Abstract:

Metal mine tailings constitute highly degraded regions marked by extreme nutrient depletion, poor structural stability, and severe heavy metal contamination, collectively generating conditions that are largely prohibitive for plant establishment and microbial colonization. These constraints pose major challenges to the efficiency and sustainability of ecological restoration in mining-affected areas. In such environments, symbiotic interactions between plants and microorganisms represent a key adaptive strategy, enhancing stress tolerance and promoting ecosystem recovery. Among the wide range of beneficial microorganisms, arbuscular mycorrhizal fungi (AMF) and nitrogen-fixing rhizobia (Rhizobium) are especially significant, given their complementary ecological functions. AMF can improve plant nutrient acquisition, particularly phosphorus uptake, and contribute to heavy metal immobilization through hyphal sequestration and modification of rhizosphere conditions. Rhizobia, in turn, enhance nitrogen availability through biological nitrogen fixation and stimulate plant growth via multiple physiological pathways. However, despite their individual benefits, the synergistic effects of AMF and rhizobia under complex co-contamination by multiple heavy metals, particularly in metal tailings, remain insufficiently understood. Therefore, in this work, vanadium-titanium magnetite tailings contaminated with copper (Cu) and chromium (Cr) were selected as the experimental substrate, and the leguminous plant alfalfa (Medicago sativa L.) was used as the phytoremediation species. A controlled pot experiment was designed to systematically evaluate the individual and combined effects of AMF inoculation and rhizobium inoculation on plant growth, tailings nutrient status, heavy metal speciation, and microbial community structure. Four treatments were established: a non-inoculated control, single inoculation with AMF, single inoculation with rhizobia, and combined inoculation with both AMF and rhizobia. To evaluate remediation performance and ecological responses, a comprehensive set of indicators was measured, including plant biomass, root nodulation characteristics, physicochemical properties of tailings, enzyme activities, and microbial community composition. To elucidate the mechanisms underlying microbial inoculation effects, particular attention was given to the interactions among plant roots, microbial symbionts, and the tailings matrix. The establishment of symbiosis can modify rhizosphere conditions through root exudation, microbial metabolism, and the development of fungal hyphal networks, thereby influencing nutrient availability and heavy metal mobility. Co-inoculation with AMF and rhizobia is expected to produce synergistic effects, with AMF enhancing phosphorus acquisition and increasing absorptive surface area via extraradical hyphae, and rhizobia providing biologically fixed nitrogen to sustain plant growth under nutrient-deficient conditions. This complementary interaction is anticipated to further promote root development and improve plant tolerance to heavy metal stress. In addition, microbial inoculation may regulate heavy metal transformation and distribution through adsorption, complexation, and redox processes, as well as by modifying rhizosphere pH, organic matter content, and microbial activity. Therefore, the impact of AMF and rhizobia was evaluated not only in terms of plant growth promotion but also from the perspective of nutrient cycling, metal stabilization, and microbial community regulation. The results indicated that elevated concentrations of heavy metals significantly inhibited plant growth, as reflected by substantial reductions in both shoot and root biomass. At the same time, heavy metal stress led to a decline in tailings nutrient status, with decreased levels of alkaline hydrolyzable nitrogen and available phosphorus, indicating a significant deterioration of substrate fertility. In contrast, microbial inoculation effectively alleviated these inhibitory effects. Among all treatments, co-inoculation with AMF and rhizobia yielded the most significant improvement in plant performance. More specifically, the shoot and root dry biomass increased by 150% and 49.3%, respectively, compared with the control. Root nodulation was also markedly enhanced in both number and fresh weight, suggesting a remarkable improvement in symbiotic nitrogen fixation capacity and root system development. Beyond promoting plant growth, microbial inoculation significantly improved the nutrient status of the tailings. The dual inoculation treatment led to an approximate doubling of alkaline hydrolyzable nitrogen and available phosphorus, accompanied by increases in soil organic matter and overall enzyme activities. These findings highlight that the synergistic interaction between AMF and rhizobia can enhance nutrient cycling processes, accelerate organic matter turnover, and create a more favourable substrate environment for plant growth. The increased enzyme activities further reflect enhanced microbial metabolic intensity and functional diversity. Moreover, microbial inoculation exerted a significant effect on heavy metal speciation and bioavailability. The combined AMF-rhizobium treatment reduced the bioavailable fractions of Cu and Cr, thereby lowering their ecological risk. Notably, the toxicity of Cr, which is highly mobile and particularly hazardous, was markedly alleviated under dual-inoculation conditions. These effects are likely driven by multiple mechanisms, including metal immobilization by fungal hyphae, complexation with organic matter, and microbial-mediated transformations. Such processes play a key role in reducing the mobility and toxicity of heavy metals, thereby improving environmental conditions for plant growth and microbial activity. Microbial community analysis further revealed that dual inoculation reshaped the microbial community structure. Beneficial microorganisms, including phototrophic bacteria and nitrogen-fixing bacteria, were significantly enriched, while the relative abundance of potentially pathogenic fungi was reduced. Additionally, the dual symbiotic system appeared to regulate competition among AMF populations and optimize resource allocation within the rhizosphere, thereby enhancing overall ecological functionality. These changes collectively contributed to the establishment of a more stable and functionally balanced microbial community under heavy metal stress. Overall, the tripartite symbiotic system consisting of alfalfa, AMF, and rhizobia demonstrated comparative advantages in enhancing plant tolerance to heavy metal stress, improving tailings nutrient status, reducing heavy metal bioavailability, and optimizing microbial community structure. This synergistic interaction not only promotes plant growth but also accelerates the ecological restoration of degraded tailings environments. These findings provide valuable theoretical insights and practical guidance for the development of efficient and sustainable bioremediation strategies for metal-contaminated tailings. The importance of integrating multi-microbial symbiosis into the design of ecological restoration strategies for degraded mining areas is also highlighted. Compared with single inoculation approaches, the combined application of AMF and rhizobia offers a more stable and resilient system capable of maintaining functionality under prolonged environmental stress. This not only improves short-term plant establishment but also contributes to long-term ecosystem sustainability by enhancing nutrient retention, reducing metal mobility, and promoting beneficial microbial succession. Future research is needed to further explore the long-term field performance of such symbiotic systems and investigate their interactions with other soil organisms and environmental factors, in order to optimize large-scale application in mine tailings remediation.

Key words: arbuscular mycorrhizal fungi (AMF), rhizobia, tailings, heavy metals, microbial community

摘要：

金属尾矿大多养分贫瘠、重金属污染严重，植物和微生物难以定殖，严重制约了生态修复进程。共生是生物应对胁迫的重要策略，丛枝菌根真菌（AMF）和根瘤菌（Rhizobium）均为重要的共生微生物，有利于提高植物在胁迫环境中的耐性。以钒钛磁铁尾矿为研究对象，以豆科植物紫花苜蓿（Medicago sativa L.）为修复植物，研究AMF和根瘤菌对Cu和Cr污染尾矿中植物生长、尾矿养分、重金属形态及微生物群落的影响。结果表明，高浓度重金属污染显著抑制了植物生长，降低尾矿养分质量分数。接菌显著缓解了重金属胁迫效应，其中丛枝菌根真菌和根瘤菌双接菌效果最优，使地上、地下部干质量最高分别增加150%和49.3%，根瘤数量和鲜质量亦明显提升；尾矿养分也得到了改善，双接菌使碱解氮和速效磷近乎翻倍，有机质和酶活性总体上升；显著降低了Cu和Cr的有效性，并缓解了Cr的环境毒性；此外，双接菌处理还促进了光养细菌席藻属及固氮细菌根瘤菌的增长，抑制潜在病原真菌的相对丰度，调节AMF竞争与资源分配，进一步优化微生物群落结构和功能。综上，紫花苜蓿-AMF-根瘤菌双共生系统在提高植物耐受、降低重金属有效性、改善微生物群落结构等方面具有显著优势，为尾矿生态修复提供了理论依据。

关键词: 丛枝菌根真菌（AMF）, 根瘤菌, 尾矿, 重金属, 微生物群落