Lupine Cultivation Affects Soil’s P Availability and Nutrient Uptake in Four Contrasting Soils

Abstract

A substantial amount of phosphorus (P) in the soil is not readily available for plant uptake. Certain species may enhance P availability from poorly soluble P forms. This study focused on improving our comprehension of the effect of two lupine species (L. albus and L. angustifolius) on soil’s P mobilization and its link with soil acidity variations, comparing the response of the lupine species in terms of plant traits (i.e., aboveground biomass and nutrient uptake) with that of oats (Avena strigosa L.) in four contrasting soils (i.e., available P in soil, soil acidity, soil fertility, and texture). The phosphorus solubilization capacity was assessed on variations of P availability (PBray1) at four points in time, comparing soils with lupine to oat-containing soils and their baseline values. Compared to soils containing oats, at harvest, lupine soils had significantly increased PBray1 concentrations; the maximum average increment was around 5.3 mg kg⁻¹, with L. albus in Sites 1 and 2, which presented higher organic matter (OM) contents than the other two sites. Lupine-induced soil acidification did not fully explain that P increase. Oats exhibited the highest increase in shoot dry weight in response to soil’s P availability, while lupine was the least affected. Nevertheless, L. albus showed similar or higher nutrient uptake than oats across all soils. The manganese (Mn) concentration was high in both lupine species’ shoot biomass; however, within each lupine species, across all soil types tested, these legumes had different Mn accumulation levels depending on the soil acidity. Lupinus albus had a higher ability to mobilize non-labile P in the light-textured soil with a high OM content, achieving comparable and higher plant P status than oats and providing N through biological N fixation (BNF), positioning it as a suitable crop for diversifying Uruguay’s agricultural crop rotation systems.

1. Introduction

The adoption of continuous cropping (CC), primarily soybean (Glycine max L. Merr.), has increased in Uruguayan agricultural systems since the early 2000s [1], replacing the traditional crop–pasture rotation [2,3]. While grazing persists in agricultural systems, its duration within the rotation has been significantly reduced. According to the Ministry of Livestock, Agriculture and Fisheries of Uruguay, the 2021 agricultural survey [4], estimated that pastures associated with wheat cropping constituted less than 8% of the total area that was planted with this winter crop. In the past 15 years, the proportion of pastures associated with winter crops has significantly decreased, from 28% in 2005 to 4% in 2021. Despite the increase in grain production due to pasture phase exclusion, recent studies have confirmed persistent wheat yield gaps in Uruguay [3], which cannot be explained by nutrient deficiency and remain unaddressed by correcting nutrient deficiencies. The issue is exacerbated because the soil quality has been compromised, which affects the sustainability of the system [2,5].

In this scenario, there is significant concern regarding the impact of a permanent negative nitrogen (N) balance in the CC rotation; thus, balancing N within this system becomes necessary [6]. In contrast to crop–pasture rotation, the CC systems have experienced an increase in the quantities of both N and P fertilizers due to agricultural intensification. However, for P in particular, due to the accumulated yield’s increased extraction of P from the soil, the amount of remaining P decreased, especially when highly extractive crops were the main components of the sequences in the rotation, such as soybean and corn [7]. Moreover, applying P fertilizer above crop requirements leads to a slightly positive soil P balance, as P gradually accumulates in the soil over time [7]. This accumulation is affected by the low P use efficiency in most crops, which typically ranges from 15 to 30% [8].

Although the majority of residual P is scarcely assimilable by plants, certain species may be able to make this P available [9,10]. The lupine genus may be able to mobilize residual P through root exudates, releasing phosphatases [11], acidifying [12], and chelating compounds or carboxylates [13,14,15], which can enhance their growth and possibly that of subsequent crops [16,17]. However, this genus has not yet been incorporated into crop rotations in Uruguay, which typically favor winter cereal crops like wheat or barley and summer crops such as soybean or maize. Additionally, further investigations [18] on lupine cultivation and its effects on soil nutrient dynamics must to be conducted.

Accordingly, for competitive crop production, the integration and diversification of functional groups—such as annual winter legumes like lupine—may be a key strategy to reverse soil deterioration processes [19,20,21]. This lupine genus is recognized for its high potential as a pulse crop in Australia, Chile, and other countries for a variety of production purposes [17,22,23,24]. Integrating crops that are capable of N fixation from the atmosphere and P solubilization from unavailable forms provides an alternative to excessive or improper fertilizer applications. Moreover, the root activity of this annual winter legume contributes to soil OM as a carbon source and enhances soil’s N content through its N-fixing ability, while also improving physical, chemical, and biological soil properties.

Given the growing interest in diversifying crops within agricultural systems and reducing fertilizer use, there is a timely opportunity to evaluate lupine cultivation. Species such as white lupine (L. albus) and narrow-leaf lupine (L. angustifolius) are renowned in regions of Australia and Chile as N fixers and P solubilizers, particularly in mildly acidic or neutral soils of light-to-medium texture [25]. Consequently, we hypothesize that the availability of plant P in contrasting soils (pH, texture, OM, and soil P concentration) increases by acidification of the soil surrounding roots by those lupine species, enhancing the solubility of P from sparingly available soil P sources. This enhanced plant P availability is expected to improve the dry matter (DM) yield and the P and N biomass content of lupine, potentially having a residual effect on subsequent crops.

This study focused on improving our comprehension of the impacts of two lupine species (L. albus and L. angustifolius) on the mobilization of P in soil and its association with soil acidification, comparing the response of aboveground biomass productivity and nutrient (i.e., P, N, base cation, Mn, and Fe) uptake effectiveness to these lupine species with the response to oats (Avena strigosa) in four contrasting (i.e., available P in soil, soil acidity, soil fertility, texture) soil types. Hence, the aims were to assess the changes in soil’s P availability and its link with soil acidification and to establish which type of lupine has a more substantial P solubilization capacity and which soil–lupine combination yields the most effective P mobilization effect.

2. Material and Methods

2.1. Soil Collection and Preparation Prior to Experiment Installation

Soil samples were collected from the topsoil layer (0–20 cm depth) from four Uruguayan agroecological areas under different soil uses with the following geographic coordinates: agricultural—Site 1 (33°59′05.4″ S and 57°43′42.7″ E); livestock grassland—Site 2 (32°49′1.20″ S and S 54°25′28.27″ O); grassland—Site 3 (34°50′15.61″ S and 56°13′21.62″ O); forestry—Site 4 (31°23′43.44″ S and 55°41′39.37″ O). The sites have contrasting soil textures, pH values, organic matter (OM) levels, and P concentrations (PBray1). Table 1 displays the collection sites and USDA soil classification [26] for each of the four soils analyzed in this study; these references (Site 1 to Site 4) were used throughout the text to identify each soil treatment.

Before planting, each soil was sieved through a 1 cm mesh sieve to homogenize the size of the aggregates and discard coarse plant material. After this screening, for each soil type, the granulometric composition (texture) and the chemical characterization (i.e., PBray1, inorganic N forms, exchangeable bases, exchangeable Al, and soil pH) were determined (Supplementary Materials, Table S1).

2.2. Greenhouse Experiment

2.2.1. Experimental Design and Plant Growth Conditions

A pot experiment was conducted under natural light conditions in a greenhouse at the Agronomy College in Montevideo (34°50′18.20″ S, 56°13 ′16.36″ W) to evaluate the efficacy of two lupine species in enhancing soil’s P availability. A total of 48 three-liter pots (15 cm in diameter) were filled with the different soil types. The treatments were arranged in a factorial design with two factors, four soil types and three species, in a completely randomized design (CRD) with four replications.

The evaluated species were Lupinus angustifolius L. var. Lavalle, a non-cluster root-forming lupine (narrow leaf lupine or blue lupine), and L. albus L. Blu25, a cluster root-forming legume (white lupin). Additionally, an annual grass, black oat (Avena strigosa L., var. Agroplanalto), was included in the study as a comparative measure to assess the increase in soil P that was induced by lupines and to evaluate BNF. The seed company Fadisol S.A. (Montevideo, Uruguay) supplied all seed species that were utilized in this study, constituting those of lupine, the single plant material available by Fadisol S.A. during the research period of the current work. The white lupine accession was provided by Seeds Baer (Temuco, Chile), a breeder who conducted formal identification and permitted using it for research purposes; blue lupine and black oat were developed and protected by Fadisol S.A. Oats were chosen as the control in our study because they are the most widely cultivated and regionally adapted cover crops in Uruguay due to their precocity and high growth rate during the winter season [27]. Furthermore, according to Wang et al. [28], this genus would be a good candidate for evaluating soil’s P utilization, given its extraordinary cover’s fibrous root system promoting key roles, such as preventing erosion and scavenging excess nutrients [29]. Both lupine species were inoculated with a non-specific commercial inoculant (Bradyrhizobium sp., strains U-612, and U620), known to be effective for these species were provided by Lage y Cía Company (Montevideo, Uruguay). The planting date was 10 July 2019, with two or three lupine seeds per pot, leaving only one plant per pot after emergence. There were five oat plants per pot.

Plants were irrigated using potable water, supplemented once a week during the first month of the experiment with a nutrient solution that contained (μM): KCl, 200; CaCl, 150; MgSO₄, 100; H₃BO₃, 2.0; MnSO₄, 0.4; ZnSO₄, 0.4; and CuSO₄, 0.2, Na₂MoO₄, 0.05; Fe Edta, 0.005, free of N and P and using 200 mL per pot. Throughout the trial, each pot was maintained around 60% of its field capacity (FC), estimated by gravimetric determination and considering the water content at FC (based on mass) of each soil type (35, 30, 28, and 13% for Sites 1 to 4, respectively). The experiment was conducted from June to October 2019, encompassing the mid-winter and early spring seasons. The average temperatures during this period varied between 8 and 25 °C. There was no need to control weeds and pests during the experiment.

2.2.2. Soil and Plant Sampling

Soil samples were collected at four different time points: 48, 76, 87, and 103 days after planting (dap). The initial soil samples were obtained on 9 September 2019, two months after the start of the experiment, when the crop was in the vegetative stage. The second sampling took place on 2 October 2019, during flowering stage, the third sampling on 14 October 2019, at the beginning of the lupine grain filling, which was the harvest time, and the final sampling on 30 October 2019. The soil pH was measured on 9 September and 14 October 2019. The soil’s exchangeable acidity was assessed to confirm any connection between changes in the solubility of inorganic P or Al and soil acidity. A hand drill, 1.9 cm in diameter, was used to collect a single soil sample from each pot, reaching a depth of 0 to 10 cm.

The plants were harvested at 87 dap at ground level using pruning shears to remove all aerial biomass per pot. At this sampling time, the oat plants had reached full maturity, while L. angustifolius was in the initial phase of grain filling, and L. albus exhibited pods in an early stage of development. Compared to L. albus, L. angustifolius had more mature pods, which accounted for around ten percent of the total P absorbed in the aboveground biomass. Consequently, the study separated the analytical determination of L. angustifolius pods from the remaining aerial plant parts. However, except for P concentration, the other analyses on these pod components were not achievable due to the sample size being insufficient for accomplishing this. The shoot dry weight was expressed per pot (g pot⁻¹), and in some instances, it was converted to its equivalent per hectare (kg ha⁻¹) based on the pot’s surface area.

2.3. Sample Processing and Analytical Determinations

2.3.1. Soil Measurements

The soil samples were dried in a forced-air oven at 40 °C for at least 48 h and were ground to a 2 mm sieve size. For the initial characterization, the determined parameters were granulometric composition, organic matter (OM), pH, labile P, mineral nitrogen (ammonium and nitrate), exchangeable cations (Ca, Mg, K, and Na), and exchangeable acidity. OM was determined by the Walkley–Black method [30], while exchangeable cations Ca and Mg were determined by atomic absorption and K and Na by flame spectrophotometry, following extraction with 1 M ammonium acetate at pH 7 [31]. Soil pH was measured in deionized water (1:2.5 soil/deionized water ratio) using a pH probe (Orion Research 701 pH electrode), while exchangeable acidity was determined using the potassium chloride method [31]. The labile P or available P content was measured using the Bray N° 1 (henceforth PBray1), method extraction system [32], which is the most widely used method in Uruguay for evaluating plants’ P availability in most agricultural soils in the country [33]. Nitrate–N (NO₃-N) concentration was determined using the Griess–Ilosvany method [34], and ammonium–N (NH₄-N) was determined using the colorimetric method [35]. Phosphorus, ammonium, and nitrate readings were taken in a spectrophotometer at 880, 650, and 540 nm, respectively, using the MRC microplate reader for the first two elements and the UNICAM spectrophotometer for nitrate. The hydrometer method [36] was employed to analyze the granulometric soil composition. During the growing season, soil samples were analyzed for PBray1 and pH using the same techniques described for soil characterization analysis.

2.3.2. Plant Measurements

The harvested plants were oven-dried at 65 °C for a minimum of 48 h until the mass remained constant. Then, initially, dried plant materials were ground with a stationary and mobile knife mill (Marconi MA-580) until the particulate size was less than 2 mm. Plant samples of this granulometric size were analyzed for total P and K contents. Afterward, previously ground subsamples were newly ground by a rotary mill (SampleTek 200 vial Rotator) to a fine powder (typically a consistency approaching that of the talcum powder), which was necessary for ¹⁵N analysis by mass spectrometry. Total C and N concentration and ¹⁵N/¹⁴N of the samples (at the natural abundance) were determined by mass spectrometry in a US laboratory “https://csi.unm.edu, accessed on 10 November 2020”). The following formula by Shearer and Kohl [37] was used to calculate the proportion of N fixed from the air (% Ndfa) for each lupine species:

$\mathbf{N}\mathbf{d}\mathbf{f}\mathbf{a}\left(\mathbf{\%}\right)=\left(\frac{{\mathsf{\delta }}^{\mathbf{15}}{\mathbf{N}}_{\mathbf{r}\mathbf{e}\mathbf{f}}-{\mathsf{\delta }}^{\mathbf{15}}{\mathbf{N}}_{\mathbf{f}\mathbf{i}\mathbf{x}}}{{\mathsf{\delta }}^{\mathbf{15}}{\mathbf{N}}_{\mathbf{r}\mathbf{e}\mathbf{f}}-\mathbf{B}}\right)\times \mathbf{100}$

where the following abbreviations are used:

• Ndfa is the proportion of plant N derived from BNF;

• δ¹⁵Nref is the δ¹⁵N value of the reference plant (non-fixing);

• δ¹⁵Nfix is the δ¹⁵N value of the fixing plant (lupine);

• B is the δ¹⁵N value of a fixing plant growing in a medium without N.

The B value was estimated as the mean δ¹⁵N value from pure lupine growing in the sand, with a value of +1.6‰ and −0.6‰ of δ¹⁵N for L. albus and L. angustifolius, respectively. The reference plant used was oat, and all δ¹⁵N values were determined under the same conditions as lupine legumes.

The total concentrations of P and potassium (K), calcium (Ca), magnesium (Mg), Mn, and iron (Fe) were also determined after calcination at 550 °C for 5 h. Phosphorus concentration was determined using the ascorbic acid method [38] after extraction with diluted HCl (20% v/v) [39]. In the ash extracts, the remaining elements (Ca, Mg, K, Fe, and Mn) were determined by spectrometry as described for soil samples. Shoot’s P and N contents per pot (mg pot⁻¹) were calculated by P or N concentrations (mg g⁻¹ dw) × shoot dry weight (g pot⁻¹), respectively. In certain instances, the plant nutrient content was also expressed per hectare, considering the pot’s surface area.

2.4. Statistics

The changes in soil PBray1, at four points in time, were evaluated by repeated measures analysis of variance using a MIXED procedure of SAS (between-subject factors were species and soil type, and within-subject factor was sampling time). Additionally, two-way ANOVAs performed at two time points (i.e., at 87 and 103 dap) were used to study the factors of interest (i.e., soil, species, and their interaction as fixed effects) on the evaluated soil and plant variables. Datasets that did not follow assumptions of normality of residuals and homogeneity of variance were log10 transformed. Two additional soil variables were estimated: 1: the difference between PBray1 (and pH) at a specific sampling time and the corresponding parameter, measured at the beginning of the experiment, is denoted as ∆PBray1−initial and ∆pH−initial, respectively; 2: for each soil parameter (PBray1 and pH values), the difference between the soil samples containing lupine and those containing oats (∆PBray1__Lup-Oat and ∆pH__Lup-Oat) was estimated.

The further analyses included orthogonal contrasts (C1: lupine vs. oat, C2: L. albus vs. L. angustifolius) that were performed to identify differences between groups of treatments and Pearson correlation and linear regression to describe and explain the association between soil and plant variables. A Tukey’s test with a confidence level of 5% was used to compare the means of treatments between species, across soils, and within each soil type. All analyses were conducted using SAS statistical software version 9.04 (SAS Institute) and R software (version 4.0.4) [40].

3. Results

3.1. Species, Soil, and Sampling Time Effects on PBray1 Concentration

The effects of species and soil type were highly significant (p = 0.001) on the soil’s PBray1, but their interaction was not (Table 2). Considering the effect of species, L. albus had the highest mean values (Figure 1), while concerning the soil factor, its effect on PBray1 reflected, on average, the initial P concentrations of each soil, and this was observed as early as 76 dap, after which the concentration tended to stabilize.

The C1 (lupine vs. oat) was significant at 48 dap, but only in the soil of Site 1 (p = 0.0029), whereas C2 (L. albus vs. L. angustifolius) was significant in Site 1 and Site 3 (p = 0.0028 and p = 0.0445, respectively). At 76 dap, both contrasts were no longer statistically significant in any of the soils, indicating that the differences between species diminished. At harvest (87 dap), C1 was significant in Site 3 (p = 0.0331), Site 4 (p = 0.029), and Site 2 (p = 0.0001), but differences between lupine species were not detected. Fourteen days after harvesting (103 dap), C1 was highly significant in all the soils, while C2 was significant in all soils except Site 4, where no differences in PBray1 between the lupine species were observed.

At harvest time (87 dap), the two-way ANOVA showed significant effects of the main factors, species and soil, but not of their interaction (Supplementary Materials, Table S2). The contrast analysis between lupine vs. oats (C1) was significant, the estimated difference being 2.6 mg kg⁻¹, while the contrast between the lupine species (C2) was not significant on this sampling date. Multiple comparisons of means using the Tukey method revealed a significant difference between the lupine species, with higher values for L. albus. For this sampling time, the difference in PBray1 compared to the initial values was almost null in the soils with lupine; with oats, meanwhile, there was a mean decrease of 2.8 mg kg⁻¹. At 87 dap, there were also statistically significant differences in the concentrations of PBray1 between soils, primarily reflecting the P analysis values of the tested soils before the start of the experiment. The concentration of PBray1 did not differ statistically between Site 1 and Site 2. However, the P levels in these soils were substantially higher and statistically different from those of the other soils (p = 0.05). In the heavy-textured soils and Site 4, the ∆PBray1-initial was minimal in this sampling period; however, Site 2 had a mean decrease of 4.0 mg kg⁻¹. Additionally, a significant percentage increase in PBray1 of 57% was observed in the loamy soil containing lupine relative to this soil containing oat, reaching 4.6 mg kg⁻¹.

At 103 dap, a significant effect of the main factors (p < 0.0001) but also of the species x soil type interaction (p = 0.0048) was detected (Figure 2; Supplementary Materials, Table S3). The C1 was significant in all the soils, the difference being 4.0, 3.6, 3.6, and 2.2 mg kg⁻¹ in Site 2, Site 1, Site 4, and Site 3, respectively. The C2 was also significant in three soils, especially for L. albus in Site 1 and Site 2, with a higher P content. Site 3 was the other soil with a significant difference between the lupine species. In this soil, however, L. angustifolius stood out over L. albus, with a difference from the oat of 3.4 mg kg⁻¹; meanwhile, the difference between L. albus and oats was statistically non-significant and estimated at 0.9 mg kg⁻¹. In summary, the ∆PBray1__Lup-Oat, depending on the lupine species and the soil, ranged from 0.9 (non-significant difference) to 5.4 mg kg⁻¹ (p < 0.0001). In addition, a significant difference was found between both species of lupine to oats only in light-textured soils (Site 2 and Site 4). In the other soils, meanwhile, that difference was significant only in one species of lupine. This was L. albus in Site 1 and L. angustifolius in Site 3.

3.2. Species and Soil Type Effects on Soil pH

The pH of the soil at harvest differed considerably between soils with lupine and those with oats, by 0.5 pH units on average (Figure 3). Compared to the pH values at 48 dap, the values were lower at harvest, although this decrease was only observed in soils with lupine (0.1 pH unit) and not in soils with oat. When the soil’s pH at harvest was compared with the initial pH of each soil, it was found that oats caused a mean 0.25 units of pH increase, while both species of lupine caused a decrease in pH, but without statistically significant differences. The effect of soil on pH changes was also highly significant, with an average increase of 0.57 units in Site 4 and a decrease of a similar magnitude (−0.62 units) in Site 2. The species x soil type interaction on soil pH measured at harvest was significant (p = 0.0365; Supplementary Materials, Table S4). As shown in Figure 3, oat-containing soils consistently recorded the lowest acidity levels. In addition, the soil pH of this species differed significantly from that of lupine in all soils except Site 1, where the differences between species were not statistically significant. Based on the statistical differences between soils across species, Figure 3 reveals that Site 2 had the lowest soil pH at harvest, while Site 3 had the highest (coincidentally with the highest initial soil pH).

At 87 dap, in Site 3, Site 4, Site 2, and Site 1, the ∆pH__Lup-Oat was −1.2, −1.1, −1.0, and −0.6 units, respectively. According to the contrast analysis, the difference between the pH values of lupine and oat (C1) was statistically significant in all soils. The C2 was only significant in Site 3, with L. angustifolius standing out with a decrease in pH of −0.6 units from the initial value. Figure 3 also shows that the ∆pH−initial fluctuated within a more negative range for soils with lupine (−0.8 to 0.5) and a more positive range for soils with oat (−0.3 to 0.9). Likewise, the exchangeable acidity values corresponded to those of the pH; when the pH decreased, the soil acidity increased. However, this increase in acidity was not associated with exchangeable aluminum but rather with an increase in the hydrogen ion concentration. On average, the highest increase in exchangeable aluminum was 0.14 cmol_c kg⁻¹ at 87 dap in Site 2; this concentration would not impose production limitations (Table 3).

Additionally, sandy soils at Sites 2 and 4 displayed more significant pH fluctuations. At Site 2, where the ∆pH−initial was the most pronounced, the difference in exchangeable acidity between soil holding lupine and soil holding oats was most noticeable and statistically significant. In contrast, at Site 4, the exchangeable acidity with oat-bearing soil was only statistically distinct from soil containing L. albus. On the other hand, the exchangeable acidity between species was not statistically significant at Sites 1 and 3 (Table 3).

3.3. Aboveground Biomass and Nutritional Status: Effects of Species and Soil Type

3.3.1. Shoots’ Dry Weight and P and N Content

As shown in Table 4, the species, soil, and their interaction significantly influenced the shoots’ dry weight and P content. The maximum shoot dry weight was produced by oat, followed by L. albus and L. angustifolius. Regarding soils, Site 4 had the lowest yield, heavier soils offered an intermediate yield, and Site 2 presented the highest yield, which was three times that of Site 4. The yield of each species was also differentially affected by the soil type (soil x species interaction; p < 0.0001). Except for Site 3, oats outyielded lupines, while L. albus produced roughly twice as much as L. angustifolius across all soil types.

The plants’ P content in oats and L. albus was not statistically different, except in Site 3, where L. albus absorbed more P than oats (Table 4). The absorption of P and the yield of the aboveground biomass varied substantially between the lupine species, with the lowest values detected in L. angustifolius. In terms of P concentrations, there were fewer differences within each species across sites, because they were already similar in all soils except for Site 4, where the concentrations were half of those that were registered in the other sites, whatever the species considered. Oats displayed the most noticeable variation in P content between sites. In soils with lupine, such differences between soils diminished, although in Site 4, the P content of lupine was substantially lower than in the other sites. The contrast analysis between lupine and oat (C1) was significant only in Site 1 and Site 2, where the plant P content in oats was markedly higher than that of lupines (Supplementary Materials, Table S5). Contrast 2 was not significant in Site 4; the remaining soils had higher P contents in L. albus than in L. angustifolius. All data that are displayed in Table 4 exclude the pod components of L. angustifolius. However, upon their inclusion, the effect of species became not significant, eliminating the difference between the P content that was reached by both lupine species in Sites 1, 2, and 4. In contrast, L. albus continued to attain a higher plant P content than L. angustifolius in Site 3. On average, the P concentration of L. angustifolius plants in Sites 1, 2, and 3, both with and without pods, was 1.6 and 1.4 mg g⁻¹ dry weight, respectively.

Similar to the shoots’ dry weight, the content of N in the biomass significantly differed between species and soils, with the lowest values in Site 4 for both lupines and in Sites 1 and 3 for oats. In contrast to the lupines’ P content, the values of N content differed less between sites, as the soil type effect was not statistically significant in terms of N concentration (Table 4). The ANOVA results revealed no significant difference between lupine species in Ndfa (Supplementary Materials, Table S6). However, there were statistically significant differences in the N biomass and the amount of N that was fixed, with average values of 347 and 237 mg N pot⁻¹, respectively, registering the highest values in L. albus. These variables, expressed as total N and N fixed content in shoots’ dry weight per hectare to determine their agronomic significance to N entering the soil system, represent approximately 190 kg N ha⁻¹ and 130 kg ha⁻¹ of N, respectively.

3.3.2. Plants’ Ca, Mg, K, and Micronutrient (Fe and Mn) Concentrations in Aboveground Biomass

The ANOVA of the plant nutrient concentration revealed that only the K concentration was similar between species. The K concentration in L. albus showed differences between soils, with the highest concentration values found in Site 3 and the lowest in Site 4. On the other hand, there were significant variations in Ca and Mg levels among species, with L. angustifolius consistently exhibiting the highest concentrations across all soil types (Table 5).

The impact of different species on the concentration of micronutrients in plants varied depending on the soil type (notably, a significant interaction effect was found between soil type x species). Across most soils, L. angustifolius exhibited the highest concentrations of Mn and Fe, with two exceptions. Specifically, L. albus had statistically higher Mn content at Site 3 compared to oats, and oats displayed the highest concentrations of Fe at Site 4 compared to lupines (Table 5). In all soils, the Mn concentration was substantially higher in lupines than in oats. The concentrations of Mn and Fe in the aboveground biomass also differed among soils within each species, with oats showing no significant differences in their Mn concentration across soils. Similarly, L. angustifolius did not show significant differences in their Fe concentration. When examining the Fe concentrations of each species across soils, both lupine species consistently exhibited higher values than oats in all soils except in Site 4, as previously mentioned.

3.4. Relationships between Soil and Plant Parameters

The correlations between the soil and plant variables revealed a moderate, yet significant (p = 0.0001) positive relationship between plants’ N and P contents (Table 6). This correlation increased to 0.90 (p = 0.0001) and 0.65 (p = 0.0068) when the legumes and oats datasets were separated. The shoots’ dry weight and shoots’ P and N contents were also positively related to the soil’s PBray1 availability at 48 dap and negatively related to the ∆pH-initial (Table 6). This last finding suggests that a pH drop, relative to its initial value, increased the P and N uptake. In addition, the relationships between ∆pH−initial and ∆PBray1−initial, depicted in Figure 4, displayed a positive association for each species, showing that when the pH decreased (soil acidification), the PBray1 at harvest diminished compared to its initial value. This outcome can be linked to the significant negative correlation (−0.59, p < 0.001) between plants’ P content and ∆pH−initial across all combinations of species and soils (Table 6) and within each species across all four soils types (Supplementary Materials, Table S7).

This study also revealed a notable inverse relationship between plants’ Fe concentration and PBray1 and a direct relationship between PBray1 and plants’ P content. Additionally, a significant positive association was detected between plants’ Fe concentration and the soil’s ∆pH−initial. The first two associations establish a positive relationship between the soil’s P availability and the plants’ P content, while indicating a negative relationship between plants’ Fe concentration and the soil’s P availability. Conversely, the final association suggests that an increase in the soil pH was associated with an increase in plants’ Fe concentration. The concentration of Mn in plants, on the other hand, exhibited a weak and inverse correlation with the soil’s ∆pH-initial, suggesting that as the soil becomes more acidic within the range of the examined pH values (4.5–6.4 units), the soil’s Mn solubility will increase (Table 6). Nevertheless, when analyzing within each species (Supplementary Materials, Table S7), the correlation was statistically significant and positive just for oats (r = 0.52, p = 0.037). Meanwhile, the soil pH at harvest only correlated with plants’ Mn concentrations for both lupines, but it was positive (r = 0.66, p = 0.006) for L. albus and negative (−0.76, p = 0.011) for L. angustifolius. In the case of oats, the significant correlations that were observed between plants’ Mn concentrations and PBray1−48 dap (r = −0.58) or plants’ Mn concentrations and plant’s P concentrations (r = −0.59) might be due to the influence of data from Site 4, in which the soil’s plant Mn concentration was more than double compared to oats that were cultivated in the other soils (Table 5). For L. albus, there was a negative trend (r = −0.5, p = 0.1, excluding Site 4) between the Mn concentration (as an index of the organic anion concentration) and plant’s P concentrations (Supplementary Materials, Table S8). In contrast, there was a significant correlation at a higher soil PBray1 at 48 dap (r = −0.63, p = 0.048, excluding Site 4, Supplementary Materials, Table S8). These correlations were not significant for L. angustifolius (Supplementary Materials, Table S8).

4. Discussion

4.1. Effects of Species and Soil Types: Variations in Soil’s PBray1 Concentration and Soil pH

The changes in the soil’s PBray1 relative to its initial concentration were agronomically negligible in soils that were planted with lupine. These results suggest that this species could solubilize plant-unavailable residual P or mobilize organic P forms, consequently maintaining an equilibrium level and not being depleted by plant absorption. In contrast, the absorption by oats reduced the amount of available P. When analyzing ∆PBray__Lup-Oat, the values ranged from 0.9 (not a significant difference) to 5.4 mg kg⁻¹, and assuming an equivalent fertilizer value of 10 kg ha⁻¹ of P₂O₅ (the average value used as equivalent fertilizer for Uruguayan soils according to Hernández and Zamalvide [41]), the highest increase in P availability due to lupines occurred in Site 2, which represents an equivalent of 54 kg ha⁻¹ of P₂O₅. This increase in soil’s PBray1 availability would significantly contribute to systems with a high proportion of P-extractive crops, leading to a considerable reduction in the application of P fertilizers. However, the species effect was also affected by the soil type, as lupine plants grew better in sites with higher PBray1 availability (Sites 1, 2, and 3) and greater soil fertility (high OM and high exchangeable cations).

The increase in PBray1 at 48 dap may be primarily attributed to the mineralization of organic P. The OM content would explain the extent of this increase, with the most significant increase in Site 1 and Site 2, which had higher OM contents than the other two sites. Interestingly, by 76 dap, the available P in the soil began to stabilize, i.e., returning to pre-experimental levels. This finding suggests that the mineralized organic P was either absorbed by the plants, retrograded in the soil, or immobilized by microorganisms [42]. The soil’s biological activity may positively influence the mobilization (accessibility) of organic P forms [43,44] or negatively hamper the P mobilization efficiency of carboxylates by way of microbial degradation [13] and biochemical factors, such as the hydrolysis rates of extracellular phosphatase enzymes [11,43,45], which play a role in plant P acquisition [46]. Other physical factors, such as variations in the soil moisture, drying conditions, and aggregate stability, may have facilitated the mineralization of organic P [46].

The higher soil buffering capacity in Site 1 and Site 3 (higher clay content, OM, and cation exchange capacity) may account for the lower PBray1 variation compared with their baseline values at 87 and 103 dap. Conversely, the minimal change in Site 4 could be due to the generally poor performance of the lupine, particularly L. angustifolius, which exhibited the lowest shoot dry weight and P uptake. The soil of Site 2, however, demonstrated the most remarkable differences in ∆PBray__Lup-Oat, which could be attributed to the higher P biomass that was attained by oats. The loamy texture, moderate clay, and OM content of Site 2 suggest a lower P adsorption capacity, leading to a higher equilibrium concentration of labile P forms in this soil compared to Sites 1 and 3, which possibly boosts the efficiency of organic anions in solubilizing inorganic P [42]. Moreover, this site had the highest PBray1 concentration at the start of the experiment, which can be partially attributed to organic P sources, ensuring a more consistent and substantial P supply for plants.

Even though lupine increased the PBray1 availability in all soils, the species effect alone could not entirely explain the increase in P by soil acidification, suggesting that other factors associated with the species could account for the differences [12]. It is crucial to recognize that variations in a species’ capacity to obtain P can be attributed to the inherent traits of each species. Furthermore, morphological modifications in the root architecture and the development of specialized structures such as proteoid or cluster roots can also contribute to these differences [14,47]. While our study did not directly measure the contribution and composition of the root exudates of organic acids, we consider this trait indirectly based on the Mn concentration, which is proposed as an index of the carboxylate concentration in the rhizosphere under low P availability conditions in soil [21,48]. The subsequent section of this paper will delve further into this matter.

As demonstrated in our study, the BNF process contributed to variations in the soil pH during the experiment, leading to higher plant Mn concentrations in lupines than in oats. Legumes also absorb more cations than anions, resulting in the rhizosphere’s exudation of protons and acidification [49]. It was further reported that P deficiency in L. albus stimulates proton release and citrate root exudation by the proteoid roots of this species, along with the inhibition of nitrate uptake [50,51]. Pearse et al. [14] showed that seven lupine species had a greater acidification capacity of the soil rhizosphere than grasses and even than other legume crops, arguing that this could be an adaptation to increase the solubility of acid-soluble Ca-phosphates. In the present study, both lupine species contributed to pH decreases in different soil types, showing comparable trends in acidification levels, except for in Site 3, where L. albus had a minimal influence on the soil pH. This discrepancy could be attributed to differences in the inherent characteristics of the species, as mentioned above. Lupinus albus is a cluster root-forming legume and grows well in strongly acidic soils and mildly acid to neutral soils; L. angustifolius, on the contrary, does not form cluster roots and is sensitive to calcareous soil, displaying a preference for acidic soil conditions. Consequently, the growth of L. angustifolius in Site 3 may have induced changes in the rhizosphere that resulted in a decrease in the soil pH by an average of 0.6 units. This decline in pH, caused by a net release of protons, may have occurred as a compensatory mechanism to counterbalance the elevated uptake of cations. The high concentration of cations that was observed in the aboveground biomass of L. angustifolius in this study was consistent with previous studies [52,53].

When oats were cultivated, all soils (except Site 2) experienced an increase in pH compared to the initial values, with the Site 4 soil experiencing an increase of nearly one pH unit. Wang et al. [28], using the rhizotron technique with Avena sativa, also found that the soil pH increased compared to the control (without plants). In that study, despite having the lowest acid phosphatase activity, oats absorbed the same amount of P as other species, a result that was attributed to the extensive root mass and high mycorrhizal colonization, which contributed to the high P absorption [28]. We did not investigate the morphology or other aspects of the roots in our study, but this explanation would be valid for our experiment. Additionally, we can partially explain the pH change trends in oat-bearing soils based on an imbalance of the cation–anion uptake (mainly influenced by the N source). In Site 2, ammonium was the predominant form of N, and its absorption by oats would account for the soil acidification in this case [54]. However, the increased uptake of N in the form of nitrate would increase the absorption of H+, consequently promoting soil alkalinization [54]; this would explain the increase in the soil pH with oats in the acidic soil of Site 4.

4.2. Effects of Species and Soil Types: Plant Growth and Nutrient Uptake

While the availability of PBray1 differed among Sites 1, 2, and 3, there were no significant differences in the shoot biomass within each lupine species across these soils. However, the P concentrations were comparable to or higher than those of oats across these soils. In addition, these sites exhibited significant differences in aboveground biomass and plants’ N and P contents for oats, indicating that the P supply and an additional factor (N availability) impacted the performance of this species across soil types differently. These findings align with a prior study that indicated that L. albus was the least impacted species by P supply, but that it exhibited the greatest P concentration in its shoots among all plant species [11].

Regarding P concentrations, whatever the species considered, there were differences across sites, particularly for Site 4, where the concentrations were, on average, half of those registered in the other sites. Previous studies have reported that the critical threshold for optimum development for L. albus and L. angustifolius is 2.2 and 2.3 mg g⁻¹ of P, respectively [55]. In our work, the shoots’ P concentrations of lupine species was below 2.0 mg g⁻¹, a level that typically stimulates cluster root development [56]. Concerning oats, the plant’s P concentration would be adequate for their mature stage, which oscillates between 1.0 and 1.5 mg g⁻¹ [57]. Furthermore, all concentrations of P, regardless of the species, were lower than 2.0 mg P g⁻¹ dry weight, implying that the shoot biomass decay will probably lead to limited P availability in the soil due to P immobilization in the microbial biomass, as Hallama et al. noted [21].

The lowest plant growth, P uptake, and BNF values were observed at Site 4, suggesting that even though lupine species have adapted to acidic soils, Site 4 had several growth-limiting factors that prevented them from thriving as well as they did at the other sites. Lupinus angustifolius exhibited reduced growth and accelerated senescence of leaves, and both lupine species showed lower P and K concentrations in plants in Site 4 compared to other sites with higher soil PBray1, OM, and K concentration. The poor performance of lupines in Site 4, which experienced severe P deficiency, can be linked to the disruption in their ability to balance the carbon costs that are involved in cluster root development and nodulation [58,59].

The proportion and amount of N in the lupine’s aerial biomass that are derived from the air fall within the reported ranges of 44 to 95% and 147 to 400 kg ha⁻¹, respectively [60]. The amount of N that is fixed by L. albus is, in most cases, higher than that of L. angustifolius, as evidenced by our research [23,61]. The wide variation between soils in the quantity of fixed N (60–240 mg pot⁻¹, or approximately 30–140 kg N ha⁻¹) reflects the varying levels of natural fertility among soils. The N that is fixed in biomass also varies between species (105–240 mg pot⁻¹, approximately 60–130 kg N ha⁻¹; p = 0.0001), indicating that lupines have different soil requirements that mainly affect the shoots’ dry weight. Besides solubilizing non-labile forms of P, the N that is derived from the BNF process is an additional benefit of the lupine genus, because this contribution of N to the system might facilitate the balancing of N losses that occur during agricultural cycles [62].

4.3. Effects of Species and Soil Types: Links between Traits of Soil and Plants

The high PBray1 at harvest in soil containing L. albus might suggest a P-sparing effect of this species. However, during the growing phase, the amount of P that was absorbed by L. albus was, as was previously mentioned, comparable to and even higher than those taken up by oats. Although higher amounts of PBray1 were present at harvest in soils under L. albus than under oats, the differences are probably not due to variations in P uptake but instead to the “rhizosphere effect” that is induced L. albus. Neumann et al. [63] stated that L.albus employs diverse mechanisms to fulfill its P requirements in nutrient-deficient environments. This species exudes organic acids (citric and malic) that are sufficient to mobilize scarcely available P sources (Ca, Al, and Fe phosphates), primarily via chelation of the bound cations to P or by competition for P adsorption sites in the soil matrix [64]. However, the role of these carboxylates was not consistent in several studies that have reported that in other species, and even L. albus, the carboxylate concentration was not explained by any variation in soil or plant traits [11,15,48], leading to the conclusion in those studies that organic anions play a minor role in P acquisition strategies.

The concentration of PBray1 at harvest showed a negative correlation (r = −0.61) with the difference in pH from the initial pH across all combinations of species and soils. This result suggests that the increase in the acidification level within the tested soil pH range (4.5 to 6.4 units) was more pronounced in soils with high PBray availability. This result was unexpected in acidic soils, given that it has been reported that the P adsorption capacity of Fe and Al oxides increases with a decreasing pH [49]. However, other studies have shown that lowering the pH of acidic soils can also increase the solubility of soil P [65]. The proposed explanation is that competitive adsorption of sulfate ions would increase with a decreasing pH, leading to a higher equilibrium P concentration [49]. In the same way, the negative relationship between the amount of P biomass and the ∆pH−initial indicated that the P absorption increased with the degree of acidification, which is consistent with other studies [12,65,66]. On average, the plant P content was higher in L. albus and oats (14 mg pot⁻¹) than in L. angustifolius (7.9 mg pot⁻¹), mainly due to the low biomass yield of the latter species. The positive association of accumulated N and P amounts with the availability of PBray1 (r = 0.50 and 0.45, respectively) at harvest (87 dap) and the negative association with the ∆pH−initial (r = −0.56 and −0.59, respectively) show that N and P absorption increased with the available soil P and with soil acidification. This last association explains the negative correlation between the ∆pH−initial and the PBray1 that is available in the soil (r = −0.61).

Lupinus angustifolius reached higher levels of base cation than L. albus, as observed by other researchers [53], although, for K, there were no statistically significant differences between species at each site. The total cation concentrations in plants within each species also differ across soils. These differences were exclusively observed in L. albus, showing the lowest concentrations in plants that were cultivated in Sites 2 and 4, whose sites corresponded to soils that were strongly desaturated in base cations. Nevertheless, these last two soils differed widely in their pH and OM content. For the plant cation concentration in the lupine genus in our experiment, and based on previous research [53,67], this would indicate that plant nutrition was adequate in Sites 1, 2, and 3, and insufficient to cover lupine’s requirements in Site 4.

The lupine-induced soil acidification remarkably impacted plants’ micronutrient concentrations such as Mn and Fe. The Mn concentrations in L. albus and L. angustifolius reached mean values of 710 and 1510 mg kg⁻¹, respectively, which represent approximately three and seven times the concentration in oats (228 mg kg⁻¹), as was reported in previous studies [47,68]. These high concentrations of foliar Mn may be deleterious to other organisms or species, but the lupine genus tolerates them [48]. It has been found that plants using a P mobilization strategy based on the release of carboxylates have elevated Mn concentrations in their leaves [68], because carboxylates mobilize both inorganic and organic P from the soil, as well as micronutrients [69]. Modifications in soil acidity or the oxidation/reduction conditions of the rhizosphere can also enhance the increased uptake of Mn [68]. It was further confirmed that lupines’ exudation of organic acid anions may or may not be linked to soil acidification, as these processes self-regulate independently [70]. Further, a negative correlation between citrate exudation and plants’ P status has been established, which has been perceived clearly with L. albus species [71]. Pearse et al. [14] and Wang et al. [72] also observed the variable response among lupine species, who found variation in both their P uptake and sensitivity to external P supply on the formation and development of proteid roots. These authors highlighted that the variations across species within the lupine genus open the possibility of selecting species with high plasticity regarding P supply [14]. In those studies, under pot experiments and using river sand as a growth medium, it was also reported that L. albus developed the most cluster roots with the lowest P concentration in shoots. Considering this context and under similar plant cultivation conditions, but using soils as a growth medium, in the present study, the Mn concentration (as an index of the organic anion concentration) in L. albus was downregulated at a greater P concentration in plants. However, this association was barely a trend (r = −0.5, p = 0.1, excluding Site 4). In addition, there was a significant correlation at a higher soil PBray1—48 dap (r = −0.63, p = 0.048, excluding Site 4), suggesting that the organic anion exudation increased when the growth medium had a low PBray1 concentration. These results are in accordance with previous studies [15,28], which found no simple linear relationships between plant and soil traits. In our work, L.albus showed a low Mn concentration in plants under severe P stress (conditions under Site 4), maximum under moderate stress (conditions under Site 3), and low again at a high P availability (conditions under Sites 1 and 2). In contrast, the L. angustifolius plants with the highest concentration of Mn were those growing in the soil of Site 2, which exhibited the highest initial PBray1 concentration. These findings suggest that the exudation of carboxylates by L. angustifolius in the soil is constitutive, similar to what was reported by Pang et al. [73] for chickpea species (Cicer arietinum L.), a non-cluster root-forming crop. In contrast, the exudation of carboxylates by L. albus would be inducible, because the plants’ Mn concentration changed in response to the soil’s PBray1 availability [72]. Monei et al. [74], who also noted a divergent response in these lupine species, found that L. albus exhibited a high release of carboxylates in conditions of P deficiency, whereas L. angustifolius responded with the highest release of carboxylates when the soil had a high P supply. Although there was no clear relationship between Mn concentrations and plants’ P contents or soil’s P availability in both lupines across all sites in the current study, there was a significant association between the soil acidity at harvest and the Mn concentration; however, it was noticeably different between the lupine species. The correlation between these last variables was statistically significant, but positive (r = 0.66) for L. albus and negative (r = −0.76) for L. angustifolius, suggesting that the exudation of organic acid anions was concomitant with proton extrusion in soils containing L. angustifolius, while in L.albus, this was the case with base cations [48].

5. Conclusions

This research contributes to understanding how the species and soil type affect P mobilization and nutrient uptake and helped us inquire about the relationships between plant and soil factors that might explain or give us a clue to increase the comprehension of the processes of controlling P demand relative to its availability in the soil. Lupinus albus had a more substantial P solubilization capacity in the light-textured soil with a high OM content. However, the plant growth and nutrient uptake of L. albus in this site were comparable to those in the heavy-textured soils. The degree of soil acidity had a direct relationship with the P and N uptake within each species, across all soil types tested. The soil pH differed among species, with the lowest values found in soils that were cultivated with lupine species. In contrast, the soils under oats tended to maintain or even increase their pH compared to the original values. Our investigation revealed a significant variation in Mn accumulation (used as an index of the organic anion concentration) within species across the tested soils. The soil acidity and changes in P supply that were induced by lupines could explain such variation, particularly in soils with L. albus. Comparing these results with those obtained with these species in the field would be necessary to determine the robustness of the identified patterns in the soil’s available P by the presence of lupine and to analyze them from the perspective of long-term stability, because the interaction and feedback with other soil–plant factors and microbial activity processes could hamper the increase in soil’s P availability that is facilitated by lupine species. Hence, field research should be carried out for at least two growing seasons to ascertain whether the advantageous impacts of lupine last with the same magnitude over time and whether these variations result in enhanced soil health and crop production. Additionally, the future recommendations that emerge from the findings of our study include the need to measure soil’s phosphatase activity, as this is an essential indicator for evaluating the effectiveness of P that is taken out by plants and analyzing the associated changes in organic P sources, such as the immobilized P within the living soil microbial biomass.

Author Contributions

Conceptualization, C.M.A. and C.P.V.; methodology, C.M.A. and A.B.G.; formal analysis, C.M.A., C.P.V., P.G.B. and A.d.P.M.; investigation, C.M.A. and A.B.G.; resources, C.M.A.; data curation, C.M.A., C.P.V., P.G.B. and A.d.P.M.; writing—original draft preparation, C.M.A., C.P.V., P.G.B., A.B.G. and A.d.P.M.; writing—review and editing, C.M.A., C.P.V., P.G.B. and A.d.P.M.; visualization, C.M.A. and A.d.P.M.; supervision, A.d.P.M. and C.P.V.; project administration, C.M.A.; funding acquisition, C.M.A., C.P.V. and A.d.P.M. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

Acknowledgments

The authors express their gratitude to N. Di Muro and B. Romano for their assistance in conducting the greenhouse experiment and laboratory work.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Tables

Figure 1. Soil PBray1 concentrations under two legumes (L. albus and L. angustifolius) and one grass (Avena strigosa) across Sites (soil type) 1 (a), 2 (b), 3 (c), and 4 (d). The vertical bars indicate the standard error, and the symbols “*” and “☨” indicate the sampling time in which Contrast 1 (lupine vs. oat) and Contrast 2 (L. albus vs. L. angustifolius) were significant (* ☨ p < 0.05; ** ☨☨ p < 0.01; *** p < 0.001), respectively.

Figure 2. PBray1 concentration according to species across Sites (soil types) 1 (a), 2 (b), 3 (c), and 4 (d) at 103 days after planting. The vertical bars indicate the standard error. Different lowercase letters indicate significant differences between species within each soil type, and different capital letters indicate differences between sites across species, according to Tukey’s test, with a p ≤ 0.05.

Figure 3. Soil pH measured 87 days after planting of the two legumes (L. albus and L. angustifolius) and the grass (Avena strigosa) across Sites (soil type) 1 (a), 2 (b), 3 (c), and 4 (d). The dotted line represents the soil’s pH value at the beginning of the experiment. Different lowercase letters indicate significant differences between species within each soil type, and different capital letters indicate differences between sites across species, according to Tukey’s test, with a p ≤ 0.05.

Figure 4. Relationships between ∆pH−initial (difference between soil pH at harvest and soil pH at the beginning of the experiment) and ∆PBray1−initial (difference between soil PBray1 at harvest and soil PBray1 at the beginning of the experiment). The dotted line splits the data from soil acidification to alkalinization relative to the initial pH. Gray squares, black circles, and white triangles represent Lupinus albus, Lupinus angustifolius, and Avena strigosa species, respectively.

Table 1

Site location, soil type, dominant geological material, physical characterization, and soil texture of the soil samples collected from four sites in Uruguay.

Site (Location)	Soil Type (USDA Classification System) †	Geological Material	OM	Sand	Silt	Clay	Texture ‡
			%
Site 1 (Colonia)	Pachic Argiudoll	Clay silt sediment/Crystalline basement	4.8	16.3	38.8	44.9	C
Site 2(T. and Tres)	Typic Dystrutepts	Crystalline basement	3.7	23.5	44.2	32.3	CL
Site 3 (Montevideo)	Typic Argiudolls	Libertad clayey silt sediment	1.7	19.3	52.3	28.4	SiCL
Site 4(Rivera)	Typic Hapludults	Colluvial material (Sandy soils)/Tacuarembó sandstones	1.4	84.7	1.4	13.9	SL

† USDA source: Keys to soil taxonomy. Soil Conservation Service. 2014. ‡ Texture: C = clay; SiCL = silty clay loam; SL = sandy loam; CL = clay loam.

Table 2

Repeated measures analysis of variance model for soil PBray1 concentration.

Treatment Effect	DF	F Value	p > F †
Species	2	23.05	<0.0001
Soil type	3	101.56	<0.0001
Species x soil type	6	1.36	0.2564
Sampling time	3	91.85	<0.0001
Species × sampling time	6	2.29	0.0518
Soil type × sampling time	9	6.66	<0.0001
Species × soil type × sampling time	18	2.17	0.0133

† Significant effects (p < 0.05) are in bold. Note: The within-subject factor was sampling time. Results are from 48, 76, 87, and 103 days after planting, and the between-subject factors were species and soil type.

Table 3

Soil’s exchangeable acidity and exchangeable aluminum according to species and soil type. Values are means ± standard error.

Site	Species	Exchangeable Acidity		Exchangeable Aluminum
		cmolc kg−1
1	Lupinus albus	0.07 ± 0.01	ns C	0.01 ± 0.03	ns C
	Lupinus angustifolius	0.08 ± 0.02	ns C	0.04 ± 0.03	ns ns
	Avena strigosa	0.07 ± 0.01	ns BC	0.04 ± 0.01	ns AB
2	Lupinus albus	0.69 ± 0.04	a A	0.18 ± 0.03	ns A
	Lupinus angustifolius	0.56 ± 0.09	a A	0.10 ± 0.03	ns ns
	Avena strigosa	0.33 ± 0.04	b A	0.13 ± 0.03	ns A
3	Lupinus albus	0.08 ± 0.03	ns C	0.06 ± 0.02	ns BC
	Lupinus angustifolius	0.05 ± 0.00	ns C	0.06 ± 0.02	ns ns
	Avena strigosa	0.03 ± 0.01	ns C	0.03 ± 0.03	ns B
4	Lupinus albus	0.32 ± 0.08	a B	0.11 ± 0.03	ns AB
	Lupinus angustifolius	0.23 ± 0.04	ab B	0.10 ± 0.03	ns ns
	Avena strigosa	0.17 ± 0.07	b B	0.08 ± 0.03	ns AB
Significance of treatment effect
Species		0.0008		0.6089
Soil type		<0.0001		0.0008
Species × soil type		0.0179		0.6339

Significant effects (p < 0.05) are in bold. Different lowercase letters within a column indicate differences between species within each soil type, and different capital letters within a column indicate differences between soils across species being significant at a p-level of 0.05.; ns means no significant difference.

Table 4

Shoots’ dry weight, plants’ P and N concentration, and plants’ P and N content of different species, as affected by soil type. Values are means ± standard error.

Site	Species	Shoots’ Dry Weight		Plants’ P Concentration		Plants’ N Concentration		Plants’ PContent		Plants’ NContent
		g pot−1		mg g−1 Dry Weight				mg pot−1
1	Lupinus albus	12.5 ± 1.5	a A	1.3 ± 0.1	a AB	29.3 ± 1.0	a ns	15.5 ± 1.7	a A	366.5 ± 46.5	a A
	Lupinus angustifolius	6.7 ± 0.3	b A	1.4 ± 0.1	a A	26.1 ± 1.6	a ns	9.0 ± 0.6	b A	174.4 ± 15.3	b A
	Avena strigosa	14.5 ± 1.1	a B	1.2 ± 0.1	b A	5.7 ± 0.2	b B	16.8 ± 1.3	a B	81.9 ± 4.8	c B
2	Lupinus albus	13.9 ± 1.3	b A	1.4 ± 0.1	a A	31.9 ± 1.3	a ns	19.5 ± 1.3	a A	436.9 ± 24.7	a A
	Lupinus angustifolius	7.8 ± 1.1	c A	1.4 ± 0.1	a A	24.5 ± 0.4	b ns	10.6 ± 1.6	b A	189.8 ± 25.8	b A
	Avena strigosa	27.0 ± 2.7	a A	0.9 ± 0.0	b B	8.4 ± 1.0	c AB	23.5 ± 1.4	a A	222.1 ± 19.1	b A
3	Lupinus albus	14.0 ± 1.3	a A	1.1 ± 0.1	c B	28.8 ± 1.9	a ns	15.4 ± 2.1	a A	402.4 ± 37.0	a A
	Lupinus angustifolius	7.0 ± 1.9	b A	1.5 ± 0.1	a A	29.4 ± 1.4	a ns	10.8 ± 2.9	b A	203.6 ± 52.9	b A
	Avena strigosa	8.1 ± 0.4	b C	1.3 ± 0.1	b A	6.5 ± 0.3	b B	10.2 ± 0.9	b C	52.9 ± 3.01	c B
4	Lupinus albus	6.5 ± 1.1	b B	0.9 ± 0.1	a C	27.5 ± 2.4	a ns	5.6 ± 1.2	ns B	180.7 ± 38.6	a B
	Lupinus angustifolius	1.7 ± 0.4	c B	0.6 ± 0.2	b B	30.2 ± 0.0	a ns	1.1 ± 0.6	ns B	38.1 ± 0.0	b B
	Avena strigosa	11.0 ± 0.4	a BC	0.5 ± 0.0	b C	10.2 ± 0.6	b A	5.3 ± 0.3	ns D	111.8 ± 2.8	ab B
Significance of treatment effect
Species		<0.0001		0.0004		<0.0001		<0.0001		<0.0001
Soil type		<0.0001		<0.0001		0.3286		<0.0001		<0.0001
Species × soil type		<0.0001		0.0017		0.0088		0.0062		0.0009

Significant effects (p < 0.05) are in bold. Different lowercase letters within a column indicate differences between species within each soil type, and different capital letters within a column indicate differences between sites across species being significant at a p-level of 0.05; ns means no significant difference.

Table 5

Plants’ Ca, Mg, K, Mn, and Fe concentration of different species, as affected by soil type. Values are means ± standard error.

Site	Species	Plants’ Ca Concentration		Plants’ Mg Concentration		Plants’ K Concentration		Plants’ Mn Concentration		Plants’ Fe Concentration
		mg g−1						mg kg−1
1	Lupinus albus	5.6 ± 0.4	b AB	1.4 ± 0.1	b AB	14.3 ± 1.7	ns B	482.6 ± 51.2	b B	97.2 ± 12.7	b BC
	Lupinus angustifolius	26.5 ± 1.6	a ns	4.7 ± 0.3	a ns	16.2 ± 0.2	ns ns	1067.0 ± 150.2	a B	214.2 ± 23.5	a ns
	Avena strigosa	3.1 ± 0.2	c B	1.2 ± 0.1	c B	15.5 ± 0.6	ns ns	216.7 ± 27.6	b ns	36.9 ± 8.6	b B
2	Lupinus albus	3.9 ± 0.4	b B	1.1 ± 0.1	b B	13.7 ± 1.1	ns B	537.6 ± 218.2	b B	54.0 ± 5.5	ab C
	Lupinus angustifolius	22.5 ± 1.1	a ns	4.4 ± 0.2	a ns	14.7 ± 1.9	ns ns	3161.0 ± 259.1	a A	142.1 ± 4.8	a ns
	Avena strigosa	2.9 ± 0.3	b B	1.5 ± 0.1	b B	13.0 ± 0.9	ns ns	214.9 ± 21.66	b ns	28.7 ± 2.6	b B
3	Lupinus albus	7.2 ± 0.1	b A	2.0 ± 0.3	b A	17.1 ± 0.7	ns A	1191.2 ± 21.51	a A	165.3 ± 21.5	a AB
	Lupinus angustifolius	26.7 ± 1.3	a ns	4.5 ± 0.2	a ns	17.4 ± 1.1	ns ns	791.9 ± 184.0	a B	214.6 ± 36.6	a ns
	Avena strigosa	4.4 ± 0.6	b AB	1.0 ± 0.2	b B	15.1 ± 0.2	ns ns	209.8 ± 96.0	b ns	47.7 ± 18.7	b B
4	Lupinus albus	4.3 ± 0.1	b B	1.2 ± 0.2	b B	13.2 ± 1.3	ns B	628.6 ± 210.0	ab B	189.0 ± 28.5	b A
	Lupinus angustifolius	12.0 ± 0.0	a ns	3.1 ± 0.0	a ns	10.8 ± 0.0	ns ns	1021.5 ± 0.0	a B	218.6 ± 0.0	b ns
	Avena strigosa	4.1 ± 0.2	a A	2.9 ± 0.2	a A	14.7 ± 0.2	ns ns	403.0 ± 47.3	b ns	440.1 ± 88.7	a A
Significance of treatment effect
Species		<0.0001		<0.0001		0.7014		<0.0001		0.0342
Soil type		0.1651		0.4564		0.0453		<0.0001		<0.0001
Species × soil type		0.0237		0.0026		0.4818		<0.0001		<0.0001

Significant effects (p < 0.05) are in bold. Different lowercase letters within a column indicate differences between species within each soil type, and different capital letters within a column indicate differences between sites across species being significant at a p-level of 0.05. ns means no significant difference.

Table 6

Matrix of Pearson correlation coefficients (r) for the relationships between soil (PBray1 and pH) and plant variables (P and N content, Fe and Mn concentration) for all combinations of species (lupines and oats) and soils at the harvest time of the experiment (87dap).

	∆pH−Initial †	PBray1−48dap	PBray1−87dap	Shoots’ Dry Weight	Plants’ N Content	Plants’ P Content	Plants’ Fe Concentration
PBray1−48dap	−0.56 ***
PBray1−87dap	−0.61 ***	0.76 ***
Shoots’ dry weight	−0.24	0.39 **	0.06
Plants’ N content	−0.56 ***	0.49 ***	0.50 ***	0.32 *
Plants’ P content	−0.59 ***	0.66 ***	0.45 **	0.83 ***	0.54 ***
Plants’ Fe concentration	0.53 ***	−0.57 ***	−0.42 **	−0.36 *	−0.17	−0.61 ***
Plants’ Mn concentration	−0.36 *	0.02	0.36 *	−0.28	0.14	−0.13	0.15

† ∆pH-initial: difference between soil pH at harvest and soil pH at the beginning of the experiment. Significant at * p < 0.05; ** p < 0.01; *** p < 0.001.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy14020389/s1: Table S1: Initial soil properties before the establishment of the experiment; Table S2: ANOVA for soil PBray1 concentration at 87 days after planting; Table S3: ANOVA for soil PBray1 concentration at 103 days after planting; Table S4: ANOVA for soil pH at 87 days after planting; Table S5: Orthogonal contrasts for biomass measurements; Table S6: Mean values of BNF proportion (% BNF) and N fixed content of lupines as affected by species and soil type; Table S7: Pearson correlation coefficients (r) within each species across all four soil types; Table S8: Pearson correlation coefficients (r) within each species across three soils types (excluding Site 4).

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