A bioluminescence assay using recombinant Nitrosomonas europaea was adopted to detect and quantify natural nitrification inhibitors in plant-soil systems. The recombinant strain of N. europaea produces a distinct two-peak luminescence due to the expression of luxAB genes, introduced from Vibrio harveyi, during nitrification. The bioluminescence produced in this assay is highly correlated with NO 2 -production (r 2 = 0.94). Using the assay, we were able to detect significant amounts of a nitrification inhibitor produced by the roots of Brachiaria humidicola (Rendle) Schweick. We propose that the inhibitory activity produced/released from plants be termed 'biological nitrification inhibition' (BNI) to distinguish it from industrially produced inhibitors. The amount of BNI activity produced by roots was expressed in units defined in terms of the action of a standard inhibitor allylthiourea (AT). The inhibitory effect from 0.22 lM AT in an assay containing 18.9 mM of NH 4 + is defined as one AT unit of activity. A substantial amount of BNI activity was released from the roots of B. humidicola (15-25 AT unit g -1 root dry wt day -1 ). The BNI activity released was a function of the growth stage and N content of the plant. Shoot N levels were positively correlated with the release of BNI activity from roots (r 2 = 0.76). The inhibitor/s released from B. humidicola roots suppressed soil nitrification. Additions of 20 units of BNI per gram of soil completely inhibited NO 3 -formation in a 55-day study and remained functionally stable in the soil for 50 days. Both the ammonia monooxygenase and the hydroxylaminooxidoreductase enzymatic pathways in Nitrosomonas were effectively blocked by the BNI activity released from B. humidicola roots. The proposed bioluminescence assay can be used to characterize and determine the BNI activity of plant roots, thus it could become a powerful tool in genetically exploiting the BNI trait in crops and pastures.

The release of chemical compounds from plant roots that suppress soil nitrification is termed biological nitrification inhibition (BNI). Determining the environmental factors that control the synthesis and release of BNI-compounds from Brachiaria humidicola (Rendle) Schweick, a tropical pasture grass that thrives on acid soils, is the focus of this investigation. Because the BNI trait is related to the N status of the plant, we investigated the possibility that the expression of this trait would be related to the forms of N found in the root environment. Plants were grown with two sources of N, NH 4 + or NO 3 -for 60 days and the release of BNI-compounds monitored. Only plants grown with NH 4 + released BNI-compounds from roots. The presence of NH 4 + and possibly the secondary effect of its uptake (i.e., acidic pH) in the root environment significantly enhanced the release of BNI-compounds. Both the NH 4 + and NO 3 -grown plants responded to the stimulus from NH 4 + in the root environment. BNI-compounds found in root tissue and their release were nearly three times greater in NH 4 + grown than from NO 3 -grown plants. The BNI-compounds released from roots composed of at least three active components-Type-I (stable to pH changes from 3.0 to 10), Type-II (temporarily loses its inhibitory effect at a pH higher than a threshold pH of 4.5 and the inhibitory effect is reestablished when the root exudate pH is adjusted to <4.5) and Type-III (inhibitory effect is irreversibly lost if the pH of the root exudate reaches 10.0 or above). A major portion of BNI-compounds released in the presence of NH 4 + is of Type-I. In the absence of NH 4 + , mostly Type-II and Type-III BNI-compounds were released. The BNI-compounds inhibited the function of Nitrosomonas europaea through the blocking of both ammonia monooxygenase and hydroxylamino oxidoreductase pathways. These results indicate that the release of BNIcompounds from B. humidicola roots is a regulated function and that presence of NH 4 + in the root environment is necessary for the sustained synthesis and release of BNI.

Using a recombinant luminescent Nitrosomonas europaea assay to quantify biological nitrification inhibition (BNI), we found that a wild relative of wheat (Leymus racemosus (Lam.) Tzvelev) had a high BNI capacity and releases about 20 times more BNI compounds (about 30 ATU g −1 root dry weight 24 h −1 ) than Triticum aestivum L. (cultivated wheat). The root exudate from cultivated wheat has no inhibitory effect on nitrification when applied to soil; however, the root exudate from L. racemous suppressed NO À 3 formation and kept more than 90% of the soil's inorganic-N in the NH þ 4 -form for 60 days. The high-BNI capacity of L. racemosus is mostly associated with chromosome Lr#n. Two other chromosomes Lr#J, and Lr#I also have an influence on BNI production. Tolerance of L. racemosus to NH þ 4 is controlled by chromosome 7Lr#1-1. Sustained release of BNI compounds occurred only in the presence of Plant Soil (2007) 299:55-64

Fertilization affects soil processes in many ways that remain unclear. The effects of N, P, and K application on plant growth and Cu and Pb accumulation were thus evaluated in a soil-maize system using five treatments: N, P, and K application; N and P application; N and K application; P and K application; and control (no fertilization). Compared to the control, fertilizer application treatments, especially N application, significantly increased maize photosynthetic rate, which further improved shoot biomass production. Root growth, root and shoot Cu and Pb concentrations, and Cu and Pb mobility also significantly altered with fertilizer application. Shoot Cu contents, root Cu uptake and translocation factors were significantly increased in the N-fertilized treatments. The significant decrease of shoot Pb concentration and root Pb uptake and increase of Cu and Pb immobilization were observed in the P-fertilized treatments. No significant correlation was observed between K application and Cu and Pb accumulation in maize. Our results show that an increase in P application and decrease in N application is recommended to reduce agro-ecological risks associated with Cu and Pb in soil-maize systems. However, the mechanisms governing the relationship between nutrients and heavy metal transformation in soil-plant systems needs further research.