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Introduction: Biological Nitrification Inhibition (BNI) is defined as the plant-mediated control of soil nitrification via the release of nitrification inhibitors. BNI of Brachiaria humidicola (syn. Urochloa humidicola) has been mainly attributed to root-exuded fusicoccane-type diterpenes, e.g., 3-epi-brachialactone. We hypothesized, however, that BNI of B. humidicola is caused by an assemblage of bioactive secondary metabolites.Methods:B. humidicola root exudates were collected hydroponically, and metabolites were isolated by semi-preparative HPLC. Chemical structures were elucidated by HRMS as well as 1D and 2D NMR spectroscopy. Nitrification inhibiting potential of isolated metabolites was evaluated by a Nitrosomonas europaea based bioassay.Results and discussion: Besides previously described brachialactone isomers and derivatives, five phenol and cinnamic acid derivatives were identified in the root exudates of B. humidicola: 2-hydroxy-3-(hydroxymethyl)benzaldehyde, vanillin, umbelliferone and both trans- and cis-2,6-dimethoxycinnamic acid. Notably, vanillin revealed a substantially higher nitrification inhibiting activity than 3-epi-brachialactone (ED50 ∼ 12.5 μg·ml−1, ED80 ∼ 20 μg·ml−1), identifying this phenolic aldehyde as novel nitrification inhibitor (NI). Furthermore, vanillin exudation rates were in the same range as 3-epi-brachialactone (1–4 μg·h−1·g−1 root DM), suggesting a substantial contribution to the overall inhibitory activity of B. humidicola root exudates. In relation to the verification of the encountered effects within soils and considering the exclusion of any detrimental impact on the soil microbiome, the biosynthetic pathway of vanillin via the precursor phenylalanine and the intermediates p-coumaric acid/ferulic acid (precursors of further phenolic NI) might constitute a promising BNI breeding target. This applies not only to Brachiaria spp., but also to crops in general, owing to the highly conserved nature of these metabolites.

Nitrification and denitrification are soil biological processes responsible for large nitrogen losses from agricultural soils and generation of the greenhouse gas (GHG) N 2 O. Increased use of nitrogen fertilizer and the resulting decline in nitrogen use efficiency (NUE) are a major concern in agroecosystems. This nitrogen cycle in the rhizosphere is influenced by an intimate soil microbiome–root exudate interaction and biological nitrification inhibition (BNI). A PANOMICS approach can dissect these processes. We review breakthroughs in this area, including identification and characterization of root exudates by metabolomics and proteomics, which facilitate better understanding of belowground chemical communications and help identify new biological nitrification inhibitors (BNIs). We also address challenges for advancing the understanding of the role root exudates play in biotic and abiotic stresses. Understanding the complex nature of root exudate and soil microbiome interaction at the root–soil interface has greatly intensified recently. Biological nitrification inhibition (BNI) can help to reduce excess nitrogen leaching into the outer soil layers, improving nitrogen use efficiency (NUE) and reducing the negative impact of excess nitrogen fertilization on the environment. Single omics analysis will not help understand complex interactions at the root–soil interface. Instead, a PANOMICS approach can determine the spatiotemporal dynamics of root exudates and microbiome. Root exudates mediate plant–microbe interactions, facilitating nutrient uptake and specifically inhibiting soil nitrification, thereby reducing our dependency on fertilizer. Metabolomics of root exudates is widely studied in different crops and germplasm under diverse environmental conditions; combining this with rhizosphere microbiota metabolomics is an upcoming trend. [ABSTRACT FROM AUTHOR]

Purpose: Rapid nitrification leads to loss of nitrogen (N) fertilizer in agricultural systems. Plant produced/derived biological nitrification inhibitors (BNIs) are an effective eco-strategy to rein-in soil nitrification to improve crop-N uptake and nitrogen use efficiency (NUE) in production systems. Sorgoleone is the major component of hydrophobic-BNI-activity in sorghum roots. However, the role of genetic differences in sorgoleone production in reducing soil nitrification and N2O emissions are not established. Methods: Two genetic-stocks of sorghum with high-sorgoleone (HS), and two with low-sorgoleone (LS) production from roots were grown using hydroponics in a plant-growth chamber, in soil in pots in a glasshouse, and in a field experiment. Release of hydrophilic-BNI activity from roots of HS and LS genetic stocks, sorgoleone levels in rhizosphere soils, soil nitrification rates, soil-nitrifier activity and N2O emissions were measured to understand the interplay involving sorgoleone release, hydrophilic-BNI release from roots, soil nitrification, plant growth and N uptake. Results: HS-producing genetic-stocks showed higher hydrophilic-BNI-capacity compared to LS- producing genetic-stocks. Biomass production and N uptake were significantly higher in HS than in LS genetic-stocks. Glasshouse and field studies suggest that HS genetic stocks had stronger suppressive impact on soil-nitrifier-populations (ammonia-oxidizing archaea and ammonia-oxidizing bacteria), soil-nitrification, and soil-N2O emissions than in LS genetic-stocks. Conclusion: These results demonstrate that HS sorghum genetic-stocks suppress soil nitrifier activity and can potentially reduce N losses from NO3 − leaching and N2O emissions more effectively than LS genetic-stocks. [ABSTRACT FROM AUTHOR]

Abstract Savannas are characterized by the coexistence of grasses and trees. Fires are critical for their coexistence, because they decrease the survival of tree seedlings and saplings and their recruitment to the adult stage. In some humid savannas, perennial grasses inhibit nitrification and trees stimulate nitrification, which likely favors coexistence between trees and grasses. However, fires may influence plant capacity to control nitrogen cycling, which could subsequently influence tree–grass coexistence and savanna nitrogen budget. Therefore, we sampled soil in a humid savanna of Ivory Coast under the dominant nitrification‐inhibiting grass species and the dominant nitrification‐stimulating tree species and under bare soil before and after (i.e., 5 days) fire during the long dry season. We quantified the total microbial and nitrifier abundances and transcriptional activities and the nitrification enzyme activity. Fire decreased soil water content, probably by increasing evaporation and, maybe, by triggering the growth of grasses, and increased soil ammonium availability likely due to ash deposition and increased mineralization. Fire did not impact the total archaeal, bacterial, or fungal abundances, or that of the nitrifiers. Fire did not impact archaeal transcriptional activity and increased bacterial and fungal total transcriptional activities. In contrast, fire decreased the archaeal nitrifier transcriptional activities and the nitrification enzymatic activity, likely due to the often reported resumption of the growth of nitrification‐inhibiting grasses quickly after the fire (and the subsequent increase in root exudation). These results pave the way for a better understanding of the short‐term effects of fire on nitrogen cycling and tree–grass competition for nitrogen.

To control agronomic N losses and reduce environmental pollution, biological nitrification inhibition (BNI) is a promising strategy. BNI is an ecological phenomenon by which certain plants release bioactive compounds that can suppress nitrifying soil microbes. Herein, we report on two hydrophobic BNI compounds released from maize root exudation (1 and 2), together with two BNI compounds inside maize roots (3 and 4). On the basis of a bioassay-guided fractionation method using a recombinant nitrifying bacterium Nitrosomonas europaea, 2,7-dimethoxy-1,4-naphthoquinone (1, ED50 = 2 μM) was identified for the first time from dichloromethane (DCM) wash concentrate of maize root surface and named "zeanone." The benzoxazinoid 2-hydroxy-4,7-dimethoxy-2H-1,4-benzoxazin-3(4H)-one (HDMBOA, 2, ED50 = 13 μM) was isolated from DCM extract of maize roots, and two analogs of compound 2, 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (HMBOA, 3, ED50 = 91 μM) and HDMBOA-β-glucoside (4, ED50 = 94 μM), were isolated from methanol extract of maize roots. Their chemical structures (1–4) were determined by extensive spectroscopic methods. The contributions of these four isolated BNI compounds (1–4) to the hydrophobic BNI activity in maize roots were 19%, 20%, 2%, and 4%, respectively. A possible biosynthetic pathway for zeanone (1) is proposed. These results provide insights into the strength of hydrophobic BNI activity released from maize root systems, the chemical identities of the isolated BNIs, and their relative contribution to the BNI activity from maize root systems. [ABSTRACT FROM AUTHOR]

Savannas are characterized by the coexistence of grasses and trees. Fires are critical for their coexistence, because they decrease the survival of tree seedlings and saplings and their recruitment to the adult stage. In some humid savannas, perennial grasses inhibit nitrification and trees stimulate nitrification, which likely favors coexistence between trees and grasses. However, fires may influence plant capacity to control nitrogen cycling, which could subsequently influence tree–grass coexistence and savanna nitrogen budget. Therefore, we sampled soil in a humid savanna of Ivory Coast under the dominant nitrification‐inhibiting grass species and the dominant nitrification‐stimulating tree species and under bare soil before and after (i.e., 5 days) fire during the long dry season. We quantified the total microbial and nitrifier abundances and transcriptional activities and the nitrification enzyme activity. Fire decreased soil water content, probably by increasing evaporation and, maybe, by triggering the growth of grasses, and increased soil ammonium availability likely due to ash deposition and increased mineralization. Fire did not impact the total archaeal, bacterial, or fungal abundances, or that of the nitrifiers. Fire did not impact archaeal transcriptional activity and increased bacterial and fungal total transcriptional activities. In contrast, fire decreased the archaeal nitrifier transcriptional activities and the nitrification enzymatic activity, likely due to the often reported resumption of the growth of nitrification‐inhibiting grasses quickly after the fire (and the subsequent increase in root exudation). These results pave the way for a better understanding of the short‐term effects of fire on nitrogen cycling and tree–grass competition for nitrogen. [ABSTRACT FROM AUTHOR]

The biological oxidation of ammonia (i.e. nitrification), results in the transformation of relatively immobile NH4+ into a highly mobile NO3-, which is vulnerable to losses through leaching and denitrification, resulting in low nitrogen-use efficiency in agricultural systems. The ability of certain plants to suppress soil nitrifier function by releasing inhibitors from roots is termed 'biological nitrification inhibition' (BNI). Using a recombinant luminescent Nitrosomonas europaea, we developed an assay to detect and quantify the inhibitory effects in plant soil systems termed, 'BNI activity', expressed in ATU (allylthiourea unit). Sorghum roots released (into water-based medium, hereafter referred to as root exudates) substantial amount of BNI activity. The BNI capacity (ATU g-1 root dry wt d-1) of roots changed with growth stage, from 60 ATU at 3 week old to 20 ATU at 45 day stage. In addition, sorghum roots released hydrophobic compounds, which can be recovered by washing with dichloromethane (hereafter referred to as DCM-root wash). One of the active constituents with BNI activity in root exudates, isolated by activity-guided HPLC fractionation, was a flavonoid, identified as sakuranetin. Using a similar approach, the major active constituent with BNI activity in DCM-root wash was isolated and identified as sorgoleone. Both sakuranetin and sorgoleone inhibited Nitrosomonas activity in a dose-response manner. Substantial genetic variability for sorgoleone production capacity was detected in sorghum. Some wild-sorghum species showed high-BNI capacity under field conditions. Potential implications of BNI in inhibiting nitrification and in reducing the nitrogen footprint from agricultural systems will be discussed.

Abstract The tropical forage grass Brachiaria humidicola (Bh) controls soil microbial nitrification via biological nitrification inhibition (BNI). The aim of our study was to verify if nitrate reductase activity (NRA) in Bh roots or leaves reflects in vivo performance of BNI in soils. NRA was measured in roots and leaves of contrasting accessions and apomictic hybrids of Bh grown under controlled greenhouse and natural field conditions. Nitrate (NO 3 −) contents were measured in soil solution and in Bh stem sap to validate NRA data. Potential soil nitrification rates (NRs) and leaf δ15N values were used to verify in vivo BNI by the NRA assay in the field study. NRA was detected in Bh leaves rather than roots, regardless of NO 3 − availability. NRA correlated with NO 3 − contents in soils and stem sap of contrasting Bh genotypes substantiating its reflectance of in vivo BNI performance. Additionally, leaf NRA data from the field study significantly correlated with simultaneously collected NRs and leaf δ15N data. The leaf NRA assay facilitated a rapid screening of contrasting Bh genotypes for their differences in in vivo performance of BNI under field and greenhouse conditions, but inconsistency of the BNI potential by Bh germplasm was observed. Among Bh genotypes tested, leaf NRA was closely linked with nitrification activity, and consequently with actual BNI performance. It was concluded that NRA in leaves of Bh can serve as an indicator of in vivo BNI activity when complemented with established BNI methodologies (δ15N, NRs) under greenhouse and field conditions. Highlights • NRA in leaves can serve as an indicator of in vivo BNI activity in soils by Bh. • Potential soil nitrification rates correlated positively with measured leaf NRA. • High BNI potentials were indicated by low leaf NRA and low leaf δ15N signatures. • Leaf NRA correlated with soil NO 3 − contents of contrasting Bh genotypes. • NRA was detected in Bh leaves rather than roots, regardless of NO 3 − availability. [ABSTRACT FROM AUTHOR]

Introduction: Biological Nitrification Inhibition (BNI) is defined as the plant-mediated control of soil nitrification via the release of nitrification inhibitors. BNI of Brachiaria humidicola (syn. Urochloa humidicola ) has been mainly attributed to root-exuded fusicoccane-type diterpenes, e.g., 3- epi -brachialactone. We hypothesized, however, that BNI of B. humidicola is caused by an assemblage of bioactive secondary metabolites. Methods: B. humidicola root exudates were collected hydroponically, and metabolites were isolated by semi-preparative HPLC. Chemical structures were elucidated by HRMS as well as 1D and 2D NMR spectroscopy. Nitrification inhibiting potential of isolated metabolites was evaluated by a Nitrosomonas europaea based bioassay. Results and discussion: Besides previously described brachialactone isomers and derivatives, five phenol and cinnamic acid derivatives were identified in the root exudates of B. humidicola : 2-hydroxy-3-(hydroxymethyl)benzaldehyde, vanillin, umbelliferone and both trans- and cis -2,6-dimethoxycinnamic acid. Notably, vanillin revealed a substantially higher nitrification inhibiting activity than 3- epi -brachialactone (ED 50 ∼ 12.5 μg·ml -1 , ED 80 ∼ 20 μg·ml -1 ), identifying this phenolic aldehyde as novel nitrification inhibitor (NI). Furthermore, vanillin exudation rates were in the same range as 3- epi -brachialactone (1-4 μg·h -1 ·g -1 root DM), suggesting a substantial contribution to the overall inhibitory activity of B. humidicola root exudates. In relation to the verification of the encountered effects within soils and considering the exclusion of any detrimental impact on the soil microbiome, the biosynthetic pathway of vanillin via the precursor phenylalanine and the intermediates p -coumaric acid/ferulic acid (precursors of further phenolic NI) might constitute a promising BNI breeding target. This applies not only to Brachiaria spp ., but also to crops in general, owing to the highly conserved nature of these metabolites., Competing Interests: 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., (Copyright © 2023 Egenolf, Schöne, Conrad, Braunberger, Beifuß, Arango and Rasche.)

African humid savannas are highly productive ecosystems, despite very low soil fertility, where grasses and trees coexist. Earlier results showed that some perennial grass species are capable of biological nitrification inhibition (BNI) while trees likely influence differently on nitrogen cycling. Here we assessed the impact of the dominant grass and tree species of the Lamto savanna (Ivory Coast) on soil nitrifying and denitrifying enzyme activities (NEA and DEA, respectively) and on the abundances of archaeal and bacterial ammonia oxidizers (AOA and AOB, respectively) and nitrite reducers. This is one of the first studies linking nitrifying and denitrifying activities and the abundances of the involved groups of microorganisms in savanna soils. NEA was 72-times lower under grasses than under trees while AOA and AOB abundances were 34- and 3-times lower. This strongly suggests that all dominant grasses inhibit nitrification while trees stimulate nitrification, and that archaea are probably more involved in nitrification than bacteria in this savanna. While nitrite reducer abundances were similar between locations and dominated by nirS genes, DEA was 9-times lower under grasses than trees, which is likely explained by BNI decreasing nitrate availability under grasses. The nir S dominance could be due to the ferruginous characteristics of these soils as nirS and nirK genes require different metallic co-enzymes (Fe or Cu). Our results show that the coexistence of grasses and trees in this savanna creates a strong heterogeneity in soil nitrogen cycling that must be considered to understand savanna dynamics and functioning. These results will have to be taken into account to predict the feedbacks between climate changes, nitrogen cycling and tree/grass dynamics at a time when savannas face worldwide threats. [ABSTRACT FROM AUTHOR]

Sorghum (Sorghum bicolor) roots are well-known for the release of Biological Nitrification Inhibitors (BNIs), such as sorgoleone (SGLN) and methyl 3–4-hydroxyphenyl propionate (MHPP). However, the deployment of sorghums with BNIs in agriculture practices has not been widely investigated and evaluated. In this study, sorghum and maize were intercropped in field and pot cultivation to investigate the Biological Nitrification Inhibition (BNI) effect of sorghum on the productivity of both crops and soil nitrification. Inorganic N content (NO 3 – and NH 4 +), N 2 O emission, and the abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in soil were analyzed. The results showed intercropping with sorghum have the potential to improve crop productivity, as indicated by the Land Equivalent Ratio greater than 1.0. Intercropping with sorghum reduced the transformation rate of NH 4 + to NO 3 – in soils and the emission of N 2 O, as compared to monoculture of maize. Additionally, the abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) under intercropping mode were reduced by 41% and 28% in field trials, and reduced by 59% and 23% in pot experiments, respectively. Incubation of the same soils with SGLN and/or MHPP showed that the combined treatment of both BNIs strongly inhibited nitrification and N 2 O emission, along with lower AOB and AOA abundances, as compared with those under the sole treatment of SGLN or MHPP. Our results indicated that the root exudates of sorghum play a significant role in inhibiting soil nitrification due to the overlapping or amplified effect of various BNIs, which contribute to the nitrogen nutrition of the neighbouring crops under intercropping mode. Therefore, intercropping with BNI crops provides an eco-friendly strategy to improve crop production with reduced N 2 O emission. • Intercropping mode of sorghum and maize reduced soil nitrification and N 2 O emission. • Intercropping mode decreased the abundance of AOA and AOB. • Effect of combined addition of SGLN and MHPP in soil was superior to sole addition. • Root exudates of sorghum were of significance in inhibiting soil nitrification. [ABSTRACT FROM AUTHOR]

Aims: Sorghum ( Sorghum bicolor) roots release biological nitrification inhibitors (BNIs) to suppress soil nitrification. Presence of NH in the rhizosphere stimulates BNIs release and it is hypothesized to be functionally associated with plasma membrane (PM) H-ATPase activity. However, whether the H-ATPase is regulated at the transcriptional level, and if so, which isoforms of the H-ATPases are involved in BNIs release are not known. Also, it is not clear whether the stimulation on BNIs release from roots is due to NH uptake or its assimilation, which are addressed in this study. Methods: Root exudates from intact sorghum plants were collected using aerated solutions of NH or methyl-ammonium (MeA); and the BNI-activity release was determined. PM vesicles were isolated from fresh roots using a two-phase partitioning system; and the hydrolytic H-ATPase activity was determined. All genes encoding PM H-ATPases were searched in sorghum genome, and their expression in response to NH or MeA were analyzed by quantitative RT-PCR in sorghum roots. Results: BNIs release and PM H-ATPase activity increased with NH concentration (≤1.0 mM) in the root-exudate collection solutions, but at higher concentrations, it did not respond further or declined in case of the PM H-ATPase activity. Twelve PM H-ATPase genes were identified in sorghum genome; and these isoforms were designated SbA1 to SbA12. Five H-ATPase genes were stimulated by NH in the rhizosphere, and have similar expression pattern, which is consistent with the variation in H-ATPase activity. MeA, a non-metabolizable analogue of NH, had no significant effects on BNIs release, H-ATPase activity, or expression of the H-ATPase genes. Conclusions: Our results suggest that the functional link between PM H-ATPase activity and BNIs release is evident only at NH levels of ≤1.0 mM in the rhizosphere. The variation in PM H-ATPase activity by NH is due to transcriptional regulation of five isoforms of the H-ATPases. The stimulatory effect of NH on BNIs release is functionally associated with NH assimilation and not just with NH uptake alone. [ABSTRACT FROM AUTHOR]

Background and aims: Nitrification and denitrification are the two most important processes that contribute to greenhouse gas emission and inefficient use of nitrogen. Suppressing soil nitrification through the release of nitrification inhibitors from roots is a plant function, and termed 'Biological Nitrification Inhibition (BNI)'. We report here the role and contribution of sorgoleone release to sorghum-BNI function. Methods: Three sorghum genotypes (Hybridsorgo, IS41245 and GDLP 34-5-5-3) were evaluated for their capacity to release sorgoleone, which has BNI-activity, in hydroponic and soil culture. Sorgoleone released was measured using HPLC; BNI-activity was determined using a luminescent recombinant Nitrosomonas europaea assay. Results: Sorgoleone production and BNI-activity release by roots are closely associated (1 μg of sorgoleone is equivalent to 1 ATU activity in assay). Purified sorgoleone inhibited Nitrosomonas activity and suppressed soil nitrification. Sorghum genotypes release varying quantity of sorgoleone; GDLP 34-5-5-3 and Hybridsorgo showed higher capacity for both sorgoleone release and BNI-activity than did IS41245. In soil culture, GDLP 34-5-5-3 released higher quantity of sorgoleone into the rhizosphere, which had higher BNI-activity, and suppressed soil nitrification to a greater extent than did by IS41245. Conclusions: These results demonstrate genetic differences for sorgoleone release and its functional link with BNI-capacity; there is potential for genetic improvement of sorghum BNI-capacity and deployment of this in low-nitrifying sorghum production systems. [ABSTRACT FROM AUTHOR]

Aims: The ability to suppress soil nitrification through the release of nitrification inhibitors from plant roots is termed 'biological nitrification inhibition' (BNI). Here, we aimed at the quantification and characterization of the BNI function in sorghum that includes inhibitor production, their chemical identity, functionality and factors regulating their release. Methods: Sorghum was grown in solution culture and root exudate was collected using aerated NHCl solutions. A bioluminescence assay using recombinant Nitrosomonas europaea was employed to determine the BNI activity. Activity-guided chromatographic fractionation was used to isolate biological nitrification inhibitors (BNIs). The chemical structure was analyzed using NMR and mass spectrometry; pH-stat systems were deployed to analyze the role of rhizosphere pH on BNIs release. Results: Sorghum roots released two categories of BNIs: hydrophilic- and hydrophobic-BNIs. The release rates for hydrophilic- and hydrophobic- BNIs ranged from 10 to 25 ATU g root dwt. d. Addition of hydrophilic BNIs (10 ATU g soil) significantly inhibited soil nitrification (40 % inhibition) during a 30-d incubation test. Two BNI compounds isolated are: sakuranetin (ED 0.6 μM; isolated from hydrophilic-BNIs fraction) and sorgoleone (ED 13.0 μM; isolated from hydrophobic-BNIs fraction), which inhibited Nitrosomonas by blocking AMO and HAO enzymatic pathways. The BNIs release required the presence of NH in the root environment and the stimulatory effect of NH lasted 24 h. Unlike the hydrophobic-BNIs, the release of hydrophilic-BNIs declined at a rhizosphere pH >5.0; nearly 80 % of hydrophilic-BNI release was suppressed at pH ≥7.0. The released hydrophilic-BNIs were functionally stable within a pH range of 5.0 to 9.0. Sakuranetin showed a stronger inhibitory activity (ED 0.2 μM) than methyl 3-(4-hydroxyphenyl) propionate (MHPP) (ED 100 μM) (isolated from hydrophilic-BNIs fraction) in the in vitro culture-bioassay, but the activity was non-functional and ineffective in the soil-assay. Conclusions: There is an urgent need to identify sorghum genetic stocks with high potential to release functional-BNIs for suppressing nitrification and to improve nitrogen use efficiency in sorghum-based production systems. [ABSTRACT FROM AUTHOR]

Aims and background: The ability to suppress soil nitrification through the release of nitrification inhibitors from plant roots is termed 'biological nitrification inhibition' (BNI). Earlier, we reported that sorghum roots release higher BNI-activity when grown with NH, but not with NO as N source. Also for BNI release, rhizosphere pH of <5.0 is needed; beyond this, a negative effect on BNI release was observed with nearly 80% loss of BNI activity at pH >7.0. This study is aimed at understanding the inter-functional relationships associated with NH uptake, rhizosphere-pH and plasma membrane H-ATPase (PM H-ATPase) activity in regulating the release of BNIs (biological nitrification inhibitors) from sorghum roots. Methods: Sorghum was grown hydroponically and root exudates were collected from intact plants using a pH-stat system to separate the secondary acidification effects by NH uptake on BNIs release. A recombinant luminescent Nitrosomonas europaea bioassay was used to determine BNI-activity. Root plasma membrane was isolated using a two-phase partitioning system. Hydrolytic H-ATPase activity was determined. Split-root system setup was deployed to understand the localized responses to NH, H-ATPase-stimulator (fusicoccin) or H-ATPase-inhibitor (vanadates) on BNI release by sorghum. Results: Presence of NH in the rhizosphere stimulated the expression of H-ATPase activity and enhanced the release of BNIs from sorghum roots. Fusicoccin, which stimulates H-ATPase activity, also stimulated BNIs release in the absence of NH; vanadate, which suppresses H-ATPase activity, also suppressed the release of BNIs. NH levels (in rhizosphere) positively influenced BNIs release and root H-ATPase activity in the concentration range of 0-1.0 mM, indicating a close relationship between BNI release and root H-ATPase activity with a possible involvement of carrier-mediated transport for the release of BNIs in sorghum. Conclusion: Our results suggest that NH uptake, PM H-ATPase activity, and rhizosphere acidification are functionally inter-connected with BNI release in sorghum. Such knowledge is critical to gain insights into why BNI function is more effective in light-textured, mildly acidic soils compared to other soil types. [ABSTRACT FROM AUTHOR]

Using a recombinant luminescent Nitrosomonas europaea assay to quantify biological nitrification inhibition (BNI), we found that a wild relative of wheat ( Leymus racemosus (Lam.) Tzvelev) had a high BNI capacity and releases about 20 times more BNI compounds (about 30 ATU g−1 root dry weight 24 h−1) than Triticum aestivum L. (cultivated wheat). The root exudate from cultivated wheat has no inhibitory effect on nitrification when applied to soil; however, the root exudate from L. racemous suppressed $$ {\text{NO}}^{ - }_{3} $$ formation and kept more than 90% of the soil’s inorganic-N in the $$ {\text{NH}}^{ + }_{4} $$ -form for 60 days. The high-BNI capacity of L. racemosus is mostly associated with chromosome Lr#n. Two other chromosomes Lr#J, and Lr#I also have an influence on BNI production. Tolerance of L. racemosus to $$ {\text{NH}}^{ + }_{4} $$ is controlled by chromosome 7Lr#1-1. Sustained release of BNI compounds occurred only in the presence of $$ {\text{NH}}^{ + }_{4} $$ in the root environment. Given the level of BNI production expressed in DALr#n and assuming normal plant growth, we estimated that nearly 87,500,000 ATU of BNI activity ha−1 day−1 could be released in a field of vigorously growing wheat; this amounts to the equivalent of the inhibitory effect from the application of 52.5 g of the synthetic nitrification inhibitor nitrapyrin (one AT unit of BNI activity is equivalent to 0.6 μg of nitrapyrin). At this rate of BNI production it would take only 19 days for a BNI-enabled wheat crop to produce the inhibitory power of a standard commercial application of nitrapyrin, 1 kg ha−1. The synthetic nitrification inhibitor, dicyandiamide, blocked specifically the AMO (ammonia monooxygenase) pathway, while the BNI from L. racemosus blocked the HAO (hydroxylamine oxidoreductase) pathway in Nitrosomonas. Here we report the first finding of high production of BNI in a wild relative of any cereal and its successful introduction and expression in cultivated wheat. These results demonstrate the potential for empowering the new generation of wheat cultivars with high-BNI capacity to control nitrification in wheat-production systems. [ABSTRACT FROM AUTHOR]

Regulating nitrification could be a key strategy in improving nitrogen (N) recovery and agronomic N-use efficiency in situations where the loss of N following nitrification is significant. A highly sensitive bioassay using recombinant luminescent Nitrosomonas europaea, has been developed that can detect and quantify the amount of nitrification inhibitors produced by plants (hereafter referred to as BNI activity). A number of species including tropical and temperate pastures, cereals and legumes were tested for BNI in their root exudate. There was a wide range in BNI capacity among the 18 species tested; specific BNI (AT units activity g−1 root dry wt) ranged from 0 (i.e. no detectable activity) to 18.3 AT units. Among the tested cereal and legume crops, sorghum [ Sorghum bicolor (L.)], pearl millet [ Pennisetum glaucum (L.) R. Br.], and groundnut [ Arachis hypogaea (L.)] showed detectable BNI in root exudate. Among pasture grasses, Brachiaria humidicola (Rendle) Schweick, B. decumbens Stapf showed the highest BNI capacity. Several high- and low-BNI genotypes were identified within the B. humidicola species. Soil collected from field plots of 10 year-old high-BNI genotypes of B. humidicola, showed a near total suppression (>90%) of nitrification; most of the soil inorganic N remained in the NH form after 30 days of incubation. In contrast, soils collected from low-BNI genotypes did not show any inhibitory effect; most of the soil inorganic N was converted to NO after 30 days of incubation. In both the high- and low-BNI genotypes, BNI was detected in root exudate only when plants were grown with NH, but not when grown with NO as the sole source of N. BNI compounds when added to the soil inhibited nitrification and the relationship was linear ( r 2 = 0.92**; n = 12). The BNI from high- and low-BNI types when added to N. europaea in pure culture, blocked both the ammonia monooxygenase (AMO) and the hydroxylamine oxidoreductase (HAO) pathways. Our results indicated that BNI capacity varies widely among and within species; and that some degree of BNI capacity is likely a widespread phenomenon in tropical pasture grasses. We suggest that the BNI capacity could either be managed and/or introduced into pastures/crops with an expression of this phenomenon, via genetic improvement approaches that combine high productivity along with some capacity to regulate soil nitrification process. [ABSTRACT FROM AUTHOR]

Nitrification, a microbial process, is a key component and integral part of the nitrogen (N) cycle. Soil N is in a constant state of flux, moving and changing chemical forms. During nitrification, a relatively immobile N-form (NH 4 + ) is converted into highly mobile nitrate-N (NO 3 - ). The nitrate formed is susceptible to losses via leaching and conversion to gaseous forms via denitrification. Often less than 30% of the applied N fertilizer is recovered in intensive agricultural systems, largely due to losses associated with and following nitrification. Nitrogen-use efficiency (NUE) is defined as the biomass produced per unit of assimilated N and is a conservative function in most biological systems. A better alternative is to define NUE as the dry matter produced per unit N applied and strive for improvements in agronomic yields through N recovery. Suppressing nitrification along with its associated N losses is potentially a key part in any strategy to improve N recovery and agronomic NUE. In many mature N-limited ecosystems, nitrification is reduced to a relatively minor flux. In such systems there is a high degree of internal N cycling with minimal loss of N. In contrast, in most high-production agricultural systems nitrification is a major process in N cycling with the resulting N losses and inefficiencies. This review presents the current state of knowledge on nitrification and associated N losses, and discusses strategies for controlling nitrification in agricultural systems. Limitations of the currently available nitrification inhibitors are highlighted. The concept of biological nitrification inhibition (BNI) is proposed for controlling nitrification in agricultural systems utilizing traits found in natural ecosystems. It is emphasized that suppression of nitrification in agricultural systems is a critical step required for improving agronomic NUE and maintaining environmental quality. [ABSTRACT FROM AUTHOR]

Nitrification, the biological oxidation of ammonium to nitrate, weakens the soil's ability to retain N and facilitates N-losses from production agriculture through nitrate-leaching and denitrification. This process has a profound influence on what form of mineral-N is absorbed, used by plants, and retained in the soil, or lost to the environment, which in turn affects N-cycling, N-use efficiency (NUE) and ecosystem health and services. As reactive-N is often the most limiting in natural ecosystems, plants have acquired a range of mechanisms that suppress soil-nitrifier activity to limit N-losses via N-leaching and denitrification. Plants' ability to produce and release nitrification inhibitors from roots and suppress soil-nitrifier activity is termed 'biological nitrification inhibition' (BNI). With recent developments in methodology for in-situ measurement of nitrification inhibition, it is now possible to characterize BNI function in plants. This review assesses the current status of our understanding of the production and release of biological nitrification inhibitors (BNIs) and their potential in improving NUE in agriculture. A suite of genetic, soil and environmental factors regulate BNI activity in plants. BNI-function can be genetically exploited to improve the BNI-capacity of major food- and feed-crops to develop next-generation production systems with reduced nitrification and N2O emission rates to benefit both agriculture and the environment. The feasibility of such an approach is discussed based on the progresses made., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)