Soil gross nitrogen transformations in forestland and cropland of Regosols

Soil gross nitrogen (N) transformations could be influenced by land use change, however, the differences in inherent N transformations between different land use soils are still not well understood under subtropical conditions. In this study, an 15N tracing experiment was applied to determine the influence of land uses on gross N transformations in Regosols, widely distributed soils in Southwest China. Soil samples were taken from the dominant land use types of forestland and cropland. In the cropland soils, the gross autotrophic nitrification rates (mean 14.54 ± 1.66 mg N kg−1 day−1) were significantly higher, while the gross NH4+ immobilization rates (mean 0.34 ± 0.10 mg N kg−1 day−1) were significantly lower than those in the forestland soils (mean 1.99 ± 0.56 and 6.67 ± 0.74 mg N kg−1 day−1, respectively). The gross NO3− immobilization and dissimilatory NO3− reduction to NH4+ (DNRA) rates were not significantly different between the forestland and cropland soils. In comparison to the forestland soils (mean 0.51 ± 0.24), the cropland soils had significantly lower NO3− retention capacities (mean 0.01 ± 0.01), indicating that the potential N losses in the cropland soils were higher. The correlation analysis demonstrated that soil gross autotrophic nitrification rate was negatively and gross NH4+ immobilization rate was positively related to the SOC content and C/N ratio. Therefore, effective measures should be taken to increase soil SOC content and C/N ratio to enhance soil N immobilization ability and NO3− retention capacity and thus reduce NO3− losses from the Regosols.

www.nature.com/scientificreports/ gross nitrification rates and lower immobilization rates, which resulted more NO 3 − losses through leaching or denitrification 9 . However, the mechanisms of different land uses influencing N transformation processes may be quite different according to soil types in subtropical regions. Therefore, more detailed studies are still needed to quantify the inherent N transformation processes under different land use soils in other important soil types in subtropical regions, which is beneficial for understanding whether those different soils can effectively conserve N.
The Sichuan Basin in Southwest China is a subtropical region characterized by numerous hills. Regosols (locally known as purple soil) are the most important and widely distributed cropland soils in this region, covering an area of more than 1.6 × 10 5 km 2 . Unlike the normally occurring soils in subtropical regions, Regosols are weakly developed mineral soils and are characterized by a shallow soil layer, coarse-textured sandy loams and good soil aeration 22 . Consequently, Regosols are susceptible to erosion and leaching, and the large N losses via leaching and overland runoff may cause local and widespread non-point source N pollution 23 . A previous study reported that annual nitrate leaching losses from sloping croplands could be up to 53.4 kg N ha −1 year −1 in this region, which was the dominant N loss pathway 24 . Mitigating this high loss rate needs to enhance the soil N retention capacity by understanding inherent soil N cycling mechanisms in Regosols. Many studies have indicated that soil N transformations predominantly regulate N forms and composition in soils 14,25 . In particular, Zhang et al. revealed that the NO 3 − proportion was mainly regulated by the process of nitrification 26 . However, the inherent soil N transformation processes are still not well understood in the Regosols of the Sichuan Basin. Wang et al. investigated the soil gross N transformations in cropland soils under different fertilization regimes in this region and found that the increased gross nitrification rates governed the increases in cumulative NO 3 − losses via interflow and overland runoff 18 . In the Sichuan Basin, the dominated land uses are forestland and sloping cropland. The difference in land uses can cause significant differences in soil properties 27 , which thus may affect soil gross N transformations. However, to date, how different land use soils influence gross N transformations in Regosols is not fully understood in this region. Understanding N transformations in Regosols and the effects of different land uses would provide important information for assessing the risks of N losses, and further provide the scientific basis for how to regulate the N transformation process to mitigate N losses in the Sichuan Basin of Southwest China.
In this study, we quantified gross N transformations in forestland and cropland of Regosols in the Sichuan Basin of Southwest China. This study aimed to (1) investigate the characteristics of gross N transformations in Regosols, (2) examine the differences in soil N transformations for different land uses in Regosols, and (3) evaluate the N conservation potential and N loss risks of Regosols under different land uses.

Materials and methods
Study region. This experiment was conducted at the Yanting Agro-Ecological Station of Purple Soil, Chinese Academy of Sciences, Yanting County of Sichuan Province (31° 16′ N, 105° 27′ E) 23 . It situates in the central Sichuan Basin and exhibits a moderate subtropical monsoon climate, with an average annual temperature and rainfall of 17.2 °C and 836 mm (30-year mean), respectively. The soil, classified as Eutric Regosols by the FAO soil classification 28 , is locally termed "purple soil" due to its purplish colour 23 . It is typically non-zonal and weakly developed mineral soil, mostly characterizing by a neutral or alkaline reaction 18,23 . Currently, forestland and sloping cropland are the main land uses in this area.

Site description.
To investigate the differences of inherent N transformations between different land use soils, sampling was conducted from the two main land uses of forestland and cropland. The forestland was initially planted with Alnus cremastogyne and Cupressus funebris in the 1970s to reforest cropland, and then, this site experienced natural succession without artificial management. It is now dominated by the representative forest type of Cupressus funebris in the study region, with a density of 1595 stems ha −1 . The selected forestland site was with an area of approximately 1.3 ha −1 . The cropland site was adjacent to the forestland site, with an area of 100 m × 100 m. It has been cultivated for more than 50 years, conventionally rotated with winter wheat (Triticum aestivum L.) and summer maize (Zea mays L.). Fertilizers in the winter wheat and summer maize seasons were applied at the same amounts of K (36 kg K 2 O ha −1 ) and P (90 kg P 2 O 5 ha −1 ), but at different rates of N (130 and 150 kg N ha −1 as ammonium bicarbonate, respectively). All the fertilizers were manually applied and incorporated into the surface soil (0-20 cm) together with harrowing (approximately 20 cm deep). No irrigation was applied during either the wheat or maize season. The forestland and cropland sites had the same soil type (Regosols) and slope (5%). Field soil sampling and N loss monitoring. For forestland and cropland sites, grids with an area of 10 m × 10 m were uniformly divided. Then eleven and eight grids were randomly selected for soil sampling from different slope positions (i.e. upslope, middle slope, and downslope) in forestland and cropland sites, respectively, to minimize the potential effect of spatial heterogeneity on the experimental results. Furthermore, to minimize the effects of fertilization on gross N transformations in cropland soils, soil samples were collected in April 2016 when closed to the wheat harvest. At each sampling grid, the organic layer was removed first if present and three soil cores were randomly taken from the surface 0-20 cm layer. Then, they were well mixed, passed through a 2-mm sieve, and ultimately separated into two sub-samples. One sample was air-dried for soil property analyses, and the other sample was stored for the incubation experiment at 4 °C for < 1 week.
To better identify how soil N transformations regulate the mechanisms involved in NO 3 − losses, field monitoring of NO 3 − loss was conducted following each rainfall event during 2016. At each site, NO 3 − concentration in surface soil (0-20 cm) was also continually monitored at least once a week. Taking the selected sloping forestland site as a whole drainage area (1.3 ha −1 ), the discharge was monitored by the triangle weir installed at the outlet and the water samples were taken from the collecting tanks installed under the weir. At the cropland site, lysimeters Scientific Reports | (2021) 11:223 | https://doi.org/10.1038/s41598-020-80395-x www.nature.com/scientificreports/ were permanently established to take both interflow and overland runoff water samples 23 . Following each runoff event, the discharges of both interflow and overland runoff were determined. For both forestland and cropland sites, water samples were collected using 500-mL polyethylene bottles for assaying NO 3 − concentration. The annual NO 3 − loss flux (Q, kg N ha −1 year −1 ) was estimated as follow: where C i is the NO 3 − concentration in the interflow and overland runoff sample (mg L −1 ), q i is the interflow and overland runoff discharges (mm), and n is the number of runoff events during the monitoring period. During the experimental period, the daily precipitation was automatically monitored by a meteorological station located at a distance of approximately 3 km from the sampling sites. . Then they were adjusted to 60% WHC (water-holding capacity). In agricultural soils, the transformation of NH 4 + to NO 3 − was generally fast 6,21 . To avoid the low NH 4 + concentrations in soils after incubation and guarantee the 15 N detection requirements 29,30 , therefore, relatively high application amounts of NH 4 NO 3 were added compared to initial NH 4 + and NO 3 − concentrations in this study. After 0.5, 12, 24, and 48 h of incubation at 25 °C, soils were extracted to measure the NH 4 + and NO 3 − concentrations and isotopic composition. Distillation with MgO and Devarda's alloy were conducted to separate NH 4 + and NO 3 − , strictly following the procedures described in previous studies 31,32 . Before separating, the recovery of NH 4 + and NO 3 − in a standard solution (1 g NH 4 + -N/NO 3 − -N L −1 ) was determined. The results showed that the recovery of NH 4 + -N and NO 3 − -N in the solution was more than 99% and 95%, respectively. Finally, the 15 N abundances of NH 4 + and NO 3 − were analyzed by an automated C/N analyser and isotope ratio mass spectrometer (IRMS 20-22, SerCon, Crewe, UK).
The widely used numerical 15 N tracing model was employed to investigate the gross N transformations in this study. For the details of this model, numerous previous studies can be referenced 18,21,31,32 . Briefly, this model mainly involved the following ten simultaneously-occurring processes ( Fig. 1 29,30 . The transformation rates were calculated by zero-order, first-order or Michaelis-Menten kinetics by minimizing misfits between the modelled and determined concentrations and 15 N enrichments of NH 4 + and NO 3 − (averages ± standard deviations). Aikaike's Information Criterion (AIC) was utilized to select the best model, and the Markov chain Monte Carlo-Metropolis algorithm (MCMC-MA) was employed for optimizing the parameter 21,30,32 . The MCMC-MA routine was conducted using MATLAB software (Version 7.2, The MathWorks Inc.). Finally, average transformation rates over a 48 h period (mg N kg −1 day −1 ) were calculated on the basis of the kinetic settings and the final parameters.
The total mineralization rates (M N ) were estimated as the sum of M Nlab and M Nrec , and the total NH 4 + immobilization rates (I NH4 ) were calculated as the sum of I NH4-Nlab and I NH4-Nrec . Nitrification capacity was expressed as the ratio of O NH4 to M N . The NO 3 − retention capacity was defined as the ratio of NO 3 − consumption (I NO3 + D NO3 ) to total nitrification (O NH4 + O Nrec ).
Soil chemical property measurements. Soil property analyses strictly followed the procedures described in Soil Agro-Chemical Analysis 33 . Soil pH was measured in a 1:2.5 (soil-to-water) suspension using  Statistical analyses. All statistical analyses were carried out in SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). The Kolmogorov-Smirnov test was conducted first to test the normality of the data. The differences in the gross N transformation rates and soil properties between the cropland and forestland soils were detected using an independent sample t test. The significance level was conventionally set at 0.05. The relationships between gross N transformation rates and soil properties were tested by Pearson correlation analysis.
Soil gross N transformation rates. The gross N mineralization (M N ) rates averaged 3.95 ± 0.51 mg N kg −1 day −1 and 4.84 ± 0.69 mg N kg −1 day −1 in the forestland and cropland soils, respectively, with no significant difference (Fig. 3a). No significant relationships were detected between the mineralization rates and the measured soil properties.

Soil NO 3 − loss.
During the whole year of 2016, six and seven runoff events were monitored in forestland and cropland sites, respectively (Fig. 6c,d). As shown, most NO 3 − losses mainly occurred in summer season (from July to August), during which the rainfall was usually heavy and contributed 51% of the annual precipitation (Fig. 6a). In particular, the heaviest rainfall event was observed at 2016/7/18 with precipitation of 162 mm (Fig. 6a). Then, the highest NO 3 − losses were monitored at 2016/7/18 in forestland and 2016/7/23 in cropland, respectively (Fig. 6c,d). The NO 3 − losses in each runoff event were all significantly higher in cropland than in forestland (P < 0.05). Significantly higher soil NO 3 − concentrations were also observed in cropland than that in forestland (P < 0.05), especially after the period of fertilization (Fig. 6b). The total NO 3 − losses were 0.25 ± 0.01 kg N ha −1 year −1 and 27.10 ± 2.54 kg N ha −1 year −1 for the forestland and cropland, respectively.

Discussion
Patterns of gross N transformations in Regosols. Different with the generally acidic and highly weathered soils in humid subtropical regions 29 , the studied Regosols inherit most properties of parent materials and characterize by coarse texture and a neutral or alkaline reaction 18,22,23 . The specific soil properties of Regosols therefore may cause different N transformation processes compared to the normally occurring subtropical acidic soils.
In current study, gross N mineralization rates in the forestland soils (mean 3.95 ± 0.51 mg N kg −1 day −1 ) were similar to those observed in subtropical zonal soils of Orthic Acrisols and Humic Planosols (FAO soil www.nature.com/scientificreports/ classification) (mean 3.67 and 3.52 mg N kg −1 day −1 , respectively) 4,34 . But they were higher than those measured in subtropical acidic Regosols of southwest China (mean 1.23 mg N kg −1 day −1 ) 35 . This difference is most likely related to the differences in soil organic matter and pH between these two Regosols 13,14 . Moreover, the measured gross NH 4 + immobilization rates in the forestland soils (mean 6.72 ± 0.74 mg N kg −1 day −1 ) were higher than those observed in above mentioned subtropical soil types (0.82 mg N kg −1 day −1 to 2.25 mg N kg −1 day −1 ) 4,34,35 . These results indicated that the alkaline Regosols might have a faster NH 4 + mineralization-immobilization turnover than other soil types in the subtropical regions.
The average total nitrification rates in forestland soils were 2.00 ± 0.57 mg N kg −1 day −1 in this study, and autotrophic nitrification contributed approximately 99.6%. This result indicated that autotrophic nitrification was the dominated NO 3 − production process in the studied Regosols. However, significantly lower total nitrification rates were reported in subtropical acid soils, and heterotrophic nitrification was their dominant process 4,34,35 . Previous studies have showed that the microbiological autotrophic nitrification would be inhibited in soil at pH values lower than 5 36 , but be stimulated at pH values higher than 7.5 37 . Moreover, high soil pH might also inhibit the existence of fungi and their activities, which were related to heterotrophic nitrification 7 . Therefore, the differences in nitrification processes were likely related to the differences in soil pH between the alkaline Regosols and subtropical acid soils. Furthermore, the nitrification capacity (i.e., O NH4 /M N ratio, mean 0.48 ± 0.12) in present forestland soils was significantly greater than that in acidic Regosols (0.02) and Orthic Acrisols (0.05) 4,35 , which may therefore promote soil NO 3 − accumulation and leaching risk in the study region [22][23][24] . This result could be verified by the high NO 3 − /NH 4 + ratio in forestland soils in this study. Gross NO 3 − immobilization and DNRA were the important NO 3 − consumption and retaining processes in soils 38,39 . DNRA generally occurred in anaerobic conditions [40][41][42] , however, it was negligible in this study due to the good aeration of Regosols. Gross NO 3 − immobilization rates in this study were also significantly lower than those in other subtropical soils 4,34 . Previous studies have indicated that NO 3 − immobilization generally needed high carbon availability 43 . The forestland soils in this study had lower soil organic C content (22.91 ± 1.37 g kg −1 ) compared to those subtropical forest soils 4,34 , which thus likely resulted the lower NO 3 − immobilization. In addition, the inhibition of fungal activities by the high soil pH might also control the NO 3 − immobilization in the studied Regosols 44 . Consequently, NO 3 − retention capacity was significantly lower in alkaline Regosols (mean 0.51 ± 0.24) than that in subtropical zonal soils of Orthic Acrisols and Humic Planosols (0.98 and 0.81, respectively) under forestland 4,34 .
As discussed above, due to the specific soil properties, the non-zonal Regosols in the Sichuan Basin of Southwest China showed greatly different N transformation processes compared to the normally occurring soils in other subtropical regions. Overall, the alkaline Regosols had a faster NH 4 + mineralization-immobilization turnover, higher nitrification rates, and lower NO 3 − immobilization rates compared to other reported subtropical soils.
Gross N transformations under different land use soils. The different land uses significantly affected the NH 4 + immobilization and autotrophic nitrification in Regosols, while no significant differences were www.nature.com/scientificreports/ observed in gross mineralization, NO 3 − immobilization and DNRA between the forestland and cropland soils (Fig. 3). In the cropland soils, the gross NH 4 + immobilization rates were significantly lower than those in the forestland soils (Fig. 3b). This result agrees with most previous findings obtained from other subtropical soils in China 9,45 . However, Zhang et al. observed similar gross NH 4 + immobilization rates between forestland and agricultural soils 4 . Soil gross NH 4 + immobilization rates were positively related to the SOC and C/N in this study (Fig. 4). Previous studies indicated that relatively higher SOC content and C/N ratio in the soils could stimulate the increase of N immobilization potentiality 9,46 . In the cropland soils, due to long-term mineral N fertilizer application and very few crop residual retention, organic matter sources are mainly dependent on crop roots 9 . Therefore, the SOC content and C/N ratio in the cropland soils significantly decreased compared to those in the forestland soils (Fig. 2c,d), which might be an important factor that significantly reduced NH 4 + immobilization. Furthermore, the gross NH 4 + immobilization rates in the forestland soils were greater than the gross mineralization rates (Fig. 3a,b), indicating that a large proportion of the NH 4 + produced from mineralization could be effectively immobilized. This rapid NH 4 + turnover in forestland soils likely reduced the accumulation of NH 4 + , thus left little available substrate for nitrifiers 19,25 . However, in the cropland soils, the gross mineralization rates were significantly greater than the NH 4 + immobilization rates (Fig. 3a,b), which might leave more available NH 4 + substrates in soils for autotrophic nitrification.
The gross autotrophic nitrification rates were significantly greater in the cropland soils than in the forestland soils (Fig. 3d). This finding is in accordance with the results of some previous studies 4,9 . Soil pH is generally viewed as the key factor influencing nitrification 4,12,13 . However, soil pH was not significantly different between the forestland and cropland in this study (Fig. 2a). Nitrogen fertilization has been considered as another important factor stimulating nitrification in the cropland soils 4,9 . Numerous studies have indicated that long-term N fertilizer applications could stimulate autotrophic nitrification rates 18,30,47,48 . Applying mineral N fertilizer could induce the rapid increases in NH 4 + concentrations for several weeks in cropland soils, thus providing sufficient available substrates for nitrification [49][50][51] . However, in this study, soil samples were collected once in April when several months had passed since the last fertilizer application. Thus, the extremely high nitrification rates following fertilization might not been considered in this study. Previous studies have shown that long-term N fertilizer application could affect the ammonia-oxidizing microbe population size and activity, thus stimulate autotrophic nitrification 18,30 . Previous studies in the same study area have revealed that application of mineral N fertilizer significantly increased soil ammonia-oxidizing bacteria (AOB) population size and changed AOB composition 49,50 . Consequently, long-term applying mineral N fertilizer might be the main factor inducing the differences in nitrification rates between the different land use soils in this study.
The ratio of soil gross autotrophic nitrification to gross NH 4 + immobilization (O NH4 /I NH4 ) can effectively indicate their relative importance in NH 4 + consumption 5,9,10 . In this study, a negative correlation was observed between the gross autotrophic nitrification rates and NH 4 + immobilization rates (Fig. 5c). In the forestland soils, the average O NH4 /I NH4 ratio was 0.32 ± 0.09, indicating that NH 4 + immobilization dominated NH 4 + consumption. Conversely, the average O NH4 /I NH4 ratio was 69.99 ± 18.91 in the cropland soils, indicating that autotrophic nitrification was the dominant NH 4 + -consuming process. These results also implied that autotrophic nitrification was greatly enhanced in the cropland soils than in the forestland soils in the study region.
Overall, in comparison to the forestland soils, gross autotrophic nitrification rates were significantly increased, while gross NH 4 + immobilization rates were significantly decreased in the cropland soils. The significant differences in soil N transformations were closely related to the long-term mineral N fertilizer application, and the significant SOC content and C/N ratio decreases in the cropland soils.

NO 3 − loss and retention driven by soil N transformations.
During the whole monitoring period, the cropland soils had significantly higher NO 3 − concentrations than the forestland soils (Fig. 6b). The large amounts of NO 3 − accumulation in the cropland soils could be easily diluted and lost during the heavy rainfall events [22][23][24] . The field monitoring results showed that the NO 3 − losses in each runoff event were all significantly greater in the cropland soils than those in forestland soils (Fig. 6c,d). Previous studies indicated that the inorganic N form and amount in soils, especially the NO 3 − accumulation, were controlled by N transformation processes 25,26 . In the cropland soils, the gross NH 4 + immobilization significantly decreased but the gross autotrophic nitrification significantly increased compared to the forestland soils, resulting in the NO 3 − /NH 4 + ratio (mean 5.79 ± 0.16) being 4 times greater than that in the forestland soils (mean 1.24 ± 0.05). This result confirmed that NO 3 − not only was the dominant inorganic N form, but also has a higher concentration in the cropland soils.
The nitrification capacity (i.e., O NH4 /M N ratio) and O NH4 /I NH4 ratio were two key indicators for the NO 3 − loss potential from the soils 52,53 . In this study, compared to the forestland soils, the cropland soils had much higher nitrification capacity and O NH4 /I NH4 ratio. The correlation analysis showed that the nitrification capacity and O NH4 /I NH4 ratio were positively correlated with the NO 3 − concentration and NO 3 − /NH 4 + ratio (P < 0.05; Fig. 5d). Moreover, the low NO 3 − retention capacity was observed in both the forestland and cropland soils (mean 0.51 ± 0.24 and 0.01 ± 0.01). Overall, the NO 3 − production rates were greater than the NO 3 − consumption rates, resulting in huge NO 3 − accumulation in Regosols (mean 3.02 ± 0.18 mg N kg −1 and 13.60 ± 0.51 mg N kg −1 for forestland and cropland), which thus caused great risks of NO 3 − losses, especially from the cropland soils 9,18,54 . NO 3 − losses caused nutrient loss and threatened the environment and human health 1,55,56 . In this study, the NO 3 − losses occurring in the cropland soils were approximately 10% of the annual N fertilization. Consequently, NO 3 − losses to the environment should be minimized by retaining NO 3 − efficiently in the soils. As discussed above, the SOC content and C/N ratio significantly influenced soil N immobilization and nitrification. Thus, increasing the SOC content and C/N ratio would be effective strategies for NO 3 − retention in the alkaline Regosols, which can potentially reduce autotrophic nitrification and enhance NH 4 + immobilization.

Conclusions
Compared to the typical zonal acidic soils in the subtropical regions, the non-zonal soils of alkaline Regosols in this study showed specifically inherent gross N transformations, i.e. the faster NH 4 + mineralization-immobilization turnover, the higher nitrification rates, and the lower NO 3 − immobilization rates. Different land use significantly affected the gross N transformation processes of autotrophic nitrification and NH 4 + immobilization in Regosols. In the cropland soils, the rates of gross autotrophic nitrification were significantly greater, but the rates of gross NH 4 + immobilization were significantly lower than those in the forestland soils. The specific soil gross N transformations resulted in low NO 3 − retention capacity and thus high NO 3 − loss risks in the Regosol croplands. The total NO 3 − losses from the cropland soils were substantial (27.10 ± 2.54 kg N ha −1 year −1 ) and much greater than those from the forestland soils (0.25 ± 0.01 kg N ha −1 year −1 ). The great differences in the N transformations between the different land use soils may be attributed to the changes of the SOC content and C/N ratio and the application of mineral N fertilizer after long-term cultivation in the cropland.