Enhanced Lead Phytoextraction by Endophytes from Indigenous Plants

: Lead (Pb) is one of the most common metal pollutants in soil, and phytoextraction is a sustainable and cost-effective way to remove it. The purpose of this work was to develop a phytoextraction strategy able to efﬁciently remove Pb from the soil of a decommissioned fuel depot located in Italy by the combined use of EDTA and endophytic bacteria isolated from indigenous plants. A total of 12 endophytic strains from three native species ( Lotus cornicolatus , Sonchus tenerrimus , Bromus sterilis ) were isolated and selected to prepare a microbial consortium used to inoculate microcosms of Brassica juncea and Helianthus annuus . As for B. juncea , experimental data showed that treatment with microbial inoculum alone was the most effective in improving Pb phytoextraction in shoots (up to 25 times more than the control). In H. annuus , on the other hand, the most effective treatment was the combined treatment (EDTA and inoculum) with up to three times more Pb uptake values. These results, also validated by the metagenomic analysis, conﬁrm that plant-microbe interaction is a crucial key point in phytoremediation.


Introduction
The overture of remediation of polluted soils for sustainable innovation is one of the critical steps in addressing current global environmental issues.
Nowadays, several physicochemical and biological solutions have been developed for the recovery of contaminated water [1][2][3] and soils [4,5], making the optimal selection a problematic yet crucial step for the success of the reclamation [6,7].
However, remediation activities also have an environmental impact since they often use chemical products or processes, with consequent consumption of raw materials and energy that could compromise the sustainability of the approach or even invalidate its beneficial aspects [8].
In the light of this, environmentally unsound technologies for the remediation of heavy metal contaminated soils (chemical extraction, chemical oxidation, stabilization/solidification, solvent extraction, etc.) should be replaced by green and sustainable technologies. The aim of the latter is not only to eliminate or reduce contamination but also to minimize the environmental impact (reduction of air emissions, minimization of energy use, decrease of waste, etc.) and create synergies between different sectors and activities (ecosystems protection, circular economy, climate change and resilience). Concerning this, sustainable phytomanagement can make a significant contribution to supporting this transition towards sustainable remediation.

Site Description and Soil Sampling
The contaminated site under investigation is located in an area of 40,000 m 2 near an urban area in Italy, previously affected for many years by a deposit for the storage of industrial wastes, active until the beginning of the 90s.
Being adjacent to a pond and at a distance of about 1 km from the sea, the site is characterized by sediments of marine and alluvial origin (silts, clayey silts, fluvio-lacustrine and marshy clays, arenaceous and conglomeratic, as well as alluvial deposits) and by a superficial layer (4-5 m depth) mainly composed of homogeneous artificial backfill soils of clayey, silty sand nature.
Previous chemical analyses performed on the soil of the area under examination revealed a relevant Pb contamination, which exceeded the contamination threshold concentrations (CSC) established by the Italian regulation (D.Lgs 152/2006) for sites intended for industrial use (1000 mg kg −1 ) [25]. The highest Pb concentration found in the area was approximately 2200 mg kg −1 .
For the soil sampling to be addressed for the laboratory activities, 6 sampling points (SP1 to SP6) were chosen within the site. The selection was based on the geological and morphological characteristics of the soils and favoring the points where previous analytical campaigns had already detected the presence of Pb.
Approximately 50 kg of soil was collected from each sampling point with an excavator to a maximum depth of 2.5 m.
The soil samples were sieved at 2 cm on-site and then transported in special containers to the laboratories for the soil characterization analysis. Based on the results of characterization, which showed that the soil samples were homogeneous, a single soil sample was prepared for the phytoextraction tests, obtained by mixing the soil aliquots (~20 kg) from each soil sample. On this soil (Pb-soil), the determination of the total and bioavailable lead content was performed again.

Soil Characterization and Pb Analysis
The soil samples were air-dried, ground, and sieved (0-2 mm) before analysis. The physicochemical characteristics of the soil were determined following the procedures reported in Methods of soil analysis [26]. Soil pH and electrical conductivity (EC) were determined by glass electrodes with a soil/water ratio of 1:2.5 and 1:2, respectively. The cation exchange capacity (CEC) was measured by exchange with barium acetate (pH 8.1) and titration with EDTA (0.05 N). Particle size distribution (sand, silt, and clay) was evaluated by the pipette method [27].
For the determination of total Pb content, soil samples were digested in a mixture of HNO 3 (65%, v/v) and H 2 O 2 (30%, v/v) using a microwave oven (FKV-ETHOS 900), according to EPA method 3051-A [28].
Potentially bioavailable concentrations of Pb in soil were determined according to an extraction procedure that sequentially involves the use of H 2 O (to extract soluble Pb), KNO 3 1 M (to extract exchangeable Pb), and EDTA 1% (to extract Pb retained with bonds also of covalent nature) with a soil/extractant ratio of 1:5 and extraction time 5 h [29,30].
EDTA was selected because it is one of the most effective mobilizing agents for Pb and can be applied effectively in various soil types [31][32][33].
A single extraction with EDTA was also performed. The concentration selected was 2 mM, frequently used in phytoextraction tests [15,31,34]. Higher concentrations of EDTA would mobilize excessive quantities of metal, which could give rise to leaching phenomena towards the aquifer [18,19,35].
The extraction was carried out by shaking the soil and extractant (ratio of 1:5) for 2 h. All extracts were centrifuged at 15,000 rpm for 15 min and filtered.
All analyses were performed in triplicate, and the mean value was recorded.

Microcosm Experimental Design
To study the role of bacterial inocula on phytoremediation processes, alone or in combination with the EDTA mobilizing agent, a microcosm experimental campaign was performed.
Each microcosm was prepared by adding 300 g of Pb-soil and sowing B. juncea var. Scala or H. annuus var. Paola, at doses of 0.5 g and 5 seeds per pot, respectively.
The experimentation lasted about 30 days and was conducted in a growth chamber (CCL300BH-AS S.p.A., Perugia, Italy). The growth conditions were the following: photoperiod of 14 h light at 25 • C and 10 h dark at 19 • C, photosynthetic photon flux density (PPFD) of 200 µmol m −2 s −1 , and relative humidity 65%. The experiment consisted of the following treatments: Pb-soil treated with 10 8 CFU (Colony-Forming Unit) per gram of soil of bacterial consortium SG_1 (PGPR) prepared as described in Section 2.6, Pb-soil treated with PGPR and EDTA 2 mM (PGPR + EDTA), and Pb-soil without any treatment (CT, control).
The study design consisted of a completely random design with three replicates per treatment. Bacterial inocula were added approximately 9 days after planting. EDTA addition was carried out after approximately 15 days, splitting the total dose over 5 days to avoid possible Pb toxic effects on plant growth resulting from rapid release and high metal mobilization [36].
Pots were thoroughly watered with tap water at field capacity, maintaining this condition throughout the experiment without fertilizer addition.
Plants were harvested 15 days after the addition of EDTA. Plant organs (roots and shoots) were washed with deionized water and then dried at 40 • C to constant weight to determine their Pb content. Roots were further treated in an ultrasonic bath (Branson Sonifier 250 ultrasonic processor, Branson Ultrasonic Corporation, Brookfield, CT, USA) to remove any residual soil.
Dried plant tissues were weighed, dry weight (DW) recorded, and powdered. Total Pb content was determined in the dried vegetal samples after overnight acid pre-digestion with HNO 3 (65%, v/v) and H 2 O 2 (30%, v/v) according to US-EPA 3052 [37]. Quantification of Pb in soil, extract, and vegetal samples was performed by inductively coupled plasma optical emission spectroscopy (ICP-OES) Liberty AX, Varian.

Test of Phytotoxicity
Soil toxicity was assessed by a phytotoxicity screening test based on germination inhibition and root extension.
The assay was performed in a Petri dish (10 cm diameter) filled with 10 g of Pb-soil moistened with deionized water to saturation, over which was placed a Whatman #1 filter and 10 seeds of Lepidium sativum L. [38]. L. sativum is a biological indicator of soil toxicity screening highly sensitive to phytotoxic compounds.
Hydrated quartz sand was used as a negative control. Five replicates were performed for each soil (Pb-soil and negative control). The closed Petri dishes were placed in a germination chamber in the dark at 25 ± 1 • C. After 72 h, the number of germinated seeds was counted, and the root length was measured. The germination index (GI%) and the radical extension inhibition (Inh%) were estimated by combining the values of seed germination and root elongation: G s and G c are the average numbers of seeds germinated in the contaminated soil samples and in the negative control, respectively. L s and L c are the radical lengths (mm) for the contaminated soil sample and negative control, respectively.

Endophyte Isolation
The contaminated site is rich in spontaneous species that have developed on the reported soil, most of which were probably already present as seed banks in the fill soil. The prevalent herbaceous vegetation has the homogeneous characteristics of the surrounding area.
For this reason, endophytic bacteria were isolated and characterized from three of the most commonly represented spontaneous species: Lotus cornicolatus, Sonchus tenerrimus, and Bromus sterilis. The soil around the roots was removed by repeatedly rinsing with tap water. To sterilize the root surface, they were treated with 70% EtOH for 5 min, with NaClO for 2 min and again 70% EtOH for another 5 min. They were then thoroughly rinsed at Soil Syst. 2021, 5, 55 5 of 14 least three times with sterile H 2 O. The roots, finely chopped with a sterile scalpel, were placed in sterile flasks containing TYEG (Trypticase Yeast Extract Glucose) medium and incubated for 16 h at 30 • C. Serial dilutions (10-4, 10-6, 10-8) of the obtained suspension were prepared, and 100 µL of each dilution (in triplicate) was spread over R2A Agar (Merck ® ) plates. Then 100 µL of the third rinsing water was plated to confirm the efficiency of sterilization. Endophyte colonies appeared after 4-5 days.
About twenty phenotypically different colonies for each root type were isolated and pure cultures were used for DNA extraction and taxonomic classification as already described in Franchi et al. [23].

PGPR Characterization
Endophytes isolated were subjected to a series of in vitro assays to assess their plant growth-promoting potential. The production of auxin indole-3-acetic acid (IAA) was estimated following the method proposed by Shahab et al. [39], siderophore molecules release was determined as described by Milagres et al. [40]. Their ability to solubilize mineral phosphorus (P) was determined by growing the strains in NBRIP (National Botanic Research Institute's Phosphate) according to the protocol developed by Nautiyal [41]. Production of exopolysaccharide (EPS) was estimated using a modified Weaver mineral medium enriched with sucrose [42]. Proteolytic activity was determined as described by Nielsen and Sørensen [43]. The production of ammonia was determined in peptone water (5 g L −1 peptone and 5% NaCl, pH 7.2), following the method proposed by Kifle and Laing [44].
The isolated strains were also tested for their capacity to form biofilm in vitro, inoculating them in glass tubes with 7 mL of LB (Luria Broth) medium. The tubes were incubated at 30 • C for 7 days without agitation. The formation of a visible layer (pellicle) at the interface between medium and air indicated a potential capability to produce biofilms. The potential capacity to fix atmospheric nitrogen was evaluated by growing the isolates with a specific nitrogen-free medium (NFb) [45]. The strains that showed at least three growth-promoting properties were selected. With the 12 potentially most promising ( Figure 1), a microbial consortium (SG_1) was prepared in the form of lyophilizate and was then used as an inoculum in the phytoremediation tests.
prevalent herbaceous vegetation has the homogeneous characteristics of the surrounding area.
For this reason, endophytic bacteria were isolated and characterized from three of the most commonly represented spontaneous species: Lotus cornicolatus, Sonchus tenerrimus, and Bromus sterilis. The soil around the roots was removed by repeatedly rinsing with tap water. To sterilize the root surface, they were treated with 70% EtOH for 5 min, with NaClO for 2 min and again 70% EtOH for another 5 min. They were then thoroughly rinsed at least three times with sterile H2O. The roots, finely chopped with a sterile scalpel, were placed in sterile flasks containing TYEG (Trypticase Yeast Extract Glucose) medium and incubated for 16 h at 30 °C. Serial dilutions (10-4, 10-6, 10-8) of the obtained suspension were prepared, and 100 μL of each dilution (in triplicate) was spread over R2A Agar (Merck ® ) plates. Then 100 μL of the third rinsing water was plated to confirm the efficiency of sterilization. Endophyte colonies appeared after 4-5 days.
About twenty phenotypically different colonies for each root type were isolated and pure cultures were used for DNA extraction and taxonomic classification as already described in Franchi et al. [23].

PGPR Characterization
Endophytes isolated were subjected to a series of in vitro assays to assess their plant growth-promoting potential. The production of auxin indole-3-acetic acid (IAA) was estimated following the method proposed by Shahab et al. [39], siderophore molecules release was determined as described by Milagres et al. [40]. Their ability to solubilize mineral phosphorus (P) was determined by growing the strains in NBRIP (National Botanic Research Institute's Phosphate) according to the protocol developed by Nautiyal [41]. Production of exopolysaccharide (EPS) was estimated using a modified Weaver mineral medium enriched with sucrose [42]. Proteolytic activity was determined as described by Nielsen and Sørensen [43]. The production of ammonia was determined in peptone water (5 g L −1 peptone and 5% NaCl, pH 7.2), following the method proposed by Kifle and Laing [44].
The isolated strains were also tested for their capacity to form biofilm in vitro, inoculating them in glass tubes with 7 mL of LB (Luria Broth) medium. The tubes were incubated at 30 °C for 7 days without agitation. The formation of a visible layer (pellicle) at the interface between medium and air indicated a potential capability to produce biofilms. The potential capacity to fix atmospheric nitrogen was evaluated by growing the isolates with a specific nitrogen-free medium (NFb) [45]. The strains that showed at least three growth-promoting properties were selected. With the 12 potentially most promising (Figure 1), a microbial consortium (SG_1) was prepared in the form of lyophilizate and was then used as an inoculum in the phytoremediation tests.   The amplification program was set up as follows: 95 • C for 10 min, followed by 25 cycles at 95 • C per 30 s, 58 • C for 30 s, and 72 • C for 20 s, a final hold time for 7 min at 72 • C and a cooling step at 4 • C.
The subsequent purification of the amplicons, the preparation, and the sequencing of the libraries followed the protocols for the Ion OneTouch™ 2 System, the Ion OneTouch™ ES, and the Ion PGM™, respectively, as previously described in Conte et al. [46].
The run was based on the workflow Metagenomics 16S w1.1 handling the Database Curated microSEQ ® 16 S and the reference Library 2013.1. The primers detected both ends to obtain 250 base pairs sequences. Alignment in Torrent Suite TM Software (version 5.8) was performed using the Torrent Mapping Alignment Program (TMAP). The sequences that occurred only once in the entire dataset were removed, and the representative sequences were defined with a 97% similarity cut-off.
After classifying the Operational Taxonomic Unit (OTU) representative sequences, the output was elaborated to obtain a relative abundance (%) of each OTU in the total amounts of the entire sample.

Quality Assurance and Quality Control
Quality assurance and quality control were performed by testing two standard solutions (0.5 and 2 mg L −1 ) for every 10 samples. CRM ERM-CC141 for soil and CRM ERM-CD281 for plants were used as certified reference materials. The values obtained for Pb were 31.8 ± 1.2 mg kg −1 for CRM ERM-CC 141 and 1.69 ± 0.20 mg kg −1 for CRM ERM-CD281, in agreement with the certified values of 32.2 ± 1.4 mg kg −1 and 1.67 ± 0.11 mg kg −1 , respectively.
The detection limit for Pb was 5 µg L −1 , and the recovery of samples with spikes (5%) ranged from 93% to 101% with a relative standard deviation (RSD) of 1.92% of the mean.

Statistical Analysis
Data referred to the phytoextraction test are reported as the mean of three replicate microcosms, and analyses were performed in triplicate, recording the mean value ± standard deviation (±SD). Data statistical analyses were performed using Statistica version 6.0 (StatSoft, Inc., Tulsa, OK, USA). Treatment effects were analyzed using a one-way analysis of variance (ANOVA, San Francisco, CA, USA). Differences between means were compared, and a post hoc analysis of variance was performed using Tukey's honestly significant difference test (p < 0.05).

Soil Analysis
Based on the data (Table 1), the soil samples under study (SP1-SP6) were homogeneous with each other, with a CEC in the range of 16-20 Cmol (+) kg −1 and an alkaline pH (~8.3 pH), which suggested a low Pb availability. Soil texture was also rather similar among the soil samples, with a predominance of the sandy fraction. The high conductivity values (on average 585 ± 54.5 µS cm −1 ) raised a particular concern initially due to possible effects on plant growth, which may have a different sensitivity to soil salinity. However, during the phytoextraction test, no growth problems due to the high salt content in the soil were observed. Therefore, the homogeneity of the main characteristics of the sampled soils allowed the composition of a single sample of contaminated soil (Pb-soil) on which to perform the phytoextraction tests.
As reported in Table 2, the soils had significant amounts of Pb, with a mean value between samples SP1-SP6 of 108 ± 3.45 mg kg −1 . The total Pb content in the Pb-soil was in the range of values found in the original samples. These values are higher than the limit of the values for public, private, and residential green use, established by the Italian legislation [25], thus implying a potential environmental risk. The sequential extraction procedure (SEP), which allowed the evaluation of potentially phytoavailable Pb samples, showed that most of the Pb was in a form not available for uptake by plants ( Table 2). The Pb amounts solubilized by H 2 O and KNO 3 were negligible, amounting to about 1% of the total Pb concentration. The results are ascribable to the basic pH of the soil, whereas the amount extractable in EDTA 1% was about 20%. Also, the extraction with 2 mM EDTA significantly increased the Pb phytoavailability, extracting on average 6.7% of total Pb.

Phytotoxicity Test
The phytotoxicity test was considered as a preliminary investigation to assess plant growth to provide supporting information for further investigations performed in this study.
GI% and Inh% data for Pb-soil, 85 ± 10.8% and 14 ± 10.5%, respectively, suggest the lack of adverse conditions for plant growth. High GI% and low Inh% values indicate reduced phytotoxicity and good quality of the contaminated soil.
The results confirmed the low values of Pb bioavailability, which did not cause significant toxic effects in the species used. Moreover, thanks to the phytotoxicity test, it was possible to verify that the high values of EC did not affect the plant germination.

Effect of PGPR and EDTA on Plant Growth and Pb Phytoextraction Efficiency
Small-scale phytoremediation experiments, performed under controlled conditions (microcosm), are essential for evaluating both the species performance and the effectiveness of treatments to be used. Therefore, the first parameter to be analyzed is biomass, which provides information about plant growth under stress conditions. B. juncea and H. annuus species showed no visible toxic symptoms during growth in the Pb-contaminated soil, even after applying the different treatments.
The results presented in Figure 2 indicate some significant differences in dry biomass of roots and shoots between treatments in the two species.
PGPR utilization positively affected the growth of both species, both when applied alone and in combination with EDTA.
In particular, the aerial biomass of H. annuus increased by 13.4% and 11.5% compared to the control when PGPRs were applied individually and combined with EDTA, respectively. In contrast, a relevant effect (36.5%) on B. juncea can be observed only in the combined treatment ( Figure 2B). As for the roots, PGPR did not significantly influence both species' biomass production, except for the combined treatment PGPR + EDTA on B. juncea (Figure 2A). Therefore, the first parameter to be analyzed is biomass, which provides information about plant growth under stress conditions. B. juncea and H. annuus species showed no visible toxic symptoms during growth in the Pb-contaminated soil, even after applying the different treatments.
The results presented in Figure 2 indicate some significant differences in dry biomass of roots and shoots between treatments in the two species. PGPR utilization positively affected the growth of both species, both when applied alone and in combination with EDTA.
In particular, the aerial biomass of H. annuus increased by 13.4% and 11.5% compared to the control when PGPRs were applied individually and combined with EDTA, respectively. In contrast, a relevant effect (36.5%) on B. juncea can be observed only in the combined treatment ( Figure 2B). As for the roots, PGPR did not significantly influence both species' biomass production, except for the combined treatment PGPR + EDTA on B. juncea (Figure 2A).
Under the experimental conditions investigated, and it is worth highlighting this, both B. juncea and H. annuus well tolerated the amounts of Pb released by EDTA.
The absence of adverse effects of EDTA on plant growth could be attributed to its ability to chelate Pb, which prevents binding between the metal and cellular components, neutralizing cytological impacts [47,48].
Pb concentrations in roots and shoots of B. juncea and H. annuus are presented in Table 3. As suggested by SEP, which did not reveal significant amounts of metal in an immediately bioavailable form, the lowest amounts of Pb were found in the control plants of both species. These minimal concentrations could be due to the radical exudates promoting the Pb solubilization. Under the experimental conditions investigated, and it is worth highlighting this, both B. juncea and H. annuus well tolerated the amounts of Pb released by EDTA.
The absence of adverse effects of EDTA on plant growth could be attributed to its ability to chelate Pb, which prevents binding between the metal and cellular components, neutralizing cytological impacts [47,48].
Pb concentrations in roots and shoots of B. juncea and H. annuus are presented in Table 3. As suggested by SEP, which did not reveal significant amounts of metal in an immediately bioavailable form, the lowest amounts of Pb were found in the control plants of both species. These minimal concentrations could be due to the radical exudates promoting the Pb solubilization.
The application of EDTA improved Pb uptake in both species with respect to the control. Indeed, significant increases in Pb concentration in the soil solution due to the EDTA addition, as indicated by the soil bioavailability results, resulted in substantial increases in Pb concentrations in the roots and shoots of both plants.
However, concerning B. juncea, the highest Pb concentrations were found in the roots and shoots of plants treated with only PGPR. The shoots of B. juncea treated exclusively with PGPR had approximately 24 times more Pb than control shoots. The PGPR + EDTA combined treatment mainly affected B. juncea roots, which showed Pb values not significantly different from plants treated with PGPR alone treatment. In contrast, the PGPR + EDTA combined treatment did not increase Pb amounts in B. juncea shoots.
In H. annuus, the highest Pb concentration was found in the roots and shoots of the plants treated with PGPR + EDTA.
The phytoextraction efficiency is the relation between the metal concentration in the plants and the biomass produced by evaluating the total accumulation (total uptake), obtained by multiplying the Pb concentration in plant tissues by the corresponding biomass EDTA combined treatment did not increase Pb amounts in B. juncea shoots.
In H. annuus, the highest Pb concentration was found in the roots and shoots of the plants treated with PGPR + EDTA.
The phytoextraction efficiency is the relation between the metal concentration in the plants and the biomass produced by evaluating the total accumulation (total uptake), obtained by multiplying the Pb concentration in plant tissues by the corresponding biomass produced. The effects of the treatments on Pb total uptake in B. juncea and H. annuus are illustrated in Figure 3. Our results indicated that treatment with PGPR, alone or in combination with EDTA, effectively improved Pb phytoextraction by B. juncea and H. annuus.
In B. juncea roots, the combined treatment PGPR + EDTA increased Pb accumulation by about 58 times compared to the control. Concerning B. juncea shoots, the highest Pb accumulation was observed in plants treated with only PGPR.
In contrast, in H. annuus, the highest Pb accumulation in shoots and roots was attributable exclusively to the PGPR + EDTA treatment. However, bacterial inocula alone increased the H. annuus root biomass to the levels of the combined treatment, without showing a significant difference.

NGS Analysis Results
The bacterial communities ( Figure 4) were mainly constituted by the family Pseudomonadaceae (maximum value of 51% in the sample 1 R), followed by the families Caulobacteraceae (maximum value of 30% in the sample 6 R), Xanthomonadanceae (maximum value of 18% in the sample 6 R) and Sphingomonadaceae (maximum value of 15% in the sample 3S). These bacteria are often correlated to hydrocarbon and metal contamination [49,50]. Considering the known PGPR strains [51] found in these communities and mainly related to Pb tolerance, the Pseudomonadaceae and Bacillaceae were isolated in Our results indicated that treatment with PGPR, alone or in combination with EDTA, effectively improved Pb phytoextraction by B. juncea and H. annuus.
In B. juncea roots, the combined treatment PGPR + EDTA increased Pb accumulation by about 58 times compared to the control. Concerning B. juncea shoots, the highest Pb accumulation was observed in plants treated with only PGPR.
In contrast, in H. annuus, the highest Pb accumulation in shoots and roots was attributable exclusively to the PGPR + EDTA treatment. However, bacterial inocula alone increased the H. annuus root biomass to the levels of the combined treatment, without showing a significant difference.

NGS Analysis Results
The bacterial communities ( Figure 4) were mainly constituted by the family Pseudomonadaceae (maximum value of 51% in the sample 1 R), followed by the families Caulobacteraceae (maximum value of 30% in the sample 6 R), Xanthomonadanceae (maxi-mum value of 18% in the sample 6 R) and Sphingomonadaceae (maximum value of 15% in the sample 3S). These bacteria are often correlated to hydrocarbon and metal contamination [49,50]. Considering the known PGPR strains [51] found in these communities and mainly related to Pb tolerance, the Pseudomonadaceae and Bacillaceae were isolated in the site and used for the inoculum. Pseudomonadaceae maintained a constant abundance in all the samples, while the abundance of Bacillaceae seemed to be affected by the inoculum, with values from 2% (both Controls) to 8% (sample 2S: PGPR B. juncea) and to 5-10% (respectively, in samples 5S: PGPR in B. juncea and 6S: EDTA + PGPR in H. annuus). Moreover, this family can be found only in the soil samples. Similarly, the family Rhodospirillaceae, mainly represented by the genus Azospirillum sp., can be found, even if only with low relative abundance (maximum value 2%), in the samples belonging to B. juncea. Continuing to consider PGPR families, also known for their tolerance to Pb, there is a slight increase of the Alcaligenaceae in the sample's roots in the treatment added with the inoculum. In detail, these families increase their relative abundance from 0% to 2% in sample 3 R (i.e., PGPR in B. juncea) and from 0% to 1-2%, respectively in the samples 5 R (i.e., PGPR in H. annuus) and 6 R (i.e., EDTA + PGPR in H. annuus). The Enterobacteraceae represents another family of interest showing its increase in the root samples treated with the mobilizing agent EDTA in the presence of both B. juncea and H. annuus (i.e., increase from 0% to 4% in sample 2 R: EDTA with B. juncea and increase from 14% to 27% in sample 6 R: EDTA with H. annuus).
ulum. In detail, these families increase their relative abundance from 0% to 2% in sample 3 R (i.e., PGPR in B. juncea) and from 0% to 1-2%, respectively in the samples 5 R (i.e., PGPR in H. annuus) and 6 R (i.e., EDTA + PGPR in H. annuus). The Enterobacteraceae represents another family of interest showing its increase in the root samples treated with the mobilizing agent EDTA in the presence of both B. juncea and H. annuus (i.e., increase from 0% to 4% in sample 2 R: EDTA with B. juncea and increase from 14% to 27% in sample 6 R: EDTA with H. annuus). The numbers represent the different treatments applied (i.e., Controls: 1 and 4; PGPR: 2 and 5; EDTA + PGPR: 3 and 6), and S stands for Soils and R for Roots. Sample 2 R was not included due to the low quality of the data required. Due to the high number of results obtained, the calculations, at the family level, were carried on by setting a cut-off < 2%. The numbers represent the different treatments applied (i.e., Controls: 1 and 4; PGPR: 2 and 5; EDTA + PGPR: 3 and 6), and S stands for Soils and R for Roots. Sample 2 R was not included due to the low quality of the data required. Due to the high number of results obtained, the calculations, at the family level, were carried on by setting a cut-off < 2%.
Expanding the attention to other PGPR families, not known for their Pb tolerance, it can be observed that both the families Flavobacteriaceae and Nocardioacea were mainly represented in the microcosms with H. annuus; the first one with higher values in the root samples, while the Nocardioacea was present almost exclusively in soil samples.

Discussion
Among soil contaminants, Pb is of particular concern due to its ability to biomagnify through the food chain, threatening food safety [47]. Lead is highly toxic for most plants, causing severe physiological damage [52]. However, some plant species have proven to be particularly resistant to lead toxicity and are widely used in the phytoremediation of Pb-contaminated soils [31,47]. The plant species tested in this experiment showed a growth capacity not affected by lead contamination. The reason for the selection of these two species is twofold: in addition to their satisfactory performances, as shown in other studies on Pb phytoremediation [53][54][55][56][57], they have recently been recognized as energy crop species, capable of providing a high level of biomass that can be reused in the bioenergy field [58][59][60][61].
This last aspect is currently very relevant as it allows further support for phytoremediation both in economic and sustainability terms.
The phytoextraction of Pb-contaminated soils with EDTA has been extensively studied. The results obtained in our study are in agreement with previous works that have shown how the addition of this complexing agent was recognized as an effective technique to increase Pb absorption by plants [31,47,62,63].
Although several studies have reported that EDTA can promote plant growth in metal-polluted soils by stimulating auxin production and aiding nutrient translocation in plants [33,64,65], the rapid release of bioavailable metal in a short period could cause harmful transient plant phytotoxicity. In this regard, a good strategy for phytoremediation of Pb-contaminated sites could be the combined use of PGPRs and EDTA to simultaneously improve metal bioavailability in soil and plant growth under metal stress.
The stress caused in plants by the presence of heavy metals triggers complex physiological and molecular mechanisms. Among these, the production of radical exudates containing, e.g., low-molecular-weight organic acids (LMWOAs) that stimulate microbial growth of the rhizosphere and solubilize essential trace elements such as insoluble or poorly soluble (e.g., phosphorus, iron and zinc), can complex some metals such as arsenic, cadmium and lead [66].
Most metals are present in the soil in the form of insoluble and non-bioavailable salts. Chemical compounds such as EDTA, dipotassium phosphate or ammonium sulfate can separate metals from complexes bound to soil particles, favoring the absorption by the plants' roots.
PGPRs can increase the bioavailability of metals by producing microbial metabolites and siderophore molecules. Many studies show that adding chelating agents (such as EDTA) would further improve plant growth and metal uptake when combined with PGPR inoculation [67,68].
In this work, we have shown that the simple addition of the microbial inoculum (PGPR) led in B. juncea to a significant increase in the absorption of lead in the aerial part. A similar result was also shown in the work of He et al. [69], where the inoculation of two Bacillus strains, isolated from the soil, improved the rhizosphere soil environment promoting absorption of Pb by plants, enhancing the dry weight of shoots of plants growing in Pb-contaminated soil, and significantly increasing the total Pb content in aerial parts.
The possibility of avoiding the addition of chemical compounds to increase the bioavailability of the metals is undoubtedly a fascinating aspect that significantly increases the sustainability of phytoremediation.
New experiments with different plant species and endophytes isolated from metal contaminated soils are underway.

Conclusions
Heavy metal contamination is an important aspect of environmental pollution, and phytoextraction is one of the most effective technologies in removing many metal species, including radionuclides.
In these contamination cases, the selection of remediation technology should be directed towards the application of phytoremediation if it is to be sustainable.
The interaction between plants and microorganisms creates a fascinating ecosystem that can lead to encouraging results. As highlighted in this study, the microbial inoculation of endophytic bacteria isolated from the roots of spontaneous plants grown on the Pbcontaminated soil allowed a significant percentage of metal phytoextraction to be obtained without chemical mobilizers.
Case studies showing positive results of assisted phytoextraction only with the support of PGPRs are limited, and research in this field must undoubtedly be supported and encouraged.