Soil sampling and preparation
In total, soils with different land-use types (Fig. 1) were collected from 15 sites, which were almost evenly distributed in the Zhejiang province (an area of 1,055,000 km2). Information on specific sampling points is presented in Table 1. The 15 sampling points covered six land-use types including orchards, single cropping rice, double-cropping rice, rice-rape rotation, rice-wheat rotation, and vegetables, generally established in the past 5 years.
Soil samples of 0–20 cm were collected from typical fields (long-term farmland with conventional fertilization by local farmers) in May 2018 during the second season of rotation systems and in other land-use types. Two samples, with three replicates, were obtained at intervals of 1000 m at each site with the same land-use type. Next, the replicates were brought back to the laboratory and mixed. Then, the mixed soil samples from each site were divided into four equal parts by the diagonal quartering method and maintained for a follow-up test. All samples were air-dried and separated into two parts: one was finely milled and sieved through a 2-mm mesh to determine basic physical and chemical properties, and the other was carefully broken into small pieces manually and passed through an 8-mm sieve for aggregate separation and Pcoll determination.
Aggregate separation and determination
Aggregate size distribution was determined for each soil sample using a modified wet sieving method [41]. Briefly, 50 g of unground soil, passed through an 8-mm sieve, was carefully placed above a nest of three sieves (2 mm, 0.26 mm, and 0.053 mm). Then, the sieves were submerged for 20 min in 2.5 L deionized water at room temperature and oscillated 300 times for 10 min with a 30-mm amplitude to separate aggregate fractions. Thus, four aggregate fractions were obtained on each sieve: large macroaggregates (2–8 mm), small macroaggregates (0.26–2 mm), microaggregates (0.053–0.26 mm), and (silt+clay)-sized particles (<0.053 mm) [42]. Aggregates of each size were carefully removed from the sieve and placed into a beaker. The water used for wet sieving was left to rest for 48 h, silt and clay particles were collected, and the supernatant was used to determine total dissolved P (TDP), truly soluble P (TSP), and Pcoll content. All aggregates were oven-dried at 65°C for 48 h, weighed, and placed in a zip lock bag. To obtain water-stable aggregates, the sediment concentration was subtracted from that obtained by wet sieving as sand was not considered a component of water-dispersible aggregates [41]. The sand content was determined by the following process: 5 g of the dry aggregates obtained above were weighed, dispersed into 30 mL 5 g L–1 hexametaphosphate solution, placed into an ultrasonic cleaner, and dispersed for 30 min. The suspension was then poured through a nest of sieves. The residue left on the 0.053-mm sieve represented the sand content of each sized aggregate. After collection, sand was dried at 65°C and weighed.
Soil pH was determined with a glass electrode pH meter (PHS-3C, Shanghai) using a soil-to-water ratio of 1:5. Soil cation exchange capacity was measured with ammonium acetate (12.5 mL 1 M NH4OAc, 2.5 g soil) [43]. Soil and aggregate-associated TP was determined by H2SO4-HClO4 digestion and evaluated using the molybdenum-blue colorimetric method [44]. Soil particle size distribution was determined by the hydrometric method according to an international soil texture classification standard. Soil- and aggregate-associated TC and TN were determined using an elemental analyzer (dry combustion with Vario MAX CNS, Elementar, Germany). Soil- and aggregate- associated Al, Fe, and Ca were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) after digestion with 5 mL HNO3 (16 M), 1 mL HClO4 (12.4 M), and 1 mL HF (23 M) for 12 h. All reagents were acquired from Sinopharm Chemical Reagent Co., Ltd.
Colloidal P was determined as described by Ilg [45]. Briefly, 10 g of unground soil was placed into a 250 mL flask, 80 mL deionized water was added. The sample was shaken at 160 rpm and 25°C for 24 h. The supernatant was pre-centrifuged at 3000 g for 10 min to remove coarse particles. After pre-centrifugation, the supernatant was filtered with a 1 μm microporous membrane, 5 mL of the primary filtrate was discarded, and the total filtrate was collected (sample I). This suspension included the colloidal and dissolved components. The filtrate was ultracentrifuged at 300,000 g for 2 h to remove colloids (Optima TL, Beckman, USA; Sample Ⅱ), and the residue at the bottom of the ultracentrifuge tube demonstrated the water-dispersible colloids. The TDP in Sample I and TSP in Sample II, in the solution, were determined after digestion with acidic potassium persulfate. The concentration of Pcoll indicated the difference between TDP in Sample I and TSP in Sample Ⅱ. Previous studies have shown that soil P through leaching and surface runoff was usually in the soluble form, that can pass through the 0.45–1 μm filter [46, 47]; therefore, in the present study, TDP including Pcoll and TSP in aggregates was defined as the potential loss P, and the Pcoll in TDP was defined as Pcoll loss potential. TDP in the supernatant after 10 min wet sieving was considered as easy loss P.
Calculation of water-stable aggregate (WSA) size fractions
The proportion of WSA in each size fraction was obtained from Equations (1) and (2), as described by Alvaro-Fuentes [48]: (see Equations 1 and 2 in the Supplementary Files)
where, i is the ith size fraction (2–8, 0.25–2, and 0.053–0.25 mm); dry soil aggregate (DSAi) is the oven-dried mass of total, non-dispersed aggregates collected on each sieve; Sand is the oven-dried mass of sand collected after dispersal in the hexametaphosphate solution on the 0.053 mm sieve; TotalSoil is the oven-dried mass of soil (50 g) for aggregate separation.
Calculation of mean weight diameter (MWD) and geometric mean diameter (GMD)
The MWD and GMD of the aggregates were obtained by Equations (3) and (4) [49]:(see Equations 3 and 4 in the Supplementary Files)
where, i is the ith size fraction (2–8, 0.26–2, 0.053–0.26, and <0.053 mm) and d is the mean diameter of each size (0.053–2 mm). WSAi include 2–8, 0.26–2, 0.053–0.25, and <0.053 mm WSA and (silt+clay) sized fractions.
Contribution of aggregate-associated Pcoll to TDP
The contribution rate (CR) was used to explore the impact of aggregate sizes on the Pcoll loss potential, calculated using Equation (5): (see Equation 5 in the Supplementary Files)
where Agg_CP is the concentration of aggregate-associated Pcoll (mg kg–1), TDP is the concentration of total dissolved P (mg kg–1), and i is the ith size fraction (2–8, 0.26–2, 0.053–0.26, and <0.053 mm).
Statistical analysis
Microsoft Excel 2016 and Origin 8.0 were used for data processing and cartography. Data were statistically analyzed using SPSS Statistics 22.0 (SPSS Inc. Chicago, USA) software. One-way ANOVA was performed using two samples from each site to examine differences of different variables as presented in Table S1, S2, and 3 and Fig S1 and S2. Pearson correlation analysis was used to identify the relationship between aggregate-associated Pcoll and other soil parameters. Stepwise linear regression was performed to evaluate the relationships between P indicators (content and loss potential of Pcoll) and soil variables (pH, TP, TC, TN, C/N, Fe, Al, Ca, MWD, and GMD).