Molecular design of hydroxamic acid-based derivatives as urease inhibitors of Helicobacter pylori.

Helicobacter pylori is the main causative agent of gastric cancer, especially non-cardiac gastric cancers. This bacterium relies on urease producing much ammonia to colonize the host. Herein, the study provides valuable insights into structural patterns driving urease inhibition for high-activity molecules designed via exploring known inhibitors. Firstly, an ensemble model was devised to predict the inhibitory activity of novel compounds in an automated workflow (R2 = 0.761) that combines four machine learning approaches. The dataset was characterized in terms of chemical space, including molecular scaffolds, clustering analysis, distribution for physicochemical properties, and activity cliffs. Through these analyses, the hydroxamic acid group and the benzene ring responsible for distinct activity were highlighted. Activity cliff pairs uncovered substituents of the benzene ring on hydroxamic acid derivatives are key structures for substantial activity enhancement. Moreover, 11 hydroxamic acid derivatives were designed, named mol1-11. Results of molecular dynamic simulations showed that the mol9 exhibited stabilization of the active site flap's closed conformation and are expected to be promising drug candidates for Helicobacter pylori infection and further in vitro, in vivo, and clinical trials to demonstrate in future.

Epiberberine: a potential rumen microbial urease inhibitor to reduce ammonia release screened by targeting UreG.

Rumen microbial urease inhibitors have been proposed for regulating nitrogen emission and improving nitrogen utilization efficiency in ruminant livestock industry. However, studies on plant-derived natural inhibitors of rumen microbial urease are limited. Urease accessory protein UreG, plays a crucial role in facilitating urease maturation, is a new target for design of urease inhibitor. The objective of this study was to select the potential effective inhibitor of rumen microbial urease from major protoberberine alkaloids in Rhizoma Coptidis by targeting UreG. Our results showed that berberine chloride and epiberberine exerted superior inhibition potential than other alkaloids based on GTPase activity study of UreG. Berberine chloride inhibition of UreG was mixed type, while inhibition kinetics type of epiberberine was uncompetitive. Furthermore, epiberberine was found to be more effective than berberine chloride in inhibiting the combination of nickel towards UreG and inducing changes in the second structure of UreG. Molecular modeling provided the rational structural basis for the higher inhibition potential of epiberberine, amino acid residues in G1 motif and G3 motif of UreG formed interactions with D ring of berberine chloride, while interacted with A ring and D ring of epiberberine. We further demonstrated the efficacy of epiberberine in the ruminal microbial fermentation with low ammonia release and urea degradation. In conclusion, our study clearly indicates that epiberberine is a promising candidate as a safe and effective inhibitor of rumen microbial urease and provides an optimal strategy and suitable feed additive for regulating nitrogen excretion in ruminants in the future. KEY POINTS: • Epiberberine is the most effective inhibitor of rumen urease from Rhizoma Coptidis. • Urease accessory protein UreG is an effective target for design of urease inhibitor. • Epiberberine may be used as natural feed additive to reducing NH3 release in ruminants.

Synergizing structure and function: Cinnamoyl hydroxamic acids as potent urease inhibitors.

The current investigation encompasses the structural planning, synthesis, and evaluation of the urease inhibitory activity of a series of molecular hybrids of hydroxamic acids and Michael acceptors, delineated from the structure of cinnamic acids. The synthesized compounds exhibited potent urease inhibitory effects, with IC50 values ranging from 3.8 to 12.8 µM. Kinetic experiments unveiled that the majority of the synthesized hybrids display characteristics of mixed inhibitors. Generally, derivatives containing electron-withdrawing groups on the aromatic ring demonstrate heightened activity, indicating that the increased electrophilicity of the beta carbon in the Michael Acceptor moiety positively influences the antiureolytic properties of this compounds class. Biophysical and theoretical investigations further corroborated the findings obtained from kinetic assays. These studies suggest that the hydroxamic acid core interacts with the urease active site, while the Michael acceptor moiety binds to one or more allosteric sites adjacent to the active site.

Synthesis and biological evaluation of triazolones/oxadiazolones as novel urease inhibitors.

Urease is the main virulence factor of infectious gastritis and gastric ulcers. Urease inhibitors are regarded as the first choice for the treatment of such diseases. Based on the triazolone/oxadiazolone skeleton, a urea-like fragment being able to specifically bind the urease activity pocket and prevent urea from hydrolysis, we designed and synthesized 45 triazolones/oxadiazolones as urease inhibitors. Eight compounds were proved to show excellent inhibitory activity against Helicobacter pylori urease, being more potency than the clinically used urease inhibitor acetohydroxamic acid. The most active inhibitor with IC50 value of 1.2 µM was over 20-fold higher potent than the positive control. Enzymatic kinetic assays showed that these novel inhibitors reversibly inhibited urease with a mixed competitive mechanism. Molecular dockings provided evidence for the observations in enzyme assays. Furthermore, these novel inhibitors were proved as drug-like compounds with very low cytotoxicity to mammalian cells and favorable water solubility. These results suggested that triazolone and oxadiazolone were promising scaffolds for the design and discovery of novel urease inhibitors, and were expected as good candidates for further drug development.

Global warming potential assessment under reclaimed water and livestock wastewater irrigation coupled with co-application of inhibitors and biochar.

The application of nitrification inhibitors (nitrapyrin) and urease inhibitors (N-(N-butyl) thiophosphoric triamide) under conventional water resources has been considered as an effective means to improve nitrogen utilization efficiency and mitigate soil greenhouse gas emissions. However, it is not known whether the inhibitors still have an inhibitory effect under unconventional water resources (reclaimed water and livestock wastewater) irrigation and whether their use in combination with biochar improves the mitigation effect. Therefore, unconventional water resources were used for irrigation, with groundwater (GW) control. Nitrapyrin and N-(N-butyl) thiophosphoric triamide were used alone or in combination with biochar in a pot experiment, and CO2, N2O, and CH4 emissions were measured. The results showed that irrigation of unconventional water resources exacerbated global warming potential (GWP). All exogenous substance treatments increased CO2 and CH4 emissions and suppressed N2O emissions, independent of the type of water, compared to no substances (NS). The inhibitors were ineffective in reducing the GWP whether or not in combination with biochar, and the combined application of inhibitors with biochar further increased the GWP. This study suggests that using inhibitors and biochar in combination to regulate the greenhouse effect under unconventional water resources irrigation should be done with caution.

Design, synthesis, and biological studies of the new cysteine-N-arylacetamide derivatives as a potent urease inhibitor.

Inhibition of Helicobacter pylori urease is an effective method in the treatment of several gastrointestinal diseases in humans. This bacterium plays an important role in the pathogenesis of gastritis and peptic ulceration. Considering the presence of cysteine and N-arylacetamide derivatives in potent urease inhibitors, here, we designed hybrid derivatives of these pharmacophores. Therefore, cysteine-N-arylacetamide derivatives 5a-l were synthesized through simple nucleophilic reactions with good yield. In vitro urease inhibitory activity assay of these compounds demonstrated that all newly synthesized compounds exhibited high inhibitory activity (IC50 values = 0.35-5.83 µM) when compared with standard drugs (thiourea: IC50 = 21.1 ± 0.11 µM and hydroxyurea: IC50 = 100.0 ± 0.01 µM). Representatively, compound 5e with IC50 = 0.35 µM was 60 times more potent than strong urease inhibitor thiourea. Enzyme kinetic study of this compound revealed that compound 5e is a competitive urease inhibitor. Moreover, a docking study of compound 5e was performed to explore crucial interactions at the urease active site. This study revealed that compound 5e is capable to inhibit urease by interactions with two crucial residues at the active site: Ni and CME592. Furthermore, a molecular dynamics study confirmed the stability of the 5e-urease complex and Ni chelating properties of this compound. It should be considered that, in the following study, the focus was placed on jack bean urease instead of H. pylori urease, and this was acknowledged as a limitation.

Ammonia volatization from conventional and stabilized fertilizers, agronomic aspects and microbiological attributes in a Brazilian coffee crop system.

We aimed to quantify the N losses through volatilization of the main conventional and stabilized N fertilizers applied in coffee plantations. Additionally, we also assessed microbiological attributes of the soil (microbial biomass carbon (MBC); microbial biomass nitrogen (MBN); microbial basal respiration (MBR); metabolic quotient (qCO2); urease, ß-glucosidase, acid phosphatase, and arylsulfatase activities) and agronomic aspects of the crop (N content in the leaves and beans, yield, and N exportation by the beans). Treatments consisted of the combination of three fertilizers (ammonium nitrate - AN, conventional urea - U, and urea with N- (n-butyl) thiophosphoric triamide (NBPT) - UNBPT, and five doses of N (0, 150, 275, 400, and 525 kg ha-1 year-1 of N), with four replicates, totalling 60 experimental plots. In the two crop seasons evaluated, daily and cumulative losses of N-NH3 from the split fertilizer applications were influenced by the N fertilizer technologies. The application of U resulted in losses of 22.0% and 22.8% for the doses of 150 and 400 kg ha-1 year-1 of N. This means that 66 and 182 kg ha-1 of N-NH3 were lost, respectively, at the end of six fertilizations with U. UNBPT reduced urease activity and N-NH3 losses compared to conventional urea, avoiding the volatilization of 15.9 and 24.3 kg ha-1 of N. As for AN, N-NH3 losses did not exceed 1% of the applied dose, regardless of the weather conditions during the fertilization. Urease activity was higher on days of maximum NH3 volatilization. There was an effect of the N sources (NS), soil sampling time (ST), and their interaction (NS × ST) on the MBN and arylsulfatase activity. The N sources also influenced the MBC and the qCO2. A substantial amount of N was removed from the system by the beans and husks of the harvested fruits. Our study showed that N fertilizer technologies are interesting options to reduce N-NH3 losses by volatilization, increase N retention in the soil, and improve microbiological attributes and the sustainability of coffee production systems.

Effects of combined natural synergists and chemical inhibitors on yield, nitrogen utilization and balance in wheat/maize rotation system.

Urease inhibitors and nitrification inhibitors can enhance nitrogen (N) fertilizer utilization efficiency and reducing N losses through regulating urea-N transformation. Common urease or nitrification inhibitors, however, are predominantly chemically synthesized and high-cost. Furthermore, their inhibitory effects are mediated by soil pro-perties, climatic conditions, and crop systems. In this study, we conducted a field experiment using natural synergists humic acid/zeolite, along with chemical nitrification inhibitor dicyandiamide (DCD) and their combination to elucidate the impacts of natural synergists combined with chemical inhibitors on annual yield, nitrogen utilization efficiency, soil nitrate-N accumulation, and nitrogen balance within the wheat/maize rotation system. The treatments included no nitrogen fertilizer application (CK), single application of urea (N), urea +DCD (ND), urea + humic acid (NH), urea + zeolite (NP), urea + urease inhibitor N-butylthiophosphoric triamide + DCD (NUD), urea + humic acid + DCD (NHD), and urea + zeolite + DCD (NPD). The results showed that, compared to the treatments NH and NP, the integration of humic acid or zeolite with DCD (NHD and NPD) significantly increased maize yield (11268 and 11397 kg·hm-2) and total annual yield (20494 and 20582 kg·hm-2), which were comparable to those of combined chemical urease and nitrification inhibitors (NUD). The NHD and NPD treatments had higher nitrogen utilization efficiency and lower soil nitrate-N accumulation in 80-100 cm soil layer across all seasons relative to the N treatment, which had no significant difference compared to the NUD treatment. Furthermore, a decline in soil nitrogen surplus by 10.7% and 13.9% was observed when comparing the NHD and NPD treatments with the NH and NP treatments, respectively. These findings suggested that combined humic acid or zeolite and chemical nitrification inhibitors could effectively enhance crop yield and N utilization efficiency and meet the requirements of the green and environmental preservation of modern agriculture.

Natural Urease Inhibitors Reduce the Severity of Disease Symptoms, Dependent on the Lifestyle of the Pathogens.

The development of new anti-ureolytic compounds is of great interest due to the newly discovered role of urease inhibitors in crop protection. Purine degradation and the generation of ammonium by urease are required for the full virulence of biotrophic and hemibiotrophic fungal plant pathogens. Accordingly, chemicals displaying urease inhibitor activity may be used as a novel class of fungicides. Several urease inhibitors belonging to different chemical classes are known, and some compounds have been developed as urea fertilizer additives. We tested whether the natural urease inhibitors p-benzoquinone (p-HQ) and hydroquinone (HQ), as well as the synthetic inhibitors isopropoxy carbonyl phosphoric acid amide (iCPAA), benzyloxy carbonyl phosphoric acid amide (bCPAA), and dipropyl-hexamino-1,3 diphosphazenium chloride (DDC), prevent or delay plant infection caused by pathogens differing in lifestyles and host plants. p-BQ, HQ, and DCC not only protected maize from infection by the hemibiotroph C. graminicola, but also inhibited the infection process of biotrophs such as the wheat powdery mildew fungus Blumeria graminis f. sp. tritici and the broad bean rust fungus Uromyces viciae-fabae. Interestingly, the natural quinone-based compounds even reduced the symptom severity of the necrotrophic fungi, i.e., the grey mold pathogen B. cinerea and the Southern Leaf Spot fungus C. heterostrophus, to some extent. The urease inhibitors p-BQ, HQ, and DCC interfered with appressorial penetration and confirmed the appropriateness of urease inhibitors as novel fungicidal agents.

Effects of nitrification and urease inhibitors on ammonia-oxidizing microorganisms, denitrifying bacteria, and greenhouse gas emissions in greenhouse vegetable fields.

Soil microorganisms and N cycling are important components of biogeochemical cycling processes. In addition, the study of the effects of nitrification and urease inhibitors on N and microorganisms in greenhouse vegetable fields is essential to reducing N loss and greenhouse gas emissions. The effects of nitrification inhibitors [2-chloro-6-(trichloromethyl) pyridine (CP), dicyandiamide (DCD)], and urease inhibitor [N-(n-butyl) thiophosphoric triamide (NBPT)] on soil inorganic N (NH4+-N, NO2--N and NO3--N) concentrations and the production rates of greenhouse gases (N2O, CH4, and CO2) in greenhouse vegetable fields were investigated via indoor incubation experiments. Polymerase chain reaction amplification and high-throughput sequencing technology (Illumina Miseq) were used to explore the community structure and abundance changes of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and denitrifying bacteria (nirK and nirS). The results showed that CP and DCD obviously inhibited NH4+-N conversion, and NO2--N, and NO3--N accumulation, NBPT slowed down urea hydrolysis and NH4+-N production, and the apparent nitrification rates of soil were in the following order: NBPT > DCD > DCD + NBPT > CP + NBPT > CP. Compared with urea treatment, the peak N2O production rate of inhibitor treatment decreased by 73.30-99.30%, and the production rate of CH4 and CO2 decreased by more than 66.16%. DCD and CP reduced the abundance of AOA and AOB, respectively. Furthermore, NBPT hindered the growth of ammonia-oxidizing microorganisms and nirS-type denitrifying bacteria, and urea and nitrification inhibitors were detrimental to the growth of Ensifer and Sinorhizobium in the nirK community. Nitrification and urease inhibitors can effectively slow down nitrification and greenhouse gas emissions, reduce N loss and improve soil quality by inhibiting the growth of ammonia-oxidizing microorganisms and denitrifying bacteria.

Nitrification and urease inhibitors mitigate global warming potential and ammonia volatilization from urea in rice-wheat system in India: A field to lab experiment.

The efficacy of alternative nitrogenous fertilizers for mitigating greenhouse gas and ammonia emissions from a rice-wheat cropping system in northern India was addressed in a laboratory incubation experiment using soil from a 10-year residue management field experiment (crop residue removal, CRR, vs. incorporation, CRI). Neem coated urea (NCU), standard urea (U), urea ammonium sulfate (UAS), and two alternative fertilizers, urea + urease inhibitor NBPT (UUI) and urea + urease inhibitor NBPT + nitrification inhibitor DMPSA (UUINI) were compared to non-fertilized controls for four weeks in incubation under anaerobic condition. Effects of fertilizers on global warming potential (GWP) and ammonia volatilization were dependent on residue treatment. Relative to standard urea, NCU reduced GWP by 11 % in CRI but not significantly in CRR; conversely, UAS reduced GWP by 12 % in CRR but not significantly in CRI. UUI and UUINI reduced GWP in both residue treatments and were more effective in CRI (21 % and 26 %) than CRR (15 % and 14 %). Relative to standard urea, NCU increased ammonia volatilization by 8 % in CRI but not significantly in CRR. Ammonia volatilization was reduced most strongly by UUI (40 % in CRI and 37 % in CRR); it was reduced 28-29 % by UUINI and 12-15 % by UAS. Overall, the urease inhibitor, alone and in combination with the nitrification inhibitor, was more effective in mitigating greenhouse gas and ammonia emissions than NCU. However, these products need to be tested in field settings to validate findings from the controlled laboratory experiment.

Effects of the Combining Straw Return with Urease Inhibitor on Ammonia Volatilization, Nitrogen Use Efficiency, and Rice Yield in Purple Soil Areas.

Straw return in rice (Oryza sativa L.) paddy has been heavily criticized for its potential to influence ammonia (NH3) volatilization loss due to irrational fertilizer N application. Therefore, improving the N fertilization strategies within residue straw systems is necessary to reduce N loss from NH3 volatilization. This study investigated how the incorporation of oilseed rape straw and the urease inhibitor affected NH3 volatilization, fertilizer N use efficiency (FNUE), and rice yields over two growing seasons (2018-2019) in the purple soil region. This study arranged eight treatments combined straw (2, 5, 8 ton ha-1, named 2S, 5S, 8S, respectively), with urea or urease inhibitor (UI, 1% NBPT) with three replicates, which included control (CK), UR (Urea, 150 kg N ha-1), UR + 2S, UR + 5S, UR + 8S, UR + 2S + UI, UR + 5S + UI, UR + 8S + UI, based on the randomized complete block method. Our results indicated that incorporating oilseed rape straw increased NH3 losses by 3.2-30.4% in 2018 and 4.3-17.6% in 2019 than the UR treatment, attributing to the higher NH4+-N content and pH value within floodwater. However, the UR + 2S + UI, UR + 5S + UI and UR + 8S + UI treatments reduced NH3 losses by 3.8%, 30.3%, and 8.1% in 2018 and 19.9%, 39.5%, and 35.8% in 2019, separately compared to their corresponding UR plus straw treatments. According to the findings, adding 1% NBPT significantly decreased NH3 losses while incorporating 5 ton ha-1 oilseed rape straw. Furthermore, adding straw, either alone or in conjunction with 1% NBPT, increased rice yield and FNUE by 0.6-18.8% and 0.6-18.8%, respectively. Otherwise, NH3 losses scaled by yield in the UR + 5S + UI treatment decreased significantly between all treatments in 2018 and 2019. These results suggest that optimizing the oilseed rape straw rate combined with 1% NBPT applied with urea efficiently increased rice yield and reduced NH3 emissions in the purple soil region of Sichuan Province, China.

The fate of nitrification and urease inhibitors in simulated bank filtration.

The application of nitrification and urease inhibitors (NUI) in conjunction with nitrogen (N) fertilizers improves the efficiency of N fertilizers. However, NUI are frequently found in surface waters through leaching or surface runoff. Bank filtration (BF) is considered as a low-cost water treatment system providing high quality water by efficiently removing large amounts of organic micropollutants from surface water. The fate of NUI in managed aquifer recharge systems such as BF is poorly known. The aim of this work was to investigate sorption and degradation of NUI in simulated BF under near-natural conditions. Besides, the effect of NUI on the microbial biomass of slowly growing microorganisms and the role of microbial biomass on NUI removal was investigated. Duplicate sand columns (length 1.7 m) fed with surface water were spiked with a pulse consisting of four nitrification (1,2,4-triazole, dicyanodiamide, 3,4-dimethylpyrazole and 3-methylpyrazole) and two urease inhibitors (n-butyl-thiophosphoric acid triamide and n-(2-nitrophenyl) phosphoric triamide). The average spiking concentration of each NUI was 5 µg/L. Experimental and modeled breakthrough curves of NUI indicated no retardation for any of the inhibitors. Therefore, biodegradation was identified as the main elimination pathway for all substances and was highest in zones of high microbial biomass. Removal of 1,2,4-triazole was 50% and n-butyl-thiophosphoric acid triamide proved to be highly degradable and was completely removed after a hydraulic retention time (HRT) of 24 h. 50% of the mass recovery for nitrification inhibitors except for 3,4-dimethylpyrazole was observed at the effluent (4 days HRT). In addition, a mild effect of NUI on microbial biomass was noted. This study highlights that the degradation of NUI in BF depends on HRT and microbial biomass.

Influence of the urease inhibitor suspension (Atmowell®) on the fluorescent dye pyranine and its spray and drift behavior in wind tunnel measurements.

Too many ammonia emissions are released into the environment from cattle farming. These damage the environment and have an impact on animal and human health. Ammonia Emissions could be reduce by urease inhibitors. Before using the urease inhibitor suspension Atmowell® in cattle farming a risk assessment is required. This includes exposure data on the animal and human in the barn. As there is no method for exposure measurements yet the approach of fluorometry was taken. The fluorescent dye pyranine shall replace Atmowell® in later studies as a tracer. Before Atmowell® can be replaced, the interaction between Atmowell® and pyranine-according to the fluorescence and storage stability under the influence of ultraviolet light, has to be observed and excluded. Also, the spray and drift behavior must be examined in the wind tunnel with three different nozzles. The results show that Atmowell® has no effect on neither the fluorescence nor the degradation rate of a pyranine-solution. Furthermore, it is shown that a pyranine + Atmowell® mixture does not differ in drift behavior from a pure pyranine-solution. Because of these findings, an Atmowell®-solution can be substituted by a pyranine-solution without any effects on the results of an exposure measurement being expected.

An overview: metal-based inhibitors of urease.

Urease is a kind of nickel-dependent metalloenzyme, which exists in the biological world widely, and can catalyse the hydrolysis of urea into ammonia and carbon dioxide to provide a nitrogen source for organisms. Urease has important uses in agriculture and medicine because it can catalyse the production of ammonia. Therefore, in this review, metal-based inhibitors of urease will be summarised according to different transition metal ions. Including the urease inhibition, structure-activity relationship, and molecular docking. Importantly, among these reviewed effective urease inhibitors, most of copper metal complexes exhibited stronger urease inhibition with IC50 values ranging from 0.46 µM to 41.1 µM. Significantly, the collected comprehensive information looks forward to providing rational guidance and effective strategies for the development of novel, potent, and safe metal-based urease inhibitors, which are better for practical applications in the future.

Identification of (N-aryl-N-arylsulfonyl)aminoacetohydroxamic acids as novel urease inhibitors and the mechanism exploration.

Thirty-three (N-aryl-N-arylsulfonyl)aminoacetohydroxamic acids were synthesized in an effort to develop novel urease inhibitors. Among these compounds, 2-(N-(3-nitrophenyl)-N-(4-tert-butylphenylsulfonyl))aminoacetohydroxamic acid (e2) exhibited excellent inhibitory activity against Helicobacter pylori urease with no perceptible cytotoxicity to mammalian cells. Compound e2 showed over 690-fold higher potency than the clinical used urease inhibitor acetohydroxamic acid, reversibly inhibiting urease with a mixed mechanism. Molecular modeling revealed that (N-aryl-N-arylsulfonyl)aminoacetohydroxamic acids may possibly bind Ni ions and two hydrophobic regions with a 'Y'-like shape.

Synthesis, evaluation and mechanism exploration of 2-(N-(3-nitrophenyl)-N-phenylsulfonyl)aminoacetohydroxamic acids as novel urease inhibitors.

Thirteen 2-(N-(3-nitrophenyl)-N-phenylsulfonyl)aminoacetohydroxamic acids which were reported for the first time were designed and synthesized as novel urease inhibitors. Most of them showed higher potency than the positive control acetohydroxamic acid, with 2-(N-(3-nitrophenyl)-N-(4-bromophenylsulfonyl)aminoacetohydroxamic acid (d7) being the most active (IC50 = 0.13 ± 0.01 µM). Compound d7 reversibly inhibits urease with mixed mechanism showing excellent binding affinity to urease active site (KD = 0.34 nM, Ki=0.065 ± 0.003 µM andKi' = 1.20 ± 0.09 µM) and very low cytotoxicity against mammalian cells (cell viability of 91.4 % against HepG2 at 250 µg/mL). These positive results indicated that d7 may be used as the lead for further research to develop urease inhibitors with promising properties.

The Influence of Fertilization and Plant Density on the Dry Matter Yield and Quality of Black Mustard [Brassica nigra (L.) Koch]: An Alternative Forage Crop.

Black mustard [Brassica nigra (L.) Koch] is mainly cultivated as a seed crop, and there is a lack of information on biomass quality and its potential for animal feeding. A 2-year field experiment was set up in a split-plot design with 2 main plots (plant densities: 46 and 76 plants m-2), 4 sub-plots (fertilization levels: control, compost, urea with and without urease and nitrification inhibitors) and 3 replications for each treatment. The highest dry matter yield (17.55-18.34 tn ha-1) was observed in high-density plots fertilized with urea fertilizer coated with double (nitrification and urease) inhibitors. In terms of the qualitive parameters of total above-ground biomass, the highest crude protein (CP) content was achieved in plots with low density and urea with double inhibitors. Moreover, the highest neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents of above-ground biomass were found under compost and urea with double inhibitors. The high ADF, NDF and relatively high CP content characterized that black mustard aerial biomass can meet the requirements of lactating animals, and therefore the production of black mustard biomass as a forage crop could be of great importance. As a conclusion, black mustard cultivated at plant densities higher than 46 plants m-2 and under inorganic fertilization, especially with urea coated with double inhibitors, could be successfully used as a novel forage crop in ruminants' diets.

Potential application of urease and nitrification inhibitors to mitigate emissions from the livestock sector: a review.

Human activities have caused an increase in greenhouse gas emissions, resulting in climate change that affects many factors of human life including its effect on water and food quality in certain areas with implications for human health. CH4 and N2O are known as potent non-CO2 GHGs. The livestock industry contributes to direct emissions of CH4 (38.24%) and N2O (6.70%) through enteric fermentation and manure treatment, as well as indirect N2O emissions via NH3 volatilization. NH3 is also a secondary precursor of particulate matter. Several approaches have been proposed to address this issue, including dietary management, manure treatment, and the possibility of inhibitor usage. Inhibitors, including urease and nitrification inhibitors, are widely used in agricultural fields. The use of urease and nitrification inhibitors is known to be effective in reducing nitrogen loss from agricultural soil in the form of NH3 and N2O and can further reduce CH4 as a side effect. However, the effectiveness of inhibitors in livestock manure systems has not yet been explored. This review discusses the potential of inhibitor usage, specifically of N-(n-butyl) thiophosphoric triamide, dicyandiamide, and 3,4-dimethylpyrazole phosphate, to reduce emissions from livestock manure. This review focuses on the application of inhibitors to manure, as well as the association of these inhibitors with health, toxicity, and economic benefits.

Global evaluation of inhibitor impacts on ammonia and nitrous oxide emissions from agricultural soils: A meta-analysis.

Inhibitors are widely considered an efficient tool for reducing nitrogen (N) loss and improving N use efficiency, but their effectiveness is highly variable across agroecosystems. In this study, we synthesized 182 studies (222 sites) worldwide to evaluate the impacts of inhibitors (urease inhibitors [UI], nitrification inhibitors [NI] and combined inhibitors) on crop yields and gaseous N loss (ammonia [NH3 ] and nitrous oxide [N2 O] emissions) and explored their responses to different management and environmental factors including inhibitor application timing, fertilization regime, cropping system, water management, soil properties and climatic conditions using subgroup meta-analysis, meta-regression and multivariate analyses. The UI were most effective in enhancing crop yields (by 5%) and reducing NH3 volatilization (by 51%), whereas NI were most effective at reducing N2 O emissions (by 49%). The application of UI mitigates NH3 loss and increases crop yields especially in high NH3 -N loss scenarios, whereas NI application would minimize the net N2 O emissions and the resultant environmental impacts especially in low NH3 -N loss scenarios. Alternatively, the combined application of UI and NI enables producers to balance crop production and environmental conservation goals without pollution tradeoffs. The inhibitor efficacy for decreasing gaseous N loss was dependent upon soil and climatic conditions and management practices. Notably, both meta-regression and multivariate analyses suggest that inhibitors provide a greater opportunity for reducing fertilizer N inputs in high-N-surplus systems and presumably favor crop yield enhancement under soil N deficiency situations. The pursuit of an improved understanding of the interactions between plant-soil-climate-management systems and different types of inhibitors should continue to optimize the effectiveness of inhibitors for reducing environmental losses while increasing productivity.