Developing indicators and characterizing direct and residual effects of biological nitrification inhibition (BNI) by the tropical forage grass Brachiaria humidicola

Nitrogen (N) losses from agroecosystems harm the environment via increased nitrate (NO3-) amounts in water-bodies and nitrous oxide (N2O) emissions to the atmosphere. Bacteria and archaea oxidize ammonium (NH4+) to NO3- under aerobic conditions. Furthermore, under mainly anaerobic conditions, microbial denitrification reduces NO3- to gaseous N forms. The tropical forage grass Brachiaria humidicola (Rendle) Schweick (Bh) has been shown to reduce soil microbial nitrification via root derived substances. Therefore, biological nitrification inhibition (BNI) by Bh might contribute to reduction of N losses from agroecosystems. The present doctoral thesis aimed at assessing the potential of the actual BNI by Bh, as well as the residual BNI effect with new developed methodologies. The overall research was based on the following major objectives: (1) characterization of the residual BNI effect by Bh on recovery of N by subsequent cropped maize (Zea mays L.) under different N fertilization rates; (2) investigate if low enzymatic nitrate reductase activity (NRA) in leaves of Bh is linked to reduced NO3- nutrition by effective BNI; (3) identify a possible link between plant delta 15N of Bh and the BNI effect of different Bh genotypes on nitrification, plant N uptake and NO3- leaching losses. The overall objective was to use and test new methodologies with a minimum of disturbance of the plant-soil system, to characterize BNI of different Bh genotypes in greenhouse and field studies. The first research study focused on the investigation of a potential residual BNI effect of a converted long-term Bh pasture on subsequent maize cropping, where a long-term maize monocrop field served as control. The residual BNI effect was characterized in terms of enhanced maize grain yield, total N uptake and 15N (labeled) fertilizer recovery. Furthermore, the impact of residual BNI effect on soil N dynamics was investigated. The residual BNI effect was confirmed for the first maize crop season after pasture conversion on the basis of lower nitrification in incubation soil, higher total N uptake and higher maize grain yields. However, the residual BNI effect did not result in higher 15N fertilizer uptake or reduced 15N fertilizer losses, nor in reduced N20 emissions. Applied N was strongly immobilized due to long-term root turnover effects, while a significant residual BNI effect from Bh prevented re-mineralized N from rapid nitrification resulting in improved maize performance. A significant residual Bh BNI effect was evident for less than one year only. In the second research study it was the aim to verify the potential of nitrate reductase activity (NRA) as a proxy for the detection of in vivo performance of BNI by selected Bh accessions and genotypes grown under contrasting fertilization regimes. NRA was detected in Bh leaves rather than in roots, regardless of NO3- availability. Leaf NRA correlated with NO3- contents in soils and stem sap of contrasting Bh genotypes substantiating its use as a proxy of in vivo performance of BNI. 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 selected Bh genotypes was observed. The third research study emphasized to link the natural abundance of delta 15N in Bh plants with reduced NO3- losses and enhanced N uptake due to BNI. Increased leached NO3- was positively correlated to rising delta 15N in Bh grass, whereas the correlation between plant N uptake and plant delta 15N was inverse. Long-term field cultivation of Bh decreased nitrification in incubated soil, whereas delta 15N of Bh declined and plant N% rose over time. Delta 15N of Bh correlated positively with assessed nitrification rates in incubated soil. It was concluded that decreasing delta 15N of Bh over time reflects the long-term effect of BNI linked to lower NO3- formation and reduced NO3- leaching, and that generally higher BNI activity of Bh is indicated by lower delta 15N plant values. Within the framework of this thesis, a residual BNI effect by Bh on maize cropping could be confirmed for one season due to the combined methodological approaches of soil incubation and 15N recovery. The development of the NRA assay for sampled Bh leaves was validated as a rapid and reliable method linked to the actual soil nitrification after NH4+ fertilizer supply. Consequently, the assay could be used for both greenhouse and field studies as BNI proxy. The gathered data from the third study indicated that decreasing delta 15N of Bh over time reflects the long-term effect of BNI linked to lower NO3- formation and reduced NO3- leaching, and that generally higher BNI activity of Bh is indicated by lower delta 15N plant values. Consequently, it was suggested that delta 15N of Bh could serve as an indicator of cumulative NO3- losses. Overall, this doctoral thesis suggests the depressing effect on nitrification by Bh might be a combined effect by BNI and fostered N immobilization. Furthermore, BNI by Bh might be altered by different factors such as soil type, plant age and root morphology of the genotypes. Finally, future studies should consider that Bh genotypes express their respective BNI potential differently under contrasting conditions.Stickstoff(N)-Verluste von Agrarökosystemen schädigen die Umwelt einerseits durch erhöhte Nitratauswaschung (NO3-), und andererseits durch Emissionen von Lachgas (N2O) in die Atmosphäre. NO3- ist das Produkt des bodenmetabolischen mikrobiellen Prozesses der Nitrifikation. Bakterien und Archaeen oxidieren Ammonium (NH4 +) unter aeroben Bedingungen zu NO3-. Das tropische Futtergras Brachiaria humidicola (Rendle) Schweick (Bh) reduziert nachweislich die mikrobielle Nitrifikation im Boden durch Abgabe von Wurzelexsudaten. Daher könnte die biologische Nitrifikationshemmung (BNI) durch Bh zur Verringerung von N-Verlusten aus Agrarökosystemen beitragen. Von genotypischen Unterschieden innerhalb von Bh in Bezug auf BNI wurde berichtet. Die Entwicklung neuer BNI-Indikatoren für den Einsatz unter Feldbedingungen könnte dazu beitragen, die Diskrepanz zwischen den unter kontrollierten Bedingungen bestimmten BNI-Potentialen und der BNI-Exprimierung im Feld zu verstehen. Die Hypothese, BNI könnte die nitrifikationsbasierte NO3- Bildung reduzieren, und folglich die Verluste durch NO3-Auswaschung verringern, wurde jedoch noch nicht überprüft. Die vorliegende Doktorarbeit basierte auf den folgenden Hauptzielen: (1) Der Charakterisierung des verbleibenden BNI-Effekts durch Bh auf die N-Aufnahme durch nachfolgenden Maisanbau (Zea mays L.) unter verschiedenen N-Düngungsraten; (2) Der Untersuchung, ob eine niedrige enzymatische Nitratreduktase-Aktivität (NRA) in den Blättern von Bh mit einer verringerten NO3- Ernährung durch einen effektiven BNI-Effekt verbunden ist; (3) Der Identifikation einer möglichen Verbindung zwischen der δ15N Pflanzensignatur von Bh Hybriden und deren BNI-Wirkung auf Nitrifikation, Pflanzen-N-Aufnahme und NO3-Auswaschungsverluste. Das übergeordnete Ziel bestand darin, neue Methoden mit einer minimalen Störung des Pflanzen-Boden-Systems zu verwenden und zu testen, um den BNI-Effekt verschiedener Bh-Genotypen in Gewächshaus- und Feldstudien zu charakterisieren. Die erste empirische Studie konzentrierte sich auf die Untersuchung eines möglichen Folgeeffekts durch BNI einer umgebrochenen Langzeit-BH-Weide auf anschließenden Maisanbau. Der BNI-Folgeeffekt wurde im Hinblick auf erhöhte Maisertragsausbeute, Gesamt-N-Aufnahme und 15N-Düngemittelrückgewinnung charakterisiert. Darüber hinaus wurde der Einfluß des verbleibenden BNI-Effekts auf die N-Dynamik untersucht. Der verbleidende BNI-Effekt wurde für die erste Maiserntezeit nach Bh-Weideumbruch auf der Basis von geringerer Nitrifikation in Inkubationsstudien, höherer Gesamt-N-Aufnahme und höheren Maiserträgen bestätigt. Der BNI-Folgeeffekt führte jedoch weder zu einer höheren 15N-Düngeraufnahme oder reduzierten 15N-Düngerverlusten. Ein signifikanter BNI-Folgeeffekt war jedoch nur für weniger als ein Jahr nachweisbar. In der zweiten Forschungsstudie wurde das Ziel verfolgt, das Potential der Nitratreduktaseaktivität (NRA) als Indikator für den aktuellen BNI-Effekt ausgewählter Bh-Akzessionen und Genotypen, die unter kontrastierenden Düngungsregimen getestet wurden, zu verifizieren. NRA wurde in Bh-Blättern, jedoch nicht in Wurzeln nachgewiesen, unabhängig von der NO3- Verfügbarkeit. Der Blatt-NRA-Test erleichterte ein schnelles Screening von kontrastierenden Bh-Genotypen in Bezug auf Unterschiede des in-vivo BNI-Effekts unter Feld- und Gewächshausbedingungen. Es wurde jedoch eine Inkonsistenz des BNI-Potentials innerhalb der ausgewählten Bh-Genotypen beobachtet. Die dritte Forschungsstudie untersuchte, ob die natürliche Abundanz von 15N (δ) in Bh-Pflanzen mit reduzierten NO3- Verlusten und einer verstärkten N-Aufnahme durch BNI verknüpft werden kann. Erhöhte NO3- Auswaschung war positiv mit steigendem δ15N in Bh korreliert, während die Korrelation zwischen Pflanzen-N-Aufnahme und δ15N in der Pflanze negativ war. δ15N-Werte in Bh korrelierten positiv mit Nitrifikationsraten in inkubierten Bodenproben. Schlußendlich konnte angenommen werden, dass die δ15N-Abnahme über die Zeit in Bh den BNI-Langzeiteffekt in Verbindung mit geringerer NO3- Bildung und reduzierter NO3- Auswaschung widerspiegelt, und dass eine allgemein höhere BNI-Aktivität von Bh durch niedrigere δ15N-Pflanzenwerte angezeigt wird. Im Rahmen dieser Doktorarbeit konnte ein verbleibender BNI-Effekt durch Bh auf darauffolgenden Maisanbau aufgrund der kombinierten methodischen Ansätze der Bodeninkubation und der 15N-Rückgewinnung für eine Saison bestätigt werden. Zudem wurde die NRA-Blatt-Methode entwickelt, welche eine schnelle und zuverlässige Analysemethode für die tatsächliche Bodennitrifikation nach NH4+-Düngung darstellt. Folglich könnte der Test sowohl für Gewächshaus- als auch für Feldstudien als BNI-Proxy verwendet werden. Die gesammelten Daten aus der dritten Studie zeigen, dass eine Abnahme von δ15N-Werten in Bh über die Zeit den BNI-Langzeiteffekt in Verbindung mit geringerer NO3- Bildung und reduzierter NO3- Auswaschung widerspiegelt. Zudem wird eine allgemein höhere BNI-Aktivität von Bh durch niedrigere δ15N-Pflanzenwerte angezeigt. Daraus läßt sich folgern, dass δ15N von Bh als Indikator für kumulative NO3- Verluste dienen kann. Insgesamt legt diese Dissertation nahe, dass die hemmende Wirkung auf die Nitrifikation durch Bh ein kombinierter Effekt von BNI und einer verstärkte N-Immobilisierung sein könnte. Darüber hinaus könnte der BNI-Effekt von Bh durch verschiedene Faktoren wie Bodenart, Pflanzenalter und Wurzelmorphologie der Genotypen variieren. Ebenfalls sollten zukünftige Studien berücksichtigen, dass Bh-Genotypen ihr jeweiliges BNI-Potential unter gegensätzlichen Bedingungen unterschiedlich exprimieren

Estimation of the optimal nitrogen dose in a Brachiaria humidicola-corn rotation system in the Colombian Eastern Plains

Improving nitrogen use efficiency (NUE) by optimizing the N fertilizer application dose is one way to reduce greenhouse gas emissions in agriculture and livestock production, especially in higher demanding crops such as corn. Taking a Brachiaria humidicola (Bh)-corn rotation system in the Colombian Eastern Plains, we seek to determine both the optimal economic dose (OED) and the optimal technical dose (OTD) of N, which allow to maximize income at producer level and minimize environmental impacts. This particular rotation system was chosen as research subject given the presence of the residual effect of Biological Nitrogen Inhibition (BNI) in permanent lots of Bh, which has positive impacts on corn production such as increased yields and better N efficiency. The data for this study was obtained from trials conducted between 2013 and 2017, where corn production in a Bh-corn rotation system (with residual BNI effect) was compared with conventional corn production (without residual BNI effect). For determining the OED and OTD of N, three response models were applied: a pseudo-quadratic model (PQM), a quadratic model (QM) and a discontinuous-rectilinear model (DRM). The results show that the PQM and DRM models turn out to be the most suitable for estimating the OTD producing a better fit for the data, thus the required N doses are not overestimated. Bh-corn treatments require lower OTD and OED compared to the control scenario, which results from the residual BNI effect. The OED is lower than the OTD in the QM and PQM models for the three treatments. Thus, for maximizing profits a lower N dose is required. Both N input and corn sales price variables determine the optimum dose for maximizing the producer's profits. In general, estimating the correct doses of N in a Bh-corn rotation system contributes to improving both efficiency in production and profitability, helping to avoid excessive application of N fertilizers and its associated negative effects on the environment

Impact of "Biological Nitrification Inhibition"on N recovery efficiency, N leaching and N2O emissions using the example of brachiaria humidicola

以线粒体Cyt b基因为分子标记,对雅鲁藏布江下游墨脱江段及察隅河的墨脱裂腹鱼进行遗传多样性及种群历史动态分析。结果显示,167尾墨脱裂腹鱼样本共检测到21个单倍型,呈现较高的单倍型多样性(h=0.768)和较低的核苷酸多样性(&pi;=0.00167)。基于单倍型构建的分子系统发育树及Network网络关系图表明,所有来自墨脱江段及察隅河的单倍型不能按照地理分布各自聚类,而是相互混杂聚在一起。不同地理种群间的遗传分化指数(F_(ST))为-0.014&mdash;0.771,其中金珠藏布(JZZB)与其他种群呈现出高度分化(F_(ST): 0.372&mdash;0.771)。分子方差分析(AMOVA)显示当JZZB种群为一组,剩余6个种群为一组时,组间遗传差异最大,表明JZZB种群与其他种群具有显著分化。相反,虽然察隅河与墨脱江段的地理距离较远,但是察隅河与墨脱江段其他种群之间(除了JZZB)的F_(ST)为0.093&mdash;0.169,仅显示中等分化水平,表明察隅河种群与雅鲁藏布江种群尚有少量的基因交流。中性检验、错配分析及BSP(Bayesian skyline plot)分析显示,雅鲁藏布江下游墨脱江段及察隅河的墨脱裂腹鱼种群在末次间冰期(0&mdash;0.137 Ma)发生过种群扩张现象。</p

Inter-microbial competition for N and plant NO3− uptake rather than BNI determines soil net nitrification under intensively managed Brachiaria humidicola

Brachiaria humidicola (syn. Urochloa humidicola) has been acknowledged to control soil nitrification through release of nitrification inhibitors (NI), a phenomenon conceptualized as biological nitrification inhibition (BNI). Liming and N fertilization as features of agricultural intensification may suppress BNI performance, due to a decrease in NI exudation, increased NH3 availability and promotion of ammonia oxidizing bacteria (AOB) over archaea (AOA). A 2-year three-factorial pot trial was conducted to investigate the influence of soil pH and soil microbial background (ratio of archaea to bacteria) on BNI performance of B. humidicola. The study verified the capacity of B. humidicola to reduce net nitrification rates by 50 to 85% compared to the non-planted control, irrespective of soil pH and microbial background. The reduction of net nitrification, however, was largely dependent on microbial N immobilization and efficient plant N uptake. A reduction of gross nitrification could not be confirmed for the AOA dominated soil, but possibly contributed to reduced net nitrification rates in the AOB-dominated soil. However, this putative reduction of gross nitrification was attributed to plant-facilitated inter-microbial competition between bacterial heterotrophs and nitrifiers rather than BNI. It was concluded that BNI may play a dominant role in extensive B. humidicola pasture systems, while N immobilization and efficient plant N uptake may display the dominant factors controlling net nitrification rates under intensively managed B. humidicola

Enlisting wild grass genes to combat nitrification in wheat farming: A nature-based solution

Active nitrifiers and rapid nitrification are major contributing factors to nitrogen losses in global wheat production. Suppressing nitrifier activity is an effective strategy to limit N losses from agriculture. Production and release of nitrification inhibitors from plant roots is termed "biological nitrification inhibition" (BNI). Here, we report the discovery of a chromosome region that controls BNI production in "wheat grass" Leymus racemosus (Lam.) Tzvelev, located on the short arm of the "Lr#3Ns(b)" (Lr#n), which can be transferred to wheat as T3BL.3Ns(b)S (denoted Lr#n-SA), where 3BS arm of chromosome 3B of wheat was replaced by 3Ns(b)S of L. racemosus. We successfully introduced T3BL.3Ns(b)S into the wheat cultivar "Chinese Spring" (CS-Lr#n-SA, referred to as "BNI-CS"), which resulted in the doubling of its BNI capacity. T3BL.3Ns(b)S from BNI-CS was then transferred to several elite high-yielding hexaploid wheat cultivars, leading to near doubling of BNI production in "BNI-MUNAL" and "BNI-ROELFS." Laboratory incubation studies with root-zone soil from field-grown BNI-MUNAL confirmed BNI trait expression, evident from suppression of soil nitrifier activity, reduced nitrification potential, and N2O emissions. Changes in N metabolism included reductions in both leaf nitrate, nitrate reductase activity, and enhanced glutamine synthetase activity, indicating a shift toward ammonium nutrition. Nitrogen uptake from soil organic matter mineralization improved under low N conditions. Biomass production, grain yields, and N uptake were significantly higher in BNI-MUNAL across N treatments. Grain protein levels and breadmaking attributes were not negatively impacted. Wide use of BNI functions in wheat breeding may combat nitrification in high N input-intensive farming but also can improve adaptation to low N input marginal areas.We gratefully acknowledge funding support from Japanese Ministry of Agriculture, Forestry and Fisheries, CGIAR Research Program on WHEAT during the execution of the research presented in this study

Exploiting crop genotype-specific root-soil interactions to enhance agronomic efficiency

Challenges of soil degradation and changing climate pose major threats to food security in many parts of the world, and new approaches are required to close yield and nutrition gaps through enhanced agronomic efficiency. Combined use of mineral fertilizers, organic inputs, improved germplasm and adaptation of these practices to local contexts through improved agronomy can promote efficiency whilst building stocks of soil organic matter (SOM). Within this framework, recent attention has turned to the nature of plant-soil interactions to increase response to mineral fertilizer inputs through utilisation of nutrients from SOM that are replenished through management. This utilisation has been shown in barley and maize to vary with genotype and to be related to root physiological traits associated with rhizodeposition. The identification of candidate genes associated with rhizodeposition takes this a step closer towards the possibility of breeding for sustainability. Here we discuss this potential and feasibility in the context of maize cropping systems, and explore the potential for a combined approach that optimises utilisation of SOM nutrients together with enhanced biological nitrification inhibition to further improve agronomic efficiency

Biological nitrification inhibition activity in a soil-grown biparental population of the forage grass, Brachiaria humidicola

Aim: Utilization of biological nitrification inhibition (BNI) strategy can reduce nitrogen losses in agricultural systems. This study is aimed at characterizing BNI activity in a plant-soil system using a biparental hybrid population of Brachiaria humidicola (Bh), a forage grass with high BNI potential but of low nutritional quality. Methods: Soil nitrification rates and BNI potential in root-tissue were analyzed in a hybrid population (117), obtained from two contrasting Bh parents, namely CIAT 26146 and CIAT 16888, with low and high BNI activity, respectively. Observed BNI activity was validated by measuring archaeal (AOA) and bacterial (AOB) nitrifier abundance in the rhizosphere soil of parents and contrasting hybrids. Comparisons of the BNI potential of four forage grasses were conducted to evaluate the feasibility of using nitrification rates to measure BNI activity under field and pot grown conditions. Results: High BNI activity was the phenotype most commonly observed in the hybrid population (72%). BNI activity showed a similar tendency for genotypes grown in pots and in the field. A reduction in AOA abundance was found for contrasting hybrids with low nitrification rates and high BNI potential. Conclusion: Bh hybrids with high levels of BNI activity were identified. Our results demonstrate that the microcosm incubation and the in vitro bioassay may be used as complementary methods for effectively assessing BNI activity. The full expression of BNI potential of Bh genotypes grown in the soil (i.e. low nitrification rates) requires up to one year to develop, after planting