Co-inoculation effect of plant-growth-promoting rhizobacteria and rhizobium on EDDS assisted phytoremediation of Cu contaminated soils
Graphical abstract
Introduction
Toxic heavy metals present in agricultural ecosystems are difficult to degrade, and thus, they can enter the food chain and be ingested by humans (Antoniadis et al., 2017; Shahid et al., 2017b). Chelant-enhanced phytoremediation, a technique that increases the solubility of toxic metals in soils and facilitates phytoextraction efficiency, has received increased interest in recent decades as a promising technology for the remediation of soils contaminated with toxic heavy metals (Luo et al., 2018; Tsang et al., 2012). S,S-ethylenediaminedisuccinic acid (EDDS), a strong and easily biodegradable chelating agent, is less toxic to plants and microorganisms than the more traditionally used ethylenediaminetetraacetic acid (Beiyuan et al., 2018b; Prieto et al., 2013; Tsang et al., 2009). Moreover, EDDS has the potential to release nutrients and subsequently improve phytoextraction efficiency (Fang et al., 2017). Therefore, EDDS is considered one of the most efficient chelants and has been widely used in metal-contaminated soils.
However, EDDS can increase the bioavailability of Cu in soils (Beiyuan et al., 2018a; Luo et al., 2015) and high Cu phytotoxicity can directly and indirectly induce oxidative injury to and metabolic perturbations in plant tissue (Mondaca et al., 2017). Moreover, the high phytotoxicity of metals can reduce plant biomass yield; thus, limit the efficiency of heavy metal phytoremediation (Antoniadis et al., 2017). The application of EDDS has been shown to decrease the biomass of plants grown in Pb-contaminated soils and the decrease is more pronounced at a high chelate dose (10 mmol kg−1 soil) (Zhao et al., 2016). EDDS can also increase the accumulation of metals in plant tissue, which subsequently induces oxidative damage by increasing the level of reactive oxygen species (ROS) and lipid peroxidation (Cestone et al., 2012; Sidhu et al., 2018). As the main source of ROS, hydrogen peroxide (H2O2) and superoxide radicals (O2−) can damage DNA, proteins, lipids, and other cellular components in plant tissues (Woodson et al., 2015). An increase in the level of malondialdehyde (MDA) generally indicates lipid peroxidation in metal-treated plants. Antioxidant enzymes can reduce oxidative damage in plants and alleviate phytotoxicity induced by excessive ROS production (Shahid et al., 2017b).
In addition, high concentrations of toxic metals in soil are detrimental to plant biomass generation, owing to a low content of nutrients (Wilson-Kokes and Skousen, 2014) and the impact on soil biological activities (Fang et al., 2017). Chelant application can result in high heavy metal concentrations in soils, which further influences soil biological activities and the microbial community composition (Asadishad et al., 2018; Beiyuan et al., 2018a). Soil enzyme activities are vital for promoting nutrient cycling and plant growth (Zhang et al., 2019) and can be indicative of heavy metal pollution (Duan et al., 2019; Yang et al., 2013). The rhizosphere, which is colonized by microbiota, is central to microbial activity, contributing to nutrient transformation, phytoremediation, and plant growth (Lundberg et al., 2012; Yergeau et al., 2014). High metal concentrations can inhibit soil enzyme activities by reacting with enzyme–substrate complexes and destroying protein activity (Belyaeva et al., 2005), which can subsequently affect specific biological processes in soil. Previous studies have shown that the application of EDDS in Cu-contaminated soils resulted in both negative and positive impacts on the diversity of rhizosphere microbial communities and enzyme activities (Yang et al., 2013; Yoo et al., 2018). Low enzyme activity, as well as reduced rhizosphere microbial activity, can limit soil nutrient cycling and plant growth and further reduce the phytoextraction capacity. Thus, it is important for effective chelant-enhanced phytoremediation to increase plant growth, the nutrient content, and the biological activity in soil.
Plant growth-promoting rhizobacteria (PGPRs) and rhizobium are used as supplements for metal phytoremediation, as they can substantially alleviate phytotoxicity and improve soil biological activities under toxic metal stress (Antoniadis et al., 2019; Sipahutar et al., 2018). PGPRs and rhizobium decrease oxidative damage to plant tissues that are exposed to heavy metal stress by enhancing antioxidant enzymatic systems (Ju et al., 2019). Additionally, PGPRs can indirectly increase plant growth by improving soil properties and biological activities (Etesami, 2018). A previous study showed that the growth-promoting phytohormone auxin (indole-3-acetic acid) alleviated the toxic effects of Pb and Zn on the growth of sunflower and increased, in combination with EDDS, the phytoextraction potential of sunflower tissue (Faessler et al., 2010). Co-application of a PGPR (Pseudomonas sp. DGS6) and EDDS has been shown to improve plant growth and increase Cu and Zn uptake by plants (Luo et al., 2015). Symbiotic relationships with metal-resistant rhizobium may improve the phytoremediation ability of legumes through various mechanisms, such as increasing the metal bioavailability and plant metal resistance, which increase the accumulation of heavy metals in plant tissues (Teng et al., 2015). Metal-resistant rhizobia can improve plant growth and affect the bioavailability of toxic metals in response to the addition of nitrogen (Pajuelo et al., 2011). PGPRs can increase N availability to plants by mineralizing organic N in soil (Etesami, 2018; Shahzad et al., 2014), and the degradation of EDDS can also provide a source of N (Fang et al., 2017). The exploration of synergistic plant–microbe–chelant relationships may be an effective strategy for enhancing phytoremediation efficiency (Hussain et al., 2019). Our previous studies have shown that rhizobacterial inoculation increased soil enzymatic activities and microbial diversity (Ju et al., 2019, Ju et al., 2020). Therefore, the combination of legume–rhizobium symbiosis and PGPRs may potentially contribute to the improvement of plant growth and soil biological activities. However, it remains unclear whether co-inoculation of PGPRs and rhizobium can regulate plant tolerance and soil quality in response to metal stress during chelant assisted phytoremediation.
Based on previous data, it is reasonable to expect that co-inoculation with PGPRs and rhizobium will alleviate Cu phytotoxicity, alter soil biological parameters, and affect the phytoremediation process in chelant-supplemented metal-contaminated soils. Alfalfa (Medicago sativa), a legume, has been used in phytoremediation studies because it is a fast-growing perennial plant, which is able to develop an extensive root system that serves as a niche for rhizosphere microorganisms (Ju et al., 2019; Pajuelo et al., 2011). Therefore, the PGPR Paenibacillus mucilaginosus and rhizobium Sinorhizobium meliloti were co-inoculated during EDDS assisted phytoremediation of a Cu-contaminated soil using alfalfa. The aim of this study was to (1) investigate the effect of rhizobacterial inoculation on the efficiency of EDDS-enhanced phytoextraction of Cu; and (2) reveal the underlying mechanisms of the positive effects of co-inoculation on plant Cu tolerance and soil quality during EDDS-enhanced phytoremediation. We anticipate that the results will provide novel insights into the effects of rhizobacterial inoculation on plant growth and soil quality during chelant assisted phytoremediation and establish an efficient phytoremediation strategy.
Section snippets
Experimental design
Soil samples were collected from the surface (0–20 cm) of a Cu smelter area in Huangshi City (30°43′ N, 114°54′ E), Hubei Province, China. After being air-dried, the samples were passed through a 2-mm sieve. The physicochemical properties of the samples, showing that Cu was the major contaminant, are presented in Table S1. A PGPR strain (P. mucilaginosus ACCC10013) and a Cu-resistant rhizobium strain (S. meliloti CCNWSX0020) (Ju et al., 2019) were used as experimental strains. P. mucilaginosus
Plant growth and soil physicochemical properties
The shoot and root biomass was 16.7% and 57.5% lower in the AE treatment than in the control (A), respectively (Table 1). However, the shoot, root, and total biomass was 22.8%, 38.8%, and 25.9% higher in the APSE treatment than in the AE treatment, respectively. The nutrient content in the shoots and roots was also higher in the APSE treatment than in the AE treatment (Table S2). The soil pH and available N were significantly higher and the total Cu content was significantly lower in the AE
Implications of co-inoculation with PGPR and rhizobium on Cu phytoextraction during EDDS assisted phytoremediation
The application of EDDS significantly decreased the plant biomass (Table 1), which could be attributed to increased toxicity and oxidative damage, resulting from high metal concentrations in the plant tissue (Fig. 1). Co-inoculation with PGPR and rhizobium increased plant biomass and improved plant growth. Additionally, the application of EDDS significantly increased the accumulation of Cu in alfalfa shoots and roots, which subsequently inhibited plant growth. The TF and BCF values were higher
Conclusions
In this study, we demonstrated that rhizobacterial inoculation further stimulated Cu phytoextraction during EDDS-enhanced phytoremediation by enhancing plant Cu tolerance and soil quality. EDDS application damaged alfalfa organs, owing to excessive Cu-induced accumulation of MDA and ROS, which subsequently inhibited the antioxidant capacity of plant organs. However, co-inoculation of PGPR and rhizobium increased the tolerance of alfalfa to Cu stress by mitigating the accumulation of MDA and ROS
CRediT authorship contribution statement
Wenliang Ju: Methodology, Investigation, Data curation, Writing - original draft. Lei Liu: Methodology, Investigation, Data curation, Writing - original draft. Xiaolian Jin: Investigation, Software, Formal analysis. Chengjiao Duan: Investigation, Software, Formal analysis. Yongxing Cui: Investigation, Software, Formal analysis. Jie Wang: Investigation, Software, Formal analysis. Dengke Ma: Investigation, Software, Formal analysis. Wei Zhao: Writing - review & editing. Yunqiang Wang: Writing -
Declaration of competing interest
This study does not involve human subjects. This manuscript is original and it has not been previously published or submitted in part or whole. No conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all authors for publication.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (41571314 and 41671406), CAS “Light of West China” Program (XAB2016A03), and Education Department Foundation of Zhejiang Province (Y201738652).
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These authors contributed equally to this work.