Free fatty acids from the pasture grass Brachiaria humidicola and one of their methyl esters as inhibitors of nitrification

Abstract

The tropical pasture grass, Brachiaria humidicola (Rendle) Schweick, produces nitrification inhibitory compounds (termed biological nitrification inhibitors or BNIs) in its shoot and root tissues and releases BNIs from its roots. In the present study, two BNI compounds were isolated and identified from the shoot tissue of B. humidicola using activity-guided fractionation. The recombinant Nitrosomonas europaea containing luxAB genes derived from the bioluminescent marine gram-negative bacterium Vibrio harveyi, were used to determine BNI activity. The BNI compounds in the shoot tissue were identified as linoleic acid (LA) and linolenic acid (LN) using authenticchemicals obtained from ©Sigma (ED 80 16.0 μg ml −1 for both LA and LN) for verification. None of the other tested free fatty acids namely stearic acid, oleic acid, arachidonic acid, and cis-vaccenic acid showed any inhibitory effect on nitrification. Among the fatty acid methyl esters (FAME) evaluated [methyl oleate, methyl linoleate (LA-ME) and methyl linoleneate (LN-ME)], only LA-ME showed an inhibitory effect (ED 80 8.0 μg ml −1 ). The inhibitory effect of LA, LN and LA-ME in the soil was stable for 120 days at 20°C. Soil treated with LA, LN and LA-ME showed a very low accumulation of NO 3 − and the maintenance of soil inorganic N in the NH 4 + form. The inhibitory effect of LA-ME on soil nitrification was greater than that of LA or LN. In addition to BNI activity, both LA and LA-ME showed a suppressive effect on urea hydrolysis in soil. Both LA and LN blocked the AMO (ammonia monooxygenase) and HAO (hydroxylamino oxidoreductase) enzymatic pathways in Nitrosomonas. Since LA and LN can be produced from vegetable oils such as soybean, flax or sunflower, they have the potential for use as nitrification inhibitors in production agriculture.

Evidence for biological nitrification inhibition inBrachiariapastures

“…Crude extract of root exudates containing BNI activity showed an inhibitory effect of similar strength on both enzymatic pathways (Table 1; Table S2), indicating that other BNIs released from roots have a mode of action different from that of brachialactone. Recently, linolenic acid, a major BNI compound present in the leaf tissue of B. humidicola, was shown to block both AMO and HAO enzymatic pathways in a manner similar to BNI activity of crude root exudates, indicating the possibility of a single inhibitor affecting both the enzymatic pathways in Nitrosomonas (29). When a fatty acid binding protein, BSA, was added (after the addition of linolenic acid) to the Nitrosomonas pure cultures, a major portion of the inhibitory effect was removed, indicating the reversible nature of the inhibitory effect from linolenic acid (29).…”

“…Recently, linolenic acid, a major BNI compound present in the leaf tissue of B. humidicola, was shown to block both AMO and HAO enzymatic pathways in a manner similar to BNI activity of crude root exudates, indicating the possibility of a single inhibitor affecting both the enzymatic pathways in Nitrosomonas (29). When a fatty acid binding protein, BSA, was added (after the addition of linolenic acid) to the Nitrosomonas pure cultures, a major portion of the inhibitory effect was removed, indicating the reversible nature of the inhibitory effect from linolenic acid (29). The reducing power generated from the oxidation of hydroxylamine by HAO is Root exudate was collected from intact BH (CIAT 679) plants (root fresh weight of Ϸ20 g) using aerated solutions of 1 mM NH 4Cl, evaporated to dryness, extracted with methanol, and evaporated to dryness and dissolved in 200 L of dimethyl sulfoxide; 1 L of the crude extract was used for the determination of BNI activity.…”

“…Mode of inhibitory action on Nitrosomonas was determined by incubating the pure cultures of luminescent N. europaea with brachialactone in the presence of hydroxylamine (i.e., inhibition of the HAO enzymatic pathway) or in the absence of hydroxylamine (i.e., inhibition of the AMO enzymatic pathway) using a previously reported protocol (23,29); 1 L of purified brachialactone (dissolved in dimethyl sulfoxide) to give 5 M in the assay medium was added to 250 L of bacterial culture and 199 L of distilled water; the contents were incubated for 10 min before the addition of 200 L of 1 mM hydroxylamine (to give 307 M) to give an assay volume of 650 L. The mean of the five bioluminescence measurements made during the subsequent 10-min incubation at 15 C was taken as the activity level. Every measurement was repeated four times, and they were considered to be replications for the calculation of the SE (Table S2).…”

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

“…The compounds with BNI activity in the aerial parts of B. humidicola are unsaturated free fatty acids, linoleic acid (LA) and a-lenolenic acid (LN; Subbarao et al, 2008), which are relatively weak inhibitors of nitrification with IC 50 values of 3 3 10 25 M, whereas the IC 50 value of the synthetic nitrification inhibitor AT is 1 3 10 27 M. Both LA and LN inhibit Nitrosomonas by blocking of both the AMO and HAO enzymatic pathways . In addition, BNIs could also disrupt the electron transfer pathway via HAO to ubiquinone and cytochrome (which need to be maintained to generate reducing power, i.e.…”

Residual effect of BNI by Brachiaria humidicola pasture on nitrogen recovery and grain yield of subsequent maize

“…First studies under controlled conditions by Subbarao et al (2008) indicated that LA maintained 50% of its inhibitory effect in terms of NO 3 − produced per g dry soil after 120 days, whereas the BNI effect of LN was stable until the end of the incubation period (4 months). Since it was observed that a major BNI effect in Bh pastures can be established within 3 years , it is also likely that next to the active release of BNI substances root decomposition could contribute to the accumulation of BNI products in soil.…”