Characterization of heavy metal-resistant rhizobia and non-rhizobia isolated from root nodules of Trifolium sp. in a lead and zinc mining area

doi:10.21203/rs.3.rs-2692408/v1

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Characterization of heavy metal-resistant rhizobia and non-rhizobia isolated from root nodules of Trifolium sp. in a lead and zinc mining area

https://doi.org/10.21203/rs.3.rs-2692408/v1

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In this study, we evaluated the ability of rhizobacteria isolated from Trifolium sp. nodules growing on a Pb/Zn mine site to produce plant growth-promoting substances, such as siderophores, extracellular enzymes, antifungal substances, their capacity to solubilize phosphate, and also their tolerance to heavy metals and salinity. Overall, the results demonstrated that bacterial isolates showed an ability to produce multiple important plant growth-promoting traits, with remarkable ability to grow up to 20% salt concentration and resist to high levels of heavy metals up to 1300 mg/L Pb⁺⁺, 1200 mg/L Zn⁺⁺, 1000 mg/L Ni⁺⁺, 1000 mg/L Cd⁺⁺, 500 mg/L Cu⁺⁺, 400 mg/L Co++, and 50 mg/L Cr^VI+. The order of resistance of isolates to heavy metals was reduced as follows: Pb⁺⁺ > Zn⁺⁺ > Ni⁺⁺> Cd⁺⁺> Cu⁺⁺ > Co⁺⁺ > Cr^VI+. All isolates had multiple metal-resistant abilities; however, the existence of the pbrA, czcD, and nccA genes responsible for resistance to Pb⁺⁺, Zn⁺⁺, Cd⁺⁺, Co++, and Ni++, respectively, was determined by PCR and were detected only on Cupriavidus paucula RSCup01. Our results also showed that the plant growth-promoting rhizobacteria strains screened in the present study could be used as a potential inoculant for the improvement of phytoremediation in heavy metal-polluted soils.

Heavy metal

Mine soil

Plant growth-promoting rhizobacteria

Resistant bacteria

Trifolium

Heavy metals (HMs) are naturally present in the environment, however, various anthropogenic activities such as: mining, surface finishing, energy and fuel production and metallurgy wastes are causing disturbances leading to the accumulation of heavy metals at concentrations higher than defined background values (Gupta et al. 2016). Heavy metals are non-degradable and therefore highly persistent compounds, they can negatively affect the behavior of living organisms present at the site or in surrounding areas and pose a serious threat to human health and the entire ecosystem when they occur in excessive amounts (Etesami 2018).

Most of heavy metals become toxic even at low concentrations and are one of the major factors limiting the growth of plants (Amari et al. 2017), however, it is well known that metalliferous sites can support growth of specific heavy metal hyper-tolerant plants, recently used as tools to develop new in situ cost-effective and environmentally friendly techniques, such as phytoremediation (Jacob et al. 2018).

Trifolium genus grows in different types of soils and climates, some of them have the ability to adapt in heavy metal-rich soils and become dominant species (Wang et al. 2018). Trifolium species establish interactions with rhizospheric bacteria, in particular a group of plant growth promoting rhizobacteria (PGPR) (Etesami 2018). PGPR are a group of free-living rhizobacterial communities colonize the plant root and benefit the plant in multiple ways by secreting a variety of phytostimulating substances (Hashem et al. 2019).

PGPR strains with remarkable heavy metals resistance properties have been isolated from the metal-contaminated rhizosphere of different crops, including vegetables that acting as filters by selecting certain bacteria that help them to develop heavy metals tolerance for survival in their habitat through various mechanisms, including nitrogen fixation, phosphate solubilization, indole-3-acetic acid (IAA) production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase synthesis, siderophore production, and antifungal activity (Etesami and Beattie 2017; Etesami 2018).

Heavy metal-resistant PGPR strains can be used as a suitable candidate for metal phytoremediation to minimize the adverse impact of metals and enhance metal accumulation capacity of plants cause it’s difficult for an external bacterial community to adapt to an already established ecosystem (Ali et al. 2013). The bacteria found in metal polluted sites develop resistance to heavy metals due to their certain resistance mechanisms such as; intracellular and extracellular sequestration, bioaccumulation, efflux system, enzymatic conversion, and resistance by impermeability (Zubair et al. 2016).

The objectives of this study were to characterize bacteria previously isolated from Trifolium sp. nodules growing on a Pb/Zn mine site in Hammam N'bail, northeastern Algeria. The isolates were screened for heavy metals and salinity resistance and their plant growth promoting traits, including siderophores production, solubilization of phosphate, extracellular enzymes production (lipase, cellulase, protease, urease, and amylase), and antifungal activity. This study also includes an analysis of three genes pbrA, czcD, and nccA responsible for resistance to lead, zinc, cadmium, cobalt, and nickel respectively.

This research will allow us to obtain a collection of metal-tolerant bacteria with PGP traits that can be used to improve phytoremediation efficiency in mining areas contaminated with multiple heavy metals. To our knowledge, this is the first study of the characterization of a plant growth–promoting rhizobacteria community isolated from Trifolium sp. nodules grown in mine soil in Algeria.

Bacterial isolates

The strains used in this study were isolated from Trifolium sp. nodules grown on a Pb/Zn mine site in Hammam N'bail, northeastern Algeria. In a previous work, we carried out a characterization and identification through 16S rRNA using universal primers fDl (5′-AGA GTT TGA TCC TGG CTC AG-3′) and rP2 (5′-ACG GCT AAC TTG TTA CGA CT-3′). The sequences were deposited in the National Center for Biotechnology Information (NCBI) GenBank database under accession numbers MW049066–MW049114 (Rahal and Chekireb 2021).

Heavy metal tolerance test on solid media

The minimum inhibitory concentration (MIC) of HMs was evaluated for all the isolates by inoculating each bacterium on tryptone yeast agar (TY) supplemented with increasing concentrations of heavy metals from 25 mg/L to 1400 mg/L including Cd⁺⁺ (CdCl₂.2H₂O), Co⁺⁺ (CoCl₂.6H₂O), Ni⁺⁺ (NiCl₂.6H₂O), Zn⁺⁺ (ZnSO₄.7H₂O), Pb⁺⁺ (Pb(C₂H₃O₂)₂), Cu⁺⁺ (CuSO₄.5H₂O) and Cr^v+(K₂CrO₇). Stock solutions of the metals were prepared in double deionized water and sterilized by 0.22 μm filter. A medium without metal and inoculated with bacterial strains was used as a control. The isolates were considered tolerant to a particular heavy metal concentration if colonies grew within 5–7 days of incubation at 28 °C (Luo et al. 2011).

Heavy metal tolerance test on liquid media

Growth studies of the selected multi-resistant bacteria was carried out in 250 mL flasks containing 50 mL TY medium supplemented with heavy metals at concentration of 1100, 1200, 1300 mg/L for Pb⁺⁺, 1000, 1100, 1200 mg/L for Zn⁺⁺, 800, 900, 1000 mg/L for Cd⁺⁺ and Ni⁺⁺, 300, 400, 500 mg/L for Cu⁺⁺ and 200, 300, 400 mg/L for Co⁺⁺.

All of the flasks were incubated including the controls were incubated at 28 °C for 8 days at 180 rpm. The bacterial growth was measured once every 24 h by measuring the OD at 600 nm (Guo et al. 2010).

Amplification of heavy metals resistant genes

The colonies of each isolate were picked from pure TY cultures, and diluted in 20 μL of sterile distilled water. To release genomic DNA, cell lysis was performed by heat shock using subsequent thermal cycling (13 cycles of 20 s at 95 °C – 20 s at 4 °C). The resulting lysate served as a template for the amplification. The czcD gene was amplified with the primers czcD1; 5-CAG GTC ACT GAC ACG ACC AT-3 (forward) and czcD2; 5-CAT GCT GAT GAG ATT GAT GAT C-3 (reverse) (Nies et al. 1989). The nccA gene was amplified using primers nccA1; 5-ACG CCG GAC ATC ACG AAC AAG-3 (forward) and nccA2; 5-CCA GCG CAC CGA GAC TCA TCA-3 (reverse) (Abou-Shanab et al. 2007). The pbrA gene was amplified with primers pbrA1; 5-ATG AGC GAA TGT GGC TCG AAG-3 (forward); pbrA2; 5-TCA TCG ACG C AA CAG CCT CAA-3 (reverse) (Borremans et al. 2001). PCR reactions were performed in a final volume of 20 μL containing: 4 μL Taq buffer (× 5), 2 μL DNA template, 0.4 μL dNTPs (2.5 mM each), 0.4 μL of each primer and 0.4 μL Taq polymerase (GoTaq, Promega). Amplification for all genes were carried out using the following conditions: initial denaturation at 95 °C for 5 min followed by 35 cycles of denaturing at 94 °C for 1 min, annealing at 57 °C for 1 min, extension at 72 °C for 1 min and a final step for extension at 72 °C for 7 min. PCR products were checked by horizontal electrophoresis on 1.5% agarose gels then purified using Wizard SV Gel and PCR Clean-Up System (Promega) according to the manufacturer’s instructions, and analyzed by Sanger sequencing (Eurofine) using the primers used to generate the PCR products.

Salt tolerance test

Salt tolerance was determined by inoculating all isolates on TY medium with NaCl at various concentrations ranging from 1.5 to 20%. A negative control without NaCl was inoculated with the same bacterial suspension. The plates were incubated at 28 °C for 5 days.

Screening of isolates for PGP traits

Phosphate solubilization

The ability of the bacterial isolates to solubilize inorganic phosphate was determined on Pikovskaya’s agar (Pikovskaya 1948). After incubation for 10 days at 28 °C, a transparent halo appeared around the colonies having the ability to solubilize the phosphates.

Siderophore production

The production of siderophores was detected using the Chrome Azurol S agar method (Schwyn and Neilands 1987). After incubation at 28 °C for 5 days, an orange halo formed around the colonies was considered as positive for siderophore production.

Production of extracellular enzymes

Amylase activity was determined by culturing the isolates on nutrient agar with 1% starch added. After incubation at 30 °C for 3 days, the plates were flooded with Lugol solution for a few minutes. A positive reaction was marked by the appearance of a clear halo around the colonies (Gupta et al. 2003).

Cellulase activity was determined on Yeast Mannitol Agar (YMA) containing 0.25% Carboxy Methyl Cellulose (CMC) for 5 days at 28 °C. After incubation, the Petri dishes were flooded with a 2% w/v Congo Red aqueous solution for 15 minutes, the staining solution was then replaced by a 1M NaCl solution and left for 30 minutes. After several subsequent rinses, the presence of a clear halo around the colonies indicated cellulase production (Gupta et al. 2012).

Protease activity was evaluated on YMA containing 5% skim milk powder. After incubation at 28 °C for 3 days, the presence of a clear halo around the colonies indicates protease production (Kavitha et al. 2013).

Esterase activity was tested on a modified Sierra’s agar (1957) as described by (Carrim et al. 2006). In this experiment, tween 80 was replaced by tween 20 after incubation at 30 °C for 3 days a white precipitate around the colonies indicated a positive result.

Urease activity was carried out on YMA medium containing 2% (w/v) urea and 0.012 g phenol red as a pH indicator. After incubation at 28 °C for 5 days, a positive reaction was indicated by a pink color around colonies (Somasegaran and Hoben 1994).

Antifungal activity

Evaluation of antifungal activity in solid medium in vitro

The antifungal activity of bacterial isolates was tested against the phytopathogenic fungus Sclerotinia sclerotiorum. Fungal plugs of 6 cm were inoculated in the center of potato dextrose agar (PDA) plates and each bacterial isolate was inoculated at a distance of 3 cm from the fungal inoculum. A negative control of the fungal strain was tested in the absence of bacteria. The plates were then incubated at 25 ± °C for 5 days and verified every day. Bacterial antifungal activity was evaluated for the presence or absence of fungal growth inhibition (Martinez-Rodriguez et al. 2015).

Evaluation of antifungal activity with plant in vitro

Medicago truncatula seeds were surface sterilized with sulfuric acid 96% for 10 minutes, then washed five times with sterile water to remove all traces of acid. The seeds were then incubated in sterile Petri dishes containing 10% water agar for 2-3 days in the dark to allow germination.

The seedlings were then transferred to sterile Petri dishes containing Fahraeus medium. Five plants were tested with each isolate and were inoculated with a 1 mL suspension of a liquid culture of isolates grown overnight in TY medium (10⁸cells ml/L). Plants were grown in a growth chamber at a constant temperature of 25 °C and a light/dark photoperiod of 16/8h. After 4 days, plants begin to grow and develop their root systems and were inoculated with 5 mL of suspension of the phytopathogenic fungus Sclerotinia sclerotiorum. Uninoculated plants were included as a control.

Statistical analysis

Principal component analysis (PCA) was performed to examine the relationships between the tolerance of isolates to high concentrations of heavy metals, salinity and their PGPR activities in vitro. Calculations, graphical display, and computer cluster analysis phenotypic variables were performed using XLSTAT software (version 2018, Addinsoft, Paris, France, http://www.xlstat.com).

Resistance of isolates to heavy metals on solid media

The minimum inhibitory concentrations (MICs) for the seven heavy metals tested are shown in table 1. Almost all isolates showed a wide range of tolerance to metals. Overall, they tolerated high concentrations of lead, but they exhibited a remarkable sensitivity to chromium at a concentration of 50 μg/mL, only 18% of isolates were resistant. The order of metal resistance of isolates was followed as follows: Pb⁺⁺ > Zn⁺⁺ > Ni⁺⁺ > Cd⁺⁺ > Cu⁺⁺ > Co⁺⁺ > Cr ^VI+.

The MIC of the isolates reached 1300 μg/mL of Pb⁺⁺, 1200 μg/mL of Zn⁺⁺, 1000 μg/mL of Ni⁺⁺, 1000 μg/mL of Cd⁺⁺, 500 μg/mL of Cu⁺⁺, 400 μg/mL of Co⁺⁺ and 25 μg/mL of Cr^VI+respectively, and were obtained from Cupriavidus isolates RSCup01- RSCup08.

Resistance of Cupriavidus paucula RSCup01 to heavy metals on liquid media

The growth of Cupriavidus paucula RSCup01 was tested in the presence of lead, zinc, nickel, cadmium, copper, and cobalt. The culture was quantified by measuring the optical density (OD_600nm) after cultivation for 8 days in TY liquid media. For each metal, three concentrations up to the respective MIC were added to the cultures. As a control, the growth of cells was analyzed in media lacking the respective metals. The growth curves showed a reduction in OD_600nm values for test cultures with rising cadmium, lead, nickel, zinc, and copper concentrations, highlighting the effect of these heavy metals on Cupriavidus paucula cultures (Fig. 1).

In the presence of lead, the culture reached an OD_600nm of 1.11 at 1100 μg/mL. In the presence of 1200 and 1300 μg/mL lead ions, the OD_600nm value slightly decreased and reached out 0.950 and 0.450 respectively. For zinc, after exposure to 1000 μg/mL and 1100 μg/mL the culture reaches an OD value of 0.9 and 0.8 respectively. In the presence of 1200 μg/mL the OD value decreased slightly and reached an OD value of 0.5. For nickel tolerance assays, growth containing 800 μg/mL and 900 μg/mL of nickel in the medium reached an OD value of 0.900 and 0.600 respectively. which were lower than the control (OD_600nm = 2.00). In cultures containing 1000 μg/mL of nickel, growth was significantly inhibited and reached a value of 0.420. For cadmium, the culture reached an OD value of 1.13 at 800 μg/mL of Cd in medium, in cultures containing 900 μg/mL the growth was inhibited and reached an OD value of 0.900; at 1000 μg/mL the growth was decreased to a value of 0.750. For copper tolerance tests, the culture reached a value of 1.3 in the presence of 300 μg/mL. In the presence of 400 μg/mL the OD value decreased slightly and reached 1.03 and at 500 μg/ml the growth was completely inhibited with an OD value at 0.500. Regarding cobalt, the isolate RSCup01 grew well at 200 μg/mL reached a value of 1.4. However, at 300 and 400 μg/mL in the medium the cultures reached OD values varying between 1.2 and 0.7 respectively.

Amplification of resistance genes in isolates

PCR amplification and electrophoresis revealed that among the selected bacteria Cupriavidus paucula RSCup01, Acinetobacter beijerinckii RSAci01, Pseudomonas putida RSPs01 and Pseudomonas sp. RSPs02 only the isolate Cupriavidus paucula RSCup 01 possessed the resistance genes czcD, pbrA and nccA that were responsible for resistance to some heavy metals. The primers used for the amplification of pbrA and czcD genes yielded both a band of approximately 500 bp and the amplification of nccA gene yielded a band of 1200 pb. The comparison and analysis of the sequences of czcD, nccA, and PbrA genes of Cupriavidus paucula RSCup01 showed 100% homology with Cupriavidus metallidurans (Fig. S1).

Resistance of isolates to salt NaCl

The salinity resistance of isolates is shown in table 1. These results showed that the growth rate of isolates decreased with the increase of the concentration of NaCl. However, 78% of isolates tolerated a concentration up to 3% NaCl and more than 50% tolerated a concentration of 6% NaCl. Above this concentration the number of tolerant isolates decreased, four highly tolerant isolates Staphylococcus xylosus RSSta01, Pseudomonas sp. RSPs02, Micrococcus yunnanensis RSMicc01, and Kocuria dechangensis RSKoc01 that can grow at 10% NaCl were also reported with the exception of one isolate Staphylococcus xylosus RSSta01 that can grow at a concentration of 20%.

Plant growth promoting traits of bacterial isolates

Screening results of PGP traits of the isolates are shown in table 2. Thirty-one isolates produced siderophore, twenty-one isolates solubilized mineral phosphate, and regarding to extracellular production cellulase production was the most common among the isolates (91.83%), followed by urease (73.46%), esterase (55.10%), amylase (30.61%) and finally protease activity which was limited to only a few isolates (20.40%). Our results showed that isolates belonging to the genera Cupriavidus, Pseudomonas and Actinetobacter had at least 4 of the 5 enzymatic properties tested.

Our results showed the ability of forty isolates to inhibit Sclerotinia sclerotiorum in vitro on solid media, and also the protective effect of the selected isolates via a direct contact with the phytopathogenic agent Sclerotinia sclerotiorum with plant. The results obtained after 6 days showed that the "Plant pathogen + isolate" batch revealed a low development of the plant pathogen in the roots compared to the "Control" batch, showing the antifungal activity of the plant growth promoting isolates (Fig. 2).

Statistical analysis

A PCA based on the set of activities present in each individual isolate (Fig. 3) indicated that the first group (blue color) included bacteria located at the rightmost position of the plot corresponded to the genus Cupriavidus includes isolates; RSCup01, RSCup02, RSCup03, RSCup04, RSCup05, RSCup06, RSCup07, and RSCup08 had a very high resistance to most of the heavy metals tested; Pb⁺⁺, Zn⁺⁺, Ni⁺⁺, Cd⁺⁺, Cu⁺⁺ and Co⁺⁺, produced 4 extracellular enzymes; urease, protease, esterase, and cellulase as well as the siderophores and antifungal molecule (Group A).

The second group (brown color) included the isolate Pseudomonas putida RSPs01 which had a very strong resistance to Pb⁺⁺ and Cu⁺⁺ and a good resistance to Zn⁺⁺, Ni⁺⁺, Cd⁺⁺, Co⁺⁺ and NaCl, produced 4 extracellular enzymes; urease, esterase, amylase, and cellulase and had antifungal activity (Groupe B).

The third group (orange color), which included the isolate Acinetobacter beijerinckii RSAci01 had a good tolerance to the majority of the tested heavy metals; Pb⁺⁺, Zn⁺⁺, Ni⁺⁺, Cd⁺⁺, Cu⁺⁺ and Co⁺⁺, produced 4 extracellular enzymes; urease, protease, esterase and cellulase as well as siderophores (Group C).

The fourth group (pink color) included Enterobacter xiangfangensis RSEnt01 and Rhizobium sp. isolates; RSRh01, RSRh02, RSRh03, RSRh04, RSRh05, RSRh06, RSRh07, RSRh08, RSRh09, RSRh10, and RSRh11 had low resistance to heavy metals, solubilized phosphate and produced urease and cellulase (Group D).

The fifth group (purple color) included Roseomonas vinaceus RSRos01, Bacillus niacini RSBac03, Acinetobacter sp. RSAci02, Pseudomonas frederiksbergensis RSPs04, RSPs05 and Pseudomonas thivervalensis RSPs06, RSPs07, RSPs08, and RSPs09 isolates had moderate resistance to heavy metals and solubilized phosphate (Group E).

The final and major group (green color) of isolates in the PCA analysis corresponded to a taxonomically heterogeneous set of bacteria (9 genera) that showed a good plant growth-promoting activities, either individually or in combination (Group F). Thus, group F isolates belong to Microbacterium foliorum RSMic01-08, Kocuria dechangensis RSKoc01, Micrococcus yunnanensis RSMicc01, Bacillus niacini RSBac02, Staphyloccocus xylosus RSStaph01, Providencia rettgeri RSPro01, Paracoccus marcusii RSPra01, Frondihabitans sp. RSFro01, and Pseudomonas sp. RSPs02. They had good resistance to NaCl and heavy metals and had antifungal activity and do not solubilized phosphate, some of these group F isolates were also able to produce siderophores and extracellular enzymes such as esterase, urease, cellulase, and amylase.

The selection of heavy metal resistant bacteria is of great practical interest. Several research projects are currently investigating the use of symbiosis between bacteria and resistant legumes as an effective means of phytoremediation against heavy metal contamination of soils (Etesami and Beattie 2017).

Hyperaccumulative plants are able of storing high concentrations of heavy metals and act as true filters of microorganisms, and structure their rhizosphere microorganisms by selecting certain rhizobia and non-rhizobia in their root nodules that support them to survive in this environment and enhance host resistance (Etesami and Beattie 2017). Isolated bacteria are studied for their ability to tolerate high concentrations of heavy metals. Determining the degree of tolerance of bacteria is an essential step in assessing the adaptation of these bacteria to different heavy metals and identifying isolates for selection in phytoremediation trials.

The results of this study showed the ability of the isolates to resist high concentrations of the heavy metals cobalt, nickel, chromium, copper, lead, cadmium, and zinc. In fact, Luo et al. (2011) showed that bacterial strains isolated from contaminated environments had higher resistance to heavy metals than strains isolated from non–contaminated areas. The order of metal resistance of isolates in this study showed that chromium was the most toxic metal, while the other metals even at relatively high concentrations were less harmful for the majority of isolates tested. Lead and zinc had a low toxicity, even at very high concentrations, the growth of isolates was not affected. These results could be explained by the fact that the concentrations of heavy metals found in soils can influence bacterial tolerance, and as a result bacteria develop resistance towards the metals that contaminate their habitat (Del Busso Zampieri et al. 2016).

Our results showed that the level of resistance to heavy metals in non-rhizobia, appears relatively higher compared to rhizobia. According to Balakrishnan et al. (2017) heavy metal resistance varies between bacteria even when isolated from the same ecological niche, proving that heavy metal tolerance is a strain- and metal-type-dependent trait. The results obtained in this study showed that the highest MICs of the isolates were obtained mainly from Cupriavidus paucula isolates RSCup01- RSCup08. Cupriavidus genus is known for its high resistance to heavy metals (Shi et al. 2020). Indeed, it has been reported that they are highly abundant in many heavy metal contaminated environments and exhibit the highest levels of heavy metal resistance (Monsieurs et al. 2011; Shi et al. 2020). This genus was identified as potential bioremediators, especially those associated with plants, promoting their growth even under environmental stresses such as heavy metal toxicity (Bravo et al. 2020). It has been shown that many species of Cupriavidus including C. pauculus C. metallidurans, C. gilardii, C. campinensis and C. neocaledonicus exhibit very high resistance to various heavy metals with very high MICs for cadmium, cobalt, chromium, nickel, copper and zinc (Zhao et al. 2012; Butler et al. 2022). Zeng et al. (2020) also demonstrated that Cupriavidus paucula 1490 strain had good resistance to heavy metals up to 400 mg /l of Cu, Co and Ni and 300 mg/l of Cd. These high levels of tolerance by the microorganisms are due to specific genetic mechanisms conferring resistance to heavy metals (Etesami 2018; Etesami and Maheshwari 2018).

Our results showed that Cupriavidus paucula RSCup01 resistance varies in both solid and liquid media with the presence of different heavy metal concentrations. The selected isolate seemed to be more sensitive in liquid media at concentrations lower than those obtained in solid media. This result was in accordance with the study by Baati et al. (2020); they showed resistance to zinc, lead, copper and cadmium was higher when incubated on a solid medium than on a liquid. Bhojiya and Joshi (2016), demonstrated that the toxicity of heavy metals in a solid medium differed from that in a liquid medium due to the complexity and availability of metals, as well as diffusion in the medium.

In the present study, various isolates showed good resistance to heavy metals, among the bacterial community, Pseudomonas and Gram-negative bacteria related to the genera Acinetobacter, Enterobacter and Rhizobium showed effective resistance in the presence of many heavy metals.

Pseudomonas genus is ubiquitous and has evolved mechanisms for adaptation and survival under various conditions. Singh et al. (2019) reported that Pseudomonas strains are dominant in heavy metal contaminated soil and can tolerate high concentrations of heavy metals. Several studies demonstrated that Pseudomonas genus can tolerate multiple heavy metals, including zinc, lead, cobalt, cadmium, nickel, and copper (Benidire et al. 2016; Fan et al. 2018).

It has also been shown that the genus Rhizobium associated with Trifolium sp. was able to grow in the presence of several heavy metals such as zinc, lead, cobalt, cadmium, nickel, and copper (Nonnoi et al. 2012). Fan et al. (2018) also showed that Pseudomonas, Rhizobium, and Enterobacter species isolated from Pb/Zn mining area have good resistance to copper, zinc, lead, and cadmium.

Gram-positive isolates mainly Bacillus, Microbacterium, Kocuria, and Staphylococcus also showed considerable resistance to heavy metals. These results were consistent with those observed by Roman-Ponce et al. (2016) who showed that bacteria of the genera Bacillus and Microbacterium isolated from a Pb/Zn mining site have high tolerances to copper, lead, and zinc. Another study by Nayak et al. (2020) reported that Bacillus and Microbacterium had good resistance to copper, lead, and zinc.

Environmental stress, such as excessive metals, exerts selection pressure on microorganisms and affects their growth, abundance and diversity leading to the selection of microorganisms that possess appropriate mechanisms to enable them to survive under these stressful conditions such as; resistance by sequestration intracellular and extracellular, bioaccumulation, efflux system, enzymatic conversion, and resistance by impermeability (Zubair et al. 2016).

In this study, PCR based on heavy metal specific primers was used to screen and detect some heavy metal resistant genes in the tested isolates (nccA, pbrA, and czcD). The pbrA gene encodes a P-type Pb(II) efflux ATPase in the lead resistance operon that involved in uptake, efflux, and accumulation of Pb(II) (Borremans et al. 2001). The CzcD is a cation diffusion facilitator (CDF) protein family transporter located in the cytoplasmic membrane and reduce Cd⁺⁺, Zn⁺⁺ and Co⁺⁺ accumulation in the cytoplasm to the periplasm (Nies 1992). The nccA gene has a similar mechanism and provides resistance to nickel, cadmium, and cobalt (Schmidt and Schlegel 1994). Cupriavidus genus currently comprise 11 species found from diverse ecological niches and have several genetic determinants for heavy metal resistance including the czc, pbr and ncc clusters, which are found in several members of the genus (Mergeay et al. 2003; Monchy et al. 2007). Our results showed that, Cupriavidus paucula had three heavy metals genes nccA, pbrA, and czcD. Butler et al. (2022), also demonstrated that Cupriavidus paucula had many heavy metals resistance genes as cnrR, cnrY, czcA-D, czcI, czcN, and nikR that code for resistance to nickel, cobalt, cadmium and zinc.

The amplification of heavy metals resistant genes for other selected bacteria did not yield any specific PCR products, probably due to the use of inappropriate primers or the presence of alternative mechanisms allowing them to survive under these conditions, including transporting HM out of the cell, metal precipitation outside or inside the cell, or HM binding through exopolysaccharides (Bruins et al. 2000; Wei et al. 2009).

In addition to resistance to heavy metals, our isolates also showed a remarkable resistance to salinity. It has been established that microorganisms that can grow well above 3% salinity are classified as halophilic bacteria (Jiang et al. 2017). Therefore, in our study, about 78% of the isolates could be considered as halophilic bacteria. Our results were consistent with those observed by Roman-Ponce et al. (2016) who isolated a salt resistant Stapylococcus up to 20% NaCl that was resistant to different heavy metals. Jiang et al. (2017) showed that bacteria from the genera Cupriavidus, Pseudomonas, Bacillus and Acinetobacter were resistant to salinity up to 7%. Recently, Abedinzadeh et al. (2018) indicated that bacteria that were resistant to heavy metals can also be resistant to NaCl and showed that these bacteria had the potential to be used in salinity–and heavy metal–polluted areas.

Plant-microorganism interactions play a key role in the adaptation to heavy metal polluted environments and thus, can be investigated in depth to improve microbe-assisted phytoremediation methods (Etesami 2018). Plant associated microorganisms, particularly, plant growth-promoting rhizobacteria (PGPR), play an important role in the phytoremediation of polluted soils and the enhancement of plant growth by different mechanisms such as production of siderophores, antifungal molecules, extracellular enzymes and inorganic phosphate solubilization were further investigated (Etesami 2018).

Phosphate solubilization is one of the main features used to select PGPR to use in phytoremediation strategies, indeed some bacteria play an important role in P solubilization, through the secretion of enzymes (phosphonates, phosphatases and C–P lyases) and organic acids (citric acid, lactic acid, gluconic acid) which convert insoluble P to a soluble form (Rafique et al. 2017). Under phosphate deficiency conditions, some heavy metal resistant isolates; facilitate phosphate mobilization which improves nutrient availability in the rhizosphere region under heavy metal stress (Arif et al. 2017). In this study, isolates affiliated to Enterobacter, Pseudomonas, Acinetobacter, and Rhizobium genera were able to solubilize inorganic phosphate with variation of halo diameters. Many studies also showed that Enterobacter, Pseudomonas, Acinetobacter, and Rhizobium strains isolated from contaminated soil were able to solubilize phosphate (Benidire et al. 2016; Sbabou et al. 2016; Ferchichi et al. 2019).

Siderophores have also an important role in the successful establishment of plants in metal-contaminated areas and play an essential role due to their ability to reduce heavy metals toxicity by binding with these toxic elements (Etesami and Maheshwari 2018). The majority of the tested isolates belonging to the different genera have the ability to produce siderophores, which is not surprising since plant-associated α, β, γ-proteobacteria, bacilli and actinobacteria have the ability to produce siderophores under low iron availability (Tian et al. 2009). In our work, Providencia, Enterobacter, and Pseudomonas were among the best siderophore producing strains. Rana et al. (2011) also reported that Providencia has a high capacity to produce siderophores. Enhanced siderophore production has also been detected in Pseudomonas and Enterobacter sp. isolated from mining soils (Ferchichi et al. 2019).

Extracellular enzyme production is another important trait of PGPRs that indirectly influences plant growth. However, little information is available on the activity of these enzymes in bacteria associated with heavy metal tolerant plants. Our study showed that all isolates were able to produce at least one enzyme activity. These results suggest that plant colonization by rhizobia and endophytes is closely related to their ability to produce extracellular enzymes (Jha and Kumar 2007). These microbial lytic enzymes are also known to play a role in antagonizing various plant pathogenic fungi by degrading their cell wall (Siddiqui 2006). Tsegaye et al. (2019) found that PGPR that synthesizes one or more of these extracellular enzymes has biocontrol ability against a range of plant pathogenic fungi and bacteria and improves crop yield.

In this work, four isolates affiliated to the genera Pseudomonas, Cupriavidus, Acinetobacter, and Providencia showed strong antifungal activity against Sclerotinia sclerotiorum. This fungus can infect many crop species worldwide (Boland and Hall 1994). It infects plant organs, such as pods, leaves, and stems at various stages of development, by penetrating the plant directly or by invading the plant by penetrating a wound site (Gerlagh et al. 1999; Kuang et al. 2011). Our research highlighted that these isolates have a beneficial effect and can be explored in the process of biocontrol of root rot diseases in Trifolium crops. The inhibition of phytopathogens by bacteria can be explained by competition with pathogens for ecological niche/substrate by the production of metabolites used as defense systems (Zhao et al. 2018).

Trifolium sp. plants growing in Pb/Zn mining soil harbored a high diversity of bacteria inside their nodules. These bacteria produced multiple PGP traits, including the production of siderophores, extracellular enzymes, solubilization of phosphate, and antifungal activity as well as being resistant to salinity and several heavy metals such as zinc, cadmium, nickel, lead, cobalt, chrome, and copper. These results showed that these isolates may protect plants against heavy metal toxicity, improve their growth and may be used for application in phytoremediation for the depollution of metals contaminated soils.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author (Sarah Rahal) on reasonable request.

Competing interests

The authors state no competing interest

Funding

This work was supported by the Ministry of Higher Education and Scientific Research of Algeria. The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data, or in writing the manuscript.

Authors' contributions

Sarah Rahal performed the experiments, Belkis Menaa performed a part of the experience. Sarah Rahal and Djamel Chekireb wrote the paper

Acknowledgements

We are grateful to Benjamin Gourion for hosting Sarah Rahal in Laboratory of Plant-Microbe Interactions (LIPM), for the help in the experiments and his extremely useful advice throughout this research. The authors would like to thank also Claire Benezech for her help and advice.

Conflict of interest

The authors declare no conflicts of interest.

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Table 1 Minimum inhibitory concentrations of heavy metals and NaCl of bacterial isolates

Strain code	Bacterial species (GenBank accession number)	NaCl	Pb⁺⁺	Zn⁺⁺	Ni⁺⁺	Cd⁺⁺	Cu⁺⁺	Co⁺⁺	Cr^VI+
Strain code	Bacterial species (GenBank accession number)	%		μg/ml
RSCup01	Cupriavidus paucula (MW049066)	4	1300	1200	1000	1000	500	400	25
RSCup02	Cupriavidus paucula (MW049067)	4	1300	1200	1000	1000	500	400	25
RSCup03	Cupriavidus paucula (MW049068)	4	1300	1200	1000	1000	500	400	25
RSCup04	Cupriavidus paucula (MW049069)	4	1300	1200	1000	1000	500	400	25
RSCup05	Cupriavidus paucula (MW049070)	4	1300	1200	1000	1000	500	400	25
RSCup06	Cupriavidus paucula (MW049071)	4	1300	1200	1000	1000	500	400	25
RSCup07	Cupriavidus paucula (MW049072)	4	1300	1200	1000	1000	500	400	25
RSCup08	Cupriavidus paucula (MW049073)	4	1300	1200	1000	1000	500	400	25
RSPs01	Pseudomonas putida (MW049082)	6	1300	300	400	100	400	300	25
RSPs02	Pseudomonas sp. (MW049083)	10	700	500	200	200	300	100	25
RSAci01	Acinetobacter beijerinckii (MW049107)	4	700	500	150	200	200	200	-
RSSta01	Staphylococcus xylosus (MW049113)	20	1200	200	300	100	200	100	50
RSMicc01	Micrococcus yunnanensis (MW049109)	10	800	150	150	50	200	100	50
RSEnt01	Enterobacter xiangfangensis (MW049105)	6	700	200	100	200	300	50	-
RSBac01	Bacillus licheniformis (MW049102)	8	700	150	200	-	200	150	25
RSPro01	Providencia rettgeri (MW049114)	8	700	150	200	200	200	100	25
RSMic01	Microbacterium foliorum (MW049074)	6	700	150	150	25	200	50	25
RSMic02	Microbacterium foliorum (MW049075)	6	700	150	150	25	200	50	25
RSMic03	Microbacterium foliorum (MW049076)	6	700	150	150	50	200	50	25
RSMic04	Microbacterium foliorum (MW049077)	6	700	150	150	25	200	50	25
RSMic05	Microbacterium foliorum (MW049078)	6	700	150	150	50	200	50	25
RSMic06	Microbacterium foliorum (MW049079)	6	700	150	150	50	200	50	25
RSMic07	Microbacterium foliorum (MW049080)	6	700	150	150	25	200	50	25
RSMic08	Microbacterium foliorum (MW049081)	6	700	150	150	25	200	50	25
RSAci02	Acinetobacter sp. (MW049108)	8	800	150	100	200	150	100	-
RSPs06	Pseudomonas thivervalensis (MW049087)	6	800	100	150	50	100	50	25
RSPs07	Pseudomonas thivervalensis (MW049088)	6	800	100	150	50	100	50	25
RSPs08	Pseudomonas thivervalensis (MW049089)	6	800	100	150	50	100	50	25
RSPs09	Pseudomonas thivervalensis (MW049090)	4	800	100	150	50	100	50	25
RSPs03	Pseudomonas frederiksbergensis (MW049084)	6	700	100	150	50	100	50	25
RSPs04	Pseudomonas frederiksbergensis (MW049085)	6	700	100	150	50	100	50	25
RSPs05	Pseudomonas frederiksbergensis (MW049086)	6	700	150	100	50	100	50	25
RSBac03	Bacillus niacini (MW049104)	3	700	100	150	-	200	50	25
RSBac02	Bacillus niacini (MW049103)	6	500	50	150	-	200	50	50
RSKoc01	Kocuria dechangensis (MW049106)	10	800	150	200	100	150	100	25
RSFr01	Frondihabitans sp. (MW049111)	3	800	150	150	200	150	150	25
RSRos01	Roseomonas vinaceus (MW049110)	3	800	50	100	-	50	100	25
RSRh07	Rhizobium sp. (MW049097)	-	400	200	100	50	50	50	25
RSRh01	Rhizobium sp. (MW049091)	-	400	150	100	50	50	50	-
RSRh02	Rhizobium sp. (MW049092)	-	400	150	100	100	100	50	-
RSRh03	Rhizobium sp. (MW049093)	-	400	150	100	100	100	50	-
RSRh04	Rhizobium sp. (MW049094)	-	500	150	100	100	100	50	-
RSRh05	Rhizobium sp. (MW049095)	-	400	150	100	100	100	50	-
RSRh06	Rhizobium sp. (MW049096)	-	400	150	100	100	100	50	-
RSRh08	Rhizobium sp. (MW049098)	-	400	100	100	100	50	50	-
RSRh09	Rhizobium sp. (MW049099)	-	500	50	100	100	100	50	25
RSRh10	Rhizobium sp. (MW049100)	-	700	150	100	100	100	100	-
RSRh11	Rhizobium sp. (MW049101)	-	700	100	100	100	50	50	-
RSPar01	Paracoccus marcusii (MW049112)	8	700	150	100	50	50	100	-

(-) no growth

Table 2 Screening of bacterial isolates with PGPR traits

Strain code	Bacterial species (GenBank accession number)	Extracellular enzymes					Siderophore production	Phosphate solubilization	Antifungal activity
Strain code	Bacterial species (GenBank accession number)	Esterase	Protease	Amylase	Urease	Cellulase	Siderophore production	Phosphate solubilization	*In vitro*	*In vivo*
RSRh01	Rhizobium sp. (MW049091)	-	-	-	+	+	-	+	-	NT
RSRh02	Rhizobium sp. (MW049092)	-	-	-	+	+	-	+	-	NT
RSRh03	Rhizobium sp. (MW049093)	-	-	-	+	+	+	+	+	-
RSRh04	Rhizobium sp. (MW049094)	-	-	-	+	+	+	+	+	-
RSRh05	Rhizobium sp. (MW049095)	-	-	-	+	+	-	+	++	NT
RSRh06	Rhizobium sp. (MW049096)	-	-	-	+	+	-	+	-	-
RSRh07	Rhizobium sp. (MW049097)	-	-	-	+	+	-	+	++	-
RSRh08	Rhizobium sp. (MW049098)	-	-	-	+	+	+	+	+	-
RSRh09	Rhizobium sp. (MW049099)	-	-	-	+	+	+	+	-	+
RSRh10	Rhizobium sp. (MW049100)	-	-	-	+	+	+	+	+	+
RSRh11	Rhizobium sp. (MW049101)	-	-	-	+	+	+	+	-	-
RSCup01	Cupriavidus paucula (MW049066)	+	+	-	+	+	+	-	+++	+
RSCup02	Cupriavidus paucula (MW049067)	+	+	-	+	+	+	-	+++	NT
RSCup03	Cupriavidus paucula (MW049068)	+	+	-	+	+	+	-	+++	+
RSCup04	Cupriavidus paucula (MW049069)	+	+	-	+	+	+	-	+++	NT
RSCup05	Cupriavidus paucula (MW049070)	+	+	-	+	+	+	-	+++	NT
RSCup06	Cupriavidus paucula (MW049071)	+	+	-	+	+	+	-	+++	NT
RSCup07	Cupriavidus paucula (MW049072)	+	+	-	+	+	+	-	+++	NT
RSCup08	Cupriavidus paucula (MW049073)	+	+	-	+	+	+	-	+++	NT
RSPs01	Pseudomonas putida (MW049082)	+	-	+	+	+	+++	-	+++	+
RSPs02	Pseudomonas sp. (MW049083)	-	-	-	+	+	++	-	+++	+
RSPs03	Pseudomonas frederiksbergensis (MW049084)	+	-	+	+	+	+++	++	+++	+
RSPs04	Pseudomonas frederiksbergensis (MW049085)	+	-	-	+	+	+++	++	+++	NT
RSPs05	Pseudomonas frederiksbergensis (MW049086)	+	-	-	+	+	+++	++	+++	NT
RSPs06	Pseudomonas thivervalensis (MW049087)	+	-	-	+	+	+++	++	+++	NT
RSPs07	Pseudomonas thivervalensis (MW049088)	+	-	-	+	+	+++	++	+++	+
RSPs08	Pseudomonas thivervalensis (MW049089)	+	-	-	+	+	+++	+++	+++	NT
RSPs09	Pseudomonas thivervalensis (MW049090)	+	-	-	+	+	+++	++	+++	NT
RSBac01	Bacillus licheniformis (MW049102)	+	-	-	-	-	++	-	-	+
RSBac02	Bacillus niacini (MW049103)	+	-	+	-	-	++	-	-	+
RSBac03	Bacillus niacini (MW049104)	+	-	-	+	-	-	+	++	+
RSMic01	Microbacterium foliorum (MW049074)	-	-	+	-	+	-	-	+++	+
RSMic02	Microbacterium foliorum (MW049075)	-	-	+	-	+	-	-	+++	NT
RSMic03	Microbacterium foliorum (MW049076)	-	-	+	-	+	-	-	+++	NT
RSMic04	Microbacterium foliorum (MW049077)	-	-	+	-	+	-	-	+++	NT
RSMic05	Microbacterium foliorum (MW049078)	-	-	+	-	+	-	-	+++	NT
RSMic06	Microbacterium foliorum (MW049079)	-	-	+	-	+	-	-	+++	NT
RSMic07	Microbacterium foliorum (MW049080)	-	-	+	-	+	-	-	+++	+
RSMic08	Microbacterium foliorum (MW049081)	-	-	+	-	+	-	-	+++	NT
RSAci01	Acinetobacter beijerinckii (MW049107)	+	+	-	+	+	+	-	+++	+
RSAci02	Acinetobacter sp. (MW049108)	+	+	-	-	+	-	++	+++	+
RSPro01	Providencia rettgeri (MW049114)	+	-	+	+	+	+++	-	+++	+
RSEnt01	Enterobacter xiangfangensis (MW049105)	-	-	-	+	+	+++	+++	+++	+
RSSta01	Staphylococcus xylosus (MW049113)	-	-	+	+	+	-	-	+++	+
RSPar01	Paracoccus marcusii (MW049112)	+	-	+	-	+	+	-	+++	+
RSKoc01	Kocuria dechangensis (MW049106)	+	-	-	+	+	+	-	++	+
RSFr01	Frondihabitans sp. (MW049111)	+	-	+	-	+	-	-	-	-
RSRos01	Roseomonas vinaceus (MW049110)	+	-	-	+	+	-	-	++	+
RSMicc01	Micrococcus yunnanensis (MW049109)	+	-	-	+	-	+	-	-	-

a (−) no activity, (+) enzyme activity detected

b (−) no production, (+) positive/weak, (++) intermediate, (+++) strong siderophore production

c (−) no solubilization, (+) positive/weak, (++) intermediate, (+++) strong solubilization of phosphate

d (−) no activity, (+) positive/weak, (++) intermediate, (+++) strong antifungal activity detected (in vitro)

e (−) no activity, (+) antifungal activity detected (in vivo), (NT) not tested

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Characterization of heavy metal-resistant rhizobia and non-rhizobia isolated from root nodules of Trifolium sp. in a lead and zinc mining area

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