Alleviation of salinity and metal stress using plant growth-promoting rhizobacteria isolated from semiarid Moroccan copper-mine soils

Phytoremediation is an eco-friendly method for rehabilitation of mine tailing. Some heavy metals and salt-tolerant plant growth-promoting rhizobacteria (PGPR) could be beneficial in alleviating soil salinity and heavy metal stress during plant growth. The aim of this work is to select PGPR that could be used in phytoremediation process. Twenty-nine rhizobacteria are examined for their ability to grow at increasing concentrations of NaCl, Zn, Pb, Cu, and Cd. The results showed that seventeen rhizobacteria displayed high salinity and metal tolerance up to 100 g L−1 of NaCl, 5 mM of Cd, 9 mM of Pb, 10 mM of Zn, and 6 mM of Cu. Moreover, almost all tested bacteria maintained their PGP traits under 10% of NaCl and multi-metal stress. Based on seedling bioassay under metallic and salt stress, using Peganum harmala L. and Lactuca sativa L., beneficial effects of seed inoculation with bacterial consortia (Mesorhizobium tamadayense, Enterobacter xiangfangensis, Pseudomonas azotifigens, and Streptomyces caelestis) have been observed in terms of root and shoot elongation. Our results show that the stress-tolerant consortium used has a great potential to sustain plants establishment in heavily disturbed soils.


Introduction
Rapid population growth and increase in urbanization relies strongly on mining activities and industrial sector in order to produce commercial materials (Kabata-Pendias and Pendias 2001). These activities involve several steps releasing different wastes, which may contain significant amounts of toxic contaminants that can pollute the air, soil, water, and could affect negatively flora and fauna (El Khalil et al. 2008;El Hamiani et al. 2010). Moreover, due to their high toxicity and persistence within the environment, heavy metals are one of the major pollutants of concern to human health (Adriano 2001). Once accumulated in soil, the toxic metals are transferred by roots to different plant parts. Consequently, they can be accumulated in the human body through food webs, causing chronic and acute disorders, and leads to serious health problems afterwards (Boularbah et al. 2006a(Boularbah et al. , 2006bEl Khalil et al. 2008;El Amari et al. 2014;El Hamiani et al. 2015). Furthermore, for sensitive plants, high level of these contaminants is extremely toxic. Indeed, high concentrations of heavy metals in soil can lead to several adverse damages in plants root system, membrane permeability, and photosynthesis process resulting in restricting of plant growth and lead to decrease of yield and quality of crops (Prabu 2009;Rizvi and Khan 2018;Jiang et al. 2019;Wang et al. 2019). In addition, several studies indicated that high level of heavy metals had various impacts on soil microbial composition and functional diversity (Boularbah et al. 1992;Benidire et al. 2016;Alam et al. 2019;Jiang et al. 2019;Lin et al. 2019;Liu et al. 2019).
Strong efforts have been made to develop an eco-friendly and cheaper technique for the restoration of heavy metals degraded soils and preventing the spread of contaminants through wind and/or water erosion. In opposition to physicochemical technologies that interfere with the soil structure, phytostabilization is one of the most environmentally friendly technologies used to decrease metal bioavailability in soil and reduce their toxicity (Testiati et al. 2013;Acosta et al. 2018;Lebrun et al. 2018). However, plants growing in metal-polluted soils usually encounter adverse growth due to edaphic and climatic factors, such as drought and salinity, which limit plant growth, biomass production, and thus influence the efficiency of the phytoremediation process (Pavel et al. 2014;Cui et al. 2020). Nevertheless, the use of plant growth promotion rhizobacteria (PGPR) may be a promising solution to overcome these problems. Indeed, it has already been demonstrated that the application of metal-tolerate PGPR may decrease the availability of metal contaminants and promote plant growth under stressed conditions (Ma et al. 2016;Benidire et al. 2020;Ju et al. 2020;Rani et al. 2021). A wide range of abiotic stress-tolerant bacteria can confer adaptive benefit to their host plant against various stress, such as heavy metals, drought, and salt through various mechanisms, including the production of plant growth promoting (PGP) substances like phytohormones (gibberellic acid, cytokinin, and indole-3-acetic acid (IAA)) involved in the regulation of root architecture, cell division and differentiation, improved seed germination, and plant stress tolerance (Ma et al. 2016;Vurukonda et al. 2016;Román-ponce et al. 2017). Several PGPR have the ability to synthesize stressalleviating enzymes such as 1-aminocyclopropane-1carboxylate (ACC) deaminase that degrades the precursor of stress ethylene in plants, thus making them more resistant and facilitating their development in harsh environments (Nascimento et al. 2012;Din et al. 2019;Kumar et al. 2021). PGPRs have also the ability to supply the necessary nutrients to plants by fixing atmospheric nitrogen, producing ammonia and siderophore, as well as solubilizing nonavailable phosphorus through the release of acids and chelating agents or via the use of enzymes (e.g., acid phosphatase) (Jha and Saraf 2015;Wei et al. 2018;Prabhu et al. 2019). In addition, it has been recently reported that improving population density of metal and osmotolerant rhizobacteria in the root zone can significantly decrease the content of toxic metal ions via binding these cations through exopolysaccharides (EPS) production leading to the decrease of available cations for plant uptake and alleviate salt and metal stress in plants (González et al. 2010;Upadhyay et al. 2011;Zhu et al. 2018;Din et al. 2019). Therefore, improving plant-PGPR interactions can enhance plant biomass production under environmental stresses (e.g., salinity, drought, and metal stresses), immobilization of heavy metals, and consequently leading to the success of the phytoremediation strategy.
Recently, PGPR have been widely used as efficient biofertilizers to increase crop productivity especially under stressed environment (Egamberdieva and Adesemoye 2016;Mushtaq et al. 2021;Kumar et al. 2021). Indigenous rhizobacteria isolated from stressed environment are reported to display high resistance capacity to stress (Upadhyay et al. 2011;Chakraborty et al. 2013;Ma et al. 2016). Thus, the effect of stress on the physiological mechanisms and adaptation strategies used by the PGPR to overcome the stressed conditions (e.g., drought, salinity, and metal stress) has been previously reported. In fact, research has shown that PGPR can maintain their ion homeostasis in response to osmotic stress by accumulating small compatible solutes such as amino-acids, proline, and sugars which serve as osmoprotectants and may also contribute as scavengers of free radicals produced by stressful conditions (Armada et al. 2015;García et al. 2017;Abbas et al. 2019;Ghosh et al. 2019).
The objectives of this study were (1) to screen rhizobacteria for their tolerance to high levels of salt, and metal stress; (2) to understand adaptive mechanisms used by rhizobacteria to overcome salinity and heavy metal stress; (3) evaluate the effect of abiotic stress on PGP traits of the studied strains; and (4) finally assess the effect of heavy metal and osmotolerant PGPR on seedling growth under osmotic and metallic stress.

Methods and materials
Screening of plant growth-promoting rhizobacteria for their abiotic stress tolerance Twenty-nine rhizobacteria used for this study were previously isolated from the rhizosphere of some metallophytes species namely Haloxylon scoparium, Peganum harmala, Aizoon canariense, Atriplex colerei, and Ononis natrix, growing on multi-metal contaminated soils of a former copper mine site located at 35 km of Marrakech city, Morocco, under semi-arid climate. The abandoned mine tailings are characterized by a very acidic pH (2.8), high electrical conductivity (2.45 mS cm −1 ), and high levels of Cu, Zn, Pb, and Cd (Boularbah et al. 2006a;Benidire et al. 2016). Bacterial strains were screened for their ability to tolerate osmotic and heavy metal stress as described below.

Bacterial growth studies under increasing levels of NaCl
Bacterial salt stress tolerance was tested on nutrient broth medium (Biokar) supplemented with five different concentrations of sodium chloride (NaCl): 20, 40, 60, 80, and 100 g L −1 . Bacterial cultures were incubated at 28°C for 6 days under shaking conditions. Positive strain growth was confirmed by measuring optical density at 600 nm. Viable cells were estimated by the appearance of recognizable individual colonies in the trypticase soy agar (TSA) (Biokar) plates after plating 100 μL of bacterial suspension from each treatment on the surface of TSA. For this test, medium containing only the bacterial strains was used as a control. The experiment was conducted in triplicate.

Metal resistance test
The effect of metals on PGPR strains was tested by inoculating each bacteria in Luria broth (LB) (Sigma) supplemented with different concentrations of single and multi-metal solutions as follows: Cu at 3,5, 6, and 7 mmol L −1 as CuSO 4 ; Zn at 3, 5, 10, and 15 mmol L −1 as ZnSO 4 ; Pb at 3, 6, 9, and 12 mmol L −1 as Pb(NO 3 ) 2 ; Cd at 2, 3, 4, 5, and 6 mmol L −1 as Cd (NO 3 ) and mixed-metal solutions of CuSO 4 /ZnSO 4 / Pb(NO 3 ) 2 /Cd (NO 3 ) at 0.2, 0.5, 1, 2 and 3 mmol L −1 . The cultures were kept in a rotary shaker for 48 h at 28°C and 150 rpm. The viability of bacteria exposed to metal stress was checked using the triphenyl tetrazolium chloride (TTC) test as described by Pandey and Bhatt (2015). A positive metal tolerance test is indicated by the appearance of a red color in the tube after the addition of TTC. Bacterial cultures in LB medium not supplemented with metal salts were used as a control. The experiments were carried out in triplicate.

Evaluation of PGP activities of osmotolerant strains under salt and metallic stress
Seventeen PGPR strains, able to tolerate the highest levels of osmotic stress (up to 100 g L −1 of NaCl), were chosen to test their PGP traits under 10% of NaCl and under mixed-metal solution of CuSO 4 /ZnSO 4 /Pb (NO 3 ) 2 /Cd (NO 3 ) at 0.5 mM for each metal.
The effect of salt and metallic stress on phosphate solubilization ability of rhizobacterial strains was evaluated in the National Botanical Research Institute's phosphate growth medium (NBRIP) (Nautiyal 1999;Singh et al. 2015) amended with 0.5% of tricalcium phosphate and 10% of NaCl or 0.5 mM of mixed-metal solutions. Triplicate 100-mL Erlenmeyer flasks containing 40 mL of NaCl-NBRIP medium were inoculated with 1 mL of fresh bacterial suspension with optical density of 0.5 at 600 nm equivalents to 3.10 8 CFU mL −1 . NBRIP media without NaCl or mixedmetal solution were used as a control. The flasks were incubated at 28°C for 5 days under constant agitation at 130 rpm. Supernatants of the bacterial cultures were collected by centrifugation at 10,000 rpm for 10 min and were used for quantitative estimation of available phosphorus by the Olsen method (Watanabe and Olsen 1965).
To assess the effect of salt and metallic stress on siderophore production, Chrome Azurol (CAS) broth medium was prepared according to Schwy and Neilands (1987) and was amended with 10% of NaCl or a mixture of four metals (Cu, Pb, Zn, and Cd) at 0.5 mM. The modified medium was inoculated with 100 μL of fresh bacterial culture and incubated at 28°C for 5 days under shaking condition at 130 rpm. Non-amended CAS medium was used as control. The development of yellow-orange color was considered as a positive result for siderophore production. The efficiency of siderophore production by bacterial strains under stress was estimated by comparing color intensity on the modified CAS medium with unmodified control by measuring absorbance at 630 nm by UV spectrophotometer.
Ammonia production was estimated according to Cappuccino and Sherman (1992). Bacterial strains were inoculated in peptone solution amended with 10% of NaCl or the mixture of four metals (Cu, Pb, Cd, and Zn) at 0.5 mM and incubated at 28°C. After 5 days of incubation, a volume of 500 μL of Nessler's reagent was added to each tube. Development of a brown to yellow color on the bacterial culture was considered as positive test for ammonia production. Bacterial cells incubated in non-modified peptone solution were used as a control.
PGPR strains were also screened for their ability to produce indole-3-acetic acid (IAA) according to Gordon and Weber (1951) under salt (10% of NaCl) and metallic stress (the mixture of four metals (Cu, Pb, Cd, and Zn at 0.5 mM). Fresh cultures were cultivated in LB broth supplemented with 1% Ltryptophan (5 mM) and 10% of NaCl or multi-metal solution for 5 days at 28°C. Medium added with only 0.5% of NaCl as usual concentration was used as control. After incubation, 1 mL of the supernatant obtained by centrifugation at 10,000 rpm for 10 min was mixed with 2 mL of Salper reagent (2% of 0.5 M FeCl 3 in 35% HClO 4 solution) and kept in the dark for 30 min. The optical density of the extracted sample and standard IAA were measured at 530 nm. For each strain, the experiment was carried out in replicate.

Bacterial response to osmotic and metallic stress
In order to explore possible mechanisms by which PGPRs mitigate the effects of abiotic stress and their potential to improve plant stress tolerance, their ability to produce EPS, sugars, amino acids, proline, and soluble proteins content were analyzed.

Exopolysaccharides production
The ability of bacterial strains to produce exopolysaccharides (EPS) was assessed both in the presence and in the absence of salt (10 g L −1 ) and metallic stress (the mixture of Cu, Pb, Cd, and Zn at 0.5 mM). A quantitative determination of EPS production was performed as described by Ghafoor et al. (2011) with few modifications. Briefly, a volume of 1 mL of supernatant, obtained from rhizobacterial cultures grown in nonamended LB and in LB supplemented with NaCl (10%) or multi-metal solution for 5 days at 28°C, was mixed with 1 mL of 2% (w/v) Congo Red solution and incubated under shaking (120 rpm) for 120 min at 28°C. Bacterial cells and bound Congo Red were precipitated by centrifugation at 10,000 rpm for 5 min. The supernatant was then collected and its optical density was measured at 490 nm. The amount of EPS produced was estimated by determining the total percentage of free Congo Red remaining in the supernatant.

Free amino acids production
Free amino acids were determined spectrophotometrically by using the Ninhydrin method as described by Ondobo et al. (2017). The bacterial cells were grown for 5 days at 28°C in non-stressed and stressed nutrient broth media with a mixture of four metals (Cu, Pb, Cd, and Zn) at 0.5 mM and 10% of NaCl, respectively. The bacterial pellets obtained after centrifugation of cultures at 10,000 rpm for 10 min were boiled in a water bath at 60°C for 45 min in the presence of 80% methanol. The resulting extract was then centrifuged and the amino acids content was estimated in the supernatant by measuring its absorbance at 570 nm. A pure analytical grade glycine was used as a standard curve at concentrations of 25, 50, 100, and 150 μmol mL −1 .

Proline production
Accumulation of proline was analyzed spectrophotometrically following the method described by Bates et al. (1973) with slight modifications. Briefly, bacterial cells obtained by centrifugation of 5 days culture grown in nutrient broth supplemented or not with10% of NaCl or mixed-metal solution (same concentration as used above) was mixed with ninhydrin in glacial acetic acid for1 h at 100°C and then the tubes were placed in an ice bath to stop the reaction. Subsequently, proline content was extracted by adding toluene and the absorbance was measured at 520 nm. The experiment was repeated three times. Proline concentration was determined using a calibration curve of pure proline as a standard at concentrations of 50, 100, 150, 200, and 250 μg mL −1 .

Soluble sugars accumulation
Total sugars content was determined on non-stressed (0.5% NaCl) and stressed (10% NaCl, and mixed-metal solution) cultures according to the procedure described by Dubois et al. (1956). The pellet of bacterial cell culture was mixed with methanol-chloroform (4:1) solution and boiled in water bath at 60°C for 20 min. The obtained supernatant was then treated with phenol (5%) and sulfuric acid (98%). The absorbance of the mixture was read at 485 nm after 20 min of incubation at 100°C. A standard curve was prepared with known concentrations of glucose at concentrations of 25, 50, 100, 150, and 200 μg mL −1 .

Proteins contents
The protein content of bacterial cells was determined by using Bradford (1976). Pellets, obtained by centrifugation of bacterial cultures grown in non-stressed and salt (10% NaCl) or multi-metal stressed LB medium, were washed vigorously with MgSO 4 (10 mM) and resuspended in 500 μL of phosphate buffer (pH 6.8). The extract was kept for 30 min at room temperature. Finally, proteins concentration in the supernatant was determined by reading the absorbance at 595 nm and using bovine serum albumin as a standard working solution.
Effect of selected PGPR strains on seedling root and shoot growth under salt and metallic stress

Bacterial inoculant preparation
In order to evaluate the effect of PGPR on seed germination and plant root elongation, a consortium formulated with four bacterial strains was used for this assay: Mesorhizobium tamadayense BKM 04, Enterobacter xiangfangensis BKM 30, Pseudomonas azotifigens BKM 07, and Streptomyces caelestis BKM 05. These bacteria were selected based on their potent tolerance to abiotic stress and their ability to maintain high levels of PGP activities under salt stress. Therefore, the two strains BKM 04 and BKM 05 were chosen for their best indole acetic acid (IAA) and siderophore production, BKM 30 for its P solubilizing capacity and BKM 04 for its ability to produce high levels of EPS under stressed condition (Tables 3  and 4). To test that there is no antagonistic activity between the chosen strains, plate confrontation tests were performed in TSA medium (Upadhyay et al. 2011). To this end, 1 mL of a bacterial culture was spread evenly over the surface of Petri plates prepared with TSA. Then, another bacterial culture was spotted on the bed of the first one. The resulting plates were incubated at 28°C for 48 h. The absence of clear bacteria-free zone around the spotted cultures indicated the absence of an antagonistic effect between the two tested rhizobacteria.
For the germination test, the bacterial inoculant was prepared as described by Whiting et al. (2001). Each rhizobacteria was grown on nutrient broth medium for 24 h at 28°C, centrifuged at 10,000 rpm for 10 min, washed twice with a sterile saline solution (0.9% (w/v) NaCl), and then resuspended in 1% of methyl cellulose solution prepared in 10 Mm of MgSO 4 . A mixture of the four chosen rhizobacteria was prepared with equal concentrations of cells of about 3.10 8 CFU mL −1 (DO 600 = 0.5). A sterile methylcellulose noninoculated with bacteria was used as control.

Seeds treatments and growth conditions
Two species were used in this study, seeds of Peganum harmala L. (wild rue) collected from Kettara mine area used as native species and seeds of Lactuca sativa L. (lettuce) from Sogemag Company used as sensitive species. Both species seeds were surface-sterilized with 70% ethanol followed by 3% sodium hypochlorite for 5 min and successively washed several times with deionized sterilized water. The surfacesterilized seeds were then soaked in both inoculated and non-inoculated methylcellulose solutions for 30 min.
Ten seeds of each plant species were placed in 50 mL polyethylene tubes filled with 20 mL of autoclaved wateragar medium, composed of (per liter): 1.2 mM K 2 HPO 4 , 0.4 mM KH 2 PO 4 , 5 mM CaCl 2 , 3.35 mg ferric citrate, 2.5 mM MgSO 4 , 2.5 mM K 2 SO 4 , 10 μM MnSO 4 , 20 μM H 3 BO 3 , 5 μM ZnSO 4 , 0.2 μM CuSO 4 , 1.5 μM CaSO 4 , 1.0 μM NaMoO 4 , and 0.8% agar with pH 6.8 (Arora et al. 2012;Tewari and Arora 2014;Román-Ponce et al. 2017). The effect of PGPR on seed germination under salt stress was evaluated in water-agar medium supplemented with NaCl ranging from 25 to 125 mM. To assess strains effect on seed germination under metallic stress, the plant growth medium was supplemented with different concentrations of Cu (CuSO 4 , 5 H 2 O), Pb (Pb(NO 3 ) 2 ), and Cd (Cd(NO 3 ) 2 ranging from 0.06 to1 mM for each metal and Zn (ZnSO 4 , 7 H 2 O) ranging from 0.25 to 2 mM. Seeds placed in tubes with medium containing neither salt nor metal were used as control. All tubes were incubated at 22°C for 2 weeks and then the length of roots and shoots was measured for 4 seedlings in each treatment. The experiment was conducted in four replicates for each treatment.

Statistical analysis
A one-way ANOVA with post-hoc Student Newman-Keuls test (P < 0.05), carried out with the SPSS program (IBM, Armonk, NY, USA, version 25.0.), was used to examine the statistical difference between the results. Student's t test was used to compare root and shoot length between inoculated seedlings and uninoculated control seedlings.

Screening of rhizobacteria for osmotic and metallic stress tolerance
Results of the tolerance of rhizobacterial strains to salt stress are shown in Table 1. Out of 29 tested bacteria, only 20 showed tolerance to 80 g L −1 of NaCl; 17 tolerated salt stress up to 100 g L −1 NaCl. Metal tolerance results of PGPR strains are shown in Table 2. All bacterial strains showed a high level of tolerance to high concentrations of heavy metals. Indeed, more than 70% of the strains were able to tolerate Cu up to 7 mM, about 48% tolerated Pb up to 9 mM, 25% could grow in medium with Zn up to 15 mM, and only 15% were able to tolerate Cd concentrations at 6 mM. However, more than 60% of the strains were able to grow well in the presence of the mixture of four metals (Zn, Cu, Cd, and Pb) at 2 mM for each metal, while only 29% could grow in medium amended with 3 mM of the mixture tested metals. Finally, the general order of the toxic effect of metals on these bacteria was classified as follows: Cd> Cu>Pb> Zn.

Effect of salt and metallic stress on PGP activities
The effect of salt and multi-metal stress on PGP activities of the selected rhizobacterial strains is presented in Table 3. All of the tested rhizobacteria were able to produce IAA under salt and heavy metal stress conditions. The indole phytohormones production in all tested rhizobacteria decreased significantly by 0.65-to 3.5-fold under stressful conditions, except for BKM 19, BKM 20, and BKM 33 strains which did not show any change in their IAA production and BKM 28 which exhibited a significant increase in IAA biosynthesis, ranging up  3 5 10 15 3 6 9 12 2 3 4 5 6 3 5 6 7 0.2 0.5 1 2 3 Advenella kashmerensis BKM 01 + + + − + + + − + + + + − + + + + + + + + −

Streptomyces enissocaesilis
Streptomyces caelestis BKM 05 + + + − + + + + + + + − − + + + + + + + + + Bacillus subtilis BKM 06  Keuls test (P < 0.05)). Different letters refer to significant differences (P < 0.05); lowercase letter show significant differences among strains for the same treatment; capital letter show significant differences between different treatments (control, salt stress, and metallic stress) for every strains according to post-hoc Student Newman-Keuls test (P < 0.05). (−), not detectable/no production; (+), positive/weak; (++), moderate; (+++), strong; (++++), very strong. *The mixture of Cu, Pb, Cd, and Zn at 0.5 mM for each metal all strains when cells were exposed to the multi-metal solution comparative to the unstressed conditions. However, the growth of BKM 06, BKM07, BKM 28, and BKM 19 with high concentration of NaCl leads to the loss of their capacity to produce ammonia as shown in the Table 3. Besides, fourteen rhizobacteria were found positive for siderophore production under control conditions. Whereas, salt stress induced a significant reduction in siderophore production compared to the control (e.g., the production of this compound by BKM 20 and BKM 18 strains was completely inhibited in response to NaCl at 10%). However, under metallic stress, the siderophore production seems to be higher for all the strains in comparison to the control conditions.

Bacterial response to salinity and metallic stress
The response of the 17 selected rhizobacterial strains to multimetal and high level of salt stress was studied by evaluating the physiological and biochemical status in terms of proline, soluble sugars, proteins, and free amino acids contents as well as their ability to produce exopolysaccharides (Tables 4 and 5) under metallic and salt stress. The results showed that all bacterial strains produce high content of EPS under osmotic stress than control. Indeed, the exposure of rhizobacteria to multi-metal or salt stress resulted in a significant increase of EPS production by an average of 4 and 2 times, respectively, if compared to salt stressed and unstressed ones. The highest amount of EPS was produced by BKM 04, BKM 07, BKM10, BKM26, and BKM 33 up of 195 μg mL −1 under heavy metal stress (Table 4). Soluble sugar contents were significantly higher when cells were exposed to metallic stress compared to salt stress and non-stressed conditions (Table 5), with the highest values recorded with five strains belonging to M. tamadayense, Ciceribacter lividus, Pseudomonas frederiks bergensis, and Pseudomonas koreensis (BKM 04, BKM 28, BKM 14, and BKM 33). Moreover, results revealed that the free amino acids levels of all bacteria increased by 2.2 times under salt stress and more than 13 times under metallic stress in comparison to the control. The highest amounts of free amino acids recorded was 570.3 μmol mL −1 , 552.3 μmol mL −1 , 598.7 μmol mL −1 , and 584.7 μmol mL −1 under metallic stress for BKM 04, BKM 06, BKM 19, and BKM 21 strains, respectively. Similarly, proline concentration also increased significantly in all rhizobacteria under salt and heavy metal stress, with the highest production that was recorded for BKM 04, BKM 05, and BKM 33 under salt stress (Table 5). However, a decrease in cell proteins content was observed in all tested bacteria grown under salt-stressed conditions compared to the control. While, the amount of proteins increased by more than 11 times when cells were exposed to heavy metal stress (Table 4).

Effect of PGPR inoculation on seedling shoot and root growth under metallic and salt stress
The effect of inoculation with consortium of four PGPR (Mesorhizobium tamadayense BKM 04, Enterobacter xiangfangensis BKM 30, Pseudomonas azotifigens BKM 07, and Streptomyces caelestis BKM 05) on root and shoot elongation of wild rue and lettuce seedlings subjected to metal and salt stress is presented in Figs. 1 and 2. In general, a significant decrease in roots and shoots growth of the two species was observed with increasing concentrations of metal or salt concentrations. In addition, bacterial inoculated plants showed significantly higher growth in terms of root and shoot length, as compared to uninoculated control under non-stress conditions. The level of seedling toxicity was higher on media supplemented with Cd followed by Cu and it was relatively lower with Pb and Zn amended media. PGPR inoculation promoted significantly the early growth of the tested plants under metal stress. Indeed, under high metal conditions, the roots and shoots length of lettuce seedlings inoculated with the bacterial consortium were increased by 1.16 to 4.73 times and 1.2 to 3 times, respectively compared with non-inoculated seeds (Fig.   1a, b). Similarly, increased growth of P. harmala seedlings grown in metal-contaminated media was observed after receiving the PGPR inoculants. The most pronounced beneficial effect on seedlings growth was observed in seeds exposed to the highest metal concentration. Root growth of lettuce seedlings was completely inhibited in the uninoculated test containing 1, 0.5 Mm of Cu and Cd while it reached about 93 mm when seeds were inoculated with PGPR strains (Fig. 1a). A significant reduction in roots and shoots length was also observed in uninoculated P. harmala seed lings exposed to high levels of tested metals. As expected, inoculation of P. harmala seeds with PGPR under metal stress caused a remarkable increase in root length by 1.28 to 6 times in comparison to uninoculated test. Shoots height was also improved by 8 times under 1 mM of Cd and 13-fold under 1 mM of Cu, 1.28 times under 2 mM of Zn, and 1.39-fold under 1 mM Pb, compared to the uninoculated seeds ( Fig. 1c and d).
Likewise, as with the metal test, PGPR treatments also increased root and shoot growth of the two studied species under salt stress induced by NaCl compared to uninoculated seedlings. Shoots of bioprimed seedlings of P. harmala increased by 1.7 times when exposed to 100 mM NaCl and up to 1.9-and 2.5-fold under 115 mM and 125 mM NaCl in comparison to the uninoculated test (Fig. 2a, b). In lettuce seedlings case, bacterial consortium induced an increase in shoot growth by 4.5-fold under 125 mM of NaCl in  ) and Peganum harmala (c, d) seedlings after two weeks of growth under different concentrations of heavy metals. Results are expressed as mean ± SD (n = 3). ns not significant. ** Significantly different than respective uninoculated control according to Student's t test (P < 0.01). +Inc: with bacterial inoculation; -Inc: without bacterial inoculation comparison to seeds grown without bacterial inoculum (Fig.  2c, d). However, the positive effect of the bacterial consortium was more pronounced in roots than in shoots. A complete inhibition of root growth was observed for uninoculated lettuce seeds subjected to the highest level of salt stress (125 mM), while a considerable improvement in its elongation (up to 0.8 cm) occurred in when treated with PGPR. For P. harmala, roots length in PGPR inoculated seedlings was increased by 3.2 and 2.7 times compared to those without bacterial treatment when seeds were exposed to 125 mM under 115 mM of NaCl, respectively. In this study, we have also outlined a more pronounced negative effect of metal stress and salinity on lettuce growth compared to P. harmala. Indeed, the results showed that with high level of metals (1, 0.5 mM of Cd and Cu and 2 mM of Zn) and salinity (125 mM NaCl) lettuce root elongation was completely inhibited, while P. harmala root growth was maintained under the same conditions and reached 43, 26, and 70 mm in length when exposed to 0.5 mM of Cu, 0.5 mM of Cd, and 125 mM of NaCl, respectively (Figs. 3 and 4).

Screening of rhizobacteria for osmotic and metallic stress tolerant
In this study, more than 56% of the tested rhizobacteria were able to tolerate high level of salinity (up to 10% NaCl). These findings may be attributed to the physicochemical conditions of the soils from which these strains were isolated. The climate conditions and high temperatures, particularly in summer in the Kettara mine, lead to high evaporation and low infiltration in the region which constitute the main factors contributing to the salinization of land in this area (Boularbah et al. 2006a;El Khalil et al. 2008;El Hamiani et al. 2015;Benidire et al. 2020). In addition, as reported by previous work (Boularbah et al. 2006a;Benidire et al. 2016), the soils of the Kettara mine presented the high values of conductivity confirming the high mineralization of the soils in Kettara mine area. Several studies reported that many bacteria isolated from salt conditions tolerate high concentration of NaCl, up to 10% NaCl (Vardharajula et al. 2011;Gururani et al. 2013;Armada et al. 2015) suggesting that natural salt environments seem to be a promising source of salinity tolerant bacteria able to alleviate salt stress in plants. In addition, among the strains tested in this study, the majority were found to be halotolerant since they could grow in media containing up to 10% of NaCl (Román-ponce et al. 2017;Khan et al. 2017;Raval and Saraf 2020).
High metal resistance was also observed, with more than 25% of the tested strains grown in media containing very high metal concentrations (up to 15 mM Zn, 6 mM Cd, 9 mM Pb, and 7 mM Cu). Previous studies indicated that long-term exposure to metal contaminants can induce the activation of adaptive mechanisms in bacteria enabling them to reduce heavy metal toxicity, such as extracellular exclusion, biosorption, enzymatic detoxification, or intracellular . ns not significant. ** Significantly different than respective uninoculated control according to Student's t test (P < 0.01). + Inc, with bacterial inoculation; -Inc, without bacterial inoculation accumulation of metals ions in non-toxic form (Boularbah et al. 1992(Boularbah et al. , 1993Aboudrar et al. 2007;González et al. 2010;Ayangbenro and Babalola 2017;Liu et al. 2018;Mitra et al. 2018).

Effect of salt and heavy metal stress on PGP activities
In order to promote plant growth under unfavorable environmental conditions, the use of stress-tolerant rhizobacteria as biofertilizers has received considerable attention in the recent years (Pandey 2009;Khan et al. 2017;Wang et al. 2019;Basu et al. 2021;Kumar et al. 2021). These beneficial microorganisms improve plant performance by using various mechanisms, such as solubilization of soil nutrients, production of plant growth hormones, and suppression of stress due to ethylene production (Ma et al. 2016;Din et al. 2019;Mahmoud et al. 2020). Moreover, due to their ability to improve plant metals tolerance and their capacity for metals immobilization in the soil, the use of PGPR for assisted phytoremediation of heavy metals contaminated soils has been widely studied (Aboudrar et al. 2013;Mitra et al. 2018;Pramanik et al. 2018;Din et al. 2019;Benidire et al. 2020). Several studies have also reported that plant-microorganism interactions influence greatly and positively crops production  (Sandhya et al. 2010;Kang et al. 2014;Khalilzadeh et al. 2018;Khan et al. 2018). In this study, we investigated the effect of salinity and metallic stress on PGPR performance. Our results showed that the PGP traits of the tested rhizobacteria, namely IAA, ammonia productions, and P solubilization, were strongly and negatively affected by the application of salt and metallic stress. Indeed, lower PGP activities were detected in strains cultivated under stressed conditions compared to non-stressed ones except for siderophore production. This decrease in PGP traits indicates that under stressful conditions, rhizobacteria were actively involved in the metabolic mechanism leading to the control of abiotic stress than other metabolic processes. Similar results were reported by Armada et al. (2015) and Sandhya et al. (2010), where multiple PGP characteristics of rhizobacteria isolated from semi-arid environment decreased significantly when exposed to osmotic stress conditions. Likewise, Karthik and Arulselvi (2017) have reported a significant decrease in siderophore, AIA, ammonia, hydrolytic enzymes production, and phosphorus solubilization ability of rhizobacteria exposed to high concentration of Cr. However, this strain was able to rapidly promote the growth of the host plants under Crinduced stress. Moreover, Deshwal and Kumar (2013) have suggested that heavy metals such as Pb, Cr, and Ni reduced microbial biomass as well as IAA, hydrogen cyanide, siderophore production, and P-solubilization capacity of Pseudomonas strains isolated from potato rhizosphere. The present study has also outlined an increase in siderophore production of rhizobacteria when exposed to metallic stress; these findings suggest that these strains might use siderophore as a tool to reduce heavy metal toxicity by chelation process. Huo et al. (2020) have reported that under high concentrations of iron, selected siderophore-producing rhizobacteria Mesorhizobium panacihumi DCY119T was able to reduce Fe-induced oxidative stress in Panax ginseng seedlings by binding toxic metals with siderophore and by activating the antioxidant system of plants.
PGPRs play an important role in improving plants performance under harsh environments, by producing various substances such as IAA and gibberellic acid which have already been identified to ameliorate seeds germination and plants growth in stressed conditions. It is also well known that IAA promote root architecture, stimulate lateral root development, and increase root absorption surface, which improves nutrient and water uptake by plants under optimal and stressed conditions (Barnawal et al. 2017;Fukami et al. 2018;Kang et al. 2020;Román-ponce et al. 2017). The ability of tested rhizobacteria to grow under extreme conditions while keeping their PGP capacities may be an interesting tool to be used to optimize the rehabilitation of areas heavily contaminated by trace elements or to enhance plant growth on metal contaminated, dry and saline environments (Upadhyay et al. 2011;Durand et al. 2016;Ma et al. 2016Ma et al. , 2019.

Bacterial response to salt and metallic stress
Results of the osmotolerant rhizobacterial cells response to stress showed a huge increase in free amino-acids, proline, and soluble sugars contents compared to the control. Indeed, these intrinsic metabolites confer to rhizobacteria a cellular adaptation to osmotic pressure, as an osmolyte function by maintaining high level of cell water status. Similar studies reported the same trend as response to osmotic stress (Sandhya et al. 2010;Gururani et al. 2013;Armada et al. 2015). Therefore, accumulation of osmolytes allows not only to improve water retention but also to alleviate oxidative damage and ameliorates membranes and enzymes stability under high level of drought and salt stress (Kang et al. 2014). In addition, soluble sugars serve as an energetic source for cells functioning; they are also used as substrate in biosynthesis procedures; contribute as a tool for signal transduction regulation; and as monitors of the gene expression (Sandhya et al. 2010).
Protein contents increased significantly in all strains under heavy metal stress. This result may be related to the increase of antioxidant enzymes expression in microbial cells, which are used to maintain the normal redox status and to support the metabolic balance by eliminating the free radicals caused by metal stress (Armada et al. 2015). However, unlike other cellular compounds, a very important decrease in protein contents in all strains while exposed to high salinity was observed, which can be considered as an indicator of bacterial cells toxicity due to the osmotic stress (Vardharajula et al. 2011). Protein hydrolysis has been reported to cause an increase in free amino acids involved in cellular osmotic adjustment, whereas proteins themselves are used for polysaccharides production (Vardharajula et al. 2011;Iqbal and Ashraf 2013). Indeed, in accordance with these findings, in our study an increase in EPS production by all bacteria was observed after their exposure to salt and heavy metal stress compared to nonstressed conditions. EPS are important components involved in bacterial biofilm formation that helps maintain hydration of the microenvironment around bacterial cells and protect them from desiccation (Zhu et al. 2018;Zhang et al. 2020). It has also been reported that EPS can bind toxic Na + cations, reducing their toxic effect on cells and alleviate osmotic stress due to salinity (Ashraf et al. 2004;Upadhyay et al. 2011;Zhu et al. 2018). Moreover, Kalpana et al. (2018) has reported that microbial tolerance to heavy metals such as Cu, Zn, Pb, and Cd is strongly related to the polysaccharides adsorption properties. In fact, due to their negatively charged hydroxyl and phosphoryl groups, these polymeric carbohydrates can reduce metals mobility and therefore increases bacterial cells viability (Boularbah et al. 1992;González et al. 2010). Our results are in line with previous studies where an increase in exopolysaccharides production was also recorded in bacteria in response to drought and salt stress (Qurashi and Sabri 2012;Tewari and Arora 2014;Din et al. 2019).

Effect of PGPR inoculation on seedlings growth under metal and salt stress
It is well known that plant candidates for phytostabilization should be metal-tolerant species which exclude heavy metals from the root apex or limit the accumulation only in their root tissues. Thus, in addition to their high metal tolerance capacity, the root system of these plants should be deeper with a large surface area to provide a high nutrient environment and to prevent heavy metal spread by erosion process over the long term (Zhang et al. 2012;Testiati et al. 2013;. In this study, we investigated the root elongation considering this parameter as a tool that can provide us with additional information on the effectiveness of the interaction of plant with the four selected PGPR strains under stressed conditions. Our results showed that, in the absence of metal stress, the mixture of the used rhizobacteria can stimulate significantly root elongation compared to non-inoculated seedlings. Furthermore, the beneficial effect is well observed when the growing media was amended with metal salts, particularly at highest concentration (0.5 mM and 1 mM of Cd and Cu with 2 mM of Zn). Under metal stress and without bacterial treatment, lettuce seeds were able to germinate but the root growth was completely inhibited few days after emergence. The four rhizobacteria used in this mixture have been characterized for their multi-metals' resistance to higher concentrations of Cu (up to 6 mM), Pb (up to 7 mM), Cd (up to 5 mM), and Zn (up to 10 mM). Moreover, they showed a high tolerance to salinity (up to 10% NaCl) and maintained their plant growth promoting traits even under high concentrations of NaCl. In previous studies, several bacterial strains belonging to the genera Pseudomonas spp. characterized by their high tolerance to Cd have also been reported to have a better capacity to stimulate plant growth under salt, drought, and metallic tress (González et al. 2010;Ma et al. 2016;Zhu et al. 2018). Similarly, a strain isolated from metalcontaminated rice rhizosphere and identified as Enterobacter sp. showed a great ability to promote rice seedling growth under Cd-induced stress by producing PGP compounds and contributed as well in reducing the oxidative damage induced by high metal concentration . Nascimento et al. (2012) and Verma et al. (2013) reported a significant improvement in the growth parameters of chickpea plants inoculated with Mesorhizobium sp. compared to uninoculated control grown under environmental constraints.
In this study, the positive effect of the studied bioinoculants on root elongation both under control and stressed conditions could be explained by their ions adsorption capacity and metals accumulation in active cells leading to the reduction of metal toxicity (Boularbah et al. 1992(Boularbah et al. , 1993Ayangbenro and Babalola 2017;Liu et al. 2018). Enhanced growth of inoculated seeds could be attributed also to the ability of r h i z o b a c t e r i a t o s y n t h e s i ze g r o w t h -s t i m u l a t i n g phytohormones, which can affect enzymes functioning such as α amylase that can ameliorate starch assimilation during the germination process and therefore promote early seed germination (González et al. 2010;Ashwini et al. 2011). The phytohormone IAA produced by PGPR can also help in increasing root surface area, root formation, and lateral root growth (Román-Ponce et al. 2017). In addition, bacterial exopolysaccharides can contribute to root growth stimulation, by protecting seeds and seedling roots against Na + and toxic metal ions through forming a polymer matrix around seeds and roots (Ashraf et al. 2004;Upadhyay et al. 2011;Zhu et al. 2018;Din et al. 2019).
The results clearly showed that P. harmala seeds sampled from Kettara mine site heavily polluted with metals are more tolerant to metal and salinity than lettuce seeds. These results might be explained by the characteristics of the growing environment of this plant. Indeed, it has been demonstrated that at the P. harmala's sampling site, the soils were highly saline and contain high concentrations of several heavy metals, especially copper and zinc (El Hamiani et al. 2015;Benidire et al. 2016). This confirms that, throughout their long-term exposure to hostile conditions in the mining region, the seeds evolved mechanisms more suited for their survival under stressed conditions.

Conclusion
In this study, most of the tested rhizobacteria displayed a high level of multi-heavy metal resistance. In addition, among the 29 tested strains, 16 strains could be classified as halotolerant bacteria due to their ability to grow in the presence of 10% NaCl. The adaptation mechanisms used by these rhizobacteria in response to osmotic stress have been highlighted, such as the accumulation of osmolytes (sugars, amino acids, and proline) and the production of exopolysaccharides. Besides their high adaptation to adverse conditions, the studied strains seem to maintain a relatively high PGP capacity even under stress conditions. The beneficial effects of osmotolerant PGPR strains on plants subjected to metal or salt stress is confirmed by an in vitro test. The results of this assay showed that inoculation of seeds with the selected strains promote a significant stimulation of lettuce and wild rue seedlings growth, compared to the control. Besides, the seedling could withstand metal and salt stress more efficiently in the presence of the bacterial mixture, as indicated by increases in root and shoot growth of inoculated stressed plants in comparison to uninoculated ones; this study revealed clearly that the consortium of PGPR used can be used as a tool to improve crop productivity in stressed condition. Therefore, the bacterial consortium studied in this paper can be used as a potential bioinoculant to assist phytoremediation of degraded soil in arid and semi-arid areas.