Evaluation of methods to measure differential 15N labeling of soil and root N pools for studies of root exudation

To study patterns of root exudation, the effectiveness of different techniques for in situ 15N labeling of Brassica napus, Centaurea jacea and Lolium perenne with ammonium nitrate was tested. Stem infiltration was found to effectively label plants with thicker stems, whereas, for grass species, cutting and immersing the leaf tips into 15N solution proved to be most effective. A microdiffusion technique to isolate ammonium, combined with conventional cation-exchange chromatography to separate nitrate from amino-N compounds thereafter, was found suitable for separation of the N fractions of plant and soil extracts for 15N determination. All three species were then cultivated in nutrient solution and labeled with 15NH4 15NO3 by stem feeding for 42 hours. Kinetics of 15N labeling of bulk roots and shoots as well as hot water extractable material were assessed, and up to 1.1 at% 15N excess (APE) was found in nutrient solutions. The main amino acids exuded by L. perenne were glycine, serine, alanine and aspartic acid. To assess the suitability of this set of methods to study root exudation in field settings, L. perenne was grown without fertiliser addition in pots containing low-nutrient soil. Plants were 15N labeled via tip immersion and 15N and N concentrations were analysed in shoots, roots and soils during a 48-h interval. Shoots reached 1.25 APE, roots and soil 0.10 and 0.005 APE, respectively. Between 4% (48 h) and 6% (24 h) of total plant 15N was exuded by roots into the soil. In roots amino acids comprised the largest proportion of the soluble 15N pool, whereas soil 15N levels were similar for amino acids and ammonium, exceeding those of nitrate. Mechanisms for the shift within N fractions from roots to soils are briefly discussed.