G. V. Subbarao

Scope and Strategies for Regulation of Nitrification in Agricultural Systems—Challenges and Opportunities

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.

Sodium—A Functional Plant Nutrient

Plant scientists usually classify plant mineral nutrients based on the concept of “essentiality” defined by Arnon and Stout as those elements necessary to complete the life cycle of a plant. Certain other elements such as Na have a ubiquitous presence in soils and waters and are widely taken up and utilized by plants, but are not considered as plant nutrients because they do not meet the strict definition of “essentiality.” Sodium has a very specific function in the concentration of carbon dioxide in a limited number of C4 plants and thus is essential to these plants, but this in itself is insufficient to generalize that Na is essential for higher plants. The unique set of roles that Na can play in plant metabolism suggests that the basic concept of what comprises a plant nutrient should be reexamined. We contend that the class of plant mineral nutrients should be comprised not only of those elements necessary for completing the life cycle, but also those elements which promote maximal biomass yield and/or which reduce the requirement (critical level) of an essential element. We suggest that nutrients functioning in this latter manner should be termed “functional nutrients.” Thus plant mineral nutrients would be comprised of two major groups, “essential nutrients” and “functional nutrients.” We present an array of evidence and arguments to support the classification of Na as a “functional nutrient,” including its requirement for maximal biomass growth for many plants and its demonstrated ability to replace K in a number of ways, such as being an osmoticium for cell enlargement and as an accompanying cation for long-distance transport. Although in this paper we have only attempted to make the case for Na being a “functional nutrient,” other elements such as Si and Se may also confirm to the proposed category of “functional nutrients.”

Evidence for biological nitrification inhibition in Brachiaria pastures

Nitrification, a key process in the global nitrogen cycle that generates nitrate through microbial activity, may enhance losses of fertilizer nitrogen by leaching and denitrification. Certain plants can suppress soil-nitrification by releasing inhibitors from roots, a phenomenon termed biological nitrification inhibition (BNI). Here, we report the discovery of an effective nitrification inhibitor in the root-exudates of the tropical forage grass Brachiaria humidicola (Rendle) Schweick. Named “brachialactone,” this inhibitor is a recently discovered cyclic diterpene with a unique 5-8-5-membered ring system and a γ-lactone ring. It contributed 60–90% of the inhibitory activity released from the roots of this tropical grass. Unlike nitrapyrin (a synthetic nitrification inhibitor), which affects only the ammonia monooxygenase (AMO) pathway, brachialactone appears to block both AMO and hydroxylamine oxidoreductase enzymatic pathways in Nitrosomonas. Release of this inhibitor is a regulated plant function, triggered and sustained by the availability of ammonium (NH4+) in the root environment. Brachialactone release is restricted to those roots that are directly exposed to NH4+. Within 3 years of establishment, Brachiaria pastures have suppressed soil nitrifier populations (determined as amoA genes; ammonia-oxidizing bacteria and ammonia-oxidizing archaea), along with nitrification and nitrous oxide emissions. These findings provide direct evidence for the existence and active regulation of a nitrification inhibitor (or inhibitors) release from tropical pasture root systems. Exploiting the BNI function could become a powerful strategy toward the development of low-nitrifying agronomic systems, benefiting both agriculture and the environment.

Detection, isolation and characterization of a root-exuded compound, methyl 3-(4-hydroxyphenyl) propionate, responsible for biological nitrification inhibition by sorghum (Sorghum bicolor)

Nitrification results in poor nitrogen (N) recovery and negative environmental impacts in most agricultural systems. Some plant species release secondary metabolites from their roots that inhibit nitrification, a phenomenon known as biological nitrification inhibition (BNI). Here, we attempt to characterize BNI in sorghum (Sorghum bicolor). In solution culture, the effect of N nutrition and plant age was studied on BNI activity from roots. A bioluminescence assay using recombinant Nitrosomonas europaea was employed to determine the inhibitory effect of root exudates. One major active constituent was isolated by activity-guided HPLC fractionations. The structure was analysed using NMR and mass spectrometry. Properties and the 70% inhibitory concentration (IC(70)) of this compound were determined by in vitro assay. Sorghum had significant BNI capacity, releasing 20 allylthiourea units (ATU) g(-1) root DW d(-1). Release of BNI compounds increased with growth stage and concentration of supply. NH4+ -grown plants released several-fold higher BNI compounds than NO3- -grown plants. The active constituent was identified as methyl 3-(4-hydroxyphenyl) propionate. BNI compound release from roots is a physiologically active process, stimulated by the presence of NH4+. Methyl 3-(4-hydroxyphenyl) propionate is the first compound purified from the root exudates of any species; this is an important step towards better understanding BNI in sorghum.

Biological Nitrification Inhibition--A Novel Strategy to Regulate Nitrification in Agricultural Systems

Human activity has had the single largest influence on the global nitrogen (N) cycle by introducing unprecedented amounts of reactive-N into ecosystems. A major portion of this reactive-N, applied as fertilizer to crops, leaks into the environment with cascading negative effects on ecosystem functions and contributes to global warming. Natural ecosystems use multiple pathways of the N-cycle to regulate the flow of this element. By contrast, the large amounts of N currently applied in agricultural systems cycle primarily through the nitrification process, a single inefficient route that allows much of the reactive-N to leak into the environment. The fact that present agricultural systems do not channel this reactive-N through alternate pathways is largely due to uncontrolled soil nitrifier activity, creating a rapid nitrifying soil environment. Regulating nitrification is therefore central to any strategy for improving nitrogen-use efficiency. Biological nitrification inhibition (BNI) is an active plant-mediated natural function, where nitrification inhibitors released from plant roots suppress soil-nitrifying activity, thereby forcing N into other pathways. This review illustrates the presence of detection methods for variation in physiological regulation of BNI-function in field crops and pasture grasses and analyzes the potential for its genetic manipulation. We present a conceptual framework utilizing a BNI-platform that integrates diverse crop science disciplines with ecological principles. Sustainable agriculture will require development of production systems that include new crop cultivars capable of controlling nitrification (i.e., high BNI-capacity) and improved agronomic practices to minimize leakage of reactive-N during the N-cycle, a critical requirement for increasing food production while avoiding environmental damage.

Suppression of nitrification and nitrous oxide emission by the tropical grass Brachiaria humidicola

Nitrification by soil nitrifiers may result in substantial losses of applied nitrogen through NO3− leaching and N2O emission. The biological inhibition of nitrification by crop plants or pasture species is not well known. This study was conducted to evaluate the ability of three pasture species, Brachiaria humidicola, B. decumbens and Melinis minutiflora to inhibit nitrification. Plants were grown in a growth chamber for sixty days, fertilized with (NH4)2SO4. After harvesting, the soil was incubated with (NH4)2SO4 for 24 days. Ammonium oxidizing bacteria (AOB), NH4-N levels, and N2O emission were monitored at 4 d intervals. Among the species studied, B. humidicola inhibited nitrification and maintained NH4-N in soil to a much greater extent than the other two species. This nitrification inhibition lasted for 12 days after initiation of soil incubation study (i.e. from 60 DAS when the plants were harvested). The AOB populations and N2O emission from the soil were significantly lower in the soils where B. humidicola has been grown compared to the other two species. Root exudates and soil extracts of B. humidicola suppressed AOB populations, whereas those of B. decumbens and M. minutiflora did not. The results are in consistence with the hypothesis that B. humidicola suppressed nitrification and N2O emissions through an inhibitory effect on the AOB population.

Potential for biological nitrification inhibition to reduce nitrification and N2O emissions in pasture crop-livestock systems

Agriculture and livestock production systems are two major emitters of greenhouse gases. Methane with a GWP (global warming potential) of 21, and nitrous oxide (N2O) with a GWP of 300, are largely emitted from animal production agriculture, where livestock production is based on pasture and feed grains. The principal biological processes involved in N2O emissions are nitrification and denitrification. Biological nitrification inhibition (BNI) is the natural ability of certain plant species to release nitrification inhibitors from their roots that suppress nitrifier activity, thus reducing soil nitrification and N2O emission. Recent methodological developments (e.g. bioluminescence assay to detect BNIs in plant root systems) have led to significant advances in our ability to quantify and characterize the BNI function. Synthesis and release of BNIs from plants is a highly regulated process triggered by the presence of NH4 + in the rhizosphere, which results in the inhibitor being released precisely where the majority of the soil-nitrifier population resides. Among the tropical pasture grasses, the BNI function is strongest (i.e. BNI capacity) in Brachiaria sp. Some feed-grain crops such as sorghum also have significant BNI capacity present in their root systems. The chemical identity of some of these BNIs has now been established, and their mode of inhibitory action on Nitrosomonas has been characterized. The ability of the BNI function in Brachiaria pastures to suppress N2O emissions and soil nitrification potential has been demonstrated; however, its potential role in controlling N2O emissions in agro-pastoral systems is under investigation. Here we present the current status of our understanding on how the BNI functions in Brachiaria pastures and feed-grain crops such as sorghum can be exploited both genetically and, from a production systems perspective, to develop low-nitrifying and low N2O-emitting production systems that would be economically profitable and ecologically sustainable.

Genetic mitigation strategies to tackle agricultural GHG emissions: The case for biological nitrification inhibition technology

Accelerated soil-nitrifier activity and rapid nitrification are the cause of declining nitrogen-use efficiency (NUE) and enhanced nitrous oxide (N2O) emissions from farming. Biological nitrification inhibition (BNI) is the ability of certain plant roots to suppress soil-nitrifier activity, through production and release of nitrification inhibitors. The power of phytochemicals with BNI-function needs to be harnessed to control soil-nitrifier activity and improve nitrogen-cycling in agricultural systems. Transformative biological technologies designed for genetic mitigation are needed, so that BNI-enabled crop-livestock and cropping systems can rein in soil-nitrifier activity, to help reduce greenhouse gas (GHG) emissions and globally make farming nitrogen efficient and less harmful to environment. This will reinforce the adaptation or mitigation impact of other climate-smart agriculture technologies.

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

AimUtilization 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.MethodsSoil 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.ResultsHigh 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.ConclusionBh 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.

Further insights into underlying mechanisms for the release of biological nitrification inhibitors from sorghum roots

BackgroundSorghum roots release two categories of biological nitrification inhibitors (BNIs) – hydrophilic-BNIs and hydrophobic-BNIs. Earlier research indicated that rhizosphere pH and plasma membrane (PM) H+ATPase are functionally linked with the release of hydrophilic BNIs, but the underlying mechanisms are not fully elucidated. This study is designed to reveal further insights into the regulatory mechanisms of BNIs release in root systems, using three sorghum genetic stocks.MethodsSorghum plants were grown in a hydroponic system with pH of nutrient solutions ranging from 3.0 ̴ 9.0. Pharmacological agents [(fusicoccin and vanadate) and anion-channel blockers (−niflumic acid (NIF) and anthracene-9-carboxylate (A9C)] were applied to root exudate collection solutions; BNI activity was determined with luminescent Nitrosomonas europaea bioassay. Sorgoleone levels in root exudates and H+ excretion from roots were determined. Two-phase partitioning system is used to isolate root plasma membrane (PM) and H+ ATPase activity was determined.ResultsA decrease in rhizosphere pH improved the release of hydrophilic-BNIs from roots of all the three sorghum genotypes, but had no effect on the release of hydrophobic-BNIs. Hydrophobic-BNI activity and sorgoleone levels in root-DCM wash are positively correlated. Fusicoccin promoted H+extrusion and stimulated the release of hydrophilic-BNIs. Vanadate, in contrast, suppressed H+ extrusion and lowered the release of hydrophilic-BNIs. Anion-channel blockers did not inhibit the release of hydrophilic BNIs, but enhanced H+-extrusion and hydrophilic-BNIs release.ConclusionRhizosphere pH has a major influence on hydrophilic-BNIs release, but not on the release of hydrophobic-BNIs. The low rhizosphere pH stimulated PM-H+ ATPase activity; H+-extrusion is closely coupled with hydrophilic-BNIs release. Anion-channel blockers stimulated H+ extrusion and hydrophilic-BNIs release. Our results indicate that some unknown membrane transporters are operating the release of protonated BNIs, which may compensate for charge balance when transport of other anions is suppressed using anion-channel blockers. A new hypothesis is proposed for the release of hydrophilic-BNIs from sorghum roots.