Final Report Summary - LEGUME ROOT IMPACT (Root longevity and the impact on soil N fertility) Efficient utilization of nutrients in agricultural systems is essential in order to meet a sustainable production of food. Grain and forage legumes add nitrogen (N) to the soil via biological N2-fixation which reduces the need for energy demanding mineral fertilizers. Management of the increased soil fertility found after legumes requires knowledge of the processes underlying soil fertility build-up. One of the most important processes in soil fertility build-up under legumes is the growth, death, and turnover of legume root systems, which is at present poorly understood. In particular there is a lack of knowledge of the longevity and turnover of legume roots in forage mixtures, due to methodological difficulties in investigating the roots of one specific species in a species mixture.The aim of the present project was to investigate the mechanisms behind build-up of N in forage mixtures with legumes in order to improve management of N fertility in the crop rotation. The research objectives were to:(1) develop and test a method for estimation of root longevity in species mixtures(2) estimate the longevity of legume roots in forage mixtures(3) quantify the temporal development in deposition of C and N from legume roots.In order to meet these objectives one semi-field experiment and two field experiments were conducted: the semi-field was conducted with white clover and perennial ryegrass to investigate the leaf-labeling procedure, and among the two field experiments one was in red clover and white clover pure stands to develop the root longevity estimation methodology, and a second was in red clover-perennial ryegrass and white clover-perennial ryegrass mixtures to test the methodology in mixtures. In all experiments leaf labeled with 14C-urea and 15N-urea mixtures was used in order to enrich the above and below ground tissue with the tracers. Following the labeling, labeled plants and non-labeled neighboring grasses were sampled in intact soil cores, which allowed sampling of intact root systems. The content of the two tracers in leaf material and different parts of the root system, and in the soil was used to answer the three objectives outlined above. The semi-field experiment:This experiment was used to test the labeling procedures used in the present and many other studies of C and N flow in the plant-soil system. In the experiment we used a phosphor screen technology to obtain detailed information of 14C and 15N distribution within white clover and grass tissue upon uptake of the tracers either through leaf-labeling or via CO2 photoassimilation. The main conclusion of the semi-field experiment was that the C-tracer labeling can effectively be obtained by use of labeled bicarbonate via leaf-labeling. Further, the study showed that the distribution of tracers was more heterogeneous than expected, although the distribution of the N-tracer was more homogeneous than that of the C-tracer. This implies that we need to be careful when making interpretation of results from this type of experiments. Additionally our finding that bicarbonate/carbonate can be used as carrier when labeling with C-tracers implies that scientist can conduct more precise and cost-effective experiment in situ. The results from this experiment have been reported in a publication in Plant and Soil (in press).The first field experiment:This field experiment was conducted in white clover and red clover pure stands, where a large number of individual plants were leaf-labeled with 15N and 14C in September, and the distribution of the two tracers were followed in the plant tissue and the soil until the following May. When sampled the roots of white clover and red clover was divided into primary and secondary roots, and nodules, which allowed us to obtain detailed information about C and N distribution in these tissues. The results show that for C, nodules behaved more like leaf tissue than as root tissue. Further, we noticed that stolons of white clover during the growing season more or less behaved as leaf tissue, whereas stolons and primary roots acted as storage organs during the winter season. In order to develop a method for estimation of root longevity we expected the concentration of the two tracers to decline with time. However, we did not observe this; after an initially higher level within the first month of experimentation the level of the tracers settled at a nearly constant level in all below ground tissues. Therefore, we unfortunately had to conclude that our aim to develop a methodology for root longevity estimation was not successful in the present project. Either we did a mistake when setting up the experiment or we did not run the experiment for sufficient time: the latter being surprising since we had expected the winter period to have a significant effect on especially the smaller roots and nodules. Nevertheless, the first field experiment gave some very interesting results in regard to tracer distribution over time within the root system to the two clover species, which will be reported in a scientific publication.The second field experiment:This field experiment was conducted in white clover and red clover mixtures with perennial ryegrass. Since the results from the first field experiment did not seem very promising in relation to estimate root longevity, we decided to work at a shorter time scale, but with a higher temporal resolution in the second experiment hoping to find the expected development in root tracer concentrations. Furthermore, this change in experimental time scale would also provide a higher resolution of the tracer deposition study, which was the third overall objective of the project. The results from the experiment again did not support the objective to find a solid method for estimating root longevity; in principle the method might work, but most likely we would have to work on time scales of years instead of the present time scale of months up to one year. And maybe roots of these plants live for that long, but the present experiment seemingly cannot answer that question. In relation to the third project objective of deposition of C and N from roots of white clover and red clover we did, however, obtain interesting new results. We expected that root exudation and turnover would be responsible for the greatest part of the build-up of C and N under grass-clover mixtures, but our results point to that leaves of the clover play a vital role in the short term deposition of C and N to the soil, as indicated by the transfer of N from labeled clover to neighbouring grasses. This finding implies that we need to reconsider our present understanding of the plant-soil interface to include a significant contribution from dying leaf tissue during the growing and off seasons to the C and N balances in the plant-soil systems. Since leaf tissue generally is more easily degradable than root tissue, this has implication for our understanding of, among other processes, production of the green house gas N2O and the presence of leachable N forms; this particularly being important when leaf tissue is lost in the autumn. The results of the second field experiment are yet to be reported in an international publication.Project outcomes and potential impact:We intended to develop a methodology for root longevity estimation in plant species mixtures, which we unfortunately did not succeed to do.However, within the framework of the project we could show:- that bicarbonate can effectively be used as a C-tracer carrier, and we visualized the distribution of both C and N tracers when using the leaf-labeling methodology- that part of the root systems of clover, especially the nodules, behaves more like leaf tissue than root tissue- that white clover stolons during the growth season should be considered as part of the above ground tissue, and during the off season as part of the below ground tissue- that leaf tissue play a much more important role in short term C and N deposition to the soil than previously expected, and that leaf deposition might even be more important than deposition from the rooting system.The enhanced understanding of C and N flow in the plant-soil system under grass-clover mixtures have potential impacts on the management strategies of these crops, as knowledge of the drivers in the system can be used to modify the management to reduce losses to water bodies and emission of green house gasses.