortium. Re-isolation of inoculated bacteria from lupine plants To verify whether the inoculations had been successful, after 4 weeks of growth, the rhizosphere, root and shoot bacteria

Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

Weyens, Gielen, Beckers, Boulet, van der Lelie, Taghavi, Carleer & Vangronsveld

Lupine bacteria and Cd phytoextraction

Table 1. Phenotypic characteristics of cultivable strains isolated from the rhizosphere, root and stem of yellow lupine growing on metal-contaminated soil. CFU g RHIZOSPHERE ? total: Pseudomonas sp. (1) (Acc No: HG423539) Pseudomonas sp. (2) (Acc No: HG423540) Pseudomonas sp. (3) (Acc No: HG423541) Bacillus sp. (1) (Acc No: HG423542) Bacillus sp. (2) (Acc No: HG423542) Bacillus sp. (3) (Acc No: HG423543) Clavibacter sp. (1) (Acc No: HG423544) Clavibacter sp. (2) (Acc No: HG423544) Clavibacter sp. (3) (Acc No: HG423544) Rhizobium sp. (1) (Acc No: HG423545) Rhizobium sp. (2) (Acc No: HG423545) Rhizobium sp. (3) (Acc No: HG423546) *? Rhizobium sp. (4) (Acc No: HG423546) Dyella sp. (1) (Acc No: HG423547) Dyella sp. (2) (Acc No: HG423547) Burkholderia sp. (1) (Acc No: HG423548) Burkholderia sp. (2) (Acc No: HG423548) Arthrobacter sp. (1) (Acc No: HG423549) Arthrobacter sp. (2) (Acc No: HG423549) Variovorax sp. (Acc No: HG423550) Luteibacter sp. (1) (Acc No: HG423551) Luteibacter sp. (2) (Acc No: HG423551) Enterobacter sp. (1) (Acc No: HG423552) Enterobacter sp. (2) (Acc No: HG423552) Caulobacter sp. (Acc No: HG423553) Agromyces sp. (Acc No: HG423554) Mycobacterium sp. (Acc No: HG423555) ROOT ? total: Pseudomonas sp. (1) (Acc No: HG423540) Pseudomonas sp. (2) (Acc No: HG423556) Pseudomonas sp. (3) (Acc No: HG423557) Pseudomonas sp. (4) (Acc No: HG423558) Pseudomonas sp. (5) (Acc No: HG423559) *? Pseudomonas sp. (6) (Acc No: HG423560) Bacillus sp. (1) (Acc No: HG423542) Bacillus sp. (2) (Acc No: HG423542) Bacillus sp. (3) (Acc No: HG423561) Bacillus sp. (4) (Acc No: HG423562) Bacillus sp. (5) (Acc No: HG423562) Bacillus sp. (6) (Acc No: HG423563) Bacillus sp. (7) (Acc No: HG423563) Rhizobium sp. (1) (Acc No: HG423546) Rhizobium sp. (2) (Acc No: HG423546) Rhizobium sp. (3) (Acc No: HG423546) Rhizobium sp. (4) (Acc No: HG423546) Rhizobium sp. (5) (Acc No: HG423546) Serratia sp. (1) (Acc No: HG423564) Serratia sp. (2) (Acc No: HG423565) Serratia sp. (3) (Acc No: HG423565) STEM ? total: Clavibacter sp. (1) (Acc No: HG423566) *? Clavibacter sp. (2) (Acc No: HG423544) Rhodococcus sp. (1) (Acc No: HG423567) Rhodococcus sp. (2) (Acc No: HG423567) Arthrobacter sp. (Acc No: HG423549)

1

5

98.01. 10 13.37 9 105 11.05 9 104 11.05 9 104 82.88 9 104 35.91 9 104 11.05 9 104 11.05 9 105 11.05 9 104 55.25 9 103 55.25 9 104 22.10 9 104 19.89 9 104 55.25 9 103 61.88 9 104 30.94 9 104 71.82 9 104 19.89 9 104 66.85 9 104 11.05 9 104 57.46 9 104 44.20 9 104 11.05 9 104 33.15 9 104 11.05 9 104 39.78 9 104 11.05 9 104 55.25 9 103 82.65 9 105 30.62 9 105 23.38 9 105 52.15 9 104 48.68 9 104 67.36 9 103 25.91 9 103 63.20 9 104 22.80 9 104 14.50 9 104 62.18 9 103 62.07 9 103 51.81 9 103 25.91 9 103 15.54 9 104 10.36 9 104 62.18 9 103 51.81 9 103 25.91 9 103 10.36 9 104 51.81 9 103 13.42 9 102 79.69 9 105 58.59 9 105 19.53 9 101 20.12 9 105 39.06 9 101 39.06 9 101

Sid

OA

IAA

ACC-D

34.6% + + + +

18.0%

49.2% + +

26.8%

+

+ + +

+

+ + +

+ + + + +

+

+ +

+ +

+

+

+ +

92.0 + + + + + + + + + + + + + + + + + + + 0.5

+

+ +

17.8

+ +

+

49.3 +

18.2

+ + + + + + +

+ + +

+ + + 73.8 + +

+ + +

+ +

+ +

+

+ +

+ +

+ +

+

74.3 + +

+ + 0.5

+ +

CMIX

++ ++ ++ ++ ++ ++ ++ ++ ++ + ++ ++ ++ + + + ++ ++ ++ ++ ++ ++ ++ ++ + ++ ++

0.4 mM Cd

0.8 mM Cd

0.6 mM Zn

1 mM Zn

12.1%

1.7%

22.0%

0.0%

+

+

++

++ + ++

+

++ ++ + ++ +

++ ++ ++

+

++ +

43.1 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++

+

++

13.7

73.3 ++ ++

+ + ++

+ + ++

++ ++

++

+

+

+ ++ ++ ++ ++ ++ ++ ++ ++ 0.2 ++

0.0

+ + ++ + ++ ++ ++ ++ 0.2 ++

+ ++ + 0.7

0.0

++

++

Acc. No.: Accession numbers: entries are available from the ENA browser at http://www.ebi.ac.uk/ena/data/view/HG423539-HG423567. Sid, siderophore production; OA, organic acid production; IAA, indole acetic acid production; ACC-D, ACC-deaminase production; CMIX, 284 medium with addition of carbon mix; 0.4 mM Cd, 0.8 mM Cd, 0.6 mM Zn and 1.0 mM Zn: 284 medium with addition of carbon mix and, respectively, 0.4 mM CdSO4, 0.8 mM CdSO4, 0.6 mM ZnSO4 and 1 mM ZnSO4. In the case of siderophore production (Sid), organic acid production (OA), indole acetic acid production (IAA) and ACC-deaminase production (ACC-D): : negative test; +: positive test. In the case of CMIX, 0.4 mM Cd, 0.8 mM Cd, 0.6 mM Zn and 1.0 mM Zn: +: growth (few colonies); ++: good growth (many colonies). The strains that were selected for inoculation are marked with *?. Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

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Lupine bacteria and Cd phytoextraction

Weyens, Gielen, Beckers, Boulet, van der Lelie, Taghavi, Carleer & Vangronsveld

0.5

a

b

25.7

4.8 1.9

14.6

73.8

Clavibacter sp.

78.7

Rhodococcus sp.

c

1.1 4.5

Arthrobacter sp.

Pseudomonas sp.

0.6 4.1

Bacillus sp.

Rhizobium sp.

Serratia sp.

Pseudomonas sp. Bacillus sp.

15.9

Clavibacter sp.

4.5

Rhizobium sp.

5.9 13.2

7.9

Dyella sp. Burkholderia sp. Arthrobacter sp.

9.4

13.0

Variovorax sp. Luteibacter sp.

9.5

10.5

Enterobacter sp. Caulobacter sp.

were isolated from three rhizosphere–root–shoot samples for each condition (for inoculated as well as non-inoculated plants) and plated on media selective for the inoculated strains (Fig. 2). Furthermore, BOX-PCR fingerprints confirmed that the strains isolated on the selective medium were identical to the inoculated strains (data not shown). As expected, the inoculated strains could be re-isolated from the inoculated, non-exposed as well as Cd-exposed plants. Besides, the higher number of CFU re-isolated from the inoculated plants (order of magnitude 106; see Fig. 2), compared with the number of CFU from the Cd-exposed plants from which the strains originated (order of magnitude 103 for rhizosphere and root strains, and 101 for the stem strain; see Table 1), suggests strong enrichment of the inoculated strains. Moreover, the number of re-isolated strains was, for all inoculation conditions, significantly higher for Cd-exposed plants in comparison with non-exposed plants (Fig. 2).

Fig. 1. Diversity of cultivable bacteria in the stems (a), roots (b) and rhizosphere (c) of yellow lupine growing on a metal-contaminated soil. Numbers indicate the relative abundance, expressed as a percentage of the total number of isolates present in the stem (a), root (b) and rhizosphere (c).

None of the inoculated strains or other naturally abundant bacterial strains could be re-isolated from the non-inoculated yellow lupine plants. This was expected, considering the selectivity of the medium and the nearly sterile growth conditions used for the inoculation experiment. Effect of Cd on growth of yellow lupine When both non-inoculated and inoculated yellow lupine plants were exposed to Cd, a significant (P < 0.01) phytotoxic effect was observed at the shoot level (Fig. 3a). Although the same phytotoxic trend was visible at the root level, the reduction in root biomass caused by Cd exposure was only significant for lupine plants inoculated with the rhizosphere strain (Fig. 3b). Probably the plant growth-promoting effect achieved after inoculation of non-exposed plants with the rhizosphere strain contributed to the phytotoxic effect of Cd at the root level, since this growth-promoting effect of the rhizosphere strain was no longer present upon Cd exposure (Fig. 3b). Cadmium uptake in roots and shoot None of the inoculations increased the Cd concentration in roots (Fig. 4b). However, after inoculation with the root endophyte, an increased Cd concentration in the shoots of yellow lupine was observed (Fig. 4a). Inoculation of the rhizosphere strain, the stem endophyte and even the consortium of all three bacteria did not affect the Cd concentration in the shoot.

Fig. 2. Number of CFU isolated from lupine plants (rhizosphere + roots + shoot). Lupine plants were inoculated with a rhizosphere strain (Rhiz Bact), a root endophyte (Root Bact), a stem endophyte (Stem Bact) or their consortium (Rhiz + Root + Stem Bact). After 4 weeks of growth, bacteria were isolated on selective medium and the number of bacteria determined and expressed per gram fresh weight. Values are mean  SE of three biologically independent replicates (significance: **P < 0.005; ****P < 0.0001 in comparison with non-exposed lupine plants inoculated with the same bacteria).

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DISCUSSION Natural selection on contaminated sites for strains that are tolerant to and/or able to detoxify the contaminants has been frequently investigated, and was first mentioned by van der Lelie (1998) and Siciliano et al. (2001). Since (facultative) endophytic bacteria mainly colonise their host plant from the rhizosphere through the roots, the bacteria present in this

Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

Weyens, Gielen, Beckers, Boulet, van der Lelie, Taghavi, Carleer & Vangronsveld

a

b

Fig. 3. Shoot (a) and root (b) biomass (fresh weight) of non-exposed and 25 lM CdSO4 exposed lupine plants non-inoculated or inoculated with the rhizosphere strain (Rhiz Bact), root endophyte (Root Bact), stem endophyte (Stem Bact) or their consortium (Rhiz + Root + Stem Bact). Values are mean  SE of at least ten biologically independent replicates (significance: ***P < 0.001 in comparison with related non-inoculated plants).

contaminated soil containing different traits to cope with metal contamination may colonise the rhizosphere and the interior of plants grown on this soil. In order to obtain yellow lupineassociated bacteria with the potential to improve Cd phytoextraction, plants were cultivated on a metal-contaminated soil originating from a field site. All cultivable bacteria were isolated, identified and characterised. Remarkably, the rhizospheric cultivable bacterial diversity was much higher than the endophytic cultivable bacterial diversity (Fig. 1). Furthermore, the total number of isolated strains was very similar in the rhizosphere, the roots and the stems (Table 1). Both observations are somewhat inconsistent with the frequently observed slightly higher diversity in the rhizosphere and total number of bacteria, which decreases from the rhizosphere to the roots to the stems by an order of magnitude (Lamb et al. 1996; Quadt-Hallmann & Kloepper 1996; Porteous-Moore et al. 2006; Weyens et al. 2009c). From the isolated root-colonising bacteria, a majority of 98.1%, including Pseudomonas spp., Bacillus spp. and Rhizobium spp., was also present in the rhizosphere (Fig. 1). Therefore these root endophytes can be expected to be facultative endophytes, while the remaining 1.9% Serratia spp. probably would be obligate endophytes. The Clavibacter spp. (73.8%) and Arthrobacter spp. (0.5%) isolated from the stem were also present in the rhizosphere, while the Rhodococcus spp. (25.7%) exclusively colonised the stem (Fig. 1). However, as none of the species colonising the stem were present in the root, the stem endophytes are expected to be obligate endophytes or to enter the plant from the phyllosphere. Previous studies revealed that, beside the root zone, the phyllosphere offers the most obvious route of entry for many endophytes. This entry may occur through natural openings in the plant surface, such as lenticels and leaf stomata, hydathodes and nectarthodes (Sharrock et al. 1991; Kluepfel 1993; Beattie & Lindow 1999). To assess whether growing the lupine plants on metal-contaminated soil could deliver plant-associated bacterial pheno-

Lupine bacteria and Cd phytoextraction

a

b

Fig. 4. Total Cd amount (mg) in the shoots (a) and roots (b) of lupine plants exposed to 25 lM CdSO4 that were non-inoculated or inoculated with the rhizosphere strain (Rhiz Bact), root endophyte (Root Bact), stem endophyte (Stem Bact) or their consortium (Rhiz + Root + Stem Bact). Values are mean  SE of at least six biologically independent replicates (significance: *P < 0.05 in comparison with non-inoculated plants).

types with the potential to improve Cd phytoextraction, all morphologically different isolated bacteria were tested for their capacity to produce siderophores, organic acids, IAA and ACC deaminase, and for their Cd and Zn resistance (Table 1). It was expected that bacteria producing (i) siderophores, organic acids, IAA and ACC deaminase would promote plant growth and/or increase the concentration of plant-available Cd resulting in a higher Cd uptake (Saravanan et al. 2007; Braud et al. 2009), and that (ii) Cd-resistant bacteria would sequester Cd in order to detoxify it for themselves but, at the same time, also for their host plant (Lodewyckx et al. 2001). High numbers of bacteria with the above-described characteristics, belonging to different taxa, were present in all plant compartments, and even strongly dominated the community of cultivable strains in the roots (Table 1). This is consistent with previous observations where metal-resistant plant growth-promoting bacteria were found on metal-contaminated sites (van der Lelie 1998; Sessitsch & Puschenreiter 2008). It is remarkable that only a minority of the stem endophytes showed any metal resistance. However, this might be due to the fact that the exposure time was too short to (i) allow the metal-resistant bacteria originating from the contaminated soil to colonise the stem, or (ii) to directly induce natural selection in the stem population. In the next experiments, a rhizosphere strain, a stem and a root endophyte were selected for inoculation (separately and together in a consortium) of Cd-exposed yellow lupine plants (Table 1: *?). Since all inoculated strains could be re-isolated from the non-exposed as well as from the Cd-exposed lupine plants, the inoculation was considered successful (Fig. 2). Further, the amount of re-isolated bacteria was significantly higher in the case of lupine plants exposed to Cd. The same increasing trend of re-isolated bacteria after exposure was observed in previous work (Weyens et al. 2010b).

Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

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Lupine bacteria and Cd phytoextraction

Weyens, Gielen, Beckers, Boulet, van der Lelie, Taghavi, Carleer & Vangronsveld

Although, based on the phenotypic characterisation, the inoculated strains were expected to decrease metal phytotoxicity, none of the strains or their consortium did this (Fig. 3). Only after inoculation of the root endophyte was an increased metal concentration in the shoot achieved (Fig. 4). These rather unexpected results clearly illustrate that characterising phenotypes of strains at a lab scale using the tests adopted in this work is not sufficient to predict the behaviour of strains, or consortia, in their in vivo interaction with their host plant. Although, based on the in vitro tests the three most interesting strains were selected, there might be other, more interesting strains within the isolated bacterial collection. Beside siderophore, organic acid, IAA and ACC deaminase production tests, additional metal mobilisation experiments including soil– bacteria interactions, as described in Kuffner et al. (2010), could contribute to predict the potential of bacteria to mobilise metals in a soil–plant–bacteria interaction. However, the potential of in vitro tests to predict the in vivo plant growth-promoting and metal-mobilising capacity of plant-associated bacteria should not be overestimated. Before applying promising strains in the field, their expected positive effect on phytoextraction should be confirmed in inoculation experiments. In an initial phase, inoculations can be performed under sterile conditions (as was the case in this work, using sterile perlite and sand as substrates) to allow one to exclusively determine the effect of the inoculated strain. In the cases where the potential to improve phytoextraction efficiency is confirmed in this first type of inoculation experiments, in a next step towards field application, the sterile substrate should be replaced by a non-sterile soil substrate. It is likely that the response of inoculants in perlite might be completely different to that in soil conditions. Only when the selected bacterial strains still confirm their capacity to improve phytoextraction, this despite the occurrence of competition with the bacteria present in the soil, can the strain be classified as a phytoextraction improver. Beside these often underestimated conditions to accurately and thoroughly test the potential of plant-associated bacteria REFERENCES Barac T., Taghavi S., Borremans B., Provoost A., Oeyen L., Colpaert J.V., Vangronsveld J., van der Lelie D. (2004) Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants. Nature Biotechnology, 22, 583–588. Beattie G.A., Lindow S.E. (1999) Bacterial colonization of leaves: a spectrum of strategies. Phytopathology, 89, 353–359. Belimov A.A., Hontzeas N., Safronova V.I., Demchinskaya S.V., Piluzza G., Bullitta S., Glick B.R. (2005) Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard (Brassica juncea L Czern.). Soil Biology & Biochemistry, 37, 241–250. Braud A., Jezequel K., Bazot S., Lebeau T. (2009) Enhanced phytoextraction of an agricultural Cr-, Hg-, and Pb-contaminated soil by bioaugmentation with siderophore-producing bacteria. Chemosphere, 74, 280–286. Chaney R.L.J., Angle S., Broadhurst C.L., Peters C.A., Tappero R.V., Sparks D.L. (2007) Improved understanding of hyperaccumulation yields commercial

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to improve phytoextraction efficiency, some other aspects still need to be considered before bacteria-enhanced phytoextraction can be applied to remediate large-scale contaminated field sites. As in previous studies (Lodewyckx et al. 2001; Barac et al. 2004; Weyens et al. 2010a,b), in this work yellow lupine was used as a model plant. However, for field application, the use of high biomass-producing (energy or feedstock) crops with moderate to high metal accumulation capacities such as willow and poplar is preferable (Ruttens et al. 2011). As phytoremediation projects often require quite long periods of time (e.g. Koopmans et al. 2007; Maxted et al. 2007; Vangronsveld et al. 2009), the production of energy crops on contaminated soil could provide an attractive economic alternative, since it combines sustainable use and improvement of contaminated sites (Van Ginneken et al. 2007; Weyens et al. 2009b; Thewys et al. 2010a,b). In conclusion, beside the environmental risks associated with contaminated sites, based on natural selection, these sites can also provide an excellent source of bacteria with potential to enhance their phytoremediation. In order to achieve accurate selection of the bacterial strains with the highest potential, screening tests at laboratory scale should be further optimised and completed. Inoculation of bacteria equipped with the appropriate characteristics is a very promising strategy to improve phytoremediation efficiency, but still more research, both at laboratory and pilot scale, should be performed before moving bacteria-enhanced phytoremediation towards full-scale field application. ACKNOWLEDGEMENTS This work has been financially supported by the UHasselt Methusalem project 08M03VGRJ and the European Commission under the Seventh Framework Programme for Research (FP7-KBBE-266124, GREENLAND). N.W. was funded by the Fund for Scientific Research Flanders (FWO-Vlaanderen) and M.G. by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).

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Lupine bacteria and Cd phytoextraction

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Bacteria associated with yellow lupine grown on a metal-contaminated soil: in vitro screening and in vivo evaluation for their potential to enhance Cd phytoextraction.

In order to stimulate selection for plant-associated bacteria with the potential to improve Cd phytoextraction, yellow lupine plants were grown on a m...
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