The Contribution of Arbuscular Mycorrhizal Fungi to Soil Enzyme Activity and the Performance of Mimosa caesalpiniaefolia in Soil Degraded by Scheelite Mining: Implications for Restoration

Abstract

Mining activity severely degrades soil, increases heavy metal contamination, and hinders ecological recovery. Arbuscular mycorrhizal fungi (AMF) offer a promising strategy for restoration, but their use in Fabaceae plants, especially in mine-degraded soils, remains underexplored. This study evaluated AMF inoculation effects on soil enzymes and Mimosa caesalpiniaefolia growth in Scheelite-mining-degraded soil. In a 10-weeks greenhouse experiment, plants were grown with different AMF species (Gigaspora margarita, Acaulospora foveata, Rhizoglomus clarum, and Mix—a combination of the three species) and without inoculation. Growth parameters, seedling quality, mycorrhizal attributes, enzymatic activity, and stoichiometry were assessed. Inoculated plants showed a greater growth compared with the control. The highest spore abundances occurred in Mix (2820), R. clarum (2261), and A. foveata (2318), with the mycorrhizal colonization highest in Mix (25.78%) and R. clarum (25.70%). The Dickson quality index was higher in R. clarum and A. foveata. The enzymatic activity varied compared with the control: β-glucosidase was higher in Mix (+62%) and A. foveata (+46%); arylsulfatase and urease increased in all AMF treatments; and acid phosphatase was highest in R. clarum (+121%). A stoichiometry and vector analysis indicated a lower P limitation in Mix and A. foveata, reflecting the trade-off between P availability and symbiotic costs. These findings highlight the biotechnological potential of AMF, particularly Mix and R. clarum, in enhancing the M. caesalpiniaefolia growth and soil enzymatic activity in mining-degraded areas.

1. Introduction

Mining plays a crucial role in the economy of many regions worldwide, but its activities can lead to significant environmental impacts, such as soil degradation and biodiversity loss [1]. Among the extracted minerals, Scheelite, composed of calcium tungstate (CaWO₄), is an important source of tungsten (W), widely used in the electrical, electronic, military, mechanical, and steel industries [2]. In Brazil, the main reserves of Scheelite are located in the semi-arid region, particularly in the states of Rio Grande do Norte and Paraíba [3]. Many mines in these areas have been decommissioned without implementing recovery strategies, resulting in serious ecological damage.

The mineral extraction process typically involves the removal of vegetation cover and large volumes of soil, leading to the degradation of the local ecosystem. This issue is exacerbated when waste is deposited on the soil surface, and exposed to the elements, facilitating the dispersion of heavy metals (HMs) such as Cd, Pb, Cr, W, and Mn into the soil and water bodies [4]. The physical, chemical, and biological characteristics of the soil, as well as the hydrological patterns of the region, are critical in choosing methods to recover areas degraded by mining [5]. Many of these areas exhibit low fertility, low organic matter contents, altered pH, and inadequate physical structure, which hinder the establishment and growth of plant species. As a result, natural restoration in these locations tends to be slow and may require long periods [6].

Soil interventions are essential to accelerate recovery. The introduction of pioneer species, especially leguminous plants that can thrive in harsh, oligotrophic environments, is crucial as they fix atmospheric nitrogen (N₂) and form associations with arbuscular mycorrhizal fungi (AMF) [7,8]. AMF are soil microorganisms that are associated with more than 80% of vascular plants, enhancing mineral nutrition and tolerance to adverse environmental conditions, such as those found in areas contaminated by heavy metals [9,10].

Recent studies have shown that AMF can accumulate heavy metals, such as manganese, in their structures, maximizing plant growth and tolerance under extreme conditions [8,11]. Additionally, an inoculation with AMF in Amorpha fruticosa increased the enzymatic activity of catalase and urease, as well as levels of C, P, N, and K, contributing positively to soil health in degraded areas by coal mining [12]. In mining environments, AMF can help alleviate hormonal imbalances in plants by raising levels of indoleacetic acid and cytokinins, reducing abscisic acid in the roots, and promoting a significant increase in plant biomass [13]. Thus, the inoculation with AMF in pioneer species can be essential for restoring degraded soils, as the lack of vegetation cover and soil degradation often result in a significant reduction of native AMF propagules [14]. This approach can facilitate the re-establishment of vegetation and contribute to the recovery of soil quality.

Despite significant findings, most research on the impacts of mining activities primarily focuses on the physical and chemical properties of the soil [4,5,15]. This highlights the need for a more strategic selection of species for ecological restoration, particularly those exhibiting a superior performance and adaptability in soils affected by mining [16]. In this context, Mimosa caesalpiniaefolia stands out as a pioneer species with rapid growth and a high capacity to adapt to degraded and low-fertility soils. In addition to belonging to the Fabaceae family and contributing to biological nitrogen fixation, this species can enhance organic matter and nutrient content through litter decomposition, facilitating the establishment of other plant species and making it a viable alternative for the revegetation of mining-impacted areas [17,18].

However, there is a notable gap in research exploring the symbiotic relationship between AMF and leguminous species, especially Mimosa caesalpiniaefolia, regarding their contributions to the revegetation of areas impacted by Scheelite mining. Therefore, this study aims to evaluate the effects of inoculating different AMF species on soil enzymatic activity and the growth of M. caesalpiniaefolia in degraded soil by mining in a semi-arid region of Brazil. We hypothesized that inoculating with AMF in M. caesalpiniaefolia enhances soil enzymatic activity and plant growth in a mining-degraded soil from a semi-arid region of Brazil, promoting a greater tolerance to adverse conditions, including high levels of W, and improving the ecological functionality of the system.

2. Materials and Methods

2.1. Growth Conditions, Experimental Design, and Plant Species

This study was conducted in the greenhouse of the Department of Soil Sciences at the Federal University of Ceará, located in Fortaleza, Brazil (coordinates: 3°45′47″ S latitude, 38°31′23″ W longitude, at an elevation of 47 m). This area experiences a tropical climate, with the annual average temperatures around 27 °C and a total precipitation of approximately 1600 mm, categorized under the Köppen climate classification as the Aw type. The soil used to fill the pots was collected from the 0–20 cm layer of an area degraded by tailings deposition from the Scheelite mineral extraction process, located in the municipality of Bodó, Rio Grande do Norte, Brazil. Immediately after the collection, we performed a sieving procedure (2 mm) and subsequently determined the soil’s physical and chemical characteristics, including heavy metal concentrations (

Table 1. The chemical and physical characteristics of soil collected in an area degraded by Scheelite mining activity, Bodó, Rio Grande do Norte, Brazil.

pH	TOC	P	Ca2+	Mg2+	Na+	K+	N
(H2O)	(g kg−1)	(mg kg−1)	(cmolc kg−1)	(cmolc kg−1)	(cmolc kg−1)	(cmolc kg−1)	(g kg−1)
7.38	2.75	22.50	2.89	2.00	3.19	0.11	0.25
W	Cd	Cr	Cu	Mo	Pb	Zn
(mg kg−1)
1294.96	0.152	50.72	67.88	2.00	5.79	42.12
Bulk density	Sand	Silt	Clay	Textural Class
(g cm−3)	(%)	(%)	(%)	-
1.37	52.48	18.70	28.82	Sandy clay loam

), following the methods of [19].

The experimental design used was completely randomized, with five treatments: (1) no AMF inoculation (control), (2) inoculated with Gigaspora margarita, (3) inoculated with Acaulospora foveata, (4) inoculated with Rhizoglomus clarum, and (5) Mix (a mixture of the three AMF species). We used 5 replications, resulting in 25 experimental units. The selected plant species for this study was Mimosa caesalpiniaefolia. This species was chosen due to its capacity to establish symbiotic relationships with arbuscular mycorrhizal fungi (AMF) and rhizobia. Furthermore, it is native to the Brazilian Caatinga biome, making it well suited to the local edaphoclimatic conditions and offering a considerable potential for research in areas affected by mining activities [16]. In addition to its role in soil improvement and green manure, M. caesalpiniaefolia also has economic potential, being used for fodder and timber production.

2.2. Experimental Process

The seedlings were grown in small pots before being transplanted. Thus, the seeds of M. caesalpiniaefolia were soaked in a 70% solution of ethyl alcohol for 30 s to facilitate the breakdown of surface tension. Afterward, the seeds were treated with a 1% sodium hypochlorite solution for 10 min to disinfect the surface. Following this, the seeds were rinsed with sterile distilled water to eliminate any residual hypochlorite [20]. The seedlings were grown in small pots (25 g of soil), with one seed placed in each pot. The substrate used was autoclaved sand (121 °C and 1 atm for 2 h). Different species of arbuscular mycorrhizal fungi (AMF) were introduced to the seedlings. For each treatment, which included G. margarita, A. foveata, R. clarum, and a mixture known as Mix, 90 spores were placed at a depth of 1 cm, close to the seeds, in line with the methodology described by [8]. In the Mix treatment, 30 spores of each species were used to total 90 spores. In the control treatments, the seeds were sown without the AMF inoculation.

After two weeks post-seedling emergence, the seedlings were carefully transplanted into larger pots, ensuring that the soil clumps adhered to the roots remained intact, following the established treatment protocols. All of the larger pots were filled with 2 kg of non-sterilized soil, sourced from an area degraded by the deposition of tailings piles from Scheelite mining. This approach was adopted to simulate the most realistic conditions possible for a potential process of revegetation and recovery of this area. The plants were irrigated daily throughout the 10-week research period.

2.3. Plant Growth Parameters

At the end of the 10-week experimental phase, plant growth was measured using the following parameters: height (H) in centimeters, stem diameter (SD) in millimeters, number of leaves (NL) per plant, and root length (RL) in centimeters. Subsequently, the plants were divided into shoot and root, stored in paper bags, and weighed after being dried in an oven with forced air circulation at 60 °C, to determine the shoot dry mass (SDM) and the root dry mass (RDM).

2.4. Dickson Quality Index

The evaluation of seedling quality was conducted using the Dickson Quality Index (DQI), a widely employed methodology for assessing the overall seedling quality. This method considers important morphological parameters, such as height, stem diameter, shoot dry mass, root dry mass, and total dry biomass. The DQI is an indicator of seedling vitality, with higher values reflecting better quality, which is crucial for their survival and success in the revegetation process. The index was calculated using the following equation: DQI = TOTAL DRY MASS/((SD/H) + (SDM/RDM)) [21].

2.5. Soil AMF Spores and Mycorrhizal Colonization

The arbuscular mycorrhizal fungi (AMF) spores in the soil were assessed by extracting 100 g of soil using the wet sieving technique [22], followed by centrifugation through a sucrose gradient [23].

To analyze mycorrhizal colonization (MC) in M. caesalpiniaefolia roots, a clarification process was performed using a 10% KOH solution [24]. The roots were then stained with 5% acidified blue pen ink (Parker Quink, Janesville, WI, USA) as described by [25]. For preservation, the roots were kept in a Lactoglycerol solution (1:1:1 v/v of glycerol, lactic acid, and distilled water). Microscope slides were prepared with 10 root segments, each about 1 cm in length, and examined under an optical microscope (Olympus–CX40, Tokyo, Japan). A total of 10 microscopic fields were analyzed for each root sample, and the percentage of MC was calculated following the methods outlined by [26].

2.6. Soil Enzymatic Activity and Its Stoichiometry

Soil enzyme activities were quantified using colorimetric assays that measure the release of ρ-nitrophenol and ammonium ions. The arylsulfatase activity was determined by measuring the release of ρ-nitrophenol after incubating soil samples with a buffered solution of ρ-nitrophenyl potassium sulfate at pH 5.8 [27]. For each sample, 4 mL of acetate buffer adjusted to pH 5.8 was used [28].

The activity of β-glucosidase was quantified by measuring the release of ρ-nitrophenol after incubation with a buffered solution of ρ-nitrophenyl-β-D-glucoside [29]. Similarly, acid and alkaline phosphatase activities were assessed using a colorimetric method that measured the release of ρ-nitrophenol after incubation with a buffered solution of ρ-nitrophenyl phosphate (PNP). For these assays, 4 mL of MUB buffer was used, adjusted to pH 6.5 for acid phosphatase and pH 11 for alkaline phosphatase [28].

Urease activity was quantified by determining the concentration of ammonium ions (NH₄⁺) after incubating soil samples with a urea solution at pH 10. At this pH, the released ammonia exists as NH₃, which reacts with water to form ammonium hydroxide (NH₄OH) and subsequently ionizes to produce ammonium ions (NH₄⁺) and hydroxyl ions (OH⁻) [30].

To determine enzyme stoichiometry, the C, N, and P-acquiring enzymes were represented by BG (β-glucosidase), U (urease), and AP (acid phosphatase), respectively. The values of C, N, and P-acquiring enzymes were then used to calculate the enzyme ratios C, P, and N. The microbial resource acquisition was estimated using the vector method [31], with vectors X and Y defined as follows: X = BG/(BG + AP) and Y = BG/(BG + U).

2.7. Statistical Analysis

The raw data were subjected to an analysis of variance using the F-test (p ≤ 0.05). Upon detecting a significant difference, the data were compared using the Scott–Knott test (p ≤ 0.05), utilizing the statistical software SISVAR (version 5.8) [32]. The principal component analysis (PCA) was conducted using data on soil enzymatic activity and stoichiometric and vector analyses, as well as mycorrhizal attributes and plant growth characteristics. We used R Studio software (version 1.3.1093).

3. Results

3.1. Plant Growth Parameters

The growth parameters of M. caesalpiniaefolia showed different responses among the AMF inoculation treatments (Figure 1 and Figure 2). Plants inoculated with AMF, regardless of the species, exhibited the highest values of SDM compared to the control treatment (Figure 2A). The increases were 94.5%, 134.7%, and 115.2% for the AMF species G. margarita, A. foveata, and R. clarum, respectively, compared to the control (Figure 2A). A similar response was also observed for RDM, except in the MIX treatment and G. margarita, where the inoculation with AMF R. clarum and A. foveata resulted in an increase in RDM of 167.8% and 107.1%, respectively, compared to the control (Figure 2B).

Regardless of the AMF species, M. caesalpiniaefolia showed the highest increases in H (Figure 2C) and SD (Figure 2D) compared to the control treatment. These increases were 115.8%, 115.1%, 113.4%, and 84.7%, respectively, for the Mix, R. clarum, A. foveata, and G. margarita treatments concerning H (Figure 2C). For SD, the Mix, R. clarum, A. foveata, and G. margarita treatments were responsible for increases of 54.5%, 76.2%, 65.9%, and 51.8%, respectively, compared to the control (Figure 2D). There were no significant differences among the experimental treatments for the variables NL (Figure 2E) and RL (Figure 2F).

3.2. Dickson Quality Index

The Dickson Quality Index (DQI) varied among the different AMF inoculation treatments (Figure 3). The treatments with R. clarum and A. foveata inoculation showed the highest DQI values, with increases of 191.4% for R. clarum and 108.6% for A. foveata compared to the control.

3.3. AMF Spore Number and Mycorrhizal Colonization

The AMF number of spores (ANS) was significantly higher in the treatments with A. foveata, R. clarum, and Mix, with increases of 1012.6%, 984.8%, and 1253.7%, respectively, in relation to the control (Figure 4A). Regarding mycorrhizal colonization (MC), the treatments with R. clarum (25.70%) and Mix (25.78%) presented the highest colonization percentages, in comparison to the control (Figure 4B). Regarding the colonization morphology in M. caesalpiniaefolia, we generally observed structures such as vesicles (Figure 4C), arbuscules, and intraradicular hyphae (Figure 4D).

3.4. Soil Enzyme Activity

The enzyme activities showed distinct responses across the AMF inoculation treatments (Figure 5). β-glucosidase activity was significantly higher in the Mix treatment, showing a 62.5% increase compared to the control (Figure 5A). Arylsulfatase activity was highest in the G. margarita treatment (17.2 µg p-nitrophenol g⁻¹ soil), and twice more in A. foveata and R. clarum treatments (Figure 5B). Acid phosphatase activity was higher in the AMF treatments, with R. clarum exhibiting the highest activity, with a 121.9% increase compared with the control (Figure 5C). In contrast, for alkaline phosphatase, the G. margarita treatment exhibited the highest activity (33.0 µg p-nitrophenol g⁻¹ soil), surpassing the control by 31.5% (Figure 5D). Urease activity was significantly higher in all AMF-inoculated treatments compared to the control, with the Mix treatment showing the greatest increase (199.7%), followed by G. margarita (114.1%) and R. clarum (111.5%), respectively (Figure 5E).

3.5. Enzyme Stoichiometry

The enzyme ratios C:N, C:P, and N:P increased significantly with AMF inoculation, except for C:N (Figure 6). Specifically, there were increases in N:P ratios in the Mix, G. margarita, and A. foveata treatments (Figure 6B), and in C:P ratios in the Mix and A. foveata treatments, compared with the control (Figure 6C).

The values of vector A were lower in the treatments with Mix and A. foveata, while vector L presented its highest values in these same treatments, compared to the others (

Table 2. Vectors A (angle) and L (unitless) in soil degraded by Scheelite mining and cultivated with M. caesalpiniaefolia inoculated with different AMF species. The values in the table represent the mean (n = 5) ± standard error. Means followed by the same letter do not differ from each other using the Scott–Knott test (p ≤ 0.05).

AMF	Vector A	Vector L
Control	44.94 ± 0.80 a	0.47 ± 0.014 b
G. margarita	41.99 ± 0.14 b	0.45 ± 0.002 c
A. foveata	43.29 ± 0.21 c	0.49 ± 0.004 a
R. clarum	44.57 ± 0.10 a	0.43 ± 0.002 c
Mix	41.36 ± 0.07 c	0.49 ± 0.005 a

).

3.6. Relationships Between Soil Enzyme Activity and Mycorrhizal Attributes on Plant Growth

The principal component analysis (PCA) explained 31.89% (PC1) and 22.16% (PC2) of the total variation, grouping the values of the plant growth variables, mycorrhizal attributes, soil enzymatic activity, and their stoichiometry (Figure 7). PCA revealed a clear separation between plants inoculated and non-inoculated with AMF. Plants inoculated with AMF, especially those combining the three AMF species (Mix), strongly correlated with β-GLU, URE, ASN, and MC (Figure 7). This pattern was also observed for SDM, ACP, ALP, RL, RDM, DQI, and SD in the treatment with R. clarum (Figure 7). On the other hand, the treatment without AMF inoculation (control) showed a clear separation, indicating that non-inoculated plants present distinct responses in relation to the analyzed variables, especially to soil enzymatic activity, mycorrhizal attributes, and plant growth (Figure 7).

4. Discussion

Revegetation is one of the main techniques employed in the recovery of degraded areas after mining activities. However, due to the low fertility of the soil and stress conditions such as heavy metal contamination, interactions between plants and soil microorganisms, especially AMF, play a crucial role in environmental recovery processes [33]. Our results confirm the hypothesis that AMF inoculation enhances the growth and quality of M. caesalpiniaefolia seedlings in soil degraded by Scheelite mining, characterized by excessive tungsten concentrations. M. caesalpiniaefolia plants inoculated with AMF, regardless of the fungal species, exhibited a higher biomass accumulation and a greater resilience to soil degradation and excessive tungsten. One possible explanation for this is the ability of certain AMF structures, such as spores, to sequester heavy metals, forming a protective barrier that limits metal toxicity [8]. By mitigating the harmful effects of excessive tungsten, AMF inoculation not only promotes a healthier root development but also facilitates a better nutrient uptake, ultimately resulting in an improved plant growth. Similar findings were reported by [34], in which mycorrhizal colonization reduced the arsenic uptake and enhanced the growth of Lens culinaris plants.

The success of revegetation projects in areas degraded by mining activities depends, in part, on the quality of the used seedlings [35]. In this context, evaluating seedling quality is a crucial parameter in designing a revegetation project for such areas. The Dickson Quality Index (DQI) is used as an indicator to assess the quality of the shoot and root of seedlings [36]. According to the criteria established by [37], seedlings with a DQI below 0.2 are considered unsuitable for field planting. Conversely, a higher DQI value indicates a better seedling quality and a greater resilience to soils contaminated with heavy metals [38]. In the present study, plants inoculated with A. foveata and R. clarum showed the highest DQI values, exceeding 0.2, compared to other treatments. This result can be attributed to the greater benefits provided by AMF of these species, which resulted in higher values for plant height, stem diameter, and the total dry mass. Consequently, the DQI was higher, as this index incorporates these parameters in its evaluation.

Degraded soils with excess tungsten can have both direct and indirect toxicological effects on plants and microorganisms [39], which may hinder mycorrhizal association. In areas contaminated by heavy metals, AMF species must exhibit a high colonization capacity to ensure the success of their application [15]. In the present study, mycorrhizal colonization was higher in treatments inoculated with R. clarum and the Mix. On the other hand, the abundance of AMF spores in the soil was greater in R. clarum, Mix, and A. foveata. This demonstrates the tolerance of these species and suggests that the synergistic effect among them may act as a form of protection for these microorganisms under adverse conditions. This adaptation could result in a more efficient symbiotic system, providing a greater protection against toxicity and stimulating the growth of M. caesalpiniaefolia under tungsten excess conditions. Importantly, the abundance of AMF spores in the soil was significantly higher for all of the species compared to some previous studies, such as those by [8,16] in manganese-contaminated soils, while the mycorrhizal colonization rate was relatively lower. This fact may be related to the higher concentrations of tungsten and phosphorus in the soil, which may have contributed to the low mycorrhizal colonization. However, there is not always a direct correlation between colonization and the abundance of AMF spores. In this case, despite the lower mycorrhizal colonization values, this did not limit the benefits provided to M. caesalpiniaefolia plants grown in Scheelite mining-degraded soil.

Here, we observed that different AMF species influence soil enzymatic activity differently. The Mix treatment showed the highest activity of β-glucosidase, an enzyme crucial for the carbon cycle and decomposition of organic matter. Meanwhile, the activity of urease, vital for nitrogen mineralization, increased in all of the treatments inoculated with AMF. According to [40], the presence of AMF can intensify enzymatic activity when interacting with microorganisms present in the soil. AMF releases exudates, such as amino acids and glucose, which transfer photosynthetic carbon to the soil, enriching it and favoring the local microbiota [41,42]. Furthermore, the large surface area of the mycelium creates niches favorable to microbial proliferation, strengthening the carbon and nitrogen cycle and soil functionality. Thus, we provide evidence that AMF inoculation resulted in an increased spore abundance and mycorrhizal colonization of M. caesalpiniaefolia. This, in turn, probably stimulated microbial growth and activity in the soil, contributing to an increased β-glucosidase activity and, more significantly, urease. Arylsulfatase activity, essential for sulfur mineralization, was higher in the G. margarita treatment. Some AMF species can stimulate the growth of specific microorganisms in the rhizosphere, such as bacteria involved in the sulfur cycle, due to the increased secretion of organic compounds, such as organic acids and sugars [43]. In a study carried out by [44], it was found that the abundance of bacteria with genes related to sulfur oxidation increased significantly after inoculation with Funneliformis mosseae, favoring the mineralization of organic sulfur in the soil and intensifying the activity of arylsulfatase. Similarly, [45] found that AMF of the genus Rhizoglomus positively influence the activity of arylsulfatase, promoting better sulfur cycling in the soil and a greater tolerance of Cajanus cajan in arsenic-contaminated soil. These results suggest that the increase in aryl sulfatase activity may depend not only on the AMF species but also on soil conditions, such as the degree of degradation and contamination, in addition to the host plant involved. In the present study, G. margarita demonstrated promise for increasing aryl sulfatase activity.

Phosphatase enzymes are essential in the breakdown of organic phosphorus compounds, thus increasing the bioavailability of phosphorus in the soil, in addition to acting as a bioindicator sensitive to changes in soil management [46]. The highest acid phosphatase activity was observed in the treatment with R. clarum, while the alkaline phosphatase activity was higher in all of the treatments inoculated with AMF, except for Mix. This more expressive effect of inoculation with AMF in increasing alkaline phosphatase activity is possibly related to the pH of the studied soil (7.38), which favors the action of this enzyme [47]. On the other hand, the acid phosphatase activity, which was only significant in the treatment with R. clarum, may indicate a unique characteristic of this species, making it more efficient in the production or stimulation of the release of acid phosphatase, even in alkaline conditions. Furthermore, the lower expressiveness of the effect of AMF inoculation on acid phosphatase activity may be related to the high concentration of phosphorus in the soil, which, in this study, was high, and may have inhibited the synthesis of this enzyme by the plant and microorganisms [48]. Interestingly, some enzymes (e.g., β-glucosidase) did not change, in relation to the control, when G. margarita was inoculated. Specifically, species from the genus Gigaspora are among the largest AMF due to their large spores, which can exceed 400 µm in diameter, and tend to have some of the largest spore sizes among AMF. Thus, Gigaspora may be more sensitive to the effects of heavy metals in the soil for several reasons, including larger spores, which can accumulate more metals due to their greater surface area and larger cell volume, which may result in higher toxicity. The lack of vesicles, unlike other AMF genera like Glomus, Gigaspora does not form vesicles for lipid storage, which may reduce its ability to withstand environmental stresses, including metal contamination. Also, the colonization strategy of Gigaspora species tends to be less aggressive in root colonization and relies more on viable propagules in the soil. Heavy metals can reduce their viability and germination, thereby impacting the stimulation of enzyme releases [49].

The inoculation with AMF also altered the soil enzymatic stoichiometry that influences the availability and limitations of C, N, and P for soil microorganisms. The increase in N:P ratios in the treatments with Mix, G. margarita, and A. foveata indicates a greater relative availability of N in relation to P, possibly due to the greater activity of enzymes related to phosphorus mineralization, promoted by AMF [46]. On the other hand, the increase in C:P ratios in the Mix and A. foveata treatments suggests that inoculation also affected the decomposition of organic matter and carbon release, favoring the allocation of microbial resources. This is important since these resources are limited in degraded areas. These results indicate that AMF not only promotes the balance between the carbon, nitrogen, and phosphorus cycles but also favors the structuring of the microbial community and the functional recovery of the soil in degraded areas.

The vectors L (length) and A (angle) are valuable indicators for assessing the extent of C limitation (represented by vector L) and the degree of P limitation relative to N (indicated by vector A) [31]. Our results demonstrated that the acquisition of microbial resources varied according to the different AMF inoculation treatments. The lower value of vector A in the Mix and A. foveata treatments suggests a lower relative limitation of P in relation to N in these treatments. This pattern may be related to the ability of AMF to improve the availability and uptake of P for the host plant, due to the extensive network of extraradical hyphae that increase soil exploration [50]. On the other hand, the higher values of the L vector in these same treatments indicate a greater limitation of C in relation to the other nutrients. This can be attributed to the greater demand for C by the mycorrhizal symbiosis, since plants allocate photosynthetic resources to sustain the metabolism of AMF [51]. This behavior is consistent with the literature, which points to a trade-off between the increase in P availability mediated by AMF and the need for C to sustain this symbiotic relationship [52]. Furthermore, they show that different species or combinations of AMF can exert distinct effects on the nutritional limitations of the system, which has practical implications for the selection of mycorrhizal inoculum in strategies for the recovery of areas degraded by Scheelite mining activities.

Overall, the PCA revealed a clear separation between inoculated and non-inoculated plants, reinforcing the beneficial effects of AMF on soil enzymatic activity. Specifically, the increased activity of β-glucosidase, urease, arylsulfatase, and phosphatase in inoculated plants indicates an improvement in nutrient cycling, leading to a greater nutrient availability in the soil [53]. This enhanced nutrient availability directly supports plant growth and increases the tolerance to adverse conditions, highlighting the potential of AMF inoculation as a sustainable strategy for restoring plant development in Scheelite mining-degraded soils in semi-arid regions. Although our results indicate that AMF contributes to plant tolerance in metal-contaminated soils, this study did not quantify heavy metal accumulation in mycorrhizal structures. This represents a limitation in directly confirming the role of AMF in metal sequestration. Future studies should focus on analyzing metal concentrations in hyphae, spores, and other fungal structures to better understand this mechanism. This study is the first to investigate the effect of tungsten on soil enzymes and stoichiometry, providing initial data on this under-explored topic. Due to the novelty of the research, the short duration focused on the immediate impacts of tungsten on soil enzymatic activity. The results contribute to a new area of research and lay the foundation for future studies, which will aim to assess the long-term stability and seasonal variations of these effects.

5. Conclusions

This study confirms the potential of AMF as a valuable tool for the recovery of soils degraded by Scheelite mining. The inoculation with AMF, especially in the Mix and Rhizoglomus clarum treatments, promoted an increased growth and a greater mycorrhizal colonization in M. caesalpiniaefolia, in addition to improving soil enzymatic activity, which may contribute to the recovery of the functionality of this ecosystem. We note that the efficacy of AMF may vary depending on the interaction between the fungal species and the host plant, indicating the importance of choosing the most suitable AMF species for each context. The results of enzymatic stoichiometry and vectors demonstrate that AMF treatments (Mix and A. foveata) could promote influence on nutrient dynamics, with a lower P limitation in relation to N and a higher C demand, reflecting the trade-off between P availability and the metabolic cost of symbiosis. These findings highlight the role of AMF in promoting a more favorable environment for plant growth in degraded soils, highlighting its potential as an efficient biotechnological tool for the ecological recovery of areas impacted by Scheelite mining in semi-arid regions.