A colourimetric microplate assay for simple, high throughput assessment of synthetic and biological nitrification inhibitors

Abstract

A simple, rapid, colourimetric method for screening biological nitrification inhibitors in plants is presented. Our approach combines the use of the Griess assay to track the rate of nitrite (NO2 −) production by pure cultures of ammonia oxidising bacteria in the presence and absence of nitrification inhibitors with a simple method for collecting root exudates from plants. NO2 − formation was tracked colourimetically on a microplate reader over 9 h of incubation. The advantage of this method is that it provides a simple, high throughput means of measuring biological nitrification inhibition in root exudates, using wild-type bacterial cultures. NO2 − formation rates and inhibition levels measured using the high through-put method were highly correlated with those measured by tracking NO2 − formation using a segmented flow analyser. The method was able to quantify inhibition of Nitrosomonas europaea by the synthetic nitrification inhibitors allythiourea (AT), dicyandiamide (DCD) and 3,4,-dimethylpyrazole phosphate (DMPP) with IC50 values similar to those reported in the literature. The method detected biological nitrification inhibition (BNI) in root exudates from Brachiaria humidicola and the lack of BNI in root exudates from wheat cv. Janz with minimal alteration of the exudates prior to testing. The results also showed that the more common soil ammonia oxidising bacterium (AOB), Nitrosospira multiformis, was much less sensitive to AT and DCD than N. europaea but had similar sensitivity to DMPP. This method provides a potentially useful way of screening large numbers of root exudate samples allowing for phenotyping of the BNI trait in crop and pasture populations which will be required for the trait to be introduced into commercial varieties.