In this study, we found that fencing had positive effects on vegetation cover, biomass and height, as also reported in other studies12,16,23,24. Fencing can enhance plant cover, biomass and height because it protects the soil seed bank and increases species composition recovery16,25. Moreover, this study showed that grazing exclusion had weak effects on the BGB in comparison to that in the GG. Conversely, previous studies have supported the hypothesis that grazing exclusion has negative effects on BGB in the 1 m soil layer12,26 or at least has no detrimental effects27 but reduces the percentage of forbs; the forb fraction also decreased in our study. The main factor that affected the plant properties in the grazed grassland: standing plant biomass was continuously removed by herbivory, after which the litter was easily lost28,29,30; this scenario would then decrease the AGB and LB. Oppositely, fencing may increase in soil coverage, plant diversity, vegetation biomass, SWC and SOC, which would increase the AGB and LB31,32.

This study showed that fencing had significant effects on SWC, BD, SOC and soil TN. Fencing increased the SWC in the 0–140 cm soil layer because the high coverage of vegetation and more plant litter may have improved the soil moisture retention and protected the soil water from evaporation23,33. Meanwhile, the soil BD in the 0–140 soil layer decreased in the FG, likely because trampling may have increased the BD in the GG but not in the FG, and an increase in plant roots and soil microorganisms may have decreased the BD in the FG33,34,35. In our study, in the soil layer from 0 to 180 cm, the SOC in the FG was greater than that in the GG, and fencing significantly increased the soil TN storage in the soil layers of 0 to 500 cm (P < 0.05). The soil in the 0 to 180 cm layer was black in the FG (Fig. 9a); this relatively deeper colour likely corresponded to a greater SOC fraction in the FG soil than in the GG soil (Fig. 9b). Previous studies showed that grazing exclusion result in significant increasing of SOC and TN as a result of perennial organic matter inputs from plant decomposition, and the lack of disturbance and formation of SOC in micro aggregates lead to the creation of fine soil particles, which causes the spatial inaccessibility of SOC and soil N for soil microbes and enzymes33,36,37.

Figure 9 Soil samples in (a) FG and (b) GG from 0 to 500 cm. Full size image

The C storages of the AGB and LB were three times and two times greater, respectively, in the FG than in the GG. Similar results were found that fencing (11 years) significantly increased the C storages of AGB and LB, respectively, comparing with the grazed grassland12, and aboveground biomass C storages were about two times greater after 8 years fencing because of fencing increases in soil coverage, plant biodiversity, biological yield, and SWC and nutrients after enclosure construction in slightly degraded steppe grasslands on the Loess Plateau12,32. In our study, the C stock of the BGB was not significantly different between the fenced grassland and grazed grassland; the possible reason for this result is that the species diversity of the GG is lower than that in the FG, and fewer roots of species were obtained in comparison to those in the FG, differing from previous studies, in which aboveground biomass C storages were significantly lower in the FG than in the GG12. Plant C and N stocks are determined by biomass, and fencing exclusion of livestock resulted in the restoration of the grassland biodiversity, and increased the plant biomass30,36. After 30 years of fencing, the nitrogen storages of the aboveground biomass and litter biomass in the FG were greater than those in the GG because of the increased plant biomass and biodiversity. In this study, fencing only affected the vegetation biomass in the 0–10 cm soil layers. In addition, plant roots in the FG were observed in the 60–80 cm soil layer, but there were no roots in the GG within the same soil layer because fencing increased the coverage and species richness of plants, while overgrazing depressed the plant diversity and the growth plant roots, and fencing stimulated plant roots to grow deeper to obtain nutrients and water38,39.

In our study, long-term (30 years) grazing exclusion significantly increased SOC in the 10 to 180 cm soil layers and soil N stock in both the GG and FG of the 0–60 cm soil layers, respectively. Previous studies showed that 30 years of fencing dramatically increased the soil C and N stocks in the 0–100 cm soil layer in temperate grassland33, and fencing for 11 years notably increased soil C storages in the 0 to 100 cm soil layers and N storages from 0 to 20 cm soil layers in comparison to those in GG12, and the C and N stocks of 0–20 soil layers significantly increased with decreasing grazing intensity39 because of the increased input of C and N into soils by litter and roots. In the fenced 26 years desert shrubland, the SOC and TN storage in the 0–30 cm soil layer increased by 13.6- and 5.4-fold, respectively40. The non-significant difference in SOC stock between the FG and GG in the 0–10 cm soil layer (Fig. 5a) and the non-significant difference in cumulative soil C stock in the 0–30 cm layer of soil between the FG and GG (P > 0.05) (Fig. 6a) were likely due to animal manure input in the GG, leading to more soil C and N accumulation, while livestock trampling also led to greater soil BD in the GG41, which led to greater C and N stocks because of increased C and N density. The manure input counteracted the soil C and N accumulation associated with long-term fencing. In the 0–10 cm soil layer, the soil C stock sequestration showed a negative value, indicating that the loss of C from the soil over the past 30 years in the FG (Fig. 7) likely occurred because the plants consumed more soil C than the soil C input by the microbial decomposition of vegetation biomass and litter. Additionally, few studies have focused on deep soil C and N in grasslands. Callesen and James found that deep roots and deep soil layers (0–300 cm) may contribute significantly to nutrient supplies and the soil C storage capacity of temperate and boreal forest ecosystems42,43. Li et al. estimated that the soil C stock in the 0–300 cm layer could be three times that in the 0–100 cm layer44. Wang et al. found that more than 50% of soil C storage occurred in the 100–300 cm layer in grasslands and deserts45. However, in our study, we estimated that the ecosystem C stocks in the 0–100 cm soil layer in the FG accounted for 41.8% and 58.1% of the ecosystem C stocks in the 0–500 cm and 0–200 cm soil layers, respectively. Meanwhile, ecosystem N stocks in the 0–100 cm soil layer in the FG accounted for 28.0% and 60.6% of the ecosystem N stocks in the 0–500 cm and 0–200 cm soil layers, respectively. Thus, using only the 0–100 cm soil layer to estimate soil C storage would lead to significant underestimation.

The cumulative soil C storages in the 0–80 cm layer, the cumulative soil C storages below 80 cm and the cumulative N storage in 0 to 500 cm soil layer increased dramatically in the fenced grassland compared to that in the grazed grassland, likely resulting from the fencing increasing the vegetation biomass and litter biomass, which supplies a suitable environment for promoting microbial activity and soil texture, and less C input from root-associated sources and possibly greater C output through heterotrophic respiration might have reduced the various SOC stocks12,30,36. Fencing promoted high soil coverage and improved the soil moisture, which increased the soil microbial biomass, specifically that of fungi, and restored soils exhibit greater rates of C and N mineralization46. Vegetation restoration decreased the C and N losses because of increased soil coverage and plant productivity47. However, previous studies showed different results, with no difference in C stocks between FG and GG areas in the 0–10 cm soil layer (P > 0.05), but the C stock was greater for FG in the underlying 10–100 cm soil layers(P < 0.001)compared to that in GG areas33. Conversely, grazing exclusion increased the soil C and N storages dramatically in the 0–20 cm soil layers, but not in 20 to 100 cm soil layers12. The annual soil C and N sequestration rates indicated that fencing may result in the accumulation of C in the 40 to 180 cm soil layer. The accumulation of soil N in the160–500 cm soil layers likely resulted from soluble N infiltrating deeper into the soil. Overall, fencing significantly improved the C and N stocks in temperate grasslands in northwest China, and fencing was supposed to be a key measure for ecological restoration of degraded grasslands.