Germination and initial growth of three legume species on managed mine substrate: effects on biomass allocation and nodulation¹

Germinação e crescimento inicial de três espécies de leguminosas em substrato de área minerada: efeitos na alocação de biomassa e nodulação

ABSTRACT

This study evaluated the germination, initial growth, biomass allocation, and association with nitrogen-fixing bacteria of three shrub legumes from rupestrian grassland, growing on bauxite mine substrates. The germinability of Senna reniformis, Chamaecrista mucronata and Centrosema coriaceum was evaluated in mine substrate after chemical and mechanical scarification. Plant survival and growth were evaluated on four substrates: mine substrate, mine substrate with the addition of 6.7% of topsoil from ferruginous rupestrian grassland, mine substrate with 3.3% of commercial substrate, and 1:1 mixture of mine and commercial substrate. Both scarification methods increased germinability of the three species. Inoculation of mine substrate with topsoil did not enhance plant nodulation. However, the moderate increase in the fertility, achieved by the adding 3.3% of commercial substrate, favored the nodulation. The 1:1 mixture of substrates increased the total plant biomass but reduced root biomass allocation and nodulation, which could reduce the success in the restoration of degraded areas.

Keywords:
Fabaceae; mine waste; nitrogen fixation; post-mining restoration; topsoil

RESUMO

Neste estudo avaliou-se a germinação, crescimento inicial, alocação de biomassa e associação com bactérias fixadoras de nitrogênio em três leguminosas arbustivas nativas dos campos rupestres. A germinabilidade de Senna reniformis, Chamaecrista mucronata e Centrosema coriaceum foi avaliada após escarificação química e mecânica. A sobrevivência e crescimento das plantas foram avaliados em substrato de área minerada (MS), MS com a adição de 6,7% de solo superficial de campo rupestre ferruginoso, MS com adição de 3,3% de substrato comercial (CS) e mistura 1:1 de MS com CS. Ambos métodos de escarificação aumentaram a germinabilidade das três espécies. A inoculação do MS com solo superficial não contribuiu para a nodulação das plantas, porém, o aumento moderado da fertilidade pela adição de 3,3% de CS favoreceu a nodulação. A mistura de substratos (1:1) reduziu a alocação de biomassa nas raízes e as nodulações, o que pode reduzir o sucesso na restauração de áreas degradadas.

Palavras-chave:
Fabaceae; fixação de nitrogênio; restauração de áreas mineradas; topsoil

Introduction

To ensure ecological restoration in degraded areas with total soil loss, such as in opencast mining sites, it is essential to combine measures that promote both revegetation and soil development. (Sheoran et al. 2010). The use of legumes capable of establishing associations with nitrogen-fixing bacteria in the revegetation of degraded areas contributes to mitigate substrate dystrophy, facilitating the establishment of other species and increasing the nutrient cycling, diversity and abundance of the soil microbiota (Spehn et al. 2005, Temperton et al. 2007, Siddique 2008).

In the Brazilian rupestrian grassland (campo rupestre), which have high concentration of areas degraded by mining and related activities (Jacobi et al. 2007, Fernandes et al. 2014, 2018, Souza-Filho et al. 2019), legumes stand out for their diversity and abundance (Oliveira et al. 2016). Legumes native to rupestrian grassland are well adapted to dystrophic soils with chemical characteristics similar to those found in areas degraded by opencast mining (Messias et al. 2013, Schaefer et al. 2015, 2016). This fact reveals the potential of these species to revegetate these impacted areas, similarly dystrophic (Sheoran et al. 2010, Whiting et al. 2010), as already observed in the few evaluations carried out (Matias et al. 2009, Figueiredo et al. 2018, Gomes et al. 2018, Silva et al. 2018, Figueiredo et al. 2024).

The first challenge for direct seeding or seedling production of native legumes for the revegetation of degraded areas in rupestrian grassland is seed germination. Most rupestrian grassland legumes have seeds with physical dormancy (Lemos Filho 1997, Silveira & Fernandes 2006, Maia et al. 2010, Silveira et al. 2014, Nativel et al. 2015, Dayrell et al. 2015, 2017), indicating the need for studies on techniques to overcome dormancy (Broadhurst et al. 2016, Kildisheva et al. 2020). In addition to cost reduction, the knowledge about techniques to overcome dormancy is important to optimize the use of leguminous seeds from rupestrian grassland. Many rupestrian grassland species present small populations, and their natural populations are usually the only source of seeds (Silveira et al. 2016).

Another important step towards the use of native legumes in the revegetation of degraded areas is the development of techniques for producing seedlings that are more suitable for survival in severely degraded areas, and that contribute, in an effective way, to the soil formation and development. It is desirable that seedlings for planting in areas degraded by opencast mining present greater biomass allocation in roots, as well as exhibit efficient associations with soil microbiota, as a way to overcome dystrophy and the water shortage of the substrate. In addition, these species should also present adaptation to the high concentrations of certain chemical elements such as Fe, Al, Mn and Cr of the substrate, in the cases of itabirite and bauxite mining areas.

Both biomass allocation in legumes and the establishment of associations with nitrogen-fixing bacteria can be affected by the availability of nutrients in the substrate (Divito & Sadras 2014, Kassaw et al. 2015, Egamberdieva et al. 2018). Thus, evaluating the production of legume seedlings on substrates with different levels of nutrient level is crucial to determine the optimal condition for achieving greater associations with nitrogen-fixing bacteria and greater root biomass allocation. In the case of rupestrian grassland plants, these evaluations are even more important since these plants are adapted to extremely dystrophic soils (Benites et al. 2007, Messias et al. 2013, Schaefer et al. 2016).

In addition to providing adequate conditions for legume-microbiota, it is important to introduce a source of microorganisms, as degraded substrates often present low microbial diversity and abundance (Sheoran et al. 2010, Prado et al. 2019, Figueiredo et al. 2023). In the case of rupestrian grassland, due to the lack of commercial inoculum availability, a low-cost and promising approach to promote associations is the use of small amounts of topsoil from preserved areas (Figueiredo et al. 2018). The use of small portions of topsoil, in addition to contributing to the inoculation of nitrogen-fixing bacteria, also promotes the association with mycorrhizal fungi. These fungi often enhance the symbiotic plant-bacteria relationship, improving the outcomes of these ecological interactions (Marques et al. 2001, Matias et al. 2009). In addition, using inoculum with local species is more likely of establish successful associations and achieve greater effectiveness in results (Emam 2016, Rúa et al. 2016). Despite the benefits and the promising results already obtained, it is necessary to refine it and evaluate its effectiveness with other species.

Senna reniformis (G.Don) H.S. Irwin & Barneby, Chamaecrista mucronata (Spreng.) H.S. Irwin & Barneby and Centrosema coriaceum Benth are shrub to sub-shrub plants with height between 0.5 and 4m, with C. coriaceum showing prostrate growth. The three species are endemic to Brazil, with emphasis on the occurrence in areas of quartzitic and ferruginous rupestrian grassland. Chamaecrista mucronata is identified as a species with potential for use in the restoration of areas degraded by mining (Lima et al. 2016). Similarly, S. reniformis and C. coriaceum have also demonstrated promising results in the post-mining restoration efforts (Matias et al. 2009, Figueiredo et al. 2021a, b).

The aims of this study were: (i) to assess methods to overcome dormancy of three legume species, and (ii) to evaluate the initial growth, biomass allocation and association with nitrogen-fixing bacteria in these species growing on mine substrate, as well as with the addition of other substrates in varying proportions.

Material and methods

Seed collection - Chamaecrista mucronata (OUPR 31516 voucher) and C. coriaceum (OUPR 31795 voucher) seeds were collected in April 2019 at the Morro do Cruzeiro Campus, Universidade Federal de Ouro Preto (20°23’S, 43°30’W), while S. reniformis seeds (OUPR 31515 voucher) were collected in August of the same year at Parque Natural Municipal das Andorinhas (20°21’S, 43°30’W). Both collection sites are areas of ferruginous rupestrian grassland, locally known as canga, located in the municipality of Ouro Preto, Minas Gerais, Brazil. Seeds were collected from at least ten individuals per species, when they were already in a natural dispersion process. After collection, fruits were dried in the shade. Subsequently, seeds were separated from fruits and impurities and stored at temperature of 8ºC (Salomão & Silva 2003) until the beginning of experiments in August 2019.

Collection and characterization of substrates - The bauxite residual mine substrate used in germination experiments and for seedling production is the substrate left in the mine area after the end of the bauxite exploitation, located at the Parque Natural Municipal das Andorinhas, Ouro Preto, Brazil. This substrate was collected from depths greater than one meter. The topsoil used as inoculum for seedling production was collected in the first five centimeters of soil in areas of ferruginous rupestrian grassland at Morro do Cruzeiro Campus, Universidade Federal de Ouro Preto. Topsoil collection took place in 10 different points, close to individuals of species evaluated in this study. After collection, the topsoil was homogenized and passed through a 4-mm sieve to remove larger soil particles and plant fragments. The commercial substrate used in this study is commonly used in seedling production and is primarily composed of sugarcane bagasse, coffee straw, peat, limestone, manure, and poultry litter. Samples of commercial substrate, topsoil, mine substrate and the 1: 1 mixture of mine substrate and commercial substrate were analyzed for fertility parameters. Mine substrate was further characterized by the total concentration of chemical elements and particle size (Table S1- supplementary material). Analyses of fertility and total concentration of chemical elements followed the methods proposed by Teixeira et al. (2017) and Andrade et al. (2012), respectively.

Germination tests - For germination experiments and determination of the weight of a thousand seeds, full seeds without signs of damage caused by insects or animals were selected. The determination of the weight of a thousand seeds followed the method proposed by Brasil (2009). The germinability of seeds was evaluated in the following treatments: (i) Control - intact seeds were placed to germinate, (ii) Chemical scarification - seeds were immersed for 20 minutes in concentrated sulfuric acid and later washed under running water (a preliminary experiment, previously carried out, indicated that 20 minutes of immersion was more effective than 10 or 40 minutes), (iii) Mechanical scarification - a small cut was performed at the opposite end of the embryonic axis of the seed.

To assess the species’germinability on degraded substrate, the germination experiments were carried out using bauxite mine substrate. For the germination tests, pots with a diameter of 30 cm and a height of 10 cm were filled with 2L of bauxite mine substrate (Figure S2 - supplementary material). Seeds were randomly scattered on the surface of the substrate, covered with a layer of one centimeter of substrate, and the pots were kept in a greenhouse under natural lighting, with a constant temperature of 25 ºC and sufficient irrigation conditions to keep the substrate moist. Germinations were assessed weekly for 60 days.

Seedling production - Seeds of the three species were placed in seedbed containing a 10 cm layer of a 1:1 (v/v) sand and vermiculite mixture. The seeds were covered with a one-centimeter layer of the sand and vermiculite mixture, and the seedbeds were kept in a greenhouse. For C. coriaceum seeds placed in the seedbed, a small cut was previously made at the opposite end to the embryonic axis to enhance germinability.

Twenty-four days after planting, seedlings were carefully removed from the substrate and transferred to 0.75-liter pots, filled with the different substrates. At the time of transplanting, 10 seedlings of each species were chosen at random to determine the total length (root and shoot) and dry biomass. For these seedlings the total length and dry biomass were: S. reniformis (8.6 cm, 0.016 g), C. mucronata (7.7 cm, 0.010 g), and C. coriaceum (7.2 cm, 0.019 g).

Seedling production was evaluated under four different conditions: (i) Mine Substrate (MS), (ii) Topsoil inoculation (TS), (iii) Commercial Substrate inoculation (CS) and (iv) Mine Substrate plus Commercial Substrate MS + CS. In the mine substrate treatment, the pots were filled with bauxite mine substrate, and the seedlings were placed in a small pit in the substrate in the center of the pot. In the topsoil inoculation treatment, the pots were also filled with mine substrate. After, the seedlings were inserted in a small pit in the center of the pot, it was then filled with 0.05 L (6.7% v/v) of topsoil. The aim of the commercial substrate inoculation was to evaluate whether the addition of inoculum facilitates plant growth due to the increased presence of microorganisms or because the enhanced fertility promoted by the addition of the topsoil. This treatment was assembled similarly to the TS one, but in this case, 0.025 L (3.3% v/v) of commercial substrate was used as the inoculum. The lower volume of commercial substrate inoculum was chosen because it is more fertile than topsoil (Table S1- supplementary material). For the mine substrate plus commercial substrate treatment a 1:1 (v/v) mixture of these two substrates was used.

For each treatment, 25 pots (replicates) were assembled and kept under natural conditions of temperature, humidity, and lighting, and with sufficient irrigation to keep the substrate moist. The experiment started in September 2019, early spring. Seventy days after transplanting of seedlings to pots, the survival rate was assessed. Then, eight individuals per treatment were selected for evaluation (Figure S2 - supplementary material). All substrate adhered to roots was carefully removed by washing with water (Figure S2 - supplementary material). Then, plants were dried at 100 ºC until they reached a constant weight for later determination of the dry biomass of shoots, roots, and nodules.

For statistical analyses, the data were first assessed for normality (Kolmogorov-Smirnov test) and variance homoscedasticity (Bartlett test). The germination data (response variable) were parametric and significant differences among treatments were assessed using one-way analysis of variance (ANOVA) followed by Tukey test. The parameters evaluated in the seedlings (dry biomass, root/shoot dry biomass and nodules dry biomass) presented non-parametric data, and the differences among the treatments were evaluated using the Kruskal Wallis followed by the Mann-Whitney tests. All statistical tests were performed at 5% significance using the MINITAB 18.0^® software.

Results and discussion

Germinability and dormancy overcoming - The three species have small seeds, with the following weight for a thousand seeds: S. reniformis (18.6 g), C. mucronata (12.1 g), and C. coriaceum (27.5 g). In the control treatment, all species exhibited low germinability (Figure 1), indicating the presence of dormancy, a characteristic commonly in many legume species, including those occurring in rupestrian grassland (Lemos Filho 1997, Silveira et al. 2005, 2014, Silveira & Fernandes 2006, Maia et al. 2010, Dayrell et al. 2015, Nativel et al. 2015, Ordóñez-Parra et al. 2023). Both chemical and mechanical scarification contributed to increasing the germinability of the evaluated species. Although S. reniformis and C. mucronata seeds treated with chemical scarification showed slightly lower absolute germination rates than those with mechanical scarification, chemical scarification remains the most suitable method for overcoming dormancy in the three species. This method is easier to perform, particularly for small seeds, when compared to mechanical scarification (Nascimento & Oliveira 1999) (Figure 1).

Figure 1
Germinability (mean ± SD) of S. reniformis, C. mucronata and C. coriaceum in bauxite mine substrate in the treatments: control (Contr), chemical scarification (Chem) and mechanical scarification (Mech.). Different letters above the columns of each species, mean significant differences between treatments. Tukey Test (p value <0.05).

Additional studies are needed to refine the chemical scarification technique, particularly to determine the optimal immersion time in sulfuric acid. It is important to assess whether seed dormancy of these species can be overcome through alternative treatments, such as seed storage, temperature variations during germination experiments, and the timing of seed harvest (Nogueira et al. 2013, Silveira & Overbeck 2013, Dayrell et al. 2015, Nativel et al. 2015).

Growth and biomass allocation - With only three exceptions - C. mucronata in TS (88%), MS + CS and MS (96%) treatments - the seedling survival rate was 100%. Even in a dystrophic and metalliferous substrate, with high concentrations of some chemical elements, such as Al, Fe, Mn and Cr (Table S1- supplementary material), all species exhibited high survival and growth rates comparable to those observed in other native legumes (Negreiros et al. 2009, Figueiredo et al. 2018), especially in the MS + CS substrate (Figure 2). These results indicate that the producing seedlings of these species for revegetating degraded areas is feasible and easy to implement, even using residual mine substrates. The use of mine substrate not only aids in obtaining seedlings better adapted to the chemical conditions of post-mining substrates, but also reduces costs and pressure on new resources by limiting the need for soil or commercial substrates.

Figure 2
Total dry biomass (a), root/shoot ratio of dry biomass (b) and dry biomass of nodules (c) (mean ± SD) of S. reniformis, C. mucronata and C. coriaceum after 70 days of planting in pots with treatments: 1:1 mixtures of mine substrate plus commercial substrate (MS + CS), addition of 3.3% of commercial substrate to mine substrate (CS), addition of 6.6% topsoil to mine substrate (TS), mine substrate (MS). Different uppercase and lowercase letters above the columns indicate, respectively, significant differences among treatments in each species and among species in the same treatment. Mann-Whitney (p value <0.05). Only one S. reniformis individual from the TS treatment had one nodule (1.2 mg).

The three species responded positively in terms of biomass increment when grown in the most fertile substrate (MS + CS), particularly S. reniformis and C. mucronata (Figure 2 and Figure S1 - supplementary material). Previous research on Chamaecrista ramosa, a legume native to rupestrian grassland, indicated no response to increased substrate fertility (Negreiros et al. 2009). However, studies on other rupestrian grassland legumes have shown that most of these species respond positively to increasing fertility, despite their adaptation to dystrophic soils (Negreiros et al. 2009, Carvalho et al. 2018, Silva et al. 2018, Bahia et al. 2020). This behavior demonstrates the phenotypic plasticity of these species with regard to soil fertility and suggests a tendency to deviate from the expected pattern, where species adapted to dystrophic environments show minimal or no response to increased soil fertility (Chapin 1980).

Although legumes from rupestrian grassland exhibit greater growth in more fertile substrates, the use of fertilizers in the restoration of degraded areas must be carefully evaluated. Nutritional enrichment of these substrates tends to favor the establishment and growth of exotic and invasive species, which are more nutrient-demanding (Barbosa et al. 2010, Hilário et al. 2011), compared to native species, adapted to dystrophic and low-productivity soils (Negreiros et al. 2014, Oliveira et al. 2015). Silva et al. (2018) demonstrated that, in substrates from ferruginous rupestrian grassland and areas degraded by iron mining, the development of legumes native to rupestrian grassland and an exotic is similar or superior to native species. However, when these substrates are fertilized, exotic legumes exhibit significantly higher growth rates compared to native ones. Consequently, fertilizing these substrates could promote the dominance of exotic species over native species, which can compromise the effectiveness of ecological restoration efforts in degraded areas.

Contrary to expectations for seedlings of species from savanna environments, the three species in this study exhibited a root/shoot biomass ratio lower than one (Hoffmann and Franco 2003) (Figure 2). However, these findings align with those from four other studies that evaluated this parameter in seedlings of legumes native to rupestrian grassland (Negreiros et al. 2009, Carvalho et al. 2018, Figueiredo et al. 2018, Silva et al. 2018). As suggested by Negreiros et al. (2009), the lower investment in roots observed in native species of rupestrian grassland may be related to the shallow soils typical of these environments, which limit root growth. Similarly, as observed in other legumes from ferruginous rupestrian grassland (Silva et al. 2018), the three species in this study increased their root investment in poorer substrates (Figure 2 and Table S2 - supplementary material). This adaptation helps to counteract the effects of environmental limitations and optimize resource acquisition (Bloom et al. 1985). It is believed that seedlings with greater root investment are better prepared to tolerate the limiting conditions of degraded areas, such as water scarcity and nutrient deficiency. In this way, producing seedlings in dystrophic substrates could be advantageous for obtaining plants better prepared to survive and growth in severely degraded areas.

Association with nitrogen-fixing bacteria - Contrary to expectations, the addition of small portions of topsoil from a preserved rupestrian grassland area did not produce significant effects on plant biomass gain or on the occurrence and biomass of nodules in any of the species. For C. coriaceum, the treatment with the addition of topsoil resulted in the lowest nodule biomass (Figure 2 and Table S2 - supplementary material). Similar experiments with Periandra mediterranea, a legume native to rupestrian grassland, showed that the addition of small portions of topsoil contributed to greater nodulation and biomass gain by plants (Figueiredo et al. 2018). Similarly, Silva et al. (2018) found that the use of topsoil from rupestrian grassland increased the number of nodules in two native legumes compared to those grown in substrate from area degraded by mining.

The inefficiency of topsoil inoculation in the present study may be related to several factors, including the relatively short duration of the experiment, potential inhibition of plant associations due to soil fertility indexes (see details below), and the possible presence of microorganisms antagonistic to nitrogen-fixing bacteria or plant pathogens (Granada Agudelo et al. 2023). The fact that topsoil was collected close to adult individuals of the species evaluated in this study would increase the probability of the presence of organisms harmful to these plants in the topsoil (Liang et al. 2016).

Chamaecrista mucronata and C. coriaceum species demonstrated a high susceptibility to association with nitrogen-fixing bacteria, as evidenced by the presence of nodules when grown in degraded mine substrate collected at depths greater than one meter, even without any inoculation method (Figure 2 and Table S2 - supplementary material). In contrast, S. reniformis presented only a structure similar to a nodulation, consistent with the findings of Faria et al. (1989), which indicated that Senna species do not form nodules. Possibly, this structure could be a product of the presence of galls, tumors, knots, hypertrophies or mycorrhizae, as suggested by Faria et al. (1989). On the other hand, S. reniformis exhibited a notable difference in dry biomass between MS + CS treatment and the other treatments (Figure 2 and Figure S1 - supplementary material). In other words, while this species appears to have a lower or null ability to establish associations with nitrogen-fixing, it demonstrated a significant increase in biomass when grown in more fertile substrate, compared to the other species in this study.

Although the association with nitrogen-fixing bacteria contributes for plants to obtain nitrogen, it demands a high energy cost (Crawford 2000). In this way, the occurrence of this association may be avoided by the plant through control mechanisms, when it does not have enough energy to establish the association, as seen in extremely dystrophic substrates (Tsvetkova & Georgiev 2003, Divito & Sadras 2014, Egamberdieva et al. 2015). Additionally, plants may avoid these associations when there is enough supply of available nitrogen in the soil (Schultze & Kondorosi 1998, Kassaw et al. 2015). Thus, in the present study, the addition of a small portion of a more fertile substrate in the CS treatment enhanced the association of C. mucronata and C. coriaceum plants with nitrogen-fixing bacteria. This slight increase in nutrient availability was sufficient to allow the establishment of these associations, but not enough to inhibit them due to the excessive availability of assimilable nitrogen forms, as observed in the MS + CS treatment (Barbulova et al. 2007, Kassaw et al. 2015).

Results similar to those observed in this study, with regard to the negative effects of excessive increase in substrate fertility on the association of nitrogen-fixing bacteria with legumes, were observed in experiments with eight native legumes aiming to recover areas degraded by iron mining (Araújo & Costa et al. 2013, Silva et al. 2018). When transplanted into degraded areas with dystrophic substrates (Machado et al. 2013, Figueiredo et al. 2016, Le Stradic et al. 2018), plants with stronger associations with nitrogen-fixing bacteria may exhibit better long-term development. This set of results suggests that excessive fertilization of mine substrate inhibits the association of nitrogen-fixing bacteria with native legume, potentially hindering the success of these plants in restoration efforts.

The species in this study demonstrate the ability to tolerate dystrophic substrates with high concentrations of metals, conditions often observed in areas degraded by mining. Seed scarification significantly enhances germinability of all the three species. Senna reniformis exhibit a higher growth rate in more fertile substrates, while the other species showed a greater ability to establish associations with nitrogen-fixing bacteria. Seedling production in more fertile substrate can accelerated initial development; however, it may reduce biomass allocation in the root system and hinder or inhibit associations with nitrogen-fixing bacteria. This could negatively impact the success of these plants in the restoration of severely degraded areas, where root development and nitrogen-fixing associations are crucial for long-term survival and growth.

Supplementary material

Figure S1
Dry biomass gain percentage of Senna reniformis, Chamaecrista mucronata and Centrosema coriaceum species after 70 days of planting in pots with treatments: 1: 1 mixture of mine substrate plus commercial substrate - MS + CS; addition of 3.3% of commercial substrate to mine substrate - CS; addition of 6.6% topsoil to mine substrate - TS; mine substrate - MS. Different letters represent significant differences between treatments (p value <0.05).

Table S1
Fertility of substrates topsoil -TS, commercial substrate - CS, mine substrate: MS, mine substrate and commercial substrate: MS + CS. Total concentration of chemical elements and percentage grain-size fractions in the mine substrate. SB: sum of bases, t: effective cation exchange capacity, T: cation exchange capacity in pH 7.0, V: base saturation, m: aluminum saturation, OM: organic matter, P-rem: remnant phosphorus, CS: coarse sand, FS: fine sand, S: silt, C: clay.

Fertility Parameters and units of measure							Total concentration of chemical elements mg/kg)					Grain size (%)
		TS	CS	MS	MS+CS		MS		MS			MS
pH		4.52	6.37	4.88	6.27		Ca	218	Al	147561		CS	10.2
P	mg/dm³	5.10	627	0.40	290		Mg	518	As	71		FS	4.8
K	mg/dm³	35	1545	0.00	753		K	354	Ba	29		S	5.8
Ca²+	cmolc/dm³	0.49	7.35	0.23	5.22		P	396	Co	19		C	79.2
Mg ²+	cmolc/dm³	0.24	8.78	0.02	4.97		S	243	Cr	346
Al3+	cmolc/dm³	0.78	0.00	0.09	0.00		Cu	17	Na	241
H+Al	cmolc/dm³	9.20	4.20	3.30	3.90		Fe	174518	Ni	49
SB	cmolc/dm³	0.82	20.1	0.25	12.1		Mn	410	Sc	14
t	cmolc/dm³	1.60	20.1	0.34	12.1		Mo	5	Sr	59
T	cmolc/dm³	10.0	24.3	3.55	16.0		Zn	36	Th	36
V	%	8.20	82.1	7.00	75.7				Ti	14095
m	%	48.8	0.00	26.5	0.00				V	325
OM	dag/kg	3.59	32	1.07	9.13				Y	24
N	dag/kg	0.11	0.54	0.03	0.19				Zr	459
P-rem	mg/L	35.5	45.0	13.3	29.0

Table S2
Average and standard deviation of number of leaves (No. of leaves), shoot length, root extension (determined by Winrhizo), dry shoot and root biomass, percentage of plants with nodules (PN (%)) and average number of nodules per plant (No. of nodules) in the different treatments. 1: 1 mixture of mine substrate plus commercial substrate - MS + CS; addition of 3.3% of commercial substrate to mine substrate - CS; addition of 6.6% topsoil to mine substrate - TS; mine substrate - MS. Data followed by different letters in the same column represent significant differences between treatments.

		Length (cm)		Dry Biomass (g)		Nodules
	No of leaves	Shoot	Root	Shoot	Root	PN(%)	No of nodules
Senna reniformis
MS+CS	5.25 ± 1.09a	15.80 ± 1.79a	604 ± 210a	0.70 ± 0.22a	0.16 ± 0.07a	0.00	0.00 ± a
CS	4.63 ± 0.48a	8.25 ± 1.30b	333 ± 90b	0.18 ± 0.08a	0.08 ± 0.04b	0.00	0.00 ± a
TS	3.38 ± 0.48b	6.25 ± 0.43c	222 ± 65b	0.11 ± 0.03a	0.06 ± 0.03b	12.5	0.13 ± 0.33a
MS	3.25 ± 0.43b	6.38 ± 0.70c	382 ± 84b	0.13 ± 0.03b	0.09 ± 0.03b	0.00	0.00 ± a
Chamaecrista mucronata
MS+CS	9.43 ± 3.02a	27.9 ± 6.69a	211 ± 86a	0.47 ± 0.18a	0.08 ± 0.03a	14.2	0.14 ± 0.35b
CS	6.86 ± 1.64ab	16.3 ± 5.62b	243 ± 44a	0.21 ± 0.08b	0.06 ± 0.02a	100	6.00 ± 3.46a
TS	6.00 ± 1.29b	13.0 ± 3.42bc	226 ± 65a	0.18 ± 0.08b	0.07 ± 0.02a	100	4.67 ± 2.05a
MS	3.86 ± 0.99b	8.00 ± 2.51c	318 ± 113a	0.12 ± 0.03b	0.07 ± 0.02a	100	4.57 ± 2.13a
Centrosema corieaceum
MS+CS	4.38 ± 0.99a	11.6 ± 2.18a	447 ± 186a	0.30 ± 0.13a	0.08 ± 0.04a	25	0.38 ± 0.70c
CS	3.75 ± 0.83a	8.38 ± 1.11b	461 ± 152a	0.13 ± 0.04b	0.07 ± 0.02ab	100	6.38 ± 3.90a
TS	2.43 ± 0.49b	7.00 ± 1.41bc	333 ± 92a	0.06 ± 0.01c	0.04 ± 0.01b	14.2	0.14 ± 0.35c
MS	2.13 ± 0.33b	5.13 ± 0.60c	382 ± 102a	0.05 ± 0.02c	0.04 ± 0.01b	100	2.88 ± 1.05b

Figure S2
Seedlings of the three species under study in a mine substrate germination experiment (a). Detail of Senna reniformis (b), Chamaecrista mucronata (c) and Centrosema coriaceum (d) individuals 70 days after planting in pots in the mine substrate plus commercial substrate treatment. S. reniformis (e) and C. coriaceum (f) individuals 70 days after planting in pots.

Figure S3
Details of nodules of nitrogen-fixing bacteria in Centrosema coriaceum (a) and Chamaecrista mucronata roots (b). Comparison of images showing the difference in investment in root biomass in C. mucronata plants. Plant grown in the 1: 1 mixture of mine substrate plus commercial substrate (c) and plant grown in mine substrate (d).

Data availability statement

The dataset for this article is available in the SciELO Dataverse of Hoehnea, at the following link: https://doi.org/10.1590/2236-8906e162024.

Acknowledgments

The authors would like to thank the Professor Jose Badini Herbarium, the Jorge Luiz da Silva Botanical Garden, and the Laboratory of Environmental Geochemistry - Universidade Federal de Ouro Preto, for providing infrastructure and assistance to carry out this work. To the Municipal Secretariat for the Environment of Ouro Preto and the entire team of the Parque Natural Municipal das Andorinhas, for the research license and support. We are grateful to Universidade Federal de Ouro Preto, Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Conselho Nacional de Desenvolvimento Científico e Tecnológico, for the financial support.

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Edited by

• Associate Editor:
  Nelson Augusto dos Santos Júnior

Publication Dates

• Publication in this collection
  21 Nov 2025
• Date of issue
  2025

History

• Received
  26 Feb 2024
• Accepted
  05 Dec 2024