Evidence for biological nitrification inhibition in Brachiaria pastures

Abstract

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 gamma-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 (NH(4)(+)) in the root environment. Brachialactone release is restricted to those roots that are directly exposed to NH(4)(+). 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.

Fig. 1.

Chemical structure of brachialactone, the major nitrification inhibitor isolated from root exudates of B. humidicola.

Fig. 2.

Inhibition of nitrification by brachialactone and contribution of brachialactone to BNI activity released from roots. (A) Inhibitory effect of brachialactone on N. europaea in an in vitro assay. (B) Contribution of brachialactone to the BNI activity released from roots (i.e., in root exudates) of B. humidicola. Root exudates were collected from intact plants using 1 L of aerated solution of 1 mM NH₄Cl with 200 μM CaCl₂ over 24 h. Each data point represents root exudates collected from hydroponically grown plants in a glasshouse during March to May of 2007 and 2008.

Fig. 3.

Soil ammonium oxidation rates (mg of NO₂⁻ N per kg of soil per day) in field plots planted with tropical pasture grasses (differing in BNI capacity) and soybean (lacking BNI capacity in roots) [over 3 years from establishment of pastures (September 2004 to November 2007); for soybean, two planting seasons every year and after six seasons of cultivation]. CON, control (plant-free) plots; SOY, soybean; PM, P. maximum; BHM, Brachiaria hybrid cv. Mulato; BH-679, B. humidicola CIAT 679 (standard cultivar); BH-16888, B. humidicola accession CIAT 16888 (a germ plasm accession). Values are means ± SE from three replications.

Fig. 4.

Influence of tropical pasture grass cultivation (in field plots, over 3 years: September 2004 to November 2007) on soil microorganism populations at 1 day after ammonium sulfate fertilization by estimating copy number of AOB amoA genes (A); AOA amoA genes (B); bacterial small-subunit (SSU) rRNA genes (C); and archaea SSU 16S rRNA genes (D). Plots are identified in Fig. 3 legend. Gene copy number was expressed as copy number per gram of dried soil and obtained through absolute quantification by using real-time PCR. Values are means ± SE from three replications.

Fig. 5.

Cumulative N₂O emissions (mg of N₂O N per m² per year) from field plots of tropical pasture grasses (monitored monthly over a 3-year period, from September 2004 to November 2007). Plots are identified in Fig. 3 legend. Values are means ± SE from three replications.