Urochloa Grasses Swap Nitrogen Source When Grown in Association with Legumes in Tropical Pastures

Abstract

The degradation of tropical pastures sown with introduced grasses (e.g., Urochloa spp.) has dramatic environmental and economic consequences in Latin America. Nitrogen (N) limitation to plant growth contributes to pasture degradation. The introduction of legumes in association with grasses has been proposed as a strategy to improve N supply via symbiotic N₂ fixation, but the fixed N input and N benefits for associated grasses have hardly been determined in farmers’ pastures. We have carried out on-farm research in ten paired plots of grass-alone (GA) vs. grass-legume (GL) pastures. Measurements included soil properties, pasture productivity, and sources of plant N uptake using ¹⁵N isotope natural abundance methods. The integration of legumes increased pasture biomass production by about 74%, while N uptake was improved by two-fold. The legumes derived about 80% of their N via symbiotic N₂ fixation. The isotopic signature of N of grasses in GA vs. GL pastures suggested that sources of grass N are affected by sward composition. Low values of δ¹⁵N found in some grasses in GA pastures indicate that they depend, to some extent, on N from non-symbiotic N₂ fixation, while δ¹⁵N signatures of grasses in GL pastures pointed to N transfer to grass from the associated legume. The role of different soil–plant processes such as biological nitrification inhibition (BNI), non-symbiotic N₂ fixation by GA pastures and legume–N transfer to grasses in GL pastures need to be further studied to provide a more comprehensive understanding of N sources supporting the growth of grasses in tropical pastures.

1. Introduction

Deforestation in the tropics has been estimated at about 0.74 million km² from 2000 to 2012 [1]. Nearly half of it occurred in the South American rainforest [1]. In the Amazon basin, most of the cleared land has been converted to pastures sown with introduced grasses (mostly Urochloa spp., formerly known as Brachiaria) for livestock production [2,3]. In Colombia alone, more than 8% of the remaining forest area has been lost since 2000, yielding one of the highest deforestation rates in South America [2,4].

The majority of tropical pastures exist in some stage of degradation [5]. Pasture degradation is understood as a marked reduction in livestock production due to a significant decrease in forage yield and nutritional quality, the invasion of non-palatable plant species leading to bare soil patches (thus increasing susceptibility to erosion), soil compaction, acidification and reduced soil microbial biomass [6,7,8]. This phenomenon has tremendous economic and ecological implications, as it leaves large areas of degraded land and promotes a trend of continuing deforestation [9]. As an example of economic implications, in Brazil, every year, about 8 million hectares of degraded pastures require considerable investment for renewal and/or recovery [3], with an estimated annual cost of USD 100 to 200 ha⁻¹, i.e., around USD 1 billion per year in total at country level [10].

The predominant soils of the deforested area in the Colombian Amazon are highly weathered Haplic Ferralsols and Haplic Acrisols [11]. These acid soils typically have low total and available phosphorus (P) contents. Overgrazing and reduced pools of available nitrogen (N) and P in soil are seen as the principal causes of pasture degradation [6,8,12,13,14]. Thus, grass-legume (GL) associations are an important alternative to grass-alone (GA) pastures because of N input from the legume through symbiotic N₂ fixation [15]. Examples of legume species sown in association with Urochloa grasses are Pueraria phaseoloides, Arachis pintoi, Desmodium ovalifolium, Centrosema spp. and Stylosanthes spp. Despite the aggressive growth of Urochloa grasses [15,16], stable Urochloa/legume associations are possible where grazing is appropriately managed [17,18]. Provided sufficient P supply, tropical forage legumes obtain at least 70% of their N through symbiotic N₂ fixation [19]. A legume proportion range from 20% to 45% of total pasture dry matter has been estimated to cover the N requirement of pasture growth in the tropics [20], similar to grass-clover swards in temperate climates [21]. GL associations also significantly improve animal (meat and milk) productivity [18,22]. Such improvements have been largely related to the production of more forage biomass (mainly in dry season) and of better quality. Mixed grass-legume diets provide highly digestible protein, less structural carbohydrates and therefore increase animal forage voluntary intake, efficiency of nutrient conversion and live weight gain more than diets based on N fertilized grass monocultures [23,24,25]. Whilst the agricultural and environmental benefits of integration of legumes in tropical pastures have repeatedly been shown in researcher-managed fields [18], the adoption of legumes by farmers has remained rather small.

In pastoral systems, the quantity and quality of plant litter inputs are crucial for nutrient cycling [26]. In addition to aboveground litter, belowground inputs composed by root and rhizodeposition may enhance nutrient cycling and availability [27]. The belowground N input of clover growing in GL mixtures under temperate climate was around 40% of aboveground N [28] and about 50% of grass N was legume-derived [29]. Grasses growing in association with legumes thus benefit from symbiotically fixed N, with decomposing legume roots most likely being the main transfer pathway [29,30]. Preliminary research work on the determination of δ¹⁵N values of shoot tissue as part of the study on pasture degradation [8] suggests that Urochloa spp. have different N acquisition strategies, resulting in N uptake from different sources, when growing alone than when growing with legumes. Indeed, Urochloa spp. have a variety of strategies, which could affect the δ¹⁵N signature of their biomass [31]. First, grasses of the genus Urochloa can obtain 20% to 40% of their total N in the plant from the atmosphere through the association with N fixing bacteria (i.e., non-symbiotic N₂ fixation) [32,33]. Secondly, U. humidicola can suppress soil nitrification by releasing inhibitors from roots [34,35], which affects the δ¹⁵N in plants [31]. Thus, the integration of legumes could advance toward improved N supply to the associated grass, either via the provision of fixed N₂, or via N sparing due to the reduced demand of mineralized soil N to support legume growth.

In temperate GL pastures, grasses and legumes mutually benefit to acquire N from diverse symbiotic and non-symbiotic sources, and transform N into biomass more efficiently than pure grass or legume fields [36]. Nevertheless, in spite of the fact that in tropical grasslands the beneficial effects of GL pastures on livestock productivity and soil fertility have been well studied, the underlying processes such as symbiotic N₂ fixation and N sources exploited by mixed pasture components have rarely been determined under farmers’ pasture management conditions.

The objectives of this work carried out under conditions of farmers’ practice, at the plot scale were to: (i) estimate the productivity and N uptake of GA vs. GL pastures; (ii) determine the symbiotic N₂ fixation by legumes; and (iii) evaluate the N sources of grasses growing alone vs. associated with legumes. We hypothesized that legumes would fix significant amounts of N, and that the associated grasses in GL pastures would benefit from the symbiotic N₂ fixation via legume N transfer, and that this legume N transfer would result in lower δ¹⁵N value of grasses in GL than in GA pastures.

2. Materials and Methods

2.1. Study Sites

The study was carried out in farms located in the Caquetá Department of Colombia, within a range of 1°19′13.2″ N to 1°44′37.51″ N, and 75°15′40.69″ W to 75°46′10.4″ W. The area is located in the Amazonia Piedmont of the eastern Andean mountain range in a landscape mostly dominated by degraded pastures. Average annual rainfall is 3758 mm and mean temperature 25.8 °C, with 1570 h of sunshine per year (adapted from IDEAM [37]).

In the study region, the landscape predominates with rolling hills of slopes lower than 25%. The soils originated from sedimentary parent material native of the Amazonic mega-basin [38]. The mineralogical fraction is constituted by gibbsite (Al(OH)₃), kaolinite (Al₂Si₂O₅(OH)₄), mica and goethite [α-Fe₃⁺O(OH)] [39]. The soils present low natural fertility, with textures ranging from silty clay to sandy clay loam, low base saturation, extreme acidity, and exchangeable aluminum saturation at toxic levels for most field crops [38]. Some soil characteristics of studied plots are provided in

Table 1. Soil chemical characteristics (0–10 cm soil depth) of grass-alone and grass-legume pastures sampled on farms in the Caquetá Department of Colombia. Each value represents the mean of ten plots for grass-alone, and of eight plots for grass-legume pastures.

Pasture Type	pH a	Total N (mg g Dry Soil−1) b	NH4+ (mg kg Dry Soil−1) c	NO3− (mg kg Dry Soil−1) c	Total C (mg g Dry Soil−1) b	Bray-II P (mg kg Dry Soil−1) d	C:N	δ15N (‰) b	δ13C (‰) b
Grass-alone	4.8 ± 0.3	2.9 ± 0.5	4.1 ± 1.0	3.1 ± 2.1	32.4 ± 5.6	1.27 ± 0.5	11.2 ± 0.7	5.9 ± 0.7	−20.6 ± 1.6
Grass-legume	4.8 ± 0.1	2.6 ± 0.7	3.9 ± 2.3	3.4 ± 4.5	28.2 ± 7.4	1.09 ± 0.6	10.6 ± 0.8	6.1 ± 1.2	−21.0 ± 1.6

^a Measured in deionized water. ^b Total N, C, δ¹⁵N, and δ¹³C determined by dry combustion using an NCS elemental analyzer coupled to an Isotope Ratio Mass spectrometer (Vario PYRO cube, Elementar, Germany and IsoPrime100 IRMS, Isoprime, United Kingdom) (precision ± 0.2‰), further details are provided in Section 2.3. ^c Mineral N extraction in 1M KCl and quantification of NH₄⁺ and NO₃⁻ following Borrero et al. [40]. ^d Bray-II extractable P [40,41].

.

Ten paired areas (from 0.22 to 3.5 ha) with grass-alone (GA) and grass-legume (GL) pastures in adjacent plots were identified in six farms. Informal interviews with the farmers indicated that pastures were aged between 16 and 32 years, and were sown using tillage with a disc harrow and applying between 0.2 to 1.0 Mg ha⁻¹ of CaMg(CO₃)₂ in five of the farms, but no-tillage or liming was used for establishing pastures in the sixth one (E1-2, Supplementary Table S1). Five of the six farmers have repeated liming since the establishment of the pastures, with intervals of several years (e.g., the plots F1-2 received lime six years before sampling). None of the farms received maintenance fertilizers or renovated pastures by re-sowing them. The grazing management of the pastures is under rotation, usually between one to three days of grazing and between 27 to 45 days of rest to permit recovery and growth of the pasture, with a dual-purpose cattle system for milk production in five farms and beef production in one farm. The establishment and management of pastures differ between farms rather than pasture types. However, sometimes more productive animals graze on GL than on GA (e.g., non-lactating cows grazing in GA), and the grazing duration gets adjusted according to forage availability. Introduced grasses evaluated in the farms were Urochloa humidicola cv. Tully (CIAT 679), U. brizantha cv. Toledo (CIAT 26110), and U. decumbens cv. Basilisk (CIAT 606). The associated forage legumes were either Arachis pintoi cv. Mani Forrajero (CIAT 17434) or Pueraria phaseoloides cv. Kudzu (CIAT 9900). Detailed information about farms management and establishment of pastures, and species found per farm is provided in Supplementary Tables S1 and S2.

2.2. Plant and Soil Sampling

In each pasture, at the end of May 2019, a 25 m² plot was fenced to impede animal grazing and deposition of excreta for 45 days before sampling. In mid-July 2019, one sampling circle of 5 m radius was delimited per plot. Topsoil samples (0–10 cm soil depth) were collected using an Eijkelkamp Edelman soil auger in the center of the circle and in other six points that were equally distant to each other in the periphery. Soil subsamples were then mixed and a composite sample per plot was air-dried for 48 h and passed through a 2 mm sieve. A PVC frame of 1 m² was placed randomly inside each sampling circle. Shoot biomass in the frames was cut to ground level, and harvested after 45 days of regrowth. This relatively long regrowth period was required, because the period fell into the rainy season, with 356, 473 and 611 mm of precipitation during May, June, and July 2019, respectively [42]. Slow rates of regrowth resulted from both, high levels of precipitation and cloudiness during the day, which may have resulted in lower level of photosynthetically active radiation [43], and it may have been partially influenced by the low cutting level (<5.0 cm) used to homogenize the pasture height before harvest. The harvested biomass was split into four botanical fractions: principal grasses (Urochloa spp.), secondary grasses (native/naturalized e.g., Homolepis aturensis and/or Paspalum spp.), legumes, and forbs. A plant litter composite sample was also collected. Plant litter was defined as dead plant parts lying on the ground including dead and completely dry grass leaves still attached to the shoot and senescent legume leaves. Plant samples were oven-dried at 60 °C for 72 h and their dry matter (DM) weight was determined. A subsample of each fraction was ground using a cutting mill (RETSCH, model SM 100) and pulverized using a home-made ball mill with a SIEMENS engine.

At the beginning of the study, we identified 10 paired GA and GL pasture plots. Nevertheless, possibly due to seasonal changes, at the time of the sampling two GL plots showed legume proportions that were lower than 3% and these two plots were not included to the total number of GL plots. Thus, the final results reported were based on 10 GA and 8 GL plots with an average legume proportion of 35% (10–60%) with respect to the total green biomass DM of the plot.

2.3. Chemical and Isotopic Analysis of Plants and Soil

Plant and soil samples were analyzed for total N and C concentration, ¹⁵N/¹⁴N and ¹³C/¹²C isotopic ratios by dry combustion using an NCS elemental analyzer coupled to an Isotope Ratio Mass spectrometer (Vario PYRO cube, Elementar, Germany and IsoPrime100 IRMS, Isoprime, United Kingdom) at ETH Zurich, Eschikon, Switzerland. The natural ¹⁵N abundance values are expressed as δ¹⁵N, i.e., per mil (‰) ¹⁵N excess or depletion over the ¹⁵N/¹⁴N ratio of the air (R_air = 367.6 × 10⁻⁵) [44]:

$${\mathsf{\delta }}^{15}\mathrm{N} (‰)=\frac{ {15}_{\mathrm{N}}/{14}_{\mathrm{N}}{ \mathrm{ratio}}_{ \mathrm{sample}} - {15}_{\mathrm{N}}/{14}_{\mathrm{N}}{ \mathrm{ratio}}_{ \mathrm{air}}}{{15}_{\mathrm{N}}/{14}_{\mathrm{N}}{ \mathrm{ratio}}_{ \mathrm{air}}} \times 1000,$$

(1)

The δ¹³C is accordingly expressed as ¹³C excess or depletion over the ¹³C/¹²C ratio of the international Vienna Pee Dee Belemnite (VPDB) standard (R_VPDB = 1123.75 × 10⁻⁵). For calibration, we used two international standards, IAEA-N-1 (δ¹⁵N = +0.4‰) and IAEA-N-2 (δ¹⁵N = +20.3‰), peptone (δ¹⁵N = +6.7‰) and glycine (δ¹⁵N = +12.2‰) for nitrogen, and peptone (δ¹³C = −15.7‰) and glycine (δ¹³C = −33.25‰) for carbon. Correction for instrumental drift was done by repeated measurement of a sulfanilamide internal standard (δ¹³C = −28‰, δ¹⁵N = −0.8‰). Repeated measurement of the sulfanilamide standard gave an analytical precision of 0.3‰ for δ¹⁵N, and 0.2‰ for δ¹³C. Calibrated pea grain was repeatedly measured as an internal quality check (δ¹³C = −24.7‰, δ¹⁵N = +2.4‰).

The P concentration in the plant tissue was determined after digestion of 0.2 g of pulverized leaf tissue with 2 mL of distilled water and 2 mL of concentrated HNO₃ using a high-pressure single reaction chamber microwave system (turboWave, MWS microwave systems) [45]. The P concentration in the extracts was determined colorimetrically at 610 nm using the malachite green method [46].

Nutrient (N and P) uptake per botanical fraction was calculated by multiplying their nutrient concentration by the biomass production per m². Total nutrient uptake was determined by summing the nutrient uptake of each botanical fraction except plant litter in 1 m².

The weighted δ¹⁵N of the swards (on a m² basis) was determined by applying the formula:

$$\begin{gathered} \mathrm{Weighted} {\mathsf{\delta }}^{15}\mathrm{N} (‰)= \\ \frac{\left[\right({\mathsf{\delta }}^{15}{\mathrm{N} }_{\mathrm{Pg}} \times {\mathrm{N} \mathrm{uptake} }_{\mathrm{Pg}})+({\mathsf{\delta }}^{15}{\mathrm{N} }_{\mathrm{Sg}} \times {\mathrm{N} \mathrm{uptake} }_{\mathrm{Sg}})+({\mathsf{\delta }}^{15}{\mathrm{N} }_{\mathrm{Leg}} \times {\mathrm{N} \mathrm{uptake} }_{\mathrm{Leg}})+({\mathsf{\delta }}^{15}{\mathrm{N} }_{\mathrm{Forbs}} \times {\mathrm{N} \mathrm{uptake} }_{\mathrm{Forbs}})]}{\mathrm{Total} \mathrm{N} \mathrm{uptake} \mathrm{of} \mathrm{the} \mathrm{plot}}, \end{gathered}$$

(2)

where Pg: principal grass, Sg: secondary grass, Leg: legumes, N uptake: N uptake in respective botanical fraction, in g m⁻². The weighed δ¹³C (‰) was calculated accordingly.

The weighted nutrient concentration of N and P of the total biomass per plot:

$$\begin{gathered} \mathrm{Weighted} \mathrm{nutrient} \mathrm{concentration} (\mathrm{mg} \mathrm{g} {\mathrm{DM}}^{-1})= \\ \frac{\left[\right({\mathrm{NC}}_{\mathrm{Pg}} \times {\mathrm{biomass}}_{\mathrm{Pg}})+({\mathrm{NC}}_{\mathrm{Sg}} \times {\mathrm{biomass}}_{\mathrm{Sg}})+({\mathrm{NC}}_{\mathrm{Leg}} \times {\mathrm{biomass}}_{\mathrm{Leg}})+({\mathrm{NC}}_{\mathrm{Forbs}} \times {\mathrm{biomass}}_{\mathrm{Forbs}})]}{\mathrm{Total} \mathrm{biomass} \mathrm{of} \mathrm{the} \mathrm{plot}} \end{gathered}$$

(3)

where NC is the nutrient concentrations (N or P, in mg g⁻¹).

2.4. Legume N Derived From the Atmosphere

The proportion of N derived from the atmosphere (%Ndfa, i.e., fixed N) was determined by the ¹⁵N natural abundance method [44] and applying the following formula:

$$\mathrm{Ndfa} (\%)=\frac{{\mathsf{\delta }}^{15}{\mathrm{N} }_{\mathrm{ref}} -{ \mathsf{\delta }}^{15}{\mathrm{N} }_{\mathrm{leg}}}{{\mathsf{\delta }}^{15}{\mathrm{N} }_{\mathrm{ref}} - \mathrm{B}} \times 100,$$

(4)

where δ¹⁵Nref: δ¹⁵N signature of the non-fixing reference plant shoots, δ¹⁵Nleg: δ¹⁵N signature of the legumes shoots, B: is the δ¹⁵N of Arachis pintoi or Pueraria phaseoloides shoots relying on atmospheric N₂ as a sole source of N and it accounts for any internal isotopic fractionation of legume plants [47].

For reference plant, we used the forbs growing in the sampling area, i.e., in association with the legume. The δ¹⁵N of non-N₂-fixing reference plant was assumed to be identical to the δ¹⁵N of soil N taken up by the legume [48]. B values used in our study were obtained from previous reports for the legume species studied or the most closely related species of the same genus. For Pueraria phaseoloides the B value used was −1.22 [49]. For Arachis pintoi we used −0.88, the mean of three values reported for Arachis hypogea [49,50,51].

The amount of N fixed was calculated for each GL plot by multiplying the N uptake in shoot DM of legumes with the respective %Ndfa.

2.5. Data Analysis

Statistical analyses and figures were performed using R v3.4.4. Significant differences between botanical fractions were assessed through a linear mixed-effects model treating pasture type and botanical fraction as fixed factors, and farm as random effect using the packages ‘lme4′ v1.1-23 and ‘nlme’ v3.1-147 in R. Multiple comparisons between botanical fractions were evaluated with TukeyHSD tests using ‘emmeans’ v1.4.8. To evaluate total differences between pasture types, weighted nutrient concentrations and isotopic signatures were calculated per farm, and a second model was built treating pasture type as fixed factor and farm as random effect. Analysis of δ¹⁵N of grass and legume species followed the same model structure as for botanical fractions, considering species and pasture type as fixed factors, and farm as random effect. Differences in %Ndfa between legume species were not statistically tested due to very high differences in sample size (n = 6 for A. pintoi, and n = 2 for P. phaseoloides). The correlation between δ¹⁵N of grasses and forbs, and the δ¹⁵N of grasses with grass biomass and N concentration were tested with the Pearson’s correlation coefficient (r). Figures were constructed using ‘ggplot2’ v2.2.1.

3. Results

3.1. Dry Matter Productivity and Nutrient Uptake

Grass-legume pastures produced more plant biomass and had greater nutrient (N and P) uptake than GA pastures. Excluding the plant litter fraction, the extent of increase in GL compared to GA swards was up to 74% for shoot DM production (g DM m⁻²: 62 in GA vs 108 in GL), while it was more than two-fold higher for N uptake (g N m⁻²: 0.8 in GA vs 2.2 in GL) and P uptake (g P m⁻²: 0.07 in GA vs. 0.14 in GL) (Figure 1). The proportion of biomass of forbs in the total plant biomass of the pastures was lower in the GL (3% of total DM) than in the GA pastures (16% of total DM).

As expected, N concentrations were significantly higher in legume than grass shoots (

Table 2. Nutrient concentrations (N, P, C) and isotopic signatures of N and C in two different pasture types for each botanical fraction. Values are mean ± standard deviation, with n = 10 for grass alone, and n = 8 for grass-legume pastures. DM = dry matter, ns = not significant.

Pasture Type	Botanical Fraction	N Concentration (g N kg DM−1)	P Concentration (g P kg DM−1)	C Concentration (g C kg DM−1)	C:N	δ15N (‰)	δ13C (‰)
Grass alone	Forbs	18.8 ± 4.1 c	1.3 ± 0.4 b	411.1 ± 5.4 a	22.7 ± 4.7 a	5.3 ± 1.1 d	−25.0 ± 5.1 b
	Principal grass	14.9 ± 3.5 b	1.1 ± 0.3 b	424.0 ± 5.4 b	30.0 ± 7.6 a	4.5 ± 3.1 c	−13.3 ± 0.5 d
	Secondary grass	15.3 ± 5.3 bc	1.4 ± 0.3 b	420.5 ± 8.1 ab	30.5 ± 11.4 a	2.5 ± 3.0 c	−20.9 ± 6.2 c
	Legumes	22.1 ± 4.8 d	1.2 ± 0.1 b	447.9 ± 9.0 c	20.7 ± 4.9 a	−0.6 ± 0.6 a	−31.0 ± 0.0 a
	Plant litter	6.8 ± 1.8 a	0.4 ± 0.1 a	424.8 ± 11.7 b	67.1 ± 20.1 b	1.9 ± 2.3 b	−16.9 ± 3.8 c
	Total *	17.4 ± 3.9 A	1.2 ± 0.3 A	426.2 ± 11.5 A	25.8 ± 7.1 A	4.6 ± 2.9 B	−17.1 ± 3.8 A
Grass legume	Forbs	18.4 ± 3.1 c	1.4 ± 0.4 b	417.4 ± 7.5 ab	23.1 ± 3.7 a	5.7 ± 2.5 d	−25.4 ± 5.3 b
	Principal grass	14.8 ± 3.5 b	1.3 ± 0.5 b	426.9 ± 5.0 b	30.1 ± 6.8 a	3.8 ± 2.9 c	−12.7 ± 0.4 d
	Secondary grass	15.4 ± 2.1 bc	1.4 ± 0.3 b	419.8 ± 5.0 ab	27.6 ± 3.7 a	3.6 ± 3.2 c	−18.0 ± 3.8 c
	Legumes	27.8 ± 3.3 d	1.3 ± 0.3 b	422.1 ± 9.2 ab	15.3 ± 1.8 a	0.4 ± 1.0 a	−29.6 ± 0.3 a
	Plant litter	7.9 ± 1.3 a	0.5 ± 0.2 a	417.1 ± 8.6 ab	53.6 ± 7.3 b	1.1 ± 2.1 b	−16.0 ± 0.9 c
	Total *	20.5 ± 3.2 B	1.2 ± 0.2 A	404.9 ± 46.2 A	20.0 ± 3.3 A	2.1 ± 1.7 A	−19.9 ± 3.5 A
Source of variation **	Pasture type	ns	ns	p < 0.05	ns	ns	ns
	Botanical fraction	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.001
	Pasture type x botanical fraction	ns	ns	p < 0.001	ns	ns	ns
	Farm (random)	p < 0.001	p < 0.001	p < 0.05	ns	p < 0.001	p < 0.001

* Calculated values of weighted nutrient concentration or isotopic signature of each botanical fraction (forbs, grasses and legumes only) by their nutrient uptake as in Equations (2) and (3). ** Sources of variation apply only for comparison of botanical fractions. For each variable, different lowercase letters indicate statistical differences of botanical fractions within and between pasture types. Uppercase letters indicate statistical differences for the total (weighted) concentrations between pasture types according to the Tukey HSD test (α = 0.05).

). In contrast, P concentrations did not differ significantly between grasses and legumes. Weighted N concentration of GL plant biomass was 18% higher than that of the biomass in GA plots. No difference was observed in weighted plant biomass P concentration between the two pasture types. Therefore, the higher P uptake observed in GL than GA pastures resulted from greater DM production.

3.2. Legume-N derived from the atmosphere

The weighted δ¹⁵N signature of the combined plant biomass of GA pastures was 4.6‰, while it was 2.1‰ for GL pastures (Table 2). The δ¹⁵N signature of forbs was similar to that of soil N, whereas that of principal grasses was on average by 1.4‰ (GA) and 2.3‰ (GL) less enriched than soil N (Table 1 and Table 2, Figure 2). Therefore, forbs were considered as more appropriate reference plants to determine Ndfa than grasses.

Arachis pintoi was the legume species found in six out of eight farms, and P. phaseoloides occurred in two farms. Average Ndfa derived for GL pastures using Equation (4) ranged from 60% to 99% (average value of 80%,

Table 3. Grass and legume species, average δ¹⁵N signature of shoots and %Ndfa ± standard deviation of the mean observed per species. Ndfa = Nitrogen derived from the atmosphere, ns = not significant.

Pasture Type	Species	δ15N (‰)	Ndfa (%)
Grass alone	U. brizantha	4.7 ± 4.2 ab	-
	U. decumbens	5.8 ± 1.1 b	-
	U. humidicola	3.2 ± 4.2 ab	-
Grass legume	U. brizantha	4.9 ± 2.7 ab	-
	U. decumbens	5.0 ± 0.0 ab	-
	U. humidicola	2.0 ± 3.3 ab	-
	A. pintoi	0.4 ± 0.9 a	83.2 ± 14.0
	P. phaseoloides	1.3 ± 0.6 ab	67.5 ± 9.2
Source of variation	Pasture type	ns	-
	Species	p < 0.05	-
	Pasture type x species	ns	-
	Farm (random)	ns	-

Different letters indicate statistical differences according to the TukeyHSD test (α = 0.05).

). A. pintoi showed on average 16% higher %Ndfa than P. phaseoloides. The amount of N fixed in the biomass of legumes ranged from 0.15 to 3.7 g N m⁻².

3.3. δ¹⁵N and δ¹³C Isotopic Signature of Pasture Components

Urochloa humidicola was the principal grass in four out of ten GA plots and three out of eight GL plots, while it was U. decumbens in four GA plots and one GL, and U. brizantha in two GA and four GL plots (Supplementary Table S2). The δ¹⁵N signature of principal grasses varied widely among plots of the same type of pasture. In three out of ten plots, the GA-principal grass showed δ¹⁵N lower than 2‰, and in two of them (A2 and F1), even lower than the corresponding GL principal grass signature (Supplementary Table S2). The average δ¹⁵N signature of shoot tissue of U. humidicola tended to be lower than that of U. decumbens and U. brizantha, however, this difference was not statistically significant (Table 3).

The δ¹⁵N of the principal grass was negatively related to the DM production of the principal grass (r = −0.5, p < 0.05. Figure 3a), and positively related to the N concentration of the principal grass shoot tissue (r = 0.5, p < 0.05. Figure 3b). However, this latter correlation was stronger in the GA pastures (r = 0.68, p < 0.05) than in GL (r = 0.26, non-significant). At N concentrations higher than 14 mg g⁻¹ in the shoot tissue, the δ¹⁵N of grasses growing in GL pastures was lower than that of GA pastures by at least 2‰ (p < 0.01).

The δ¹⁵N values of the principal grass and the forbs were closely related (r = 0.76, p < 0.01. Figure 2). The δ¹⁵N value of the principal grass was lower than that of the forbs, on average by 0.8‰ for GA pastures, and by 1.9‰ in GL pastures (Table 2, Figure 2).

The δ¹³C signature of grasses was significantly higher (less negative) than that of forbs and legumes (Table 2). The type of pasture had no significant effect on δ¹³C of the botanical fractions and soil organic matter (Table 1 and Table 2, respectively).

4. Discussion

4.1. Legumes Improve Pasture Productivity and Nutrient Uptake

The average biomass production of 62 g m⁻² in GA pastures was in the range obtained by Fonte et al. [26] on farms in the same study region (average values of 101 g m⁻² for Urochloa spp. GA pastures which farmers had characterized as productive, and 47 g m⁻² for degraded pastures). Moreover, during the rainy season, as in our study and Fonte et al. [26], Gomez et al. [43] harvested from fertilized Urochloa spp. GA pastures on average 250 g m⁻² after 42 days of regrowth of the pasture. Pasture biomass production was reduced by a factor of two to three during the wettest months compared to drier months [43], and June 2019 had a very high amount of precipitation. Lower biomass production during the wettest months was probably due to low soil oxygen levels caused by waterlogging and/or lower rates of net photosynthesis associated with a lower level of photosynthetically active radiation, which may have been driven by higher cloudiness [52].

The pastures containing legumes (i.e., GL) had a significantly higher biomass production (Figure 1a). This is in agreement with earlier results obtained from on-station experiments. For instance, the DM production of GL pastures composed by U. decumbens and Calopogonium mucunoides was 1.25 times greater than that of GA pastures containing U. decumbens alone in an experiment near Campo Grande, Brazil [53], and DM production was doubled in experiments with U. decumbens and A. pintoi association, as compared to U. decumbens alone in Carimagua, Colombia [17].

Because Urochloa grasses obtain atmospheric N₂ via non-symbiotic N₂ fixation [32,33,54], we used the δ¹⁵N of the forbs as an indicator of the δ¹⁵N of available soil N. The strong depletion of δ¹⁵N in legumes compared to the forbs and grasses indicated that legumes largely relied on atmospheric N (Table 2 and Table 3) [44]. The average value of 35% legume DM in the total pasture biomass is within the range of 35–45% proposed by Thomas [20] required to maintain a balanced N cycle of tropical GL pastures with a herbage utilization of 50–70% by grazing animals. The proportion of N derived from the atmosphere (%Ndfa) observed with the legumes in this study was about 80% and this value is at the high end of the range reported for tropical legumes [19,55]. This is remarkable because available P concentrations of less than 2 mg kg soil⁻¹ are considered as plant growth-limiting for both grasses and legumes [56,57] and could, therefore, limit N₂ fixation of legumes [19].

The amount of N fixed in the shoots of legumes observed in this study was 0.15 to 3.7 g N m⁻² for 45 days of regrowth. Thus, the average increase in N uptake of 1.4 g m⁻² in GL pastures than GA pastures was largely due to N₂ fixing ability by the legume (Figure 1b). Because on average around 80% was derived from N₂ fixation, some legume N transfer to grasses might still explain the overall greater N uptake in GL than GA pastures. In temperate GL meadows, about 50% of the grass N was derived from the associated legumes [29]. Trannin et al. [30] suggested that the mineralization of root residues from Stylosanthes guianensis was the major source of legume-N transferred to the associated Urochloa decumbens. Moreover, greater N deposition through litter has been reported for GL pastures than GA pastures [58].

The P uptake was doubled in GL compared to the GA pasture (Figure 1c), at similarly low available P status of the soil (Table 1). It was reported that legumes such as A. pintoi acquire more P from less available P pools from acid soil with its smaller root system than Urochloa grass [59]. Thus, the increase of 72 mg P m⁻² acquired by GL pasture can be attributed to the superior performance of legume towards improved P cycling in the system through soil P pools [60]. According to the farmers’ information, the GL pastures are grazed by more productive animals than the GA pastures, and more P may hence be exported via animal products from plots containing legumes, as suggested by Oberson et al. [61] for Urochloa-Kudzu pastures. Thus, legumes may take up more soil P and stimulate biological P cycling (through plant litter, animal excreta and microbial turnover) to keep it in available P forms. Although this could increase the risk of soil P mining [62], the strategic application of small amounts of P fertilizer (10 kg ha⁻¹ every two years as maintenance fertilizer) may overcome the risk for soil P mining in grazed pastures [63].

4.2. What N Sources are Exploited by Grasses in Each Pasture Type

The δ¹⁵N of the principal grasses, all of which were Urochloa spp. grasses was higher than that of the legumes except in two cases (Table 3, Supplementary Table S2). At the same time, their δ¹⁵N was lower than that of the associated forbs (Table 2, Figure 2). This observation indicates that the grasses, on average, were benefiting less from atmospheric N₂ than the legumes, irrespective of the underlying process.

The N concentrations of Urochloa spp. grasses observed in our study were higher than previous reports [64], although it was not sufficient to sustain higher plant growth, as indicated by the lower value of biomass production. At N concentrations higher than 14 mg g⁻¹, the δ¹⁵N of the principal grasses of GL was lower than that of GA pastures by at least 2‰. This suggests that legume N transfer was a process involved in the provision of atmospheric N to grasses. Atmospheric N₂ fixation seems to make a significant contribution resulting in low δ¹⁵N values of the Urochloa grasses in both GA and GL pastures (Table 3). Still, the contribution from non-symbiotic N₂ fixation in GA may not be adequate to sustain grass growth without the supply of N from the soil through mineralization. This finding is consistent with earlier reports that suggested no more than 20–40% of N in Urochloa grasses was derived from the atmosphere via non-symbiotic N₂ fixation [32,33].

C₄ grasses typically have δ¹³C higher than −20‰, whereas C₃ legumes usually have δ¹³C lower than −20‰ due to differences in C isotopic fractionation during CO₂ assimilation [65]. In our study, the δ¹³C and δ¹⁵N of soil was not statistically different between pasture types, around −20‰ and 6‰, respectively (Table 1 and Table 2). While the former forest C₃ vegetation still affects the isotopic composition of soil organic matter C [66], the contribution of legume residues seems to not have been enough to enrich the total soil N pool.

Although the grasses with the lowest δ¹⁵N and N concentration in shoot tissue were mostly U. humidicola (Figure 3b), no clear pattern of distribution was observed among Urochloa species, either for plant biomass production or N concentration (Figure 3a, b). We consider that the distribution of grass species observed in our study is representative of the pastures in the region. However, to draw valid conclusions on N uptake and utilization at the species level, a more balanced design with an equal number of observations per species will be needed in future research.

Low ¹⁵N natural abundance in the shoot tissue of U. humidicola has been interpreted as an indicator of high capacity of biological nitrification inhibition (BNI) in that grass [31]. Indeed, in our study, U. humidicola showed the lowest δ¹⁵N values, both in GA and GL pastures, but this grass was found to obtain a relatively significant proportion of N through non-symbiotic N₂ fixation [33]. Our results rather suggest that low δ¹⁵N of the grasses is an indicator of low N availability in soil [67], and grasses adapted to N depleted soils can cope with either through BNI ability [31] and/or with non-symbiotic N₂ fixation [33].

5. Conclusions

In farmers’ long-term tropical pastures established in the forest margins of Colombia, legumes associated with grasses (GL pastures) resulted in greater pasture biomass production than grass-alone (GA) pastures. Legumes derived on average 80% of their N from symbiotic N₂ fixation, despite low fertility acid soils with low plant-available P content. Legumes significantly increased both N and P uptake by the pasture biomass. The greater N uptake by legumes could be assigned mostly to N fixed from the atmosphere. The δ¹⁵N signatures of grasses in GA vs. GL pastures suggested that sources of grass N are affected by legumes integrated in the pasture. While lower δ¹⁵N values of grasses growing in GL than GA pastures suggest that grasses could obtain fixed N via legume N transfer, exceptionally lowδ ¹⁵N values of grasses in GA pastures indicate significant potential for N input via non-symbiotic N₂ fixation from the atmosphere. Overall, this study indicates that Urochloa grasses are capable to swap N sources when these grasses are grown in association with legumes. The role of different soil-plant processes such as BNI or N₂ fixation from the atmosphere need to be further studied under field and also controlled conditions. This missing knowledge is critical to define the sources of N for grass growth, either in GA or GL pastures in the tropics.