Biological Nitrification Inhibition in Urochloa Genotypes and Implications for Biomass Production and Nitrogen Uptake

Abstract

The identification of forage species with Biological Nitrification Inhibition (BNI) capacity is a promising strategy to inhibit soil nitrification and reduce nitrogen (N) losses. This study evaluated the BNI capacity of five Urochloa genotypes (Camello, Cayman, Marandú, Mulato II, Talismán) and their impact on biomass yield and nitrogen uptake (NU). The BNI capacity, biomass yield, N content, and NU of five Urochloa genotypes were compared. Significant differences in BNI capacity were observed between genotypes (p < 0.009). Cayman and Marandú presented the highest BNI values (87.41 and 87.21%, respectively), higher than those of Mulato II, Talismán and Camello (78.20, 81.77 and 82.63%, respectively). Regarding biomass yield, Cayman and Marandú stood out with 3093.5 and 2911.7 kg DM ha⁻¹, respectively. Talismán and Camello showed higher N concentrations in the biomass (1.64 and 1.63%). In terms of NU, Cayman recorded the highest efficiency (47.32 kg N ha⁻¹), surpassing Marandú, Camello, Talisman and Mulato II (42.83, 42.77, 41.53 and 37.23 kg N ha⁻¹, respectively; p < 0.0001). BNI capacity influences biomass yield and nitrogen uptake. The Cayman genotype is positioned as a promising forage alternative for the development of more efficient and sustainable livestock systems by promoting more efficient N use.

1. Introduction

Biological nitrification inhibition (BNI) is the ability of certain plant roots (i.e., Oryza sativa) to suppress nitrification by producing and releasing exudates known as BNIs [1,2]. These compounds can block the ammonia monooxygenase and hydroxylamine oxidoreductase (HAO) pathways of ammonium-oxidizing bacteria and archaea in the soil, which are responsible for converting ammonium (NH₄⁺) to nitrate (NO₃⁻) [3,4], reducing the real nitrification rates in agricultural soils [5]. However, the expression and effectiveness of BNI is not solely a plant-intrinsic trait; rather, it is strongly modulated by edaphic conditions (i.e., moisture) that influence both the production and release of inhibitory exudates [6]. These interactions highlight the ecological complexity of BNI and the need for further research to understand its variability across species, genotypes, and environmental contexts.

Nitrate is highly mobile and susceptible to leaching, which can potentially lead to environmental pollution that affects both groundwater and human health. In addition, it can be transformed into nitrous oxide (N₂O) through denitrification [1,2,3,4,7], N₂O is a greenhouse gas significantly more potent than carbon dioxide, and its global warming potential is approximately 300 times greater [4,8,9]. Anthropogenic activities account for about 43% of total global N₂O emissions, and nitrogen additions in agriculture represent the dominant share of this contribution, generating approximately 52% of anthropogenic N₂O emissions [10]. These emissions contribute to global warming, putting the resilience of agri-food systems worldwide at risk and highlighting the need for livestock systems to transition towards more sustainable practices, such as agroecological and low-emission approaches [11]. BIN can attenuate these emissions by slowing the conversion of ammonium to nitrate, thereby reducing the availability of NO₃⁻ as a substrate for denitrification and ultimately decreasing N₂O production [12].

Nitrogen (N) losses in the form of N₂O and NO₃⁻ represent about 70% of fertilizer nitrogen use worldwide, equivalent to more than 81 million dollars per year, with a nitrogen uptake (NU) of less than 30% [7,8,13]. Recent evaluations of tropical forage grasses indicate that nitrogen use efficiency (NUE) in these systems is particularly low, with limited recovery of applied nitrogen even under controlled fertilization regimes [14]. This reduces the availability of soil nitrogen for pastures, which can affect their biomass production, crude protein content and consequently can negatively impact cattle productivity [15]. Therefore, uncontrolled excess nitrification can lead to significant N losses, environmental and economic problems [16].

As a mitigation strategy, BNI has been proposed to recover N losses in livestock systems and contribute to food production with low environmental impact [14]. Among the grass species with BNI capacity, Urochloa humidicola (genus Urochloa, formerly Brachiaria), secretes exudates such as brachialactone, with reported BNI capacity ranging from 60 to 90% [8]. Furthermore, Urochloa is one of the most widely used forage species in tropical livestock systems. Despite its agronomic relevance, information on BNI capacity across Urochloa species remains limited, although recent studies suggest substantial variability in nitrification inhibition potential among cultivars [5,16,17]. In this inhibition range, nitrogen remains longer in the form of ammonium in the soil, which allows plants to improve their nitrogen uptake and biomass yield [14,18].

Since the discovery of BNI capacity in U. humidicola and given its importance in livestock systems as fodder for cattle feeding, as well as in the control of soil erosion and carbon capture [15,19], the International Center for Tropical Agriculture has been developing new genotypes with this capacity as an environmental benefit. Among the Urochloa genotypes such as Mulato II, Cayman, Talisman, Camel and Marandú, which could have better BNI capacity. However, there is still little information about its biological nitrification inhibition ability and its impact on biomass production and nitrogen uptake. Urochloa mosambicensis has gained attention for its adaptability to semi-arid environments, high tillering ability, stoloniferous growth habit, and strong regrowth capacity under water deficit [20,21,22]. The species is used for grazing, hay production, and integrated systems, showing promising biomass productivity and drought tolerance across fertilization and cutting regimes. These agronomic traits highlight its potential for sustainable forage production and justify further investigation into its biological nitrification inhibition (BNI) capacity [23].

Despite the recognized BNI potential in certain Urochloa species, there is still a lack of systematic characterization of genotypic variability in BNI capacity and its relationship with nitrogen use efficiency and biomass production. Recent studies have shown that BNI capacity can vary up to threefold among genotypes within the same species, as observed in Sorghum bicolor and B. humidicola [5,24]. Moreover, the expression and effectiveness of BNI are strongly influenced by plant–soil–microbiota interactions, highlighting the importance of field-based evaluations to capture the real expression of the trait under tropical conditions [12]. Addressing this gap through comparative field evaluation could help identify superior genotypes that enhance sustainability and productivity in tropical forage systems. Understanding such variability under field conditions is essential to identify genotypes combining high productivity and enhanced nitrogen uptake.

The study aimed to evaluate Urochloa genotypes for their impact on biomass production and nitrogen uptake, as a first step toward identifying grass genotypes with traits potential associated with biological nitrification inhibition and more sustainable nitrogen utilization in tropical forage systems. We hypothesized that Urochloa genotypes have differentiated capacities for biomass production and effective use of nitrogen due to their variability in their capacity for biological nitrification inhibition.

2. Materials and Methods

2.1. Study Site and Experimental Design

This study was carried out under greenhouse conditions at the Biological and Agricultural Sciences Campus of the Autonomous University of Yucatán, located in the municipality of Xmatkuil, at Merida, Yucatán, Mexico. The study had a completely randomized experimental design with six treatments (five Urochloa genotypes and one control) and 10 replications each. The treatments evaluated were five genotypes of Urochloa (Mulato II, Cayman, Talismán, Camello and Marandú) and one control treatment consisting of pots with soil only (no grass cultivation). The experimental unit consisted of pots filled with 3 kg of red soil. The soil used was classified as red Leptosol, with the following physicochemical properties: pH (H₂O) of 7.5, electrical conductivity of 0.48 dS·m⁻¹, cation exchange capacity of 14.32 cmol·kg⁻¹, available phosphorus (Mehlich 1) of 86.72 mg·kg⁻¹, total N 44.25 mg kg⁻¹, and exchangeable potassium (K⁺) of 0.05 cmol·kg⁻¹. Twenty seeds of each Urochloa genotype were sown per pot and thinned to three plants after 30 days.

2.2. Management of Experimental Units

Plants were watered every two days with 1.8 L per pot, maintaining soil moisture at 60%, considered optimal for the nitrification process [8]. Mineral nitrogen was applied at 100 kg N ha⁻¹ using diammonium phosphate ((NH₄)₂HPO₄) (DAP). This rate was selected based on previous studies reporting optimal biomass responses of tropical forage grasses at similar nitrogen levels [21]. Given the low phosphorus availability typical of karst soils, diammonium phosphate was used to prevent P limitation and allow the genotypes to express their maximum growth potential. The application rate was kept constant across treatments to minimize differential P effects on experimental outcomes. The first application was 3 months after establishment, then it was applied monthly for 12 months.

2.3. BNI Capacity

To estimate the biological nitrification inhibition capacity of Urochloa genotypes, 50 g of soil were collected from each experimental pot, 15 days after nitrogen application and 180 days after establishing. Soil samples (30 g per pot) were collected at 0–15 cm depth, homogenized by treatment and stored in plastic bags. Soil samples were then oven-dried for 24 h at 105 °C. Subsequently, soil samples were sieved through a 2 mm mesh to remove plant debris and coarse particles. Ammonium content was determined in triplicate at 667 nm and the nitrate content at 410 nm by UV–visible spectroscopy, using a Synergy HT spectrophotometer (BioTek Instruments, Santa Clara, CA, USA), to the Soil, Plant and Water Analysis Laboratory of the UADY.

The BNI capacity of each Urochloa genotype was calculated (Equation (1)), using the average nitrate concentration of soils cultivated with grasses and without grass cultivation, based on the equation used by [25], who used the nitrification rate of the soils.

$$\mathrm{B}\mathrm{N}\mathrm{I} \mathrm{c}\mathrm{a}\mathrm{p}\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{t}\mathrm{y}\left(\%\right)=\left({\displaystyle \frac{{\mathrm{N}\mathrm{O}}_3^{-}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-{\mathrm{N}\mathrm{O}}_3^{-}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{{\mathrm{N}\mathrm{O}}_3^{-}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\right)\left(100\right)$$

(1)

where NO₃⁻ control was the average nitrate concentration of the soil without grass cultivation and NO₃⁻ shows the average nitrate concentration of the soil of the tested grass.

2.4. Biomass Yield Estimation

Three months after establishment, all grass genotypes were cut by hand to 5 cm above ground level. Subsequently, the aboveground biomass of each genotype was harvested every 30 days for 12 months. The biomass was weighed fresh, and a subsample of approximately 150 g was taken for dry matter determination. This subsample was dried at 55 °C in a forced-air oven for 48 h to constant weight and dry matter yield was calculated and expressed as kg DM ha⁻¹.

2.5. Determination of Total N in Biomass

Biomass samples were ground in a Thomas Wiley mill (Thomas Scientific, Swedesboro, NJ, USA) to pass a 1 mm sieve and stored until the samples were sent to the Animal Nutrition Laboratory at the Faculty of Medicine Veterinary and Animal Sciences, Yucatan University. Then, triplicate analyses of nitrogen content were performed. Nitrogen content was determined by the micro-Kjeldahl digestion method using a Velp Scientifica™ A00000169 unit (Velp Scientifica, Usmate, Italy) [26].

2.6. Nitrogen Uptake (NU)

The NU of each grass genotype was determined by calculating the total N uptake. This was calculated as dry matter yield (kg DM ha⁻¹) multiplied by N concentration (%/100) [14].

2.7. Statistical Analysis

The evaluated variables ammonium, nitrate, total available nitrogen, soil nitrification (%), biomass yield, nitrogen uptake (%), N content in biomass, and NU were subjected to statistical tests to verify assumptions of normality (Shapiro–Wilks test) and homoscedasticity (Levene test). Subsequently, they were analyzed by analysis of variance (ANOVA) for a completely randomized design. When significant differences were detected (p < 0.05), means were compared using the least significant difference (LSD) test at a 5% error level. The analyses were performed using InfoStat, (InfoStat, Universidad Nacional de Córdoba, Argentina), version 2008 [27].

3. Results

3.1. Biological Nitrification Inhibition (BNI) Capacity

Table 1 shows that the concentration of ammonium, nitrate, total available nitrogen and the% nitrification in the soils of the genotypes of Urochloa and uncultivated soil showed statistically significant differences (p < 0.005, p < 0003, p < 0.0001, respectively). The highest nitrification rate was observed in uncultivated soil (91.4%), reflecting a high conversion of NH₄⁺ to NO₃⁻. In contrast, soils of the Cayman and Marandú genotypes showed lower nitrification (32.4% and 38.6%, respectively), with a higher biological nitrification inhibition capacity (87.4% and 87.2%, respectively). On the other hand, the soils of the Camello, Talisman and Mulato II genotypes presented a higher concentration of NO₃⁻, with nitrification levels of 53.4%, 57.9% and 69.3%, which suggests a lower capacity for biological inhibition of nitrification (82.6%, 81.8% and 78.2%, respectively).

Table 1. Concentration of mineral nitrogen in soil of Urochloa genotypes and their biological nitrification inhibition (BNI) capacity, based on the formation of NO₃⁻.

Treatments	NH4+ (mg kg−1 Soil)	NO3− (mg kg−1 Soil)	Available N (mg kg−1 Soil)	Nitrification (%)	Ability % BNI
Mulatto II	8.91 d	20.71 b	29.62 c	69.35 b	78.20 b
Cayman	25.05 a	11.96 c	37.00 b	32.40 d	87.41 a
Talisman	12.53 c	17.31 b	29.85 c	57.95 c	81.78 b
Camello	14.47 c	16.50 b	30.97 c	53.42 c	82.63 a
Marandú	19.44 b	12.14 c	31.58 c	38.58 d	87.22 a
Control	8.91 d	95.01 a	103.92 a	91.43 a
SEM	1.19	1.36	0.99	3.10	1.35
p-value	0.0005	0.0003	0.0001	0.0001	0.009

Means followed by different letters in each column indicate statistical differences according to the LSD (p ≤ 0.05). SEM, standard error of the means.

3.2. Biomass Yield, Total Nitrogen Content, and Nitrogen Uptake

The Urochloa genotypes exhibited significant differences in biomass yield. Cayman and Marandú achieved the highest production levels, with 3093.55 and 2911.75 kg DM ha⁻¹, respectively. Followed by Mulato II, Camello and Talismán, with reductions of 10.68%, 15.94% and 18.10%, respectively. Talismán recorded the lowest yield, with an 18.10% decrease compared to Cayman.

The results showed statistically difference in the percentage of N in biomass among the genotypes analyzed. In Table 2, the genotypes Marandú, Mulato II, and Cayman presented similar average values (1.51% and 1.52%), with no significant differences among them. Camello and Talismán showed values of 1.63% and 1.64%, respectively, and were statistically different (p < 0.05) from the rest.

Table 2. Biomass yield, total nitrogen content, and nitrogen uptake of five different Urochloa genotypes.

Treatments	Yield (kg MS ha−1)	Nitrogen Content (%)	Nitrogen Uptake (kg MS ha−1)
Cayman	3093.55 a	1.52 b	47.32 a
Marandú	2911.75 a	1.51 b	42.83 b
Mulato II	2763.06 b	1.51 b	37.23 c
Camello	2600.46 b	1.63 a	42.77 b
Talismán	2533.55 c	1.64 a	41.53 b
SEM	70.16	0.02	0.78
p-value	0.005	0.027	0.001

Means followed by different letters in each column differ significantly according to LSD (p ≤ 0.05). Values are presented as mean ± standard error.

Statistical analysis indicated significant differences in N uptake between Urochloa genotypes. Table 2 shows that Mulato II had the lowest N uptake, with an average of 37.23 kg N ha⁻¹, while the genotypes Talismán, Camello and Marandú exhibited similar values, around 41.53–42.83 kg N ha⁻¹, with no significant differences between them. Cayman recorded the highest N uptake with an average of 47.32 kg N ha⁻¹, establishing a statistical difference with respect to the other genotypes.

4. Discussion

4.1. Biological Nitrification Inhibition Capacity (BNI)

Biological nitrification inhibition is a fundamental mechanism by which certain plants release radical exudates capable of suppressing the activity of nitrifying bacteria, limiting the conversion from NH₄⁺ to NO₃⁻ in the soil [8]. This process represents a sustainable alternative to improve the uptake of nitrogen use and reduce its loss through leaching and denitrification.

In addition, Ref. [28] reported that application of linoleic acid, a compound exuded by U. humidicola, resulted in a conversion of only 6.1% of the available N to NO₃⁻, while 93.9% remained as NH₄⁺; the inhibitor’s efficacy was 87.5% in a volcanic soil with pH 6.0, underlining its potential as an BNI agent. Consistent with this evidence, the present study showed that soils cultivated with Urochloa genotypes Cayman and Marandú, where the transformation of NH₄⁺ to NO₃⁻ was significantly lower compared to uncultivated soil. The estimated BNI capacities in these genotypes were 87.4 and 87.2%, respectively, reinforcing the hypothesis that certain Urochloa genotypes play a key role in regulating nitrification.

In contrast, our results exceed the inhibition levels reported by [29] in fresh Andosol soils (67–76%, pH 5.9), since the Mulato II, Talismán, and Camello genotypes presented higher BNI values (78.2%, 81.7%, and 82.6%, respectively). These levels are comparable to those found by [30], who documented an BNI of 80% at 360 days after planting U. humidicola in oxisol soils with a pH of 4.3. Such differences across studies can be attributed, in part, to variations in soil type, pH, and genetic factors, as noted by [31]. In particular, pH plays a determining role: according to [32], the effectiveness of exudates remains stable between pH 3 and 10, but decreases temporarily around pH 4.5, and is completely lost above pH 10. Likewise, Ref. [33] highlights that pH directly affects the BNI secretion mechanism in the rhizosphere, and [17] also note that BNI effectiveness and persistence are strongly conditioned by soil physicochemical properties, including pH.

In this study, soil pH ranged from 7.4 to 7.8; it has been reported that soil nitrification is highly sensitive to pH, and alkaline and neutral soils have much faster nitrification rates than acidic soils [34]. Therefore, under these karstic conditions with pH > 7.0, nitrification remains consistently high and stable, reducing the likelihood that pH differences contributed to the variability in BNI observed among genotypes. Likewise, soil moisture also modulates the release of compounds with BNI activity, as demonstrated by [6], who observed that in Sorghum bicolor, water stress reduced the abundance of AOB to a lesser extent than under irrigated conditions, thus preserving a greater reserve of NH₄⁺ in the soil, available for absorption by crops, as also supported by [5], who reported that sorgoleone markedly suppresses nitrifiers and limits nitrate accumulation.

Beyond these factors that can influence BNI variability under field conditions, some discrepancies between the theoretical expectations and the results observed in the field (i.e., high %IBN values without a proportional accumulation of NH₄⁺) could also be attributed to genotypic variation among Urochloa species. Previous studies have reported a range in the nitrification rate among U. humidicola genotypes, from 1.80 up to 3.86 mg N–NO₃⁻ kg⁻¹ soil [35]. Such variability could reflect differences in the quantity and composition of root exudates with inhibitory activity, which ultimately determine the magnitude and persistence of the BNI effect.

Additionally, DAP temporarily increases soil pH and enhances NH₄⁺ availability as it dissolves, followed by acidification as NH₄⁺ is nitrified [36]. This sequence can shift nitrifier community structure/activity [37] and interact with genotype-specific BNI and N uptake, yielding outcomes that deviate from simple theory. Such selective responses among Urochloa genotypes and soil microbiota provide a plausible explanation for the discrepancies between %IBN and measured NH₄⁺/NO₃⁻ in some treatments, particularly in genotypes with contrasting BNI capacities. This is supported by [24], who showed that genotypic differences in BNI expression directly influence nitrifier activity and the resulting NH₄⁺/NO₃⁻ dynamics.

4.2. Total N in Biomass and N Uptake (NU)

The data obtained on biomass yield indicate that certain Urochloa genotypes, such as Cayman and Marandú with values of 3093.5 and 2911.7 kg DM ha⁻¹, respectively, compared to the other genotypes evaluated. These results exceed those reported by [14], who reported yields of 2.47, 2.24, 1.47 and 1.57 t DM ha⁻¹ cut⁻¹ for the genotypes Cayman, Cobra, Basilisk and Tully, respectively, when fertilized with calcium ammonium nitrate. In contrast to that study, the present work was carried out under controlled greenhouse conditions, which may have favored greater environmental control and, consequently, a more efficient expression of the productive potential of the outstanding genotype. Furthermore, diammonium phosphate was used as a source of nitrogen and phosphate fertilization, which may have contributed to both initial growth and efficient N and P uptake by plants, in contrast to calcium ammonium nitrate used by [14] under open field conditions.

Regarding NU, the Cayman genotype showed a recovery of 47.9 kg N ha⁻¹ cut⁻¹, a value very similar to that obtained in the present study 47.32 kg N ha⁻¹. Ref. [15] reported that, among 118 accessions of Brachiaria hybrids, currently classified under the genus Urochloa, the accession Uh08/1149 presented the highest NU, with an absorption of 31.6 kg N cut⁻¹, followed by UhCIAT/16888 with 25.7 kg N cut⁻¹. Regarding forage quality, Ref. [14] also observed increases in crude protein content associated with the type of fertilizer, which reinforces the importance of jointly evaluating biomass and quality.

In the case of Megathyrsus maximus, Ref. [38] identified that, among 119 accessions evaluated, the one with the greatest capacity to inhibit the transformation of NH₄⁺ to NO₃⁻ was also the one that demonstrated the highest N absorption in biomass, with 200.2 mg N per pot. Similar results were observed by [39] who reported up to 124 kg N ha⁻¹ recovered in biomass when evaluating 27 accessions of M. maximus, in soils where the conversion of NH₄⁺ to NO₃⁻ was reduced (0.7 mg NO₃⁻ kg⁻¹ soil day⁻¹), which suggests a functional link between nitrification inhibition and absorption efficiency.

According to [40], the higher NU observed in plant N recovery is partly explained by the inhibition of the nitrification process in the soil. This mechanism allows a longer permanence of nitrogen in its ammoniacal form, which is more stable in the soil profile and less susceptible to losses by leaching or denitrification, compared to the nitric form. This pattern is consistent with [14], who showed that forage grasses with higher NUE also exhibited lower N₂O emission intensities, reflecting reduced nitrogen losses under optimized N uptake.

Plant assimilation of NH₄⁺ requires less energy than that of NO₃⁻, while the reduction of NO₃⁻ to NH₄⁺ during plant assimilation consumes approximately 20 ATP molecules per mole of N, direct assimilation of NH₄⁺ requires only about 5 ATP molecules [34]. Therefore, under conditions where ammoniacal N predominates, plants can incorporate this nutrient at a lower energy cost, which translates into greater physiological and agronomic efficiency.

From an environmental and economic perspective, these results acquire additional relevance. As highlighted by [15], higher NU is associated with lower emissions of N₂O a potent greenhouse gas. In their study, cultivars with higher NU (e.g., Cayman with calcium ammonium nitrate) also showed lower emissions intensities per unit of biomass. This aligns with [12], who emphasized that strengthening biological nitrification inhibition can reduce nitrogen losses, improve NUE, and mitigate N₂O emissions in agricultural soils. Consequently, the use of N-recovery-efficient genotypes not only improves forage productivity but can also reduce nitrogen losses and costs associated with excessive fertilizer use, contributing to the sustainable intensification of tropical livestock systems.

5. Conclusions

The results reveal substantial variability in BNI capacity among the evaluated genotypes, with Cayman and Marandú standing out due to their high inhibitory efficiency (87%) and biomass yields exceeding 2900 kg DM ha⁻¹. These genotypes not only exhibited superior agronomic performance but also higher nitrogen uptake (NU), positioning them as promising options for sustainable forage-based systems. In contrast, Talisman and Camello showed lower BNI rates yet presented higher nitrogen concentrations in their biomass, a trait that may positively influence forage nutritional quality. Overall, these findings underscore the importance of jointly considering BNI capacity, agronomic performance, and nutritional attributes when selecting Urochloa genotypes aimed at enhancing nitrogen use efficiency and promoting sustainable tropical livestock systems. Additionally, enhanced nitrogen use efficiency reduces the conversion of ammonium to nitrate, thereby minimizing nitrogen losses and improving grass uptake. This optimization enables pasture systems to achieve higher productivity while mitigating environmental impacts by suppressing nitrification and lowering N₂O emissions.