Red clover belowground N input affected by the nutrient availability and its fate during two years of clover-grass sward cultivation

Di nitrogen (N) fixation of legume-rhizobia symbiosis represents a relevant N source for agricultural systems and has the potential to reduce the input of synthetic N fertiliser. Legumes in mixture with grass constitutes an important component of cropping systems of crop-livestock farms. In recent years, these mixtures became an interesting alternative in stockless farms as feedstock for biogas production. Clover growing in association with grass receives 80% of its N from symbiotic N2 fixation. Estimations of fixed N have usually considered neither belowground clover N nor clover N transferred to the associated grass. One reason might be that the assessment of belowground N is laborious, since roots have to be recovered from the soil and N derived from rhizodeposition has to be estimated. Rhizodeposition N, composed of particulate and non-particulate compounds, is released into the soil from living roots via exudation and root turnover. Rhizodeposition has been studied by stable isotope 15N enrichment techniques. Likewise, N transfer from clover to grass has been assessed with 15N techniques using both the 15N enrichment and 15N natural abundance method. Fixed N contained in above- and belowground N of clover-grass swards potentially presents a significant N input in both conventional and organic cropping systems. These systems differ in fertilisation and crop protection strategies. The amount and the quality of applied fertilisers result in differences in soil nutrientavailability. Organically cropped soils often have a greater soil microbial biomass both in size and in activity compared to conventionally cropped ones. There is a lack of understanding on how these factors affect the input of symbiotically fixed N and on the fate of legume N remaining in the soil. To examine these relations, a microplot study was conducted in the DOK long-term field experiment, since 33 years of organic vs. conventional cropping with respective fertilisation and plant protection strategies resulted in soils with a gradient in nutrient-availability, soil microbial biomass both in size and in activity, and 15N natural abundance of soil N. Red clover (Trifolium pratense L.) above- and belowground N was quantified in model clover-grass swards. Swards were cultivated in microplots over two consecutive years. Microplots were located in DOK field plots with different fertilisation strategies. In general, the fertilisation increased in the order zero-fertilisation control (NOFERT), organic cropping system with half dose fertilisation (BIOORG1), organic cropping system with regular dose fertilisation (BIOORG2), and conventional cropping system (CONMIN2) with regular dose fertilisation. The organic cropping system at level 1 and 2 was fertilised with farmyard manure (manure and slurry) while the conventional cropping system was supplied by mineral fertiliser exclusively. Red clover rhizodeposition N was determined by 15N multiple-pulse leaf labelling (chapter 1). All roots were carefully removed from the soil. Subsequently, the fate of N derived from rhizodeposition being still present in the soil was investigated at different points of time using a sequential extraction. This procedure allows to extract soluble N and microbial biomass N from the soil. The remaining soil organic matter pools were separated in low and high density occluded particulate organic matter and silicate and quartz mineral-organic associations using a sequential density fractionation. Cropping system related effects from the size and activity of the microbial biomass on the General introduction 4 incorporation of N derived from rhizodeposition in soil pools and soil organic matter pools were assessed (chapter 2). Furthermore, different 15N natural abundance and 15N enrichment procedures were compared to determine the transfer from red clover N to perennial ryegrass (Lolium perenne L.). Subsequently, the 15N natural abundance method was verified with cropping systems under identical environmental and management conditions but differing in 15N natural abundance of the applied N fertilisers. One 15N natural abundance procedure was selected to quantify red clover N and symbiotically fixed red clover N being transferred to perennial ryegrass (chapter 3). Finally, the results from chapter 1 to 3 were scaled up from the microplot level to the field plot level of the DOK experiment. For this, N yields and clover proportions of clover-grass swards of field plots were used. Results were synthesised to present an overall picture on symbiotically fixed N in the plant-soil system of clover-grass swards under different cropping systems (general discussion). Red clover belowground N developed proportional to aboveground N at a ratio of 0.4 to 1 irrespective of the fertilisation strategy and the time of cultivation. However, the ratio N derived from rhizodeposition to root N changed with time. More than 90% of rhizodeposition N was incorporated into soil organic matter pools. Thereof, about 40% was found in silicate mineral-organic associations, 40% to 50% in both occluded particulate organic matter fractions, and the remainder in quartz mineralorganic associations irrespective of the fertilisation strategy related organic matter input. The proportions of grass N transferred from red clover N being estimated by 15N natural abundance procedures were within the range of that being estimated by 15N enrichment based procedures. About 40% of perennial ryegrass N was transferred from red clover in the low N fertilised swards (< 50 kg mineral N ha-1 a-1) NOFERT, BIOORG1 and BIOORG2. This corresponds to more than one third of perennial ryegrass N if related to symbiotically fixed red clover N. Up scaled from the microplot level to the field plot level of the DOK experiment, the sward plant-soil system obtained 300 to more than 400 kg N ha-1 from the symbiotic N2 fixation of clover after two years of cultivation. From that symbiotically fixed clover N, nearly 50% was present in the clover yield, about one third was transferred to grass, nearly 15% was accumulated in soil organic matter pools, 5% remained in the stubble and the root, and less than 1% was found in the both soil pools soluble N and microbial biomass N. Subsequent to the last harvest at the end of the 2nd year, on average 100 kg ha-1 symbiotically fixed clover derived N remained in the stubble and the roots of clover and grass and in the soil pools. Remaining N increased in the order NOFERT, CONMIN2, BIOORG2, and BIOORG1 at a ratio of 0.9 to 0.9 to 1.1 to 1.2 related to the average of the four cropping systems. In conclusion, red clover belowground N could be estimated from aboveground N by a factor of 0.4 irrespective of the fertilisation strategy and the cultivation time. Allocation of red clover rhizodeposition N to the silicate mineral-organic association was the dominant process N derived from rhizodeposition remaining in the soil was exposed. This process proceeded within months, but remained unaffected by the cropping system related microbial size and activity. 15N natural abundance procedures resulted in somewhat less variable estimates of N transfer from red clover to perennial ryegrass than 15N enrichment General introduction 5 procedures. Cropping systems affected the amount of red clover and perennial ryegrass N accumulation, red clover N derived from rhizodeposition, and N transfer to grass, but hardly affected the partitioning of N derived from rhizodeposition to soil pools. The partitioning of red clover N between above- and belowground and the partitioning within soil organic matter pools was not affected by cropping systems in the studied plant-soil system. Up scaled from the microplot to the field plot level of the DOK experiment, about 50% of symbiotically fixed clover N was deposited into the soil (≈15%) or transferred to grass (≈35%) at the end of the 2nd cultivation year. Hence, clover belowground N and N transfer to grass may represent important and considerable N inputs to agricultural systems