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Front Microbiol. 2017; 8: 1385.
Published online 2017 July 25. doi:  10.3389/fmicb.2017.01385
PMCID: PMC5524726

Bacteria Associated to Plants Naturally Selected in a Historical PCB Polluted Soil Show Potential to Sustain Natural Attenuation


The exploitation of the association between plants and microorganisms is a promising approach able to boost natural attenuation processes for soil clean-up in vast polluted areas characterized by mixed chemical contamination. We aimed to explore the selection of root-associated bacterial communities driven by different plant species spontaneously established in abandoned agricultural soils within a historical polluted site in north Italy. The site is highly contaminated by chlorinated persistent organic pollutants, mainly constituted by polychlorobiphenyls (PCBs), together with heavy metals and metalloids, in variable concentrations and uneven distribution. The overall structure of the non-vegetated and root-associated soil fractions bacterial communities was described by high-throughput sequencing of the 16S rRNA gene, and a collection of 165 rhizobacterial isolates able to use biphenyl as unique carbon source was assayed for plant growth promotion (PGP) traits and bioremediation potential. The results showed that the recruitment of specific bacterial communities in the root-associated soil fractions was driven by both soil fractions and plant species, explaining 21 and 18% of the total bacterial microbiome variation, respectively. PCR-based detection in the soil metagenome of bacterial bphA gene, encoding for the biphenyl dioxygenase α subunit, indicated that the soil in the site possesses metabolic traits linked to PCB degradation. Biphenyl-utilizing bacteria isolated from the rhizosphere of the three different plant species showed low phylogenetic diversity and well represented functional traits, in terms of PGP and bioremediation potential. On average, 72% of the strains harbored the bphA gene and/or displayed catechol 2,3-dioxygenase activity, involved in aromatic ring cleavage. PGP traits, including 1-aminocyclopropane-1-carboxylic acid deaminase activity potentially associated to plant stress tolerance induction, were widely distributed among the isolates according to in vitro assays. PGP tested in vivo on tomato plants using eleven selected bacterial isolates, confirmed the promotion and protection potential of the rhizosphere bacteria. Different spontaneous plant species naturally selected in a historical chronically polluted site showed to determine the enrichment of peculiar bacterial communities in the soil fractions associated to the roots. All the rhizosphere communities, nevertheless, hosted bacteria with degradation/detoxification and PGP potential, putatively sustaining the natural attenuation process.

Keywords: soil pollution, natural attenuation, bacterial diversity, rhizosphere, polychlorobiphenyls, plant growth promotion, bphA gene


Polychlorinated biphenyls (PCBs) are highly stable, hydrophobic and persistent organic pollutants. Due to their lipophilic nature, they bioaccumulate and biomagnify through the food web and can have a broad range of toxic effects on humans (Turrio-Baldassarri et al., 2009; Letcher et al., 2010; Quinete et al., 2014; IARC, 2015). Physico-chemical remediation techniques of PCB polluted soils are not sustainable environmentally and economically for extended contaminations and the properties of these molecules make them recalcitrant to biodegradation, impairing the efficacy of bioremediation technologies. However, PCB pollution is a worldwide problem associated to their past production and utilization in industrial facilities (Passatore et al., 2014). PCBs strongly bind to the soil organic matter, resulting into low bioavailability both for plant uptake and microorganism metabolism (Sinkkonen and Paasivirta, 2000). Nonetheless, several studies showed that PCB-polluted soils host bacterial communities endowed with aerobic degradation abilities and harboring the biphenyl dioxygenase (bph) operon, the most studied and widespread pathway sustaining the aerobic biodegradation ability (Abraham et al., 2002; Leigh et al., 2006; Leigh et al., 2007; Kurzawova et al., 2012). Plants establish mutual beneficial interactions with selected bacterial populations, which promote nutrient uptake and enhance stress tolerance against pollutants, such as PCBs, in turn decreasing their phytotoxicity (Ma et al., 2011; Abhilash et al., 2016). Root exudates, besides creating a selective ecological niche for specific bacteria, may contain molecules that can induce the expression of genes involved in the PCB degradation pathways, such as the bph operon, in turn stimulating PCB degradation in the rhizosphere (Toussaint et al., 2012; Pham et al., 2015). Rhizoremediation, the exploitation of such positive interactions between plants and bacteria for reclamation of polluted soils, represents a sustainable alternative for clean-up of recalcitrant organic pollutants in extended areas (Vergani et al., 2017). Soil natural attenuation potential therefore relies on the presence, diversity and activity of the resident microbiota, driven by the selecting activity of autochthonous plants.

In this work, we studied the phylogenetic and functional diversity of the microbiota associated to the root-soil fractions of the three spontaneous plant species Medicago sativa, Centaurea nigrescens, and Dactylis glomerata, naturally established in a former agricultural field located within the heavily polluted SIN (National Priority Sites) site Caffaro in north Italy. This is a large area polluted by the activity of the former Caffaro chemical factory and includes more than 100 ha of former agricultural areas (Di Guardo et al., 2017). It presents a mixed and uneven contamination of chlorinated persistent organic pollutants, mainly PCBs, heavy metals and metalloids and several other contaminants including dioxins, furans, tetra-chloromethane (Turrio-Baldassarri et al., 2007; Di Guardo et al., 2017). PCBs were produced by Caffaro for 46 years and banned in Italy in 1984. To avoid pollutant exposure to humans, economic activities, including agriculture, have been banned in 2002 in the SIN Caffaro area. In the last 12 years only natural attenuation processes occurred, with the establishment of spontaneous plants resisting phytotoxic effects. Remediation strategies for such a large and complex site represent a challenge raising the interest for the soil self-depuration potential, which could be exploited and boosted by phyto-rhizoremediation approaches. A basic question is whether PCB degradation potential is present in the bacterial communities enriched by plants able to grow in the polluted soil, mediated by the selection force determined by the rhizosphere effect. We compared the bacterial communities in the root-associated soil fractions with that of the non-vegetated soil, through 16S rRNA gene phylogenomics and the amplification of the bphA gene, encoding for the α sub-unit of the biphenyl dioxygenase enzyme, a proxy for the PCB degradation ability (Iwai et al., 2010; Sylvestre, 2013; Vergani et al., 2017). By looking to the culturable bacterial fraction, we assessed whether bacteria represent a potentially exploitable resource for rhizoremediation purposes, by assessing their PGP capacity and PCB biodegradation potential.

Materials and Methods

Site Description, Plants and Soil Sampling

Non-vegetated and root-associated soil fractions were collected in the SIN Caffaro, a site located in Northern Italy (Brescia municipality), within the sampling area A, a former agricultural grassland field that was previously chemically characterized and contained PCBs and other chlorinated pollutants in concentrations often exceeding the safety limits (Di Guardo et al., 2017). The root system of three spontaneous plant species was collected from triplicate specimens for each plant species. Plants were identified basing on their morphological traits as M. sativa L. (MS), C. nigrescens Willd. (CN), and D. glomerata L. (DG). These plant species were among the most widely widespread in the meadows present in the agricultural areas of the SIN Caffaro, according to a recent survey (Armiraglio et al., 2009). Non-vegetated bulk soil (B) was collected in triplicate in the same sampling station. Samples were transferred to the laboratories and the different soil fractions (as defined below) were separated within 4 h from collection as described in Marasco et al. (2012). Soil fractions were stored at -20°C for molecular analyses and at 4°C for bacterial isolation.

DNA Extraction and Analyses of the Bacterial Community Structure

Total DNA extraction was performed from 0.25 g of each of the three non-vegetated bulk soil (B), root surrounding soil (S) and rhizosphere soil (R) replicates, using the PowerSoil DNA kit (MoBio) according to the manufacturer’s protocol. Illumina tag screening of the V3–V4 hypervariable regions of the 16S rRNA gene was applied on DNA of R, S, and B triplicate samples using the primers 341F and 785R (Klindworth et al., 2013). The obtained sequences were analyzed using a combination of the UPARSE v8 (Edgar, 2013) and the QIIME v1.8 (Caporaso et al., 2010b) softwares. Briefly, raw forward and reverse reads for each sample were assembled into paired-end reads considering a minimum overlapping of 50 nucleotides and a maximum of one mismatch within the region using the fastq-join algorithm1. The paired reads were then quality filtered, the primer sequences were removed and the individual sample files were merged in a single fasta file. This file was imported in UPARSE where operational taxonomic units (OTUs) of 97% sequence similarity were formed and chimeras were removed using both de novo and reference-based detection. For reference chimera detection, the “Gold” database containing the chimera-checked reference database in the Broad Microbiome Utilities2 was used. Taxonomy was assigned to the representative sequences of the OTUs in QIIME using UClust (Edgar, 2010) and searching against the latest version of the Greengenes database (McDonald et al., 2012). After the removal of reads affiliated to chloroplast, Archaea and unassigned sequences, a total of 1319653 high-quality merged paired-end reads with an average length of 450 bp were obtained. All the samples analyzed presented Good’s coverage values ranging from 90 to 99 capturing sufficient diversity with an adequate sequencing depth (Supplementary Table 1).

The OTU table, composed by 3587 OTUs, and the phylogenetic tree were calculated with FastTree2 (Price et al., 2010) using default parameters and the PyNast-aligned (Caporaso et al., 2010a) representative sequences as an input. The OTU table and the phylogenetic tree were used as inputs for the subsequent analyses of alpha- and beta-diversity. Bray–Curtis distance matrix on the log transformed OTU table was used to perform a Principal Coordinates Analysis, Canonical Analysis of Principal coordinates (CAP) and to conduct a permutational multivariate analyses of variance (PERMANOVA). Statistical analyses were conducted in PRIMER v. 6.1, PERMANOVA++ for PRIMER routines (Anderson et al., 2008) to test differences in bacterial community composition among the three soil fractions and among the three plant species. A first analysis was conducted in a one-way anova to explore difference among the fraction considering as a factor “Fraction” as fixed and orthogonal (three levels: rhizosphere/root surrounding soil/non-vegetated bulk). A second analysis was conducted exploring difference of the microbial assemblage among plants and fractions using the factor “Plant” (three levels: M. sativa, C. nigrescens and D. glomerata) and the factor “Fraction” (two levels: rhizosphere and root surrounding soil) both as a fixed and orthogonal, and their interaction (Fraction × Plant). Prior to perform the statistical analysis we verify that the data were not over-dispersed using PERMDISP for the factor “Fraction” (F1,18 = 3.61, p = 0.21) and the factor “Plant” (F2,15 = 9.36, p = 0.06). The shared OTUs among different fractions and plant species have been defined by Venn-diagram analysis using the software available at Diversity indexes were calculated using PAST (Hammer et al., 2001) and their statistical difference was evaluated with the analysis of variance considering the index as response variable and “Fraction” and “Plant” as explanatory categorical variable (see above for details). Raw sequences have been deposited at the ENA European Read Archive under accession number from SAMN07167786 to SAMN07167806, BioProject PRJNA388028, Submission ID SUB2728610.

16S rRNA gene quantitative PCR (qPCR) was performed in a reaction volume of 15 μl with 1 μl of template using universal primers 27F and 1492R and the SsoAdvancedTM Universal SYBR® Green Supermix (Bio-Rad). Thermal protocol was set up as follow: 98°C (3 min), then 40 cycles at 98°C (15 s), 58°C (30 s) and 72°C (1 min). Starting DNA concentration (ng/μl) was measured using a PowerWave HT Microplate Spectrophotometer (BioTek) and the number of 16S rRNA copies obtained with qPCR was normalized with the DNA concentration.

Bacteria Isolation and Identification

For bacterial isolation, the R samples obtained from the triplicate plants of the same species were pooled and homogenized. One gram of the resulting soil was suspended in 9 ml of physiological solution (0.9% NaCl), diluted in 10-fold series and plated onto mineral medium (Uhlik et al., 2011), adding biphenyl crystals on the plate lid as unique carbon source. Colonies were randomly picked after 1 week of incubation at 30°C, after the appearance of a stable number of colonies, and were spread three times on the same medium to obtain pure bacterial cultures. A collection of 165 biphenyl-utilizing rhizobacterial strains was established (52 isolates from MS, 56 from DG and 57 from CN) and cryopreserved in 25% glycerol at -80°C. Strain code includes a different number according to the plant of origin (1: MS, 2: CN, 3: DG).

The genomic DNA of each isolate was extracted through boiling cell lysis (Ferjani et al., 2015). Bacterial strains were identified through 16S rRNA gene amplification and partial sequencing (Macrogen, Rep. of South Korea) as described by Mapelli et al. (2013). 16S rRNA nucleotide sequences were subjected to BLAST search (using the blastn suite) and were deposited in the ENA database under accession numbers LT838007LT838169.

bphA Gene Detection, Quantification and Sequencing in Soil Metagenomes and Strain Genomes

The presence of the genes encoding for biphenyl dioxygenase α subunit (bphA) was assessed in the metagenome of soil samples (B, S, and R) through PCR as described by Iwai et al. (2010), using the primers BPHD-F3/R1, and further confirmed using 512F and 674R primer set (Leewis et al., 2016b). The latter primer set was also used for a bphA gene qPCR assay as described by Leewis et al. (2016b) and the relative abundance of bphA gene copies was expressed as a ratio over the total community 16S rRNA gene copy number. Bacterial isolates were grown overnight in Tryptic Soy Broth medium and then subjected to CTAB – phenol chloroform DNA extraction (Chouaia et al., 2010). BphA gene amplification was performed with 2 μl of DNA template in a final volume of 30 μl with primers 463F/674R (Petric et al., 2011), at the following conditions: buffer 1X, MgCl2 1.8 mM, dNTPs 0.2 mM, primers 1 μM, Taq 1.5 U per reaction. PCR thermal protocol was set up as follows: 10 min at 95°C, then 40 cycles of 95°C (15 s), 65°C (1 min), 72°C (2 min) and a final elongation step of 10 min at 72°C. All PCR reactions were performed utilizing FastStartTM High Fidelity PCR System (Roche). Genomic DNA of the model PCB-degrading strain Paraburkholderia xenovorans LB400 (DSMZ, Germany), was used as positive control for all bphA PCR reactions. PCR results were visualized on 1.2% agarose gel and PCR products that did not show the presence of aspecific bands were sequenced at Eurofins Genomics (Italy). Sequences were then identified using the BLASTn suite of the NCBI website3 and a Neighbor-Joining phylogenetic tree was then built using MEGA 5.1 (Tamura et al., 2011), computing the evolutionary distances using the Jukes–Cantor method. Nucleotide sequences were deposited in the ENA database under accession numbers LT840193LT840239.

In Vitro Characterization of Bacterial Isolates for PGP Traits and Bioremediation Potential

In vitro screening for the presence of activities related to plant growth promotion (PGP) was performed for the entire bacteria collection. Inorganic phosphate solubilization and the production of indole-3-acetic acid (IAA), ammonia, protease and exopolysaccharides (EPS) were assessed as described by Cherif et al. (2015); 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity was evaluated according to Belimov et al. (2005). Strains were also tested for abiotic stress resistance, namely the ability to grow at 4 and 42°C, and in presence of 5% NaCl and 20% polyethylene glycol (PEG) supplemented to the medium (Cherif et al., 2015). Catechol 2,3-dioxygenase activity was tested following the protocol described by Margesin et al. (2003).

In Vivo Assessment of Plant Growth Promotion of Tomato

Tomato seeds were sown in commercial non-sterile soil placed in trays and, after 1 week, uniform-sized seedlings were selected, each transplanted in separated 0.3 kg commercial soil pots and maintained under greenhouse conditions (≈110 photons m-2 s of light for 12 h during the day and average temperature of 25°C). A subset of 11 rhizobacteria strains, selected based on a cluster analysis performed using MVSP (Kovach, 1999) combining the PGP activities and the abiotic stress tolerance traits, were inoculated separately on tomato plants. One week after transplantation, the tomato plants (n = 5 for each treatment) were fertilized once with a bacterial suspension of the selected strains at a final concentration of 108 cells g-1 of soil (fresh weight). The inocula have been prepared according to Rolli et al. (2015) resuspending the bacterial cells in sterile tap water. Non-inoculated plants (n = 5) were irrigated with the same amounts of sterile tap water and used as control. After 30 days, plants were harvested for the measurement of shoot/root length and weight. Statistical analysis was performed by a pairwise comparison between each bacterial strain inoculated with the negative control, using Student’s t-test.


Diversity and Degradation Potential of Bacterial Communities Associated to Non-vegetated and Root-Associated Soil Fractions in the SIN Caffaro

The β-diversity of bacterial communities colonizing non-vegetated bulk soil (B) and root-associated soil fractions (rhizosphere, R, and root surrounding soil, S) in the SIN Caffaro was significantly different (Figure Figure1A1A, PERMANOVA, F2,18:3.48, p = 0.0007, Supplementary Table 2A). Considering only the R and S soil fractions associated to the three plant species M. sativa (MS), C. nigrescens (CN) and D. glomerata (DG), the bacterial population assemblage was significantly driven by both plant species and soil fraction (Figure Figure1B1B, PERMANOVA, F1,12 = 4.72, p < 0.001 and PERMANOVA, F2,12 = 3.21, p < 0.05, respectively, Supplementary Table 2B) but not by their interaction (PERMANOVA, F2,17 = 1.67, p > 0.05, Supplementary Table 2B). Quantification of the individual factors’ contributions to the observed bacterial community variations, determined by PERMANOVA of Bray–Curtis, showed that the two factors “Fraction” and “Plant species” equally contributed to determine the observed bacterial microbiome variation, explaining 21 and 18% of the variation, respectively (Supplementary Table 3), followed by the interaction of these two factors (11%). For the three fractions, not significant differences have been retrieved considering the OTU richness, but among the rhizosphere the MS and CN plants presented a lower number of OTUs (Figure Figure1C1C). OTU evenness was instead significantly different among fractions (ANOVA, F2,20 = 21.63, p < 0.001) but not between plants (Figure Figure1D1D). OTU diversity, calculated as inverse Simpson diversity index, was significantly lower in R compared to B and S (ANOVA, F2,20 = 4.7, p < 0.05). While not significant differences have been observed among the different plants in S fraction, plant species determined a strong effect in R fraction where DG hosted the significantly highest diversity, followed by CN and MS (Figure Figure1E1E).

Analysis of bacterial community’s structure and diversity of soil samples associated to spontaneous plants at SIN Caffaro. (A) Constraint (CAP) and (B) Unconstraint analysis of principal coordinates of the OTU97 obtained from 16S rRNA gene sequencing ...

The three soil fractions collected in the SIN Caffaro showed (i) low number of specific OTUs typical of each fraction, and (ii) a consistent group of shared OTUs (2831/3587) accounting the 97% of the 16S rRNA sequences (Figure Figure2A2A and Supplementary Table 4A). The rhizosphere and root surrounding soil associated to the three plant species shared a total of 1922/3436 and 2558/3436 OTUs, respectively, accounting up to 98% of relative abundance in S (Figures 2B,C and Supplementary Tables 4B,C). The high number of shared OTUs in non-vegetated and plant-associated soils indicated that all the fractions hosted bacterial communities with similar phylogenetic composition and different in the structure, as demonstrated by the analysis of Beta and Alpha-diversity (Figure Figure11).

Shared microbiome and taxonomic diversity of bacterial communities associated to non-vegetated bulk soil and root-associated soil fractions of plants grown in the SIN Caffaro. Venn diagrams showing the shared and exclusive bacterial OTUs of (A) the three ...

The taxonomic affiliation of OTUs (Figure Figure2D2D and Supplementary Tables 5, 6) revealed that the soil fractions hosted 37 bacterial phyla, 105 classes (99.3% sequences classified), 153 orders (89% classified), 167 families (67% sequences classified) and 162 genera (23% sequences classified). The most represented phyla were present in all the soil fractions (R, S, and B): Proteobacteria 34% (among these Gammaproteobacteria 16% and Alphaproteobacteria 10%), Actinobacteria 30%, Chloroflexi (8%), and Acidobacteria (8%). The three soil fractions were nevertheless dominated by different phyla/classes. In the non-vegetated soils Proteobacteria (on average 35%, mainly represented by Alphaproteobacteria and Betaproteobacteria) was the prevalent phylum followed by Acidobacteria (22%) and Bacteroidetes (12%) (Figure Figure2D2D). Proteobacteria dominated also the rhizospheres with Gammaproteobacteria (26%) and Alphaproteobacteria (9%) as the main Proteobacteria-classes, while in S fraction the Actinobacteria was the dominant phylum (30%).

Moving from the root-associated fractions (R and S) to the non-vegetated bulk soil, a strong reduction of the Actinobacteria phylum was observed in the bacterial communities, from an average of 33% in R to an average of 6% in B (Supplementary Table 5). An opposite trend has been observed for Acidobacteria, Bacteroidetes, Verrucomicrobia, and Gemmatimonadetes phyla that were enriched in the non-vegetated bulk soil and strongly limited in R and S fractions (Supplementary Table 5). Finally, while Firmicutes bacteria were enriched in R (7%) respect to the other two fractions (1%), Chloroflexi were more prevalent in S (10%) respect to R (7%) and B (4%). Difference in taxa enrichment and selection were observed also according to the plant species in both R and S fractions (Supplementary Table 7). The MS and CN bacterial communities in R fractions were dominated by Gammaproteobacteria (52%) and Actinobacteria (50%) respectively, determining a higher dominance index value (0.32 and 0.28) in these communities respect to the DG one (0.15), while DG hosted a more equally distributed bacterial community with Evenness index of 0.62. An opposite trend has been observed in the S fraction, where DG was dominated by Actinobacteria (37%) while MS and CN presented several taxa equally distributed within the bacterial communities (Evenness 0.72 and 0.69, respectively, Supplementary Table 7).

The bphA gene was detected, by means of qualitative PCR using different primer sets, in the metagenomes of all B, S and R samples. Despite that, the quantification of bphA gene with primers 512F and 674R was possible only for two replica of the CN rhizosphere (bphA/16S rRNA ratio, CN R2: 0.56 ± 0.03; CN R3: 0.50 ± 0.08) and one replica of the DG S (bphA/16S rRNA ratio, DG S2: 0.08 ± 0.004), showing the highest bphA/16S rRNA gene ratio in the CN rhizosphere.

Identification of Rhizosphere Biphenyl-Utilizing Bacterial Isolates

One-hundred sixty-five bacterial strains have been isolated from the rhizosphere of the three plant species MS, CN, DG in mineral medium supplemented by biphenyl as unique carbon source, hence considered as potential biphenyl degraders. The strains belonged only to 3 phyla, Actinobacteria (75, 51, and 52% in MS, CN, and DG collections, respectively), Proteobacteria (17, 30, and 32% in MS, CN, and DG collections, respectively) and Bacilli (8, 11, and 13%, in MS, CN, and DG collections, respectively). All the isolates belonged to phyla that were present in all the metagenome 16S rRNA high-throughput sequencing libraries, even though with a different relative abundance. All the isolates exhibited sequence identity with the closest described species in NCBI database higher than 97% (Supplementary Table 8) and their phylogenetic affiliation at the genus level is represented in Supplementary Figure 1. Among Gammaproteobacteria, Pseudomonas species were widespread in the three sub-collections, and constituted 15, 23 and 13% of MS, CN, DG rhizosphere, respectively. Acinetobacter was isolated only from CN and DG rhizospheres (5 and 16%, respectively). Among the phylum Bacilli, the genus Bacillus was the most represented, isolated from the rhizosphere of all plant species (7, 13, and 6% in CN, DG, and MS, respectively). Seventy-three percent of all the isolates from the rhizosphere of the three plant species belonged to three genera: Arthrobacter (18, 14, 10 strains from DG, MS, CN rhizospheres, respectively), Microbacterium (7, 24, 11 strains from DG, MS, CN rhizospheres, respectively), Pseudomonas (7, 8, 13 strains from DG, MS, CN rhizospheres, respectively). MS rhizosphere showed the lowest number (6) of cultivable genera within the biphenyl-utilizing bacteria, widely dominated by Microbacterium (44% of the isolates) and Arthrobacter (29% of the isolates), compared with CN and DG rhizosphere that included 11 genera.

Functional Characterization of the Cultivable Rhizosphere Biphenyl-Utilizing Bacteria

The isolate collection has been screened in vitro for PGP activities, bioremediation potential and abiotic stress resistance (results are detailed for each isolate in Supplementary Table 8). Production of IAA and proteases were the PGP related activities most represented among the collection, present in more than 50% of the strains, in similar percentages among sub-collections isolated from each plant species (Figure Figure33). A large fraction of the collection produced ammonia (21, 33, and 39% of the MS, CN, and DG strains, respectively), EPS (13, 25, and 14%, respectively) and siderophores (10, 24, and 24%, respectively). ACC deaminase activity was detected in higher percentage among CN (65%) than MS (37%) and DG (36%) sub-collections. Phosphate solubilization activity was present in a small proportion among CN (4%) and DG (11%) isolates, while it was absent in MS sub-collection. Concerning the biodegradation potential, catechol 2,3-dioxygenase activity was widespread in the entire collection with 83, 70, and 63% of the MS, CN, and DG active isolates, respectively. Biphenyl dioxygenase α subunit gene (bphA) was detected in the genomic DNA of 65, 81, and 68% of the MS, CN, and DG isolates, respectively. PCR products presenting one single band of the expected size (211 bp) in agarose gel electrophoresis were sequenced, obtaining 45 partial bphA gene sequences (9, 19, and 17 from MS, CN, and DG isolates, respectively). Eighty percent of the sequences showed high nucleotide identity (>99%) with Pseudomonas pseudoalcaligenes KF707 bphA, and the remaining ones showed >99% nucleotide identity with Rhodococcus wratislaviensis strain P13 bphA1 and R. opacus bphA1 genes (Supplementary Table 9). The bphA sequences were clustered in a phylogenetic tree with bphA sequences of reference strains having demonstrated PCB degradation ability. The sequences were clustered in two groups, both including known PCB-degraders (Supplementary Figure 2): Pseudomonas KF707-like sequences clustered together with Paraburkholderia xenovorans LB400, while Rhodococcus-like sequences grouped separately.

Characterization of the bacteria collection for plant growth promotion (PGP) traits and degradation capacity. Percentage of strains isolated from MS (blue), CN (black), DG (gray) rhizosphere that resulted positive to PGP and degradation screenings. Among ...

All strains were able to cope with the osmotic stress induced by the addition of 20% of PEG in the growth medium (Supplementary Table 2). The majority of the strains (71% on average) from each plant species proved to grow in presence of salt in the growth medium, while a percentage comprised between 58 and 71% tolerated high (42°C) and low (4°C) temperatures for growth (Figure Figure33 and Supplementary Table 8).

A total of 11 strains has been selected basing on PGP activities and abiotic stress tolerance traits (Supplementary Figure 3) and have been tested in vivo on tomato as a model plant. The strains were affiliated to different species belonging to the genera Pseudomonas, Acinetobacter, Arthrobacter, and Curtobacterium, and presented an array of different in vitro PGP traits, abiotic stress tolerance and bioremediation potential (Supplementary Table 10).

Despite showing only 1 and 2 in vitro PGP traits, respectively, strains 2–30 and 2–50, both isolated from CN plant and affiliated to different species of the genus Arthrobacter (Supplementary Table 10), significantly promoted plant growth compared to the non-inoculated control (Figure Figure44). Both strains mainly affected shoot development, significantly increasing shoot length (p < 0.01) and fresh weight (p < 0.001). While 2–30 did not showed effect on the root development, 2–50 significantly promote also the root length (p < 0.05). Strain 2–50 possesses, moreover, both the tested bioremediation potential traits (Supplementary Table 10).

Results of In vivo plant growth promoting test on tomato under greenhouse conditions, according to (A) shoots length, (B) root length, and (C) fresh weight measurement. Screened strains and plant measurement results are reported on the X-and Y-axes, respectively. ...


Plant Influence on Soil Bacterial Community Structure and Degradation Potential

In vegetated soils physico-chemical properties, together with plant root exudation and turnover, shape the structure of the microbial community. Resulting in the so-called “rhizosphere effect,” every plant species presents a specific pattern of root exudation and interacts with the soil community selecting a specific microbiota recruited from the surrounding soil (Haichar et al., 2008; Berg and Smalla, 2009). Here we compare the bacterial communities inhabiting the rhizosphere and root surrounding soils of three spontaneous plant species with a non-vegetated bulk soil from a site heavily contaminated with chlorinated persistent organic pollutants and heavy metals, demonstrating that both the soil fraction and the plant species cooperated in the selection of a specific bacterial assemblage in the root-associated soils. The bacterial communities inhabiting all soil fractions hosted the typical phyla that dominate soil ecosystems, i.e., Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, and Proteobacteria (Bulgarelli et al., 2013). R and S fractions, that are directly under plant root influence, showed an increase of Actinobacteria and Proteobacteria. Within the Actinobacteria, the order Gaiellales was retrieved at higher percentage in all the plant-associated fractions and in particular in the CN rhizosphere. This bacterial order was previously detected in the root system of rice (Hernández et al., 2015) and maize planted on heavy metals polluted soil (Touceda-Gonzalez et al., 2015), however, it is still poorly studied and includes only one described species (Albuquerque et al., 2011). Plant exudates enriched also the orders Sphingomonadales and Pseudomonadales in the DG and MS rhizospheres, respectively. Members of these orders have already been retrieved in PCB polluted soils, involved in biphenyl degradation and for some genera also in PCB degradation (Leigh et al., 2007; Hu et al., 2015; Jayanna and Gayathri, 2015; Leewis et al., 2016b). The phylum Chloroflexi, comprising a well known group of anaerobic PCB degraders (Jugder et al., 2015), was retrieved at relative abundance higher than 1% exclusively in the S fractions, being particularly enriched in MS1. It is possible that an anoxic microniche occurred in this sample, allowing the selection of Chloroflexi members, in agreement with previous finding of Chloroflexi enrichment in the rhizosphere of M. sativa during a PCB polluted soil phytoremediation trial (Tu et al., 2011) and of Sparganium sp. during biostimulation of the autochthonous microbiota in historically PCB polluted sediments (Di Gregorio et al., 2013).

Studies on a hydrocarbon-contaminated soil proved that the pollutant concentration rather than rhizosphere effect of planted willows had a major role in shaping the bacterial community (Bell et al., 2014; Yergeau et al., 2014). In a previous work (Di Guardo et al., 2017) we suggested that soil pollutant profiles of different former agricultural fields in the SIN Caffaro acted as drivers of the bacterial community composition. Here we demonstrated that in one of the previously analyzed fields, the rhizosphere effect of spontaneous plants adapted to the occurring soil contamination significantly influenced bacterial assemblages in the root-associated soil fraction, R and S. We speculate that in the rhizosphere an enhanced pollutant degradation, according to the higher abundance of taxa previously associated to PCB metabolism, contributed to shape bacterial diversity (Leewis et al., 2016a; Ridl et al., 2016). Our results indicated that autochthonous vegetation, not specifically selected for rhizoremediation efficiency, but rather naturally adapted to counteract phytotoxicity and the specific polluted soil conditions, can establish a strong relationship with the soil bacterial community, potentially sustaining PCB detoxification and in turn the natural attenuation process. The diversity of R bacterial communities was significantly influenced by the three plant species, which selected a root-associated microbiome having a peculiar structure. Despite this, the occurrence of a high percentage of OTUs shared among the different soil fractions, may suggest a strong selection force toward specific taxa constituted by the high pollution level, and possibly related to contaminant detoxification.

Different plant species, including MS, were previously demonstrated to enrich and stimulate the PCB-degrading bacterial communities, inducing an increase of bphA gene copy number and its expression levels in the rhizosphere compared to the non-vegetated bulk soil (Tu et al., 2011; Li et al., 2013; Pagé et al., 2015). In this work, the bphA gene was detected in the metagenome of all the soil fractions and could be quantified in CN rhizosphere and DG soil surrounding roots. The copy number ratio between this gene and the 16S rRNA bacterial gene was higher in R than in S samples, leading to speculate that bacterial populations harboring degrading potential were enriched in the rhizosphere fraction. However, the spread of bphA gene in the bacterial microbiota of the SIN Caffaro soil indicated an intrinsic PCB attenuation potential in this site, regardless of soil fraction or plant species, suggesting a stronger role of edaphic conditions rather than vegetation.

Isolates Potential for Rhizoremediation

The root system of plant growing on other PCB polluted sites, was shown to host PCB metabolizing bacteria (Leigh et al., 2006; Ionescu et al., 2009). Hence, we applied a cultivation approach to identify indigenous bacteria in the rhizosphere of plants naturally able to cope with the high level of pollution in the SIN Caffaro, having PGP and PCB degrading potentials. The synergistic effect of these traits has indeed the potential to sustain rhizoremediation approaches, in which both plants and bacteria are involved in remediation (Vergani et al., 2017). The cultivation approach applied to MS, CN, DG rhizospheres, due to the selective conditions determined by biphenyl as unique carbon source, led to isolate only bacteria species belonging to three of the thirty-seven phyla identified in the soil metagenome by 16S rRNA high-throughput sequencing (Actinobacteria, Proteobacteria, and Firmicutes). The high taxonomic similarity shown by the three sub-collections, in which four species over the 21 detected accounted for the 78% of the whole collection, suggests that the cultivable biphenyl-utilizing bacteria likely were mainly selected by the soil characteristics occurring at the SIN Caffaro site rather than the plant species. The detection of bphA gene in 72% of the isolates indicates that these strains harbor the genetic information to initiate the upper pathway of PCB degradation. Moreover, the large majority of the isolates belonged to the genera Arthrobacter, Microbacterium, and Pseudomonas, taxa previously isolated from the rhizosphere of plants growing in PCB contaminated soils and known for their capacity to grow on biphenyl and, for some strains, to degrade PCB (Gilbert and Crowley, 1997; Leigh et al., 2007; Uhlik et al., 2011; Kurzawova et al., 2012). Most of the bphA sequences amplified from the rhizobacteria collection showed higher similarity with the biphenyl dioxygenase α subunit of Pseudomonas pseudoalcaligenes KF707, a model strain studied for its ability to metabolize PCBs through 2,3-dioxidation (Furukawa, 1994). This large group of sequences clusters together with the bphA sequence of the reference strain Paraburkholderia xenovorans LB400. The functional diversity between Paraburkholderia xenovorans LB400 and Pseudomonas pseudoalcaligenes KF707 is determined by minor differences in the gene sequence (Furukawa and Fujihara, 2008), whose detection was not possible in our analysis due to the short sequence coverage of the used primer set. Functional redundancy for PCB degradation in the SIN Caffaro soil is supported by bphA sequences displaying sequence divergence. A second cluster of sequences was affiliated with a gene previously sequenced in Rhodococcus spp., including the reference strain R. jostii RHA1, which shows a different range of substrates and a low degree of homology compared with the bphA proteins produced by Pseudomonas KF707 and Paraburkholderia LB400 (Masai et al., 1995). The bphA sequences of the SIN Caffaro isolates were not related to the phylogenetic identification of the isolates, reflecting that bph genes are frequently associated to mobile elements and can be spread through horizontal gene transfer (Pieper and Seeger, 2005). Catechol 2,3-dioxygenase activity was widespread throughout the collection and all the isolated strains harboring bphA gene also presented this activity. Biphenyl dioxygenase and catechol 2,3-dioxygenase are enzymes involved in the metabolism of several root-derived and xenobiotic aromatic compounds, so these results support the hypothesis that plants foster organic contaminant degradation by their root-associated bacteria (Fuchs et al., 2011). In particular, since catechol is a toxic metabolite produced by the degradation of biphenyl in the benzoate lower pathway, its degradation capability is essential for a complete mineralization of PCBs that can occur through co-metabolism by bacteria harboring the upper and/or lower degradation pathways (Leewis et al., 2016b).

Plant growth promotion activities have been frequently reported in bacteria from polluted soils (Croes et al., 2013; Thijs et al., 2014; Franchi et al., 2016), according to the ability of plants to select beneficial bacteria when growing under phytotoxic and stress conditions. Likewise, IAA production and ACC deaminase activity are PGP traits well represented in the cultivable biphenyl-utilizing rhizobacteria hosted by all the three plant species. Bacterial IAA production can influence root proliferation and elongation, thereby affecting nutrient and water uptake by plants (Lambrecht et al., 2000) besides phytoextraction and phytostabilization in soils contaminated by heavy metals (Ma et al., 2011). Moreover, indole and its derivatives are considered inter-kingdom signal molecules and play a role as biofilm regulators, a further feature in PGP bacteria-plant interactions (Lee et al., 2015). ACC deaminase activity is known to lower ACC level in plant cells interfering with ethylene biosynthesis and thereby decreasing plant stress response potentially deriving from chemical phytotoxicity (Glick, 2010). Therefore, the isolated strains showed a notable potential in supporting plant adaptation and growth in the highly polluted soil of the SIN Caffaro. Moreover, a significant fraction of isolates tolerated moderate saline, osmotic and temperature stresses. These phenotypes are not directly related to PGP or biodegradation activity, but could confer to the strains a higher fitness in soils with complex and uneven pollutant fingerprints and subjected to seasonal changes (Di Guardo et al., 2017).

Two strains belonging to the genus Arthrobacter, one of the most abundant in our collection, also proved to promote the growth of a model plant (tomato) under greenhouse conditions. Since in vitro screening alone is not always sufficient to evaluate the actual PGP potential of bacterial strains (Cardinale et al., 2015), this result is encouraging for further in vivo tests with plant species selected for rhizoremediation trials, considering that these bacteria are also well adapted to the heavy pollution of the SIN Caffaro soil.


In this work we reported that three spontaneous plant species selected in the strongly PCB-polluted soil of a historical contaminated site, differentially affected the composition of the bacterial community in the root-associated soil fractions. The selective pressure imposed by the persistent chlorinated organic pollutants, heavy metals and metalloids in the soil, putatively affected the soil shared microbiome and determined an intrinsic functional potential for natural attenuation in the root-associated soil fractions. Rhizosphere bacterial strains harboring bphA gene and displaying catechol dioxygenase and PGP abilities, such as IAA production and ACC deaminase activity, are a potential resource for the improvement of plant growth and the detoxification in the heavily contaminated soils of SIN Caffaro, possibly exploitable for future rhizoremediation interventions.

Author Contributions

Conceived and designed the experiments: LV, FM, and SB. Selected the sampling site and collected the samples: RM, SA, AD, and SB. Performed the experiments: LV, RM, FM, EC, and MF. Analyzed the data: LV, RM, MF, and FM. Contributed reagents/materials/analysis tools: DD and SB. Wrote the paper: LV, FM, RM, SB, and DD. All authors critically revised the manuscript.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The authors wish to thank the collaboration of members of the “Caffaro Working Group”: Elisa Terzaghi, Elisabetta Zanardini, Cristiana Morosini, Giuseppe Raspa, Simone Anelli, Vanna M. Sale, and Paolo Nastasio. F. Mapelli acknowledges personal support from “Piano di Sostegno della Ricerca 2015–2017: Linea 2 - Dotazione annuale per attività istituzionali” in the projects “Microbial Interactions in complex ecosystems (MIRACLE)” and “Microbial diversity and Antibiotic resistance in Polluted Soils (MAPS)”. Part of the experiments performed in this study were done in the ambit of the course ‘FACILIS-2014 Microbially-driven facilitation systems in environmental biotechnology’ supported by the EU-US Task Force in Environmental Biotechnology. Students and organizers of the summers school “FACILIS-2014” are acknowledged.

Funding. Authors acknowledge King Abdullah University of Science and Technology (KAUST) baseline research funds to DD.


  • Abhilash P. C., Dubey R. K., Tripathi V., Gupta V. K., Singh H. B. (2016). Plant growth-promoting microorganisms for environmental sustainability. Trends Biotechnol. 34 847–850. 10.1016/j.tibtech.2016.05.005 [PubMed] [Cross Ref]
  • Abraham W.-R., Nogales B., Golyshin P. N., Pieper D. H., Timmis K. N. (2002). Polychlorinated biphenyl-degrading microbial communities in soils and sediments. Curr. Opin. Microbiol. 5 246–253. 10.1016/S1369-5274(02)00323-5 [PubMed] [Cross Ref]
  • Albuquerque L., Franc L., Rainey F. A., Schumannc P., Nobred M. F., da Costa M. S. (2011). Gaiella occulta gen. nov., sp. nov., a novel representative of a deep branching phylogenetic lineage within the class Actinobacteria and proposal of Gaiellaceae fam. nov. and Gaiellales ord. nov. Syst. Appl. Microbiol. 34 595–599. 10.1016/j.syapm.2011.07.001 [PubMed] [Cross Ref]
  • Anderson M. M. J., Gorley R. N., Clarke K. R., Clarke R. K. (2008). PERMANOVA + for PRIMER: Guide to Software and Statistical Methods. Plymouth: PRIMER-E.
  • Armiraglio S., Caccianiga M., Micheli E., Capretti A. (2009). Analisi preliminari sulla dinamica della vegetazione nel SIN Brescia-Caffaro. Ann. Mus. Civ. Sc. Nat. Brescia 2009 263–267.
  • Belimov A. A., Hontzeas N., Safronova V. I., Demchinskaya S. V., Piluzza G., Bullitta S., et al. (2005). Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard (Brassica juncea L. Czern.). Soil Biol. Biochem. 37 241–250. 10.1016/j.soilbio.2004.07.033 [Cross Ref]
  • Bell T. H., Hassan S. E.-D., Lauron-Moreau A., Al-Otaibi F., Hijri M., Yergeau E., et al. (2014). Linkage between bacterial and fungal rhizosphere communities in hydrocarbon-contaminated soils is related to plant phylogeny. ISME J. 8 331–343. 10.1038/ismej.2013.149 [PMC free article] [PubMed] [Cross Ref]
  • Berg G., Smalla K. (2009). Plant species and soil type cooperatively shape the structure and function of microbial communities in the rhizosphere. FEMS Microbiol. Ecol. 68 1–13. 10.1111/j.1574-6941.2009.00654.x [PubMed] [Cross Ref]
  • Bulgarelli D., Schlaeppi K., Spaepen S., Ver E., van Themaat L., Schulze-Lefert P. (2013). Structure and functions of the bacterial microbiota of plants. Annu. Rev. Plant Biol. 64 807–838. 10.1146/annurev-arplant-050312-120106 [PubMed] [Cross Ref]
  • Caporaso J. G., Bittinger K., Bushman F. D., Todd Z., DeSantis T., Andersen G. L., et al. (2010a). PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics 26 266–267. 10.1093/bioinformatics/btp636 [PMC free article] [PubMed] [Cross Ref]
  • Caporaso J. G., Kuczynski J., Stombaugh J., Bittinger K., Bushman F. D., Costello E. K., et al. (2010b). QIIME allows analysis of high–throughput community sequencing data. Nat. Methods 7 335–336. 10.1038/nmeth.f.303 [PMC free article] [PubMed] [Cross Ref]
  • Cardinale M., Ratering S., Suarez C., Zapata Montoya A. M., Geissler-Plaum R., Schnell S. (2015). Paradox of plant growth promotion potential of rhizobacteria and their actual promotion effect on growth of barley (Hordeum vulgare L.) under salt stress. Microbiol. Res. 181 22–32. 10.1016/j.micres.2015.08.002 [PubMed] [Cross Ref]
  • Cherif H., Marasco R., Rolli E., Ferjani R., Fusi M., Soussi A., et al. (2015). Oasis desert farming selects environment-specific date palm root endophytic communities and cultivable bacteria that promote resistance to drought. Environ. Microbiol. Rep. 7 668–678. 10.1111/1758-2229.12304 [PubMed] [Cross Ref]
  • Chouaia B., Rossi P., Montagna M., Ricci I., Crotti E., Damiani C., et al. (2010). Molecular evidence for multiple infections as revealed by typing of Asaia bacterial symbionts of four mosquito species. Appl. Environ. Microbiol. 76 7444–7450. 10.1128/AEM.01747-10 [PMC free article] [PubMed] [Cross Ref]
  • Croes S., Weyens N., Janssen J., Vercampt H., Colpaert J. V., Carleer R., et al. (2013). Bacterial communities associated with Brassica napus L. grown on trace element-contaminated and non-contaminated fields: a genotypic and phenotypic comparison. Microb. Biotechnol. 6 371–384. 10.1111/1751-7915.12057 [PMC free article] [PubMed] [Cross Ref]
  • Di Gregorio S., Azaizeh H., Lorenzi R. (2013). Biostimulation of the autochthonous microbial community for the depletion of polychlorinated biphenyls (PCBs) in contaminated sediments. Environ. Sci. Pollut. Res. 20 3989–3999. 10.1007/s11356-012-1350-x [PubMed] [Cross Ref]
  • Di Guardo A., Terzaghi E., Raspa G., Borin S., Mapelli F., Chouaia B., et al. (2017). Differentiating current and past PCB and PCDD/F sources: the role of a large contaminated soil site in an industrialized city area. Environ. Pollut. 223 367–375. 10.1016/j.envpol.2017.01.033 [PubMed] [Cross Ref]
  • Edgar R. C. (2010). Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26 2460–2461. 10.1093/bioinformatics/btq461 [PubMed] [Cross Ref]
  • Edgar R. C. (2013). UPARSE: highly accurate OUT sequences from microbial amplicon reads. Nat. Methods 10 996–998. 10.1038/nmeth.2604 [PubMed] [Cross Ref]
  • Ferjani R., Marasco R., Rolli E., Cherif H., Cherif A., Gtari M., et al. (2015). The date palm tree rhizosphere is a niche for plant growth promoting bacteria in the oasis ecosystem. BioMed Res. Int. 2015:153851 10.1155/2015/15385 [PMC free article] [PubMed] [Cross Ref]
  • Franchi E., Agazzi G., Rolli E., Borin S., Marasco R., Chiaberge S., et al. (2016). Exploiting hydrocarbon-degrading indigenous bacteria for bioremediation and phytoremediation of a multicontaminated soil. Chem. Eng. Technol. 39 1676–1684. 10.1002/ceat.201500573 [Cross Ref]
  • Fuchs G., Boll M., Heider J. (2011). Microbial degradation of aromatic compounds — from one strategy to four. Nat. Rev. Microbiol. 9 803–816. 10.1038/nrmicro2652 [PubMed] [Cross Ref]
  • Furukawa K. (1994). Molecular and evolutionary relationship of PCB-degrading bacteria. Biodegradation 5 289–300. 10.1007/BF00696466 [PubMed] [Cross Ref]
  • Furukawa K., Fujihara H. (2008). Microbial degradation of polychlorinated biphenyls: biochemical and molecular features. J. Biosci. Bioeng. 105 443–449. 10.1263/jbb.105.433 [PubMed] [Cross Ref]
  • Gilbert E. S., Crowley D. E. (1997). Plant compounds that induce polychlorinated biphenyl biodegradation by Arthrobacter sp. Strain B1B. Appl. Environ. Microbiol. 63 1933–1938. [PMC free article] [PubMed]
  • Glick B. R. (2010). Using soil bacteria to facilitate phytoremediation. Biotechnol. Adv. 28 367–374. 10.1016/j.biotechadv.2010.02.001 [PubMed] [Cross Ref]
  • Haichar F. Z., Marol C., Berge O., Rangel-Castro J. I., Prosser J. I., Balesdent J., et al. (2008). Plant host habitat and root exudates shape soil bacterial community structure. ISME J. 2 1221–1230. 10.1038/ismej.2008.80 [PubMed] [Cross Ref]
  • Hammer Ø., Harper D. A. T., Ryan P. D. (2001). PAST-Palaeontological Statistics, ver. 1.89. Oslo: University of Oslo.
  • Hernández M., Dumont M. G., Yuan Q., Conrad R. (2015). Different bacterial populations associated with the roots and rhizosphere of rice incorporate plant-derived carbon. Appl. Environ. Microbiol. 81 2244–2253. 10.1128/AEM.03209-14 [PMC free article] [PubMed] [Cross Ref]
  • Hu J., Qian M., Zhang Q., Cui J., Chunna Y., Su X., et al. (2015). Sphingobium fuliginis HC3: a novel and robust isolated biphenyl and polychlorinated biphenyls-degrading bacterium without dead-end intermediates accumulation. PLoS ONE 10:e0122740 10.1371/journal.pone.0122740 [PMC free article] [PubMed] [Cross Ref]
  • IARC (2015). Polychlorinated and Polybrominated Biphenyls Volume 107 IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Lyon, France. Available at:
  • Ionescu M., Beranova K., Dudkova V., Kochankova L., Demnerova K., Macek T., et al. (2009). Isolation and characterization of different plant associated bacteria and their potential to degrade polychlorinated biphenyls. Int. Biodeterior. Biodegradation 63 667–672. 10.1016/j.ibiod.2009.03.009 [Cross Ref]
  • Iwai S., Chai B., Sul W. J., Cole J. R., Hashsham S. A., Tiedje J. M. (2010). Gene-targeted-metagenomics reveals extensive diversity of aromatic dioxygenase genes in the environment. ISME J. 4 279–285. 10.1038/ismej.2009.104 [PMC free article] [PubMed] [Cross Ref]
  • Jayanna S. K., Gayathri D. (2015). Degradation of 2,4 dichlorobiphenyl via meta-cleavage pathway by Pseudomonas spp. consortium. Curr. Microbiol. 70 871–876. 10.1007/s00284-015-0800-3 [PubMed] [Cross Ref]
  • Jugder B.-E., Ertan H., Lee M., Manefield M., Marquis C. P. (2015). Reductive dehalogenases come of age in biological destruction of organohalides. Trends Biotechnol. 33 595–610. 10.1016/j.tibtech.2015.07.004 [PubMed] [Cross Ref]
  • Klindworth A., Pruesse E., Schweer T., Peplies J., Quast C., Horn M., et al. (2013). Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 41:e1 10.1093/nar/gks808 [PMC free article] [PubMed] [Cross Ref]
  • Kovach W. L. (1999). MVSP–A Multivariate Statistical Package for Windows, ver. 3.1. Pentraeth: Kovach Computing Services; 137.
  • Kurzawova V., Stursa P., Uhlik O., Norkova K., Strohalm M., Lipov J., et al. (2012). Plant–microorganism interactions in bioremediation of polychlorinated biphenyl-contaminated soil. N. Biotechnol. 30 15–22. 10.1016/j.nbt.2012.06.004 [PubMed] [Cross Ref]
  • Lambrecht M., Okon Y., Van de Broek A., Vanderleyden J. (2000). Indole-3-acetic acid: a reciprocal signalling molecule in bacteria–plant interactions. Trends Microbiol. 8 298–300. 10.1016/S0966-842X(00)01732-7 [PubMed] [Cross Ref]
  • Lee J.-H., Wood T. K., Lee J. (2015). Roles of indole as an interspecies and interkingdom signaling molecule. Trends Microbiol. 23 707–718. 10.1016/j.tim.2015.08.001 [PubMed] [Cross Ref]
  • Leewis M. C., Uhlik O., Fraraccio S., McFarlin K., Kottara A., Glover C., et al. (2016a). Differential impacts of willow and mineral fertilizer on bacterial communities and biodegradation in diesel fuel oil-contaminated soil. Front. Microbiol. 7:837 10.3389/fmicb.2016.00837 [PMC free article] [PubMed] [Cross Ref]
  • Leewis M.-C., Uhlik O., Leigh M. B. (2016b). Synergistic processing of biphenyl and benzoate: carbon flow through the bacterial community in polychlorinated-biphenyl- contaminated soil. Sci. Rep. 6:22145 10.1038/srep22145 [PMC free article] [PubMed] [Cross Ref]
  • Leigh M. B., Pellizari V. H., Uhlık O., Sutka R., Rodrigues J., Ostrom N. E., et al. (2007). Biphenyl-utilizing bacteria and their functional genes in a pine root zone contaminated with polychlorinated biphenyls (PCBs). ISME J. 1 134–148. 10.1038/ismej.2007.26 [PubMed] [Cross Ref]
  • Leigh M. B., Prouzová P., Macková M., Macek T., Nagle D. P., Fletcher J. S. (2006). Polychlorinated biphenyl (PCB)-degrading bacteria associated with trees in a PCB-contaminated site. Appl. Environ. Microbiol. 72 2331–2342. 10.1128/AEM.72.4.2331-2342.2006 [PMC free article] [PubMed] [Cross Ref]
  • Letcher R. J., Bustnes J. O., Dietz R., Jenssen B. M., Jørgensen E. H., Sonne C., et al. (2010). Exposure and effects assessment of persistent organohalogen contaminants in arctic wildlife and fish. Sci. Total Environ. 408 2995–3043. 10.1016/j.scitotenv.2009.10.038 [PubMed] [Cross Ref]
  • Li Y., Liang F., Zhu Y., Wang F. (2013). Phytoremediation of a PCB-contaminated soil by alfalfa and tall fescue single and mixed plants cultivation. J. Soils Sediments 13 925–931. 10.1007/s11368-012-0618-6 [Cross Ref]
  • Ma Y., Prasad M. N. V., Rajkumar M., Freitas H. (2011). Plant growth promoting rhizobacteria and endophytes accelerate phytoremediation of metalliferous soils. Biotechnol. Adv. 29 248–258. 10.1016/j.biotechadv.2010.12.001 [PubMed] [Cross Ref]
  • Mapelli F., Marasco R., Rolli E., Barbato M., Cherif H., Guesmi A., et al. (2013). Potential for plant growth promotion of rhizobacteria associated with Salicornia growing in Tunisian hypersaline soils. Biomed Res. Int. 2013:248078 10.1155/2013/248078 [PMC free article] [PubMed] [Cross Ref]
  • Marasco R., Rolli E., Ettoumi B., Vigani G., Mapelli F., Borin S., et al. (2012). A drought resistance-promoting microbiome is selected by root system under desert farming. PLoS ONE 7:e48479 10.1371/journal.pone.0048479 [PMC free article] [PubMed] [Cross Ref]
  • Margesin R., Gander S., Zacke G., Gounot A. M., Schinner F. (2003). Hydrocarbon degradation and enzyme activities of cold-adapted bacteria and yeasts. Extremophiles 7 451–458. 10.1007/s00792-003-0347-2 [PubMed] [Cross Ref]
  • Masai E., Yamada A., Healy J. M., Hatta T., Kimbara K., Fukuda M., et al. (1995). Characterization of biphenyl catabolic genes of gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1. Appl. Environ. Microbiol. 6 2079–2085. [PMC free article] [PubMed]
  • McDonald D., Price M. N., Goodrich J., Nawrocki E. P., DeSantis T. Z., Probst A., et al. (2012). An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 6 610–618. 10.1038/ismej.2011.139 [PMC free article] [PubMed] [Cross Ref]
  • Pagé A. P., Yergeau E., Greer C. W. (2015). Salix purpurea stimulates the expression of specific bacterial xenobiotic degradation genes in a soil contaminated with hydrocarbons. PLoS ONE 10:7 10.1371/journal.pone.0132062 [PMC free article] [PubMed] [Cross Ref]
  • Passatore L., Rossetti C., Juwarkard A. A., Massacci A. (2014). Phytoremediation and bioremediation of polychlorinated biphenyls (PCBs): state of knowledge and research perspectives. J. Hazard. Mater. 278 189–202. 10.1016/j.jhazmat.2014.05.051 [PubMed] [Cross Ref]
  • Petric I., Bru D., Udikovic-Kolic N., Hrsaka D., Philippot L., Martin-Laurent F. (2011). Evidence for shifts in the structure and abundance of the microbial community in a long-term PCB-contaminated soil under bioremediation. J. Hazard. Mater. 195 254–260. 10.1016/j.jhazmat.2011.08.036 [PubMed] [Cross Ref]
  • Pham T. T., Pino Rodriguez N. J., Hijri M., Sylvestre M. (2015). Optimizing polychlorinated biphenyl degradation by flavonoid-induced cells of the rhizobacterium Rhodococcus erythropolis U23A. PLoS ONE 10:e0126033 10.1371/journal.pone.0126033 [PMC free article] [PubMed] [Cross Ref]
  • Pieper D. H., Seeger M. (2005). Bacterial metabolism of polychlorinated biphenyls. J. Mol. Microbiol. Biotechnol. 15 121–138. 10.1007/s00253-004-1810-4 [PubMed] [Cross Ref]
  • Price M. N., Dehal P. S., Arkin A. P. (2010). FastTree 2 – approximately maximum-likelihood trees for large alignments. PLoS ONE 5:e9490 10.1371/journal.pone.0009490 [PMC free article] [PubMed] [Cross Ref]
  • Quinete N., Schettgen T., Bertram J., Kraus T. (2014). Occurrence and distribution of PCB metabolites in blood and their potential health effects in humans: a review. Environ. Sci. Pollut. Res. 21 11951–11972. 10.1007/s11356-014-3136-9 [PubMed] [Cross Ref]
  • Ridl J., Kolar M., Strejcek M., Strnad H., Stursa P., Paces J., et al. (2016). Plants rather than mineral fertilization shape microbial community structure and functional potential in legacy contaminated soil. Front. Microbiol. 7:995 10.3389/fmicb.2016.00995 [PMC free article] [PubMed] [Cross Ref]
  • Rolli E., Marasco R., Vigani G., Ettoumi B., Mapelli F., Deangelis M. L., et al. (2015). Improved plant resistance to drought is promoted by the root-associated microbiome as a water stress-dependent trait. Environ. Microbiol. 17 316–331. 10.1111/1462-2920.12439 [PubMed] [Cross Ref]
  • Sinkkonen S., Paasivirta J. (2000). Degradation half-life times of PCDDs, PCDFs and PCBs for environmental fate modeling. Chemosphere 40 943–949. 10.1016/S0045-6535(99)00337-9 [PubMed] [Cross Ref]
  • Sylvestre M. (2013). Prospects for using combined engineered bacterial enzymes and plant systems to rhizoremediate polychlorinated biphenyls. Environ. Microbiol. 15 907–915. 10.1111/1462-2920.12007 [PubMed] [Cross Ref]
  • Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28 2731–2739. 10.1093/molbev/msr121 [PMC free article] [PubMed] [Cross Ref]
  • Thijs S., Van Dillewijn P., Sillen W., Truyens S., Holtappels M., D’Haen J. et al. (2014). Exploring the rhizospheric and endophytic bacterial communities of Acer pseudoplatanus growing on a TNT-contaminated soil: towards the development of a rhizocompetent TNT-detoxifying plant growth promoting consortium. Plant Soil 385 15–36. 10.1007/s11104-014-2260-0 [Cross Ref]
  • Touceda-Gonzalez M., Brader G., Antonielli L., Ravindran V. B., Waldner G., Friesl-Hanl W., et al. (2015). Combined amendment of immobilizers and the plant growth-promoting strain Burkholderia phytofirmans PsJN favours plant growth and reduces heavy metal uptake. Soil Biol. Biochem. 91 140–150. 10.1016/j.soilbio.2015.08.038 [Cross Ref]
  • Toussaint J.-P., Pham T. T., Barriault D., Sylvestre M. (2012). Plant exudates promote PCB degradation by a rhodococcal rhizobacteria. Appl. Microbiol. Biotechnol. 95 1589–1603. 10.1007/s00253-011-3824-z [PubMed] [Cross Ref]
  • Tu C., Teng Y., Luo Y., Sun X., Deng S., Li Z., et al. (2011). PCB removal, soil enzyme activities, and microbial community structures during the phytoremediation by alfalfa in field soils. J. Soils Sediments 11 649–656. 10.1007/s11368-011-0344-5 [Cross Ref]
  • Turrio-Baldassarri L., Abate V., Alivernini S., Battistelli C. L., Carasi S., Casella M., et al. (2007). A study on PCB, PCDD/PCDF industrial contamination in a mixed urban-agricultural area significantly affecting the food chain and the human exposure. Part I: soil and feed. Chemosphere 67 1822–1830. 10.1016/j.chemosphere.2006.05.124 [PubMed] [Cross Ref]
  • Turrio-Baldassarri L., Alivernini S., Carasi S., Casella M., Fuselli S., Iacovella N., et al. (2009). PCB, PCDD and PCDF contamination of food of animal origin as the effect of soil pollution and the cause of human exposure in Brescia. Chemosphere 76 278–285. 10.1016/j.chemosphere.2009.03.002 [PubMed] [Cross Ref]
  • Uhlik O., Strejcek M., Junkova P., Sanda M., Hroudova M., Vlcek C., et al. (2011). Matrix-assisted laser desorption ionization (MALDI)–time of flight mass spectrometry- and MALDI biotyper-based identification of cultured biphenyl-metabolizing bacteria from contaminated horseradish rhizosphere soil. Appl. Environ. Microbiol. 77 6858–6866. 10.1128/AEM.05465-11 [PMC free article] [PubMed] [Cross Ref]
  • Vergani L., Mapelli F., Zanardini E., Terzaghi E., Di Guardo A., Morosini C., et al. (2017). Phyto-rhizoremediation of polychlorinated biphenyl contaminated soils: an outlook on plant-microbe beneficial interactions. Sci. Total Environ. 575 1395–1406. 10.1016/j.scitotenv.2016.09.218 [PubMed] [Cross Ref]
  • Yergeau E., Sanschagrin S., Maynard C., St-Arnaud M., Greer C. W. (2014). Microbial expression profiles in the rhizosphere of willows depend on soil contamination. ISME J. 8 344–358. 10.1038/ismej.2013.163 [PMC free article] [PubMed] [Cross Ref]

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