Mining activities and contamination introduced by these activities are severe environmental issues in China. Bioremediation mediated by plants and microorganisms is an effective method for alleviating mining contamination. The roots connecting soils and plants are hotspots for interactions between microorganisms and the environments. Although microbial communities in mining-contaminated sites have been extensively characterized, less attention has been focused on the roots, especially the root endosphere. In this study, microbial communities in plant compartments, including the bulk soil, rhizosphere soil, and the root endosphere, were characterized and compared. Their interactions with geochemical conditions were also analyzed and discussed.
4.1. Bacterial diversity was influenced by soil contamination level
Endosphere species richness was lower than the bulk and rhizosphere soil richness, indicating the non-uniform and selective colonization of plant roots (Beckers et al., 2017). It is well known that the root microbiome is primarily assembled from external soil microbes (Edwards et al., 2015), which may migrate to the rhizosphere due to the attraction of rich nutrients like root exudates (Zhalnina et al., 2018), resulting in the rhizodeposition of various microbes. However, root-colonizing bacteria are highly competitive in order to ensure successful colonization. Therefore, root-colonizing bacteria possess traits that favor resistance to harsh environments, such as chemical toxicity and pathogen disease (Mendes et al., 2011; Berendsen et al., 2012). Thus, bacteria inside the root endosphere are much less rich and diverse than in the rhizosphere soil, as confirmed in this study, with the exception of the SL root endosphere samples (Fig. S3), which had the highest Shannon and Simpson diversity indices when compared to the bulk and rhizosphere soils (Fig. S3d, e). These findings may be attributed to the long-term impact pollution on Cyp roots in the surrounding SL tailing, which may affect Cyp development, shape root-associated microbes, and change acquisition patterns in the root endosphere (Edwards et al., 2015; Chaparro et al., 2014).
Based on the bacterial richness and diversity indices, we found that the alpha diversity was highly dependent on the sample site. The SL samples had the lowest richness and diversity, regardless of sample type, followed by the GD samples, while the SD samples possessed the highest alpha diversity (Fig. S3). These results implied that the contamination level of the Pb/Zn mines greatly affected and shaped plant-associated microbes. The concentrations of metal(loid)s, including Cu, Cd, Pb, Zn, As, and Sb, in the SL bulk soil were significantly higher than those in SD bulk soil, which exerted persistent selection pressure on the assemblage of soil microbes (Fig. 2b). Additionally, soil characteristics, including pH and the nitrate and sulfate contents in the SL bulk soil, were significantly higher compared to those of the SD bulk soil (Fig. 2a), which have been reported to closely correlate with soil bacterial communities (Gagnon et al., 2020; Xiao et al., 2016; Chen et al., 2013). In contrast, the GD site was being actively mined and its contamination of the surrounding soils was less severe. The concentrations of metal(loid)s in the GD bulk soil were lower compared to the SL bulk soil, but were still significantly higher than in the SD bulk soil (Fig. 2b). Moreover, a significantly higher pH and lower SOC were also observed (Fig. 2a). These factors likely modulate the population and composition of plant-associated microbes, resulting in the loss of bacterial richness and diversity. Additionally, the PCoA analysis showed site-dependent clustering of the bulk soil, rhizosphere soil, and root endosphere communities (Fig. 4a). This result is consistent with the alpha diversity results, confirming that contamination conditions greatly affected microbial assemblages. As for specific sites (i.e., the GD, SL, and SD), the majority of bulk and rhizosphere soil samples clustered together (Fig. 4b), suggesting that bacterial diversity was comparable between the bulk and rhizosphere soil samples. In contrast, the root endosphere samples were sporadically distributed, indicating distinct patterns of bacterial communities.
4.2. Structural distribution of the dominant bacterial communities
Bacterial structures and compositions were dependent on the sampling site and Cyp compartment. However, phylum Proteobacteria (mostly Alphaproteobacteria and Gammaproteobacteria) consistently dominated bulk soil, rhizosphere soil, and root endosphere samples, followed by Acidobacteria and Actinobacteria. These phylotypes have been commonly identified in the soil microbiome surrounding mining areas (Gao et al., 2019; Sun et al., 2018b). It was previously reported that the ratio of Proteobacteria and Acidobacteria could be an indicator of soil trophic levels (Castro et al., 2010; Smit et al., 2001; Gottel et al., 2011; Beckers et al., 2017), in which, Acidobacteria and Proteobacteria were associated with nutrient-rich and nutrient-poor soils, respectively. The relative abundance of Proteobacteria (mostly Alphaproteobacteria) increased from rhizosphere soil to the root endosphere, while Acidobacteria decreased in the GD and SD samples, indicating nutrient-rich conditions of the root endosphere compared to rhizosphere and bulk soils. Similar results were observed in other plants, including poplar (Beckers et al., 2017; Gottel et al., 2011; Shakya et al., 2013) and rice (Edwards et al., 2015). In the SL samples, however, contrasting results were observed, where the relative abundance of Proteobacteria (mostly Alphaproteobacteria and Gammaproteobacter) decreased remarkably from rhizosphere soil to the root endosphere, while Acidobacteria increased slightly, which was similar to previous reports on grasslands (Singh et al., 2007) and soybeans (Xu et al., 2009).
These findings suggested that heavy contamination (i.e., high contents of metal(loid)s) in the SL site likely changed the soil nutrient status and thereby shaped bacterial assemblages. Similar results were previously reported by Sun et al. (2018a) who found that plant rhizosphere communities strongly correlated with Cr and V in Pb/Zn mining sites. Moreover, Acidobacteria and Proteobacteria in the bulk and rhizosphere soil samples from the GD and SD sites were comparable, indicating an intermediate nutrient level. This result is consistent with the alpha diversity results (Fig. S3). As for the SL samples, Proteobacteria were enriched in the rhizosphere soil when compared to the bulk soil, which may be attributed to the nutrient-rich conditions of the rhizosphere soil. This result is likely due to the production of root exudates under the selection of metal(loid)s in polluted soils (Kozdrój and van Elsas, 2000; Qin et al., 2007), as well as the increased pollution resistance and/or tolerant Proteobacteria, such as Alphaproteobacteria, in this study (Sandaa et al., 1999). In addition to metal(loid)s, soil pH was previously found to correlate with overall bacterial community composition and phylogenetic diversity, which affect bacterial relative abundances, such as Acidobacteria (Lauber et al., 2009; Qi et al., 2018). Unfortunately, the pH values of the rhizosphere soil and the root endospheres were not directly measured in this study due to a lack of samples. Rhizosphere pH is mediated by plant roots (i.e., exudates of organic acids) in response to environmental constraints (Hinsinger et al., 2003; Sasse et al., 2018). As a result, plant-associated microbial communities in contaminated mining sites are largely shaped by various factors, including plant genotype and soil conditions.
Additionally, phylum Tenericutes was exclusively found in the SL root endosphere samples with a relative abundance of 14.1%, which was exclusively consisted of genus Acholeplasma. Previous studies found that Acholeplasma belongs to wall-less, saprophytic, and free-living bacteria (Zhao et al., 2015; Mitter et al., 2017), which is transmitted to plants and colonize in the vascular tissues as a sap-sucking endophyte (Blain et al., 2017). Phylum Deinococcus Thermus (mostly Meiothermus) was remarkably richer in SL samples than in GD and SD samples. This result can be ascribed to the heavy contamination conditions of SL sites, as Meiothermus species are always present in extreme environments (Thokchom et al., 2017). Consistently, the dominance of Meiothermus has been observed in mining tailings (Sun et al., 2018b). Thus, it was proposed that Meiothermus may have the potential to oxidize and reduce As.
4.3. Core members of the root bacterial microbiome
Within the core bacterial microbiome, similar site-specific distributions were observed. At the genus level, the GD and SD bulk and rhizosphere soil communities were dominated by Gp6, Spartobacteria genera incertae sedis, Bradyrhizobium, Gp16, and Gaiella, of which, Gp6 and Bradyrhizobium also dominated root endosphere assemblages. This finding suggested that the root endosphere communities consisted of a subset of specific microbes from the corresponding rhizosphere soil, which was consistent with the findings of previous reports (Hallmann et al., 1997; Miliute et al., 2015). Similar results were observed in SL rhizosphere soil and root endospheres, which were both primarily dominated by Halomonas, Pelagibacterium, and Chelativorans, and their rhizosphere relative abundances were almost one order of magnitude higher. Bradyrhizobium is a common endophyte with higher abundance in the root endosphere than in the bulk and rhizosphere soils, which has been reported to improve plant growth in the heavy metal-contaminated environment (Wani et al., 2008; Seneviratne et al., 2016). In contrast, overall distinct compositions of root core microbes between SD and SL samples, which could be ascribed to the selection pressures of the surrounding soil conditions (described above). Halomonas of class Gammaproteobacteria is capable of indole acetic acid production and phosphate solubilization in the presence of high concentrations of heavy metals and high-salinity, which thereby promotes plant growth (Desale et al., 2014). Additionally, Halomonas species isolated from mangrove Avicennia marina rhizosphere soil increased exopolysaccharide production under arsenic stress, thereby enhancing rice growth (Mukherjee et al., 2019). Pelagibacterium of class Alphaproteobacteria has been found in Tamarix ramosissima root-associated communities and is tolerant to salinity (Taniguchi et al., 2015). Some species have been found to grow well in high-salinity environments, such as seawater (Li et al., 2013; Xu et al., 2011; Wang et al., 2017), lake water (Lu et al., 2018), and deserts (Yang and Sun, 2016). Several Chelativorans species of class Alphaproteobacteria have been isolated from EDTA-enriched soils (Bohuslavek et al., 2001). These species grew with EDTA as carbon, nitrogen, and energy sources (Doronina et al., 2010). This microbe was relatively abundant in SL rhizosphere soil, which may be due to EDTA pollution from mining. Chelativorans species have also been found in seawater (Evans et al., 2018). A recent study reported that Chelativorans species were abundant in marine sponge-associated microbes in the Palk Bay of India, which has suffered from sewage wastewater, salt pan, and heavy metal pollution (Meenatchi et al., 2020). In this study, it was interesting to find that the core bacterial microbes in SL rhizosphere soil (i.e., Halomonas, Pelagibacterium, and Chelativorans) were salinity- and metal-tolerant communities, which could be ascribed to long-term selection under heavy soil pollution in the SL site.