Seed and soil provenance in the production and survival of Melanoxylum brauna seedlings

Abstract

Melanoxylum brauna Schott is an endangered Atlantic Forest tree species whose seedling production is constrained by low survival under nursery conditions. This study evaluated seedling survival, morphophysiological performance, biochemical traits, and arbuscular mycorrhizal fungi (AMF) occurrence in seedlings produced from seeds collected at two locations and cultivated under different substrate combinations. Treatments included organic substrate, subsoil, soil collected near mother trees, and mixtures of sand, subsoil, mother-tree soil, and ground branches. Seedlings grown in 100% soil collected near mother trees showed the highest survival rate (97.5%), superior Dickson quality index, greater shoot and root growth, and enhanced photochemical efficiency (F_v/F_m), regardless of seed origin. These soils exhibited higher AMF spore density and the presence of Glomus macrocarpum, Sclerocystis spp., and Gigaspora spp., whereas organic substrates lacked mycorrhizal propagules and resulted in complete seedling mortality after 60 days. Biochemical analyses revealed increased accumulation of carbohydrates, proteins, lipids, and starch in seedlings grown in mother-tree soils, indicating improved metabolic performance. Overall, the results indicate that soil collected near mother trees functions as a natural bioinoculum source and is associated with improved seedling growth, physiology, and survival, possibly mediated by native microbial communities, including AMF.

Introduction

Melanoxylum brauna Schott., a member of the Fabaceae family, is a native tree species of the Atlantic Forest biome, occurring mainly in the northeastern and southeastern regions of Brazil¹. Its wood is highly valued due to its high density (approximately 1.05 g cm^{− 3}, durability, and mechanical resistance², which historically supported its extensive use in civil construction, shipbuilding, and furniture manufacturing³. As a consequence of intense and prolonged exploitation, the species is currently classified as vulnerable on the Brazilian List of Endangered Species⁴.

Commercial propagation of M. brauna has been proposed as an alternative strategy to reduce pressure on natural populations and support conservation and restoration programs⁵. However, seed-based propagation remains challenging due to difficulties in locating reproductive individuals, high seed predation by borer insects that compromise endosperm integrity, reduced seed viability during storage^6–8, and high seedling mortality during nursery production.

In addition, genetic variability associated with seed origin can influence seedling establishment and early development. Seedlings derived from different mother trees may exhibit substantial intra-specific variation in growth, physiology, and stress tolerance⁹. Environmental conditions at the seed source can further induce phenotypic plasticity, leading to adaptive physiological and biochemical responses to local climatic factors^10–12.

Substrate composition is another critical factor influencing seedling performance during nursery cultivation¹³. An adequate substrate must ensure physical stability, water retention, nutrient availability, and gas exchange¹⁴. Although commercial substrates are widely used due to their standardized composition and high nutrient content¹⁵, they generally lack the biological complexity of natural soils, particularly symbiotic microorganisms that play key roles in plant development¹⁶. In this context, the interest in arbuscular mycorrhizal fungi (AMF) is linked to the biological and physicochemical characteristics of soils collected near mother trees, which may differ substantially from commercial substrates and influence seedling establishment. Studies with woody species, including recent evidence for M. brauna, have shown that seedling establishment may vary according to the physicochemical and biological characteristics of the substrate, especially when native soils are compared with conventional commercial substrates^17,18.

Natural soils collected near mother trees are characterized by an active rhizosphere, where complex interactions occur between roots and soil microorganisms, including AMF. These microorganisms have been reported to enhance nutrient acquisition, modulate hormonal signaling, and improve stress tolerance, potentially influencing seedling establishment and survival^19–22. However, critical gaps remain regarding (i) the role of native AMF in the early development of M. brauna seedlings, (ii) the influence of seed origin on seedling establishment and survival, and (iii) the comparative performance of commercial substrates versus soils collected near mother trees.

In woody species, AMF have been widely reported to enhance nutrient acquisition, particularly phosphorus and nitrogen, improve biomass accumulation, and increase photosynthetic efficiency under nutrient-limited conditions^19–21. Experimental studies in forest trees demonstrate that AMF colonization can modulate carbon allocation patterns, stimulate root development, and improve stress tolerance during early establishment, especially in low-fertility or degraded soils^20,23. Moreover, plant responsiveness to AMF has been associated with ecological strategy and functional traits, including growth rate and successional status, suggesting that mycorrhizal dependency may vary among woody species^24,25. In tropical systems, native AMF communities have been shown to contribute significantly to seedling establishment and field performance, reinforcing the importance of plant, microbe interactions in forest regeneration dynamics²⁶.

Recent experimental evidence further demonstrates that plant responses to arbuscular mycorrhizal fungi (AMF) in woody species are strongly species- and context-dependent. For example, enriching native AMF communities in non-sterile forest soil altered physiological traits and growth in three woody species, with contrasting outcomes among species, including increases in photosynthetic parameters and biomass in some cases²⁷. Under field restoration conditions, prior inoculation with selected native mycorrhizal fungi was reported to be associated with higher survival and improved recovery following low water availability in a woody tropical species²⁸. In Atlantic Forest woody species, AMF colonization and growth responses differed between pioneer and late secondary groups, and mycorrhizal plant responses were reported as inversely related to seed mass²⁹. At broader scales, a large meta-analysis reported AMF benefits to plants across multiple global change factors (including drought and nitrogen addition), supporting the ecological relevance of AMF-plant interactions while emphasizing response variability²². Collectively, these studies indicate that woody seedlings may respond to AMF inoculum sources, although responses are not universal and require species-specific evaluation.

Despite the recognized ecological relevance of AMF in woody plants, no studies have evaluated the occurrence of native AMF communities associated with M. brauna mother trees or their potential contribution to seedling performance under nursery conditions. The absence of such information represents a critical gap for this endangered Atlantic Forest species, particularly given the high mortality observed during conventional seedling production. Therefore, understanding whether rhizospheric soils function as natural reservoirs of effective microbial inoculum may provide new strategies to improve propagation and conservation programs.

We hypothesized that soils collected near M. brauna mother trees would promote greater seedling survival and superior morphophysiological and biochemical performance than commercial substrate or subsoil, likely due to differences in native microbial communities, including arbuscular mycorrhizal fungi, and soil attributes. This study evaluated the effects of different soil and substrate combinations, as well as seed origin, on the establishment of M. brauna seedlings. Specifically, we aimed to: (i) compare seedling survival and morphophysiological performance under contrasting soil and substrate compositions; (ii) assess the biochemical responses of shoots and roots under these cultivation conditions; and (iii) characterize the chemical, physical, and microbiological attributes of the soils/substrates, including the occurrence of arbuscular mycorrhizal fungi in mother-tree soils.

Materials and methods

Seeds and soil samples were collected from two locations in the state of Espírito Santo, Brazil: Laranja da Terra (19°52′44.6″ S, 40°56′50.6″ W), a natural forest remnant, and Jerônimo Monteiro (20°46′26.93″ S, 41°23′53.00″ W), a managed plantation area (Fig. 1). At each site, seeds were collected from 10 healthy and reproductively mature mother trees, selected based on accessibility, absence of visible disease symptoms in the plant, and active fruit production.

Fig. 1.

Geographic location of the two seed and soil sampling sites of Melanoxylum brauna in the state of Espírito Santo, Brazil: Laranja da Terra (19°52′44.6″ S, 40°56′50.6″ W), a natural forest remnant, and Jerônimo Monteiro (20°46′26.93″ S, 41°23′53.00″ W), a managed plantation area. These figures were prepared by the authors themselves using ArcGIS 10.5³⁰, and Inkscape 0.92.5 Software³¹.

Soil samples were collected near the mother trees, at a distance of 30 cm from the main stem and at a depth of 20–40 cm, using a mattock. This distance and depth were chosen to target the active rhizosphere zone, where fine roots, microbial activity, and arbuscular mycorrhizal fungal propagules are most abundant. Soil samples were transported to the forest nursery in plastic bags, where treatments were prepared.

Seeds were subjected to a germination process in a Biochemical Oxygen Demand (BOD) chamber regulated at 25 °C, under a 12 h photoperiod. The seeds were placed on two sheets of germitest paper in Gerbox^® containers for up to eight days, and germination was defined as radicle protrusion ≥ 2 mm. After radicle emergence, germinated seeds were transferred to 280 cm³ plastic tubes containing the different soil and substrate combinations described in Table 1.

Table 1.

Composition of soils and substrate, and the location of seed and soil used in the production of M. brauna seedlings.

Treatments	Substrate composition	Location
		Seeds	Soil
1	Organic substrate	LT*	–
2	Subsoil	LT	–
3	Soil near the mother tree with roots (100%)	LT	JM
4	Sand (49.5%) + soil near the mother tree with roots (49.5%) + ground branches** (1%)	LT	JM
5	Sand (33%) + subsoil (33%) + soil near the mother tree with roots (33%) + ground branches (1%)	LT	JM
6	Sand (33%) + subsoil (32%) + soil near the mother tree with roots (33%) + ground branches (2%)	LT	JM
7	Sand (33%) + subsoil (30%) + soil near the mother tree with roots (33%) + ground branches (4%)	LT	JM
8	Organic substrate	JM	–
9	Subsoil	JM	–
10	Soil near the mother tree with root (100%)	JM	LT
11	Sand (49.5%) + soil near the mother tree with roots (49.5%) + ground branches (1%)	JM	LT
12	Sand (33%) + subsoil (33%) + soil near the mother tree with roots (33%) + ground branches (1%)	JM	LT
13	Sand (33%) + subsoil (32%) + soil near the mother tree with roots (33%) + ground branches (2%)	JM	LT
14	Sand (33%) + subsoil (30%) + soil near the mother tree with roots (33%) + ground branches (4%)	JM	LT

^*LT: Laranja da Terra town, ES; JM: Jerônimo Monteiro town, ES. ^**Ground branches were obtained by milling branches in a Wiley-Tecnal^® mill, and the same procedure was used for all treatments.

The proportion of ground branches incorporated into the substrate mixtures (1%, 2%, and 4%) was defined based on traditional silvicultural practices reported by local rural communities in the region, who commonly incorporate decomposing plant residues collected near adult trees into nursery substrates to enhance seedling establishment. These empirical practices were used as a reference framework to establish low-percentage organic additions that would not substantially alter the physicochemical structure of the substrate while allowing the evaluation of potential biological effects. The access to associated traditional knowledge followed Brazilian legislation (Law 13.123/2015) and was registered in SISGEN (No. A81745F).

The following materials were used as raw materials for preparation of the treatments: commercial organic substrate Tropstrato^® (pine bark, vermiculite, PG Mix 14-16-18, potassium nitrate, simple superphosphate and peat); mother tree soil (soil collected near the mother tree with the presence of fine M. brauna roots); sand (autoclaved at 121 °C, 1 atm for 30 min); branches (branches of the mother tree ground in a Wiley-Tecnal^® mill); and subsurface soil samples (passed through 2 mm sieves) (Table 1).

After being placed in the tubes, the germinated seeds were kept in the greenhouse. The tubes were positioned on suspended iron benches (12 m × 0.64 m) installed 80 cm above the ground, ensuring adequate drainage and air circulation. The greenhouse was covered with 150 μm plastic film, and the sides were protected with an anti-aphid screen. Greenhouse environmental conditions were characterized by mean temperatures ranging from 22 to 32 °C, relative humidity between 60 and 80%, and natural photoperiod conditions. Seedlings from all treatments were irrigated manually whenever necessary to maintain adequate moisture.

Seedling growth

The first assessment of the seedlings was carried out 40 days after sowing, analyzing the shoot length (cm), collar diameter (mm), the ratio of the shoot to collar diameter and the number of leaves. For this evaluation, all seedlings within each replicate (n = 15 seedlings per replicate) were individually measured, using a graduated ruler for shoot length and a digital caliper for collar diameter. The final evaluation was carried out 150 days after the experiment was set up, where the following variables were analyzed: survival (%), shoot length (cm), shoot growth rate (cm) (shoot growth rate = C150 − C40, where: C150 is the shoot length at 150 days; C40 is the shoot length at 40 days), collar diameter (mm), collar diameter growth rate (mm) (collar diameter growth rate = D150 − D40, where: D150 is the collar diameter at 150 days; D40 is the collar diameter at 40 days), number of leaves, root length (cm), shoot/collar diameter ratio, dry matter of the shoot (g) and dry matter of the roots (g). Root length was measured after careful removal of the substrate and washing of the root system under running water. And finally, the Dickson quality index (DQI) using the equation DQI = TDM/((SH/D) + (DMS/DMR)), where: TDM: total dry matter of the plant, SH: shoot height, D: collar diameter, DMS: dry matter of the shoot, DMR: dry matter of the roots³². The DQI is a robust and widely used metric for evaluating forest seedling quality and morphological balance³².

The dry matter of the shoot and the root were obtained by drying in a forced air circulation oven, maintained at approximately 60 °C until constant weight, and quantified using an electronic analytical balance (0.0001 g). Mean values per replicate were used for statistical analyses.

Chlorophyll index a and b and chlorophyll fluorescence a

Two evaluations were conducted. The first at 40 days after the experiment was set up, and the final at 150 days. The chlorophyll indices a and b were measured using Clorofilog^® at 9 a.m. The maximum quantum yield of photosystem II (F_v/F_m) was measured using a fluorimeter (FMS2, Hansatech, Norfolk, UK). Before taking the measurements, the leaves were kept in the dark for 30 min using tweezers. The maximum quantum yield of photosystem II was obtained using the equation F_v/F_m = (F_m −F₀)/F_m³.

Biochemistry in seedlings

Samples of the shoot and root of the seedlings from each treatment were kept in an oven at 45 °C and then grinded. The extraction was carried out according to Bligh and Dyer³⁴ using 0.250 g of material, with four replicates for each treatment, initially extracted in an ethanolic series (98, 80, and 50%) and then in chloroform. For this extraction, the samples had to be kept in a water bath at 80 °C for 20 min and then centrifuged at 10,000 rpm for 10 min, resulting in a hydrophilic and a hydrophobic liquid phase.

The precipitated hydrophobic part of the extraction was used to measure the lipid content. The total soluble sugar content was measured by adding 80% ethanol and 0.2% anthrone to the hydrophilic solution, then placing the samples in a water bath at 100 °C for 15 min and reading them on a spectrophotometer at a wavelength of 620 nm, with results expressed as mg g^− 1 dry weight.

For the analysis of the total soluble phenols, the Folin-Ciocalteau 10% and sodium carbonate 4% reagents were used, keeping the samples in a dark chamber for 30 min for reading on a spectrophotometer at a wavelength of 760 nm. The protein content was analyzed by adding 0.2 M potassium hydroxide (KOH) to the remaining pellet from the initial extraction, then placing it in a water bath at 75 °C for 2 h, followed by centrifugation at 10,000 rpm for 10 min to remove the supernatant. The extract was then diluted in 0.2 M KOH and Bradford solution, leaving the samples to stand for 10 min. Readings were taken on a spectrophotometer at a wavelength of 595 nm.

The starch was quantified by adding 3% hydrochloric acid (HCl) to the pellet obtained from the protein extraction, requiring the samples to be incubated in a dark chamber for three hours. After incubation, the samples were centrifuged at 10,000 rpm for 10 min, and the supernatant was used for spectrophotometric readings at a wavelength of 620 nm. In turn, the fiber content was calculated by drying the pellet resulting from the starch extraction in an oven at 60 °C until constant weight.

Physical, chemical and basal respiration analysis of substrates

The substrate samples from the different treatments used in the development of M. brauna seedlings were dried and sieved (2 mm mesh). For the chemical, physical, and basal respiration analyses, four samples were evaluated per treatment (n = 4). Soil pH was measured in a 1:2.5 (soil: water) suspension, according to Teixeira et al.³⁵.

To determine the exchangeable Ca, Mg and Al contents, 10 cm³ of soil was mixed with 100 mL KCl 1 M), followed by shaking and resting for 16 h, according to Teixeira et al.³⁵. Then, 0.5 mL of extract was mixed with 10 mL of SrCl₂ to determine the exchangeable Ca and Mg contents, using the atomic absorption spectrophotometer. To quantify Al, three drops of bromothymol blue indicator (1%) were added to the extract (25 mL) and titrated with NaOH (0.025 M).

To determine H + Al, 5 cm³ of soil was mixed with 75 mL of calcium acetate (0.5 M, pH 7.0), followed by shaking and 16 h of rest, according to Teixeira et al.³⁵. Subsequently, two drops of phenolphthalein were added to 25 mL of extract, which was titrated with NaOH (0.025 M).

For the extraction of P, Na, K, Fe, Cu, Zn and Mn, 10 cm³ of soil was mixed with 100 mL of Mehlich^− 1 extracting solution (HCl 0.05 M, H₂SO₄ 0.0125 M), shaken and left to rest for 16 h, according to Teixeira et al.³⁵. Na and K were quantified by flame photometer. To determine the P content, 5 mL of extract was mixed with 5 mL of working reagent (200 mL of 725 solution, 1.6 g of ascorbic acid, distilled water for 1000 mL) and determination was made by colorimetry (wavelength 725 nm). Fe, Cu, Zn and Mn were analyzed directly from the extract using the atomic absorption spectrophotometer. Organic matter (OM), expressed in dag kg^− 1, was quantified by wet carbon oxidation with potassium dichromate in an acidic medium³⁵.

The physical analysis of the soil (particle size analysis) was in accordance with Embrapa³⁶ and IAC - Technical Bulletin 106³⁷.

Basal soil respiration was quantified from 100 g of soil, incubated for 168 h in a closed system, at a controlled temperature of 25 ± 2 °C, with the C-CO₂ captured in a 0.5 N sodium hydroxide solution. Barium chloride (50%) and phenolphthalein were subsequently added to the solution, followed by titration with 0.5 N HCl³⁸. C–CO₂ (mg kg⁻¹ h⁻¹) was determined according to Stotzky³⁹.

Characterization of arbuscular mycorrhizal fungi (AMF) communities in the soil

After the collection, the soil samples were packed in plastic bags and sent to the laboratory, where they were sieved (2 mm mesh). The soil was stored in a refrigerator at 4 °C for later extraction of arbuscular mycorrhizal fungi (AMF) spores, determination of the number of spores and identification of the AMF species.

The AMF spores were extracted according to Gerdemann and Nicolson⁴⁰, followed by centrifugation in water and sucrose (45%). The spores were counted and then separated according to their morphological characteristics of color and size and stored frozen at -20 °C in 2 mL Eppendorf tubes. The spores from each Eppendorf tube were poured into a Petri dish and divided into two groups, which were transferred to spots on a glass slide for microscopy using a Pasteur pipette. In this case, one group was fixed with PVLG (polyvinyl alcohol-lactoglycerol) and the other with PVLG + Melzer’s reagent (1:1)⁴¹. The spores fixed with PVLG + Melzer’s reagent was subjected to a small amount of pressure on the coverslip to break down the walls, which allowed the internal walls to be stained, serving as an identification parameter. After the assemblage, the slides were kept at room temperature for initial drying and then taken to the oven (± 40 °C) for complete drying of the PVLG.

The AMFs were identified at Embrapa Agrobiologia using an optical microscope (Leica^® DM750) equipped with a micrometric eyepiece. The Schenck and Pérez⁴² manual, original articles describing the species and characterizations of the species provided on the website of the “International Culture Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi”⁴³ were used to support the identification work.

Statistical analysis

The experiment was completely randomized design, consisting of 14 treatments (substrate compositions) with four repetitions of 15 seedlings each, totaling 60 seedlings per treatment (Table 1). For statistical purposes, each repetition consisted of 15 seedlings from each treatment, and the average number of these seedlings was considered as a single experimental unit. The data was subjected to an analysis of variance and F test (p < 0.05), compared using the Scott-Knott average grouping test (p < 0.05) and Pearson’s correlation to compare the parameters. All statistical analyses were carried out using the R statistical program⁴⁴, the analyses were performed using the emmeans package⁴⁵, all plots were generated using the ggplot2 package⁴⁶ and used the package corrplot⁴⁷ to run the correlations.

Results and discussion

Forty days after transplanting, significant differences were observed among treatments for all growth variables analyzed (Fig. 2). Shoot length was higher in treatment 5 and did not differ statistically from treatment 12, both composed of similar substrate proportions, differing only in seed origin and soil collected near the mother tree (Fig. 2a). A similar pattern was observed for treatments 1 and 8, which showed comparable shoot length despite differing seed origins, both using organic substrate.

Fig. 2.

Growth parameters of Melanoxylum brauna seedlings at 40 days after transplanting under different substrate treatments. (a) Shoot length (cm); (b) collar diameter (mm); (c) Ratio between shoot length and collar diameter; (d) number of leaves. Bars represent mean ± standard error. ¹Different letters indicate significant differences among treatments according to the Scott-Knott test (p < 0.05). These figures were prepared by the authors themselves using R⁴⁴ Software.

Collar diameter was significantly higher in treatment 3, which consisted of 100% soil collected near the mother tree from Laranja da Terra combined with seeds from Jerônimo Monteiro (Fig. 2b). In contrast, the smallest collar diameters were observed in treatments 1 and 8, both based on organic substrate. Organic substrate promoted higher values for the shoot-to-collar diameter ratio and number of leaves, with treatments 1 and 8 showing statistically superior values for these variables (Fig. 2c,d).

These early growth differences reflect substrate-dependent allocation patterns. Although organic substrate stimulated greater shoot elongation and leaf production, treatments containing soil collected near mother trees exhibited more balanced structural development, indicating that early morphological expansion does not necessarily predict long-term performance^48–52.

At 40 days after transplanting, the SPAD index and chlorophyll a and b contents were significantly higher in treatment 10 (Fig. 3a–c). In contrast, the maximum quantum yield of F_v/F_m was significantly higher in treatments 3 and 10, both using 100% soil collected near the mother tree, regardless of soil origin (Fig. 3d).

Fig. 3.

Physiological parameters of Melanoxylum brauna seedlings at 40 days after transplanting under different substrate treatments. (a) SPAD index; (b) chlorophyll index a; (c) chlorophyll index b; (d) maximum quantum yield of photosystem II (F_v/F_m). Bars represent mean ± standard error. ¹Different letters indicate significant differences among treatments according to the Scott–Knott test (p < 0.05). These figures were prepared by the authors themselves using R⁴⁴ Software.

Higher F_v/F_m values observed in treatments using soil collected near mother trees indicate improved photochemical efficiency under these substrate conditions. This suggests that soil biological attributes influenced early physiological performance beyond chlorophyll accumulation alone^48–52.

Although treatment 10 also exhibited higher chlorophyll indices, the decoupling between chlorophyll content and F_v/F_m in some treatments highlights that pigment accumulation alone does not necessarily translate into improved photochemical efficiency, reinforcing the importance of soil-plant-microorganism interactions in determining early seedling physiological performance.

At 150 days after sowing, the highest survival rate of M. brauna seedlings (97.50%) was observed in treatments 3 and 10, both composed exclusively of soil collected near the mother tree (Fig. 4a). In contrast, seedlings cultivated in organic substrate (treatments 1 and 8) began to show drying symptoms and leaf abscission after 50 days, and complete mortality was recorded by 60 days. Although organic substrate treatments promoted greater early shoot expansion and leaf production at 40 days, these seedlings exhibited reduced structural balance, absence of mycorrhizal association, and complete mortality after 60 days, indicating that early morphological stimulation was not translated into long-term physiological stability or survival.

Fig. 4.

Survival, growth, and quality parameters of Melanoxylum brauna seedlings at 150 days after sowing under different substrate treatments. (a) Survival (%); (b) shoot length (cm); (c) shoot growth rate (cm); (d) collar diameter (mm); (e) collar diameter growth rate (mm); (f) number of leaves; (g) root length (cm); (h) ratio between shoot length and collar diameter; (i) shoot dry matter (g); (j) root dry matter (g); (k) Dickson Quality Index (DQI). ¹Different letters indicate significant differences among treatments according to the Scott–Knott test (p < 0.05). These figures were prepared by the authors themselves using Excel Software⁵⁴.

Shoot length was higher in treatments 5, 12, and 13, with treatments 5 and 12 sharing similar substrate proportions and differing only in seed origin and mother-tree soil origin (Fig. 4b). Collar diameter and root length were significantly higher in treatments using 100% soil collected near the mother tree (treatments 3 and 10) (Fig. 4d,g), indicating superior structural development. The number of leaves was also higher in treatment 3 and in treatment 6 (Fig. 4f).

Dry matter accumulation in both shoots and roots was significantly higher in treatment 3, which combined seeds from Laranja da Terra with soil collected near the Jerônimo Monteiro mother tree (Fig. 4i,j). Consequently, seedling quality, assessed by the DQI, was significantly higher in treatments 3 and 10 (Fig. 4k).

The superior growth and DQI observed in treatments based on mother-tree soils indicate improved structural balance and biomass allocation. These results are consistent with enhanced nutrient acquisition efficiency associated with arbuscular mycorrhizal presence in these soils^50–53.

At 150 days after sowing, the SPAD index, chlorophyll b content, and the maximum quantum yield of F_v/F_m were significantly higher in treatment 10, in which seedlings were cultivated exclusively in soil collected near the M. brauna mother tree (Fig. 5). These results indicate that the physiological advantages associated with mother-tree soils were maintained throughout seedling development.

Fig. 5.

Physiological parameters of Melanoxylum brauna seedlings at 150 days after sowing under different substrate treatments. (a) SPAD index; (b) chlorophyll index a; (c) chlorophyll index b; (d) maximum quantum yield of photosystem II (F_v/F_m). ¹Different letters indicate significant differences among treatments according to the Scott–Knott test (p < 0.05). These figures were prepared by the authors themselves using Excel Software⁵⁴.

The sustained increase in F_v/F_m at 150 days reinforces the association between soil origin and long-term physiological stability. Treatments containing mother-tree soils maintained greater photosynthetic efficiency, which was reflected in improved survival and growth^50–52.

At 150 days after sowing, biochemical analyses revealed significant differences among treatments in the shoot of M. brauna seedlings (Fig. 6). Lipid, protein, and starch contents in the shoot of seedlings from both seed origins were statistically higher in treatment 10 (Fig. 6a,d,e), whereas phenolic compounds were higher in treatment 3 (Fig. 6c), in which 100% soil collected near the mother tree was used.

Fig. 6.

Biochemical composition of shoots of Melanoxylum brauna seedlings at 150 days after sowing under different substrate treatments. (a) Lipids (%); (b) carbohydrates (mg g⁻¹); (c) phenolic (mg g⁻¹); (d) proteins (mg g⁻¹); (e) starch (mg g⁻¹); (f) fibers (%). ¹Different letters within each panel indicate significant differences among treatments according to the Scott–Knott test (p < 0.05). These figures were prepared by the authors themselves using Excel Software⁵⁴.

The higher accumulation of lipids, proteins, and starch in treatment 10 indicates favorable metabolic conditions associated with reserve formation and growth, reflecting improved physiological performance under biologically active soil conditions^55,56. In contrast, the predominance of phenolic compounds in treatment 3 suggests a metabolic shift toward defense-related pathways, which may be associated with abiotic constraints or biotic interactions within the rhizosphere⁵⁷. Together, these results demonstrate that shoot metabolic allocation in M. brauna seedlings is strongly influenced by substrate origin, with soils collected near mother trees promoting distinct biochemical strategies, either reserve accumulation or enhanced phenolic metabolism, both compatible with improved seedling performance at later developmental stages.

At 150 days after sowing, the biochemical composition of M. brauna seedling roots differed significantly among treatments (Fig. 7). Lipid content was statistically higher in treatment 12, with an average value of 35.49% (Fig. 7a). For carbohydrates and phenolic compounds, treatment 10 showed the highest average values, 44.90 mg g^− 1 and 4.11 mg g^− 1, respectively (Fig. 7b,c), both using soil collected near the mother tree.

Fig. 7.

Biochemical composition of roots of Melanoxylum brauna seedlings at 150 days after sowing under different substrate treatments. (a) Lipids (%); (b) carbohydrates (mg g⁻¹); (c) phenolic (mg g⁻¹); (d) proteins (mg g⁻¹); (e) starch (mg g⁻¹); (f) fibers (%). ¹Different letters within each panel indicate significant differences among treatments according to the Scott–Knott test (p < 0.05). These figures were prepared by the authors themselves using Excel Software⁵⁴.

These contrasting biochemical patterns indicate that root metabolic allocation is highly responsive to substrate composition. Soil microbial systems can influence the persistence and dynamics of allelochemicals, including phenolic compounds, thereby modulating plant metabolic profiles within root-associated environments⁵⁸. Such biologically mediated adjustments help shape root biochemical status in seedlings grown in mother-tree soils and contribute to the physiological stability reflected in higher survival, vigor, and photochemical efficiency observed in the best-performing treatments (Figs. 4 and 5).

These patterns suggest a trade-off between metabolic investment in growth-related reserves and defense-related compounds, in which improved photochemical efficiency reduces metabolic costs and favors carbon allocation toward structural and storage compounds rather than stress mitigation^55,56,58.

Treatments 1 and 8, which used organic substrate, presented statistically higher concentrations of P, K, S, Ca, Mg, Zn, B, and Na, as well as higher organic matter (OM) content and higher soil basal respiration rate (SBR) (Table 2). Despite this greater nutrient availability, the survival of M. brauna seedlings was higher in treatments 3 and 10, which used 100% soil collected near the mother tree, with pH values ranging from 6.5 to 4.6, respectively (Fig. 4). These results indicate that seedling survival was not directly related to the total nutrient content of the substrate.

Table 2.

Chemical, physical, and basal respiration characteristics of the soils and substrates used in the different treatments for the production of Melanoxylum brauna seedlings.

Treat.	P	K	S	Ca	Mg	Al	H + Al	RBS	pH	OM	Fe	Zn	Cu	Mn	B	Na	SB	t	T	Sand	Silt	Clay
	mg dm− 3			cmolc dm− 3				C–CO2 mg kg−1h− 1		dag kg− 1	mg dm− 3						cmolc dm− 3			g kg− 1
1 and 8	197.5 a1	547.5 a	201.0 a	8.7 a	2.5 a	0.0 f	3.7 c	0.6 a	5.8 b	15.8 a	113.5 h	7.1 a	0.6 a	79.8 b	0.7 a	46.8 a	13.0 a	13.0 a	16.5 a	408.5 h	536.5 a	65.0 e
2 and 9	7.7 b	40.0 c	20.2 b	2.7 c	0.6 b	0.0 f	1.6 f	0.3 b	6.3 a	0.5 e	37.0 j	2.2 e	0.2 c	29.0 h	0.2 f	16.0 e	3.6 c	3.5 c	5.1 d	382.5 i	351.5 b	275.0 a
3	7.5 b	55.0 b	13,0 c	3.4 b	0.5 c	0.0 f	1.9 f	0.3 b	6.5 a	2.2 c	183.5 b	0.6 g	0.3 c	37.3 g	0.2 f	7.5 f	4.1 b	4.1 b	6.3 c	727.0 b	96.8 e	210.0 b
4	4.2 d	28.7 c	13.2 c	1.4 e	0.3 d	0.0 f	1.7 f	0.3 b	5.7 b	1.1 d	221.5 a	0.8 g	0.3 b	89.0 a	0.2 f	4.8 g	1.7 e	1.7 d	3.6 f	758.0 a	111.0 e	138.8 d
5	7.0 b	33.2 c	17.2 b	1.8 d	0.3 d	0.0 f	1.8 f	0.3 b	5.7 b	1.0 d	124.0 g	0.7 g	0.7 a	62.0 d	0.1 g	20.3 c	2.2 d	2.2 d	4.1 f	660.0 e	162.5 c	170.0 c
6	8.5 b	39.0 c	13.5 c	1.5 e	0.4 d	0.0 f	2.0 e	0.3 b	5.7 b	1.0 d	167.0 c	1.1 f	0.3 c	87.5 a	0.2 f	10.8 f	2.0 d	2.0 d	4.0 f	675.5 d	135.5 d	182.5 c
7	7.2 b	44.5 b	11.5 c	1.5 e	0.5 c	0.0 f	2.1 e	0.3 b	5.7 b	1.6 d	163.0 d	1.0 f	0.4 b	86.5 a	0.2 f	9.3 f	2.0 d	2.0 d	3.9 f	656.0 e	157.5 c	220.0 b
10	2.7 d	34.2 c	6.2 c	0.4 h	0.2 e	1.0 a	6.6 a	0.3 b	4.6 d	3.1 b	3.2 k	4.8 b	0.0 d	68.5 c	0.5 b	18.5 d	0.8 g	1.8 d	7.4 b	584.0 g	132.5 d	275.0 a
11	4.0 d	33.7 c	9.5 c	0.6 h	0.3 e	0.7 b	5.5 b	0.3 b	4.8 d	2.8 b	89.3 i	4.2 c	0.1 d	63.1 d	0.4 c	20.3 c	0.9 g	1.6 d	6.4 c	586.0 g	129.8 d	229.8 b
12	5.2 c	40.7 c	12.2 c	0.9 g	0.5 c	0.5 c	3.6 c	0.3 b	5.3 c	2.1 c	122.3 g	3.3 d	0.2 c	52.8 e	0.4 c	21.5 c	1.3 f	1.8 d	5.1 d	624.5 f	122.8 d	190.8 c
13	5.7 c	50.2 b	11.7 c	1.1 f	0.4 d	0.4 d	2.8 d	0.3 b	5.5 c	1.5 d	136.3 f	1.3 f	0.3 c	43.9 f	0.3 e	23.3 b	1.6 e	1.7 d	4.3 e	708.0 c	124.5 d	170.0 c
14	5.7 c	50.0 b	11.2 c	1.2 f	0.2 e	0.3 e	2.2 e	0.3 b	5.5 c	1.5 d	140.2 e	1.2 f	0.2 c	44.5 f	0.3 e	24.0 b	1.7 e	1.8 d	4.4 d	701.8 c	120.0 d	176.8 c

Treat., treatment; OM, organic matter; SB, sum of bases; t, effective cation exchange capacity; T, cation exchange capacity at pH 7.0; RBS, basal soil respiration. ¹Means followed by the same letter in the column do not differ by the Scott-Knott test (p < 0.05). Four samples were evaluated per treatment (n = 4).

Soils collected near the mother tree, regardless of location, were characterized by high acidity, low fertility, and low organic matter content. Low pH values can negatively affect biological processes and nutrient cycling, particularly for elements such as phosphorus, nitrogen, and sulfur^59,60. However, these limitations may be mitigated by biological interactions in the rhizosphere. Many soil fungi contribute to nutrient uptake^22,61, establish symbiotic associations with plant roots²³, and provide protection against soil pathogens⁶², which may explain the higher survival observed in treatments 3 and 10 despite lower nutrient concentrations.

Together, these results indicate that seedling performance was primarily influenced by biological soil attributes rather than total nutrient availability, supporting the role of mother-tree soils as functional bioinoculants.

The arbuscular mycorrhizal fungi (AMF) community associated with soils collected near M. brauna mother trees was composed of the genera Glomus, Sclerocytis, and Gigaspora, among which was the species Glomus macrocarpum (Table 3). These fungal taxa were detected exclusively in soils collected in the presence of roots, whereas no AMF spores were observed in soils from the remaining treatments.

Table 3.

Number of spores from the different treatments regardless of the origin of the soil and the Melanoxylum brauna seeds.

Treatments	Number of spores
Organic substrate	0 b1
Subsoil	0 b
Mother-tree rhizospheric soil	29 a
Mixture	0 b

¹Averages followed by the same letter in the column within each variable, between the different treatments, do not differ by the Tukey test (p < 0.05). *Sand (33%) + subsoil (32%) + soil near the mother tree with roots (33%) + ground branches (2%).

The occurrence of these AMF genera indicates a biologically active rhizospheric environment shaped by long-term plant-microorganism interactions. Species belonging to Glomus and Gigaspora are widely recognized for their role in enhancing nutrient uptake, improving plant growth, and modulating physiological responses in woody species^22,61. In addition, symbiotic associations formed by these fungi contribute to plant protection against soil-borne pathogens and environmental stress^23,62.

The exclusive presence of AMF in soils collected near mother trees reinforces the importance of these substrates as sources of beneficial microbial inoculum, which is consistent with the superior survival, growth, and seedling quality observed in treatments based on mother-tree soils, although root colonization was not directly assessed.

The abundance of arbuscular mycorrhizal fungi (AMF) spores was significantly higher in soils collected near the M. brauna mother tree, regardless of geographic origin (LT or JM). In contrast, soils from the remaining treatments showed no detectable AMF spores, indicating a strong dependence of mycorrhizal occurrence on the rhizospheric environment of mother trees.

This pattern reflects the long-term interaction between M. brauna roots and soil microorganisms, which shapes the biological structure of the rhizosphere. Arbuscular mycorrhizal fungi play a key role in nutrient acquisition, physiological regulation, and plant development, particularly under conditions of low soil fertility and high acidity^21,22,61. In addition, these symbiotic associations contribute to plant protection against soil-borne pathogens and environmental stress^23,62.

The higher abundance of AMF spores observed in mother-tree soils indicates a greater availability of mycorrhizal propagules in these substrates. This pattern is consistent with the superior survival, growth, and physiological performance of M. brauna seedlings cultivated in these treatments; however, as root colonization was not directly quantified, the potential contribution of AMF remains inferential. Rhizospheric soils may therefore function as reservoirs of diverse beneficial microorganisms, whose combined biological effects could have supported seedling establishment and performance.

Survival, shoot length, root length, collar diameter, and number of leaves showed a strong positive correlation with the maximum quantum yield of F_v/F_m, with correlation coefficients of 0.88, 0.90, 0.96, 0.92, and 0.95, respectively (Fig. 8). The increase in F_v/F_m was consistently associated with higher values of these morphological and survival-related variables, indicating a strong interaction between physiological performance and seedling development.

Fig. 8.

Pearson’s correlation coefficient between growth and physiological variables of Melanoxylum brauna seedlings after 150 days in the different treatments. SUR, survival (%); SL, shoot length (cm); CD, collar diameter (mm); NL, number of leaves; R, root length (cm); SL/CD, ratio between shoot length and collar diameter; DMS, dry matter of the shoot (g); DMR, dry matter of the root (g); DQI, Dickson quality index; SPAD, SPAD index; CHLA, chlorophyll a index; CHLB, chlorophyll b index. These figures were prepared by the authors themselves using R⁴⁴ Software.

These correlations demonstrate that photochemical efficiency plays a central role in determining seedling vigor and structural development. The close association between F_v/F_m and growth-related parameters supports the interpretation that seedlings cultivated in soils collected near the mother tree achieved greater physiological stability, which was directly reflected in improved survival and morphological traits. Such integration between physiological and morphological responses highlights the importance of soil-plant-microorganism interactions in sustaining plant performance under contrasting substrate conditions^28,63.

The strong coupling between photosynthetic efficiency and growth variables reinforces the role of the rhizospheric environment as a regulatory system influencing multiple physiological pathways. Similar integrative responses have been reported for woody species, in which interactions with soil microbiota contribute to coordinated metabolic and developmental regulation⁶⁴.

The integration of morphological, physiological, biochemical, and microbiological variables highlights the strong influence of soil origin on the performance of M. brauna seedlings (Fig. 9). Soils collected near mother trees (treatments 3 and 10) differed from commercial organic substrates primarily by the presence of arbuscular mycorrhizal fungal propagules (29 spores per 100 g^− 1) and by distinct chemical attributes, including pH, organic matter content, and soil basal respiration. These conditions resulted in seedlings with higher photosynthetic performance (F_v/F_m), greater vigor, and increased reserve accumulation, as reflected by higher DQI values (Fig. 9).

Fig. 9.

Integrated model of how mother-tree soil and its microbiota enhance physiological, biochemical, and survival traits of Melanoxylum brauna seedlings. AMF, Arbuscular mycorrhizal fungal; SUR, survival; DQI, Dickson quality index; SL, shoot length; CD, collar diameter; NL, number of leaves; R, root length; OM, organic matter; SBR, soil basal respiration rate. Bars: 1 cm. These figures were prepared by the authors themselves using R⁴⁴ Software, Excel Software⁵⁴, and Inkscape 0.92.5 Software³¹.

Seedlings cultivated in soils collected near the M. brauna mother tree, regardless of geographic origin, exhibited higher seedling quality, characterized by greater morphological balance and more expanded, greener leaves. In contrast, seedlings grown in organic substrate showed progressive drying of the stem apex and leaf tips after 40 days, followed by complete mortality at 60 days of cultivation (Fig. 9).

These integrated responses indicate that seedling performance was influenced not only by nutrient availability, but also by combined soil chemical and biological attributes. Although higher AMF spore density and distinct taxa were detected in soils collected near mother trees, root colonization in seedlings was not directly quantified. Therefore, the proposed AMF-mediated effects on physiological stability and metabolic balance should be interpreted as a plausible inference based on inoculum presence rather than as direct evidence of symbiotic establishment. Comparable responses have been reported in other woody species; for example, Toprak et al.⁶⁵ demonstrated that indigenous arbuscular mycorrhizal fungi improved biomass accumulation and nutritional status of Elaeagnus angustifolia and Robinia pseudoacacia seedlings under nursery conditions. In addition, other soil biological components, including potential nitrogen-fixing or plant growth, promoting microorganisms, may have contributed to the observed responses, as these were not specifically evaluated in the present study. Thus, soils collected near mother trees likely function as sources of native microbial communities that support seedling establishment and resilience, reinforcing their potential application in conservation and restoration strategies for this vulnerable forest species^28,66.

Conclusions

Soil origin was the main factor determining the survival and quality of Melanoxylum brauna seedlings. Soils collected near mother trees consistently promoted higher survival, improved morphological balance, and better physiological performance, whereas organic substrate did not sustain seedling establishment under nursery conditions. These results indicate that rhizospheric soils function as biologically active substrates that support seedling development, likely due to their native microbial communities. Therefore, the use of soil collected near mother trees represents a viable strategy for improving the production and conservation of this vulnerable forest species.

Author contributions

Conceptualization Ingridh Medeiros Simões and Rodrigo Sobreira Alexandre; Data curation: Ingridh Medeiros Simões and Rodrigo Sobreira Alexandre; Formal analysis: Ingridh Medeiros Simões, Edilson Romais Schmildt; Funding acquisition: Rodrigo Sobreira Alexandre; Investigation, Methodology: All authors; Supervision: Ingridh Medeiros Simões; Writing – original draft and review & editing: All authors.

Funding

This work was supported by the Coordination for the Improvement of Higher Education Personnel  (CAPES)/ Espírito Santo Research and Innovation Support Foundation (FAPES) - Postgraduate Development Program (PDPG), to the project “Consolidation of Graduate Programs in the area of Agricultural Sciences in the State of Espírito Santo - Forestry Sciences” for the financial support of the research, through FAPES/CNPq Notice N° 23/2018 - PRONEM (Grant Term 131/2021 and Process N° 2021-FDGS5) and, FAPES Notice N° 21/2022 Support For Research Infrastructure, Development And Innovation In Interdisciplinary Laboratories.

Data availability

Data will be made available upon request to Tamyris de Mello (tamyrisdemello@gmail.com) or Rodrigo Sobreira Alexandre (rodrigosobreiraalexandre@gmail.com).

Declarations

Competing interests

The authors declare no competing interests.