Metal pollution of soils is a significant problem worldwide; it undermines the quality and fertility of soils and is an obstacle to sustainable development (Tchounwou et al., 2012). Soils can be natural as well as anthropogenically enriched with trace metals, which can be taken up by plants, and thereafter spread through the food chains (Walker et al., 2012). Metal ions, even at low concentrations, can hamper living organisms by disturbing cell metabolism, damaging the anatomical structure of tissues, leading to growth reduction, accelerated senescence, and necrosis, which may result in a reduction of population size, density, and genetic variability (Jaishankar et al., 2014). Consequently, metal pollution acts as a natural selection power on non-fit organisms and favors the survival and reproduction of individuals that carry adaptive traits (Macnair, 1997).

Certain plants have evolved ways to deal with high metal pollution (Viehweger, 2014; Wójcik et al., 2017; Nikalje and Suprasanna, 2018), and are associated with specific microbial communities that can be valuable in the remediation of polluted areas (Weyens et al., 2009a,b; Thijs and Vangronsveld, 2015; Yan et al., 2020). Metal accumulators possess the ability to store metal ions in aerial tissues (Lange et al., 2017), while excluders retain metals in their roots, thereby preventing metal translocation into the shoots (Shackira and Puthur, 2019; Zgorelec et al., 2020). Microorganisms inhabiting the rhizosphere of such plants can be of significant importance for their application in phytostabilization (Thijs and Vangronsveld, 2015; Wang et al., 2020). The beneficial effects of plant growth-promoting (PGP) bacteria on the metal tolerance of their hosts can be attributed to a lowered external or internal availability of metals due to incorporation of metals in the bacterial cell walls, metal sequestration inside bacterial cells, efflux and precipitation of metal ions on bacterial cell walls, or exopolysaccharide production. PGP bacteria can also indirectly improve plant growth due to traits like enhancing nutrient availability, synthesis of growth hormones, increasing the antioxidative status of plants, and/or protecting the plants from diseases and pathogens (Hayat et al., 2010; Abhilash et al., 2012; Oleńska et al., 2020a). Hence, identification of the most effective plant-microbe association is of significant importance for phytostabilization.

Legumes (Fabaceae) are commonly found as pioneer plants on metal-polluted sites, also on waste deposits of metal ore mining and processing in southern Poland (Nowak et al., 2011). They are well suited for site stabilization since they possess extensive root systems to protect soil from erosion, improve aeration for microbial activity, increase soil humus content, synthesize substantial amounts of biomass rich in proteins, and co-exist in symbiosis with rhizobia providing ammonium in the process of atmospheric nitrogen reduction (Padilla and Pugnaire, 2006; Li et al., 2007; Fterich et al., 2014; Roa-Fuentes et al., 2015). In southern Poland, some sites are extremely polluted with metals from the anthropogenic origin, including postindustrial waste deposits, where the soils are additionally highly deficient in water and nutrients (Wójcik et al., 2014). White clover (Trifolium repens), a member of the legume family, is found frequently on the more than 100-year-old Zn-Pb waste heap Bolesław in southern Poland (Nowak et al., 2011; Oleńska and Małek, 2013b). Rhizobium leguminosarum bv. trifolii bacteria were identified as inhabitants of plant root nodules (Oleńska and Małek, 2015), and this partnership might be a promising plant-microbe association for site stabilization. Earlier results (Oleńska and Małek, 2013b) revealed that R. leguminosarum bv. trifolii of the Bolesław waste heap carries genes whose products are involved in metal ion exclusion, metal tolerance as well as the production of exopolysaccharides with specific sugar composition (Oleńska et al., 2021). Moreover, Oleńska and Małek (2015, 2019) found that R. leguminosarum bv. trifolii populations inhabiting root nodules of white clover established on the Bolesław Zn-Pb waste heap show a moderate level of genetic diversity, while studies of other European R. leguminosarum bv. trifolii populations from metal-polluted sites show a drastic reduction of genetic diversity of these populations, which were not able to fix nitrogen (e.g., in Woburn, United Kingdom), and even lack any rhizobial symbionts (e.g., in Braunschweig, Germany) (Chaudri et al., 1993; Giller et al., 1998; Lakzian et al., 2002).

Since the level of genetic polymorphism of bacterial populations stands for adaptability and tolerance of bacteria to changing environmental conditions, T. repens nodule microsymbionts from the Bolesław waste heap might be interesting partners for symbiosis. It is worth noting that a symbiosis may also be established between T. repens and mycorrhizal fungi (Li et al., 2005). Indeed, endo- or ectomycorrhizal fungi were found to improve the growth of many plant species under metal stress conditions (Luo et al., 2014; Ważny et al., 2021). For example, symbiotic mycorrhizal fungi can sequester metal ions in their hyphae, acting as a barrier toward metals, and thus indirectly protect plant roots. Hence, white clover fitness in metal-polluted areas, such as the Bolesław waste heap, might be increased due to the joint action of rhizobia and mycorrhizal fungi.

In this study, we characterized the R. leguminosarum bv. trifolii—arbuscular mycorrhizal fungi (AMF)—white clover association as a potential tool in phytostabilization of metal-polluted soils. To investigate the activities of the T. repens nodule microsymbionts, R. leguminosarum bv. trifolii strains were isolated from root nodules of white clover growing on the Bolesław waste heap as well as on the reference grasslands of Bolestraszyce (Przemyskie Fothills). The rhizobial strains were studied for their Zn, Pb, and Cd tolerance, nodulation ability, the polymorphism of nodA genes encoding proteins involved in the nodulation process, nitrogenase enzyme activity, as well as for their PGP traits. To determine the effects of R. leguminosarum bv. trifolii on white clover growth, morphological parameters of plants, as well as biochemical ones of T. repens inoculated with the waste heap and reference site rhizobia, were studied. To define the strategy of the plants to deal with metals (accumulator vs. excluder), metal concentrations in leaves and roots were determined. Both light and transmission electron microscopy (TEM) analysis of root nodules were used to evaluate the adaptation of T. repens to metals. Soil nutrients (total N and nitrate, ammonia) and micro/macro-element concentrations were determined. The presence of mycorrhiza in white clover roots was investigated using TEM, and the genetic diversity of mycorrhizal populations was estimated using ARISA (automated ribosomal intergenic spacer analysis) fingerprinting.

Five T. repens root and leaf samples, soil samples, as well as forty-two R. leguminosarum bv. trifolii strains that were previously isolated (Oleńska and Małek, 2015) from nodules of T. repens that originated from the more than 100-year-old Zn-Pb waste heap in Bolesław (50°17′N 19°29′E, Silesia-Krakow Upland, Poland) and a grassland in the Bolestraszyce (49°48′N 22°50′E, Przemyskie Foothills, Poland) reference area, were used in this study (Supplementary Table 1).

The results of this study demonstrate that R. leguminosarum bv. trifolii bacteria isolated from nodules of T. repens growing on the polluted Bolesław waste heap harbor traits that can be beneficial to plant growth and health. Rhizobia, together with their host plant, which is a metal excluder, enter into a symbiosis with AMF, which have (AMF means arbuscular mycorrhizal fungi, fungi is a plural form) the potential to be adopted in phytostabilization. Chaudri et al. (1993) and Giller et al. (1998) reported that, on metal-polluted areas (Woburn, United Kingdom) R. leguminosarum bv. trifolii bacteria formed an ineffective symbiosis with clover, and were classified into only a few genotypes. In this study, we show that rhizobia from an old metal-polluted waste heap are tolerant to metals, enter into symbiosis with white clover, exhibit substantial nitrogenase activity, and are equipped with nodulation genes (i.e., nodA). The nodA gene is a member of the canonical core nodABC genes and encodes an enzyme that is involved in the transfer of an N-acyl residue into a lipochitooligosaccharide Nod factor, which plays a crucial role in nodule development. The Nod molecule is a host-specificity determinant that triggers, i.a., the plant cell developmental program resulting in the formation of a nodule, the entry of rhizobia into plant cells, and the progress in nodule formation (Franche et al., 2009). Moreover, we found that rhizobia from the waste heap area do not differ in nodulation ability as well as nitrogenase activity from rhizobia from the non-polluted area (Table 1 and Supplementary Table 1). Consequently, metal-tolerant rhizobia from the metal-polluted area can be considered as effective microsymbionts of leguminous plants on the extremely high-metal-polluted Bolesław waste heap.

Bacteria-induced improvement of plant fitness on metal-polluted soils may be achieved by direct interactions of microorganisms with metals, i.e., modifications of the cellular barrier permeability, preventing the transfer of metal ions into the cytoplasm, efflux of metal ions out of the cell, enzymatic reduction of metal ions, extracellular sequestration of metal ions by bacterial metabolites, and intracellular sequestration of ions (Ji and Silver, 1995; Silver and Phung, 1996; Bruins et al., 2000; Oleńska and Małek, 2013a) or/and by indirect beneficial effects on plant growth on metal-polluted sites (Oleńska et al., 2020a). It was demonstrated that R. leguminosarum bv. trifolii bacteria from the metal-polluted waste heap are equipped with some adaptation mechanisms to metals. For instance, they possess a gene, whose product may be potentially involved in chemiosmotic removal of Cd (Oleńska and Małek, 2013b). Moreover, it has also been shown recently that the R. leguminosarum bv. trifolii bacteria from the waste heap produce exopolysaccharides that differ substantially from the exopolysaccharides of rhizobia from the reference soil in both qualitative and quantitative compositions, suggesting a significant role of these compounds in direct interactions of rhizobia with metals (Oleńska et al., 2021). In biofilm studies, rhizobia from the waste heap show a significantly higher survival rate in non-exposed as well as metal-exposed conditions in comparison with strains from the non-polluted area, implying the adaptation of the waste heap strains to metals (Oleńska et al., 2021).

The present study shows that R. leguminosarum bv. trifolii bacteria from the metal-polluted waste heap possess traits that have the potential to indirectly improve the tolerance of their host to toxic metals through a beneficial influence on plant growth (Table 3). The percentage of rhizobia isolated from nodules of plants growing on the metal-polluted waste heap showing acetoin production is higher than for those from the non-polluted area. Acetoin (3-hydroxy-2-butanone) is considered as a significant inducer of induced systemic resistance (ISR) (Wang et al., 2009) and as a volatile organic compound (VOC), playing a role in the bacterial life cycle (e.g., regulation of bacterial motility, antibiotic resistance, biofilm formation) and as a compound participating in bacterial association with host plants (e.g., increases of plant biomass, fruit yield, seed production, lateral root and root hair formation, nutrient uptake, and photosynthetic activity) (Sharifi and Ryu, 2018a,b; Morcillo et al., 2020a,b). Oleńska et al. (2020b) reported that in in vitro studies also other bacterial taxa exhibit traits that can promote plant growth in conditions of toxic metal concentrations. For example, Bacillus thuringiensis, Chryseobacterium lathyri, Pseudomonas putida, Bacillus cereus, Stenotrophomonas maltophilia, which are also inhabitants of nodules of T. repens growing on the Bolesław waste heap, showed able to, e.g., synthesize acetoin, siderophores, IAA, ACCD, fix N₂, solubilize phosphates, as well as to tolerate exposure to increased metal concentrations. Furthermore, Sánchez-López et al. (2018) revealed that, in case of high concentrations of Zn/Pb/Cd in soil, the Methylobacterium sp. strain Cp3 possesses potentially beneficial traits, i.e., plant growth promotion and metal tolerance. It is also known that bacteria producing siderophores, which are low molecular (400–1,500 Da) weight chelators, with a high affinity for unavailable Fe(III) and transforming it into Fe(II), supply plants with iron that is crucial for chlorophyll synthesis, maintenance of chloroplast structure and function, DNA synthesis, respiration, and is a constituent of many enzyme prosthetic groups (Rout and Sahoo, 2015; Jian et al., 2019). Shi et al. (2017) reported enhanced siderophore synthesis in Pseudomonas aeruginosa under Cd(II) and Zn(II) stress, while Złoch et al. (2016) showed synthesis of hydroxamate-, catecholate-, and phenolate-type siderophores production by Streptomyces sp. from Betula pendula and Alnus glutinosa growing in the presence of Cd(II). The Bacillus spp. strain PZ-1 synthesized hydroxamate-type siderophores when exposed to toxic Pb(II) concentrations and enhanced the storage of this metal in the underground tissues of Brassica juncea (Yu et al., 2017; Jinal et al., 2019). Auxins are key regulators of plant development, e.g., cell division, expansion, and differentiation (Paque and Weijers, 2016). Increased expression of IAA was reported, for instance, for Pseudomonas grimontii strain Bc09, Pantoea vagans strain So23, Pseudomonas veronii strain E03, and Pseudomonas fluorescens strain Oj24 that positively influenced biomass production of switchgrass under Cd(II) stress (Begum et al., 2019). Similarly, Leifsonia xyli strain SE134 under Cu exposure showed an enhanced IAA synthesis (Kang et al., 2017). In plants inoculated with ACCD-producing bacteria, longer roots and higher resistance to pathogens were reported (Ravanbakhsh et al., 2017; Ghosh et al., 2018; Saikia et al., 2018; Gupta and Pandey, 2019). Han et al. (2015) showed a positive effect of ACCD-producing Pseudomonas stutzeri A1501 strain on rice biomass. Solubilization of inorganic phosphates by bacteria is predominantly performed as the result of production of organic acids (Zhao and Zhang, 2015; Naraian and Kumari, 2017). Oves et al. (2017) revealed that, under metal stress, Ensifer adhaerens strain OS3 is a phosphate solubilizer and a chromium reducer. Taking into consideration that the rhizobia from the metal-polluted waste heap exhibit positive traits in vitro, we may assume that these bacteria may accomplish a substantial role in improving plant growth, including these able to accumulate toxic metals and being useful for remediation purposes.

Rhizobia from both, the waste heap and the reference area, influence positively the growth of T. repens (Table 1 and Figure 2). No significant differences in chlorophyll a and b, the sum of chlorophylls, the chlorophyll a to b ratio, except protein concentration, were found between white clover inoculated with rhizobia from the waste heap and the reference area. Therefore, it can be assumed that R. leguminosarum bv. trifolii bacteria originating from the waste heap can be proposed as endophytes efficient to improve the growth of T. repens in conditions of high metal pollution, where phytostabilization is required. Bidar et al. (2007) suggested that T. repens can be used as a phytostabilizing plant species in metal-polluted areas; Oleńska et al. (2020b) suggested that white clover has the potential to be used for phytostabilization on the Bolesław waste heap. The results of the present study demonstrate that T. repens accumulates metals in roots and leaves, and that the root is the predominant location of metal accumulation (Table 4). Light and transmission electron microscopy investigation of nodules of T. repens growing on the waste heap revealed morphological adaptations of plants to toxic metals that are manifested predominantly as apoplast modifications (Figures 3, 4). Suberinization of the cortex cell walls, as well as the presence of granules in their vacuoles, suggest redirection of stored material (conceivably polyphenols and metals connected with organic acids) into the apoplast, what was found exclusively in plants from the waste heap and can be considered as adaptations to metal stress. Redirection of ions in extracellular (i.e., cell walls) and intracellular (i.e., vacuole) spaces is the first line of plant defense against toxic metals (Hasan et al., 2017). After entering into root cells, metal ions can form complexes with different ligands, e.g., organic acids and as carbonate, sulfate, or phosphate precipitates, which are accumulated among others in vacuoles, preventing the accumulation of free metal ions in a cytosol (Ali et al., 2013; Yan et al., 2020).

Ważny et al. (2021) claimed that mycorrhiza may serve as a significant, sufficient constituent of plant adaptations to metal-polluted areas. Mycorrhizal fungi can enhance the metal tolerance of their host plants by increasing the uptake of water, nutrients, as well as plant growth-promoting traits, e.g., by binding metals in the mycelium (Luo et al., 2014). In mycorrhized plants under metal stress, Turnau (1998) reported preferential accumulation of metals in intraradical fungal structures rather than in root tissues. Yang et al. (2016) reported a higher Pb uptake in Robinia pseudoacacia roots mycorrhized with Rhizophagus intraradices compared to non-mycorrhizal plants. Also, biomass as well as nutrients (i.e., N, P, S, and Mg) uptake were reported to increase in case of AMF presence in roots. In addition, these authors found that the presence of other mycorrhized legume herbs, i.e., Trifolium pratense and Medicago sativa increased the mycorrhization of R. pseudoacacia, possibly due to a common signaling pathway turned on by mycorrhiza and rhizobia. Leguminous plants also lowered soil pH, resulting in higher availability of Pb ions. Dhalaria et al. (2020) reported a substantial role of mycorrhizal fungi vesicles in the accumulation of metals as well as the synthesis of glycoprotein chelators (e.g., glomalin) by mycorrhizal fungi and enhancing the antioxidative responses in metal stress conditions. Our microscopic analyses of T. repens roots revealed the presence of AMF only in plants from the metal-polluted waste heap (Figure 5). This may suggest that these AMF fulfill an important role in the adaptation of the plants to the extreme conditions on the waste heap. Martínez-Hildago and Hirsch (2017) reported a substantial role for AMF in the selection of bacterial nodule residents, and Rodríguez-Caballero et al. (2017) showed changes in the plant bacterial communities under influence of AMF. It was also demonstrated that AMF may trigger plant metabolism, e.g., induce a systemic response through leaf protein expression (Lingua et al., 2012). Moreover, Scheublin et al. (2004) reported arbuscular mycorrhizal fungi invasion of legume nodules, and that AMF communities vary depending on plant species as well as parts of a root system.

The present study shows that the Bolesław waste heap, besides the occurrence of very high metal concentrations, is also deficient in nutrients, including bioavailable forms of nitrogen, i.e., ammonium and nitrate ions (Table 5). It is obvious that the presence of rhizobia, effectively fixing nitrogen and reducing it into ammonia that is available to leguminous plants, is of significant importance for their hosts, inhabiting such challenging metal-polluted and nutrient-poor areas. Also, the presence of genetically diverse AMF communities in roots and nodules of T. repens, originating from the metal-polluted waste heap area, is promising in the function of protection of leguminous plants against toxic metals. However, the taxonomy and characteristics of these AMF still must be studied more in detail. Taking into consideration that leguminous plants, entering in symbiotic interaction with rhizobia fixing N₂, are pioneers on nutrient-poor soils (Padilla and Pugnaire, 2006; Li et al., 2007; Fterich et al., 2014; Roa-Fuentes et al., 2015), we may conclude that R. leguminosarum bv. trifolii, mycorrhizal fungi, and T. repens, together constituting a metaorganism (Thijs et al., 2016), can be a promising tool for phytostabilization of Zn-, Pb-, and Cd-polluted soils.

The data presented in the study are deposited in the NCBI GenBank repository, accession numbers MZ231019-23.

EO contributed to conceptualization, data curation, project administration, resources, and writing the original draft. EO, MS-R, ST, SS, and OA contributed to formal analysis. EO and JV contributed to funding acquisition. EO and ST investigated the study and contributed to software. TW, ST, SS, EO, and MS-R contributed to methodology. JV and WM contributed to supervision. MW, IS, and WM contributed to validation. EO, ST, and MS-R contributed to visualization. JV, MW, WM, and MS-R contributed to writing, reviewing, and editing. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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