Introduction

Heavy metal pollution of soils as a result of anthropogenic activities drastically affects soil functions, microbial activities, biodiversity, and growth, vegetation growth, and biodiversity, in addition to the human food chain (Yaashikaa et al. 2022; Biswal 2022; Ogundola et al. 2022). Lead (Pb) is one of the most toxic non-biodegradable metals; its presence in soil interferes with plants’ functions, including chlorophyll production, cell division, root elongation, seed germination, and plant growth. Exposure of plants to this metal also leads to the production of reactive oxygen species (ROS) and lipid peroxidation, destroying the plant’s cell membrane and organelles. However, the effects depend on Pb levels, exposure time, and plant species (Zulfiqar et al. 2019; Dalyan et al. 2020).

Excessive lead concentration produces many chronic health consequences, damaging cardiovascular, brain, and gastrointestinal systems or even causing the mortality of living species via food chain pollution (Meena et al. 2020; Mousavi et al. 2022; Srivastava et al. 2022; Yang et al. 2022). The bioremediation approach provides effective measures for the treatment of a wide range of pollutants. Among the several known bioremediation processes, phytoremediation, rhizoremediation, and bioremediation by microbes might be effective techniques for reducing soil lead contamination (Saha et al. 2021).

Inherently, contaminated regions harbor microorganisms with a remarkable capacity to endure a wide array of pollutants (Nzila et al. 2016; Oubohssaine et al. 2022a; Chettri et al. 2022; Annamalai et al. 2022; Chatterjee et al. 2022). Specifically, Actinobacteria have been widely identified as potential bioremediation agents among environmental microorganisms. They have been shown to be common in polluted soils and to be capable of metabolizing pollutants to thrive, including pesticides and heavy metals (Mawang et al. 2021; Farda et al. 2022; Behera et al. 2022; Raimondo et al. 2022). In addition to their ability to tolerate heavy metals, they can also contribute to the remediation process through their plant growth-promoting (PGP) properties and heavy metal-induced stress alleviation. The implementation of useful plant growth-promoting rhizobacteria (PGPR) in soil contributes to plant health, development, and nutrition by a variety of processes, including phosphate solubilization, nitrogen fixation, production of phytohormones, ammonia siderophores, and 1-amino-cyclopropane-1-carboxylic acid deaminase (ACCD) (Govindasamy et al. 2022; Oubohssaine et al. 2022a). Furthermore, some Actinobacteria have the ability to chelate excessive Pb and reduce its accessibility by producing active compounds, such as exopolysaccharides (EPS), and forming biofilm under stressful conditions. All of these properties are involved in critical physiological processes that guarantee bacterial survival in the presence of heavy metal contamination (Li et al. 2021a, 2021b; Homero et al. 2021).

One of the most common genera encountered in contaminated soils is Rhodococcus, a promising group of bacteria suitable for bioremediation. Rhodococci are a group of remarkable bacteria known for their extraordinary ability to thrive under mild, moderate, or even severe stresses across diverse environments. They have been discovered in various settings, including those characterized by harsh environmental conditions (Pátek et al. 2021; Oubohssaine et al. 2022a, 2022b). These Gram-positive, aerobic, non-sporulating bacteria possess a high G+C content (Guevara et al. 2019; Donini et al. 2021) and exhibit unique cellular features. Their cell walls contain peptidoglycan, while their cell envelopes consist of arabinogalactan and a lipid bilayer of mycolic acids (Malas 2021). The outer mycomembrane plays a vital role in protecting these cells against various toxic compounds (de Carvalho and da Fonseca 2005; de Carvalho 2012).

Moreover, Rhodococcus strains possess an extensive array of enzymatic activities that prepare them to handle numerous natural and xenobiotic organic compounds, enabling them to degrade substances such as alkanes, cycloalkanes, aromatic compounds, phenols, polycyclic aromatic hydrocarbons, halogenated hydrocarbons, and polychlorinated phenyls (Cappelletti et al. 2019, 2020). For these reasons, Rhodococcus strains are considered one of the best candidates for bioremediation strategies. Furthermore, it can be assumed that the use of tolerant Rhodococcus strains possessing several plant-growth-promoting mechanisms can have a great potential for the enhancement of the efficiency of heavy metal removal from contaminated soils, especially lead, and can serve as an intelligent and eco-friendly way for strategic implementation of phytoremediation interventions.

Here, we set out to examine Sulla spinosissima L.’s ability to phytoremediate heavy metals in association with a selected rhizospheric bacterium, Rhodococcus qingshengii strain LMR356, that was previously isolated from abandoned mining sites situated in Oujda region (Eastern Morocco). This plant species is a perennial native multi-tolerant legume known for its contribution to soil fertility as a result of its symbiotic nitrogen fixation capacity. Furthermore, it exhibits invasive traits as it naturally spreads within the previously mentioned mining sites, attributed to its remarkable ability to withstand and accumulate heavy metals in its roots (Sbabou et al. 2016). Therefore, the current study aimed to (a) examine the plant growth-promoting (PGP) traits of LMR356 in the presence of lead (Pb) pollution, (b) investigate the effects of inoculation with this strain on the growth of Sulla spinosissima L., and (c) reveal its impact on plants growth in a contaminated soil and try to elucidate the mechanistic role of plant growth-promoting rhizobacteria (PGPRs) in enhancing phytoremediation. The final objective is to provide critical knowledge for the prospective scaling-up of this technology to preserve the environment and rehabilitate polluted areas.

Material and methods

Origin of strain LMR356

For this study, we selected a strain isolated from the rhizosphere of Acacia cyanophylla, a legume tree species introduced into an abandoned mining site (Touissit) located in Oujda, Eastern Morocco. Oubohssaine et al. (2022a) previously reported the physico-chemical characteristics of the soil at this site, revealing extensive and varied contamination by several heavy metals, including chromium (Cr), copper (Cu), lead (Pb), zinc (Zn), and arsenic (As).

Molecular identification of strain LMR356

The genomic DNA of the isolate was extracted following the method described by Oubohssaine et al. (2022a, 2022b). Subsequently, the 16S rDNA gene was amplified using the polymerase chain reaction (PCR) technique and specific primers, namely 27f (5-AGAGTTTGATCCTGGCTCAG-3) and FGPS1509 (5-AAGGAGGGGATCCAGCCGCA-3). The PCR products were then sent for nucleotide sequencing at Genoscreen in France. The obtained 16S rDNA sequences were subjected to BLAST analysis in the NCBI database to identify model strains showing high sequence homology. To visualize the evolutionary relationships, a phylogenetic tree was constructed using the MEGA X software and the neighbor-joining method, with bootstrap analysis performed with 1000 repetitions to assess the robustness of the phylogeny.

Behavior of the strain LMR356 faced with lead stress

Pb tolerance of strain LMR356

To assess the strain’s tolerance to lead, we quantified it by measuring the optical density (OD) at 600 nm after 6 days of growth at 28 °C in nutrient broth (NB) medium supplemented with different concentrations of lead (ranging from 0 to 35 mM of Pb (Pb (NO3)2)). The selection of lead concentrations was based on the elevated levels of this metal previously measured at the Touissit site (Oubohssaine et al. 2022a).

Bioaccumulation of Pb

A volume of 100 ml of bacterial cultures, grown in tryptone yeast extract (TY) medium containing 5 g tryptone, 1 g yeast extract, and 0.65 g CaCl2, was prepared in 250 ml flasks. After 48 h of incubation at 37°C, the metal (30 mM of Pb(NO3)2) was introduced into the bacterial cultures, and they were re-incubated under the same conditions. The cell mass was then separated from the medium through centrifugation (8000 rpm for 10 min at 4°C). To distinguish the cells (pellet) from the medium, two specimens of the pellet were washed twice with sterile distilled water (the pellets were re-suspended in sterile distilled water and centrifuged for 6 min at 7000 rpm), while the other two specimens of the pellet were washed twice with 0.25 M of acid ethylenediaminetetraacetic (EDTA) (the pellets were re-suspended in 0.25 M EDTA, left for contact time, and then centrifuged at 7000 rpm for 6 min). After centrifugation, the supernatant represented the membrane compartment (metals extracted from cell membranes by EDTA), while the pellet corresponded to the intracellular compartment.

The pellets of the strain cultivated in the presence of the metal were obtained after centrifugation and dried at 52 °C for 48 h. Subsequently, the dried pellets were subjected to analysis by Inductively coupled plasma - optical emission spectrometry (ICP-OES) after digestion with sulfuric acid (H2SO4), nitric acid (HNO3), and perchloric acid (HClO4) following the method described by El Aafi et al. (2015).

Motility Assay

Following the protocol outlined by Turnbull and Whitchurch (2014), we conducted the bacterial motility assay. The assessment of bacterial motility was performed on a semi-solid tryptone yeast extract (TY) (0.3%) medium under both normal and lead stress conditions (0–30 mM of Pb). The evaluation involved measuring the diameter of the halo formed as an indicator of bacterial motility.

Exopolysaccharide production and biofilm assay

The strain was initially cultured for 30 h at 28 °C in Man-Rogosa and Sharpe (MRS) broth medium containing sucrose as the carbon source. After centrifugation at 10,000 rpm for 20 min, the supernatant was collected and mixed with three times its volume of cold acetone. The mixture was left overnight at 4 °C for precipitation, resulting in the recovery of the extracellular polysaccharides (EPS). The EPS obtained were then subjected to the phenol sulfuric acid assay to estimate their quantity (DuBois et al. 1956).

To study biofilm formation, pre-sterilized 96-well plates were used following the procedure described by O'Toole (2011) and Sahal et al. (2020). A cell suspension with an optical density of 0.5 at 600 nm was inoculated with 10 μl into each well containing 190 μl of Luria-Bertani (LB) medium, and 200 μl of autoclaved distilled water was added. After 16 h of growth at 37 °C, the cells were immobilized using 99% methanol. Following this, the plates underwent two washes with phosphate buffer saline and were left to air-dry. Subsequently, a 0.2% crystal violet solution was introduced into each well, and after a 5-min incubation, any surplus crystal violet was eliminated through two additional washes, followed by air-drying. To dissolve the crystal violet within the biofilm, 100 μl of 95% ethanol was added to each well, enhancing the reading sensitivity with a spectrophotometer. The growth of the biofilm was monitored by measuring the OD570 using a microplate reader (Rivas et al. 2007; Flores-Treviño et al. 2014; Shukla and Rao 2013; Shukla and Rao 2014).

PGP trait evaluation under normal and lead stress conditions

To assess the production of indole-3-acetic acid (IAA), strain LMR356 was cultured in Yeast Extract Mannitol (YEM) medium supplemented with tryptophan (0.5 mg mL−1), which serves as the precursor of IAA. The quantification of auxin production followed the methods described by Gordon and Weber (1951), Sheng et al. (2008), and Oubohssaine et al. (2022a).

The ability of strain LMR356 to solubilize phosphate was evaluated using a Pikovskaya medium containing 0.5% insoluble phosphate, following the procedures outlined by Pikovskaya (1948) and Oubohssaine et al. (2022a). For the estimation of siderophore production, the Chrome Azurol-S (CAS) analytical method was employed, as described by Modi et al. (1985), Schwyn and Neilands (1987), Manjanatha et al. (1992), and Oubohssaine et al. (2022a).

To measure ACC deaminase activity, the ACC-deaminase activity assay from Penrose and Glick (2003) and Oubohssaine et al. (2022a) was employed. To estimate the ammonium production by strain LMR356, the technique described by Cappuccino and Sherman (1992) was employed. In triplicate, the strain was inoculated into peptone water broth (containing 10 g L−1 Peptone, 5 g L−1 NaCl, and pH 7.0 ± 0.2) and then incubated at 28° ± 2°C with continuous shaking at 200 rpm. Following the incubation period, 1 ml of the cell-free supernatant was combined with 1 ml of Nessler’s reagent.

All the plant growth-promoting (PGP) traits were assessed under both normal and lead stress conditions (30 mM Pb).

Sulla spinosissima experiments

Inoculum and seed preparation

An overnight inoculum of LMR356 was cultured in NB medium, starting with an initial OD600 of 0.05, and incubated at 28 °C with agitation at 180 rpm for 24 h. After incubation, the bacterial culture was harvested by centrifugation at 8000 rpm at 4 °C for 10 min. Afterwards, the supernatant was removed, and the cell pellets were subjected to two washes with sterile distilled water. They were then re-suspended in sterile distilled water to attain an OD600 of 0.1, which corresponds to approximately 108 cells mL−1.

Previously collected seeds of Sulla spinosissima from the Oujda region were manually scarified, then surface sterilized in 70% ethanol for 1 min, followed by five rinses with sterile distilled water. The treated seeds were then germinated on 9% agar plates at 25°C in a dark environment.

Hydroponic experiments

Tolerance of Sulla spinosissima to lead

The primary objective of the initial experiment was to assess the tolerance level of Sulla spinosissima plants to lead (Pb). Uniform and aseptic pre-germinated seeds of Sulla spinosissima were transplanted into vessels filled with nutrient solution following the Hoagland and Arnon (1950) method. The seedlings were allowed to grow under normal conditions for 15 days. Subsequently, the young seedlings were transferred to nutrient solutions containing varying concentrations of Pb(NO3)2 (ranging from 500 to 1000 μM) and were left to grow for 30 days. To ensure controlled growth conditions, the vessels were randomly arranged within a growth chamber set at a temperature of 25/20°C (day/night) with a photoperiod of 16 h of light and 8 h of darkness. To maintain optimal nutrient conditions, the nutrient solutions were renewed every 5 days.

Effect of LMR356 inoculation on Sulla spinosissima

The second experiment aimed to investigate the impact of inoculating the Rhodococcus strain on the growth and development of plants under both normal and Pb-stressed conditions. Three distinct treatments were tested:

  • Sulla plants are grown without Pb and without inoculation (control).

  • Inoculation with Rhodococcus under normal growth conditions at a rate of 108 cells per seedling.

  • Rhodococcus inoculation at the same rate under the previously identified Pb minimal inhibitory concentration for Sulla spinosissima.

All plants were cultivated under the same conditions as described earlier.

Pot experiment

The seedlings were planted in earthen pots of uniform size, filled with sand or soil depending on the specific experiment. The arrangement of the pots followed a completely randomized design (CRD).

Effect of inoculation with LMR356 on Sulla spinosissima growth under normal conditions

To evaluate the influence of the Rhodococcus inoculum on plant growth, the inoculated plants were grown in sand that was autoclaved at 121°C for 1 h. The strain and seeds were prepared following the previously described method.

The plants were cultivated in pots, each containing 100 g of sterilized sand. One plant was placed per pot, and there were 12 repetitions for each treatment. Each plant received an inoculation of 1 mL of the Rhodococcus inoculum at the same rate as before. Additionally, uninoculated control plants were also set up for comparison.

All the pots were placed in a controlled growth chamber, maintaining the same environmental conditions as previously mentioned. Regular watering with a nutritive mineral solution was provided to the plants throughout the 100-day duration of the experiment.

Effect of inoculation with the Rhodococcus strain on Sulla spinosissima growth under lead stressing conditions

In this particular experiment, we utilized the heavily lead-contaminated soil from the Touissit site in the Oujda region, as previously studied by Oubohssaine et al. (2022a). Aseptic pre-germinated seeds were planted in pots filled with this soil, and the experimental conditions were kept consistent with the preceding assay, except for the watering process. In this case, the plants were watered with sterile distilled water instead of the mineral solution. The preparation and application of the bacterial inoculant were carried out in the same manner as in the previous experiment.

Measurements of biomass and chlorophyll/carotenoid content

Following a growth period of 30 days for hydroponic experiments and 100 days for pot assays, the Sulla plants were harvested, and the shoots and roots were carefully separated. In the soil experiment, the roots were meticulously removed from the soil by thorough water washing. Afterward, both the roots and shoots were dried with blotting paper. The length and biomass of the plant shoots and roots were subsequently measured.

A portion of the fresh leaves was utilized to estimate the chlorophyll and carotenoid contents, following the method outlined by Mackinney (1941).

Antioxidant enzyme assays

The measurement of ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD) activities was conducted using the procedures outlined by Chen and Asada (1989), Nakano and Asada (1981), Aebi (1984), and Chance and Maehly (1955), respectively.

Statistical analysis

The data underwent analysis using the analysis of variance (ANOVA) statistical package within XL STAT for social sciences. Subsequently, multiple treatment levels were compared using the Duncan significant difference test at a significance level of P ≤ 0.05.

Results

Identification of strain LMR356

By comparison of 16S rDNA sequences in BLAST analysis, the strain LMR356 was shown to have 99.7% homology with a Rhodococcus qingshengii strain (GenBank No. NR_115708.1) (Fig. 1). The nucleotide sequence of LMR356 has been registered in the NCBI database with accession number OQ991161.

Fig. 1
figure 1

Neighbor-joining phylogenetic tree of partial 16S rDNA sequence of LMR356

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Rhodococcus strain lead tolerance

As shown in Fig. 2, the growth of strain LMR356 was reduced progressively when lead concentration in the growth medium increased. This strain can tolerate up to 30 mM of Pb, thus the minimal inhibiting concentration (CIM) of strain LMR356 can be established between 30 and 35 mM of Pb. 

Fig. 2
figure 2

The effect of different lead concentrations on Rhodococcus qingshengii LMR356 growth in nutrient broth medium

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Bioaccumulation of lead

The primary objective of this study was to assess the bioaccumulation of Pb by the Rhodococcus strain, aiming to characterize the distribution of lead at the subcellular level and evaluate the strain's potential for bioremediation of mining sites. The results obtained showed that the strain accumulated 55 ppm of Pb inside the cell, against 8490 ppm outside of the cell (Table 1).

Table 1 Bioaccumulation of lead in Rhodococcus qingshengii LMR356 growing in tryptone yeast extract (TY) medium containing 30 mM Pb
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Motility test

The motility was assessed in the presence of different concentrations of Pb ranging between 0 and 30 mM. Halo measurements indicated that the strains’ motility was significantly increased with the augmentation of the Pb concentration, and the maximum value of 35 mm was measured at 20 mM Pb (Fig. 3).

Fig. 3
figure 3

Motility of strain LMR356 assessed by changes in colonies’ size measured on soft agar (0.3 %) under different concentrations of lead (0–30 mM)

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Exopolysaccharide production and biofilm assay

The phenol-sulphuric acid assay showed that the Rhodococcus strain was able to secrete a high amount of exopolysaccharides under normal conditions (1952.28 mg mL−1). In the presence of 30 mM of lead, EPS production was increased to 3637.72 mg mL−1 (Table 2). The strain was also able to form a biofilm in the presence of Pb that was dependant on the concentration of lead in the medium (10 mM, 20 mM, 25 mM) (Fig. 4).

Table 2 PGP activities and EPS production measurements under normal and Pb stress conditions
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Fig. 4
figure 4

Quantitative assessment of biofilm formation using the crystal violet assay

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Plant growth-promoting characteristics

The amounts of IAA produced by the strain LMR356 not exposed and exposed to Pb stress are shown in Table 2. The production of IAA was drastically reduced by the incorporation of lead in the culture medium, 2.65 μg mL−1 of IAA instead of 21.17 μg mL−1. Lead also affected negatively the amount of phosphorus solubilized by the strain grown in PVK liquid medium, 8.22 mg L−1 of P against 33.60 mg L−1 in non-stressed conditions. In contrast to the two previous PGP activities, lead stimulated siderophores production and ACC deaminase activity of the Rhodococcus strain tested (83.7% instead of 69% for siderophores and 71.37 nmol mg−1 h−1 α-ketobutyrate against 38.17 for ACC deaminase). Moreover, ammonium production by the strain was not affected by the presence of lead in the medium. Globally, the results of PGP activity measurements demonstrate that the Rhodococcus LMR356 studied may be a good candidate to be tested with plants growing under normal and Pb-contaminated conditions.

Sulla spinosissima growth under Pb stress in hydroponic conditions

Effect of elevated Pb concentrations on plant growth parameters

During this experiment, Sulla plants were exposed to various concentrations of Pb (500, 750, and 1000 μM) in a hydroponic setup. The results presented in Fig. 5 demonstrate that lead had varying effects on different growth parameters of the plants. The control plants exhibited the highest shoot length, followed by the treatments with 750 μM and 1000 μM of Pb, with shoot lengths of 125 mm plant−1, 80 mm plant−1, and 65 mm plant−1, respectively. However, lead did not have a significant impact on root length, as there were no noticeable differences observed between the control group and both Pb treatments (750 μM and 1000 μM). Interestingly, the plants treated with only 500 μM of Pb displayed the lowest root and shoot lengths, indicating that they were most affected by lead. These results were also reflected in the measurements of shoot and root dry weights.

Fig. 5
figure 5

Effect of lead on Sulla plants grown under hydroponic conditions (growth parameters, total chlorophyll and carotenoids content, and antioxidant enzymes were measured). The growth parameters were determined by calculating the means from 10 replicates. As for the pigment parameters, the results are presented as means ± SE (n = 3). The mean values of antioxidant enzymes were calculated based on four replicates. Subsequently, an ANOVA test was conducted, and the Duncan test revealed significant differences (P < 0.05) among the mean values of different treatments, as denoted by distinct letters

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Furthermore, the impact of high Pb concentrations on chlorophyll content was evident, as the production of chlorophyll decreased with increasing Pb concentration. On the other hand, the carotenoid content in the shoots showed an increase with rising Pb concentration, although these differences were not statistically significant.

The measurements of oxidative enzymes in the plants revealed notable variations. Root and leaf APX activities were significantly higher in Sulla plants treated with 1000 μM of Pb, recording 10.58 U mg−1 protein and 2.68 U mg−1 protein, respectively, compared to the control plants with 3 U mg−1 protein and 0.44 U mg−1 protein, respectively. At this Pb concentration, roots’ POD activity was also significantly higher, recording 20.21 U mg−1 protein instead of 3.57 U mg−1 protein in control plants. However, in leaves, the maximum POD activity was recorded in plants treated with 750 μM of Pb, showing 1.52 U mg−1 protein compared to 0.27 U mg−1 protein in control plants. It is noteworthy that all the plants studied did not exhibit any CAT activity at the root level, but in the leaves, higher levels of CAT activity were recorded in plants treated with 750 μM and 1000 μM of lead compared to the control plants (Fig. 5).

Effect of Rhodococcus inoculation on the growth of Sulla spinosissima

In a controlled hydroponic environment, the inoculation of plants with the LMR356 strain exhibited beneficial effects on the plants growing under lead stress, as depicted in Fig. 6. The Pb-stressed inoculated plants demonstrated shoot and root lengths higher than non-inoculated Pb-stressed plants but comparable to the control plants grown under normal conditions. Moreover, the inoculation had a positive influence on the shoot and root dry weights of plants subjected to lead stress, with no significant differences observed between control and inoculated Pb-stressed plants. Additionally, Sulla plants inoculated with strain LMR356 showed partial recovery in chlorophyll and carotenoid content levels compared to plants treated with 1000 μM of Pb, although they performed significantly less efficiently than non-stressed plants.

Fig. 6
figure 6

Effect of Rhodococcus qingshengii LMR356 inoculant on Sulla plants grown under hydroponic conditions in the presence of lead (growth parameters, total chlorophyll and carotenoids content, and antioxidant enzymes, were measured). The growth parameters were determined by calculating the means from 10 replicates. As for the pigment parameters, the results are presented as means ± SE (n = 3). The mean values of antioxidant enzymes were calculated based on four replicates. Subsequently, an ANOVA test was conducted, and the Duncan test revealed significant differences (P < 0.05) among the mean values of different treatments, as denoted by distinct letters

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Regarding the enzymatic activities, both root and leaf APX activities were significantly higher in Pb-stressed plants inoculated with strain LMR356, measuring 10.82 U mg−1 protein and 4.96 U mg−1 protein, respectively, compared to non-inoculated non-stressed control plants, which recorded 5 U mg−1 protein and 0.96 U mg−1 protein, respectively. These values were also higher than those of non-inoculated plants treated with 1000 μM of Pb. On the other hand, POD activity was substantially greater in the roots of plants treated with 1000 μM (4.87 U mg−1 protein) compared to both control plants (0.57U mg−1 protein) and those inoculated with LMR356 (2.79 U mg−1 protein). Conversely, in leaves, POD activity was higher in inoculated plants (2.2 U mg−1 protein) than in control plants (0.27 U mg−1 protein). Moreover, CAT activity was not detectable in roots but was measured in leaves of inoculated plants (0.01 U mg−1 protein).

Evaluation of the effect of LMR356 on Sulla plants grown in sand pots

In a controlled environment using sterilized sand, the inoculation with strain LMR356 exhibited a positive influence on Sulla plants. This was evident by the higher shoot dry weights measured (68.07 mg plant−1) compared to the control plants (58.88 mg plant−1). However, it was noted that the root dry weights were lower for the inoculated plants in comparison to the control group. The roots of the latter plants were also longer than inoculated plants, but no significant difference was recorded between the shoot lengths of both types of plants (60 mm plant−1 for inoculated plants versus 54.28 mm plant−1 for the control).

Pigment measurements corresponded to the aboveground biomass, with Sulla-inoculated plants showing higher values of chlorophyll and carotenoids. Additionally, inoculated plants exhibited higher root and leave APX activities (35.19 U mg−1 protein and 15.01 U mg−1 protein, respectively) than control plants (7.82 U mg−1 protein and 12.9 U mg−1 protein, respectively). The same tendency was observed in root and leaf POD activities. As for CAT activities, inoculated plants displayed statistically higher values in leaves than control plants (0.03–0.05 U mg−1 protein). Conversely, the root catalase (CAT) activities in the inoculated plants were found to be statistically indistinguishable from those in the control plants (Fig. 7).

Fig. 7
figure 7

Effect of Rhodococcus qingshengii LMR356 inoculant on Sulla grown in sterilized sand (growth parameters, total chlorophyll and carotenoids content, and antioxidant enzymes were measured). The growth parameters were determined by calculating the means from 14 replicates. As for the pigment parameters, the results are presented as means ± SE (n = 3). The mean values of antioxidant enzymes were calculated based on four replicates. Subsequently, an ANOVA test was conducted, and the Duncan test revealed significant differences (P < 0.05) among the mean values of different treatments, as denoted by distinct letters

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Effect of Rhodococcus inoculation on Sulla spinosissima growth in a metal-contaminated soil

The bacterial isolate LMR356 demonstrated a significant impact on the growth of plants in the highly contaminated soil, as compared to the control non-inoculated plants (Fig. 8). Specifically, Sulla plants inoculated with LMR356 exhibited higher biomass production compared to the control group. This effect was also observed in terms of root biomass, shoot length, and root length. Significant differences were observed in all growth parameters (shoot length, root length, root dry weight) between the control group and the inoculated plants, with a valid significance level of P < 0.05 for all parameters except for shoot dry weight. Moreover, Sulla inoculation also resulted in a significantly higher chlorophyll content (1.49 mg g−1 FW) in comparison with control plants. Conversely, the carotenoid content of inoculated plants was lower than control plants.

Fig. 8
figure 8

Effect of Rhodococcus qingshengii LMR356 inoculant on Sulla plants grown in the heavy metal contaminated soil of Touissit (growth parameters, total chlorophyll and carotenoids content, and antioxidant enzymes were measured). The growth parameters were determined by calculating the means from 14 replicates. As for the pigment parameters, the results are presented as means ± SE (n = 3). The mean values of antioxidant enzymes were calculated based on four replicates. Subsequently, an ANOVA test was conducted, and the Duncan test revealed significant differences (P < 0.05) among the mean values of different treatments, as denoted by distinct letters

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Root and leave APX activities were noticeably lower in plants inoculated with LMR356 (47.4 U mg−1 protein and 26.13 U mg−1 protein, respectively) than in control plants (53.8 U mg−1 protein and 30.1 U mg−1 protein, respectively). The same trend was observed for roots and leaves POD activities. However, a different result was recorded for CAT activity, as neither inoculated nor control plants showed any activity in the roots. In contrast, in leaves, the inoculation of plants significantly lowered the CAT activity (0.02 U mg−1 protein) compared with control plants (0.041 U mg−1 protein).

Discussion

Mining sites, whether currently operational or abandoned, exert significant pressure on surrounding ecosystems, leading to pollution and ecological degradation. In Morocco, for instance, approximately 200 abandoned mining sites stand as stark reminders of this environmental challenge. Addressing the contamination of these areas emerges as a paramount concern for nations committed to sustainable development agendas. The remediation of such contaminated sites necessitates robust strategies aimed at mitigating the adverse effects of heavy metal toxicity, thereby safeguarding ecosystems for present and future generations. Among the array of remediation approaches available, biological solutions stand out for their effectiveness and relatively lower environmental risks. Leveraging agro-biotechnological methods, which capitalize on natural processes, offers a promising avenue for the elimination and stabilization of toxic metals in polluted environments. To accomplish phytorestoration of contaminated sites, this bioremediation approach employs either microorganisms or products of their metabolism, or higher plants and their rhizospheric bacteria. With this goal in mind, the present study seeks to explore the capabilities of Pb-tolerant PGPR Rhodococcus qingshengii LMR356 in promoting the growth of Sulla spinosissima and mitigating the effects of Pb-induced stress.

Notably, this strain exhibits multiple characteristics associated with plant growth promotion, enhancing its suitability for assisting in phytoremediation endeavors. Numerous strains within the Rhodococcus genus have been identified as possessing both PGP features and resistance to heavy metals such as lead (Pb). Examples include Rhodococcus erythropolis AV96 (Navazas et al. 2022), Rhodococcus hoagii (MG432495) (Jinal et al. 2019), Rhodococcus sp. NSX2 (Wang et al. 2020), Rhodococcus jostii B12 (Vergani et al. 2019), and Rhodococcus sp. (González Henao and Ghneim-Herrera 2021). These findings underscore the potential of Rhodococcus strains as valuable allies in the quest for sustainable remediation of metal-contaminated sites.

Lead tolerance of Rhodococcus qingshengii LMR356 and possible involved mechanisms

The strain under the current investigation exhibited pronounced resistance to lead at a concentration of 30 mM. However, it was observed that bacterial survival declined progressively under the influence of lead stress, aligning with earlier research findings (Gikas et al. 2009). Lead is recognized for its capacity to instigate structural destabilization of the cytoplasmic membrane, thereby leading to a reduction in the bacterial population. Moreover, the slowing of the onset of growth is a very common aspect of the effects of sub-lethal metal concentrations on bacteria (Aljerf 2018).

According to Li et al. (2021a, 2021b), exopolysaccharide (EPS) production is one of the mechanisms of heavy metal tolerance in bacteria. It prevents heavy metals’ entrance into bacterial cells through the nonspecific binding of heavy metals and EPS. This is the case of strain LMR356 that produced a large amount of EPS and was able to form biofilm at 30 mM of lead. EPS may also decrease metal toxicity in plants regarding the adhesive properties of EPS that assist bacteria in combining with soil particles and heavy metals (Zhang et al. 2006; Gupta and Diwan 2016; Bhagat et al. 2021). Polysaccharides, according to BeMiller (2019), are acidic and have a significant affinity to particular ions. These EPS help to build bacterial aggregates, which improve soil aeration and root development (Bhagat et al. 2021).

Biofilm formation is another characteristic of the studied Rhodococcus strain that is important for lead resistance. In this respect, it was found that higher levels of Pb increased Rhodococcus biofilm development. Studies have reported that bioremediation performed by biofilm cells surpasses that of planktonic cells due to the biofilm cells’ ability to adapt and thrive in stressful conditions, being shielded within the matrix. This phenomenon highlights the multifaceted advantages conferred by biofilm in the context of environmental stressors such as lead contamination (Rodrigues and de Carvalho 2015; Adhami et al. 2017). In addition to EPS and biofilm formation, Pb-tolerant bacterial strains can employ other sophisticated distinct mechanisms to efficiently survive in a Pb-contaminated environment. Several mechanisms have been identified for reducing Pb toxicity, including biosorption, Pb efflux, the formation of metal chelators like metallothionein and siderophores, as well as bioaccumulation (Najm-ul-Seher Ahmad et al. 2021; Pande et al. 2022). The last mechanism was estimated for strain LMR356, and we found that the strain has a high Pb bioaccumulation capacity. The metals associated with the intracellular compartment (cytoplasm) were measured in bacterial pellets (after washing with water) and those associated with the extracellular compartment (membranes and polysaccharides) were determined after washing with EDTA, a chelator that is utilized to capture and remove metallic components adsorbed on the surface of bacterial cells (El Aafi et al. 2015). EDTA washing demonstrated that the overall metal accumulation was consistently higher than the accumulation inside the cell, suggesting that a significant portion of the metal was adsorbed onto the cell surface. This is may be due to the high concentration of polysaccharides produced by the studied strain, where the metals could be trapped and thus adsorbed on these polysaccharides found on the cell surface. El Aafi et al. (2015) reached the same conclusion. These results indicate that the Rhodococcus strain LMR356 is not only capable of tolerating lead but also accumulating high levels of this metal, particularly on the cell surface, which indicates that this strain is suitable for bioremediation in heavy metal-contaminated soils or waters. Alternatively, this type of bacteria can be employed in rhizoremediation by inoculating them to plant roots to increase and improve their tolerance to polluted environments. Moreover, the studied Rhodococcus strain showed a chemotaxis response under Pb stress. Many studies showed similar results such as Rhodococcus sp. BAP-1 (Li et al. 2014), and Rhodococcus ruber and Rhodococcus pyridinivorans (Wang et al. 2018). Aroney et al. (2021) reported that chemotaxis systems, which respond to abiotic stresses, tend to govern bacterial motility, enabling bacteria to migrate toward optimal environments.

Rhodococcus strains not only grew in the presence of lead (Pb) but also in the presence of other heavy metals. In a study conducted by Wevar Oller et al. (2013), Rhodococcus erythropolis AW3 demonstrated growth in high concentrations of arsenite and arsenate. The bacterial strains exhibited resistance of up to 24 mM for arsenite and up to 400 mM for arsenate. In a related study by González Henao and Ghneim-Herrera (2021), a Rhodococcus strain exhibited resistance to high concentrations of arsenite. In another study conducted by our team, Oubohssaine et al. (2022a, 2022b) Rhodococcus strains demonstrated the ability to tolerate high concentrations of lead (Pb), zinc (Zn), and arsenic (As).

Plant growth-promoting traits of Rhodococcus qingshengii LMR356

The strain LMR356 is able to produce significant levels of ammonia, IAA, and siderophores, to solubilize rock phosphate, and synthesize ACC deaminase. Some of these activities were reduced under lead stress, while others were increased and others were not affected.

Phosphorus is an important nutritional source for plants; nevertheless, P deposited in soil is generally inaccessible to plants (He et al. 2021). As a result, isolating bacteria that can use these soil P reserves is of significant interest. Bacteria with P-solubilization ability may solubilize soil inorganic P that plants are unable to utilize, which is an excellent strategy to increase plant P absorption (Ahmad et al. 2022; Adetunji et al. 2022). Strain LMR356 is a phosphate-solubilizing bacteria, but its capacity was reduced under Pb stress. Similar results were reported for a strain of Enterobacter under Cd stress (Li et al. 2022a). The strain LMR356 was also able to produce ammonia even under Pb stress. Rhizobacteria producing ammonia are crucial to plant’s growth and health and play an important role in nutrient cycling (Hayat et al. 2010; Kumar and Verma 2018; Imran et al. 2021).

Among the PGP traits of rhizobacteria, IAA is often researched, especially under stressful conditions. IAA belongs to a significant auxin family and plays a pivotal role in plant root initiation, cell division, and cell enlargement (Borah et al. 2019; Wang et al. 2022a; Wang et al. 2022b; Robas Mora et al. 2022). As a result, PGPR that produces IAA can significantly enhance plant biomass, stimulate rooting and germination, and foster the growth of root hairs and cotyledon cells. The production of IAA is associated with root growth and structural changes in response to stress (Vacheron et al. 2013; Adeleke et al. 2022). In the current research, IAA secretion by strain LMR356 decreased considerably at 30 mM of lead. Li et al. (2022a) elucidated that heightened levels of lead exposure can induce a reduction in the activity of the IAA synthetase system, accompanied by a depletion of the synthetic tryptophan pool. This dual effect may significantly hinder the biosynthesis of indole-3-acetic acid (IAA), a crucial plant growth regulator. This finding underscores the intricate mechanisms through which lead exerts its inhibitory influence on IAA production, shedding light on potential pathways for mitigating its adverse effects on plant development and physiology. Unlike IAA, siderophores production was promoted when Rhodococcus qingshengii LMR356 was exposed to high amounts of Pb. Similarly, Sinha and Mukherjee (2008) discovered that Pseudomonas aeruginosa KUCd1 produced siderophores in response to high Pb exposure.

ACC deaminase activity is one of the PGP attributes that was enhanced strongly in LMR356 exposed to high amounts of lead. This activity is very important in the context of heavy metal stresses. PGPR isolates degrade ACC, the precursor of ethylene, into ammonia and α-ketobutyrate. As a result, the negative effects of ethylene are reduced, and germination improves, resulting in increased plant development under lead circumstances (Glick et al. 2007; Ghosh et al. 2018; Haldar et al. 2022).

Thus, Rhodococcus qingshengii LMR356 shows different PGP characteristics even when exposed to Pb, which provides this bacterium an edge over other competitors in the rhizosphere of plants (Lopez et al. 2022).

Sulla spinosissima behavior under lead stress in hydroponic system

In situ investigations were performed in hydroponic conditions, utilizing the plant species Sulla spinosissima. This species was selected owing to its inherent capacity to thrive naturally in soils heavily contaminated with multiple heavy metals within the Oujda mining region.

We started by looking at how lead concentration affected plant growth, chlorophyll and carotenoid levels, and antioxidant enzyme activities. We found that as the concentration of lead increased, the growth of Sulla plants decreased as compared to control plants and variable responses across the three concentrations tested (500, 750, and 1000 μM) were recorded for the different parameters used.

Excessive concentrations of heavy metals are widely recognized for their detrimental effects on plant biomass, exerting negative influences across various critical stages, including germination, growth, development, and key physiological processes such as photosynthesis (Yaashikaa et al. 2022; Singhal et al. 2022; Podar and Maathuis 2022). Plants’ fast and rapid synthesis of reactive oxygen species (ROS) following Pb exposure is regarded as an early defense response (Shahid et al. 2014; Berni et al. 2019). The development of Sulla plants can be attributed to a complex interplay involving reactive oxygen species (ROS) and biomolecules, coupled with challenges such as inefficient water and nutrient absorption and electrolyte loss due to altered membrane permeability. The diminished chlorophyll content observed in Sulla plants may result from elevated ROS levels or the inactivation of enzymes crucial for chlorophyll synthesis (Batool et al. 2019). The presence of heavy metals is recognized for its impact on diminishing the levels of photosynthetic pigments, predominantly by influencing the integrity of cell walls and thylakoid membranes, and also through alterations in proteins and DNA caused by ROS interference (Ajmal et al. 2022; Noor et al. 2022; Sharma et al. 2022a). This intricate cascade of events underscores the multifaceted impact of heavy metal exposure on plant physiology, emphasizing the importance of understanding these mechanisms for devising effective strategies to mitigate heavy metal-induced stress in plants like Sulla. Carotenoids content increased in response to Pb stress, which can be explained by the presence of nitrogen, as reported by Gurpreet et al. (2012). Whereas the increase in CAT, APX, and POD activities may be related to the activation of plant defense systems that confer resistance to heavy metals stress in plants (Ajmal et al. 2022). These antioxidant enzymes catalyze free radical conversion, and their increased activity leads to plant bioprotection against abiotic stresses. These findings, together with those of Sbabou et al. (2016) and Lamin et al. (2020), indicate that Sulla plants are phytostabilizing plants due to their root structure, which endures at high lead concentrations.

Rhodococcus qingshengii LMR356 effect on Sulla spinosissima growing under Pb stress in hydroponic

The Rhodococcus strain utilized in this study increased the vegetative development of Sulla plants under 1000 μM Pb as compared to uninoculated plants. Many possible traits of the strain can be implicated in such improvement, IAA biosynthesis, inorganic phosphate solubilization, siderophores production, and others. A reduced level of IAA can impact the bioavailability of heavy metals (HMs) in the rhizosphere microenvironment, leading to an increase in HM absorption (Li et al. 2022a, 2022b) and a significant enhancement in root development (Patten and Glick 2002). Additionally, the application of IAA was found to elevate the activity of H+-ATPase in the root plasma membrane of Medicago sativa L., promoting H+ secretion from root tips and resulting in a lowered rhizosphere pH (Wang et al. 2018). Moreover, P-solubilizing bacteria are involved in the production of organic acids, which leads to a decrease in pH, cation chelation, and competition with phosphate for soil adsorption sites (de Freitas Duarte et al. 2022; Bhardwaj et al. 2022). Additionally, the secretion of organic acids by P-solubilizing bacteria was reported to play a role in the dissolution of HMs (Gupta and Diwan 2016). Various organic acids, including ketogluconic acid, gluconic acid, citric acid, oxalic acid, tartaric acid, succinic acid, and others, undergo protonation and conversion into low-molecular-weight organic acids, potentially leading to alterations in soil pH and redox potential, thereby promoting the dissolution of HMs (Li et al. 2022a). Additionally, the secretion of organic acids by siderophore-producing bacteria can modify the bioavailability of heavy metals like Pb in the rhizosphere (Saha et al. 2016; Podar and Maathuis 2022; Pandey et al. 2022; Sharma et al. 2022b). Moreover, this process may facilitate the interaction of siderophores with heavy metal ions, forming metal-siderophore chelates, which enhance heavy metal activity in the plant rhizosphere, consequently increasing their accumulation (Barman and Jha 2021; Mitra et al. 2021; Wang et al. 2022a). Studies by other researchers have demonstrated that bacterial siderophore synthesis can mitigate oxidative stress and ethylene stress, while also providing essential mineral nutrients for plant growth and development (Nazli et al. 2020a; Devi et al. 2022; Koza et al. 2022). Rhodococcus’ mobility also allows bacterial cells to escape a harsh environment, giving them a survival benefit. According to Bashan and Holguin (1994) and Williams (2011), motility is an active process involved in effective colonization. Bacterial motility toward root exudates and the capacity to form EPS and biofilms are essential for rhizosphere and rhizoplane colonization (Lucero et al. 2020).

Effect of LMR356 on Sulla spinosissima growing in sterilized sand

Inoculation of Sulla plants growing in sterilized sand improved plant growth and chlorophyll and carotenoid content. These findings are consistent with the strain’s in vitro PGP features, in particular improved P bioavailability via inorganic phosphate solubilization, siderophores production that improves plant iron uptake, and phytostimulation by auxin production. Plants that were inoculated displayed shoot lengths and dry weights comparable to those of control plants. The slight rise in antioxidant enzyme activities, such as peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT), observed in certain instances might be attributed to the stimulation of plant defense mechanisms by the inoculated Rhodococcus strain (Oubohssaine et al. 2022b).

Impact of inoculation on Sulla spinosissima growth in multi-heavy metal-polluted soil

The Touissit mine soil used in this experiment is heavily contaminated with various heavy metals, particularly As and Pb (Oubohssaine et al. 2022a). Inoculation significantly enhanced shoot and root lengths, as well as the chlorophyll content of the plants growing in this soil. Numerous studies have provided evidence that the inoculation with PGPR strains can elevate the concentration of photosynthetic pigments under conditions of heavy metal stress (Jinal et al. 2019; Zainab et al. 2021; Kaur et al. 2021; Navazas et al. 2022). This effect may be attributed to increased nutrient absorption through phosphate solubilization by the inoculated strain and the release of active substances that play a critical role in the production of photosynthetic pigments (Santoyo et al. 2021; Saeed et al. 2021; Das et al. 2022). Carotenoids, renowned for their ability to scavenge ROS, play a crucial role in shielding the plant’s photosynthetic machinery from photo-oxidative disruptions (Khanna et al. 2019; García-Caparrós et al. 2021; Zandi and Schnug 2022; Kaur and Goyal 2022). Furthermore, characteristics such as phosphate solubilization, siderophore production, IAA (Indole-3-acetic acid) synthesis, EPS (extracellular polymeric substances) production, biofilm development, and nutrient availability contribute to strain’s capacity to support plant growth under heavy metal stress conditions (Khoshru et al. 2020; Nazli et al. 2020b; Syed et al. 2021; Zainab et al. 2021; Singh et al. 2022).

The outstanding performance of LMR356 observed in different plant inoculation experiments (hydroponic or pot) may be attributed to its origin and properties. Being isolated from Touissit’s soil, the strain is well-adapted to polluted environments, tolerating high levels of lead. In the context of this investigation, the level of Pb in soils appears to be a crucial factor. Strain LMR356 produces IAA, ACC deaminase, and EPS, and it has the ability to solubilize phosphate, form biofilm, and exhibit mobility.

When comparing our study with others, conducted under multi-heavy metal-contaminated conditions of Touissit’s soil, the results demonstrate that plants inoculated with a Pb-tolerant PGPR Rhodococcus strain exhibited reduced antioxidant enzyme activities, especially in the aerial parts of the plants. It is well recognized that plants employ a detoxifying antioxidative system consisting of various antioxidant enzymes, such as APX, CAT, and POD, to maintain an optimal level of ROS. The activity of these enzymes can vary depending on factors such as metal concentration, exposure duration, metal ion, and plant species (Sharma et al. 2019; Raza et al. 2021; Hasanuzzaman et al. 2021).

Inoculated plants retained high levels of biomass, chlorophyll, and carotenoid content. Consequently, the decrease in antioxidant enzyme activities may be linked to the action of tolerant PGPR Rhodococcus in the rhizosphere in relation to heavy metals, as documented in a study by Li et al. (2022b). In recent studies, the importance of interactions between plants and tolerant PGPR in heavy metal-contaminated soils has been emphasized, as they can expedite phytoremediation and safeguard plants against the detrimental effects of metals (Deb et al. 2020; Yan et al. 2020; Zainab et al. 2020; Zainab et al. 2021; Oladoye et al. 2022).

In a study by Wang et al. (2022c), it was demonstrated that phytoextraction of cadmium (Cd), zinc (Zn), and lead (Pb) using Vetiveria zizanioides, Brassica juncea, Lolium perenne, Solanum nigrum, and Sedum alfredii was significantly enhanced by inoculation with ABA-catabolizing Rhodococcus qingshengii. This strain notably improved the biomass of the aforementioned hyperaccumulators, thus markedly enhancing the capacity for remediation of Cd, Zn, Pb, and copper (Cu). In another study conducted by Kathi (2022), the phytoextraction of heavy metals and crude oil contaminants from the soil was assessed using the native grass Cynodon dactylon. The results indicated that crude oil contamination caused a reduction in plant biomass across all treatments. The degradation order of metals was found to be Pb, Zn, Cu, and Cd. Higher percentages of degradation of heavy metals and crude oil were observed in Rhodococcus ruber-inoculated treatments compared to other treatments, attributed to the effectiveness of the combination of R. ruber and C. dactylon in soil.

Lu et al. (2020) also confirmed that R. qingshengii improved the phytoextraction efficacy of Cd, Zn, and nickel (Ni). Du et al. (2022) demonstrated that Rhodococcus qingshengii facilitates the phytoextraction of Zn, Cd, Ni, and Pb from soils by Sedum alfredii Hance. Two Rhodococcus erythropolis strains (S4 and S10) isolated from the rhizosphere of Mesembryanthemum crystallinum treated with 10 mM Cd confirmed their positive impact on plant growth and biomass yield, mainly through phosphate and zinc solubilization (Supel et al. 2022). All these studies collectively demonstrate the ability of the genus Rhodococcus to remediate heavy metal-contaminated soils.

Same, our study demonstrates that inoculating with a locally selected Pb-tolerant PGPR Rhodococcus strain promotes the growth of Sulla plants in highly polluted soil, suggesting it as a promising alternative for soil remediation. The inoculation of Sulla plants with this Rhodococcus strain counterbalanced the adverse impacts of lead stress and led to an increase in physiological parameters, including chlorophyll content. These findings align with previous research, which demonstrated that PGPB inoculation promotes enhanced chlorophyll production and accelerates plant growth (Oubohssaine et al. 2022b).

Conclusions

Our study presents compelling evidence of the efficacy of utilizing a multi PGP traits Rhodococcus strain, sourced from a metal-polluted habitat, as a bio-inoculant for enhancing the growth of Sulla spinosissima in heavy metal-contaminated soils. By demonstrating significant improvements in various growth parameters, including biomass production, photosynthetic pigment levels, and antioxidant enzyme activity, our findings underscore the potential of such microbial interventions in facilitating phytoremediation processes. Moreover, our results emphasize the importance of selecting indigenous microbial strains that exhibit resilience to heavy metal stress and possess diverse phytobeneficial properties. This approach not only enhances the efficacy of bio-inoculants but also ensures their compatibility with local environmental conditions, thereby promoting sustainable remediation practices. Furthermore, our study contributes to the growing body of knowledge on the application of plant growth-promoting rhizobacteria (PGPR) in phytoremediation. By highlighting the unique capabilities of PGPR in mitigating heavy metal toxicity and improving plant health, we advocate for the integration of microbial-based strategies into broader remediation initiatives. Importantly, our research underscores the novelty and significance of these findings within the context of environmental science and biotechnology. By elucidating the practical benefits of PGPR inoculation in heavy metal-contaminated soils, we offer valuable insights that can inform future research and guide the development of innovative remediation technologies.

In conclusion, our study not only advances our understanding of microbial-assisted phytoremediation but also underscores the critical role of indigenous microbial communities in sustainable environmental management. By harnessing the potential of microbial bio-inoculants, we can pave the way for more effective and environmentally friendly approaches to soil remediation and ecosystem restoration.