Changes in nitrogen pools in the maize-soil system after urea or straw application to a typical intensive agricultural soil: A 15N tracer study

A 15N maize pot experiment was conducted to compare the N value of fertilizer alone and fertilizer combined with straw at an equivalent N rate. The four treatments were control (CK), 15N-urea, 15N-urea plus straw, and 15N-straw plus urea. Soil N pools, maize N and their 15N abundance were determined during maize growth. At maturity 26.0% of straw N was assimilated by maize in the urea plus straw treatment. From the eighth leaf stage to maturity, urea plus straw had a significantly (P < 0.05) higher concentration and percentage of exogenous substrate N present as soil total N (TN), particulate organic N (PON), and mineral associated total N (MTN) in bulk and rhizosphere soils than the urea-only treatment. From silking to maturity in the urea plus straw treatment, rhizosphere soil significantly (P < 0.05) increased the percentage of exogenous substrate N present as inorganic N (Inorg-N) and MTN, and significantly (P < 0.05) decreased that present as PON and microbial biomass N (MBN) compared with the bulk soil. From the eighth leaf stage to maturity, rhizosphere soil significantly (P < 0.05) increased the percentage of straw N present as Inorg-N and MTN except for MTN at the silking stage, and significantly decreased (P < 0.05) that present as PON compared with the bulk soil. Overall, straw was an available N source to the crop, and the increase in straw N availability needs to be considered from the interaction of fertilization practices and the crop rhizosphere.


Introduction
were removed from the pot and shaken to remove the loosely attached soil with roots, then the soil adhering to the root system was placed in a paper bag, vigorously shaken, and 174 brushed to collect the closely adhering soil with roots. The soil adhering to the roots was regarded as rhizosphere soil, and the remaining soil in each pot was mixed thoroughly and 176 regarded as bulk soil. Any visible roots in either soil fraction were removed.  Soil microbial biomass N (MBN) was determined using the CHCl 3 fumigation-K 2 SO 4 192 method as described by Brookes et al. (1985) [26]. Briefly, fresh soil was fumigated with CHCl 3 for 24 h at 25 ℃ and then the N in fumigated and unfumigated samples was extracted 194 with 0.5 M K 2 SO 4 (1:4 w / v, 0.5 h). 20 ml fumigated and unfumigated solution was analyzed using the Kjeldahl method. Soil microbial biomass N was calculated as the difference in N 196 concentration between fumigated and unfumigated samples divided by a conversion coefficient of 0.45 (Jenkinson 1988) [27]. Soil dissolved organic N (DON) was calculated by 198 subtracting Inorg-N from the N concentration in unfumigated solution extracted with 0.5 M K 2 SO 4 [9,28]. The 15 N abundance of fumigated and unfumigated solution was determined as 200 15 N abundance of Inorg-N as described above.
Particulate organic matter and mineral associated matter were fractionated as described 202 by Bronson et al. (2004) [29]. Briefly, fresh soil at each maize stage, equivalent to 25 g oven-dried soil (< 2 mm), was dispersed in 5% sodium hexametaphosphate solution 204 (soil:solution ratio 1:4, w / v) on a reciprocal shaker for 60 min. The slurry was sieved using a 53-μm mesh until the deionized water became clear. The ＜ 53 and ≥ 53 μm soil fractions 206 were transferred to beakers separately and oven-dried at 60 ºC. Nitrogen in both isolated soil fractions is defined as particulate organic N (PON, > 53 μm) and mineral associated total N 208 (MTN, < 53 μm) and their N concentrations were determined using an elemental analyzer after particulate organic matter and mineral associated matter were passed through a 0.15-mm 210 sieve (Macrocube, Elementar, Hanau, Germany). The 15 N abundance of PON and MTN was determined as total 15 N as described above.

214
The concentration of exogenous substrate N present as MBN was calculated by the difference between the fumigated N concentration and the unfumigated N concentration 216 derived from urea or straw, and then divided by a conversion coefficient of 0.45.
Correspondingly, DON derived from urea or straw was calculated by the difference between 218 unfumigated N content derived from urea or straw and Inorg-N derived from urea or straw. where Ndfx is N derived from urea or straw, CON is the concentration of N (mg N kg -1 ), the subscript P is the soil or plant N pool, APE is 15 N atom percent excess, calculated by 226 subtracting the 15 N natural abundance from exogenous substrate N (0.365%) and 15 N abundance of the control treatment from the applied exogenous substrate N treatments. The 228 subscript ex is the exogenous substrate N, which is the applied urea N or straw N.
Here, the recovery rate in the fractions is the percentage of labeled exogenous substrate

Statistical analysis
Data are presented as the mean of three replicates. All the variables were analyzed using 234 the SPSS 16.0 software package (SPSS Inc., Chicago, IL) at the 0.05 level. Student's t-test was used at each growth stage to compare the differences in all variables of specific N pools 236 between urea-only and urea plus straw treatments as well as between bulk soil and rhizosphere soil, and least significant difference (LSD) at the 0.05 protection level was used 238 to assess the differences in mean values of the recovery rate among 15 N-labeled urea, 15 N-labeled urea plus straw and 15 N-labeled straw plus urea treatments at maturity.

Exogenous substrate N uptake in different plant parts
Under the equivalent N amendments the urea plus straw treatment significantly (P < 244 0.05) decreased exogenous substrate N uptake in shoots, grains, and roots at different growth stages except for shoots at the V8 stage compared with the urea-only treatment ( Fig. 1a & b) 246 because of the lower biomass in the urea plus straw treatment (Fig. S1). Conversely, urea plus straw treatment significantly (P < 0.05) increased the percentage of exogenous N present as N 248 uptake in shoots or roots at the V8 stage over the urea-only treatment, and the opposite trend occurred in shoots, roots, and grains from stages R1 to R6 (Fig. 1c & d).

Exogenous substrate N distribution in different soil N pools
Compared with the urea-only treatment, urea plus straw treatment significantly (P < 0.05) 254 increased the concentration of exogenous substrate N present as TN in the bulk and rhizosphere soils from stages R1 to R6 except for the opposite trend in rhizosphere soil at the 256 R1 stage ( Fig. 2a & b). Similarly, compared to urea-only treatment, urea plus straw treatment significantly (P < 0.05) increased the percentage of exogenous substrate N present as TN in 258 the bulk soil from stages V8 to R6 and in rhizosphere soil at R3 and R6 stages ( Fig. 2c & d).

Discussion
With or without straw amendment 308 The combination of straw and chemical fertilizer increases N recovery and decreases the loss of chemical fertilizer N [31,32]. At the equivalent N rate in the 15 NU-S treatment (Table   310 2), the N recovery (the sum of root, shoot and soil) was significantly higher at 5.4% than in the urea-only treatment at the R6 stage. However, the significantly (P < 0.05) higher N use 312 efficiency of maize in the urea-only treatment than the other two treatments is attributed to the small amount of available N in the urea plus straw treatments in our pot experiment ( Fig.   314 3). Some studies show that crop biomass increased with increasing crop N uptake up to the optimum N rate [33,34]; in the urea plus straw treatment, the shortage of N supply resulted 316 in a significantly (P < 0.05) lower shoot biomass from stages R1 to R6 and grain biomass from stages R3 to R6 compared with the urea-only treatment (Fig. S1). Conversely, the lower 318 N supply stimulated an increase in maize root biomass to meet maize N demand [35,36], this resulted in the significant (P < 0.05) increase of maize roots biomass at stages R1 and R3 in 320 the urea plus straw treatment than the urea-only treatment (Fig. S1). In our study, 26.0% straw N recovery was shown in maize at the maturity, moreover, a substantial amount of recalcitrant N in the straw [37,38] under this equivalent N amendment experiment was mainly responsible for the significantly (P < 0.05) lower 39.5 -47.8% exogenous substrate N 324 uptake in the shoots from the R1 stage, 11.6 -29.1% of exogenous substrate N uptake in the roots from the V8 stage in the urea plus straw treatment compared with the urea-only 326 treatment ( Fig. 1a & b), and the lower straw N recovery in different maize parts in the 15 NS + U treatment than the other two treatments (Table 2).

328
Straw has a high C/N ratio and it provides C sources for microbial N turnover and promotes the immobilization of Inorg-N. Moreover, the recalcitrant N in straw is an 330 important stable N source in soils [39,40], Thus, the urea plus straw treatment significantly (P < 0.05) increased the percentage of exogenous substrate N present as TN by 0.3-2.4 times 332 in bulk soil from stages V8 to R6 and 0.5 -0.7 times at stages R3 and R6 in rhizosphere soil compared to the urea-only treatment (Fig. 2). The same explanation accounts for the  (Table 2). Conversely, the characteristics of the organic N in straw under equivalent N amendment conditions was responsible for the significantly (P < 340 0.05) higher concentration and percentage of exogenous substrate N present as Inorg-N in the urea-only treatment compared with the urea plus straw treatment at stages V8 and R1 in the 342 bulk soil and rhizosphere soil (Fig. 3). However, at the R3 stage in the bulk soil and the rhizosphere soil the significantly (P < 0.05) higher concentration and percentage of 344 exogenous substrate N present as Inorg-N in the urea plus straw treatment than that in the urea-only can be attributed to the re-mineralization of the labile organic N by the  Table S1). In parallel, during rapid crop growth (V8), both inorganic N and low-molecular-weight compounds can be assimilated by

364
As maize growth proceeded, a shortage of available N in the soil enhanced the 366 competition for N between plants and microorganisms, further resulting in significantly (P < 0.05) less urea N present as MBN in rhizosphere soil than that in the bulk soil under the urea 368 plus straw treatment, as shown by the concentration of urea N present as MBN in rhizosphere soil being significantly (P < 0.05) lower 6.7 -77.0% than that in the bulk soil from stages V8 370 to R6 (Fig. 5, Table S1). Moreover, N insufficiency can stimulate the decomposition of native soil N to meet the demand of the maize and microorganisms for N [52][53][54]. These conclusions are supported by the observed decrease in the concentration and percentage of exogenous substrate N present as PON and the increase in the percentage of straw N present as Inorg-N 374 in rhizosphere from stages R1 to R6 in comparison with the bulk soil ( Fig. 6b & d, Table S1).

Maize growth stages
At the V8 stage the significantly (P < 0.05) higher 18.6% of exogenous substrate N 378 uptake in roots in the urea-only treatment than the urea plus straw treatment, and the non-significant difference in exogenous substrate N uptake in the shoots in both treatments 380 may be attributed to the rapid growth of maize accompanied by the N transfer from roots to shoots [4] and the higher available N supply in the urea-only treatment than the urea plus 382 straw treatment (Fig. 3). As plant growth proceeded, shoot N uptake from exogenous substrate showed a stable trend from stage R3 to R6 while root N uptake from exogenous 384 substrate showed decrease from stage R1 because of some of the roots senescing and further leading to a decrease in root biomass after stage R1 (Fig. S1)  The combination of urea and straw affected the distribution of exogenous substrate N in soil-crop system. Compared to the urea-only treatment at an equivalent N application rate the 396 urea-N recovery increased by 5.4% in the urea plus straw treatment and straw recalcitrant N in the urea plus straw treatment limited exogenous substrate N uptake in maize despite the 398 26.0% straw N recovery in maize at the maturity (Fig. 1, Table 2). Moreover, straw labile C in the urea plus straw treatment promoted the immobilization of urea N and the contribution 400 of exogenous substrate N to different soil N pools as shown by the significantly (P < 0.05) higher percentages of straw N and urea N in DON, MBN, PON, or MTN during the maize 402 life cycle in most cases (Figs. 3-7). Rhizosphere soil further altered the contribution of exogenous substrate N to different N pools, e.g. rhizosphere soil showed a higher percentage 404 of straw N and urea N present as Inorg-N and MTN in each treatment compared with the bulk soil from R1 to R6 stages (Fig.3 c, d; Fig. 7c, d). Overall, straw N availability needs to be 406 considered after the combined application of chemical fertilizer and straw in agroecosystems, and the increase in straw N availability should be take into account the interactions between 408 fertilization practices and the rhizosphere.