Nitrous oxide (N₂O) is a potent greenhouse gas with a significant 100-year global warming potential that is 265 times higher than that of carbon dioxide on a per-molecule basis (IPCC, 2013). In addition, N₂O depletes stratospheric ozone, which protects the earth from biologically damaging ultraviolet radiation (Ravishankara et al., 2009). Notably, the concentration of atmospheric N₂O has increased from 270 ppb during the pre-industrial era to 335.55 ppb in 2022, with an average annual increase rate of 0.90 ppb over the last 2 decades (Lan et al., 2022). Agriculture reportedly emitted approximately 4.1 Tg N₂O-N year⁻¹, accounting for approximately 66% of total global anthropogenic N₂O emissions (UNEP, 2013). Using the dynamic land ecosystem model, Dangal et al. (2019) estimated that the net N₂O emission from the global grasslands was 2.2 Tg N₂O-N year⁻¹, which was responsible for 54% of the total agricultural N₂O emissions.

To meet the increasing food demands, nitrogen (N) fertilizer and agricultural land are growing substantially (Foley et al., 2011). The global synthetic N fertilizer consumption has increased from 12 to 112 Tg N while that has risen from 0.8 to 24 Tg N in China during the 1961–2020.¹ However, the N-use efficiency (NUE) in China was only 28–35%, which is much lower than the global average (Liu et al., 2013; Han et al., 2015; Zhang et al., 2015).The heavy reliance of N fertilizers in agriculture has contributed to the stimulation of nitrifier activity and the trend toward high-nitrifying soil environments (Poudel et al., 2002; Bellamy et al., 2005).

Nitrification is closely related to N utilization and loss, and has become a key process to improve NUE and reduce N pollution (Subbarao et al., 2006; Beeckman et al., 2018). Nitrification is a microbes-driven process of oxidizing ammonia (NH₃) to nitrite and further to nitrate (NO₃⁻) and producing N₂O as a byproduct (Stein, 2020). The NO₃⁻ produced during nitrification serves as a substrate and denitrification further reduces NO₃⁻ to dinitrogen and produces N₂O as an intermediate product (Coskun et al., 2017a). Nitrification inhibitors can depress the activities of nitrifiers in soil, thereby delaying NH₃ oxidation and reducing N₂O emissions and NO₃⁻ production (Rodgers, 1986; Coskun et al., 2017b). To date, a few synthetic nitrification-inhibiting compounds have been efficiently adopted in the field, such as nitrapyrin, dicyandiamide, and 3,4-dimethyl pyrazole phosphate (Weiske et al., 2001; Zerulla et al., 2001; Niu et al., 2018). Meta-analysis revealed that the combination application of nitrification inhibitors and urea reduced NO₃⁻ leaching by 48% and N₂O emissions by 44% (Burzaco et al., 2014), and increased crop yields by 7.5% and NUE by 12.9% (Abalos et al., 2014a). However, synthetic nitrification inhibitors have certain limitations such as low cost-effectiveness, application challenges, poor biological stability, and environmental pollution risks (Subbarao et al., 2012; Coskun et al., 2017b; Wang et al., 2021).

Natural compounds with biological nitrification inhibition (BNI) have been found from litters, root exudates, tissue extracts, and rhizosphere of plants such as grasses, trees, and crops (Wang et al., 2021), including methyl 3-(4 hydroxyphenyl) propionate (MHPP), sorgoleone and sakuranetin from sorghum (Subbarao et al., 2013), 1,9-decanediol from rice (Sun et al., 2016) and brachialactone from Brachiaria humidicola grass (Subbarao et al., 2009). Some root-secreted biological nitrification inhibitors (BNIs) like sorgoleone, sakuranetin, and brachialactone as well as linolenic acid and linoleic acid found in B. humidicola can inhibit both ammonia mono-oxygenase and hydroxylamine oxidoreductase activities (Coskun et al., 2017b), while 1,9-decanediol and MHPP only inhibits activity of ammonia mono-oxygenase (Zakir et al., 2008; Nardi et al., 2013, 2020; Sun et al., 2016; Lu et al., 2019). Up to date, the functional validation of the BNIs is mainly performed in the pure culture system of a single strain Nitrosomonas europaea, and the effect in the complicated soil system remains to be tested (Subbarao et al., 2015). For example, sakuranetin isolated from sorghum shows a strong inhibitory activity in vitro-cultural bioassay but losses the inhibitory effect in soil-assay (Subbarao et al., 2013). Gopalakrishnan et al. (2009) found that the inhibition effect of BNIs is affected by soil type, and the BNIs derived from B. humidicola in Cambisol can inhibit 90% nitrification with comparable effects to dicyandiamide (50 mg kg⁻¹ soil), but are less effective in Andosol during the 60-day incubation.

Forage grasses with biological nitrification inhibition (BNI) capacity exhibit approximately 2-fold greater productivity than those lacking such capacity in nutrient-limited ecosystems, based on an estimate of a newly developed model (Lata et al., 1999; Boudsocq et al., 2009). The B. humidicola, reportedly exhibits the strongest BNI function among tropical grasses reduces the NH₃ oxidation rate and N₂O emissions significantly during a 3-year field experiment, when compared with soybean-planted or plant-free soils (Subbarao et al., 2009). During a short-term (29 days) monitoring period in Colombia, cumulative N₂O emissions from a B. humidicola cv. Tully field was decreased by approximately 60% when compared with that in a Brachiaria hybrid cv. Mulato field under bovine urine amendment (Byrnes et al., 2017). In contrast, no significant effect on N₂O emissions of the two forage genotypes was observed under cattle dung amendment in the same experimental site (Lombardi et al., 2022).

Latosol is a most widely distributed soil and covers 51.26% of the total area in Hainan Province, China. In the present study, a 2-year field experiment was conducted in a Latosol cultivated with B. humidicola and a native grass species, Eremochloa ophiuroides. We hypothesized that in situ N₂O emissions from grasslands under cultivation with Brachiaria with higher BNI capacity are lower than in those cultivated with Eremochloa. The objectives of the present study were: (1) to determine whether the N₂O emissions from a B. humidicola field are lower than those from an E. ophiuroides field in tropical Hainan Province, China; and (2) to evaluate the mitigation effects of B. humidicola on yield-scaled N₂O emissions under the different N application rates. We also established an incubation with soils from the field experiment using a ¹⁵N tracing technique to evaluate the influence of B. humidicola on the N transformation process rates and N₂O production rates via nitrification and denitrification (Xie et al., 2022).

Annual N₂O emissions from this tropical grassland varied from 1.35 to 9.54 kg N₂O-N ha⁻¹, which was within the 0–29.1 kg N₂O-N ha⁻¹ range in grasslands as reported previously (Mosier et al., 1996; Wolf et al., 2010; Merbold et al., 2014; Luo et al., 2017). Out of expectation, N₂O emissions from the B. humidicola field were 1.3–2.6-fold higher than those from the E. ophiuroides field under N fertilization. Additionally, the N₂O emission factor of the N applied was increased to 0.74–0.98% under B. humidicola from 0.11–0.47% under E. ophiuroides. Our results suggest that cultivation of exotic, tropical forage grass B. humidicola with BNI capacity by replacing native E. ophiuroides stimulated N₂O emission. To our knowledge, this is the first study to find the stimulation effect of B. humidicola on N₂O emissions in the field when compared with the native grass. Apparently, more field studies are required to evaluate the impact of plants with BNI capacity on N₂O emissions at the ecosystem and global scale, as suggested by Lata et al. (2022).

Previous study suggested that Brachiaria genotype with high BNI capacity reduced almost 50% of N₂O emission when compared with soybean or plant-free soils (Subbarao et al., 2009). Byrnes et al. (2017) reported that B. humidicola cv. Tully with high BNI capacity reduced approximately 60% of N₂O emissions in the field when compared with the Brachiaria hybrid cv. Mulato having low BNI capacity during the 29-day monitoring period. Planting B. humidicola with high BNI capacity reduced soil N₂O emissions by 18.3% when compared with B. humidicola with low BNI capacity in a 21-day pot experiment (Teutscherová et al., 2022). The active substances with BNI capacity, such as methyl-p-coumarate, methyl ferulate, and brachialactone, have been identified from exudates of B. humidicola (Gopalakrishnan et al., 2009; Subbarao et al., 2009). Brachialactone can simultaneously block the enzymatic pathways of ammonia monooxygenase and hydroxylamino oxidoreductase (Subbarao et al., 2009). The inhibitory potential reportedly increases with an increase in grass root density (Subbarao et al., 2007; Boudsocq et al., 2009). Subbarao et al. (2009) estimated that B. humidicola roots can potentially release 2.6 × 10⁶–7.5 × 10⁶ ATU (allylthiourea units) ha⁻¹ day⁻¹ BNI activity in the South American savannas, which is equivalent to the application of 6.2–18 kg ha⁻¹ nitrapyrin based on 1 ATU being equal to 0.6 μg of nitrapyrin. Karwat et al. (2017) demonstrated that B. humidicola, like dicyandiamide, significantly suppresses soil nitrification potential. In a previous study, using the ¹⁵N tracing incubation with soils collected from the B. humidicola and E. ophiuroides plots at the field experiment end, we found that B. humidicola decreased the autotrophic nitrification rate and N₂O production rate via nitrification by 27.3 and 14.7%, respectively when compared with E. ophiuroides (Xie et al., 2022). This indicated that in the test soil, B. humidicola efficiently inhibited nitrification and N₂O production via nitrification.

Subbarao et al. (2009) observed that cultivation of B. humidicola reduced the abundance of both ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a Vertisol with pH 7.40 when compared with soil cultivated with soybean. Hink et al. (2018) further reported that although both AOA and AOB were capable of N₂O production under high NH₄⁺-N concentrations, the contribution of AOB was greater in a soil with pH 6.50. In the test acid soil with pH 5.42, it is likely that both AOA and AOB participated in NH₃ oxidation and N₂O production. Further investigations are required to determine the relative importance of AOA and AOB in N₂O production, and the suppression effects of B. humidicola on AOA and AOB activity (Nuñez et al., 2018).

Byrnes et al. (2017) suggested that by increasing N uptake, B. humidicola with high BNI capacity more efficiently decreased soil NO₃⁻-N availability and potential denitrification than B. humidicola with low BNI capacity, thereby reducing N₂O emissions. Abalos et al. (2014b) reported that mixed cultivation of Folium perennial L. and Poi trivialize L. decreased soil NO₃⁻-N concentrations and consequent N₂O emissions when compared with either monoculture at an N application rate of 60 kg N ha⁻¹. They suggested that the trends were attributed to L. perennial taking up N using the “scale strategy” by increasing root biomass, and P. trivialize absorbing N via the “precision strategy” by providing access to N hotspots that were not emptied by L. perennial. In the present study, although B. humid cola cultivation increased N uptake, N₂O emissions were positively correlated with pasture yield and N uptake, indicating that increased N uptake by B. humid cola did not reduce N₂O emissions. In the present study, the mean soil NO₃⁻-N concentration under N fertilization ranged from 5.60 mg N kg⁻¹ in the BN1 treatment to 8.45 mg N kg⁻¹ in the BN3 treatment, which was higher than the 5 mg N kg⁻¹ threshold for occurrence of denitrification (Dobbie and Smith, 2003), and indicated that although B. humid cola efficiently increased N uptake and partially inhibited nitrification, soil NO₃⁻-N under N fertilization was still higher than the threshold value for denitrification in the test field.

Using ¹⁵N paired incubation (¹⁵NH₄NO₃ and NH₄¹⁵NO₃), we found that the N₂O production rate during denitrification in the B. humid cola soil increased by 7.7-fold when compared with the E. ophiuroides soil and the contribution of denitrification to N₂O emissions sharply enhanced from 9.7% in the E. ophiuroides soil to 47.1% (Xie et al., 2022). In the present study, B. humidicola biomass was 3–6-fold greater than that of E. ophiuroides and SOC was more efficiently increased under B. humidicola. Horrocks et al. (2019) also observed that 1-year cultivation of B. humidicola increases SOC content and improves aggregate stability in Colombia. Fisher et al. (1994) and Amézquita et al. (2004) attributed the SOC enhancement to rapid accumulation of B. humidicola roots and exudates. Plant reportedly release as much as 40% of net photosynthetic C into the rhizosphere (Marschner, 2011), which in turn provides more labile C substrates for denitrifiers (Wu et al., 2017). Enhanced SOC at least exhibits two stimulation effects on denitrification. Firstly, enhanced SOC promotes the formation of anaerobic microsites for denitrification by stimulating aggregation (Bollmann and Conrad, 2004). Secondly, increased organic C availability reduces the soil moisture threshold for the occurrence of denitrification (Rochette et al., 2000; Van Groenigen et al., 2004; Chantigny et al., 2013) resulting in increased denitrification potentials. Our results indicate that cultivation of exotic B. humidicola with a much higher biomass compared with E. ophiuroides stimulated N₂O production during denitrification by providing more organic C, which in turn masked N₂O reduction by inhibiting nitrification, thereby enhancing N₂O emissions.

Comparing yield-scaled N₂O emissions has been suggested to be an effective way of evaluating the tradeoff between production and environmental impacts and determining the economic feasibility of N₂O emission mitigation methods (van Groenigen et al., 2010; Grassini and Cassman, 2012). In the present study, yield-scaled N₂O emissions from B. humidicola field with and without N fertilization during two seasons were 128.80–183.02 and 93.02 g N kg⁻¹ biomass, respectively, which were significantly lower than the corresponding values under E. ophiuroides cultivation (171.07–221.62 and 186.93 g N kg⁻¹ biomass, respectively). In addition, we observed interannual shifts in yield-scaled N₂O emissions in both grasslands, which was primarily driven by changes in annual N₂O emission in B. humidicola fields, whereas they were driven by changes in biomass yield in E. ophiuroides fields. The lower yield-scaled N₂O emissions under B. humidicola cultivation compared with under E. ophiuroides indicated that although B. humidicola increased annual N₂O emissions, it was more environmentally friendly based on its higher forage productivity and NUE.

In the present study, B. humidicola exhibited higher yields and NUE, and in contrast and unexpectedly, induced higher soil N₂O emissions when compared with E. ophiuroides. Although cultivation of B. humidicola with high BNI capacity reduced N₂O production rate via nitrification, however, it more efficiently enhanced N₂O production rate than E. ophiuroides via denitrification due to increased SOC and exudate concentrations, thereby increasing N₂O emissions (Figure 6). When compared with under E. ophiuroides, however, the lower yield-scaled N₂O emissions under B. humidicola cultivation indicated that although B. humidicola increased annual N₂O emissions, it was more environmentally friendly based on its higher forage productivity and NUE.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

WD and DL: conceptualization. LX, YN, and DL: field experiment. LX and ZC: data analysis. LX, WD, and LM: writing. WD: funding acquisition. All authors contributed to the article and approved the submitted version.

This work was financially supported from the National Natural Science Foundation of China (41730753, 41977049 and 42077029), the International Partnership Program of Chinese Academy of Sciences (151432KYSB20200001), and International Atomic Energy Agency coordinated research project (D15020).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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