4.1. 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. 2006; Benidire et al. 2016), the soils of the kettara mine presented the high values of conductivity confirming the high mineralization of the soils in kettra 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 (Egamberdieva et al. 2009; Román-ponce et al. 2017; Khan et al. 2017; Raval et al. 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 accumulation of metals ions in non-toxic form (Boularbah et al. 1992; Boularbah et al. 1993; Aboudrar et al. 2007; Gonzàlez et al. 2010; Ayangbenro and Babalola 2017; Liu et al. 2018; Mitra et al. 2018).
4.2. 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 a biofertilizers has received considerable attention in the recent years (Pandey 2009; Singh et al. 2015; Wang et al. 2019; Khan et al. 2017). These beneficial microorganisms improve plant performance by using various mechanisms, such as solubilization of soil nutrient, 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 under salt and drought conditions (Egamberdieva et al. 2009; Sandhya et al. 2010; Kang et al. 2014). 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 stress conditions compared to non-stressed ones except for siderophores production. This decrease in PGP traits indicates that under stressful conditions, rhizobacteria were actively involved on the metabolic mechanism leading to the control of abiotic stress than other metabolic process. The same 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 et al. (2017) have reported a significant decrease of siderophores, AIA, ammonia, hydrolytics enzymes productions and phosphorus solubiliszation ability of rhizobacteria exposed to high concentration of Cr. However, this strain was able to rapidly promote the growth of the host plants under Cr-induced stress. Moreover, Deshwal et al. (2013) have suggested that heavy metals such as Pb, Cr and Ni reduced microbial biomass as well as IAA, hydrogen cyanide, siderophores productions and P-solubilization capacity of Pseudomonas strains isolated from potato rhizosphere. The present study has also outlined an increase in siderophores production of rhizobacteria when exposed to metallic stress, these findings suggest that these strains might use siderophores 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 DCY 119T was able to reduce Fe-induced oxidative stress in Panax ginseng seedlings by binding toxic metals with siderophores 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 (Asghar et al. 2002; Rajkumar et al. 2006; Idriss et al. 2007; Kang et al. 2009; 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 area heavily contaminated by trace element or to enhance plant growth on metal contaminated, dry and saline environments (Updhyay et al. 2011; Durand et al. 2016; Ma et al. 2016; Ma et al. 2019).
4.3. 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. 2012; 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 tool for signal transduction regulation and as monitors of the gene expression (Sandhya et al. 2010).
Proteins 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 et al. 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 non-stressed 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 (Becker et al, 1998; 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 et al. 2012; Tewari and Arora 2014; Din et al. 2019).
4.4. Effect of PGPR inoculation on seedlings growth under metal and salt stress
It is well known that plants candidates for phytostabilization should be metal-tolerant species which exclude heavy metals from the root apex or limit the accumulation only in their roots 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; Zou et al. 2012; Testiati et al. 2013; Shackira 2017 a, b). 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 and 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 plants growth under salt, drought and metallic tress (Gonzàlez et al. 2010; Ma et al. 2016; Zhu et al. 2018). Similarly, a strain isolated from metal contaminated 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 (Mitra et al. 2018). 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. 1993; Ayangbenro and Babalola 2017; Liu et al. 2018). Enhanced growth of inoculated seeds could be attributed also to the ability of rhizobacteria to synthesize growth stimulating 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 (Bharathi et al. 2004; 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 on 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 (Benidire et al. 2016; El Khalil et al. 2008; El Hamiani et al. 2015). 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 the stressed conditions.