Nitrogen addition increases the contents of glomalin-related soil protein and soil organic carbon but retains aggregate stability in a Pinus tabulaeformis forest

Study area

The experiment was conducted in the Songyugou watershed in the Loess Plateau region, Shaanxi Province, China (35 390N, 110 060E), the elevation and slope of the study area were 1,000–1,200 m and 20°–25°. The region has a classic mainland monsoon-type climate with a mean annual precipitation of 584.4 mm, mean annual temperature of 9.7 °C, and frost-free period of 180 days. The soil is gray forest soil (Gray Luvisols, FAO soil classification) with a surface soil (0–20 cm) bulk density of 1.1 g/cm³. The soil total phosphorus (P) content is 1.42 ± 0.38 g/kg, and the soil pH is 8.6. P. tabulaeformis was planted in 1960. The current stand density is 1,400–1,800/ha. The average canopy density is 0.7. The average diameter at breast height is 10.0 cm. The average height of the tree is 11.2 m. The forest stand volume is 75.5 m³/ha. The leaf area index is 6.34. The biomass of tree layer, shrub layer, and herbaceous layer are 112.96, 3.56, and 8.28 t/hm², respectively, the plant diversity index of the community is 0.51 (calculated using the Simpson method). Shrubs are mainly Elaeagnus pungens Thunb, Rosa xanthina Lindl, Spiraea Salicifolia L, Lonicera japonica Thunb, and Viburnum dilatatum Thunb. The shrub coverage is 30%. Herbs are mainly Carex lanceolata Boott, and the herb coverage is 30–50%.

Experimental design

In China, the N deposition rate in 2010 was ranged from 0.1 to 7.43 g N m⁻² y⁻¹, with an average of 2.11 g N m⁻² y⁻¹ (Liu et al., 2013). The ambient N deposition rate in the region was approximately 1.2 g N m⁻² y⁻¹, and N deposition is increasing at an annual rate of 0.041 g N m⁻² y⁻¹ (Lü & Tian, 2007). Moreover, most studies added 0–12 g N m⁻² y⁻¹, which is sufficient to change soil N content from limitation to saturation for plant growth in most ecosystems (Wang et al., 2018; Jing et al., 2017a). Thus, we designed four levels of N addition treatments (0, 3, 6, and 9 g N m⁻² y⁻¹) in the form of urea (Fumin Agriculture Product Company, Xi’an, China). In each treatment, six 10 × 10 m plots were randomly established for N addition. A total of 24 plots were subjected to the same climate, stand age, and terrain conditions. In 2014 and 2015, urea was dissolved in 10 L distilled water and evenly sprayed in each plot before the rain in early April, June, August, and October every year. Soil samples were collected after N addition in September 2015, and N was added for two years.

Soil sampling

In September 2015, soil was collected from 0 to 20 cm soil layer (remove litter layer), because the short-term N addition directly affected the physical and chemical properties of the surface soil. Three undisturbed soil samples were randomly collected in each plot. The undisturbed soil samples were carefully transported back to the laboratory. The samples were gently broken apart and all the soil samples were air-dried after passed through 8 mm sieve. The three soil samples of each plot were mixed up after air-drying. Finally, all soil samples were sealed in plastic bags without squeezing until physical and chemical analysis.

Aggregate distribution

Soil aggregates were classified by wet sieving method (Six et al., 1998). Air-dried samples (100 g) placed in a 250 μm sieve and submerged in deionized water for 5 min, and the surface of the water submerged the soil samples. Aggregates were separated by moving the sieve up and down for 3 cm with 50 repetitions/min for 2 min. Aggregates retained in the sieve (>250 μm) were macro-aggregates. The soil and water passed through the 250 μm sieve and then were sieved again as above using a 53 μm sieve for 3 min. Aggregates retained in the sieve (>53 μm) were micro-aggregates, and those that passed through the sieve were fine materials. All separated soil and water were collected, oven-dried at 45 °C, and weighed and stored for chemical analysis.

GRSP analyses

Glomalin-related soil protein content was determined according to the method of Wright & Upadhyaya (1998). T-GRSP was extracted from soil sample (1 g) with 8 mL of 50 mmol/L citrate solution at pH 7.0 and then autoclaved at 121 °C for 60 min. The supernatant was removed after centrifugation at 10,000×g for 10 min. This procedure was repeated five times on the same sample until the solution was straw-colored. The supernatant was pooled together and stored at 4 °C. EE-GRSP was extracted from soil sample (1 g) with 8 mL of 20 mmol/L citrate solution at pH 7.0 and autoclaved at 121 °C for 30 min. The supernatant was removed after centrifugation at 10,000×g for 10 min and stored at 4 °C. The T-GRSP or EE-GRSP in the supernatant was determined by Bradford analysis spectrophotometrically at 590 nm light (Wright & Upadhyaya, 1996; Rillig, 2004; Wright & Upadhyaya, 1996).

SOC analyses

Soil organic carbon content was measured by dichromate oxidation procedure (Nelson & Sommers, 1982).

Statistical analyses

Aggregate stability was evaluated by mean weight diameter (MWD) millimeter and geometric mean diameter (GMD) millimetr. MWD is a comprehensive index for the evaluation of aggregate stability, and GMD is an index of the main grain size distribution of aggregates. The larger value of MWD and GMD indicate higher aggregate stability (Bedini et al., 2009). MWD and GMD were calculated using the following equation: $\text{MWD}={\displaystyle \sum _{i=1}^n{x}_i}{w}_i\text{and}\text{GMD}=\text{EXP}\left[\frac{{\displaystyle {\sum }_{i=1}^n{w}_i}\text{ln}{x}_i}{{\displaystyle {\sum }_{i=1}^n{x}_i}}\right]$ x_i is the mean diameter of each class aggregate (mm), w_i is the percentage of each class aggregate (%), and n is the number of aggregate fraction classes.

Differences between any two N addition treatments in aggregate percentage, MWD, GMD, GRSP content, and SOC content were tested with one-way ANOVA on SPSS 20.0 statistical software package (SPSS Inc., Chicago, IL, USA). Linear regression analyses were used to test the relationships among GRSP, SOC, and MWD.

Effects of N addition on soil aggregate distribution and stability

The percentage of aggregate varied with size classes, and macro-aggregates were more dominant than the other size aggregate fractions in all samples (Table 1). Across all N addition treatments, the values of MWD were in the range of 1.38–1.70 mm, and GMD was in the range of 0.86–1.05 mm. N addition had no significant effects on aggregate distribution, MWD, and GMD. However, the percentage of macro-aggregate, MWD, and GMD increased first and then decreased with increasing N addition levels, whereas the percentage of micro-aggregate and fine material decreased first and then increased with increasing N addition levels.

Effects of N addition on GRSP content

Figure 1 shows that the contents of EE-GRSP and T-GRSP were in the range of 0.099–0.551 and 0.315–4.058 mg g⁻¹, respectively. In the control treatment, GRSP content varied among bulk soil and aggregate fractions, and GRSP content increased in the order of macro-aggregate<bulk soil<micro-aggregate<fine material. Across all N addition treatments, EE-GRSP content in the macro-aggregates and bulk soil increased first and then decreased with the increasing N addition levels and had a top value at 6 g N m⁻² y⁻¹. Compared with that of control treatment, N addition changed the EE-GRSP content of the macro-, micro-, fine materials, and bulk soil by −24.8% to 222.1%, −30.8% to 22.0%, 22.8–40%, and −8.4% to 134.4%. Across all N addition treatments, T-GRSP contents in all size aggregates and bulk soil increased with the increasing N addition levels and had a top value at 9 g N m⁻² y⁻¹. Compared with that of control treatment, N addition changed the T-GRSP content in macro-, micro-, fine materials, and bulk soil by 412.7–872.4%, 203.1–266.9%, −0.3% to 14.3%, and 259.3–468.0%.

Effects of N addition on SOC content

Soil organic carbon content ranged from 6.478 to 21.627 mg g⁻¹. Across all aggregate fractions, macro-aggregate had the highest content of SOC and fine material had the lowest content of SOC (Fig. 2). Compared with that of control treatment, N addition significantly increased SOC content in all size aggregate fractions and bulk soil, and the change ranges of SOC content in the macro-, micro-, fine materials, and bulk soil were 71.7–79.5%, 75.5–91.5%, 49.6–53.5%, and 47.6–74.4%.

Relationships among GRSP, SOC and MWD

Table 2 shows that the contents of SOC, EE-GRSP and T-GRSP in aggregate fractions and bulk soil have no significant relationship with MWD (except for the relationship between SOC content in micro-aggregates and MWD).

Effects of N addition on GRSP content

The contents of EE-GRSP and T-GRSP were in the range of 0.099–0.551 and 0.315–4.058 mg g⁻¹, respectively. This finding is consistent with that of the studied on the GRSP contents of different vegetation communities (0.6–5.8 mg g⁻¹) (Singh, Singh & Tripathi, 2013). In this study, GRSP content varied among aggregate fractions and bulk soil, and N addition had different effects on GRSP distribution in aggregate fractions and bulk soil. The research in farmland system found that tillage management and manure amendment had different effects on GRSP distribution in aggregate fractions (Wright, Green & Cavigelli, 2007; Xie et al., 2015). Long-term manure amendments redistributed the GRSP in the macro-aggregate fractions. These results indicated that GRSP distribution in aggregate fractions is susceptible to environmental changes. EE-GRSP was produced by AMF most recently, and EE-GRSP was proved to be more sensitive to environmental changes than T-GRSP (Singh, Singh & Tripathi, 2013). In our study, the content of EE-GRSP in bulk soil increased first and then decreased with the increasing N addition levels and reached the peak (0.403 mg g⁻¹) at 6 g N m⁻² y⁻¹. The variation in EE-GRSP content was mainly attributed to the changes in plant growth and soil microbial activities (Xie et al., 2015), which were induced by N addition. In our study, plant growth and soil microbial activities might be increased at 3 g N m⁻² y⁻¹ and inhibited at 9 g N m⁻² y⁻¹. Thus, N addition of 6 g N m⁻² y⁻¹might be the threshold value between increased and inhibited GRSP production. Xie et al. (2015) found that the highest EE-GRSP contents were not obtained at the highest amount of organic manure. Our results indicated that low-level N addition could increase the EE-GRSP contents, but high-level N addition inhibited the EE-GRSP contents. The highest EE-GRSP contents were observed at 6 g N m⁻² y⁻¹, which indicated a proper N addition level can maximize the EE-GRSP content in this region. These results supported our first hypothesis. Furthermore, N addition had different effects on EE-GRSP content in aggregate fractions. The EE-GRSP content in macro-aggregates increased initially and then decreased with the increasing N addition levels and had a peak value at 6 g N m⁻² y⁻¹. Meanwhile, the EE-GRSP content in micro-aggregates had a low value at 6 g N m⁻² y⁻¹. These results were inconsistent with our first hypothesis. Previous studies showed that organic manure significantly increased the EE-GRSP content in all soil aggregate fractions except micro-aggregates in farmland ecosystems (Xie et al., 2015), and increasing soil depth significantly increased the EE-GRSP content in 0.5–1.0 and 1–2 mm aggregates without other size aggregate fractions in citrus rhizosphere (Wu et al., 2013). These different results indicated that the distribution of EE-GRSP in aggregate fractions varied with environmental conditions, which resulted in the changes of EE-GRSP in bulk soil. In addition, the percentage of macro-aggregates was higher than those of other size aggregate fractions. N addition has the same effects at EE-GRSP content in macro-aggregates with that in bulk soil, which indicated that the EE-GRSP in macro-aggregates was the main factor that determined the EE-GRSP in bulk soil. T-GRSP contained easily and hardly extractable parts. Compared with EE-GRSP, T-GRSP can reflect the accumulation of glomalin in the soil (Singh, Singh & Tripathi, 2013). In our study, N addition significantly increased the T-GRSP content in bulk soil and had a peak value at 9 g N m⁻² y⁻¹, which was different from the changes of EE-GRSP content in bulk soil. A large number of studies found that environmental changes had different effects on T-GRSP and EE-GRSP contents in bulk soil as well (Wright, Green & Cavigelli, 2007; Xie et al., 2015; Antibus et al., 2006; Zhang et al., 2015). An explanation for this result in our study is that high-level N addition can quickly relieve N limitation in the soil and promote the microbial activities at the beginning of the experiment, which increase GRSP production in the short term. Given the increasing time, high-level N addition may cause N saturation in the soil and inhibit microbial activities that decrease the GRSP production (Yu et al., 2013). However, the T-GRSP content under high N addition level was higher than that under low N addition level for a period of time due to the cumulative effect. The T-GRSP content in aggregate fractions increased significantly with the N addition, this result supports our explanation. While the continued responses of T-GRSP depend on the time and rate of N addition. Another explanation is that the conventional Bradford method we used in this study is often hardly to separate T-GRSP from total protein (Roberts & Jones, 2008). So continuing the study for three to five more years with infrared spectroscopy for analysis of the glomalin (Jiří et al., 2017) would be very essential. Besides, the micro-aggregates had the highest contents of T-GRSP in our study, which was consistent with previous research in this region (Jing et al., 2017b). However the distribution of T-GRSP in aggregates was variable based on different tillage management treatments in farmland (Dai et al., 2015). These results suggest that the distribution of GRSP in aggregates was affected by soil types, vegetation, climate, and other factors.

Effects of N addition on SOC content

In this study, SOC content ranged from 6.478 to 21.627 mg g⁻¹. Across all aggregate fractions, macro-aggregate had the highest SOC content, followed by the micro-aggregate, and those in fine materials is the lowest. These results were consistent with that of the previous study (Zhang et al., 2014). However, some studies showed that the highest SOC content was in fine material, and the lowest was in macro-aggregate (Gao et al., 2013; Tang et al., 2016). Different results might be caused by soil type, plant community, and experimental conditions (Wei et al., 2011). N addition for two years significantly increased SOC content in bulk soil, which indicated that N addition could quickly increase plant growth and decomposition in N limitation soil and increase soil C storage (Nave et al., 2009). Interestingly, N addition significantly increased SOC content in all size aggregate fractions. Compared with other size aggregate fractions, macro-aggregate had the highest percentage and SOC content, with the greatest contribution to the soil C storage. Meanwhile, the SOC in fine material had the smallest contribution to the soil C storage. These results were different from our hypothesis. Zhong et al. (2017) found that N addition (0–300 kg N ha⁻¹ y⁻¹) for one year had no significant influence on SOC in any size aggregate fractions in a subtropical forest. These different results might be related to soil type and initial N content. For example, macro- and micro-aggregate fractions had the highest SOC content among the size aggregate fractions in loamy soils, while fine material fraction had the highest SOC content than those of other size aggregate fractions in sandy soil (Gao et al., 2013). Our results indicated that the SOC content varied among aggregate fractions, and N addition had different effects on SOC in aggregate fractions. The effects were not only affected by N addition level but also by soil type and initial N content.

Relationships among GRSP, SOC and aggregate stability

GRSP and SOC were insignificantly correlated with MWD. This result fails to support our hypothesis. A large number of studies found that GRSP and SOC were significantly correlated with MWD because both can bind soil particle for aggregate formation (Rillig, 2004; Wright, Green & Cavigelli, 2007; Baldock, Kay & Schnitzer, 1987). However, recent studies found that GRSP and/or SOC may have no significant relationship with MWD (Wu et al., 2013; Li et al., 2005; Leelamanie & Mapa, 2015) because aggregate stability was not only affected by GRSP and SOC contents but also by soil texture, human disturbance, and other factors (Wei et al., 2011; Tang et al., 2016). In our study, the contents of GRSP and SOC significantly changed, but MWD insignificantly changed with N addition levels which caused insignificant relationships among MWD, GRSP and SOC. N addition significantly increased the contents of GRSP and SOC and insignificantly increased MWD, indicating that increasing GRSP and SOC contents does not necessarily increase aggregate stability rapidly. Aggregate formation requires not only binding agents but also physicochemical effects, but the underlying mechanism still needs further study.