The potential of stable carbon and nitrogen isotope analysis of foxtail and broomcorn millets for investigating ancient farming systems.

Foxtail and broomcorn millets are the most important crops in northern China since the early Neolithic. However, little evidence is available on how people managed these two crops in the past, especially in prehistory. Previous research on major C3 crops in western Eurasia demonstrated the potential of stable carbon and nitrogen isotope analysis of charred archaeobotanical remains to reveal the management of water and manure, respectively. Here, we evaluate the feasibility of a similar approach to C4 millets. Foxtail and broomcorn millet plants grown in pots in a greenhouse under different manuring and watering regimes were analysed to test the effects of management on stable carbon and nitrogen isotope values of grains. Stable nitrogen isotope values of both millets increased as manuring level increased, ranging from 1.7 ‰ to 5.8 ‰ in different conditions; hence, it appears a feasible tool to identify manuring practices, in agreement with results from recent field studies. However, the two millets exhibit opposing trends in stable carbon isotope values as watering level increased. The shift in stable carbon isotope values of millets is also smaller than that observed in wheat grown in the same experimental environment, making it difficult to identify millet water status archaeologically. In addition, we charred millet grains at different temperatures and for varying durations to replicate macro-botanical remains recovered archaeologically, and to evaluate the offsets in carbon and nitrogen isotope values induced by charring. We found that the stable nitrogen isotope values of foxtail millet and broomcorn millet can shift up to 1-2 ‰ when charred, while the stable carbon isotope values change less than 0.3 ‰. Overall, we demonstrate that stable nitrogen isotope values of charred foxtail and broomcorn millet seeds could provide insight into past field management practices, and both carbon and nitrogen isotope values can together inform palaeodietary reconstruction.

Filling gaps in models simulating carbon storage in agricultural soils: the role of cereal stubbles.

Carbon (C) input is a prerequisite for the formation of soil organic matter and thus for soil organic C (SOC) sequestration. Here we used the C-TOOL model to simulate SOC changes in a long-term field experiment (1932-2020) at Askov, Denmark, which involved four different levels of nutrients added in mineral fertilizer (0, 0.5, 1, 1.5 NPK) and a four-crop rotation. The C input into soils consists of belowground and aboveground plant biomass and was estimated using allometric functions. The simulation showed that modelled SOC based on standard allometric functions of C input from crop residues did not adequately matched measured SOC contents. However, applying modified allometric functions based on current and the previously measured results for aboveground and belowground C inputs in winter wheat and grass clover in rotations provided much better match between simulated and measured SOC contents for fertilized treatments at normal and high level of fertilization. This improved indicators of C-TOOL model performance (e.g. yielding RMSE of 2.24 t C ha-1 and model efficiency of 0.73 in 1.5 NPK treatment). The results highlight that standard allometric functions greatly overestimates the amount of C in winter wheat stubble left after harvest in treatments dressed with NPK compared with modified functions. The results also highlight further needs for improvement of allometric functions used in simulation models for C-accounting in agroecosystems.

Land-use and agriculture in Denmark around year 1900 and the quest for EU Water Framework Directive reference conditions in coastal waters.

The EU Water Framework Directive (WFD) aims to protect the ecological status of coastal waters. To establish acceptable boundaries between good and moderate ecological status, the WFD calls for reference conditions practically undisturbed by human impact. For Denmark, the nitrogen (N) concentrations present around year 1900 have been suggested to represent reference conditions. As the N load of coastal waters relates closely to runoff from land, any reduction in load links to agricultural activity. We challenge the current use of historical N balances to establish WFD reference conditions and initiate an alternative approach based on parish-level land-use statistics collected 1896/1900 and N concentrations in root zone percolates from experiments with year 1900-relevant management. This approach may be more widely applicable for landscapes with detailed historic information on agricultural activity. Using this approach, we find an average N concentration in root zone percolates that is close to that of current agriculture. Thus, considering Danish coastal waters to be practically unaffected by human activity around year 1900 remains futile as 75% of the land area was subject to agricultural activity with a substantial potential for N loss to the environment. It appears unlikely that the ecological state of coastal waters around year 1900 may serve as WFD reference condition.

Short-term changes in soil pore size distribution: Impact of land use.

Changes in land use affect the pore size distribution (PSD) of the soil, and hence important soil functions such as gas exchange, water availability and plant growth. The objective of this study was to investigate potentially damaging and restorative soil management practices on soil pore structure. We quantified the rate of change in PSD six years after changes in land use taking advantage of the Highfield land-use change experiment at Rothamsted Research. This experiment includes short-term soil degradation and restoration scenarios established simultaneously within long-term contrasting treatments that had reached steady-state equilibrium. The land-use change scenarios comprised conversion to grassland of previously arable or bare fallow soil, and conversion of grassland to arable and bare fallow soils. In the laboratory, we exposed intact soil cores (100 cm3) to matric potentials ranging from -10 hPa to -1.5 MPa. Based on equivalent soil mass, the plant available water capacity decreased after conversion from grassland, whereas no change was observed after conversion to grassland. Structural void ratio decreased after termination of grassland and introduction of grassland in bare fallow soil, while no change was seen when changing arable to grassland. Consequently, it was faster to degrade than to restore a complex soil structure. The study illustrates that introducing grassland in degraded soil may result in short-term increase in soil density.

Soil degradation and recovery - Changes in organic matter fractions and structural stability.

The combination of concurrent soil degradation and restoration scenarios in a long-term experiment with contrasting treatments under steady-state conditions, similar soil texture and climate make the Highfield land-use change experiment at Rothamsted Research unique. We used soil from this experiment to quantify rates of change in organic matter (OM) fractions and soil structural stability (SSS) six years after the management changed. Soil degradation included the conversion of grassland to arable and bare fallow management, while soil restoration comprised introduction of grassland in arable and bare fallow soil. Soils were tested for clay dispersibility measured on two macro-aggregate sizes (DispClay 1-2 mm and DispClay 8-16 mm) and clay-SOM disintegration (DI, the ratio between clay particles retrieved without and with SOM removal). The SSS tests were related to soil organic carbon (SOC), permanganate oxidizable C (POXC) and hot water-extractable C (HWC). The decrease in SOC after termination of grassland was greater than the increase in SOC when introducing grassland. In contrast, it was faster to restore degraded soil than to degrade grassland soil with respect to SSS at macro-aggregate scale. The effect of management changes was more pronounced for 8-16 mm than 1-2 mm aggregates indicating a larger sensitivity towards tillage-induced breakdown of binding agents in larger aggregates. At microscale, SSS depended on SOC content regardless of management. Soil management affected macroscale structural stability beyond what is revealed from measuring changes in OM fractions, underlining the need to include both bonding and binding mechanisms in the interpretation of changes in SSS induced by management.

Bacterial Preferences for Specific Soil Particle Size Fractions Revealed by Community Analyses.

Genetic fingerprinting demonstrated in previous studies that differently sized soil particle fractions (PSFs; clay, silt, and sand with particulate organic matter (POM)) harbor microbial communities that differ in structure, functional potentials and sensitivity to environmental conditions. To elucidate whether specific bacterial or archaeal taxa exhibit preference for specific PSFs, we examined the diversity of PCR-amplified 16S rRNA genes by high-throughput sequencing using total DNA extracted from three long-term fertilization variants (unfertilized, fertilized with minerals, and fertilized with animal manure) of an agricultural loamy sand soil and their PSFs. The PSFs were obtained by gentle ultrasonic dispersion, wet sieving, and centrifugation. The abundance of bacterial taxa assigned to operational taxonomic units (OTUs) differed less than 2.7% between unfractionated soil and soil based on combined PSFs. Across the three soil variants, no archaeal OTUs, but many bacterial OTUs, the latter representing 34-56% of all amplicon sequences, showed significant preferences for specific PSFs. The sand-sized fraction with POM was the preferred site for members of Bacteroidetes and Alphaproteobacteria, while Gemmatimonadales preferred coarse silt, Actinobacteria and Nitrosospira fine silt, and Planctomycetales clay. Firmicutes were depleted in the sand-sized fraction. In contrast, archaea, which represented 0.8% of all 16S rRNA gene sequences, showed only little preference for specific PSFs. We conclude that differently sized soil particles represent distinct microenvironments that support specific bacterial taxa and that these preferences could strongly contribute to the spatial heterogeneity and bacterial diversity found in soils.

Soil texture analysis revisited: Removal of organic matter matters more than ever.

Exact estimates of soil clay (<2 µm) and silt (2-20 µm) contents are crucial as these size fractions impact key soil functions, and as pedotransfer concepts based on clay and silt contents are becoming increasingly abundant. We examined the effect of removing soil organic matter (SOM) by H2O2 before soil dispersion and determination of clay and silt. Soil samples with gradients in SOM were retrieved from three long-term field experiments each with uniform soil mineralogy and texture. For soils with less than 2 g C 100 g-1 minerals, clay estimates were little affected by SOM. Above this threshold, underestimation of clay increased dramatically with increasing SOM content. Silt contents were systematically overestimated when SOM was not removed; no lower SOM threshold was found for silt, but the overestimation was more pronounced for finer textured soils. When exact estimates of soil particles <20 µm are needed, SOM should always be removed before soil dispersion.

Consolidating soil carbon turnover models by improved estimates of belowground carbon input.

World soil carbon (C) stocks are third only to those in the ocean and earth crust, and represent twice the amount currently present in the atmosphere. Therefore, any small change in the amount of soil organic C (SOC) may affect carbon dioxide (CO2) concentrations in the atmosphere. Dynamic models of SOC help reveal the interaction among soil carbon systems, climate and land management, and they are also frequently used to help assess SOC dynamics. Those models often use allometric functions to calculate soil C inputs in which the amount of C in both above and below ground crop residues are assumed to be proportional to crop harvest yield. Here we argue that simulating changes in SOC stocks based on C input that are proportional to crop yield is not supported by data from long-term experiments with measured SOC changes. Rather, there is evidence that root C inputs are largely independent of crop yield, but crop specific. We discuss implications of applying fixed belowground C input regardless of crop yield on agricultural greenhouse gas mitigation and accounting.

Appearance of ß-lactam Resistance Genes in Agricultural Soils and Clinical Isolates over the 20th Century.

Debate exists about whether agricultural versus medical antibiotic use drives increasing antibiotic resistance (AR) across nature. Both sectors have been inconsistent at antibiotic stewardship, but it is unclear which sector has most influenced acquired AR on broad scales. Using qPCR and soils archived since 1923 at Askov Experimental Station in Denmark, we quantified four broad-spectrum ß-lactam AR genes (ARG; bla(TEM), bla(SHV), bla(OXA) and bla(CTX-M)) and class-1 integron genes (int1) in soils from manured (M) versus inorganic fertilised (IF) fields. "Total" ß-lactam ARG levels were significantly higher in M versus IF in soils post-1940 (paired-t test; p < 0.001). However, dominant individual ARGs varied over time; bla(TEM) and bla(SHV) between 1963 and 1974, bla(OXA) slightly later, and bla(CTX-M) since 1988. These dates roughly parallel first reporting of these genes in clinical isolates, suggesting ARGs in animal manure and humans are historically interconnected. Archive data further show when non-therapeutic antibiotic use was banned in Denmark, bla(CTX-M) levels declined in M soils, suggesting accumulated soil ARGs can be reduced by prudent antibiotic stewardship. Conversely, int1 levels have continued to increase in M soils since 1990, implying direct manure application to soils should be scrutinized as part of future stewardship programs.

Increase in soil stable carbon isotope ratio relates to loss of organic carbon: results from five long-term bare fallow experiments.

Changes in the (12)C/(13)C ratio (expressed as δ(13)C) of soil organic C (SOC) has been observed over long time scales and with depth in soil profiles. The changes are ascribed to the different reaction kinetics of (12)C and (13)C isotopes and the different isotopic composition of various SOC pool components. However, experimental verification of the subtle isotopic shifts associated with SOC turnover under field conditions is scarce. We determined δ(13)C and SOC in soil sampled during 1929-2009 in the Ap-horizon of five European long-term bare fallow experiments kept without C inputs for 27-80 years and covering a latitudinal range of 11°. The bare fallow soils lost 33-65% of their initial SOC content and showed a mean annual δ(13)C increase of 0.008-0.024‰. The (13)C enrichment could be related empirically to SOC losses by a Rayleigh distillation equation. A more complex mechanistic relationship was also examined. The overall estimate of the fractionation coefficient (ε) was -1.2 ± 0.3‰. This coefficient represents an important input to studies of long-term SOC dynamics in agricultural soils that are based on variations in (13)C natural abundance. The variance of ε may be ascribed to site characteristics not disclosed in our study, but the very similar kinetics measured across our five experimental sites suggest that overall site-specific factors (including climate) had a marginal influence and that it may be possible to isolate a general mechanism causing the enrichment, although pre-fallow land use may have some impact on isotope abundance and fractionation.

Higher temperature sensitivity for stable than for labile soil organic carbon--evidence from incubations of long-term bare fallow soils.

The impact of climate change on the stability of soil organic carbon (SOC) remains a major source of uncertainty in predicting future changes in atmospheric CO2 levels. One unsettled issue is whether the mineralization response to temperature depends on SOC mineralization rate. Long-term (>25 years) bare fallow experiments (LTBF) in which the soil is kept free of any vegetation and organic inputs, and their associated archives of soil samples represent a unique research platform to examine this issue as with increasing duration of fallow, the lability of remaining total SOC decreases. We retrieved soils from LTBF experiments situated at Askov (Denmark), Grignon (France), Ultuna (Sweden), and Versailles (France) and sampled at the start of the experiments and after 25, 50, 52, and 79 years of bare fallow, respectively. Soils were incubated at 4, 12, 20, and 35 °C and the evolved CO2 monitored. The apparent activation energy (Ea) of SOC was then calculated for similar loss of CO2 at the different temperatures. The Ea was always higher for samples taken at the end of the bare-fallow period, implying a higher temperature sensitivity of stable C than of labile C. Our results provide strong evidence for a general relationship between temperature sensitivity and SOC stability upon which significant improvements in predictive models could be based.

Abundance of 13C and 15N in emmer, spelt and naked barley grown on differently manured soils: towards a method for identifying past manuring practice.

The shortage of plant-available nutrients probably constrained prehistoric cereal cropping but there is very little direct evidence relating to the history of ancient manuring. It has been shown that the long-term addition of animal manure elevates the δ(15)N value of soil and of modern crops grown on the soil. We have examined the δ(15)N and δ(13)C values of soil and of the grain and straw fractions of three ancient cereal types grown in unmanured, PK amended and cattle manured plots of the Askov long-term field experiment. Manure increased biomass yields and the δ(15)N values of soil and of grain and straw fractions of the ancient cereal types; differences in δ(15)N between unmanured and PK treatments were insignificant. The offset in straw and grain δ(15)N due to manure averaged 7.9 and 8.8 ‰, respectively, while the soil offset was 1.9 ‰. The soil and biomass δ(13)C values were not affected by nutrient amendments. Grain weights differed among cereal types but increased in the order: unmanured, PK, and animal manure. The grain and straw total-N concentration was generally not affected by manure addition. Our study suggests that long-term application of manure to permanently cultivated sites would have provided a substantial positive effect on cereals grown in early agriculture and will have left a significant N isotopic imprint on soil, grains and straw. We suggest that the use of animal manure can be identified by the (15)N abundance in remains of ancient cereals (e.g. charred grains) from archaeological sites and by growing test plants on freshly exposed palaeosols.

The natural abundance of 13C, 15N, 34S and 14C in archived (1923-2000) plant and soil samples from the Askov long-term experiments on animal manure and mineral fertilizer.

The Askov field experiment (Denmark), established in 1894, provides a unique opportunity to examine long-term effects of animal manure and mineral fertilizer on soil organic matter quality and turnover. This sandy loam soil is classified as Alfisol (Typic Hapludalf). Soil C, N, S, 13C, 15N, 34S and 14C contents were measured in a selection of archived soil samples (1923, 1938, 1945, 1953, 1964, 1976, 1985, 1996 and 2000) from unfertilized (O), animal manure (1 AM) and mineral fertilizer (1 NPK) treatments. These treatments are imbedded in a four-course crop rotation of winter cereals, root crops, spring cereals and a clover/grass mixture. The contents of C, N, S, 13C, 15N and 34S in selected crop samples (1953-1996) and in contemporary samples of animal feed and manure were also determined. Temporal soil nutrient and isotope trends between fertilizer treatments were significantly different, except for S content in 1 AM and 1 NPK. The total soil C and S was higher in 1 AM and 1 NPK than in the O treatment. The total soil N content (1 AM>1 NPK>O) and the delta15N content (1 AM>1 NPK and O) were also different. Analyses of plant, animal feed and manures confirmed that differences in soil 15N values were related to delta15N values of added source inputs. Soil and crop delta13C values were similar, but manures had slightly lower values. The variation of soil delta34S (and total S) from 1923 to 1996 was larger in the O than 1 AM and 1 NPK plots reflecting changes in atmospheric S inputs. The total contents of soil C, N and S were significantly correlated, but their isotopic signatures were not, suggesting that the C, N, S turnovers in soil are subject to different controls. The 14C content was generally higher in the 1 AM than 1 NPK and O, with bomb-14C incorporation modelling indicating that mean residence time (MRT) was ca. 170 years in the 1 AM, but closer to 250-290 years in the 1 NPK and O treatments. The measured trends in soil C and 14C during 1923-1996 were successfully modelled using the RothC model. The OM accumulation in the Askov soils was generally dominated by microbial decomposition products rather than by recalcitrant components of the various inputs.