Inoculum potential of arbuscular mycorrhizal fungi in iron ore tailings areas under rehabilitationABSTRACT
Mining has long been important for Brazil’s economy, but it causes severe environmental damages. The Fundão Dam disaster in Minas Gerais, for example, harmed ecosystems and soil biota, particularly arbuscular mycorrhizal fungi (AMF). These fungi are essential for soil and plant health, but mining reduces their colonization. Revegetation with proper management and AMF inoculation can accelerate ecological recovery. This study evaluated the inoculum potential of AMF in mining-impacted areas at different revegetation stages in Mariana, Minas Gerais. Soil samples were collected for chemical analyses, spore and mycorrhizal colonization quantification, and soil inoculum-efficiency tests. Our results showed that areas with five years of revegetation had higher spore viability and colonization, indicating the onset of ecological stabilization. However, these levels remained lower than those observed in the reference area. Longer revegetation periods increased colonization and symbiotic efficiency. Overall, revegetation contributed to the chemical and microbiological recovery of soils, but full recovery of mycorrhizal function requires more time and more favorable environmental conditions.
Index terms:
Revegetation; mining; arbuscular mycorrhiza
RESUMO
A mineração no Brasil, apesar de sua importância histórica e econômica, causa impactos ambientais significativos, como o desastre da barragem de Fundão (MG), que afetou ecossistemas e a biota do solo, especialmente os fungos micorrízicos arbusculares (FMAs). Estes fungos são essenciais para a saúde do solo e das plantas, e sua colonização é reduzida pela mineração. No entanto, a revegetação com manejo adequado e inoculação micorrízica pode acelerar a recuperação ecológica, tornando os FMAs indicadores importantes da eficiência da reabilitação ambiental. Este estudo avaliou o potencial de inóculo de FMAs em áreas impactadas e em diferentes estágios de revegetação em Mariana, Minas Gerais. Amostras de solo foram coletadas para análises químicas, quantificação de esporos, colonização micorrízica e testes de eficiência do solo-inóculo. Os resultados indicaram que áreas com cinco anos de revegetação apresentaram maior viabilidade de esporos e colonização, sugerindo início de estabilização ecológica, embora ainda inferior à área de referência. O tempo de revegetação influenciou positivamente a colonização e a eficiência simbiótica. Conclui-se que a revegetação contribui para a recuperação química e microbiológica dos solos, mas a restauração completa da funcionalidade micorrízica requer mais tempo e condições ambientais favoráveis.
Termos para indexação:
Revegetação; mineração; micorrizas arbusculares
Introduction
Metal mining in Brazil has played an important role since before independence, attracting the interest of the Portuguese crown after the decline of the sugarcane industry in the Northeast. The gold rush was so influential that it prompted the transfer of the colonial capital from Bahia to Rio de Janeiro, fostering the development of mining towns and establishing mining as a key economic activity (Figueirôa, 1994). Today, Brazil remains a major mineral exporter, with both legal and illegal extraction, including artisanal mining in the Amazon (Magno, 2015; Souza Junior, Moreira, & Heineck, 2018).
The collapse of the Fundão Dam in Mariana, Minas Gerais, on 5 November 2015, caused by operational negligence, resulted in severe environmental and socioeconomic consequences (Freitas et al., 2019). Local biodiversity was drastically affected, compromising ecosystems, human communities, microclimates, and particularly soil biota (Espíndola et al., 2016; Neto et al., 2009). Among the most severely impacted organisms are arbuscular mycorrhizal fungi (AMF), which form mutualistic associations with vascular plants and perform essential ecological functions (Brundrett & Tedersoo, 2018; Soares et al., 2020).
AMF colonize the roots of approximately 80% of terrestrial plant species, enhancing the uptake of water and nutrients, especially phosphorus, in exchange for photosynthetically derived carbohydrates and lipids produced by plant roots (Moreira & Siqueira, 2006). They also improve tolerance to environmental stress, contribute to soil aggregation, and support plant biodiversity (Gong, Wang, & Li, 2025). This symbiosis indirectly benefits other soil organisms by stimulating organic matter decomposition and nutrient cycling (Pouyú-Rojas & Siqueira, 2000).
In areas affected by dam failures, microbial biomass and activity decline sharply, including changes in AMF density and community composition. These alterations reflect both the severity of mining impacts and the potential for ecosystem resilience (Mergulhão et al., 2010). Such disturbances can severely reduce AMF propagules and root colonization (Kiernan, Hendrix, & Maronek, 1983). Nevertheless, studies indicate that revegetation can be successfully established in degraded substrates when appropriate management strategies are applied (Melloni, Siqueira, & Moreira, 2003), although thick layers of tailings may represent a long-term constraint to soil recovery.
Recent studies have shown advances in the use of mycorrhizal inoculation as a restoration strategy. Zanchi et al. (2021) demonstrated that herbaceous plant consortia inoculated with Acaulospora morrowiae Spain & N.C. Schenck enhance dry matter production and mycorrhizal colonization. Zanchi et al. (2021) reported that herbaceous species facilitate the establishment of tree species such as Enterolobium contortisiliquum (Vell.) Morong, which contribute to phytoextraction and improve soil attributes. Such practices accelerate microbiota reactivation and promote ecological succession.
Mergulhão et al. (2010) observed in gypsum mining areas that certain AMF species, such as Glomus sp., are more resilient to environmental disturbances. Similarly, Bi et al. (2021) documented significant increases in AMF diversity less than one year after forest revegetation in a coal tailings area, underscoring the influence of soil type and mining context on microbial dynamics. In deforested areas of northeastern Brazil, Silva, J. et al. (2023) found that AMF ecological succession proceeds slowly and is strongly influenced by soil characteristics. Therefore, the distinct properties of mining tailings require tailored management strategies, and AMF dynamics can serve as reliable indicators of revegetation success.
Given the importance of revegetation for restoring degraded environments, the role of AMF as facilitators of ecological regeneration and soil restructuring is particularly relevant (Teixeira, A. et al., 2017). This study is based on the hypotheses that (i) the propagation potential of AMF was affected by the deposition of iron mining tailings, and (ii) revegetation effectively contributes to the environmental rehabilitation of affected areas. The objective is to evaluate the inoculum potential of AMF in areas impacted by mining tailings deposition under revegetation in Mariana, Minas Gerais.
Material and Methods
Study area
The study area is located in the municipality of Mariana, Minas Gerais, along the banks of the Gualaxo do Norte River within the Doce River Basin, in regions affected by the deposition of iron mining tailings from the collapse of the Fundão Dam, which occurred in the district of Bento Rodrigues, Mariana, MG (Figure 1).
Location of the dam with the affected stretch of the Doce River (A) and the district of Bento Ribeiro before (B) and after (C) the mining tailings deposition. Source: Armada (2021).
The region exhibits typical climatic characteristics of Southeastern Brazil,’ with a high-altitude tropical climate characterized by a rainy summer season and a dry winter season. It is also influenced by large-scale climatic phenomena such as El Niño and La Niña (Alvares et al., 2013). Native vegetation includes forest formations from the Atlantic Forest and Cerrado biomes. Common species consist of trees such as Cecropia spp., Schizolobium parahyba (guapuruvu), Handroanthus spp. (ipês), and Jacaranda spp.; shrubs such as Tibouchina spp. (quaresmeiras) and Rosmarinus spp. (rosemary); and herbaceous and aquatic plants, including riparian vegetation along watercourses (Neto et al., 2009; França & Stehmann, 2013). This riparian vegetation plays an essential role in supporting the health of aquatic and terrestrial ecosystems by providing a wide range of ecosystem services (Pfeifer, Restello, & Zakrzevski, 2022).
The soils of the region are predominantly clayey, ranging from Cambisols to Latosols, with medium to high fertility depending on the specific location (Silva, T. 2023; Diniz et al., 2021). After the disaster, iron mining tailings composed mainly of fine sand and silt with extremely low organic matter content and high concentrations of silicon, iron, and manganese oxides were deposited across the landscape. This material substantially altered soil properties, such as infiltration capacity, bulk density, and organic matter content, and consequently affected other chemical characteristics, leading to their classification as Technosols (Botelho & Melo, 2019; Lacaz, Porto, & Pinheiro, 2017).
A major challenge linked to the deposited tailings is that this material hampers ecological recovery. To mitigate these impacts, revegetation strategies have been implemented. One approach involves planting trees, shrubs, vines, epiphytes, and grasses selected according to site-specific conditions to restore the original environment. Initially, an emergency seed mixture composed of leguminous and grass species was applied to promote rapid ground cover (Engel & Parrotta, 2001; Rodrigues, Brancalion, & Isernhagen, 2009; Brancalion et al., 2015).
In addition to environmental degradation, the disaster generated significant social impacts, including the displacement of communities, loss of livelihoods, and negative effects on the health and well-being of local populations. These consequences underscore that human presence is an integral component of the ecosystem and is deeply affected by environmental disturbances (Lopes, 2016).
Field sampling
Field sampling was conducted in 2023 at representative sites to evaluate the behavior of arbuscular mycorrhizal fungi (AMF) under different management practices implemented after the deposition of mining tailings. Three revegetated areas at 3, 4, and 5 years of restoration were selected, along with a reference area exhibiting vegetation characteristics similar to those prior to the disaster. Revegetation in the impacted areas was carried out using plant species identified in botanical surveys conducted before the accident.
Sampling was performed along transects spaced 50 m apart (A, B, and C), each 100 m in length. In each transect, four composite samples were collected, each composed of four subsamples taken within a 1-m radius and spaced 12 m apart, totaling 48 samples (Figure 2). This sampling design followed a 4x12 structure, consisting of four treatments and twelve replicates. After collection, the samples were transported to the Microbiology Laboratory at the Federal University of Lavras and stored for subsequent analyses.
Sampling scheme in the revegetation areas and in the reference area. A- years; B- 4 years; C- 5 years; D- reference.
For the chemical characterization of the collected soil, pH in water, available phosphorus, and potassium were determined using the Mehlich-1 extractor. Exchangeable calcium, magnesium, aluminum, and sodium were extracted with a KCl solution. Micronutrients (zinc, iron, manganese, copper, boron, and sulfur) and soil organic carbon were quantified by wet oxidation. Subsequently, base sum, potential and effective cation exchange capacity, organic matter content, and base and aluminum saturation levels were calculated (Table 1) (Teixeira, P. et al., 2017).
Total number of AMF spores in field samples
To determine AMF spore density, spore extraction was carried out using the wet-sieving method (Gerdemann & Nicolson, 1963). A 50 g soil sample was mixed with water and shaken to separate spores from soil particles. The soil-water suspension was then passed through 0.710 mm and 0.053 mm sieves to retain the spores while removing residual soil, and the procedure was repeated several times.
The material retained on the sieves was washed with water to remove adhering soil particles. The spores were then transferred to Falcon tubes and centrifuged for 3 min at 3000 rpm. After decanting the excess water, a 50% sucrose solution was added, and the samples were centrifuged again for 2 min at 2000 rpm. The resulting supernatant was passed through a 0.053 mm sieve and rinsed with water to remove the sucrose.
Spore counting was performed by standardizing all samples to 20 mL and transferring them to a grooved counting dish divided into quadrants (A and B). Observations were made under a binocular stereomicroscope (up to 80x), counting spores groove by groove. Counts were conducted at least twice, and when variation exceeded 10%, a third count was performed; the final value was then recorded. When dilutions were made, the corresponding dilution factor was applied.
Trap culture for multiplication
To assess soil conditions and interactions with organisms such as AMF, multiplication was conducted in a greenhouse using trap cultures (Figure 3) following Moorman and Reeves (1979). This approach enables the observation of AMF responses to the tailings material and to the revegetation process. Pots with a 1000 mL capacity were prepared with autoclaved sand, and 50 g of field-collected material (reference soil or tailings) was placed on the surface. The trap plants used were Brachiaria brizantha cv. BRS Piatã, Helianthus annuus L., and Glycine max L., representing a Poaceae, an Asteraceae, and a Fabaceae, respectively. All seeds were previously disinfected and sown at high density.
Thus, a total of 48 pots were prepared and distributed across four treatments (3, 4, and 5 years of revegetation, and the reference area). Partial fertilization was applied, with phosphorus supplied at 50% of the recommended rate based on the average recommendation for the three trap crops, according to the soil analyses from the study areas (Table 1). This procedure was implemented to maximize sporulation in both the revegetated tailings areas and the reference soil.
During the greenhouse experiment, periodic irrigation was carried out; however, water supply was intermittently suspended to induce stress, as was the cutting of Brachiaria, for the same purpose. After four months, the experiment was terminated, and the soil inoculum and roots were separated for subsequent re-evaluation of spore density and assessment of mycorrhizal colonization.
Determination of mycorrhizal colonization
After dismantling the greenhouse experiment, all roots were separated, and 0.5 g of fine roots were subsampled for colonization analysis. Staining procedures followed Phillips and Hayman (1970), with modifications by Koske and Gemma (1989). Roots were cleared in 2.5% KOH at 90 °C for 10-60 min, rinsed, and acidified in 1% HCl for two hours. Subsequently, they were stained with 0.05% trypan blue in acidified glycerol and heated at 90 °C for 10-30 min. When immediate processing was not possible, roots were stored in 70% ethanol and rinsed before restaining.
Mycorrhizal colonization was quantified using the gridline intersect method (Giovanetti & Mosse, 1980). Stained roots were evenly distributed in petri dishes over a grid, and each intersection between roots and gridlines was examined for mycorrhizal structures. The percentage of colonization (%MC) was calculated based on the proportion of intersections presenting fungal structures.
Statistical analyses
After data collection, statistical analyses were performed using STATISTICA version 7. Spore density and potential colonization data obtained from both the field experiment and trap cultures were analyzed by calculating treatment and area means, followed by comparison using ANOVA with Tukey’s test at a 5% significance level to assess differences between revegetated areas and the reference area.
Field spore density data were further examined using principal component analysis (PCA). Prior to PCA, sphericity was verified using Bartlett’s test (p < 0.05), and sampling adequacy was evaluated through the Kaiser-Meyer-Olkin (KMO) index. Multivariate analysis was then applied to explore the relationships among soil chemical attributes and spore density, supported by correlation analysis.
To compare propagation potential based on spore densities obtained from field samples and trap cultures for each treatment, Student’s t-test was used, considering comparisons between two means.
Results and Discussion
In Figure 4, the spore density behavior in field soils from areas impacted by iron mining tailings and from the reference area is presented. High spore numbers were observed across all areas, which can be attributed to the high biodiversity of the Atlantic Forest biome, both in the natural reference site and in the restoration plots. This biome is among the most diverse globally, with a wide range of plant species capable of forming associations with AMF. These findings are consistent with Teixeira, A. et al. (2017), who also reported high AMF spore abundance in mining areas of the Iron Quadrangle.
Mean spore density in the reference area and in areas affected by tailings under revegetation in Mariana, MG. Ref: reference; Rev1: 3 years of revegetation; Rev2: 4 years of revegetation; Rev3: 5 years of revegetation. Different letters among treatments indicate statistically significant differences at the 5% probability level according to Tukey’s t-test.
However, the impacted and revegetated areas exhibited higher spore counts than the reference area, with statistically significant differences. This pattern is expected, as the introduction of new soil conditions alters ecosystem functioning, and the rehabilitation of such environments requires long timeframes. This scenario aligns with Silva, J. et al. (2023), who reported marked differences in AMF dynamics between mature Atlantic Forests (>60 years) and secondary forests (<38 years).
AMF produce spores as a survival mechanism, either under natural conditions, supporting species perpetuation and completing the fungal life cycle, or under stress, which also stimulates sporulation for persistence (Ramirez et al., 2023). Several factors influence this process, particularly changes in vegetation and soil characteristics. Unsustainable modifications in these environments may intensify sporulation, while shifts in plant community structure can either reduce or enhance diversity, consequently altering spore abundance (Hooker & Black, 1995). Pioneer plant species, typically dominant in revegetated areas and characteristic of early successional stages, frequently establish strong associations with AMF, which may contribute to the elevated spore production observed (Munyanziza, Kehri, & Bagyaraj, 1997).
The analysis of soil chemical attributes presented in Table 2 reveals marked differences between the reference area and revegetated plots, demonstrating that the deposition of mining tailings significantly modified soil composition. Among the evaluated variables, pH, aluminum (Al), iron (Fe), base saturation (V%), organic matter (OM), and spore density (SD) were the most sensitive to tailings influence and to the time since revegetation.
In the revegetated areas (Rev1, Rev2, and Rev3), soil pH showed a significant increase compared to the reference area, where values were around 4.0, a condition classified as highly acidic. In contrast, the revegetated areas presented pH values ranging from 5.06 to 5.70, with statistically significant differences (p < 0.05). This increase may be related to the presence of natural amendments in the tailings or to the influence of revegetation plants and AMF activity in the rhizosphere (Dias et al., 2008; Salim et al., 2025). Besides directly affecting nutrient availability, pH is a key determinant of soil microbial structure and is positively correlated with AMF spore density (Prado et al., 2019).
Aluminum (Al) content also exhibited pronounced differences. In the reference area, Al reached 1.98 cmolc kg⁻¹, whereas in the revegetated areas values were nearly zero. This reduction is beneficial, as aluminum toxicity in acidic soils limits plant development and impairs nutrient uptake by both roots and microorganisms (Hildebrandt, Regvar, & Bothe, 2007; Salim et al., 2025). The decrease likely reflects the combined effect of increased pH and organic matter dynamics, which promote Al complexation.
Iron (Fe) concentrations ranged from 0.42 to 1.05 cmolc kg⁻¹. The highest value occurred in Rev1 (1.05b), while Ref (0.42a) and Rev2 (0.42a) showed the lowest levels, with Rev3 presenting an intermediate content (0.63a), statistically similar to the reference. This variation suggests that Fe availability does not follow a linear recovery pattern, reflecting the heterogeneity of the tailings and the progressive establishment of vegetation (Mergulhão et al., 2010). Although revegetation enhances nutrient cycling, high Fe concentrations may pose oxidative stress risks to plants and fungi, particularly in the soluble Fe³⁺ form (Hildebrandt, Regvar, & Bothe, 2007).
Base saturation (V%) increased substantially in the revegetated areas (57-61%) compared to the reference (4.92%), indicating improved soil fertility and contributing to mycorrhizal establishment and microbiota recovery (Prado et al., 2019; Orłowska et al., 2010). Organic matter (OM), although still lower in the revegetated areas, exhibited a pattern consistent with revegetation time; Rev3 showed the lowest OM (16.3 g kg⁻¹), possibly due to greater microbial decomposition or reduced plant residue input (Dias et al., 2008).
Regarding spore density (SD), revegetated areas showed markedly higher values, particularly Rev1 (1,317 spores/50 mL) and Rev2 (1,189 spores/50 mL), compared with the reference (665 spores/50 mL). This result indicates a reorganization of AMF communities driven by revegetation and supports the hypothesis that soil attributes such as pH, V%, and OM directly influence AMF recovery (Prado et al., 2019; Salim et al., 2025).
Potassium (K) exhibited a slight decrease in the revegetated areas, without evidence of detrimental effects. Manganese (Mn) and sulfur (S) also varied, with S showing lower contents in the revegetated areas, potentially related to reduced leaching or lower organic matter incorporation.
Overall, the results demonstrate that mining tailings profoundly modified soil chemical composition; however, revegetation has gradually promoted the recovery of soil fertility and microbiological functioning, highlighted by increased pH, reduced Al, and greater AMF spore production. The positive AMF response, even in soils influenced by heavy metals, reinforces their potential as indicators of ecological recovery and facilitators of restoration in degraded environments (Teixeira, A. et al., 2017; Verma & Verma, 2016; Orłowska et al., 2010).
Additionally, the presence of other materials associated with the tailings (Figure 5) may compromise spore germination by altering their development cycle and viability. The observation of spores coated with an orange-colored material of unknown composition supports this hypothesis, as such a coating may act as a physical or chemical barrier affecting germination processes.
Residues found in the deposition areas with tailings under revegetation and spores covered by unknown orange-colored material.
Given this scenario, the hypothesis is reinforced that marked alterations are occurring in the ecosystem where arbuscular mycorrhizal fungi (AMF) establish and develop. Although spores are present in all treatments, there is a tendency for spore density to decline over time, suggesting that revegetation may be exerting a negative effect on sporulation. This pattern, also reported by Caproni et al. (2007), may reflect an adaptive response of AMF to the environmental stress imposed by tailings deposition. Disturbance events often trigger an initial peak in sporulation, followed by a subsequent decline, which may indicate the onset of system stabilization and the advancement of ecological succession.
A notable aspect identified through visual assessment was the high proportion of non-viable spores in the revegetated areas (Figure 6). This condition may compromise local mycorrhizal diversity when compared to the reference area, evidencing the persistence of stress in these environments, particularly due to adverse edaphic conditions. The reduced presence of native plant species limits the availability of suitable hosts for AMF, restricting the establishment of effective symbiotic associations. Under conditions of low plant compatibility and degraded soil structure, many spores tend to lose viability or fail to complete their development cycle (Mergulhão et al., 2007).
Spores found in the studied areas. On the left, spores from the reference area; on the right, spores from the revegetated area with tailings deposition. The data indicate a positive correlation between spore density and the levels of pH, Na, and Mn.
In the correlation analysis (Figure 7), a pattern that may reflect a defense mechanism of AMF under conditions of environmental stress. Such correlations reinforce the idea that shifts in soil chemistry modulate AMF survival strategies, particularly in disturbed environments.
Correlation analysis of chemical attributes and spore density in the reference area and areas affected by tailings under revegetation in Mariana, MG. T: effective CEC; SDF: field spore density; V%: base saturation; OM: organic matter. Gradient from lightest to darkest indicates the degree of correlation from weak to strong, respectively.
Figure 8 presents comparative data between spore density observed in the field (SDF) and after the multiplication experiment in the greenhouse (SDG). Although significant differences among treatments were evident in the field, statistical analysis using Tukey’s test (p ≤ 0.05) showed that these differences tend to diminish under controlled greenhouse conditions. This homogenization of responses suggests a limitation in the viability or infective capacity of a some of the spores originating from the impacted areas, indicating a dissociation between field sporulation and actual symbiotic functionality. Such behavior reinforces the idea that high sporulation in stressed environments does not necessarily correspond to effective propagule potential, particularly when environmental conditions restrict spore development and colonization.
Comparison of spore density in the areas and after multiplication. Ref: reference; Rev1: 3 years of revegetation; Rev2: 4 years of revegetation; Rev3: 5 years of revegetation.
Identical lowercase letters within the same treatment indicate no significant difference at the 5% probability level according to Student’s t-test. Identical uppercase letters across treatments indicate no significant difference at the 5% probability level according to Tukey’s test.
The application of Student’s t-test (p ≤ 0.05), comparing spore density between field and greenhouse conditions for each treatment, confirmed that despite the initially high spore density observed in the revegetated areas, there was a significant reduction in the number of viable spores after multiplication, particularly in the Rev1 and Rev2 treatments. This pattern supports the hypothesis that part of the spores quantified in the field represent non-viable structures or morphological responses to environmental stress rather than active propagules. In contrast, the reference area (Ref) showed a 25% increase in spore density after multiplication, demonstrating greater viability and functional integrity of its original mycorrhizal community, consistent with findings from Hildebrandt, Regvar and Bothe (2007) in non-impacted soils.
The analysis of percentage variation reinforces these interpretations: Rev1 exhibited a 64% reduction, Rev2 a 21% reduction, and Rev3 an 11% reduction. The smaller decline observed in Rev3 may indicate the onset of functional restructuring of the mycorrhizal community, associated with increasing symbiotic stability. The multiplication capacity of AMF under controlled conditions is a useful indicator of symbiotic viability and of the ecological recovery potential of degraded areas (González-Chávez et al., 2002).
The principal component analysis (Figure 9), which considered the first two components (PC1 and PC2), responsible for 68.54% of the total data variability (14.94% for PC1 and 53.60% for PC2), highlighted the interrelation between spore density scores (in field - SDF - and greenhouse - SDG) and the factors pH, manganese (Mn), and base saturation (V%), especially associated with the area under five years of revegetation (Rev3). Samples were grouped based on multivariate similarity of these soil chemical attributes and spore density, indicating an environment with lower chemical stress that favors sporulation of arbuscular mycorrhizal fungi (AMF), which may positively reflect on symbiotic activity. Similar behavior was observed in another principal component analysis, where pH and magnesium scores showed an inverse relationship to diversity, suggesting that certain chemical conditions affect microbial composition and functionality (Santos et al., 2024).
Principal Component Analysis (PCA) of soil chemical attributes and spore density in the reference area and in areas affected by mining tailings under revegetation in Mariana, MG. Ref: reference; Rev1: 3 years of revegetation; Rev2: 4 years of revegetation; Rev3: 5 years of revegetation; T: effective cation exchange capacity (CEC); SDF: spore density in the field; SDG: spore density in the greenhouse; V%: base saturation; OM: organic matter; PC1: principal component 1; PC2: principal component 2.
The reference area (Ref), represented by black circles, formed a well-defined cluster in the left quadrant of the graph and was associated with organic matter (OM), sulfur (S), aluminum (Al), and lead (Pb). These attributes are characteristic of soils with high environmental quality and preserved Atlantic Forest ecosystems, indicating partial maintenance of edaphic and microbiological integrity. The presence of Al and Pb reflects natural geochemical properties rather than anthropogenic contamination.
In contrast, the revegetated areas showed distinct patterns. The three-year revegetation site (Rev1) was positioned in the upper right quadrant and strongly associated with iron (Fe) and sodium (Na), indicating the persistent influence of mining waste rich in iron oxides. This association suggests that, even after three years, the chemical signature of the tailings continues to affect soil composition. Its partial proximity to Al may reflect residual characteristics of the waste, although Al was more strongly linked to the reference area.
The four-year revegetation site (Rev2) exhibited a central and more dispersed pattern, reflecting greater variability among samples. This dispersion indicates a transitional phase in soil and microbial composition, consistent with intermediate stages of functional recovery. Rev2 was associated with nickel (Ni) and, to a lesser extent, with spore density in the field (SDF), suggesting that residual stressors still constrain arbuscular mycorrhizal fungi (AMF) activity.
The five-year revegetation site (Rev3) grouped near vectors representing pH, manganese (Mn), base saturation (V%), and greenhouse spore density (SDG). This grouping suggests progressive improvement in soil conditions that promote AMF development, particularly under controlled conditions. SDG correlated positively with pH, Mn, and V%, indicating that these attributes support greater sporulation, consistent with findings that increases in pH and organic matter in areas revegetated for four to five years enhance root colonization and spore production (Prado et al., 2019; Salim et al., 2025).
Collectively, the data indicate that revegetation has induced substantial changes in soil chemical attributes and microbial functionality. However, the revegetated areas have not yet converged with the reference condition, suggesting that full recovery of edaphic properties and mycorrhizal symbiosis requires additional time and possibly complementary management practices. The trajectory observed in Rev3 demonstrates a positive trend, reinforcing revegetation age as a key factor in ecological restoration. Similar patterns have been reported in coal-mined soils, where the use of native AMF combined with compost and lime increased colonization rates (60-100%), raised pH, and improved nitrogen and phosphorus uptake (Salim et al., 2025). In zinc and bauxite tailings, increases in microbial biomass and spore density after four years of revegetation further support the role of AMF as indicators of functional soil recovery (Dias et al., 2008).
Figure 10 displays the percentages of mycorrhizal colonization in plants grown in soils from areas revegetated for three, four, and five years, along with the non-impacted reference soil. The observed differences among treatments highlight the strong influence of revegetation time on AMF symbiotic activity.
Analysis of mycorrhizal colonization in the reference area and in areas affected by mining waste under revegetation in Mariana, MG. Ref: reference; Rev1: 3 years of revegetation; Rev2: 4 years of revegetation; Rev3: 5 years of revegetation. Different letters within columns indicate significant differences at the 5% probability level according to Tukey’s test.
The area with four years of revegetation (Rev2) exhibited the highest mycorrhizal colonization index (51.45%), which was statistically higher than that of Rev1 (26.89%) and the reference area (29.07%). This result suggests a more efficient microbial reorganization after four years of management, promoting plant-fungus symbiosis. This pattern aligns with studies reporting the positive influence of revegetation time on soil biological activity (Dias et al., 2008; Juge et al., 2021).
The area with five-years of revegetation (Rev3) showed a colonization rate of 38.79%, a value statistically similar to all other treatments, as indicated by the “ab” grouping. This may represent a phase of symbiotic stabilization, in which mycorrhizal functionality is maintained but without the peak observed in Rev2. The absence of significant differences between Rev2 and Rev3 may reflect local variability in soil conditions or plant community composition.
The Rev1 area, with only three years of revegetation, had the lowest colonization value (26.89%), possibly due to limiting factors such as elevated iron and sodium levels and low organic matter content, which may suppress microbial activity (Prado et al., 2019). These findings are indicative of an early stage of soil and microbial symbiosis restructuring. The reference area showed colonization levels similar to Rev1, which may be related to low compatibility between native AMF and the host plant used, suggesting that symbiotic efficiency depends not only on soil chemical conditions but also on plant-fungus specificity (Orłowska et al., 2010; Salim et al., 2025).
Conclusions
The five-year revegetated area (Rev3) showed stable spore viability and mycorrhizal colonization, suggesting initial ecological stabilization, though still distant from reference conditions. Revegetation time enhanced mycorrhizal colonization, with the four-year site (Rev2) presenting the highest symbiotic efficiency. While revegetation improves chemical and microbiological soil properties, full recovery of mycorrhizal functionality depends on longer timeframes, suitable environmental conditions, and management practices that integrate plant and microbial components, including proper inoculation and substrate selection to enhance AMF activity.
Acknowledgment
Minas Gerais State Agency for Research and Development (FAPEMIG)- provision of resources for carrying out the research. Universidade Federal de Lavras (UFLA) - technological and logistical support for research development.
Data Availability Statement
Data available upon request to authors.
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Editor de seção:
Renato Paiva
